Reliable, real-time heart and respiratory rates are key vital signs used in evaluating the physiological status in many clinical and non-clinical settings. Measuring these vital signs generally requires superficial attachment of physically or logistically obtrusive sensors to subjects that may result in skin irritation or adversely influence subject performance. Given the broad acceptance of ingestible electronics, we developed an approach that enables vital sign monitoring internally from the gastrointestinal tract. Here we report initial proof-of-concept large animal (porcine) experiments and a robust processing algorithm that demonstrates the feasibility of this approach. Implementing vital sign monitoring as a stand-alone technology or in conjunction with other ingestible devices has the capacity to significantly aid telemedicine, optimize performance monitoring of athletes, military service members, and first-responders, as well as provide a facile method for rapid clinical evaluation and triage. Heart rate (HR) and respiratory rate (RR) are essential vital signs for evaluating the physiologic status of children and adults in clinical and non-clinical settings. These two vital signs constitute the initial measurements in acutely ill patients and provide the basis for clinical severity stratification , 2 as weThere are numerous methods for monitoring HR and RR, but most require the attachment of superficial sensors to the body. HR can be monitored using electrical methods such as electrocardiogram (ECG), optical methods such as photoplethysmography , or mechanical methods such as ballistocardiography. RR can be monitored directly using trans-thoracic plethysmography and expired gas analysis approaches, or indirectly using advanced processing methods applied to PPG . All of We present initial proof-of-concept experiments in a porcine model to show that HR and RR can be measured simultaneously and with high fidelity from within the GI tract using a single acoustic waveform. Using an endoscopically-guided miniature electret microphone, we measured acoustic data along the GI tract from the mouth to the colon. We evaluated the impact device contact with GI tissue and previously ingested food had on acoustic data quality. We then developed a robust signal processing algorithm to analyze these raw waveforms. Our results support that an ingestible, ultra-miniature acoustic monitoring device could accurately measure vital signs. This technology is likely to be adaptable to a wide range of clinical and non-clinical uses.2 analysis), and a superficial microphone positioned directly above the heart. A total of ~407 minutes each of HR and RR data were collected from all segments of GI tract over the course of four experimental days in six Yorkshire pigs. Specifically, 80.3, 66.7, 149.7, 54.3, and 60.3 minutes of raw audio data were collected from the oral cavity, esophagus, stomach, proximal duodenum and rectum respectively. A schematic of our experimental setup and a representative dataset is shown in We performed physiological monitoring experiments in six sedated Yorkshire pigs using an endoscopically-guided electret microphone to collect acoustic waveforms along the GI tract. We concurrently recorded physiological waveforms using a standard veterinary vital signs monitor, including external 3-lead ECG, PPG, capnography , and duodenum: the HR is detected within 5 bpm 97%, 95%, 98% of the time, respectively. The RR is detected within 5 breaths per minute 84%, 82%, 88% of the time in these locations, though many errors in these locations are due to the AMDF algorithm’s valley-finding which very predictably doubles the respiratory rate see  below. RAn ingestible monitoring device must be able to function under a variety of common GI environmental conditions, including fasted and fed states and device contact with tissue. To demonstrate our system’s applicability to these heterogeneous GI environments, we measured waveforms in the gastric content , and in contact with the gastric wall . All these areas demonstrated HR and RR values in good agreement with external vital sign monitor values. The HR median absolute percent error was noted to be 0% in the proximal third, gastric pool, and distal third. For RR, the median percent error was 4.3%, 10%, and 6.7% in the proximal third, gas bubble, and distal third. Additional median percent errors are noted in non-wearable formats has seen little development. Wearable systems can be associated with skin irritation from allergic responses or repetitive abrasion during extended use in high physical activity settings, as well as from constriction, and all are subject to the logistical burdens of user compliance. Furthermore, wearable systems are not always capable of directly measuring some key physiological variables externally, namely core temperature.Physiological status monitoring is central to the clinical evaluation of patients and increasingly used in non-clinical settings for safety  and performance monitoring . Though significant development has focused on low-profile external vital sign monitoring systems, extended monitoring in in vivo in a large animal model at various locations along the GI tract. Signal fidelity was maintained with and without microphone tissue contact, as well as within solid and liquid food material. Our signal processing algorithm robustly detected HR and RR in excellent agreement with measurement from standard external vital signs monitors (PPG and capnography) in the majority of the GI tract.To address these limitations, we have established a new technique for physiologic status monitoring using technologies and methods compatible with ingestion. Our method is capable of sensing primary vital signs (HR and RR) in a heterogeneous set of environments in the GI tract using a single sensing modality. We have demonstrated measuring HR and RR using this technique in vivo, yet non-implantable medical devices able to monitor difficult-to-measure variables (such as core temperature) simultaneously with HR and RR. Burdens associated with implantable devices, namely the need for a surgeon to introduce the device and potential negative health outcomes , have prevented their wide adoption, and which ingestible devices overcome.Ingestible devices have a number of advantages over wearable systems. The ergonomic profile of an ingestible-sized device is minimal; unless a patient has a specific contra-indication for such a device , the user will remain asymptomatic for the duration of monitoring. Such small devices will be similar in size to a bolus of food or pharmaceutical capsule  and will remain free-floating in the GI tract; with an appropriate material choice for the device capsule, this will minimize any abrasive irritation of the GI mucosa. There is also the possibility of implementing this sensor concept as a dissolvable electronic device, thereby eliminating contra-indications. Furthermore, ingestible electronic systems offer the possibility for Our signal processing algorithm produced accurate HR estimates for the majority of the GI tract. Overall HR performance is very strong in the esophagus, stomach, and duodenum: concordance with external PPG is detected within 5 bpm 97%, 95%, 98% of the time, respectively. While we did measure an acoustic waveform in the mouth and colon, agreement with standard vital sign monitoring was poor. Because of the excellent performance of the sensor and algorithm proximal to the heart and lungs, we suspect these sites were too distant from the heart and lungs for the sensitivity of the particular microphone chosen (-45dB ±4dB). Higher sensitivity electret or MEMS microphones may be chosen, but must also be small enough for ingestion and have sufficient frequency sensitivity (from 10 to 40 Hz for both HR and RR).Percent error histograms  show simTo statistically measure our algorithm’s performance, we performed an equivalency test between HR and RR derived from our acoustic approach and the gold-standard value based upon the median difference between matched pairs. We similarly performed a test for correlation significance between acoustic estimates and gold-standard values.  for the esophagus, proximal stomach, and stomach with food, but we were unable to demonstrate significant correlation. As mentioned above, the full histograms see  imply paOne considerable limitation to our proposed system is that GI transit time varies considerably among healthy individuals . Our iniin situ treatment delivery devices to provide remote, automated systems for rapid diagnosis and treatment of high-risk patients.Extended vital sign monitoring via ingestible devices could be applied in emergency triage settings in the field, for post-operative patients, outpatient telemedicine monitoring, and performance measurements . In this study we measured respiratory and heart rates ranging from 6–56 breaths/min and 48–128 beats/min with high correlation to standard monitoring devices. Further testing to the extremes encountered in active  or pathophysiologic states will be required to fully delineate the limitations of this technology for monitoring, but are the focus of current hardware and algorithm development efforts. Continuous auscultation, when coupled with more advanced signal processing algorithms focusing on anomaly detection or machine learning-based classifiers, could provide advanced diagnostic tools for pulmonary  or cardiac  pathologies. Future vital sign device designs may even include coupling with In vivo porcine studies were performed in 6 Yorkshire pigs weighing between 50 and 65kg. All animals were female and between 6–7 months of age. For evaluations free of food material, the pigs received a liquid diet for 48 hours prior to the procedure. Otherwise animals were fasted overnight. On the day of the procedure, the morning feed was held and the animal was sedated and intubated. Following induction of anesthesia with intramuscular injection of Telazol (tiletamine/zolazepam) 5mg/kg, xylazine 2mg/kg, and atropine 0.05 mg/kg, the pigs were intubated and maintained on isoflurane 1–3%. An endoscope guiding a miniature electret microphone  was introduced in the esophagus and recordings taken from the mouth, esophagus, stomach, and duodenum with and without tissue contact. Additionally an enema was administered and recordings were taken from the colon. All procedures were conducted in accordance with protocols approved by the Massachusetts Institute of Technology Committee on Animal Care Protocol #0113-009-16. After data collection, all animals were returned to the animal colony and used in other experimental protocols.Raw acoustic data was sampled at 44.1 kHz. We built a custom level-shifting device (shifting from 0-5V to 0–1.6V) capable of interfacing with existing medical monitoring equipment attached to the animal  and with a multi-channel A/D converter (Roland Octa-Capture™) capable of handling all 5 input streams  and outputting via a USB connection to a laptop running Audacity audio collection software.Our algorithm was specifically designed for implementation in a low size, weight, and power device. First, the 44.1 kHz signal is segmented into 20s segments and copied into two parallel processing tracks; one track ultimately estimates HR, and the other RR. All acoustic data is highpass filtered with a 100dB attenuation Chebyshev Type II filter with a transition band from 5-10Hz to eliminate motion artifacts caused by mechanical compressions rather than acoustic vibrations. This filter is digitally implemented and would not necessarily be part of the final design. The signal in both HR and RR tracks is filtered with an emulated analog RC bandpass filter constructed from a low pass filter with a 3 dB cutoff frequency at 40 Hz and a high pass filter with a 3 dB cutoff frequency at 10 Hz. Finally a 60Hz comb filter is applied to reduce line noise and its harmonics. Both signals are then decimated by a factor of 450 to a sampling rate of 98 Hz to emulate minimal data collection, storage, and processing capabilities.x[n], is normalized to a maximum amplitude of -1 or 1 and is denoted x[n]norm. Then, a sliding window over the 20 second frame computes the energy, E[n], according to Nwindow = 25 samples or approximately 0.25 seconds, and the RR track uses a window of Nwindow = 98 samples or 1 second.The analog filtered signal, D[n] similar to the output from an autocorrelation operation, but without using any multiplications [NE is the number of samples in the frame of the energy signal . d varies from 0 to 196 (2 seconds) or 0 to 980 (10 seconds) for HR or RR estimation, respectively, because normal cardiac and respiration cycles have periods less than these upper bounds.The average magnitude difference function (AMDF) computes a waveform ications   As the segments slide over each, the heart beat or breath that was well aligned in each segment becomes misaligned, and the AMDF value increases. However, eventually the segments will have slid far enough apart that the original heart beat or breath will overlap with the second heart beat or breath in the frame. Because of the consistency in heart rate and breath morphology as an output from the energy stage, the AMDF function will compute a small, non-zero value before increasing once again. If several heart beats are within a frame, the AMDF function will appear to have a ripple or sinusoidal pattern as the first heart beat overlaps each of the successive heart beats. The ripple period is the period of the vital sign to be estimated.Ratealgorithm is the result of the method above, and Rategold-standard is the rate derived from PPG or capnography for HR and RR, respectively. Histograms of these values, normalized by the total counts for each anatomic site, are reported in We compared the results from the signal processing algorithm above with the “gold-standard” PPG and capnography signals to determine algorithm performance. Data from these gold-standard methods was resampled to 98 Hz using MATLAB’s  resample function, which applies an anti-aliasing filter, and then processed through the AMDF and valley detection stages as described earlier. Only HR values between 40 and 200 beats per minute and RR values below 60 breaths per minute were accepted as physiologically reasonable. In the rare event (10/1229 frames for RR and 6/1234 frames for HR) that the valley finding function did not return an estimate from the acoustic data but the gold standard estimate was valid, the frame was excluded from analysis. For each 20s segment, an absolute percent error is calculated, defined asWe used two methods to statistically determine output similarity between the gold-standard methods (PPG and capnography) and our acoustic approach. The first was a median difference between matched pairs for repeated samples equivalency test, which checks for bias while still being robust to outlier failures from period doubling; the second was a test for correlation significance, which quantitatively assesses whether our acoustic based estimates track the corresponding gold-standard vital signs.For the equivalency test, the null hypothesis is that a non-tolerable difference exists between the acoustic and gold standard estimates, and the alternative hypothesis is that the median difference is equivalent to 0 within the tolerance limits (at the 95% confidence level). We chose a non-tolerable difference of +/- 5 bpm for both the HR and RR median differences. We performed a bootstrap procedure for the equivalency test given that our data is non-normal and has few independent samples , though we have repeated samples for each pig at a given location. For each location, we randomly sampled one measurement pair  from each pig that had data for that location. We computed the difference for the pair and took the median of the differences for all the pigs to create one bootstrapped datum. We repeated this procedure 10000 times to create a distribution of median difference errors, and then computed the 5 percentile and 95 percentile points. If both the 5 percentile and 95 percentile points lie within the tolerable difference region of +/- 5 bpm, we reject the null hypothesis (that the median is significantly different than 0 within the tolerance bounds) in favor of the alternative. Note that 5 and 95 percentile points are used for a 95% confidence level instead of 2.5 and 97.5 percentiles because both ends of the confidence interval must be within the tolerance bounds to reject the null hypothesis.A correlation test was also performed with a similar bootstrapped approach. For each location, one measurement pair was taken for each pig. The Pearson correlation among pairs was computed to create one bootstrapped datum. We repeated this procedure 10000 times to create a distribution of correlations, and then computed the 2.5 and 97.5 percentile points. If the 2.5 to 97.5 percentile range did not include 0, then we reject the null hypothesis (that our acoustic measurements are uncorrelated with the gold standard) in favor of the alternative that data are correlated at the 95% confidence level.In the case of only one pig being available for the stomach with food condition, the samples were treated as independent and half the samples were randomly picked to create a bootstrap datum. In one case, the gold-standard vital sign was constant, and consequently, we can only report on median difference error and not report a correlation value.


Overall, the failure to finish rate in New Zealand, 2.88 per 1000 horse starts (95% CI 2.64–3.12), was lower than international figures for race day catastrophic injury. Racing and environmental variables such as horse experience, race distance and season were associated with failure to finish a race. Catastrophic injury accounted for approximately half the failure to finish events. Jockey falls were positively associated with less experienced jockeys and horses.The objective was to describe the incidence of failure to finish a race in flat-racing Thoroughbreds in New Zealand as these are summary indicators of falls, injuries and poor performance. Retrospective data on six complete flat racing seasons  of all Thoroughbred flat race starts from 1 August 2005 to 31 July 2011 were obtained. The incidence of failure to finish events and binomial exact 95% confidence intervals were calculated per 1000 horse starts. The association between horse-, rider- and race-level variables with the outcomes failure to finish, pulled-up/fell and lost rider were examined with a mixed effects Poisson regression model. A total of 544 horses failed to finish in 188,615 race starts with an overall incidence of 2.88 per 1000 horse starts (95% CI 2.64–3.12). The incidence of failure to finish horses across each race year showed little variability. In the univariable analysis race distance, larger field size, season, and ratings bands showed association with failing to finish a race. The overall failure to finish outcome was associated with season, race distance and ratings bands (horse experience and success ranking criteria). In the multivariable analysis, race distance and ratings bands were associated with horses that pulled-up/fell; season, apprentice allowances and ratings bands were associated with the outcome lost rider. The failure to finish rate was lower than international figures for race day catastrophic injury. Racing and environmental variables were associated with failure to finish a race highlighting the multifactorial nature of race-day events. Further investigation of risk factors for failure to finish is required to better understand the reasons for a low failure to finish rate in Thoroughbred flat races in New Zealand. In recent years, there has been much attention focused on the quantification of catastrophic and musculoskeletal injury, and risk factors for these in Thoroughbred flat racing . EpidemiWhilst variation between racing jurisdictions may prevent the application of a single global solution, it does provide the opportunity to examine the moderating effects of the racing industry structure on risk factors for, and rates of, injury. To fully interpret these moderating effects it is important to describe the complexity and structure of the racing system within each jurisdiction. Within New Zealand, the production process preceding race training (growth and development leading to yearling preparation) and the training milestones leading up to the first race start have been well described ,8,9,10, The interaction of the production process with the progression through training , and theIt is important to consider each event reported  as part of an integrated continuum of the interaction of cyclic load  and environmental challenge, and not as discrete entities ,19. The Racing and racing injuries could therefore be regarded as part of a complex system. A complex system is a term used to describe how relationships between parts give rise to the collective behaviors of a system and how the system interacts and forms relationships with its environment . An examMany racing jurisdictions present detailed racing event data from which the horses failing to finish a race can be identified. As part of the rules of racing the Racing Integrity Unit are responsible for producing a stipendiary stewards’ report for each race, detailing any events that occurred during the race. Failure to finish data incorporate a spectrum of events ranging from jockeys “pulling-up” a horse because it was failing to “run on its merits” and suspected injury through to catastrophic injury and jockey falls. As such, failure to finish data provides a holistic system based measure that includes components of racehorse welfare, jockey safety and confidence of the betting public. Thus failure to finish provides an opportunity to screen the performance of a racing jurisdiction for the reliability and consistency of racing. There is little published information on the number of horses failing to finish races in New Zealand or possible associations relating to horses not finishing races. Additionally, there appears to be limited analysis of risk factors for failure to finish data in racehorse populations and there is an opportunity to gain greater understanding of the events that occur during a race that prevent horses from finishing. The aim of this study was to describe the incidence of failure to finish a race and investigate risk factors for failure to finish events pulled up and lost rider in flat-racing Thoroughbreds in New Zealand. A retrospective cohort study was used to investigate all Thoroughbred flat race starts in the six years from 1 August 2005 to 31 July 2011; data were obtained from New Zealand Thoroughbred Racing (NZTR). Data available included race date, race track, race number, race class, race distance, track condition (or ‘going’), penetrometer reading, horse name, horse age, horse sex, trainer, trainer location (city), finishing position in race, barrier draw (position in the starting gates), carded weight , carried weight  and domestic rating . i.e., the starting gate was released) and failed to cross the finish line. Failure to finish was classified as: pulled up (when the jockey pulled the horse out of the race), fell (when the horse fell during the race), lost rider (when the jockey was dislodged from the horse), brought down , and ran off (when the horse ran off to the outside of the racecourse). A horse start occurred whenever a horse entered the starting gate for a race and the gate was released. Any horse involved in an incident that occurred prior to the release of the starting gate and was deemed a late scratching (non-starter) by race day officials was excluded from the study. A horse could contribute several starts and more than one failure to finish event over the study period. Failure to finish occurred whenever a horse started in a race  and screened for errors using exploratory data analysis. Continuous data were assessed for normality using the Shapiro-Wilks test. The continuous variables that were categorised into groups included: weight carried (quartiles), race distance (quartiles) and field size (number of starters in the race) (quartiles). New variables were created for race year, season , field size and whether or not the jockey had an apprentice allowance. Apprentice jockeys in New Zealand claim a weight reduction  on the handicap weight of horses they ride depending on previous experience. Under current rules an apprentice that has 0–5 career wins claims 4 kg, 6–30 wins claims 3 kg, 31–60 wins claims 2 kg and 61–100 wins claims 1 kg . Ratingsp < 0.2) with the outcomes were analysed in multivariable mixed effects Poisson regression models (for each outcome separately) fitted in a backwards step-wise fashion. A postestimation goodness-of-fit test was performed to test for overdispersion in each multivariable model using the Pearson chi-square and deviance chi-square test, then a random effect for horse was added to each model to adjust for clustering at the horse-level. Biologically plausible interaction terms were assessed in the final models. A Kaplan-Meier curve was used to graphically present the failure to finish by race distance. Statistical significance was set at p < 0.05 and analysis conducted in STATA 12 .The incidence of failure to finish and corresponding binomial exact 95% confidence intervals were calculated and reported as events per 1000 horse starts for all variables. Failure to finish events were sub-categorised into a pulled-up outcome and a lost rider outcome. Poisson regression was used to estimate incidence rate ratios (IRR) with 95% confidence intervals (95% CI) for the outcomes failure to finish, pulled-up, and lost rider within the univariable analysis. Variables showing association (n = 17) and ran off (n = 7).There were 188,616 race starts for 16,646 individual horses during the study period. The data represented 6072 2-year-old starts, 43,228 3-year-old starts and 139,316 4-year-old and older starts. During the study period the horses contributed a median of 7 (IQR 3–16) race starts. There were 544 failure to finish events providing an overall incidence of 2.88 per 1000 horse starts (95% CI 2.64–3.12). There was little variation in the incidence of failure to finish between racing years. The lowest incidence rate was 2.66 per 1000 starts (95% CI 2.13–3.28) in the 2009/10 racing year and the highest was 3.10 per 1000 starts (95% CI 2.52–3.78) in the 2007/08 racing year. There was no significant effect of horse age associated failure to finish, and no significant difference between ages in the older horse category of 6 years old and older. Of the 544 failure to finish events, 507 (93.2%) horses had single events, 17 (6.2%) horses contributed two events, and one (0.6%) horse had three events. Overall there were 269 (49.4%) pulled-up, 72 (13.2%) fell and 179 (32.9%) lost rider events, other failure to finish events were brought down  with both failure to finish and pulled-up. There was a greater failure to finish rate in autumn compared to spring, and at all rating bands 55 or greater compared to rating band 50–54. The rate of pulled-up was significantly lower in rating bands 66–75 and 86–115 compared to rating band 50–54. Multivariable mixed effects Poisson regression models of variables significantly associated with the outcomes failure to finish, pulled-up and lost rider are presented in Season, apprentice allowance and rating band were associated with lost rider in the multivariable model . The ratn = 16,646) prevented the use of Cooks Distance to test for model validity. The addition of the horse as a random effect improved the goodness of fit of the model. The Poisson model was the best fit for the data rather than a negative binomial regression. The large number of horses included as a random effect  of the New Zealand failure to finish records. Internationally, within the literature, musculoskeletal injury reported on race day ranges from 3.1 per 1000 starts  to 4.4 pvs. 3.7–4.4/1000 for the United Kingdom/ Ireland and France [Compared to international studies there is also a low incidence of race day falls by flat race jockeys in New Zealand (2.2/1000)  vs. 3.7–d France  and Austd France , which sd France , where tSeason (Spring) and race grade (maiden/lower rating horses) were positively associated with lost rider. The race grade effect was also supported by a study of predictors of jockey falls in flat racing in Australia and followed up with the study on early career jockeys ,32. The per se/individually are not uniquely different from other major racing jurisdictions with similar range of field sizes and number race starts reported for flat racing horses in the UK [Contributing factors to the low failure to finish rate may relate to the structure and the type of racing within New Zealand. Racehorses within New Zealand typically run over 1400 m in races with 11 horse fields and have a median of 5 (IQR 2–8) starts per racing season . These pn the UK  and USA n the UK . Distribn the UK ,30,35. GThe apparent low level of race starts, despite a relatively low cost racing structure, may be due to the use of trials  by racehorse trainers in New Zealand for education and in the final stages of race preparation. A cross-sectional survey of 2-year-old training practices identified the use of trials for education and training milestones within this age group as well as a strong emphasis in 2-year-old training being for education and conditioning purposes rather than with the primary objective of obtaining a race start . The presence of the rating band 50–54, which is associated with maiden and 2-year-old racehorses, within the multivariable model indicated that despite these potentially positive characteristics of New Zealand racing there is still an increased rate of failure to finish, and specifically lost rider, associated with younger and more inexperienced horses. This pattern of greater risk with the less experienced/lower grade horses has been reported with jockey falls in Australia . Within Previous studies have shown that racehorse trainers in New Zealand provide horses with voluntary breaks from training (a “spell”) in order to allow the horse time to “strengthen and develop” ,36. The Despite racing on turf tracks within a temperate climate, racetrack surfaces in New Zealand are consistently reported in the good to dead range of going . It is only in winter that the median going decreases to heavy (penetrometer reading 4.3) . HoweverOverall, the failure to finish rate was lower than international figures for race day catastrophic injury. Racing and environmental variables such as horse experience, race distance and season were associated with failure to finish a race, highlighting the multifactorial nature of race-day events. Investigation of the biological and industry based drivers of the risk factors, particularly season and horse experience are required to identify pragmatic management changes to reduce the risk of failure to finish. Further investigation of risk factors for failure to finish is required to better understand the reasons for a low failure to finish rate in Thoroughbred flat races in New Zealand.


In face-to-face bargaining tasks human adults almost always agree on an equal split of resources. This is due to mutually recognized fairness and equality norms. Early developmental studies on sharing and equality norms found that egalitarian allocations of resources are not common before children are 5 or 6 years old. However, recent studies have shown that in some face-to face collaborative situations, or when recipients express their desires, children at much younger ages choose equal allocations. We investigated the ability of 3.5 and 5-year-olds to negotiate face-to-face, whether to collaborate to obtain an equal or an unequal distribution of rewards. We hypothesized that the face-to-face interaction and interdependency between partners would facilitate egalitarian outcomes at both ages. In the first experiment we found that 5-year-olds were more egalitarian than 3.5-year-olds, but neither of the age classes shared equally. In the second experiment, in which we increased the magnitude of the inequality, we found that children at both ages mostly agreed on the unequal distribution. These results show that communication and face-to-face interactions are not sufficient to guarantee equal allocations at 3–5 years of age. These results add to previous findings suggesting that in the context of non-collaboratively produced resources it is only after 5 years of age that children use equality norms to allocate resources. Early research on the development of fairness found that it is only from 5–6 years onwards that children exhibit inequality aversion. In the majority of these early studies, subjects were interviewed and asked how they would allocate rewards between two hypothetical partners –3. In soMore recently, economic games that investigate altruism and equality have found that, when subjects are involved and make real decisions, winning more or less rewards depending on their choices, children at younger ages (4–5 years of age), sometimes choose to altruistically share resources with others, and that they even share low-value resources in an egalitarian way ,7. SimilFehr, Bernhard and Rockenbach  reportedIn the studies reviewed above subjects make solo decisions on how to distribute the resources, but a few studies have also investigated the development of equality in interactive sharing situations. Birch & Billman  took a fA more recent line of studies in which peers interact face-to-face and both individuals in a dyad can influence the final outcome has found that collaboration or joint acquisition of resources enhances equality. In these studies pairs of 3-year-olds shared equally  when they had acquired the resources collaboratively by simultaneously pulling on a rope in order to move a baited board within reach. However, they did not share equally when they pulled independently of each other , or did not pull at all, since the rewards were already reachable from the start of the trial  .Previous developmental studies looking at face-to-face bargaining have generally investigated children older than six –22. For In the present study we were interested in the level of equality exhibited by pairs of 3.5 and 5-year-olds when they are given the choice between an equal and an unequal distribution of rewards. How do children solve a conflict of interests in which one child’s best possible outcome is associated with a worse deal for the partner? From which age can they overcome self-serving biases and agree on mutually acceptable and fair outcomes? It has recently been suggested  that youWe presented dyads of 3.5- and 5-year-old children with two baited boards, one holding 2 rewards for each child and the other holding 3 (or 6 in Experiment 2) rewards for one child and 1 reward for the other child. Children were required to pull simultaneously to move the board within reach and were instructed to choose only one of the boards. We chose a collaboration task to give both children in the dyad bargaining power and create interdependency between partners. However, it is important to emphasize that the negotiation process took place prior to collaboration. The study resembles previous studies, in which children could choose between two predetermined options with equal or unequal resource distribution –12, but We chose 3.5-year-olds since the recent collaboration studies mentioned above have shown that 3-year-olds can occasionally share equally. In addition, we tested 5-year-olds since many studies have shown that with increasing age children become increasingly prosocial and more egalitarian when distributing resources , 25, 26.Our hypothesis was that at 5 year of age, if not earlier, children would be able to agree on an equal and egalitarian outcome. Since previous research has shown that from 3 years of age children react negatively to disadvantageous inequality ,15 and cAlternatively, based on previous studies showing that children younger than 8 are rarely willing to pay a cost to reduce advantageous inequality, and in the present task choosing the equal option is costly (children would lose 1 and 4 rewards in Experiments 1 and 2 respectively) one could also hypothesize that they would struggle to agree on the equal option.Since gender, familiarity and friendship between partners have been found to play a role in how children’s make decisions about rewards’ allocations, we also included these variables in the analysis. Gender differences have not always been found but when they emerge it is normally girls that behave more prosocially . RegardiM = 3.9 years, range = 3.5–3.10 years) and the other half (N = 12) 5-year-olds . An additional nine dyads with 3.5-year-olds were tested, but these were not included in the final sample due to equipment failure  and not passing the pre-test within 9 trials .We tested 48 preschoolers in same-sex dyads . Half of the dyads (N = 12) were 3.5-year-olds  with whom they spent a lot of time playing in the same class during day care (as reported by the teachers) or with an unfamiliar partner (“unfamiliar”) they did not know, but who also attended the same day-care centre. Unfamiliar partners were thus not disliked peers but strangers or acquaintances at the most, whereas familiar partners were friendsThe cooperation apparatus was adapted from studies with chimpanzees , 30 but The two apparatuses were placed 40–70 cm apart in a quiet room at the day-care centre, in such a way that both apparatuses were equidistant from the door through which the children entered.Warm-up phase: This phase consisted of first playing with each child separately in their playgroup. Then, children were asked if they wanted to participate in a game together with a partner. Children were then paired up with the partner  and went with the experimenters (E1 and E2) into a room, which was next to the testing room. To make the children feel more comfortable with their new surroundings—and, in half of the cases, with a new partner—E1 and E2 looked at a picture book together with the children. Afterwards, E1 showed the children a bag of gummy bears and told them that they could play a game in the room next door, in which they could win lots of gummy bears.Demonstration phase: The two experimenters sat in front of the apparatus and demonstrated how to move the board within reach by holding onto both ends of the rope and pulling them simultaneously. Meanwhile the children were sitting or standing between E1 and E2 and just watching the demonstration. This demonstration was conducted with both apparatuses. During the demonstration the plates in both apparatuses were filled with the same amount of gummy bears (3 gummy bears per plate). After the demonstration children went out of the testing room with E1.Introduction phase: It was important ensuring that children understood the game and paid attention to the contents of the 2 apparatuses before making a choice. Therefore, to acquaint children with the procedure, we introduced them to all important information little by little (E1 becoming increasingly passive in the introduction and staying outside the testing room during the pretest phase). The children waited with E1 outside the testing room while E2 baited the apparatuses. After E2 returned, the children entered the testing room together with E1. The introduction phase was split into two parts. In the first 2 trials, E1 helped the children with the order of events in the game by (1) checking with them both apparatuses and the amount of gummy bears in each apparatus and on each plate; (2) encouraging children to decide together which apparatus they wanted; and (3) after reaching a joint decision and the children pulling on the rope together, telling the children that each child would get the gummy bears of her/his plate, and that they shouldn’t split differently the gummy bears obtained (“Each keeps her own gummy bears”). It was important to tell the children that they should not share the gummy bears obtained, since we were interested in testing whether or not they would come up with an egalitarian solution before collaboration  and told to place their gummy bears into the tube to take with them afterwards.n et al.  have alrPre-test phase: This phase was designed to ensure that children understood and adhered to the game rules and were motivated to obtain the largest possible amount of gummy bears. Children waited outside the testing room while E2 baited the apparatuses. Both plates in each apparatus were baited with the same amount of gummy bears. However, one apparatus had a larger amount of gummy bears than the other apparatus. There were three types of trials depending on the number of gummy bears per plate and apparatus: 1–1 vs. 2–2; 1–1 vs. 3–3; and 2–2 vs. 3–3. The side of the apparatus containing the largest amount was counterbalanced across trials. Pairs were required to succeed in pulling the apparatus with the largest amount of gummy bears in three consecutive trials. All pairs participated in a minimum of three and a maximum of nine trials. If after nine trials subjects were still inconsistently choosing different apparatuses (or consistently choosing the apparatus with less gummy bears) they were excluded from the test phase. On average 3.5-year-old dyads passed this pre-test after 5.3 trials and 5-year-old dyads after 3.9 trials (eight 3.5-year-old dyads were excluded as mentioned above).Test phase: In this phase children were faced with the choice between an equal apparatus (2 gummy bears per plate) and an unequal apparatus (1 on one plate and 3 gummy bears on the other plate). The position of the apparatus (left or right) containing the unequal split was counterbalanced across trials and within dyads. The position of the plate (left or right) in the unequal apparatus holding the larger amount was also counterbalanced across trials and dyads. A trial started when children entered the testing room and ended either if they failed to retrieve the gummy bears (i.e. pulling out the rope or failing to succeed for 120 seconds) or after they had succeeded in retrieving the gummy bears. Each dyad participated in four test trials.A number of behavioural measures were coded within every trial. A “no agreement” occurred if children could not come to an agreement within two minutes, or if at least one child tried to pull the rope alone and it became unthreaded from the wheels, thus making the rope ineffective, or if the children argued so harshly that E1 had to intervene and stop the test. For “agreements” we discriminated between successful and unsuccessful agreements. Successful agreements were those cases in which children agreed on pulling one apparatus and actually succeeded in obtaining the gummy bears. Unsuccessful agreements also included those cases in which children had obviously agreed  to pull one apparatus but were not successful in getting the gummy bears because of coordination problems with the rope.To investigate the distribution of gummy bears within the dyads, we coded the amount of gummy bears each child obtained (successful agreements) per trial. Furthermore, we coded which apparatus  the children chose and obtained. Additionally, we coded the protest behaviour between children in the negotiating phase of each trial. The coding phase started when children entered the testing room and lasted until the children started pulling the rope together. We observed and categorized as protest any of the following behaviours: verbal protests , gestural protests  and physical protest (e.g. pushing away the child from the favoured side of the apparatus). We only considered protests against a small amount in the unequal tray or the equal tray and did not distinguish whether one dyad used several types of protests within a single trial . We coded whether or not the final decision of which tray dyads pulled was the result of any kind of protest or negotiation (“change of tray”). All coding was done from videotape by A.F. and A.P. M. A minimum of 20% of the trials were coded independently by a second person who was blind to the hypotheses tested .Effects of age, gender and familiarity of the partner on the equality of the rewards’ distribution: The two response measures, one being the absolute difference of gummy bears within the dyads and the other the number of trials where the apparatus with an unequal distribution of gummy bears was chosen, were analysed using Generalized Linear Models (GLMs) separately for these two dependent variables in R (Version 2.10.1). For both GLMs the predictors were age group, gender and familiarity  we used a Monte Carlo simulation . We ran two separate Monte Carlo simulations. For both analyses we simulated dyads making random decisions in favour of either of the two alternatives , with the number of decisions equalling the number of successful trials per dyad, as revealed in the experiment. For the first analysis we determined for each dyad its absolute deviation from 50% decisions for one option (the random expectation) and then averaged these across dyads. The entire process was repeated 10,000 times. If this revealed significance, then the dyads had pure strategies. Having a pure strategy means that a certain dyad did not choose between the two trays randomly, but that instead chose significantly more often than expected by chance (50%) the equal or the unequal tray. However, the pure strategies were not necessarily the same across dyads. Therefore, we conducted a second analysis in which we determined the proportion of decisions for the unequal apparatus per dyad and then averaged these across dyads. The entire process was also repeated 10,000 times. If this reveals significance, pairs’ strategies were the same across dyads . In both simulations we determined significance as the proportion of simulations revealing a score at least as far away from the expectation as the original data. The simulations were run in a script written for R [en for R  by R. MuTo investigate whether, when children decided to pull the unequal apparatus, the overall distribution of gummy bears between the children was equal , we compared (using an exact Wilcoxon signed-rank test) the absolute difference of gummy bears (per dyad) in the original data with an expected absolute difference of gummy bears (per dyad). In order to calculate the expected absolute difference (per dyad) we started by determining the number of decisions for the unequal apparatus (per dyad) and all possible distributions of the gummy bears for this number of sequences. For example, if the children decided twice to go for the unequal apparatus then there were the following possibilities: Either Child A or Child B got the big amount twice, or Child A got the big amount first and then Child B did, or vice versa. For all the possible distributions per dyad we determined the absolute difference of gummy bears received and then averaged them .In order to compare the overall distribution of rewards between children depending on how often they agreed to cooperate with the equal  apparatus we plotted for each dyad (and for each age group) the proportion of gummy bears obtained by the “highest earner” child against the proportion of trials in which the dyad cooperated to obtain the unequal split. The highest earner was the child obtaining the largest amount of gummy bears within the dyad. We calculated two different potential theoretical distributions of rewards depending on whether or not, whenever dyads agreed to pull the unequal tray, there was alternation between individuals with regards to who obtained the larger amount  Inequality pattern: One child obtained the big reward in the majority of the trials ; 2) Equality pattern: Children chose the equal split in the majority of the trials ; 3) Reciprocal pattern: Both children per dyad obtained the large reward as many times as the small reward; 4) Mixed pattern: Children chose different options so that no clear pattern could be defined.Both 3.5 and 5-year-olds agreed to cooperate by pulling one board in the majority of the trials . More specifically, there was an age effect and a gender tendency on the overall difference of gummy bears within dyads. The difference of gummy bears within the 3.5-year-olds was greater than among the 5-year-olds , and it tended to be greater among the girls than the boys  differed significantly from chance (50%). Second, we investigated whether children took turns getting the large reward when pulling the unequal tray, since this would be another solution leading to equality.We ran two Monte Carlo simulations to look at whether children’s decisions to pull one or the other apparatus  differed significantly from chance. In a first analysis we looked at whether dyads had pure strategies, that is, whether within individual dyads choices differed from 50% chance choosing equal or unequal. We found that 3.5-year-olds did not differ from chance (p = 0.45), whereas 5-year-olds did (p = 0.003). This means that within individual dyads 3.5-year-olds were not choosing one or the other tray significantly above chance levels, whereas 5-year-olds were. In order to see whether the strategies were the same across dyads, we ran a second Monte Carlo simulation. This revealed that the pure strategies of the 5-year-olds were not all in the same direction (p = 0.694), with some dyads agreeing on the equal and some dyads agreeing on the unequal apparatus. To investigate whether or not there was an overall fair distribution of rewards when children chose the unequal apparatus , we compared the observed difference of gummy bears (per dyad) with an expected difference of gummy bears using a Wilcoxon signed-rank test  by each dyad and found that 3.5-year-olds chose the equal apparatus less often than expected by chance , whereas the choices of the 5-year-olds did not differ from chance  (p = 1). Girls also chose the equal apparatus less often than expected by chance  (p = 0.038), whereas boys did not . Finally, familiar partners also chose the equal apparatus less often than predicted by chance , whereas unfamiliar partners did not differ from chance levels .At a more descriptive level we were interested in the number of dyads exhibiting a pattern of behaviour that could be characterized as: “inequality”, “equality”, “reciprocal” and “mixed”. The characterization of the dyads revealed that nine dyads of 3.5-year-olds exhibited mostly inequality, whereas the other three behaved in a mixed way. Among the 5-year-olds, six out of the 12 dyads behaved in an equal way, five showed inequality and only one dyad behaved in a mixed way .Finally, also at a descriptive level, we analysed two other measures (protests and changes in the decision of which tray to pull), that could shed light on how children negotiated and solved the conflict of interests. On average, 3.5-year-olds protested in 42% of trials  and 5-year-olds in 36% of trials . In most of the cases these protests were against the small amount (75% and 83% of protests among the 3.5-year-olds and 5-year-olds respectively). A small proportion of the protests were also against pulling the equal tray (25% and 17% of protests among the 3.5-year-olds and 5-year-olds respectively), which often occurred after the partner had obtained the large amount from the unequal apparatus in previous trials.+ = 30, n = 15, seven ties, p = 0.10). Note that in the 21% of the trials in which dyads accepted on pulling the unequal tray, one of the children managed to convince the partner to leave the equal tray in favour of the unequal tray  which percentage of unequal/equal outcomes (per dyad) was preceded by some kind of protest vs. which percentage of trials was the result of both individuals immediately pulling or one child following the other one, without suggesting or commenting anything else (no protest), (2) which percentage of the unequal/equal outcomes was the result of children changing the tray which one of them initially suggested to pull. The levels of immediate acceptance of a tray  and proportion of successful negotiation, leading to a change of trays, was not significantly different between the two age groups . Children at both ages tended to immediately accept the equal and unequal tray at similar levels . However, the proportion of equal outcomes due to dyads changing the tray that initially one of them intended to pull tended to be higher than the proportion of unequal outcomes due to a change of trays . The age difference seems to have been due mainly to the higher number of dyads among the 5-year-olds that suggested right from the beginning to pull the equal tray, and not necessarily due to their better negotiation skills.Although children could have chosen to pull the equal apparatus obtaining 2 gummy bears each, 3.5-year-olds agreed mostly on the unequal split and individuals obtaining the larger amount did not seem to care that their partner obtained a smaller amount of gummy bears even though he/she had contributed to obtaining the rewards. Five-year-olds exhibited more equality than the 3.5-year-olds, but they still did not behave in a purely egalitarian way .These results are particularly striking considering that children often protested at and did not seem happy with the unequal and disadvantageous split. Furthermore, children who were going to receive the small amount could have refused to pull until the partner agreed to pull for the equal split. One possibility is that the magnitude of the inequality presented in this experiment was not high enough to make children care that much, and therefore they just tended to follow the partner independently of what (s)he wanted to obtain. It is possible that a higher level of inequality would motivate children, including 3.5 year-olds, to behave more fairly or motivate partners to refuse to pull in order to punish selfish partners and convince these to pull the equal apparatus. This was the rationale for the next experiment.In this experiment we increased the magnitude of the inequality by placing six gummy bears instead of three on one of the plates of the unequal tray. We hypothesized that when presented with these 2 options, children at both ages would react more strongly to the inequality and would more likely agree on pulling the equal tray. Alternatively, another strategy to maximize the gains would be essentially to take turns regarding who obtains the large amount (6 pieces), thus over time benefiting both individuals in the pair .M = 3.9 years, range = 3.5–3.10 years) and the other half (N = 12) 5-year-olds . An additional five dyads were tested, but these were not included in the final sample due to experimenter errors  or because they did not pass the pre-test phase within 9 trials . Children who participated in this study had not participated in the previous one.As in the previous experiment we tested 48 preschoolers in dyads with a same-sex partner . Half of the dyads (N = 12) were 3.5-year-olds .The only difference was that in the test phase children were faced with the choice between an equal apparatus containing 2 gummy bears per plate and an unequal apparatus, containing 1 gummy bear on one plate and 6 gummy bears on the other plate.The coding and analyses were performed in exactly the same way as in Experiment 1.Both 3.5 and 5-year-olds agreed to cooperate by pulling one board in the majority of the trials . More specifically, there were effects of gender and familiarity level, but no effects of age on the overall difference of gummy bears within dyads. The difference of gummy bears among the boys was greater than among the girls, and the difference was also greater among familiar partners than non-familiar partners .We conducted the same two analyses than in Experiment 1. First, we looked at whether children’s decisions to pull one or the other apparatus  differed significantly from chance (50%). Second, we investigated whether children took turns getting the large reward when pulling the unequal tray, since this would be another solution leading to equality.The Monte Carlo simulations revealed that at both ages children did not choose the apparatus  randomly, and exhibited a preference for one or the other apparatus (3.5 and 5-year-olds: p = 0.0001). The second Monte Carlo simulation revealed that at both ages children chose the unequal apparatus significantly more often than expected by chance . The final distribution of gummy bears did not differ significantly from chance levels in either age group . This is because there were 5 and 4 dyads of the 3.5-year and 5-year-olds respectively, in which there was some alternation between individuals regarding who obtained the large amount.The more unequal outcome of this experiment was also revealed in the analysis that looked at the first choices made. We found that at both ages, at trial 1, children chose the equal apparatus less often than expected by chance . If we look at the data and split the results based on gender and familiarity of the partner, there is also no difference. Both boys and girls and familiar partners and non-familiar partners chose the equal apparatus significantly less often than expected by chance .The characterization of the dyads’ general pattern as “inequality”, “equality”, “reciprocal” and “mixed” revealed that eight dyads of 3.5-year-olds exhibited mostly inequality, one dyad chose mostly “equal” and three reciprocally. Among the 5-year-olds, nine out of the 12 dyads exhibited inequality, one equality and two behaved reciprocally .First, we looked at children’s protests at a descriptive level and found that on average, 3.5-year-olds protested in 52% of trials (only 1 out of 12 dyads never protested) and 5-year-olds in 42% of trials . In most of the cases these protests were against pulling for the small amount (92% and 95% of protests among the 3.5-year-olds and 5-year-olds respectively). A small proportion of the protests were also against pulling the equal tray (8% and 5% of protests among the 3.5-year-olds and 5-year-olds respectively), which often occurred after the partner had obtained the large amount from the unequal apparatus in previous trials.In this experiment we couldn’t analyse the negotiation process as in Experiment 1  since only one dyad at each age group agreed on pulling the equal tray. Children accepted the unequal tray immediately in 42% and 40% of the trials (3.5- and 5-year-olds respectively). In the remaining 58% and 60% (3.5- and 5-year-olds respectively) of the trials that ended in an unequal outcome there was some communication or discussion between partners.Dyads at both ages agreed in the majority of the trials. However, levels of agreement were slightly lower than in the previous experiment, in particular among the 5-year-olds. Three and 5-year-olds agreed almost exclusively on the unequal split, and they did this from trial 1. When looking at the distribution of rewards after pulling the unequal apparatus, we found that both age groups did not differ from chance, meaning that they were not completely unfair  but also not completely fair (taking turns). This is because, despite the majority of dyads agreeing on a rather unequal deal, a small number of dyads at both ages took turns obtaining the large amount of the unequal tray. Thus, the higher inequality of this experiment did not lead to pure equal outcomes among the 3.5 or 5 year-olds. Children in the advantageous position neither suggested pulling for the equal split, nor did children in the disadvantageous position manipulate their partners into pulling the equal tray by refusing to pull. Basically, we can conclude that the behaviour of the 3.5-year-olds was similar to that of the previous experiment, whereas 5-year-olds exhibited more inequality. Very few dyads in both age groups (3 per age group) developed an alternating fair strategy.We found a gender and familiarity effect, with boys and familiar partners sharing less fairly than girls and unfamiliar partners respectively. The gender effect in this experiment would be in line with the results from several developmental studies that have reported that girls behave more pro-socially than boys , 32, 33.The results of these two experiments show that children of 3.5 and 5 years of age do not choose an equal allocation of rewards in a face-to-face bargaining situation. This seems to be the case even when children depend on each other and have the option of communicating and negotiating with the partner by way of face-to-face interaction. In Experiment 1, 3.5-year-olds agreed mostly on an unequal outcome, whereas 5-year-olds exhibited less inequality than 3.5-year-olds but still did not preferentially agree on the equal outcome. In Experiment 2 children at both ages exhibited mostly inequality and some few pairs developed a turn-taking and fair strategy.The inequality observed in Experiment 2 among the 5-year-olds  could be due to the higher costs involved at giving up the unequal tray in this last experiment. In Experiment 1 the cost to keep the partner happy was smaller (1 reward) than in Experiment 2, where one child had to forego 4 rewards to maintain equality. Several other studies have also found that equality is often influenced by the costs subjects must endure to maintain it , 12.The results of this study show that in conflict-of-interest situations in which 3.5 and 5-year-olds have a choice between an unequal and an equal split, the outcome is mostly unequal. Children in the most favourable position were willing and happy to obtain the large amount, independently of the consequences that this had for their partner, showing no aversion to advantageous inequality , 14, 36.In any case, the results are particularly surprising considering the fact that children were required to collaborate by way of face-to-face interaction with peers from the same school and to do so repeatedly, which is supposed to influence pro-sociality positively. Whenever there is a possibility for future interaction and in particular if subjects are dependent on each other , subjects might behave pro-socially because of the expectation of future benefits (or future help needed) from the partner. Of course, this argument could be an explanation for children’s acceptance to pull the unequal and disadvantageous split. Actually, there were trials in which children convinced their partners to leave the equal tray and pull the unequal tray, or change positions in the unequal tray to their advantage. This interpretation would also be supported by the findings that outcomes among familiar partners were more unequal than among unfamiliar partners. However, it was never the disadvantaged child’s initiative, and we cannot know whether children were motivated by friendship considerations or were simply trying to avoid conflict. Since children never agreed or promised each other to share the resources after the test or outside the test room, we can also rule out the hypothesis that children accepted the disadvantageous splits expecting future sharing from the partner.Furthermore, there were also those trials in which children expressed their unhappiness about obtaining just one gummy bear, physically competing over the position in front of the large amount, or verbally expressing that they also wanted a large amount and did not want the small amount. Surprisingly, in these cases children very rarely suggested to pull the equal tray. Only among the 5-years-olds did one girl suggested to pull the equal tray saying that “that was the fair thing to do”. So, despite children protesting and communicating in various ways their preference for a larger reward , their pSo, why can’t children agree on pulling the fair split in this task? First, although children were required to pull together, the decision whether to acquire the unequal or the equal distribution of rewards was made prior to collaboration, resembling more a windfall situation, in which individuals are confronted with non-collaboratively produced resources. This would support previous findings and theories suggesting a different developmental trajectory for sharing resources acquired in different ways . In studThere are still a number of aspects that deserve further investigation to fully understand what promotes and limits equality at these young ages. First, would the results be any different if they first worked together obtaining the resources and then had to make a choice between equal and unequal allocations? If this was the case, this would further support the collaboration explanation. If not, it would suggest that there are additional variables influencing how children share at these ages, and joint acquisition of resources does not automatically guarantee equality.It is also possible that the present task is cognitively more complicated than actively dividing a pile of clumped resources after collaboration or restoring equality by giving up one reward after a 3–1 split ,18. In tIn the future, we will also need dominance data that allow us to test whether children obtaining the large amount were also more dominant ones. Some previous studies measuring dominance have shown that dominance does indeed predict levels of resource control and utilization at these young ages –44. DomiTo sum up, the present study shows that in face-to-face bargaining situations in which children have to choose between an equal or an unequal distribution of non-collaboratively produced rewards they choose unequal allocations. Despite interdependence between partners and the possibility to protest, communicate, and refuse to pull, equality does not emerge at these young ages.S1 Fig(TIF)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.


Phosphoinositides are master regulators of multiple cellular processes: from vesicular trafficking to signaling, cytoskeleton dynamics, and cell growth. They are synthesized by the spatiotemporal regulated activity of phosphoinositide-metabolizing enzymes. The recent observation that some protein modules are able to cluster phosphoinositides suggests that alternative or complementary mechanisms might operate to stabilize the different phosphoinositide pools within cellular compartments. Herein, we discuss the different known and potential molecular players that are prone to engage phosphoinositide clustering and elaborate on how such a mechanism might take part in the regulation of intracellular trafficking and signal transduction. PIs are the phosphorylated products of phosphatidylinositol. The reversible phosphorylation of the inositol ring at positions 3, 4, and 5 gives rise to the seven PI isoforms identified in eukaryotic cells , the FYVE , the PX (Phoxhomology), the ANTH and ENTH , and the FERM  modules.Phosphoinositides (PIs) are essential phospholipids that control, either directly or indirectly, multiple cellular functions including membrane trafficking, signal transduction, cell growth, cytoskeletal dynamics, lipid transport/exchange between organelles, and the regulation of transmembrane proteinsc cells . Inter-clls (3 . The actthers5 . Proteins5 (6 . There a7. Lipid molecules freely diffuse in the 2D membrane plane (D ~2.6 × 10-7 cm2·s-1)8 and interact with protein effectors based on their association (Kon) and dissociation (Koff) rates. As a result, lipid-protein interactions are, in general, highly dynamic and thus strongly depend on their respective local concentration.Cellular membranes are highly heterogeneous composites built of different types of lipids and proteins. For instance, in eukaryotic cells, more than 1000 different lipid species build up the different membrane compartments7, yet they work as unique docking sites for the multiple PI effectors on membranes, which in turn either compete or cooperate with each other to interact with downstream partners and elicit specific responses. Thus, what are the driving mechanisms that ensure such a thorough spatiotemporal recognition and membrane association of host PI-binding motifs?PIs constitute less than 1% of the steady-state cell lipids5. PI pools within the same compartment are locally synthesized thanks to the spatiotemporal regulation of different PI-metabolizing enzymes5. In addition, small GTPases of the ARF and RAB family also contribute to the generation and regulation of PI turnover on membranes9.An attractive hypothesis is that PIs might be organized as specialized membrane subdomains with distinct organelle localizations10. The unstructured basic cluster on the effector domain of the MARCKS protein is able to bind up to at least three PIP2 molecules by means of nonspecific electrostatic interactions at physiologic pH. The Ca2+/calmodulin complex reversibly controls the association of MARCKS with the plasma membrane11. Interestingly, a growing number of studies report the local enrichment of PI subdomains independently of the catalytic activity of PI-metabolizing enzymes. Jahn and co-workers have shown that the SNAP receptor protein syntaxin-1A co-clusters with PIP2via electrostatic interactions with its juxtamembrane polybasic sequence12. The segregation of PIP2 microdomains by syntaxin-1A has been proposed to work as a molecular beacon at sites of synaptic vesicle docking during exocytosis13. Similar polybasic clusters to that of the MARCKS protein or syntaxin-1A are found in the cytosolic membrane interface of many plasma membrane proteins15, including the epidermal growth factor receptor (EGFR) and the NMDA receptor as well as the voltage-gated potassium and calcium ion channels11.In vitro studies have shown that divalent cations such as Ca2+ are also capable of clustering together PIP2 molecules, although the exact correlation with the activity of ion channels inside the cell has yet to be established. Followingin vitro approaches on giant unilamellar vesicles (GUVs), clustering of PIP2 was initially reported for ezrin16. Later on, using the yeast endocytic F-BAR/BAR domains, Lappalainen and co-authors have shown that the scaffolding effect of these proteins leads to the formation of stable PIP2 microdomains with reduced lateral diffusion in the membrane plane18. Since then, the list of proteins involved in the formation of PIP2 clusters has been extended to other endocytic proteins such as Epsin2, AP180, and the N-BAR domain proteins amphiphysin1 and BIN119. So far, the formation of PI clusters has been mainly restricted to PIP2, possibly owing to its multiple regulatory functions at the plasma membrane. In addition, PIP2 is more abundant than other more elusive PI isoforms and has therefore been the focus of many studies for several years. However, we recently reported that the monophosphate PIs PI4P and PI5P can also be clustered19.Considering the diffusion coefficient of lipid molecules within the membrane plane, it is likely that complementary mechanisms need to operate in order to spatially preserve the turnover of different PI subdomains. Indeed, several mechanisms have been reported in the literature to play roles as selective and reversible PI sinks by locally sequestering and releasing PIs. This is the case for the myristoylated alanine-rich C-kinase substrate (MARCKS) protein and the growth-associated protein 43 (GAP43)17. The number of PI molecules that interact with basic residues is determined by the negative net charge of the PIs at a given pH. For instance, the charge of the PIP2 molecules at pH 3 is −1.5e, whereas at pH 7.4, which is close to the pH of the cytosol (7.2), it is −4e20. For a N-BAR homodimer of charge +8e, one could estimate that at cytosolic pH, the stoichiometry of PI-interacting molecules per protein module is 2:1, which gives an estimated 1.5-fold increase of local PIP2. However, experimental studies have shown that the binding of the N-BAR module on PI-containing membranes induces a local enrichment of at least 10-fold19. How could such a difference in the local PIs’ enrichment be explained?PI clustering has initially been proposed to originate from electrostatic interactions and, to a lesser extent, from hydrogen bonding between PI headgroups. PI molecules appear thus sequestered beneath positively charged surfaces, which results in a significant reduction of lateral diffusion in the membrane plane21. This phenomenon is the result of the combination of electrostatic interactions and an entropic effect. Upon protein-membrane binding, charged lipids diffuse in the plane of the membrane towards the protein surface to preserve charge neutrality P2.Theoretical studies have shown that the binding of a positively charged protein with a negatively charged membrane induces lipid demixing near to the protein surfacetrality . In the 23 and lipid dynamics17. This is in agreement with recent molecular simulations that show that clustering of lipids such as PIs and GM3 correlates with membrane curvature8.PI demixing has been reported in both flat and curved membranes. In the latter case, the segregation of PI molecules is likely to be amplified by membrane curvature since it is reported to significantly reduce protein diffusion24. PI clustering has been reported for proteins with F-BAR, BAR, N-BAR, and I-BAR modules19. The tendency of multivalent PIs to engage lipid demixing over the monovalent PS provides BAR proteins with some specificity to generate PI subdomains at the plasma membrane, where PIP2 and PIP3 are the predominant affected PI isoforms. According to the structural homology within members of the BAR superfamily, it is likely that the formation of PI-enriched microdomains could be a general feature of any protein hosting a BAR module. Combination of the BAR module with PI-binding motifs within the same protein might provide an additional layer of regulation and, possibly, production of monophosphate PI pools in other organelles than the plasma membrane, as observed in the case of BIN119. This suggests that the property of PI clustering might be extrapolated to some members of the sorting nexin (SNX) family holding a BAR module and a PX motif25, although this link has yet to be established.Local segregation of PIs into submicron domains has been mostly described for proteins with the intrinsic property to polymerize on membranes, such as the BAR domain family. Proteins of the BAR family can sense and generate membrane curvature, owing to the scaffolding structure that results from the homodimerization of the BAR module. Association of BAR proteins with membranes takes place through electrostatic interactions between positively charged amino acids on the concave/convex face of the dimeric module and acidic phospholipids19. Therefore, PI clustering seems to be a general property of proteins that directly interact with PIsvia electrostatic interactions with more or less specificity for a given PI isoform. Accordingly, natively unstructured polybasic protein domains have also been shown to induce local segregation of PIs at the plasma membrane, as observed for MARCKS, GAP43, CAPS23, and syntaxin-1A13. The number of proteins that associate with acidic lipids at the plasma membrane through polybasic sequences is large15. For instance, several small GTPases have been shown to interact with plasma membrane PIP3 and PIP2 by means of polybasic clusters26.The clustering of PIs is, however, not necessarily associated with the intrinsic ability of proteins to self-assemble. Indeed, the transient segregation of PIs is likely to generate a positive feedback loop. As a result, proteins that selectively interact with PIs can locally accumulate on PI-enriched areas, independently of their ability to polymerize, as observed for the ENTH and ANTH domains28. The polymerization of actin cytoskeleton was also shown to promote segregation of lipid phases inin vitro models29. These observations are in agreement with the “picket fence” model, which predicts that the cytoskeletal network might act as a diffusion barrier for lipids and proteins30. The exact partition of PIP2 into Lo-Ld domains is not yet clear, but the depletion of cholesterol with methyl-β-cyclodextrin was shown to reduce PIP2 levels at the plasma membrane31. The partition of PIP2 to cholesterol-dependent domains was also reported using the targeting of a 5-phosphatase32. In addition, the sequestration of syntaxin-1A microdomains at sites of synaptic vesicle exocytosis in the plasma membrane was shown to require the formation of cholesterol and PIP2-mediated clusters, which are both distinct from lipid “rafts”33. An interesting observation is that Ld domains were found to align along actin fibers independently of the lipid phase to which actin was pinned28. This might be explained by local changes in membrane curvature induced by the actin network. Indeed, Ld domains appear to favor lipid sorting and membrane deformation34. Recently, numerical simulations have shown that clustering of lipids such as PIP2 correlates with membrane curvature8. The exact contribution of membrane curvature itself in PI clustering is not yet established, but lipid packing defects associated with membrane curvature might favor a better exposure of PIP2 headgroups35. Here, one will have to take into account in future experiments the nature of the fatty acids present on PI molecules, which might also impact on the rigidity and shape of the lipid bilayers to which they belong.PI clustering might be solely limited to ionic protein-lipid interactions, although it is tempting to speculate that alternative or complementary mechanisms might take on the stabilization of PI pools. For instance, recent studies have shown that the pinning of the cytoskeleton on membranes preserves liquid-ordered and liquid-disordered (Lo-Ld) phase coexistence at physiological temperatures (37°C)19. This suggests that PI clusters are more dynamic than initially anticipated and that a given PI cluster could sequentially interact with different effectors. Thus, PI clustering induced by an upstream protein could favor the recruitment of a downstream PI-binding partner, providing a mechanism to coordinate trafficking or signaling events.Importantly, after PI clustering, protein-PI dissociation can still take place independently of the initial concentration of PIs36. All of these protein modules have been shown to engage local segregation of PIP219, which is the key PI isoform in CME. Therefore, PI clustering could participate in the spatiotemporal regulation of CME based on the affinity constant of the different protein intermediates and their interaction with PIP2. A hypothetical example of how PI clustering might operate in CME is shown in2 effectors implicated in CME is much larger . Indeed, the F-BAR, ANTH, ENTH, and N-BAR domains are present in central molecular players involved in CMErger see. The pol67. For instance, the N-BAR protein Arfaptin 1 has been shown to participate in the biogenesis of secretory storage granules through the interaction with PI4P at thetrans-Golgi network68. The ArfGAP ASAP1 also carries a BAR module along with a PI-binding motif and has been shown to provide a platform to regulate Arf4 and Rab8/Rab11-mediated targeting of rhodopsin transport carriers to cilium69. Finally, some members of the SNX family also hold a BAR module in addition to the characteristic PX domain, which typically binds to PI3P6. The SNX-BAR proteins are implicated in tubule-based endosomal sorting70. This includes the two retromer subunits SNX1 and SNX2, SNX5, and SNX6 or SNX4 among others72. One may speculate that the formation of PI clustering together with the binding affinity for different PI effectors might be linked to the ability of SNX-BAR proteins to define tubular endosomal subdomains.It is tempting to propose that the coordinated action of PIs and scaffolding protein complexes, in particular BAR proteins, is a general feature of the biogenesis of transport vesicles274. Indeed, natively unstructured polybasic protein domains have been shown to engage PIP2 clustering11. The interaction of the EGFR with PIP2 is required for the activation and downstream signaling of the receptor at the plasma membrane and seems also to regulate its fate in the endosomal compartments. The first observation that PI4P 5-kinase activity generating PIP2 pools was associated with the EGFR and required for appropriate activation and downstream signaling originates from the early 90s75. Later studies demonstrated that PIP2 clustering induced by the binding and antiparallel dimerization of the juxtamembrane segments of two associated EGFRs can lead to the activation of the receptor even in the absence of ligand76. This property was suggested to be important at a high density of EGFR monomers (>800/µm2), as is often observed in aberrant activation of the receptor in cancers77. In this condition, formation of EGFR nanoclusters takes place as a result of the electrostatic interaction between PIP2 molecules at the plasma membrane and the juxtamembrane region of the receptor78.PI clustering could also play an important role in the coordination of signaling events. Interestingly, the juxtamembrane segment of the EGFR, which is implicated in the activation of the receptor, is also composed of a cluster of basic residues that interact with PIP2 generated on endosomes is required for the appropriate sorting of active EGFR towards multivesicular bodies and further termination of the signal. This process relies on the recruitment of the endosomal type Iγ PIP kinase, PIPKIγi5, that gets targeted to early endosomes by association with SNX5, an effector of PIP2. The kinase will then increase local pools of PIP2, also required for association of SNX5 with Hrs proteins that will then interact with ubiquitinated EGFR and ensure its proper sorting79.Recent evidence demonstrates that PIP76. Although PI clustering being a general feature of membrane-associated polybasic domains provides an attractive hypothesis to activate receptors and trigger signaling, work is still needed to define whether it is a broad mechanism or applies only to some specific proteins.It is noteworthy that most of the tyrosine kinase receptors of the EGFR family harbor a polybasic juxtamembrane domain that could play the same role in terms of ligand free activation or sorting and signal transduction 80. Irvine and co-authors also showed that the maintenance of the steady-state pool of PIP2 at the plasma membrane does not require localization of its synthetic precursor PI4P on the same cellular compartment81. It is tempting to speculate that PI clusters might work as potential platforms to coordinate PI-mediated protein interactions or as molecular beacons, as previously proposed13. Nevertheless, the myriad of protein modules capable of engaging PI clustering is becoming broad. Based on structural homologies, one might predict that the list will progressively increase. An interesting feature to point out is that PI clustering seems to be a general mechanism for either multivalent or monophosphate PIs19. The precise regulatory role of PI clustering in trafficking and signal transduction has still to be established, but it certainly opens up exciting perspectives in the field. For instance, PI clustering might orchestrate the different steps in carrier biogenesis. Also, the ability of cellular receptors to engage PI clustering might determine their sorting to the appropriate compartment. The physiological implication of PI clustering in living organisms has yet to be established. Recent studies have already shown that the oligomerization of Sec14-nodulin proteins controls the localization of PIP2 and signaling landscape in polarized membrane morphogenesis inArabidopsis thaliana root hairs83. Despite the role of PIs in many cellular processes, certain PI isoforms and functions have often been elusive due to the lack of detection or labeling strategies, which is typically limited to the use of PI-binding motifs with all of the associated side effects. The development of novel experimental strategies capable of detecting the intrinsic dynamics of PIs or of exploiting the recently developed sub-100nm life cell imaging techniques84 will be key to unraveling the regulatory role of PI clustering.The spatiotemporal remodeling of PI pools within distinct organelles is an intrinsic feature that makes possible the orchestration of PI-mediated cellular functions. Indeed, PIs are constantly subjected to the activity of PI-metabolizing enzymes and must be in addition accessible to effectors. Because the lateral diffusion of lipid molecules within the membrane plane is extremely fast, PI clustering comes up as a realistic mechanism to locally preserve newly metabolized PI pools on cellular membranes. Indeed, Balla and co-workers already anticipated that PI4P replenishment from the Golgi was not essential to preserve the plasma membrane pool, although it does contribute to its formation


Currently there is a lack of effective treatment options for patients with calciphylaxis. There is anecdotal evidence that non-calcium based phosphorus binders may offer some benefit. The aim of this pilot study is to determine if lanthanum carbonate is effective in inducing remission of calciphylaxis lesions and demonstrate an improved DLQI .This is a multi-site exploratory pilot study conducted through the Wisconsin Network for Health Research (WiNHR), a collaboration of health services researchers across the state of Wisconsin. Dialysis patients were recruited from in-center dialysis units, clinics and hospital admissions over a period of 24-months.Due to the low inclusion rate, the trial was terminated after which 4 patients were prospectively analyzed. Dose of lanthanum carbonate was escalated to 3750 mg divided into 3 meals and titrated according to level of serum phosphorus. Gastrointestinal symptoms were the most common adverse effect. All 4 patients achieved complete remission by definition of skin re-epithelialization. Secondary outcome measurements showed a significant improvement in serum albumin  and a significant improvement in overall DLQI score .Lanthanum carbonate appears to be efficacious as an adjunctive therapy to improve calciphylaxis lesions and symptom burden. More prospective clinical trials are warranted to determine the feasibility of this novel treatment strategy. Since the proposed etiologic mechanism of injury and vascular calcification of calciphylaxis is predominantly hyperphosphatemia, elevated serum PTH, and hypercalcemia, lanthanum carbonate would be an ideal pharmacologic agent to utilize in this extremely enigmatic disease [Calciphylaxis or calcific uremic arteriolopathy is an infrequently occurring, debilitating vasculopathy seen primarily in patients with end stage renal disease (ESRD) which almost always affects the skin. It has a prevalence rate of ~1-4% in long-term hemodialysis patients, with 1-year survival of 45% and an 8-fold risk of death as compared to the general dialysis population . Despite disease . Further® is a potent phosphate binder that was approved for use by the FDA in October, 2004. It is indicated to reduce serum phosphate levels in patients with end stage renal disease. It is not metabolized and is not a substrate or inhibitor of CYP450. It inhibits intestinal absorption of phosphate by forming highly insoluble complexes, thereby lowering serum phosphorus and calcium-phosphorus product. Well designed, landmark studies have shown that doses of 375 mg/day up to a maximum of 2250 mg/day were effective in reducing phosphorus levels and calcium-phosphorus product as compared to placebo [Lanthanum carbonate or FOSRENOL placebo . Adverse placebo . Two mor placebo ,10.This is a preliminary open-label pilot study to test the ability of Wisconsin Network for Health Research (WiNHR) collaborative sites to collect information and data to determine if lanthanum carbonate will be efficacious in the treatment of calciphylaxis. The goal of the study is to:Determine if lanthanum carbonate is effective in eliciting remission  in calciphylaxis skin lesionsSecondary aims include:Evaluate the ability of each site to identify and enroll dialysis subjects with confirmed calciphylaxis into the study Correlate the relationship of lanthanum carbonate phosphorous control and calciphylaxisEvaluate additional information including laboratory parameters of intact PTH, calcium-phosphorous product levels, and albumin , as well as demonstrate an improved DLQI  . PrelimiThis pilot study (NCT01289626) had an intended enrollment of 12 patients and was approved by University of Wisconsin Health Sciences Institutional Review Board for all sites conducting the research. In addition, the FDA granted approval for the Initial New Drug application prior to initiation of the trial. This is a phase II clinical trial designed to evaluate efficacy. The principal investigator (MRC) had complete access to data acquired during the course of the pilot study and was responsible for the interpretation and analysis of the data and writing the manuscript. This study was performed from January 1, 2011, to December 31, 2012.Patients who were included in the study required the following criteria: (1) Participants will have provided informed consent; (2) Participants will be greater than or equal to 18 years of age; (3) Chronic renal failure receiving hemodialysis or peritoneal dialysis; (4) A diagnosis of calciphylaxis proven by skin biopsy or initial dermatology visit within the previous 5 years; and (5) Serum phosphorus > 4.5 mg/dL.Patients were excluded from consideration if they had the following criteria: (1) Participants are not able to understand or provide written informed consent; (2) The research team deems that the participant may not be able to follow the study protocol; (3) Non-dependent HD patients receiving dialysis while in acute kidney injury; (4) Pregnant dialysis patient; (5) Active gastrointestinal obstruction or bleed; (5) Active inflammatory bowel disease; (6) Acute arteriovenous graft occlusion; (6) Known hypersensitivity to lanthanum carbonateThis study was conducted following all local laws and regulations in the conduct of research, as well as the International Conference on Harmonisation’s Good Clinical Practice Guidelines. These guidelines provide a unified standard for designing, conducting, recording, and reporting trials that involve the participation of human subjects.This is an open-label pilot study to assess the efficacy of lanthanum carbonate in patients with calciphylaxis divided into 2 phases; a washout period of 4-weeks if patient was previously on lanthanum carbonate and a treatment period lasting 12-weeks.All patients had a baseline physical examination, a recent history of dermatology consult, and photograph of calciphylaxis involved skin lesions. Laboratory parameters included intact PTH, phosphorus, calcium, and albumin .All long-term medications patients received were documented. Lanthanum carbonate was administered orally in a dose of 1500- 3750 mg daily in divided doses with meals. Dose escalation was adjusted carefully to a target dose of 3750 mg daily or as designated by clinician over a 4-week period from start of study. The drug was administered for 12 weeks. This dose was increased over 4-weeks to 3750 mg total in divided doses  to keep phosphorus levels between 3.5-5.5 mg/dl. The clinician increased the dose from 500 mg to 750 mg the second week, 750 mg to 1000 mg the third week and finally 1250 mg the fourth week. The level of phosphorus was measured at the second month as part of study protocol. If the phosphorus level was below 3.5 mg/dl, then the dosage was decreased by 20% to 3000 mg daily in divided doses. If the phosphorus level was still below 3.5 mg/dl at the third month, the dose was decreased by 25% to 2250 mg daily in divided doses. If the phosphorus level was still below 3.5 mg/dl at the fourth month of the study, the dose was decreased by 33% to 1500 mg daily in divided doses. If levels of phosphorus continued to be elevated above 5.5 mg/dl, then the clinician could add non-calcium based binders of choice according to standard clinical practice. The study drug is an established and widely prescribed drug currently in dialysis patients. Guidelines for drug inventory and management was established by the University of Wisconsin Pharmaceutical Research Center and utilized at all study sites. The drug is used in standard practice and clinicians are accustomed to increasing dosages.Patients were evaluated monthly to determine clinical response to treatment during a period of 3 months. A final photograph was taken to document response to therapy at the conclusion of the treatment period. The subject then completed a final telephone follow-up four weeks after treatment discontinuation. We reviewed all adverse events for 30 days post-last dose of study drug .Twelve patients total were intended to be enrolled through WiNHR sites  of skin lesions to medication. Remission was based on a reduction in ulcer size and depth as well as an assessment of the degree of undermining of the ulcer edge. Complete wound closure and therefore complete remission was defined as skin re-epithelialization without drainage or dressing requirements confirmed at two consecutive study visits as cited in the FDA’s Guidance for Industry Chronic Cutaneous Ulcer and Burn Wounds — Developing Products for Treatment booklet. Partial remission was defined as skin re-epithelialization that still exhibits drainage or dressings. We expected at least a 50% complete remission (CR) or partial remission (PR) of skin lesions to consider the treatment effective.The primary driving force for vascular calcification is elevated calcium and phosphorus which were measured as outcomes. We expected that since phosphorus control would improve based on efficacy data from multiple clinical trials, an improvement in secondary hyperparathyroidism and increased albumin levels should also improve as a result of improvement in overall health, nutritional and inflammatory status. We anticipate that there will be an improvement in quality of life compared to historical data. The DLQI survey is validated for dermatological conditions such as calciphylaxis .Sample size and power were calculated based on number of recruited subjects that were expected to achieve CR or PR. A sample size of 12 was required under the assumption of 50% of patients having CR or PR, such that the lower 95% confidence level would extend to 0.283. Mixed effect linear regression models were used to examine the changes in DLQI variables associated with lanthanum treatment, while taking into account the within-person correlations. We also examined which laboratory values were affected by the treatment of lanthanum, controlling for baseline variables. For all statistical tests, two-sided alpha levels were set a priori at 0.05. The statistical analyses were performed using Stata MP 12.1.Five patients were enrolled in the study during the 24-months of recruitment but only four completed the entire treatment period of 12-weeks. All four patients were women and ranged in age from 32 to 74 years (mean 51.8 ± 17.3). The ethnicity of all patients was white. There was a variety of causes of ESRD including IgA nephropathy, diabetes mellitus, systemic lupus erythematosus and hypertension. These patients used a variety of medications for mineral bone disorders, including sevelamer carbonate, sevelamer hydrochloride, calcium acetate and cinacalcet, however per protocol, dosages did not change during treatment period. Caloric intake and nutritional status did not change during treatment period. There were no serious adverse events. The most common side effects were gastrointestinal and musculoskeletal occurring in two patients.The primary outcome of complete remission of calciphylaxis skin lesions was seen in all four patients enrolled in the study. Before and after photos of skin lesions are shown in two patients in different stages of lesion progression  and 2. CThe DLQI is calculated by summing the score of each question (survey of 10 questions) resulting in a maximum of 30 and a minimum of 0. The higher the score, the more quality of life is impaired. The DLQI can also be expressed as a percentage of the maximum possible score of 30. Meaning of DLQI Scores:0-1 = no effect at all on patient’s life2-5 = small effect on patient’s life6-10 = moderate effect on patient’s life11-20 = very large effect on patient’s life21-30 = extremely large effect on patient’s lifeThe DLQI questionnaire showed an improvement in median scores after patients received treatment with lanthanum carbonate as demonstrated in Data are expressed as standardized regression coefficient (β) with 95% Confidence Intervals and P-values.Laboratory data at the end of the 12-week treatment period for study subjects are listed in Data are expressed as standardized regression coefficient (β) with 95% Confidence Intervals and P-values.To date, this is the first open-label pilot study to assess the efficacy of treating calciphylaxis in patients with ESRD. Even though the disease process has been described since 1898, the pathophysiology and treatment remains elusive ,13. We rOur results showed for the first time an improvement in skin lesions documenting complete remission over a 12-week treatment period. The patients also showed an improvement in the DLQI questionnaire which is a validated tool in dermatologic studies. The intact PTH, phosphorus and calcium did not change significantly during the trial likely due to the short duration of treatment. However, serum albumin levels significantly improved during the trial. Higher serum albumin is a surrogate marker of improved nutrition and possibly reduced inflammatory status.There were certain limitations to this study given its observational nature, including the inability to reach original target sample size, patients’ demographic factors, hemodynamic status, and medications. These confounding factors may all play critical roles in the pathogenesis and pathology of calciphylaxis.In conclusion, it is feasible to study the effect of short-term treatment with lanthanum carbonate on dialysis patients with calciphylaxis. In view of the proven safety and tolerability of this medication in this pilot study and many others, we propose that a multicenter randomized, placebo-controlled trial be performed to determine the safety and efficacy of this potentially novel therapeutic regimen for dialysis patients with calciphylaxis.