This study aimed to evaluate changes in tibial bone speed of sound (SOS) over time, in preterm and term infants during infancy, in addition to identifying factors influencing the development of tibial SOS during infancy. Preterm (n = 155) and term (n = 65) infants were enrolled in this study. Tibial bone SOS was measured using quantitative ultrasonography (QUS) on the left tibia of newborn infants after birth (within 7 days), at 1 month old, and then every 2 months until subjects were approximately 12–15 months old. Follow-up checks included anthropometric measurements and tibial bone SOS. Mean tibial bone SOS at birth was significantly higher in term infants  than in preterm infants (2912.2 ± 122.6 m/s). Values of follow-up tibial bone SOS declined for the first 4 months, and then increased gradually until 12–15 months old. This increasing trend was greater in preterm infants after 2 months of corrected age than in term infants. There were no significant differences by 12–15 months of age between preterm and term infants. A longitudinal mixed-effect model controlling for internal correlations and other covariates in the two groups showed that age and the SOS value at birth were important factors affecting the tibial bone SOS in both preterm and term newborn infants during infancy. There are significant differences in the pattern of change in tibial bone SOS values between preterm and term infants during the first 12–15 months of life. Age and SOS value at birth were important factors affecting the pattern of tibial bone SOS change in both preterm and term newborn infants during infancy. Given that the rates of osteoporosis is reportedly increasing in all demographic groups , 2, it iMeasuring bone development precisely in newborns and infants is a methodological challenge. Dual-energy X-ray absorptiometry (DEXA) is now well defined as an assessment method for the accretion of minerals in adults, and is the current standard for whole-body measurement of minerals . HoweverAlthough there have been a number of reports on QUS-related assessment of bone status in children and adolescents –18, few 3) [Term  and preterm (GA < 37 weeks) infants admitted to the baby room or neonatal ward of Kaohsiung Medical University Hospital between October 2007 and July 2015 were enrolled in this study. Infants with major congenital anomalies or congenital chromosomal disorders were excluded. Following enrollment, a birth chart including gestational age, birth body weight, and birth height was recorded. Infants with weight below the 10th percentile for GA  and above the 90th percentile for GA (large for GA), based on intrauterine growth charts in Taiwan , and mulQUS measurement of the tibial bone SOS was performed using an appropriate sized probe with the Sunlight Omnisense 7000S Quantitative Ultrasound scanner , which is based on the technique of ultrasound critical angle reflectometry, according to the manufacturer’s instructions. There are three different size probes with a frequency of 1.25 MHz , chosen appropriately for infants of different body weight. The CS probe is used for infants weighing less than 3 kg, the CR probe for infants weighing 3–5 kg, and the CM probe for infants weighing more than 5 kg. Infant weight is used when choosing the probe because the thickness of soft tissue can result in unsuccessful measurements (lack of recording of SOS during a measurement cycle) if the probe used is inappropriately sized for infant weight. The same technician performed all measurements. System quality verification of the Sunlight Omnisense 7000S Quantitative Ultrasound scanner was performed each day using a phantom, provided by the manufacturer, and the coefficients of variation for the CS, CR, and CM probes were 0.24%, 0.42%, and 0.31%, respectively. Five neonates weighing less than 3 kg (CS probe), 5 infants weighing 3–5 kg (CR probe), and 5 infants weighing more than 5 kg (CM probe) were measured 5 times each to determine the precision of each probe. Coefficient variation for the CS, CR, and CM probes were 0.92%, 1.31%, and 1.31%, respectively. In this longitudinal study, all enrolled infants were measured with at least 2 different probes and, and in most cases, with all 3 probes. In order to validate the consistency of SOS measurements using 3 different probes, paired SOS measurements were performed by using CS and CR probes in 7 infants weighing 1.5–3.1 kg, and CR and CM probes in 7 infants weighing 3.5–4.1 kg. There was strong correlation between CS and CR probes , and between CR and CM probes .Tibial bone SOS was measured at the middle of the left tibia. The first measurement was performed within seven days after birth . Infants returned to the hospital after discharge for a routine follow-up at 1 month old, and then every 2 months until they were 12–15 months old. Follow-up checks included measurements of body weight, height, and tibial bone SOS. For preterm infants, corrected age (CA) is calculated as postnatal age (PNA) reduced by the number of weeks born before 40 weeks of gestation .This study was approved by the Institutional Review Board (IRB) of Kaohsiung Medical University Hospital (number KMUH-IRB-980555). Informed consent was documented by the use of a written consent form approved by the IRB and signed by the parents of all infants prior to enrollment. The investigator gave the parents adequate opportunity to read it before it was signed.2) that was able to reflect the pattern of change in bone SOS with age. Univariate analysis was used to identify possible factors influencing values of tibial bone SOS in both groups. A longitudinal mixed-effect model was further used to test for associations between bone SOS and possible influencing factors, an analytic approach that permits full use of available data while controlling for internal correlations and other covariates in two groups of infants. This model treats each bone SOS data from each enrolled infants as a separate observation and adjusts for within-infant correlations and correlations with their prior bone SOS data. Subjects were treated as random effects, and the analysis could be adjusted to each infant’s bone SOS data. A first-order autoregressive error structure accounted for within-infant correlation. A 2-tailed p value less than 0.05 was considered statistically significant. All statistical analyses were performed using the SAS® statistical software version 9.3 .Continuous variables are presented as mean and standard deviation (SD), and categorical variables as number and percentage. Chi-square tests and t-tests were used to compare basic characteristics among groups. Polynomial regression analysis was used to identify the best fitting model  preterm infants with birth weight less than 1000 g, whose corresponding gestation was 24–28 weeks. Sixty-two (40.0%) preterm infants weighed 1000–1499 g at birth, with corresponding gestation of 26–31 weeks. The remaining 56 (36.1%) preterm infants weighed more than 1500 g at birth with corresponding gestation of 29–36 weeks. The QUS procedure was well tolerated in all infants during the study period, and no adverse effects were observed. There was a total of 1352 tibial bone SOS and anthropometric measurements taken from enrolled infants between birth and 12–15 months old.Clinical characteristics of infants enrolled in the study are shown in There was no mortality among 155 preterm infants. Mean length of hospitalization was 69.6 ± 37.8 days for preterm infants. Fifty-five preterm infants (35%) were diagnosed as having bronchopulmonary dysplasia, which was defined as oxygen dependency at postmenstrual age 36 weeks. Seventy-three preterm infants (47.1%) were prescribed diuretics (furosemide/thiazides) more than 7 days. The body weight, body length and bone SOS in preterm infants during hospitalization were 2.1 ± 0.9 kg, 42.3 ± 5.6 cm, and 2811.8 ± 113.6 m/s at the first month, 2.5 ± 0.9 kg, 43.6 ± 5.1 cm, and 2759.0 ± 119.8 m/s at the second month, and 3.7 ± 0.8 kg, 48.5 ± 5.4 cm, and 2747.3 ± 308.0 m/s at the third month, respectively.The relationship of bone SOS with age in months was evaluated by polynomial regression analysis for curve fitting. The best fitting model was a third degree polynomial in both groups by sex. Trends in age-related changes for tibial bone SOS in term and preterm infants are shown in Univariate analysis to identify possible factors influencing values of tibial bone SOS in both groups revealed that advanced age class (term infants in PNA and preterm infants in CA), body weight, height at the measured time, and SOS at birth positively affected tibial bone SOS development after birth, whereas sex and maternal smoking during pregnancy did not. PI and status of breastfeeding at the measured time were negatively associated with tibial bone SOS in both preterm and term infants. In preterm infants, GA, birth body weight and birth body height, but not PI at birth, were also significant factors influencing development of tibial bone SOS during infancy. In term infants, PI at birth was a negative factor for development of tibial bone SOS.We further preceded multivariate analysis by using a longitudinal mixed-effect model to test for associations between bone SOS and possible influencing factors. Collinearity may occur among body weight, body height, and PI  in regression; therefore, we selected PI as an anthropometric status measure for the multivariate analysis. The variables of age class, sex, GA, SOS value at birth, anthropometric status at birth, season of birth, status of breastfeeding at the measured time, and PI at the measured time were included in the multivariate analysis for both preterm and term infants. The results revealed that age and the SOS value at birth remained significant positive factors influencing tibial bone SOS development during infancy for term infants. For preterm infants, PI at the measured time was an important factor with a negative effect on increased tibial bone SOS during infancy. Status of breastfeeding at the measured time and season of birth in multivariate analysis did not show significant effects on tibial bone SOS during infancy in both groups.The present study provides important information on the postnatal changes in tibial bone SOS for preterm and term infants from birth to 12–15 months of age. There are significant differences in the pattern of change in tibial bone SOS values between preterm and term infants during infancy. Furthermore, we showed that the most important factors associated with bone strength as a reflection of tibial bone SOS in both preterm and term infants were age in months, and the tibial bone SOS value at birth.Prevrhal et al. have shown that the tibial bone SOS measurement depends on both the thickness and density of the tibia, and is influenced by the density of the cortex near the surface, rather than the internal structure . Given tThe differences in factors influencing tibial bone SOS between preterm and term infants may be explained by maturity of the fetus. The majority of calcium accretion occurs during the third trimester, with peak accretion rates between 36 and 38 weeks of gestation , 23. WheThe present study revealed that there were no sex-based differences in results of both univariate and multivariate analysis. In our previous study on bone status at birth , we showInterestingly, we found a decreasing trend in tibial bone SOS for both groups after birth, until 4 months old, which was more evident in preterm infants. After this period of decrease, tibial bone SOS values were observed to increase gradually until 1 year of life. The same phenomenon has been reported in Caucasian  and ChinIn our study, we used PI to approach body mass. Although PI and body mass index are poor predictors of adiposity at birth , PI is sThe limitation of our study is that we did not account for other potential confounders, such as complementary feeding, sunlight exposure, activity and hormonal status of the enrolled infants. Moreover, subcutaneous fat and probe size can confound the QUS measurements. Tibial bone measurement by the same probe cannot be used due to growth status during a longitudinal study. Infant size appears to significantly affect bone SOS measurements at the tibia, with the smaller probe tending to be unsuccessful in infants with larger weights. Hence, validity of QUS data include SOS as surrogate for bone status remains to be defined.The strength of this study is that we used a longitudinal mixed-effect model analysis with a first-order autoregressive error structure to test for associations between bone SOS and possible influencing factors while accounting for within-infant correlation. Even though there were limitations to this study, our results provide important information on tibial bone SOS values, according to age in months and sex during infancy, for both term and preterm infants. We also observed differences in the pattern of postnatal tibial bone SOS changes between groups. Given the limited data in the literature pertaining to these trends, this study could be useful for evaluating bone status in both preterm and term infants during their infancy.This study provided information of postnatal changes in tibial bone SOS for preterm and term infants during infancy. There are significant differences in the pattern of change in tibial bone SOS values between preterm and term infants during the first year of life. For both preterm and term infants, age and the SOS value at birth are important factors affecting the pattern of tibial bone SOS change during infancy.


Epidemiological asthma research has relied upon self-reported symptoms or healthcare utilization data, and used the residential address as the primary location for exposure. These data sources can be temporally limited, spatially aggregated, subjective, and burdensome for the patient to collect.First, we aimed to test the feasibility of collecting rescue inhaler use data in space–time using electronic sensors. Second, we aimed to evaluate whether these data have the potential to identify environmental triggers and built environment factors associated with rescue inhaler use and to determine whether these findings would be consistent with the existing literature.We utilized zero-truncated negative binomial models to identify triggers associated with inhaler use, and implemented three sensitivity analyses to validate our findings.Electronic sensors fitted on metered dose inhalers tracked 5,660 rescue inhaler use events in space and time for 140 participants from 13 June 2012 to 28 February 2014. We found that the inhaler sensors were feasible in passively collecting objective rescue inhaler use data. We identified several environmental triggers with a positive and significant association with inhaler use, including: AQI, PM10, weed pollen, and mold. Conversely, the spatial distribution of tree cover demonstrated a negative and significant association with inhaler use.Utilizing a sensor to capture the signal of rescue inhaler use in space–time offered a passive and objective signal of asthma activity. This approach enabled detailed analyses to identify environmental triggers and built environment factors that are associated with asthma symptoms beyond the residential address. The application of these new technologies has the potential to improve our surveillance and understanding of asthma.Su JG, Barrett MA, Henderson K, Humblet O, Smith T, Sublett JW, Nesbitt L, Hogg C, Van Sickle D, Sublett JL. 2017. Feasibility of deploying inhaler sensors to identify the impacts of environmental triggers and built environment factors on asthma short-acting bronchodilator use. Environ Health Perspect 125:254–261; http://dx.doi.org/10.1289/EHP266 Despite efforts to address the substantial health and economic burden of asthma in the United States and around the world, it remains a significant health issue. Previous epidemiological studies trying to identify environmental triggers of asthma have relied upon aggregated and infrequently-reported asthma outcome measures, such as emergency department visits or hospitalizations, which lack temporal and spatial resolution due to aggregation to an annual basis and grouping to a ZIP code or county level. Other studies have used patient self-reported data to assess the location and frequency of symptoms; however, patient diaries have been demonstrated to be fraught with missing data, errors and are burdensome for the patient . StudiesPatients with inadequately controlled asthma are at particularly high risk of exacerbations, hospitalization, and death, and they often have severely impaired quality of life . AsthmatThis feasibility study had three distinct objectives. First, we aimed to evaluate the feasibility of collecting rescue inhaler use data in space–time using inhaler sensors. Second, we aimed to evaluate whether these collected data could identify environmental triggers and built environment factors associated with rescue inhaler use, and whether these findings would be consistent with the existing literature. These two objectives were the primary focus of this paper. Third, the study aimed to assess the feasibility of using inhaler sensors and a mobile health platform to improve asthma outcomes for participants, such as the frequency of rescue inhaler use and symptom-free days. These clinical objectives and results are discussed elsewhere and will not be the focus of this paper .The city of Louisville, located in Jefferson County, Kentucky, ranks among the top 20 “most challenging places to live with asthma” in the United States . Asthma a) were under the age of 4; b) did not speak English; c) did not have access to the internet; or d) had substantial respiratory co-morbidity such as chronic obstructive pulmonary disease. The study was reviewed and approved by the Copernicus Group Independent Review Board, and received written informed consent from all participants, including written consent from guardians on behalf of children under 18. Demographic data were collected via surveys. The parents of children < 12 years old assisted with completing the survey. In general, the parents of children < 18 years old were also trained on the sensors and mobile health platform.The program recruited participants through convenience sampling from community events, clinics and retail pharmacies in Jefferson County. Participants were eligible if they reported a physician diagnosis of asthma and had a current prescription for a compatible inhaled short-acting bronchodilator medication. Participants were excluded if they Rescue inhaler use events were captured using a wireless inhaler sensor . The Propeller sensor and platform comprise a U.S. Food and Drug Administration (FDA) cleared digital therapeutic that combines inhaler sensors, mobile applications, and predictive analytics for patients and clinicians . The senThe digital health platform enables a participant to self-report information on perceived symptoms and triggers associated with a rescue inhaler use event, and report whether the inhaler was used pre-emptively. Before our analysis, we removed all rescue inhaler events marked as pre-emptive.The Propeller Health System is considered an electronic Metered Dose Inhaler (MDI) Accessory according to the Code of Federal Regulations  and requ2), ozone (O3), sulfur dioxide (SO2), and particulate matter with aerodynamic diameter ≤ 2.5 μm (PM2.5) and ≤ 10 μm (PM10). We also collected Air Quality Index (AQI) data from the available monitoring stations . It is a piecewise linear function of a pollutant concentration, and it ranges from < 50  to over > 400 . If multiple pollutants are measured at a monitoring site, then the highest pollutant level is reported for AQI at that location  algorithm to estimate pollutant concentrations for locations of rescue inhaler use per hour or per day using the AQS monitoring data  was calculated using all known monitoring sites  concentration measurements (ijtc) (Equation 1):Environmental triggers assessed in this study included air pollutants, pollen, mold and meteorological factors. We acquired air pollutant data from the U.S. Environmental Protection Agency’s (EPA) Air Quality System (AQS) for the following criteria pollutants: nitrogen dioxide (NOstations . AQI is location . The AQSing data . The spajtc = Σi (ijw × ijtc)/Σij,w [1]ijw = 1/ijd and ijd is the distance between known monitoring station i and rescue inhaler use location j. For rescue inhaler use events without location data, daily or hourly regional mean statistics were used.where The pollen data for the study period were collected in Louisville by the staff at Family Allergy & Asthma from a monitoring station on the roof of the clinic , and incWe downloaded daily meteorological data, including wind speed, relative humidity, temperature, and atmospheric pressure, from U.S. EPA AQS sites . The IDWWe identified built environment factors that might influence rescue inhaler use, such as land use, land cover, and property characteristics. We acquired land use and property data from the Louisville/Jefferson County Information Consortium for 2014. For each rescue inhaler use location, corresponding land use characteristics  were calculated within a 250-m buffer, an area representing local influence on an individual .We also acquired land cover data from the National Land Cover Database for 2011 at a spatial resolution of 30 m. The land cover classes for vegetation included forest , shrub land, and grassland/herbaceous cover. Our primary interest in including land cover was to examine the potential protective  or causal  effect of vegetation on rescue inhaler use. Pollen counts were measured at the regional level and were temporally resolved (daily), while land cover vegetation classification data were spatially resolved using the 250-m buffer.We first explored bivariate relationships between daily number of rescue inhaler use events and corresponding daily pollutant concentrations and meteorological conditions with time series plots and scatterplots. To be consistent with subsequent statistical modeling, we used the ratio of rescue inhaler use events each day to the number of active participants on that day. We then assessed the association of environmental triggers and built environment factors with inhaler use by implementing zero-truncated negative binomial models, and validating these results using three sensitivity analyses, which we describe in further detail.First, we identified the environmental triggers that might have an impact on rescue inhaler use through an unadjusted zero-truncated negative binomial model. Our goal was to model the number of events of rescue inhaler use of all active participants per day using average environmental exposures within the same day (Equation 2):iY)) = β0 + β1 × iX + log(iA) + εi, [2]log(E (iY) and iX are respectively the expected number of inhaler use events and the environmental predictor at the ith day. Negative binomial models were applied because initial data exploration identified that the data for rescue inhaler use were highly overdispersed, with the variance about 10 times the mean. We used rate ratios (RR) in interquartile range (IQR) increments and corresponding 95% confidence intervals (CI) to identify the degree of impact of environmental triggers on rescue inhaler use. RR is a relative difference measure used to compare the incidence rates of events. For environmental triggers that were spatially resolved, a single mean statistic averaged from all the locations of rescue inhaler use each day was used. iA is the total number of active participants at the ith day, representing the participants who were tracking their inhaler use on that day. An active participant was defined as having a sensor that was on and capable of transmitting data, however not all active participants used their rescue inhaler on any given day. We used the number of active participants each day as an offset to adjust for bias. εi is the model error term.where E :iY)) = β0 + β1 × X1i + β2 × X2i + β3 × X3i + log(iA) + εi, [3]log. We used each discrete rescue inhaler event’s time and location to determine the environmental exposure. For pollutants with hourly data available, the concentrations at the specific space–time for which rescue inhaler use occurred were assigned. For pollutants with only daily data available, the interpolated daily concentrations at the locations of rescue inhaler use were used. In our zero-truncated negative binomial modeling process, the exposure values from all rescue inhaler events occurring in a single day were averaged to daily means. For assessing time-lagged exposures, lagged exposures were calculated by averaging concentrations measured from all the locations of rescue inhaler use that occurred on the respective preceding 1–3 days.We included property and land cover data in the models to identify the impacts of built environment characteristics, such as transportation infrastructure, land use or vegetation, on rescue inhaler use. We used the subset of rescue inhaler use data with geographic location data assigned to each event. A location of inhaler use at a specific time and day was linked to the land cover data based on the latitude and longitude information recorded by participants’ smartphones. Similar to the identification of environmental triggers of rescue inhaler use, we built unadjusted models to assess associations of rescue inhaler use with property types and land cover characteristics using Equation 2.We tested the reliability of our findings by implementing three sensitivity analyses. To test the feasibility of using the individual observation as the unit of analysis, we used a subgroup of 80 participants for which we had demographic data and conducted two sensitivity analyses to identify associations of environmental triggers with rescue inhaler use. For each participant, we tracked their start date, end date, and the intervening active days the participant was in the program. First, we developed a generalized linear mixed model with repeated measures. For those active days on which an individual experienced a rescue inhaler use event (response = 1), we extracted the corresponding environmental exposure values based on the time and location of the event. If location data were not available, regional mean statistics at the time of exposure were applied. For those active days on which an individual did not experience a rescue inhaler use event (response = 0), we extracted the corresponding regional mean exposure statistics for each day. We tried to control for person-level confounding by including race/ethnicity, sex, smoking status, and pet ownership. Studies show that race/ethnicity and smoking status are significantly correlated with disease severity and medication adherence and could be treated as a proxy for such measures . The secWe recruited 140 volunteer participants across Jefferson County who passively recorded their rescue inhaler use from 13 June 2012 to 28 February 2014. Of the 140 participants, 80 of them provided demographic information via surveys. Participants self-reported their race/ethnicity as African American (31.3%), Non-Hispanic White (57.5%), Hispanic (1.3%), Native American (2.5%) or other (7.5%) . Among tThe inhaler sensors collected a total of 10,475 actuations, which were grouped into 5,660 unique rescue inhaler use events. Of the rescue inhaler events logged, 100% successfully recorded information on the date, time, and the number of actuations. A subset of the inhaler use events were assigned GPS locations (23%), which enabled a kernel density analysis of the geographic distribution of the rescue inhaler use events . Of all r) between environmental triggers and found that air pollutants had r values between 0.28 and 1.00, while their correlations with pollens from grass, tree, weed, and mold were < 0.20 (see Table S1). AQI was heavily impacted by PM10 (r = 1.0) and PM2.5 (r = 0.75). We overlaid the daily number of rescue inhaler use events with daily pollutant concentrations and temperature in time series plots , and explored the relationship between rescue inhaler use and temperature .We first assessed the correlation coefficients ], and NO2 [1.033 ] were positively associated with inhaler use, but O3 had a negative association [0.913 ] for immediate exposures, all significant at the 0.05 level. We also found that weed pollen [1.005 ] and mold [1.153 ] had positive associations but tree pollen [0.982 ] and grass pollen [0.992 ] had negative associations. In addition, we identified that the impacts of time-lagged exposures were similar to the immediate impact of daily exposures for all variables (see Table S2).We also identified the feasibility of using sensor-collected data to detect possible associations with environmental triggers, including air pollution, pollen, and mold, in the unadjusted zero-truncated negative binomial models (see Table S2). All the modeling results are expressed as RR (95% CI) through corresponding IQR increments. We found that AQI [1.201 ], PM10 [1.054 ] were still positively and significantly associated with rescue inhaler use. O3 had a positive association for lag exposures of 1 day [1.082 ] and 2 days [1.073 ]. Tree pollen maintained a negative association [0.991 ], but grass pollen [1.012 ] and weed pollen [1.005 ] demonstrated a positive association.Next we built an adjusted environmental trigger model that also included meteorological variables (see Table S2). After including these additional environmental factors, AQI [1.055 ] and PM10 were found to maintain their positive and significant association with rescue inhaler use (see Table S3). Tree pollen maintained a negative association while weed pollen and mold maintained a positive association, all significant. In the second sensitivity analysis (time-lagged exposures), these relationships were also maintained (see Table S3). In the third sensitivity analysis , the results were also largely maintained, except that the association with O3 was positive and significant, which was opposite of the result from the original analysis (see Table S4).The three sensitivity analyses demonstrated associations that largely aligned with the zero-truncated negative binomial models. In the first sensitivity analysis (subgroup of 80 participants with demographic data), after controlling for race/ethnicity, sex, smoking status and pet ownership, AQI, and PM10, and weed pollen were found to be positively and significantly associated with rescue inhaler use in all five models. Mold was found to be positively and significantly association with inhaler use in four of the five models. Additionally, tree pollen demonstrated a negative and significant association in all five models.To summarize the consistent results from the two primary analyses and three sensitivity analyses, AQI, PMIn the unadjusted models of built environment factors (see Table S5), we found that vegetation cover [0.829 ], which included trees, herbaceous and shrubland, was negatively associated with rescue inhaler use. When examined individually, tree cover [0.825 ] and herbaceous cover [0.901 ] were negatively associated with rescue inhaler use, but shrubland [0.993 ] exhibited no significant associations.We also found that industrial land use [0.992 ] and residential land use categories [0.880 ] had significant and negative associations, while public utilities [1.009 ] and exempt land use categories [1.153 ] had positive and significant associations. Public utilities included areas such as railroads, pipelines, electricity delivery, and other utilities. Exempt properties include those owned by communities, churches, hospitals, colleges, cities, counties, state or federal authorities. Upon further investigation into the detailed exempt properties, we found that educational institutions [1.008 ], religious gathering places [1.094 ], residential condominium master lots  [1.014 ], and metro government sites [1.054 ] were all significantly and positively associated with rescue inhaler use.In this study, we tested the feasibility of using inhaler sensors to collect rescue inhaler use data in space–time, and to use these data to detect environmental triggers and built environment factors associated with asthma symptoms. We found that the sensors are reliable in collecting medication use data: 100% of the 10,475 rescue inhaler actuations successfully recorded the date and time. Geographic data were not captured on all events primarily because participants without smartphones used alternative devices or wireless hubs (35.1%), which are not capable of collecting GPS data. It was known before the study started that these participants would not be collecting GPS data, but the study chose not to exclude participants based on lack of smartphone ownership. Additionally, we chose to exclude a subset of GPS data points to be conservative. If the data transmission between sensor and smartphone occurred more than 10 min after the rescue inhaler event, we disregarded the geolocation because sufficient time had passed to allow movement away from the original location of rescue inhaler use and could misrepresent the location. Lastly, geolocations were not captured if the smartphone was not communicating fully with the sensor . In these scenarios, all actuations and their date and time data were still collected and stored on the sensor for future transmission, so these data were never lost. Smartphone ownership was the key predictor of GPS data collection; there were no differences in other predictors such as sex, race, smartphone type or time of day.The geographic data enabled analyses that were not possible previously, such as a kernel density analysis of inhaler use, which highlighted those neighborhoods with the most frequent rescue inhaler use. Additionally, these data also enabled an analysis of the associations of built environment factors with rescue inhaler use. These data could improve upon the limitations of previous studies by identifying nonresidential locations of exposures and even the protective effects of built environment factors.10, mold, and weed pollen were found to be positively and significantly associated with rescue inhaler, while tree pollen and tree cover demonstrated a negative and significant association.Negative binomial regression models, including unadjusted and adjusted models, have been used in previous studies to identify asthma exacerbations . We testIn our primary analysis, we did not use the individual rescue inhaler use event as the unit of analysis because we needed to control for the differences across individuals in asthma severity and sensitivity to environmental triggers, but did not have these data. Instead, we used the number of active participants per day as an offset to address the impacts of individual user variability. The offset controls for situations when similar exposures between two different days result in dissimilar number of rescue inhaler use events due to differences in participant composition. In our sensitivity analysis using just the subset of the data with demographic information, we could control for variability across individuals using race/ethnicity, sex, smoking status, and pet ownership information and found consistent results. In future studies, we will have access to demographic, socioeconomic, asthma severity, and self-management information on all participants, which will allow us to use the individual rescue inhaler use event as the unit of analysis and control for variability across individuals, which will greatly enhance the utility of these data.10 have been found to be positively and significantly associated with rescue inhaler use  PDFClick here for additional data file.


This study aims to produce optimized biochar from oil palm empty fruit bunches (OPEFB), as a green, low cost adsorbent for uptake of zinc from aqueous solution. The impact of pyrolysis conditions, namely, highest treatment temperature (HTT), heating rate (HR), and residence time (RT) on biochar yield and adsorption capacity towards zinc, was investigated. Mathematical modeling and optimization of independent variables were performed employing response surface methodology (RSM). HTT was found to be the most influential variable, followed by residence time and heating rate. Based on the central composite design (CCD), two quadratic models were developed to correlate three independent variables to responses. The optimum production condition for OPEFB biochar was found as follows: HTT of 615°C, HR of 8°C/min, and RT of 128 minutes. The optimum biochar showed 15.18 mg/g adsorption capacity for zinc and 25.49% of yield which was in agreement with the predicted values, satisfactory. Results of the characterization of optimum product illustrated well-developed BET surface area and porous structure in optimum product which favored its sorptive ability. Heavy metal pollution is one of the most serious environmental issues, faced worldwide today –3. IntenDifferent methods and techniques have been introduced for controlling water and wastewater pollution by heavy metals such as ion exchange, adsorption, precipitation, coagulation, membrane technologies, and reverse osmosis. Among these methodologies, adsorption has been demonstrated to be an economically practicable alternative method for uptake of metals from water system , 8. The In recent years, biochar from different agrobased and municipal waste materials has been shown to be a potential low cost alternative for separation of heavy metal from water system , 12. DifAnnually, enormous amounts of biowastes from oil palm mills are produced around the world which contribute to great environmental concern, and many researchers have been focused on conversion of these wastes into value-added products. Oil palm empty fruit bunches account for 23% per ton of fresh fruit bunch which should be handled properly . CurrentProduction of efficient sorbent for this purpose has been always a concern. Among the sorbent characteristics, surface area, surface functionalities, and acceptable level of yield are important in adsorption process design . These cDesign of experiment (DOE) enables engineering researchers to alleviate the costs by rationalizing the experiments and improving productivity and product quality. Response surface methodology, one of the DOE techniques, is applied for experimental design, statistical modeling, and optimization of a process. It is a helpful tool in studying the effect of factors and their interactions on specific response to optimize the response of interest .RSM has been widely used in optimization of experimental conditions of various processes; however, its application in production of biochar is very rare in literature. Some previous studies focused on applying RSM in determining the effect of different parameters during production of adsorbent and batch adsorption experiments, on removal of hazardous contaminants such as chromium , 22, copTo our knowledge, no study has been performed on optimized production of biochar from oil palm empty fruit bunches, applying RSM. Therefore, the focus of the present study is to produce biochar from oil palm empty fruit bunches and to optimize the preparation conditions using central composite design for yield and its adsorption capacity towards zinc. The influence of three numerical variables, namely, highest treatment temperature, heating rate, and residence time, on the responses was considered, simultaneously.3/min was used during pyrolysis process to wash tarry vapors away from the surface of biochar.Oil palm empty fruit bunches were collected from Ulu Langat Palm Oil Mill, a local oil palm mill in Malaysia; Seri Sdn Bhd . The biomass samples were dried in oven at 105°C for 24 h to get constant weight. Afterward, the samples were cut into smaller parts (2 nm) to ease the pyrolysis process. The pyrolysis process was performed by placing samples in vertical stainless steel reactor and heating from room temperature to predetermined temperature with specific heating rate and they were kept at highest temperature for specific duration. Purified nitrogen (99.995%) with flow rate of 150 cmn factorial runs, 2n axial runs, and nc center points in which n is the number factors.Response surface methodology (RSM) is a statistical technique which utilizes the quantitative data of the experiments for purpose of determining the regression model and optimum operation conditions . It applx1 as highest treatment temperature (HTT), x2 as heating rate (HR), and x3 as residence time (RT). Two responses in this work were yield (Y1) and adsorption capacity of OPEFB biochar (Y2). Number of runs based on CCD method for three independent variables is equal to 20 experiments, including 8 factorial points, 6 axial points, and 6 center points based on the following equation [N is the total number of experiments and n is the number of factors. The center points are useful in considering the errors of experiments and reproducibility of the model. For all three factors the ranges have been entered based on “−alpha” and “+alpha” level where alpha is the distance of axial points from center points, intending not to have any unreachable level for the factors. These variables and their respective ranges were selected based on screening tests in preliminary studies. Levels of independent variables are given in Three numerical factors have been considered for this purpose, namely, equation :(1)N=2n+b0 is the constant, bi is the linear coefficient and bii is the quadratic coefficient, and xi and xj are the coded values of pyrolysis conditions.In which, The analysis of experimental data was performed using Design Expert version 7  for regression analysis to fit the empirical models and statistical significance evaluation of the models.2  in Millipore water with purity of 99.99% to concentration of 2000 ppm. The batch adsorption experiments were conducted in 20 sets of 250 ml Erlenmeyer flasks. In a typical experiment, 0.4 g of biochar was added to 50 ml of heavy metal solution with concentration of 200 ppm and the pH of solution was set at 6. Subsequently the mixture was agitated for 24 hours for equilibration and then filtered employing Whatman filter paper. The equilibrium time was selected based on preliminary studies as the time when the zinc concentration remained constant. The resultant solution was analyzed for concentration of zinc utilizing A Analyst 400 PerkinElmer atomic absorption spectrometer device. The adsorption capacity of biochars was calculated by the following equation: Zinc stock solution was prepared by dissolving appropriate amount of ZnClCi and Ce are the initial and equilibrium concentration of zinc (mg/l), respectively. Q (mg/g) is the adsorption capacity of biochar, m is the dry mass of biochar (g), and v is for volume of solution (l).In the previous equation, The yield of biochar was calculated according to the following equation:N2 adsorption at 77 K was performed for surface area and pore volume estimation of biochar, using Sorptomatic 1990 system (Thermo Finnigan). Surface functional groups of biochars were determined with aid of Fourier Transform Infrared spectroscopy, using Nicolet Nexus 6700 FTIR spectrometer. The KBr pellet was prepared by mixing well grinded biochar samples with KBr powder at ratio of 1 : 100 approximately.The surface structure of biochar was analyzed by means of scanning electron microscopy utilizing Zeiss scanning electron microscope (Carl Zeiss Germany). The complete design matrix of experiments with the obtained results for both responses is presented in Y1) and zinc adsorption capacity of biochar (Y2) in terms of coded variables are presented in , and coefficient of variation (CV) were used to evaluate the quality of developed model. R2 values for . The ANOVA for quadratic model for yield of biochar is given in F-value was 46.44 and p value was less than 0.0001, reflecting that model was significant. Regarding the model terms, p value less than 0.05 implies that model term was significant. According to x1, x2, x3, and x12 were significant model terms, whereas x1x2, x2x3, x1x3, x22, and x32 were insignificant terms to the model.Positive sign in front of the terms represents the synergistic influence while negative sign represents antagonistic influence. Values of coefficient determination, lues for  and 6)  R squareF-value of 52.95 with p value less than 0.0001 indicates the significance of the model. x1, x2, x3, x12, x22, and x32 are significant model terms. On the other hand, the interactions of factors x1x2, x1x3, and x2x3 were insignificant terms to the response.The result of ANOVA for quadratic model for adsorption capacity of biochar is presented in The predicted versus experimental values for yield and adsorption capacity of OPEFB biochar are illustrated in Figures Referring to the yield of biochar, HTT had the greatest influence on the response followed by the HR and RT. Figures  Conocarpus wastes biochar by increasing the highest treatment temperature, specifically when the temperature increased more than 200°C. This may be due to the destruction of cellulose and hemicellulose and combustion of organic matters [Similar trend was also reported in other works, studying the influence of production parameters on char yield. Al-Wabel et al. reported reduction in yield of matters . Angin f matters . McBeath matters . This is matters . Ronsse  matters .In this work, all three production variables correlated negatively with biochar yield. The interaction effect of production parameters on biochar yield was not significant. The increase in pyrolysis temperature resulted in releasing of more volatile compound, primary decomposition of parent material, and possible secondary decomposition of produced biochar. Increasing in heating rate may cause severe heat and mass transfer rate which led to lower biochar yield. The influence of residence time on biochar yield was not also significant.F-value of 33.4246 was the most influential factor. Figures Based on the ANOVA, all three variables and their quadratic effect were found to be significant on the adsorption capacity of OPEFB biochar; however, HTT with Surface plot of the effect of highest treatment temperature and residence time on adsorption capacity of OPEFB biochar when heating rate was fixed at zero level is presented in Getting high yield is an important factor in producing biosorbents, but adsorption capacity determines the quality of product. Therefore, both high yield and high adsorption capacity is desirable for economics feasibility of the product. However, optimizing both of these responses is very difficult as their affecting factors are opposite, which means adsorption capacity of OPEFB biochar increases while the yield decreases and vice versa. Consequently, in order to compromise between the two responses, the function of desirability has been employed using Expert-Design software version 7 . The experimental conditions which showed the highest desirability were chosen for verification. The yield and adsorption capacity of the biochar prepared under optimum conditions compared to the predicted values are presented in As it can be observed from Figures 2/g surface area and 0.15 cm3/g total pore volume.In this study, novel, low cost adsorbent from empty fruit bunches were synthesized by pyrolysis. The influence of pyrolysis conditions on the yield and adsorption capacity of oil palm empty fruit bunches biochar for zinc was studied employing RSM. Through analysis of developed response surfaces, HTT was found to have the most significant influence on both responses. The optimum biochar was obtained at highest treatment temperature of 615°C, heating rate of 8°C/min, and residence time of 128 min which demonstrated 15.18 mg/g zinc adsorption capacity and 25.49% yield. The resultant biochar produced under optimum conditions demonstrates 421.26 m


Epilepsy is a neurological condition that affects 1% of the world population. Conventional treatments of epilepsy use drugs targeting neuronal excitability, inhibitory or excitatory transmission. Yet, one third of patients presents an intractable form of epilepsy and fails to respond to pharmacological anti-epileptic strategies. The ketogenic diet is a well-established non-pharmacological treatment that has been proven to be effective in reducing seizure frequency in the pharmaco-resistant patients. This dietary solution is however extremely restrictive and can be associated with complications caused by the high [fat]:[carbohydrate + protein] ratio. Recent advances suggest that the traditional 4:1 ratio of the ketogenic diet is not a requisite for its therapeutic effect. We show here that combining nutritional strategies targeting specific amino-acids, carbohydrates and fatty acids with a low [fat]:[proteins + carbohydrates] ratio also reduces excitatory drive and protects against seizures to the same extent as the ketogenic diet. Similarly, the morphological and molecular correlates of temporal lobe seizures were reduced in animals fed with the combined diet. These results provide evidence that low-fat dietary strategies more palatable than the ketogenic diet could be useful in epilepsy. However, approximately 30% of patients display intractable forms that do not respond to antagonists of sodium or calcium channels, glutamate receptors, nor to enhancers of gamma-aminobutyric acid (GABA) mediated inhibition1. In such cases, a common solution is the ketogenic diet (KD), which was developed in the early 1920s and designed to mimic the metabolic mode of fasting. KD causes body fat to be converted into ketone bodies, which can then be used as a source of energy. To achieve this, the diet must comply to 4 portions of lipids for 1 portion of carbohydrates + proteins, which typically leads to 90% of the energy being provided by fat, 7–8% by proteins and only 2–3% by carbohydrates2. Despite the effectiveness of the KD, which reduces seizures by at least 50% in approximately half of the enrolled patients, this dietary solution is very demanding and difficult to follow, such that it is almost exclusively applied to the treatment of children experiencing intractable epilepsy2. In these instances, KD is indeed a valuable way of controlling seizures during critical periods of development. However, KD is associated with systemic complications, including growth retardation, nephrolithiasis, and hyperlipidaemia3.Epilepsy is a neurological condition that affects ~65 million people worldwide. The development of antiepileptic drugs in the second half of the 204. This implies that it is not ketone bodies but rather the drastic reduction in glucose, which shuts down the LDH metabolic pathway, that is protective. Consistent with this, the relationship between seizure control and serum levels of β-hydroxybutyrate (βHB) and acetoacetate (ACA), the two main ketones produced in KD, is far from clear5. Most interestingly, several other dietary strategies have been explored to modify or replace KD. One of the first modifications of the KD consisted in replacing part of the lipids typically provided as long-chain fatty acids with medium-chain fatty acids6. These indeed present a higher ketogenic potential as they are absorbed more effectively and carried directly to the liver by the portal blood. The original version of this diet contained 60% of medium-chain triglycerides, a proportion ensuring ketosis. However, such high amounts of medium-chain triglycerides were found to often provoke abdominal pain and gastrointestinal disorders7, so that it is now advised to use medium- and long-chain triglycerides in equal measures8. The use of polyunsaturated fatty acids may also be beneficial against seizures, as shown for omega-3 fatty acids both in animal models10 and in humans12, although their efficacy is not obvious in patients14. An alternative approach has been to target carbohydrates, not through restriction but by making sure they do not elevate blood sugar levels. Such low glycaemic index treatment has provided good protection against seizures in the small number of clinical trials reported15, despite weaker and more variable levels of ketosis than the KD. Accordingly, the efficacy of the low glycaemic index treatment was found not to correlate with the degree of ketosis but rather with the decrease in blood glucose16. Also supporting this approach, glycolytic inhibition with 2-deoxyglucose administration has proved effective in reducing seizure occurrence in epilepsy models18. Finally, interesting data suggest that the proportion of branched-chained (BCAA) vs aromatic amino acids (AAA) in the blood can influence excitability of central neuronal networks20. The BCAAs leucine, isoleucine and valine would indeed favour ketosis and GABA synthesis while reducing glutamate levels21. Thus, a higher BCAA/AAA blood ratio has been proposed to decrease brain excitability, thereby favouring seizure control20. Accordingly, L- and D-Leucine have been found to exert a potent anti-seizure effect22.Although the mechanisms by which KD protects against seizures remain poorly understood, it has been proposed that reducing the activity of the glycolysis enzyme lactate dehydrogenase (LDH) would be sufficient to reduce seizure occurrence23. Antiepileptic efficacy of the CD was then tested on susceptibility to seizures and on the chronic kainate (KA) mouse model of epilepsy. Cytological and molecular correlates of seizure occurrence in KA mice were also compared between diets.In the current study, we sought to design a healthy and more palatable diet with effective anti-epileptic properties by combining different nutritional strategies. This new combined diet (CD) comprises medium-chain fatty acids, polyunsaturated fatty acid, low glycaemic index carbohydrates, and a high BCAA/AAA ratio. We first ascertained whether CD could induce the downregulation of excitatory drives associated with the traditional KDWe aimed at composing an antiepileptic diet with a ratio of [fat]:[proteins + carbohydrates] which moderately deviates from a standard diet (SD), by combining macronutrients reported to reduce neuronal excitation. Hence, the fatty acid content of our combined diet was enriched in both medium-chain and polyunsaturated fatty acids. Nutritional analyses showed that they respectively provided 31.9% and 17.2% of the lipid energy content of the CD. In addition, 38.5% of the carbohydrate caloric supply was provided by a powder of low glycaemic index vegetables. Finally, the dietary proteins were chosen so that they showed a high BCAA/AAA ratio, yielding respectively 20.6% and 6.81% of the diet energy content Fig. . The resad libitum showed no change in body mass assessed every third day upon 3 weeks of diets . To investigate whether such downregulation could be due to high level of ketone bodies, blood samples from mice fed with the different diets were tested for markers of ketosis β-hydroxybutyrate (βHB) and acetoacetate (ACA), as well as markers of glycolysis - glucose, lactate and pyruvate , while markers of glycolysis metabolism, i.e. glucose , lactate  and pyruvate , significantly decreased . In order to ascertain that this change in presynaptic excitability influences presynaptic release of neurotransmitters, we assessed paired pulse facilitation, a form of short-term plasticity sensitive to changes in release probability25. Paired-pulse profiles of KD and CD fed mice were significantly different from controls , with a higher facilitation at the shortest inter-stimulation interval , thus indicating lower synaptic glutamate levels in the CA1 hippocampal area of mice fed with CD and KD.Finally, to investigate whether the decreased presynaptic function resulted in reduced glutamate levels at the synapse we used γ-d-glutamylglycine (γ-DGG), a low-affinity competitive antagonist of the AMPARs, at a non-saturating concentration (0.5 mM) at which its potency depends on glutamate levelAltogether, these data indicate that CD reduces neuronal excitability and glutamate release at excitatory synapses, as does KD, thus suggesting that a combined non-ketogenic dietary treatment may be an alternative protective strategy against epileptic seizures.A-receptor antagonist pentylenetetrazol  or KD , indicating that mice fed with these diets were less prone to trigger paroxysmal events. Furthermore, the frequency of interictal events was also found to be reduced by ~30% in the CD and KD groups compared to controls . However, the severity of PTZ-induced convulsive behaviour was comparable between the groups as most paroxysmal episodes were scored ≥ grade 4 in SD (100%), CD (~90%) and KD (100%) groups. Together, these data show that both CD and KD decrease latency to paroxysmal events, but not severity.To investigate whether CD could indeed protect against seizures, we first acutely induced paroxysmal events and convulsions by injecting the proconvulsant GABAin vivo assay enabling evaluation of the sensitivity of mice to proepileptic agents. However, it is not a model of chronic epilepsy, which most closely resembles the human epileptic condition. One experimental model of chronic epilepsy is KA-induced temporal lobe epilepsy obtained by performing a unilateral intrahippocampal injection of KA . Respectively ~38% (5 out of 13) and ~12% (1 out of 8) of the CD and KD fed mice even showed no EEG detected seizures in the recorded period while 100% of SD fed mice displayed epileptic events (10 out of 10). However, the mean seizure duration was similar for all three groups  into the CA1 area of the hippocampus of 6 weeks old C57BL/6j males Fig. , and the2, 144 = 71.409, p < 0.001 for both diets).We then tested whether this protective effect of the CD could be due to a diet-induced reduction in excitability in the epileptic hippocampus. To assess presynaptic excitability in epileptic KA mice, we measured the fibre volley amplitude as a function of stimulation intensity. This analysis revealed a significant reduction in presynaptic excitability of CD and KD fed mice as compared to controls fed with SD .Gliosis is a typical hallmark in chronic temporal lobe epilepsy and may contribute to the disease phenotype. Molecular correlates of chronic epilepsy were assessed by examining the levels of glial fibrillary acidic protein (GFAP) in the cortical regions from which electrophysiological recordings were obtained Fig. . Consist3,15 = 5.26, p < 0.05 for both treatments). Cell loss in CA1 and CA3 hippocampal areas was also limited in both CD and KD groups .Finally, neuroanatomical correlates of recurrent seizures were also considered by assessing dispersion of the granule cell molecular layer of the dentate gyrus, as well as cell loss in the hippocampal CA1 and CA3 areas, typical consequences of chronic temporal lobe epilepsy Fig. . SD fed As a whole, our study indicates that the CD strategy decreases excitatory drive, lessen the propensity to paroxysmal events, and reduces anatomical and histological markers of epileptogenicity.The current study aimed at ascertaining whether preferential intake of specific nutrients could downregulate excitatory neurotransmission and yield protection against seizures. To our knowledge, this is the first attempt to assess the effect of combining several antiepileptic nutritional strategies aiming at dampening excitatory transmission and seizure occurrence. Evaluation of synaptic function at Schaffer-collaterals CA3 to CA1 hippocampal synapses indicated that mice fed with this combined diet indeed show significantly reduced excitatory neurotransmission attributable to a decreased presynaptic excitability and hence, a reduced probability of glutamate release. As well, CD fed mice are much less susceptible to PTZ induced seizures and are largely protected against chronic seizures as well as cytological and molecular changes in the KA model of epilepsy.in vitro studies showing no inherent effect of βHB or ACA on synaptic transmission30, although an acute application of these compounds overlooks the chronic effects likely occurring in these protracted treatments. Kim and collaborators indeed showed that chronic administration of ketone bodies is sufficient to reduce seizures in the kcna1-null mouse model of epilepsy31. Interestingly, most investigations that examined the intrinsic effect of ketone bodies by direct application in glucose containing artificial cerebrospinal fluid (ACSF) found no effect on synaptic function30 while replacement of extracellular glucose with βHB or ACA reduces neurotransmission32. Therefore, a conspicuous yet recurrent question in KD research is whether the reduction in excitation is attributable to lipid metabolism or to dormant glucose metabolism. The latter possibility is supported by several investigations showing that glycolytic inhibition alone does reduce epileptic events in rodent models of epilepsy33. Moreover, a recent study provides some ground for these effects by demonstrating that blocking the glycolysis metabolic enzyme lactate dehydrogenase (LDH) is sufficient to hyperpolarize neurones and protect against seizures4. Our findings are also in line with this, as mice maintained on CD showed no increase in ketone blood content and nonetheless showed a similar decrease in neurotransmission as KD fed mice. The decrease in lactate observed in both CD and KD groups also favours this interpretation as it may be due to lower LDH activity in converting glucose and pyruvate to lactate. This is indeed a possible consequence of a low glycaemic index diet34, enriched BCAA35, as well as high levels of medium-chain triglycerides36 and polyunsaturated fatty acids37. Importantly, although synaptic glutamate concentrations have previously been proposed to be reduced in KD23, we here further suggest that such reduction is, at least in part, due to a decreased presynaptic excitability leading to less glutamate release. These findings are in line with previous in vivo investigations reporting a decreased hippocampal excitability in calorie-restricted and KD fed rodents38. Interestingly, the reduction in fibre volley we report in CD and KD fed epileptic mice is more profound than in control CD and KD fed mice. This differential effect might be explained by cellular alterations and homeostatic changes that occur following kainate injection even when cell death is not prominent. Such effect may not be seen in control SD fed epileptic mice as the increase in presynaptic excitability would increase the fibre volley while neurodegeneration would decrease it, eventually resulting in no change. Insofar as both CD and KD induce similar changes in neurotransmission, and that these are consistent with the neuronal hyperpolarization showed by Sada et al. (2015)4, it is indeed likely that the nutritional strategies employed in our CD concur to downregulate the LDH metabolic pathway, a component of the astrocyte-neurone lactate shuttle. It also remains probable that other mechanisms act in parallel to dampen neuronal excitation, such as for instance reduced glutamate level in the brain that could result from a high BCAA/AAA ratio20, or neuronal hyperpolarization induced by polyunsaturated fatty acids though modulation of voltage gated ion channels39, or the increase in adenosine receptors activation under medium-chain triglycerides treatment, which reduces probability of neurotransmitter release40.Most interestingly, unlike KD fed mice, animals under CD did not enter ketosis and still displayed comparable changes in neurotransmission and susceptibility to seizures. These results support the view that ketone bodies do not fully explain the decrease in excitation associated with the classical KD. A lack of correlation between ketone bodies concentration and protection against seizures has indeed been reported 42. This side effect is however reversible upon interruption of the KD. In this regard, the combined nutritional strategy we propose herein would most likely be beneficial as, besides its much lower fat content, it provides several nutrients with beneficial effects. Indeed, polyunsaturated fatty acids provided in rapeseed oil were found to improve lipid content of children with hypercholesterolaemia43 and a polyunsaturated fatty acids rich olive oil based KD showed reduced dyslipidaemia44. Medium-chain fatty acids have also been found to reduce cholesterol, triglycerides and lipoproteins levels36. Interestingly, several studies also suggest that preferential intake of low-glycaemic index carbohydrates reduces lipid blood content46. Growth retardation is also a detrimental side effect often observed in children on KD. It has been suggested that the risk of growth impairment might be lower on diets allowing higher protein consumption and decreased fat intake47. Although no study is available to confirm this hypothesis, if it held true, the more balanced combined strategy used in our study might avert concerns about growth.Although the classical KD is less deleterious in terms of general health than one might expect in regard to its high fat content, various side effects of the diet have been reported. These include defects in growth (in children), lipid levels, cardiac function, kidney stones, haematology, immunology, metabolism , as well as gastric issues such as constipation, diarrhoea, nausea and vomiting48. To circumvent this issue, Kossoff et al. developed a less restrictive protein rich KD called “modified Atkins diet” (MAD) with the aim of ameliorating diet palatability. Such strategy proved to be efficient as approximately half of the patients displayed reduction in seizure frequency >50%49. Yet, although the diet is well tolerated among patients, hyperlipidaemia is still commonly observed50. The nutritional strategy proposed herein may be more palatable than the traditional KD, as it comprises a higher proportion of carbohydrates (~15% vs. ~10% in the MAD) and similar proportions of proteins and fat. Given the beneficial impact of polyunsaturated fatty acids and low glycaemic index46 on health parameters including blood lipid content, and the manageability of medium-chain triglycerides gastrointestinal side effects, it is also reasonable to anticipate that the CD approach may be well tolerated by patients.The unpalatability leading to difficult adherence mostly explains why KD is less commonly used in adult treatments52. Given that epileptic conditions are often deleterious to cognitive functions, besides its protection against seizures, the CD approach may also offer appreciable procognitive effects. This assumption is supported by our finding that CD decreases presynaptic excitability. Indeed, although the well accepted synaptic plasticity and memory hypothesis suggests that higher neuronal excitability would favour the long-lasting changes in the strength of synaptic connections thought to underlie memory formation53, several investigations suggest that decreasing neuronal excitability would actually be beneficial to cognition as it enhances the dynamic range for plasticity and reduces the signal-to-noise ratio. Perhaps the most direct evidence of this effect is the enhancement of cognitive functions in both humans and rats by cathodal transcranial direct current stimulations, known to decrease excitability by hyperpolarizing neurons55. Similarly, caloric restriction, which decreases excitability38, has been found to improve cognitive performances in humans and mice57. Finally, BCAA have been found to help cognitive recovery following traumatic brain injury58 and may therefore show similar recuperation properties on seizure-induced lesions.Other positive features of the proposed dietary strategy stem from recent investigations showing that medium-chain triglycerides and polyunsaturated fatty acids have a beneficial impact on cognition, mostly by slowing down its decline in pathology or ageingIn summary, by comparing the effect of the KD with the combination of several dietary antiepileptic strategies on basal excitatory neurotransmission and their consequences in terms of susceptibility to acute or chronic seizures, our study provides the proof of concept that the association of specific nutrients into the same non-ketogenic regimen confers robust protection against seizures. Such diet is more balanced (lower [fat]:[proteins + carbohydrates] ratio) than the classic KD or MAD and contains nutrients providing several beneficial impacts on general health, including cardiovascular health and cognitive functions. Although side-effects and toxicology tests are required, it would now be of prime interest to perform clinical trials using a similar combined approach.Experiments were carried out according to the guidelines of European Community Council Directives of 01/01/2013 (2010/63/EU) and our local animal care committee (Center for Interdisciplinary Research in Biology in College de France). Experiments were performed on 6 to 12 weeks old wildtype C57BL/6j male mice. All efforts were made to minimize the number of used animals and their suffering.Experiments were approved and carried out in accordance with the national ethical committee for animal experimentation number 59, authorization number 2015071010466740.Mice were fed with standard diet (SD), CD, or KD. SD was provided by the Teklad 2018 diet. KD was provided by the rodent Bioserv F3666 specific diet. PILEJE Laboratoire provided the CD.Levels of glucose, lactate, pyruvate, β-hydroxybutyrate, and acetoacetate were measured on whole blood  deproteinized (with one volume of perchloric acid) using spectrophotometric enzymatic methods as described in ref. 61. Briefly, mice were sacrificed by cervical dislocation and decapitation. The brain was rapidly removed and placed in chilled (1–4 °C) ACSF composed of (in mM): 119 NaCl, 2.5 KCl, 2.5 CaCl2, 1.3 MgSO4, 1 NaH2PO4, 26.2 NaHCO3, 4 glucose, 7 sucrose). Hindbrain and forebrain were excised and the brain laid flat on the rostral aspect. Fine curved blunt forceps were used to remove the midbrain and the majority of the white matter. The brain was then laid dorsally and the hippocampi dissected out. Slices were maintained at room temperature in a storage chamber containing ACSF saturated with 95% O2 and 5% CO2 for at least 1 h before the experiments.Acute transverse hippocampal slices (400 μm) were prepared as previously describedstratum radiatum region of the hippocampus. Field excitatory postsynaptic potentials (fEPSPs) were recorded in the presence of the GABA-receptor inhibitor picrotoxin (100 µM) with glass pipettes (2–5 MΩ) filled with 1 M NaCl. A cut was made between CA1 and CA3 to prevent the propagation of epileptiform activity  microscopy and were perfused with ACSF (2 ml/min). All experiments were performed in CA1 ity Fig. . PostsynEEG experiments were carried out using wireless ETA-F10 transmitters  for chronic EEG recording and video monitoring. After anaesthesia , a 1 cm midline sagittal incision was made starting above the skull midline and extending along the neck to create a pocket for subcutaneous placement of the transmitter along the dorsal flank of the animal. The flexible recording electrodes were implanted subdurally through small holes drilled in the skull  and held in place with dental cement. Mice were allowed to recover for 7 days before recording.EEG signal was collected through DSI radiofrequency receivers placed under each cage. EEG data were acquired at a sampling rate of 200 Hz using the DSI Dataquest A.R.T. system, version 4.33.status epilepticus. Protective properties of diets were assessed by feeding the mice with KD, CD or SD (controls) for 3 weeks before the PTZ experiment, scoring status epilepticus grades according to Ono et al.62 and evaluating the latency to ictus as well as the frequency of interictal events. The Racine’s scale used to grade status epilepticus was as follows: 0, behavioural arrest; 1, rhythmic mouth and facial movements; 2, head nodding; 3, forelimb clonus; 4, rearing and bilateral forelimb clonus; 5, rearing and falling or jumping. Interictal events were spikes or spike trains with a minimal amplitude of 1.5X baseline, and a maximal duration of 5 seconds. The number of detected interictal events was then counted manually.Nine weeks old mice implanted with wireless ETA-F10 DSI EEG electrodes were injected i.p. with 50 mg/kg of PTZ and placed in an open arena for video and EEG recording of the 64. The number of seizures was averaged per day. Artefacts were automatically detected using a high detection threshold and removed manually.Six weeks old C57BL/6j mice were injected unilaterally with kainate  at a rate of 0.05 µl/min in the CA1 area of the right hippocampus  by mean of a stereotaxic apparatus and a cannula (0.29 mm diameter) connected to a precision pump. Mice were then left on SD for 3 weeks for settling of the chronic epileptic phenotype. To assess therapeutic potential of the diets, KA mice were then fed for 3 weeks with KD, CD or SD (controls) and recorded with ETA-F10 DSI EEG electrodes for 72 h in their home cage. Analysis of electrographic seizures, i.e. seizures detected on the EEG recording, was performed using NeuroScore software, and defined as spike trains with a minimal amplitude of 1.5X baseline, and a minimal duration of 5 seconds at a frequency ≥1 HzCortical tissue surrounding the subdural electrode Fig.  was sampSix weeks after intrahippocampal injection of KA, mice were anesthetized, perfused transcardially with PBS 0.1 M and their brains were rapidly removed and frozen in isopentane cooled at −30 °C. Coronal sections (20 μm) were then cut on a cryostat (Microm Cryo-Star HM 560), collected on slides, and fixed with 4% paraformaldehyde in PBS for 30 min at 4 °C. Slices were then processed for staining with the nucleus marker DAPI, mounted with fluoromount and left overnight to set before imaging with a confocal laser-scanning microscope  with a 10X and 60X objective. The fluorescent images were analysed with ImageJ. CA1 and CA3 areas as well as the dentate gyrus (DG) of the hippocampus were independently outlined, and an intensity threshold was set to identify pixels that were DAPI-positive. Cells were then counted in areas of interest using the ImageJ built-in particle analyser and their density (number of cells per mm²) was determined.All data are expressed as mean ± SEM and n represents the number of independent experiments. Statistical significance was determined by one-way and two-way ANOVA using Statistica 6.1 (Statsoft Inc.) and Statview 5.0 . When relevant, a Newman-Keuls post hoc analysis was performed.Supplementary information


Human cytomegalovirus is a ubiquitous infectious agent that affects mainly immunosuppressed, fetuses, and newborns. The virus has several polymorphic regions, in particular in the envelope glycoproteins. The UL55 gene encodes the glycoprotein B that has a variable region, containing a furin cleavage site and according to the variability different genotypes are characterized. Here we investigated variability and existence of selective pressure on the UL55 variable region containing the furin cleavage site in 213 clinical sequences from patients worldwide. We showed the occurrence of positive selective pressure on gB codons 461 and 462, near the furin cleavage site. Cleavage analysis of synthesized peptides demonstrated that most mutations confer better cleavage by furin, suggesting that evolution is acting in order to increase the efficiency cleavage and supporting the hypothesis that gB processing is important in the host. We also demonstrated that peptides containing sequences, that characterize genotypes gB2 and 3, are differentially cleaved by furin. Our data demonstrate for the first time that variability in the cleavage site is related to degree of gB processing by furin. Human Cytomegalovirus (HCMV) is a ubiquitous human pathogen, present in 40–100% of the global population. In most cases, viral replication is controlled by the host immune system and infection is asymptomatic. HCMV-associated pathogenesis, therefore, predominantly affects immunocompromised individuals such as transplant recipients and AIDS patients , while cAs an opportunistic agent, it is evident that the mechanisms that allow the virus to escape the humoral and cellular immune responses contribute to pathogenesis . MoreoveThe effect of genetic variability within the HCMV glycoproteins on many aspects of the viral life cycle has been investigated. One of the most explored polymorphic regions is the UL55 gene that encodes glycoprotein B (gB) . gB is aThe gB protein has 906 amino acids, containing a cytoplasmic domain (Cy), a transmembrane region (TM), a membrane proximal region (MP), and a ectodomain (ED) . It has in vivo and in other cell types was not been determined.The clinical relevance of these different genotypes has been evaluated by several studies, in different parts of the world, which investigated a correlation between gB genotypes and tropism, viral loads or clinical disease. While a number of studies found some possible association between gB genotype and infection course , other sPopulations of organisms, including viruses, have genetic variability among their individuals, which can be selected by different environmental pressures. In the context of viral infection, the pressures imposed by the cellular environment positively select the variations that confer a higher fitness to the virus. The selective pressure can be estimated site by site using nucleotide multiple alignment of target regions among different populations and analyzing how it translates to amino acids in the protein primary structure. The identification of genomic regions under positive selection can be measured by estimating the non-synonymous (dN) and synonymous (dS) nucleotide substitutions and their ratio (ω = dN/dS). Rates of (ω) = 1 mean neutral evolution, (ω) < 1 purifying or negative selection, and (ω) > 1 diversifying or positive selection. Biologically, sites under positive selection could indicate regions that might be important for adaptation of the virus, augmenting viral fitness.In this work, we performed an extensive analysis of the occurrence of positive selection in the variable region of the UL55 ORF that contains the furin cleavage site. We report the occurrence of sites under positive selection pressure. Analysis of the cleavage efficiency by furin, using FRET peptides, indicates that the all mutations on residue 462 (gB2) increase the quality of gB as a furin substrate in terms of processing velocity and afinity while the mutation on residue 461 (gB3) increased the processing velocity, but decreased the affinity. Moreover, sequences that characterize different gB genotypes are differentially cleaved.In order to determine the gB genotypes present in clinical strains obtained from Brazil, and to establish their relationship to sequences from different geographical locations, DNA amplification of the gB variable region was performed in blood samples obtained from 22 renal recipients, 2 allogeneic transplant recipients, 1 premature newborn and tumor tissues tissue from 6 glioblastoma patients , followed by gB3 (18%) and gB1 (4%). Genotypes gB4, gB5, gB6, and gB7 were not found.Figure S4).In addition to the sequences obtained from Brazilian patients, 181 sequences from different groups of patients (HIV positive and transplant recipients) from distinct geographic regions were retrieved from GeneBank. The total 213 sequences were aligned and the 100% homologous sequences, at nucleotide level, were excluded  and 5, 6, and 7 (dots).The cleavage by furin occurs between an arginine (R) and a serine (S) at residues 5 and 6 in the sequence, as indicated by an arrow. As observed the canonical sequence recognized by furin RXR(K)R/S is well conserved, however, mutations outside this region occur frequently and characterize the different genotypes.Figure 2), gB6 and gB7 are close related to gB1 and gB3  was used to evaluate the strength and direction of selection. Codons with higher B+/α rates are under a stronger positive selective pressure than codons with lower B+/α rates .Figure 1), which corresponds to codons 461 and 462 of the gB.The output revealed the occurrence of positive selection on codons 6 and 7 on the alignment (Table 1).Notably, codon 461 is under a significantly stronger positive pressure than codon 462 as demonstrated by its higher B+/α rate , according to the protease-substrate model from Figure 1). The other regions in the sequence did not exhibit significant positive selection.These two codons are located near the furin cleavage site and correspond to the positions P1′ and P2′, respectively .In order to strength our findings, a second analysis of selective pressure was performed codon by codon for each gB genotype by Bayesian Inference (BI), using the software MrBayes . The BI Supplementary Figures S1–S3). The kcat/km ratio relates to the catalytic efficiency, meaning how fast in M/s the enzyme reacts with the substrate once it encounters it. As a control, a peptide which contains a mutation previously shown to interfere with furin cleavage .In order to see if the mutations have an influence in cleavage, peptides containing the original reference sequences and their derivative containing positive selected mutations, were synthesized and tested against recombinant human furin. All substrates were cleaved after the double basic residues RR or KR as analyzed by Liquid Chromatography mass spectrometry (LC-MS) . The differences in km are notable, in particular in the S462R substitution, which increases 31-fold the kcat/km ratio. This mutation was found in two allogeneic transplant recipients and eight renal transplant recipients. Mutation S462M was found in one renal transplant recipient and S462A in eight real transplant recipients. All these patients are from São Paulo, Brazil (Supplementary Table kcat) in comparison to the reference sequences.As shown in The HCMV genome presents polymorphisms at both intra and inter host levels, and variability is found in many regions, in particular in genes that encode glycoproteins . The mutHere we analyzed the occurrence of positive selection in the gB variable region that contains a furin cleavage site, in sequences from different populations worldwide.Figure 3). Notably, mutations that characterize positive selection were not found in regions outside the specific cleavage site.Our analysis shows that the furin cleavage site is highly conserved in patient samples. Nevertheless, positive selection was evident in two codons at positions P1′ (aa 461) and P2′ (aa 462) , and the better-cleaved reference peptide among the genotypes is gB3. Furthermore, three positively selected mutations in genotype 2 significantly increase the efficiency of cleavage, while one mutation in genotype 3 decreases it (Table 2). This data reveal that variability in this regions affect cleavage and that mutations under positive selective pressure are indeed modifying the quality of the gB as a furin substrate, either increasing or decreasing the km/kcat ratio.kcat/km ratio are better cleaved by furin, suggesting that gB processing is important for viral replication in vivo. We also can speculate that in other context substrates for which the furin have high affinity and low reaction velocity, such as gB3_26, may trap the enzyme, preventing the cleavage of host substrates by furin, which may somehow favor to the virus. In this way, gB could act as a competitive inhibitor of furin. In fact, Positively selected mutations which alter the gB cleavage kinetics can indicate an augment of the viral fitness. Substrates with an increased in vivo is not recognized. A study by in vitro replication of the RVHB5 strain in fibroblasts. Analysis in other herpesviruses such as pseudorabies virus (PRV), Epstein-Barr virus (EBV), and BHV-1 gB also concluded that cleavage is not required for virus replication in vitro but is important for cell-to-cell fusion . In gB1, gB3, gB4, gB6, and gB7, glycine (G), a small non-polar residue, is the only hydrophobic residue present in this region. These observation suggests that in HCMV gB this region is not directly related to host-cell membrane fusion, as its hydrophobicity is low when compared to viruses which have fusion proteins with hydrophobic P3′–P6′ regions. In fact, previous analysis indicate that the HCMV gB fusion loops comprise aa 174, 179, 259, 261, and 262 , asparagine (N), and Threonine (T) . The accession numbers are shown in Supplementary Table A total of 213 sequences corresponding to the gB variable region of HCMV were analyzed in this study. Thirty two sequences were obtained through DNA sequencing of blood from 22 renal transplant, 3 allogeneic transplant recipients, 1 premature newborn and tumor tissues of 6 glioblastoma patients from Brazil. The sequences KX859110 to KX859141, were deposited in NCBI’s GenBankThis study has approval of the research ethics committee (REC) no. 04171312.0.0000.5415 of the Medical School of São José do Rio Preto- FAMERP – São Paulo. The patients were anonymized and the written consent was waived by REC. This study also has approval of the REC 0654/10 of the Neurology Center of Clínicas Hospital – São Paulo. The patients were informed of the procedure and signed a consent form.® Genomic DNA Purification KitWizard (PromegaTM) and DNA from glioblastoma tumor tissues was extracted using the Pure Link genomic DNA mini kit (Invitrogen), according manufacturer’s instructions.DNA from peripheral blood was extracted using the 2, 2.5 U Platinum Taq Polymerase High Fidelity (Invitrogen), 200 μM dNTPs, and 0.15 μM of each primer.To amplify the gB variable region a hemi-nested PCR (nPCR) was performed using primers previously described by The thermo cycling program was set as 94°C for 14 min, 35 cycles at 95°C for 30 s; 56.2°C for 30 s; 72°C for 45 s; 72°C for 10 min. All the reactions were performed at least three times for confirmation and to avoid false positive and negative results. No positive controls were used during the HCMV detection in nPCR to minimize the possibility of laboratory contamination with HCMV DNA. As a positive control for cellular DNA, primers for the Glyceraldehyde 3-phosphate dehydrogenase gene (GAPDH) gene were used (5′-ACC CAC TCC TCC ACC TTT GAC-3′ and 5′-CTG TTG CTG TAG CCA AAT TCG T-3′).TM) at the Serviço de Sequenciamento de DNA–SSDNA IQUSP. Confirmation of the HCMV sequences, and the determination of the gB genotypes were performed using the National Center for Biotechnology Information (NCBI-USA) Blast tool.Amplified products were purified using the Illustra GFX PCR DNA and Gel Band Purification Kit  and sequenced using the BigDye Terminator v3.1 Cycle Sequencing kit with an ABI-3130  or synonymous (dS) substitutions. The algorithm attributes different weights for each substitution, according to the probability of occurrence and their impact in the protein translation. In this manner scores are calculated for synonymous (α) and non-synonymous (β+) substitutions and the ratio is used to estimate the selective pressure site by site. A second analysis of the probability of selection was done for each gB genotype by BI, using the software MrBayes .The search for positive selection was performed using the Mixed Effects Model Evolution (MEME)  via Data MrBayes  with a 8Recombinant human furin was expressed and purified as previously described . The enze = 17,300 M-1.cm-1). The quantum yields of the FRET peptides and the products of their hydrolysis were unchanged in all tested conditions.Seven synthetic peptides were obtained by solid-phase strategy, as previously described  using thThe FRET peptides were assayed in 10 mM MES, 1 mM CaCl2, pH 7.0, using a F-2500 spectrofluorimeter , at 37°C , as prevkm), the catalysis constant (kcat) and the kcat/km ratio. The kinetic parameters km and kcat were calculated by non-linear regression using Grafit software . km, kcat, and standard errors (less than 10%) were calculated using Grafit software  with a non-linear least squares Michaelis-Menten fit associated with a matrix invertion fitting routine to refine kcat, km and allowing estimation of the errors. Standard deviations greater than 10% are due to low values obtained.Three kinetic parameters from the Michaelis-Menten model were analyzed, representing enzyme-substrate (All the authors contributed with: substantial contributions to the conception or design of the work; or the acquisition, analysis, or interpretation of data for the work; drafting the work or revising it critically for important intellectual content; final approval of the version to be published; agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Tetrao urogallus) and other grouse species represent conservation concerns across Europe due to their negative abundance trends. In addition to habitat deterioration, predation is considered a major factor contributing to population declines. While the role of generalist predators on grouse predation is relatively well known, the impact of the omnivorous wild boar has remained elusive. We hypothesize that wild boar is an important predator of ground-nesting birds, but has been neglected as a bird predator because traditional morphological methods underestimate the proportion of birds in wild boar diet. To distinguish between different mammalian predator species, as well as different grouse prey species, we developed a molecular method based on the analysis of mitochondrial DNA that allows accurate species identification. We collected 109 wild boar faeces at protected capercaillie leks and surrounding areas and analysed bird consumption using genetic methods and classical morphological examination. Genetic analysis revealed that the proportion of birds in wild boar faeces was significantly higher  than indicated by morphological examination (3.8%). Moreover, the genetic method allowed considerably more precise taxonomic identification of consumed birds compared to morphological analysis. Our results demonstrate: (i) the value of using genetic approaches in faecal dietary analysis due to their higher sensitivity, and (ii) that wild boar is an important predator of ground-nesting birds, deserving serious consideration in conservation planning for capercaillie and other grouse.Capercaillie ( Tetrao urogallus) is one of the most threatened and best studied species among this group of ground-nesting birds [Grouse  have long been a conservation concern . Many pong birds . Conservng birds , mesopreng birds  and reinng birds . Populatng birds  and in sng birds . Nonetheng birds ,8.Martes martes), raccoon dog (Nyctereutes procyonoides), red fox (Vulpes vulpes) and goshawk (Accipiter gentilis) [Sus scrofa) has been largely neglected and the species is at best considered as an occasional nest predator [In Estonia, the overall predation pressure and number of different predators are high in capercaillie protection zones in the early breeding season . The maentilis) –10, wherpredator . The diepredator . Althougpredator –18 and wpredator ,20, low predator  with potpredator ,22. Yet,We hypothesize that: (a) the proportion of birds in wild boar faeces is underestimated by common morphological methods, and (b) wild boar has significant impact on ground-nesting birds. Our aims were: (1) to develop a non-invasive genetic method to identify both the predator and the bird species by the analysis of mitochondrial DNA isolated from faeces, an approach preferable to stomach contents because non-invasive collection can be carried out also in areas where hunting is prohibited, and 2) to determine the frequency of occurrence of ground-nesting birds, particularly the capercaillie, in wild boar diet. We analysed wild boar faeces collected at capercaillie lek sites during spring-early summer, which is the capercaillie lekking and nesting period, when birds spend a lot of time on the ground and are most vulnerable to predation.Seven protected capercaillie lekking areas were monitored during three years (2013-2015) from the end of March to the middle of June—the period of capercaillie displaying and breeding in Estonia. Three areas were located in Central Estonia , three in Eastern Estonia  and one in Southern Estonia (Karula). All of the Central Estonian and one lekking area in Eastern Estonia were situated in a forest-wetland habitat; the rest of the areas were characterised by open coniferous forests. Daily visits to the lekking areas were conducted in order to minimize disturbance to the birds. Wild boar faeces, which are morphologically easily distinguishable from other mammals in Estonia, were systematically collected by searching lekking areas and the surrounding forest. Faeces were put into plastic bags and stored at -80°C before analysis to prevent further degradation of DNA in samples.Samples were collected during the time when movement was restricted in the protected areas of capercaillie. A special permit (No 1–4.1/13/241) was obtained from the Estonian Environmental Board for searching capercaillie lekking areas and the surrounding territories for faeces left by potential predators of capercaillie.DNA was extracted from 180–220 mg of faeces using QIAamp DNA Stool Mini Kit (Qiagen) according to the manufacturer’s instructions, as described in the protocol “Isolation of DNA from Stool for Human DNA Analysis”. For molecular identification of wild boar scats and prey species, taxon-specific primers  were desPCR reactions were carried out in a total volume of 20 μl with 1x Phusion HF Buffer , 0.2 mM dNTP, 0.25 μM of each primer and 0.4U Phusion Hot Start II DNA Polymerase and 2 μl of purified DNA. The PCR mixture was initially denatured at 98°C for 30 s, followed by 10 touchdown cycles for 10 s at 98°C, 20 s at 60°C (reducing the temperature 1°C per cycle) and 30 s at 72°C, followed by 30 cycles of 10 s at 98°C, 20 s at 50°C and 30 s at 72°C.PCR products were checked using 2% 1xTAE gel-electrophoresis and visualized under UV radiation using ethidium bromide. GeneRuler Ultra Low Range DNA Ladder (ThermoFisher Scientific) was used as a molecular-size marker. In each PCR batch both a negative (reaction mixture without DNA) and a positive control were included to monitor contamination and check for the right amplicons.PCR products were purified using the Exo-SAP method: to degrade primers and dephosphorylate dNTPs, 1 μl mixture consisting of 1U Exonuclease I  and 1U Alkaline Phosphatase  (ThermoFisher Scientific) was added to 10 μl PCR product. The reaction mixture was incubated for 30 minutes at 37°C, followed by 15 minute enzyme inactivation at 80°C.http://blast.ncbi.nlm.nih.gov/) was used to identify mammal and bird taxa.DNA-sequencing reactions were conducted for the samples that gave positive results in mammal and/or bird detection, using the same primers as for PCR . SequencFaeces were washed on a metal sieve (0.8 mm mesh size) and identifiable objects were divided into eight categories: greens , roots, supplementary (grain from feeding sites), invertebrates , mammals , birds , birds/reptiles , other .Va% is the percentage volume of a food category, ∑Va is the volume of objects in a category, and V is the total volume of objects in all categories.To quantify diet composition, frequency of occurrence  and volume of each food category were measured. Items were placed into a measuring cylinder by category, and the volume (ml) of each category was measured by water displacement . Owing to the difference in faeces size, relative volume percentage was calculated, using the formula Tetrao tetrix) and 1 hazel grouse (Tetrastes bonasia). Due to partial sequences, the other four bird-positive samples were determined to genus level: three to Tetrao and one to Corvus. In comparison, the morphological analysis of wild boar faeces (N = 52) identified only two cases of bird consumption (FO = 3.8%), containing both feathers and bone fragments of adult birds, and failed to detect birds in six samples where bird consumption was confirmed with genetic analysis. In two cases the molecular method helped to specify morphological category bird/reptile, one of which turned out to be black grouse. Thus, the sensitivity of genetic analysis is over 4.5 times higher compared to the morphological approach ; and the remaining three consisted of bird/reptile, carrion  or invertebrate.Of 109 wild boar faeces analysed with the molecular method, 49 were identified as wild boar and of these in 6 we identified bird DNA. Moreover, among the samples that gave negative result in mammalian PCR, the genetic method identified bird DNA in three samples. Although the genetic analysis could not identify the predator for three faecal samples, either because the sample had degraded or included inhibitors that prevented DNA amplification, they clearly belonged to wild boar (based on morphology). Thus, including the three samples in which the genetic analysis failed to identify the mammalian predator, in total we identified 9 faeces with bird DNA from 52 wild boar samples  was also frequently detected in wild boar faeces see . CapercaWe conclude that for efficient conservation of ground-nesting birds, the wild boar should be considered as an important predator of capercaillie and other grouse, especially in areas where wild boar densities are high. Compared to the traditional morphological analysis, non-invasive genetics proved to be considerably more efficient in identifying birds consumed by wild boars. This suggests that many previous studies based on morphological methods may have underestimated the rate of bird consumption by omnivorous mammalian predators and can have important consequences on conservation decisions that depend on the results of dietary studies. Based on the results of this study, we recommend using genetic methods in all trophic studies where the food category of interest is not easily detectable or identifiable due to high level of degradation.S1 TableFO % – percentage of frequency of occurrence; Volume % – percentage volume of different food categories. The smaller dataset (N = 52) includes wild boar faeces that gave a positive result with the molecular analysis, the larger dataset (N = 109) includes all samples that were collected and morphologically identified as belonging to wild boar.(PDF)Click here for additional data file.