Batrachochytrium dendrobatidis (Bd), and for which there is an urgent need to develop mitigation methods. We treated tadpoles of the common midwife toad (Alytes obstetricans) with antifungal agents using a capture-treat-release approach in the field. Antifungal treatment during the spring reduced the prevalence of Bd in the cohort of tadpoles that had overwintered and reduced transmission of Bd from this cohort to the uninfected young-of-the-year cohort. Unfortunately, the mitigation was only transient, and the antifungal treatment was unable to prevent the rapid spread of Bd through the young-of-the year cohort. During the winter, Bd prevalence reached 100% in both the control and treated ponds. In the following spring, no effects of treatment were detectable anymore. We conclude that the sporadic application of antifungal agents in the present study was not sufficient for the long-term and large-scale control of Bd in this amphibian system.Emerging infectious diseases can drive host populations to extinction and are a major driver of biodiversity loss. Controlling diseases and mitigating their impacts is therefore a priority for conservation science and practice. Chytridiomycosis is a devastating disease of amphibians that is caused by the fungal pathogen  An increasing number of emerging fungal diseases is causing host mass mortality, extirpating local populations, and threatening biodiversity2. These diseases can cause population declines because all host individuals can become infected before host population size falls below a threshold that would halt an epizootic2. Chytridiomycosis is an emerging infectious fungal disease of amphibians which has become a model system for the study of disease-induced host population decline and extinction3. The etiological agent of chytridiomycosis is the chytrid fungus Batrachochytrium dendrobatidis (Bd) (Chytridiomycota), which can infect larval, metamorph, and adult amphibians4 (a second pathogenic chytrid fungus was recently described5). Bd infects a wide range of amphibian species, some acting as a reservoir for the pathogen6. Even though Bd is responsible for amphibian mass mortality, population extirpations, global extinctions of species, and the loss of phylogenetic diversity8, there is no established method to mitigate the effects of the disease in wild populations12. However, it is known from agriculture that fungal pathogens of plants can be controlled using fungicides. Fungicides are known to reduce Bd infection13 and two proof-of-concept studies have shown that fungicides can be used to control or even eradicate Bd in the wild15.Emerging infectious diseases have been recognized as important drivers of the loss of biodiversityBd infection prevalence of the different host life history stages and the sensitivity of the host population growth rate to stage-specific disease-induced mortality16. Bd does not seem to affect the survival of tadpoles, which allows this stage to act as an intraspecific reservoir for Bd19. In contrast, Bd reduces survival of post-metamorphic individuals21, the life history stage to which the amphibian population growth rate is highly sensitive24. Fungal treatments that clear the infection or that reduce the infection load in tadpoles before metamorphosis may therefore reduce mortality in post-metamorphic juveniles and increase population viability16. Such treatments may further reduce disease prevalence in amphibian species where Bd transmission occurs between overlapping generations of tadpoles.The efficacy of mitigation depends on the Alytes obstetricans) is a good model system for studying the epidemiology and control of Bd because this host is highly susceptible and commonly infected with this pathogen26. This species has suffered Bd-driven population declines and extirpations and is red-listed in some European countries29. The tadpoles of this species overwinter so that in the summer there are often multiple cohorts of tadpoles present in the same pond. After overwintering, Bd prevalence is often very high, which may lead to pathogen transmission to young-of-the-year tadpoles30. In a previous laboratory study, we had shown that treatment with an antifungal agent  could reduce Bd prevalence and load in A. obstetricans tadpoles31. The purpose of the present study was to use antifungal agents to reduce the prevalence and infection intensity (zoospore load) of Bd in natural populations of the common midwife toad. We predicted that antifungal treatment of tadpoles would reduce Bd prevalence and load, allow tadpoles to metamorphose into a disease-free state, and reduce Bd transmission between overlapping cohorts of tadpoles.The common midwife toad  or the young-of-the-year cohort (n = 1484), hereafter referred to as the 2010 and 2011 cohort, respectively. The tadpoles in the 2010 cohort were captured in the months of May, June, and July 2011, whereas the tadpoles in the 2011 cohort were captured in the months of August 2011 to April 2012. The 2010 and 2011 cohorts were analysed separately because they occurred in the pond at different times of the year.A total of 2096 Bd if it had a Bd zoospore load >0.01. The Bd prevalence refers to the proportion of tadpoles that were infected with Bd. In the statistical analysis of micro-parasite infections, it is standard practice to separately analyse pathogen prevalence and pathogen load (or pathogen burden). The analysis of Bd prevalence was based on all of the tadpoles in the data set (n = 2096 tadpoles), whereas the analysis of Bd zoospore load (see below) was based on the subset of tadpoles that were classified as being infected with Bd.A tadpole was classified as infected with Bd in the tadpoles was lowest in August and September and highest in the winter in most ponds . Similarly, for most of the statistical analyses below, the treatment status of a tadpole (fungicide versus control) depends upon its pond of origin and not whether that particular individual was treated with fungicide or not. For this reason, these statistical analyses are labelled as occurring at the pond level. This approach is justified if fungicide treatment of a fraction of the tadpole population in the treatment ponds reduces the risk of Bd transmission to the untreated tadpoles (i.e. herd immunity).The prevalence of nds Fig. . In the Bd as a function of the fungicide treatment , the covariate time, and their interaction. Here all tadpoles belonging to the same pond were assigned the same fungicide treatment status . The 2010 and 2011 cohorts were analysed separately because they occurred at different times of the year. The interpretation of the parameter estimates is as follows: the intercept refers to the prevalence of Bd in the control ponds on the intercept date (see below for the definition of the intercept date). The fungicide treatment is the difference in Bd prevalence between the treated ponds and the control ponds on the intercept date. The covariate time is a slope that indicates whether the prevalence of Bd increases or decreases over time. A significant interaction between fungicide treatment and time indicates that the difference in Bd prevalence between the control ponds and treated ponds changes over time. In the case of a significant fungicide treatment:time interaction, it is not possible to make a general statement about the effect of the fungicide treatment.We used generalized linear mixed effects models with binomial errors to model the prevalence of 2) to account for the curvature of the relationship between Bd prevalence and time , it became necessary to include a quadratic effect of time , the Bd prevalence was lower in the treatment ponds (compared to the control ponds) and therefore it had to increase rapidly over the fall to reach the same high level  in the winter . The seasonal pattern in the Bd zoospore load resembled the seasonal pattern of the Bd prevalence , the covariate time, and their interaction. All tadpoles belonging to the same pond were assigned the same fungicide treatment status . The 2010 and 2011 cohorts were analysed separately because they occurred at different times of the year. The interpretation of the parameter estimates is the same as described for the prevalence of Bd. The intercept date for the 2010 cohort is May 5, 2011, whereas the intercept date for the 2011 cohort is December 27, 2011  with normal errors to model the log10-transformed Bd-infected tadpoles in the 2010 cohort, examination of the model selection results , July 2011 , and October 2011 to April 2012 (recapture of 2011 cohort tadpoles marked 0 or 1 times in September 2011).In the three treatment ponds , tadpoles were marked with visible implant elastomer (VIE) tags to indicate whether they had been treated with fungicide once or twice. The VIE tags allowed us to test whether the number of times that an individual tadpole had been treated with fungicide influenced its 2, 113 = 18.29, p < 0.001). The fungicide treatment decreased the Bd zoospore load of the tadpoles in the SagO and Herg ponds but increased the Bd zoospore load in the SagU pond . Similarly, for the 2011 cohort tadpoles, the fungicide reduced the Bd zoospore load in individual tadpoles but the effect was not significant . We point out that in Fig. Bd zoospore load follows the expected pattern in 5 of the 6 panels (the SagU pond in the top row is the exception). In summary, tadpoles that had been treated with fungicide once or twice, generally had lower zoospore loads than the tadpoles that had never been treated but the effect was not significant.For the 2010 cohort tadpoles captured in June 2011, the interaction between pond and fungicide treatment was significant  and it is therefore a challenge to separate their effects. We tried to model the temporal dynamics of Bd infection as thoroughly as possible to make the analysis as conservative as possible with respect to detecting an effect of temperature .We had originally wanted to include pond temperature as a covariate in the preceding analyses. Unfortunately, the temperature data logger failed for the SchU pond. In the preceding analysis, we decided that it was more important to include the SchU pond than temperature. The purpose of the present analysis was to estimate the effects of temperature on Bd prevalence of the 2011 cohort, there was strong support for a positive linear effect of time  rather than clear infection.Developing treatments against fungal diseases is a priority for both humans and wildlifeBd zoospore load was weaker and inconsistent , the Bd zoospore load was actually higher in the treated ponds than the control ponds. Overall, the results are consistent with the interpretation that the antifungal treatment cleared weak infections (low Bd zoospore load) but had no measurable effect on the heavy infections (high Bd zoospore load). The analysis at the individual level generally found that the median Bd zoospore load declined with the number of fungicide applications, but the results were not statistically significant . By December, almost 100% of all the tadpoles were infected with Bd in both the control ponds and the treated ponds. The monotonic rise in the Bd prevalence from summer to winter suggests that there is a very high force of infection in these tadpole populations. The observation that Bd prevalence and Bd zoospore load remained high throughout the winter . In the present study, the Bd infection spread rapidly during the months of August, September, and October, when pond temperatures were in the range of the optimal growth conditions  and winter (December and January), the Bd zoospore load remained high but did not change in the tadpole population 33. Lam et al.45 assumed that p ≈ 0.8 which led Woodhams et al.9 to suggest that R0 ≈ 5 for Bd. Since R0 for Bd is unknown, it is hard to predict whether successful control of Bd might be achieved (reservoir hosts and persistence of zoospores in the environment make control even more difficult). Thus, estimating R0, a basic epidemiological parameter, would be of great importance to better inform the science and practice of disease control.The present study has shown that a capture-treat-release approach using antifungals can be used to transiently decrease the prevalence of Bd treatments have been tested in the laboratory12 but the number of successful attempts at Bd mitigation is still very low12. It is therefore important to do more field trials of mitigation methods15 that are ethically and legally acceptable12. Since the Bd-host interaction is strongly context-dependent46, mitigation strategies should be tested under a variety of environmental conditions to determine whether a method can be used only locally or whether it is transferrable to other systems. Last but not least, even if recent studies suggest that Bd mitigation in natural populations may be possible, prevention is better than cure47. Thus, preventing the emergence of pathogens in naïve wildlife populations is the best way to protect biodiversity against emerging infectious diseases48.Many potential anti-A. obstetricans and Bd30 were selected: Sagerhüsli upper (SagO), Sagerhüsli lower (SagU), Hergiswald (Herg), Schauensee upper (SchO), Schauensee lower (SchU), and Hinter Rohren (HiR). These ponds were similar in elevation (range = 558–798 m), surface area (6–30 m2), and depth (0.5–2.5 m). The water temperature of each of the six ponds was measured six times per day over the duration of the study using HOBO H8 temperature data loggers . The tadpoles in SagO, SagU, and Herg were treated with an antifungal agent whereas SchO, SchU, and HiR served as control ponds.The study was carried out in canton Lucerne in central Switzerland. At four different sites, six ponds that were known to contain ® antifungal agent for 8 days in 100 L cattle tanks that were filled with 50 L of tap water. We used a concentration of 0.6 ml of General Tonic® per litre of water31. After 4 days, the tanks were filled with a fresh solution of General Tonic® water. Cattle tanks were placed near the ponds and covered with a shade cloth. Fungicide-treated tadpoles were marked with visible implant elastomer (VIE) tags before being released into their original ponds49. A different VIE tag colour was used for the fungal treatments of May, June, July and September 2011. Tadpoles were tagged every time that they were captured and treated with antifungals.The study took place from May 2011 to April 2012 and involved two different cohorts of tadpoles: 2010 and 2011. For the 2010 cohort, most of the tadpoles (98.2% = 612/623) were captured in the months of May, June, and July  and this cohort left the ponds as metamorphs by August 2011. For the 2011 cohort, most of the tadpoles (98.5% = 1484/1507) were captured from August 2011 to April 2012 . Tadpoles in the 2010 cohort were treated with fungicide on three separate occasions , whereas tadpoles in the 2011 cohort were treated once (September). Field-captured tadpoles were treated with General TonicA. obstetricans tadpoles were captured each month except in February 2012 because the ponds were frozen. Tadpoles were captured using dip nets and tested for Bd infection by swabbing the mouthparts with sterile rayon-tipped plain swabs using a plastic applicator . In the treatment ponds, the surveyors swabbed a maximum of 30 previously treated tadpoles (VIE tag present) and 30 untreated tadpoles (no VIE tag) each month. In the control ponds, the surveyors swabbed a maximum of 30 untreated tadpoles each month. We obtained a total of 2132 mouthpart swab samples that were stored at −20 °C until analysis.Over the duration of the study (May 2011 to April 2012), ® Ultra Sample Preparation Reagent Protocol (Applied Biosystems by Life Technologies). The tip of each Bd swab (1–2 mm) was cut off using a sterile and single-use surgical carbon blade (Paramount®). The tips were placed into 60 µL of PrepManTM Ultra (Applied Biosystems by Life Technologies) with 0.03–0.04 g Zirconial/Glas-beads . The samples were shaken at 30 Hz for 45 seconds, using a TissueLyser Adapter Set 2 × 24 (QIAGEN®) then centrifuged at 13,000 rpm for 1 minute using an ALC Multispeed refrigerated centrifuge. The last two steps were repeated before placing the samples into an Eppendorf Thermomixer Comfort® at 99 °C for 10 minutes followed by centrifugation at 13,000 rpm for 3 minutes47. The resultant supernatant was placed into an Eppendorf tube and stored at −20 °C.DNA was extracted from the tadpole mouthpart swabs using the PrepManBd in the tadpole mouthpart swabs following the protocol of Boyle et al.50. The specific primers ITS1–3 Chytr and 5.8S Chytr and the minor groove binder probe Chytr MGB2 were used to amplify and detect the 5.8S rRNA gene and the flanking internal transcribed spacer (ITS) of the Bd pathogen50. Each qPCR reaction had a total reaction volume of 20 µL and contained 10 µL of 2x Master Mix , 0.9 µM of each primer, 0.25 µM of the MGB2 probe, and 5 µL of DNA (diluted 1:10 in water). The thermocycling conditions were as follows: a preincubation step at 95 °C for 10 minutes followed by 50 cycles at 95 °C for 15 seconds and 60 °C for 1 minute50. The qPCR was performed using a LightCycler® 96 Real-Time PCR System (Roche Applied Science). All DNA samples were run in duplicate. Each 96-well plate contained 3 negative controls, 3 positive controls, and 4 standards  run in triplicate to quantify the Bd zoospore load. The four standards contained 101, 102, 103, and 104 copy numbers of the target gene.Quantitative PCR (qPCR) was used to estimate the zoospore load of Bd zoospore load was very high for a sample of 606 DNA extractions that had been run in duplicate for the qPCR assay .The repeatability of the Bd zoospore load > 0.01 were defined as being infected with Bd. We analysed two different response variables: the proportion of tadpoles infected with Bd (n = 2096 tadpoles), and the log10-transformed Bd zoospore load for the subset of infected tadpoles (n = 1638 tadpoles). We used generalized linear mixed effects (GLME) models with binomial errors and linear mixed effects models (LME) with normal errors to model the Bd prevalence and the log10-transformed Bd zoospore load, respectively. The 2010 and 2011 cohorts were analysed separately because they occurred at different times of the year . The fixed effects structure included the fungicide treatment , the covariate time, the covariate time2 (only for the 2011 cohort), and their interactions. For the 2011 cohort, the covariate time2 was included because the temporal change in the Bd infection resembled a negative quadratic function. The random effects structure included the categorical factors pond, month, and their interaction. In this study, we expected that there would be no difference between ponds at the beginning of the experiment. The fungicide treatment was expected to gradually reduce the Bd prevalence over time in the tadpoles. Thus, the treatment-by-time interaction was expected to be more informative than the main effect of treatment.The 36 tadpoles that were captured outside the dominant sampling period for their cohort (13 tadpoles for 2010 cohort and 23 tadpoles for the 2011 cohort) were eliminated from the analysis. The final sample size was therefore 2096 tadpoles: 612 and 1484 tadpoles for the 2010 and 2011 cohorts, respectively. Tadpoles with a 2 were highly correlated, which caused problems with model convergence and parameter estimation. These variables were therefore transformed using the poly function in R so that they were orthogonal to each other. The origin (t = 0) of this transformed time scale is day 136 , and day 148 . After transformation, time2 was a negative quadratic (concave down). A positive slope for the transformed time2 variable therefore meant that the Bd prevalence (or Bd zoospore load) was highest in the winter and lower in the fall and spring.Each cohort had its own time scale: day 1  to day 68  for the 2010 cohort, and day 1  to day 246  for the 2011 cohort. For each cohort, the covariate time was expressed in months by dividing the number of days by 30. For the 2011 cohort, time and timeBd infection  over time. We expected the dynamics of the Bd infection to be different between the treatment ponds and the control ponds. In other words, we expected an interaction between the fungicide treatment and time, which indicates that the rate at which tadpoles acquire or lose Bd infections is different between the treatment ponds and the control ponds. In this study, we expected that the interaction term would be more important than the main effect of the fungicide treatment. For each cohort, the meaning of the contrast in the Y-intercept between the control and treated ponds depends on how the time covariate was rescaled. For the 2010 cohort, this contrast measures pre-existing differences in Bd infection between the two types of ponds on May 5, 2011, which was prior to the application of the fungicide treatment. For the 2011 cohort, this contrast compares the Bd prevalence (or log10-transformed Bd zoospore load) between treated and control ponds on December 15, 2011 .We monitored the epidemiology of the 51.We used a model selection approach based on the Akaike information criterion (AIC) to find the most parsimonious model. Models were ranked according to their AIC values and the Akaike weights were calculated for each model. The support for a given explanatory variable of interest is the sum of the Akaike weights of all the models in the set that include that particular explanatory variable. The support for a given explanatory variable can range from low (0.0%) to high (100.0%). We also used the Akaike weights to calculate the model-averaged parameter estimates and their 95% confidence limitsBd zoospore load. We used linear models to test whether the number of fungicide treatments applied to an individual tadpole influenced its log10-transformed Bd zoospore load. This analysis was restricted to the tadpoles in the three experimental ponds . The 2010 and 2011 cohorts were analysed separately. For the 2010 cohort, the analysis was further split for the months of June and July because the number of fungicide applications differed between these two months. For the 2010 cohort, tadpoles captured in June (n = 172) had been treated never or once, whereas the tadpoles captured in July (n = 167) had been treated never, once, or twice. For the 2011 cohort, tadpoles captured in the months following the single fungicide treatment in September (n = 569) had been treated never or once.In the three treatment ponds , the marking of the tadpoles with VIE tags allowed us to test whether the number of times that an individual tadpole had been treated with fungicide influenced its Bd prevalence and the log10-transformed Bd zoospore load as a quadratic function of the pond temperature (°C) and a quadratic function of time (days). The random effects structure was similar to the previous models. We restricted these analyses to the 2011 cohort because there were only 3 time points for the 2010 cohort. Due to a data logger failure, the analysis was restricted to five ponds because mean monthly pond temperature data were not available for SchU. As before, time (range: day 1 to day 246) and time2 were rescaled as orthogonal contrasts, and the same was done for temperature (range: 2.2 °C to 18.2 °C) and temperature2. For the 2011 cohort, the origins of the transformed time scale and the transformed temperature scale are day 137  and 9.48 °C , and day 151  and 8.75 °C . After transformation, time2 was a negative quadratic  was highest in the winter and lower in the fall and spring (the reverse was true for the transformed temperature2 variable).We used GLME and LME models with binomial and normal errors to model °C Table  for all °C Table  for the c Figure  whereas c Figure . A posit52. We used the glmer function and the lmer function in the lme4 package to run the generalized linear mixed effects models with binomial errors and normal errors, respectively. We used the model.sel function and the model.av function in the MuMIn package to create the model selection tables and the model-averaged parameter estimates. We used the confint function in the base package to calculate the 95% confidence intervals (CI) for the model-averaged parameter estimates. We used the poly function in the base package to rescale time and time2 (or temperature and temperature2) so that they were uncorrelated with each other.We used R for all statistical analysesExperiments were performed in accordance with the relevant regulations . The study was conducted under the animal experimentation permit number 75/2009 by the veterinary office of the canton Zürich; the permit included an approval of the methods. Capture permits (as required by the nature conservation law) were provided by the nature conservation office of canton Lucerne.Supplemental Material


Passer domesticus) were exposed to a chronic stress protocol. We took physiological and behavioral measurements before, during, and after the protocol. Blood samples were used to assess four aspects of hypothalamic-pituitary-adrenal (HPA) axis function: baseline corticosterone, stress-induced corticosterone, negative feedback, and the maximal capacity to secrete corticosterone. We also assessed bacterial killing capacity and changes in uric acid concentration. Neophobia trials were used to assess behavioral changes throughout the protocol. We found no significant changes in HPA axis regulation in any of the four aspects we tested. However, we found that uric acid concentrations and neophobia significantly decreased after only four days of the chronic stress protocol, while bacterial killing capacity did not decrease until after eight days of exposure. These results indicate that different components of the stress response can be impacted by chronic stress on different timescales. Our results further indicate the importance of assessing multiple aspects of both physiology and behavior in order to understand how exposure to chronic stress may influence ability to cope with future challenges.Despite decades of research, we still lack a complete understanding of what factors influence the transition of the necessary and adaptive acute stress response to what has become known as chronic stress. This gap in knowledge has illuminated the necessity for studies that examine the thresholds between these two sides of the stress response. Here, we determine how repeated exposure to acute stressors influences physiological and behavioral responses. In this repeated measures study, house sparrows ( Passer domesticus) to this threshold in order to identify key transition points in physiological and behavioral indices.In recent decades, the stress response has become widely studied across many disciplines including ecology e.g., , conservThe stress response involves several behavioral and physiological systems e.g., . We focuCORT helps regulate downstream effects of the stress response, including metabolic, immunological, and behavioral effects . CORT isGallus gallus domesticus) (Sturnus vulgaris)  , but remulgaris) . Additioulgaris) , while oulgaris) . Finallyulgaris)  and incrulgaris)  in rodenThe goal of this study was to expose house sparrows to repeated acute stressors over the course of several days to push them just to the edge of experiencing serious symptoms of chronic stress. Prior studies have shown that different aspects of HPA axis function and regulation are significantly altered after eight to ten days of simulated chronic stress in laboratory and field settings . While wTo test the physiological and behavioral effects of repeated exposure to stressors, 22 wild free-living house sparrows  were caught June 1–3 2016 in Medford, MA, USA. Individual birds were randomly assigned to one of two treatment groups and housed in cages in separate rooms according to this distinction. Randomization occurred such that each group contained both males and females and such that no group contained birds that were all captured on the same day. Birds were housed in pairs and/or in view of other birds in cages (45 cm × 37 cm × 33 cm) and kept on a natural 15L:9D light cycle. An acclimation period occurred for at least 2 weeks during which birds were disturbed only for routine husbandry. This period was determined based on previous research involving house sparrows, which showed hormonal acclimation occurring after 2 weeks in captivity .This study was a repeated measures design in which the responses of individual birds during the experiment were compared to pre-stress samples. At the end of the initial 2-week acclimation period, a pre-experiment blood sample was taken . Assay sensitivity was 1 ng/ml; baseline and negative feedback samples were often below the level of detection (20 samples) and therefore assigned this floor value. The inter and intra-assay CVs were 7.6% and 3.5% respectively.Radioimmunoassays (RIA) were used to quantify CORT concentrations in the baseline, stress-induced, negative feedback, and Max plasma samples . Brieflyin vitro analyses of bacterial killing capacity. All samples were assayed within 10 days of sampling as longer periods of freezing have been shown to dramatically affect killing  at 37 °C for 30 min. Then, 250 µL of tryptic soy broth (TSB) was added to each tube, followed by an additional 12-hour incubation at 37 °C. Positive controls with only E. coli and TSB were also created. Finally, a NanoDrop spectrophotometer  was used to quantify absorbance of each sample at 300 nm. Killing capacity was calculated as 1 –(absorbance of sample / absorbance of positive control). All samples were run in triplicate when possible; only 4 samples out of all those that were assayed had to be run in duplicate. The inter-assay CV, based on the positive controls, was 11.5%.Baseline plasma samples were also used for  killing . This as killing . Briefly® Red Uric Acid/Uricase Assay Kit . This assay relies on the conversion of uric acid to hydrogen peroxide, which subsequently reacts with Amplex® Red reagent in the presence of horseradish peroxidase (HRP) to form a red-fluorescent product. Briefly, plasma samples were diluted with 200 µL of the provided reaction buffer and a uric acid standard curve was generated using the provided reagents. To have enough reagents to run all samples, the Mastermix was slightly altered to include 150 µL of Amplex® Red reagent and 85 µL of the reaction buffer. To account for this change, spectrophotometric readings were taken at 30 and 45 min at 560 nm. No statistical differences were found between these samples and therefore all further analysis was completed using the 30-minute sample. A negative control, lacking uric acid, and a maximum-reading positive control were also included on the 96-well plates. Four separate assays were completed and samples were run in triplicate. The inter-assay CV from the positive controls was 7.3%.Baseline plasma samples were used to quantify uric acid using the AmplexIn addition to the physiological data gathered from plasma sampling, changes in behavior were assessed by measuring the birds’ responses to novel objects. Three neophobia trials were conducted prior to any stimuli, after five days of the chronic stress protocol, and after seven days of the chronic stress protocol. Because blood and behavioral samples could not be taken the same day, we were limited which days the neophobia trials could occur. Beginning five days prior to the first neophobia trial, food dishes were removed each night (within 1 h before lights off) and replaced each morning (within 1 h after lights on) to acclimate the birds. At this time, the cage liners were also replaced to remove any excess food was removed from the bottom of each cage; this was to ensure that birds would reliably return to their food in the morning.On the night directly before a trial, food dishes were removed and opaque blinders were placed between each cage to prevent the birds from seeing the neighboring trial, which could alter their own behavior . The folThree control trials were also conducted (one each day) prior to each neophobia trial to ensure that the stress treatments did not affect the birds’ normal feeding behavior. During these control trials, food dishes were returned in the morning without a novel object. Neophobia and control trials were conducted on days separate from blood sampling.All statistical analyses were conducted in RStudio . Before F-tests with Kenward-Roger adjusted degrees of freedom. Interactions were checked using the same method, but with an interaction term between group or sex and sample.Before conducting further analyses, we tested whether there was an effect of group or sex in any of the datasets. This was done in order to check if differences in sex or stimulus order (whether cage rolling or cage tapping was experienced first) caused significant effects to physiological or behavioral responses. We used linear mixed effects models (LMM) with individual bird identity as a random effect and group and sample as main effects in separate models  and three negative feedback samples (n = 63) did not contain enough plasma to be run. One sample from the stress-induced data was dropped because that bird did not mount an acute stress response to the restraint stimulus and a second sample did not contain enough plasma (n = 64). The different CORT responses measured here  play complicated activation, inhibition, permissive, and preparative roles and interact with different physiological systems  while two samples did not contain enough plasma to be run in the uric acid assay (n = 64). A single LMM was used to assess changes in killing capacity and uric acid concentrations over the course of the chronic stress protocol; again, individual bird identity was included as a random effect. Tukey’s multiple comparison post-hoc analyses were conducted as indicated above.One sample did not contain enough plasma to be run in the BKA (n = 59). Interactions between total perch hops and group, sex, and object were checked with individual LMMs and again a separate LMM was completed to compare activities across sampling periods. Separate models were used to assess changes in the control trials for both the feed latency and perch hopping data sets.Birds that failed to approach the novel object in the allotted time were assigned a ceiling value of 20 min. Interactions between feed latency and novel object were assessed with separate LMMs and a final LMM was run to compare feed latencies across the sampling protocol. Perch hops were counted during the last 15 min of the neophobia videos. The first 5 min were discounted to avoid confounding effects of experimenter presence. All videos were coded by the same person. The movements of two birds were obscured from where the camera was placed and one sample from an additional bird was removed from analysis because they did not display any perch hopping in the allotted time, making log transformation impossible . There were also no significant interactions between group and sample . Therefore, stimulus order was removed from any further analyses of those datasets. We conducted the same check for sex effects and interactions and found none ; sex was therefore removed from any further analyses in all datasets, except for the perch hopping data where we did detect a significant effect of sex.There was no main effect of group in the datasets for stress-induced, negative feedback, and maximum capacity CORT, bacterial killing, uric acid, or neophobia datasets  or group B  across the chronic stress protocol. We also did not detect any statistically significant differences in CORT levels at any of the other three bleed types  over the course of the chronic stress protocol . There was, however, a significant main effect of object . Regardless of object type, feed latency decreased significantly after 4 days of the chronic stress protocol  and sample  nor were there significant interactions between object and perch hops . Perch hopping significantly decreased after the initiation of the stress protocol and remained reduced through the remainder of the protocol , however neither females  nor males  significantly changed their activity during these control trials. We also found a significant interaction between group and perch hopping  during the control trials when no novel objects were present. Further inspection of graphs revealed that this interaction was driven by birds in group A (experienced cage rolling first) increased their perch hopping at the final sample point, while those in group B (experienced cage tapping first) decreased their perch hopping  significantly reduced feedback strength  . Birds tAlthough our original hypotheses for the behavioral measurements were based on prior studies connecting CORT and behavior, there is an alternative interpretation arising from the reactive scope model that could explain the decreases. The data suggest that chronic stress induced house sparrows to take additional risk when foraging. Rather than delaying their approach towards a novel, potentially dangerous, object, these birds more readily risked potential harm to forage. In certain circumstances this could be quite dangerous, particularly in the wild where novel objects might not be so innocuous as a colored food dish, ribbon, or plastic egg.Many behavioral ecology studies have attempted to link CORT to broader indices of behavior, including explorative behavior and foraging activity e.g., . The resWe have shown that both physiology and behavior can significantly change over the course of a short chronic stress protocol. Most importantly, there are different timescales at which the effects of chronic stress are being reflected in the different indices measured here. Additionally, birds became less neophobic and more active. These results suggest that the overall physiology of the birds is entering a new state, perhaps indicative of “chronic stress.” It is possible that these metrics indicate that these birds are closer to the overload threshold of the reactive scope framework. Interestingly, eight days of the chronic stress protocol did not appear sufficient to significantly alter HPA function, although a longer period of stress may have elicited an effect. Furthermore, these results provide important support for the idea that CORT is not the best indicator of chronic stress, particularly considering its complicated relationship with so many other physiological and behavioral systems. Despite decades of research on stress, we still lack the ability to predict under what circumstances pathological symptoms may develop. We hope that these data provide initial steps towards being able to better interpret results pertaining to the vertebrate stress response and their relation to overall individual animal health and fitness.10.7717/peerj.4961/supp-1Data S1Click here for additional data file.


Food-based dietary guidelines are promoted to improve diet quality. In applying dietary recommendations, such as the MyPlate, the number of servings in a food group is the unit of measure used to make food selections. However, within each food group, different foods can vary greatly in their nutritional quality despite often having similar energy  values. This study aimed to develop a novel unit of measure that accounts for both the quantity of energy and the quality of nutrients, as defined by caloric and micronutrient density, respectively, in foods and to demonstrate its usability in identifying high quality foods within a food group.A standardized unit of measure reflecting the quality of kilocalories for nutrition  was developed through a mathematical function dependent on the energy content  and micronutrient density of foods items within a food group. Nutrition composition of 1806 food items was extracted from the USDA nutrient database. For each food item analyzed, qCaln ratios were calculated to compare qCaln to its caloric content. Finally, a case example was developed comparing two plates adapted from the MyPlate.Examples of food items with highest and lowest qCaln ratios were displayed for five food groups: vegetables, fruits/fruit juices, milk/dairy products, meats/meat alternatives, and breads/cereals. Additionally, the applicability of the qCaln was presented through comparing two plates, adopted from the USDA MyPlate, to show differences in food quality.The newly developed qCaln measure can be used to rank foods in terms of their nutrient density while accounting for their energy content. The proposed metric can provide consumers, public health professionals, researchers, and policy makers with an easy-to-understand measure of food quality and a practical tool to assess diet quality among individuals and population groups.The online version of this article (doi:10.1186/s12937-016-0215-4) contains supplementary material, which is available to authorized users. Identifying best methods to measure food and diet quality is an ongoing scientific and policy-level challenge facing public health professionals, researchers, and decision-makers. Over the years, several approaches and methods have been proposed to assess food and diet quality, including diet quality indices and various nutrient profiling methods.Diet quality indices were developed to measure conformance of individuals and population groups to international and national dietary guidelines and recommendations, such as the U.S. dietary guidelines (MyPlate) . The HeaThe nutrient profiling method, defined as the science of ranking foods based on nutrient composition , 5, reprChallenges are faced when interpreting currently used diet quality indices and nutrient profiling measures, particularly in non-research settings, when consumers are requested to meet food-based dietary guidelines (FBDGs) and nutrition recommendations. For example, in applying dietary recommendations, such as the US MyPlate , the numIn order to address this problem, the present study aimed to create a novel unit of measure that accounts for both the quantity of energy  and the quality of nutrients, as defined by micronutrient density, in foods and to demonstrate the usability of this novel measure to facilitate the assessment of food quality. Specific objectives of the study included 1) developing a standardized unit of measure reflecting the quality of kilocalories for nutrition  that can account for the caloric and nutrient density per food item, 2) comparing food items per food groups based on the newly developed qCaln measure, and 3) testing the applicability of qCaln measure through demonstrating different food plates with various qCaln ratio scores.Findings from this study can provide a new measure that allows for merging multiple nutritional parameters into one simple, standardized unit. In addition, this newly proposed measure could provide researchers and public health professionals with a relatively easier tool to assess the diet quality of individuals and population groups in comparison to other nutrient profiling tools and diet quality indices.Generally, food energy has been measured in kilocalories , which accounts for the energy in the macronutrients  included in the sum total of ingredients in any food item. The quality of the food, however, is more often than not, determined by the amount and distribution of key micronutrients in food. Energy, macronutrient and micronutrient content of all food items within the Nutritionist Pro software  were included in the computations. The food composition database in this software was based on the United States Department of Agriculture (USDA) National Nutrient Database for Standard Reference, release 27 . In addii is the weighting for each micronutrient I, “Si” is the micronutrient score for each micronutrient “I”, and n is the amount of micronutrients included in the calculation. The value wi is the relative weighting for importance to each of the micronutrients.The qCaln is a function of the caloric content (per 100 g) from macronutrients and the amount and distribution of micronutrients (per 100 g) in a particular food such that:Equation wi of 0.10 as the weighting scores indicate that each micronutrient is equally important in a diet. For the purposes of this study, weightings for the selected micronutrients were considered identical, and weightings for micronutrients that were not included in the calculation were assumed to be “0”. Saturated fat, total sugar, and sodium were negatively weighted in the qCaln calculation, indicating that these micronutrients need to be limited to prevent cardio-metabolic risk factors, including elevated blood sugar, cholesterol, or blood pressure levels [Thus, each micronutrient was assigned an equal weighting e levels –33.Equation i” was calculated. The calculation is based on the amount of the selected micronutrient in the food item relative to other food items in the same food group to ensure proper comparison. The distribution of each selected micronutrient among all foods within each food group was tested for normality and were found to be close to a normal distribution. Given a normal distribution, the value of Si will depend on the standard score  of the micronutrient. The equation for the standard score of a micronutrient amount relative to the rest of the food group is given by:For each food item, and for each micronutrient “i”, a micronutrient score “Si of a micronutrient i was estimated based on z-score tables such that (0 ≤ Pi ≤ 1). Then, for each micronutrient i,Equation i was calculated for each micronutrient i, Eq. th percentile (0.5 < Pi ≤ 1.0) will have a greater qCaln value than the listed value of Cal indicating higher quality food, and foods with lower than the 50th percentile (0.0 ≤ Pi < 0.5) have qCaln values less than the listed Cal, indicating less quality. Also, it follows from Eqs. max = 2 * Cal).Once Si for each micronutrient, a qCaln value per 100 g was calculated for 1806 different food items from five different food groups. These 5 main food groups were: 1.) breads and cereals, 2.) dairy products, 3.) fruits and juices, 4.) vegetables, 5.) and meat and meat alternatives. Using the weightings from Eq. After calculating STo show the applicability of the qCaln ratio, two separate plates were compared. Each plate was based on FBDG recommendations from USDA MyPlate  and consOf the 1806 total food items analyzed, 534 had qCaln ratio values > 1.0 when compared to kilocalorie  content, and 1272 had ratios < 1.0. Ratios that are > 1.0 can be interpreted as higher quality food per calorie, whereas qCaln ratios < 1.0 represent lower micronutrient content per food item compared to its caloric content. Figures Figure Figure Figure Figure Figure In the case of vegetables, the highest 10 qCaln ratios included spinach, leeks, green peppers, collards, and turnip (greens), whereas those of the lowest qCaln ratios, reflecting lower micronutrient content to caloric content, were sweet pickles, pickled eggplants, canned capers, and red cabbage. For fruits, food items with the highest qCaln ratios were dried mixed fruits , dry cranberry beans, wild raspberries, cherries, and blackberries, and those of the lower qCaln ratios were canned juices and fruits canned in syrup such as papaya, plums, and loquats. These results are in line with dietary recommendations that highlight the high nutrient yet low energy density of green leafy vegetables and other fresh fruits and vegetables that are not preserved in salt or sugar. It is worth noting that the sugar content of canned fruits and fruit juices was a major contributor to lower qCaln ratios for fruit items. When analyzing the dairy products, food items with the highest quality were low-fat, non-fat, and low sodium varieties of milk and cheese as these items score lower in terms of fat and thus caloric content and have lower sodium levels, which is found in abundance within dairy products and could contribute to elevated sodium intake levels.Figure Finally, two separate meals were compared that would be considered good diet choices for each plate according to the MyPlate guidelines . Note thThis study aimed to develop a new unit of measure that accounts for both the caloric and micronutrient density of foods and to demonstrate the usability of this novel measure in assessing food quality. The qCaln was the standardized unit developed based on the caloric density and the amount and distribution of micronutrients per 100 g in a particular food. Results showed that food items in any food group can be analyzed based on their qCaln values. In addition, through calculating a qCaln ratio for each food item, the caloric and micronutrient contents of a food were compared with the food’s energy density  in one single measure. Using the qCaln ratio, higher quality foods could be differentiated easily from lower quality foods. More specifically, the qCaln ratios of food items within each of the food groups, including vegetables, fruits/fruit juices, milk/dairy products, breads/cereals, and meats/meat alternatives, were compared to each other in this paper.The advantages of the proposed approach over other existing food quality measures and nutrient profiling approaches lies in simplifying complex nutrient composition data into a singular based score that reflects both nutrient and energy density. The definition of nutrient dense foods have expanded over the years due to numerous attempts by researchers, regulatory agencies, and food companies to identify adequate criteria for ranking foods based on their nutrient composition. Some of the models highlighted the importance of limiting certain nutrients, such as total fat, saturated fat, sugar, or sodium, that were associated with adverse health outcomes, when defining nutrient-density , 35. OthThe selection of the 10 micronutrients that were included in the calculation of the qCaln accounted for two groups of nutrients: those that are not consumed in sufficient amounts and contribute, in part, to micronutrient deficiencies across various population groups –17 and lIt is worth noting that the percent daily value (%DV) of each micronutrient was not included in the calculation for qCaln within the present study for a number of reasons. First, Eqs. The proposed qCaln allows for a better interpretation of the food quality not only by researchers but also consumers. The qCaln can be linked to food-based dietary guidelines depicted visually through the MyPlate, MyPyramid, and the Mediterranean Diet Pyramid among other education models used by health care professionals to educate consumers about healthy dietary choices. For example, promoting the consumption of low-fat and low-sodium dairy products that have higher qCaln ratios , can assist consumers in meeting their beneficial nutrient requirements while avoiding less desirable nutrients. Similarly, promoting the consumption of fresh fruits like oranges rather than orange juice from frozen concentrate or sweetened fruit juices can help in making smarter food choices.Training consumers to select foods with higher qCaln values, where appropriate, can help improve diet quality and meet dietary recommendations. Currently, dietary guidelines are presented to the public as recommendations in terms of serving sizes of food groups that are promoted to be consumed, such as whole grains, low fat dairy products, and foods to avoid including those high in sugar and fat. These recommendations are usually based on studies highlighting the daily optimal intakes of energy, and macro- as well as micro-nutrients among humans . Despite the extensive efforts exerted by nutrition professionals to relay dietary guidelines in simple and interactive manners, consumers still face challenges in interpreting these recommendations. Difficulties arise when consumers are asked to make healthy dietary choices from a wide array of food products available on the market and with various nutrition facts labeling systems. In addition, deciphering the labeling systems, which include the amount of calories, macronutrient, and micronutrient content of foods, requires health and nutrition literacy at the consumer end, which may be lacking by various population groups and in numerous contexts. This is further complicated with the diversity of units, serving sizes, and nutrients listed on nutrition facts labels in differing countries, in addition to front-of-package labels along with other competing marketing slogans and techniques. Furthermore, fresh produce and certain types of meats and dairy products lack nutrition labels and the choice among the myriad of produce on the market leaves consumers confused as to which of the food items within each of the food groups are the most nutritious. The qCaln measure can merge the multiple nutritional measures including, caloric content of food, and the macro- and micronutrient content of food into one simple, standardized unit. In addition, the qCaln can be linked to optimal daily nutrient intakes of individuals and allows for daily meal construction. This linkage will allow consumers to make food purchases with conscious dietary knowledge rather than just preference, taste, convenience, and cost. The qCaln, if deemed valid in various contexts, could be used in addition to food labeling either on the front or the side of products, and on menus at various points of purchase with the potential for expanding nutrition education and supporting nutrition labeling systems that are already in place. Future research should evaluate the experiences of consumers towards the use of the qCaln and their success in attempting to make healthier dietary choices at different points of sales .Using the qCaln, as a composite score that takes into consideration energy and nutrient density, is not only valuable for consumers but also researchers and health professionals who seek to assess the diet quality of individuals and population groups. The proposed measure can provide a simple method to assess nutrient profiles of individual food items within the same food category while also providing investigators with a relatively simple tool to assess the overall nutrient profiles of individuals . The latter measure can be then linked to other global measures of diet quality, such as the HEI and diet diversity scores.One of the main benefits and applications of the qCaln is to help individuals make better food item choices and improve their overall dietary intake. In fact, nutrition programs that attempt to increase awareness and proper consumption of micronutrients achieve some of the highest benefit-cost ratios, even in the short term . HoweverFindings from this paper need to be interpreted in light of a number of limitations. First, equal weights were assumed for all included micronutrients in the calculation for the qCaln. Although the use of equal-weighted scores has been used extensively by other researchers when developing nutrient profiling methods , targeteAnother limitation of the qCaln calculations in the present study is the lack of inclusion of bioavailability data and the effect of food preparation on micronutrient content of foods. For example, animal-based protein sources are more readily digested and absorbed allowing for higher bioavailability of essential micronutrients compared to plant-based sources, and cooked vegetables that are prepared through steaming have different micronutrient contents compared to boiled or baked vegetables . To addrThe proposed unit of measure reflects a novel nutrient profiling approach that accounts for both the quantity of energy  and the quality of nutrients in foods into one standardized metric and demonstrates the usability of this new measure to facilitate the assessment of food quality. Results show that the qCaln can be used to rank foods in terms of their nutrient and energy content simultaneously, making food selections within a food group easier to understand. This easy-to-use tool can be applied by public health professionals, regulatory agencies, and the food industry to promote the selection of healthier nutrient-dense yet low in energy content foods and assist consumers in meeting dietary guidelines and nutrition recommendations. In addition, this novel metric could be further validated by researchers in comparison to other nutrient profiling and diet quality measures to test its usability in assessing diet quality of individuals and population groups. This metric can have other applications and can be further expanded beyond the nutrition research serving as the impetus for nutrition-sensitive agriculture through tracking of food quality from production to consumption.


SCN5A is expressed in cardiomyocytes and gastrointestinal (GI) smooth muscle cells (SMCs) as the voltage-gated mechanosensitive sodium channel NaV1.5. The influx of Na+ through NaV1.5 produces a fast depolarization in membrane potential, indispensable for electrical excitability in cardiomyocytes and important for electrical slow waves in GI smooth muscle. As such, abnormal NaV1.5 voltage gating or mechanosensitivity may result in channelopathies. SCN5A mutation G615E – found separately in cases of acquired long-QT syndrome, sudden cardiac death, and irritable bowel syndrome – has a relatively minor effect on NaV1.5 voltage gating. The aim of this study was to test whether G615E impacts mechanosensitivity. Mechanosensitivity of wild-type (WT) or G615E-NaV1.5 in HEK-293 cells was examined by shear stress on voltage- or current-clamped whole cells or pressure on macroscopic patches. Unlike WT, voltage-clamped G615E-NaV1.5 showed a loss in shear- and pressure-sensitivity of peak current yet a normal leftward shift in the voltage-dependence of activation. In current-clamp, shear stress led to a significant increase in firing spike frequency with a decrease in firing threshold for WT but not G615E-NaV1.5. Our results show that the G615E mutation leads to functionally abnormal NaV1.5 channels, which cause disruptions in mechanosensitivity and mechano-electrical feedback and suggest a potential contribution to smooth muscle pathophysiology. In all, the current-clamp data suggest that loss of mechanosensitivity in G615E NaV1.5 would lead to a loss of mechanically induced excitability in WT NaV1.5.Shear stress accelerated the spiking upstroke for WT Na faster) . Howeverest (I)] . The effV1.5 mechanosensitivity and its role in cellular mechano-electrical feedback. We used a unique SCN5A mutation G615E that is associated with acquired long-QT syndrome [V1.5 had mostly intact voltage-dependent function – normal voltage-dependence of activation, and either no or minor changes in voltage-dependence of inactivation.The goal of the current study was to examine Nasyndrome , sudden syndrome , and irrsyndrome . In prevsyndrome ,17 and iV1.5. We tested both constructs in whole cell and patch, using shear stress and patch pressure as mechanical stimuli, respectively. For WT NaV1.5, we saw force produce several changes to voltage-dependent function, similar to previous studies on NaV1.5 [V1.4 [V1.5. Thus, G615E NaV1.5 joins previously identified disease-associated mutations with abnormal NaV1.5 mechanosensitivity. These associated with long QT syndrome [V1.5 with abnormalities in voltage-dependent function that were further accentuated by mechanical forces. However, to our knowledge, this is the first NaV1.5 mutation that has disrupted mechanosensitivity, while voltage-sensitivity remained mostly intact. Our findings may be relevant for understanding channelopathy mechanisms in cases when NaV1.5 mutations fail to reveal functional changes using voltage-dependence protocols. In such cases, and as we see with G615E NaV1.5, the functional impact may be on mechanical [On the other hand, we found dramatic differences in mechanosensitivity of voltage-dependent gating between WT and G615E Nan NaV1.5 ,12,16,22.5 [V1.4 , but thesyndrome  in the hchanical  sensitivV1.5 mechanosensitivity. First, G615E is located on the intracellular linker connecting DI and DII, which is a novel location for a missense mutation to impact mechanosensitivity. Other NaV1.5 channelopathies like G298S have loss-of-mechanosensitivity and are in linkers as well. However, how these linkers contribute to the mechanism of NaV1.5 mechanosensitivity remains unclear. Second, our findings suggest that mechanisms of voltage- and mechano-sensation by NaV1.5 may be distinct. This is surprising since mechanical stimuli are well established to modulate voltage-sensitivity of voltage-gated channels [V1.5 lost one but not both mechanosensitive responses – it lacked a perfusion-induced current increase but maintained a negative shift in the voltage-dependence of activation. This would suggest that mechanosensitive increases in peak Na+ current may be mechanistically distinct from mechanically induced shifts in voltage-dependence of activation and availability. Fourth, the findings suggest that the mechanosensitivity of the voltage-dependence of activation may be separate from that of availability. Previous and current studies show that mechanical stimuli accelerate the kinetics of activation and inactivation by the same constant. If the acceleration of activation by force is the rate-limiting step [V1.5 demonstrates an intact mechanosensitivity of activation but a loss of mechanosensitivity of inactivation, suggesting separate mechanisms. In all, our results shed important light on the mechanism of NaV1.5 mechanosensitivity and show that NaV1.5 mechano- and voltage-sensitivity may be targeted separately.Our results provide intriguing mechanistic insights on Nachannels ,12,25 buchannels ,12. Thiring step , it may V1.5 mechanosensitivity impacts SMC excitability, also called mechano-electrical feedback [V1.5 channels at the upstroke. But given the acceleration of inactivation, NaV1.5 stretch also results in a more significant current decrease during inactivation [V1.5 mechanosensitivity on SMC function is difficult to judge only from the voltage-dependent operation. We designed current-clamp protocols in a reductionist system, a HEK-293 cell that expressed only WT NaV1.5 or G615E NaV1.5. Similar to previous studies [V1.5 had decreased mechanosensitivity of both spontaneous and elicited firing, suggesting that NaV1.5 mechanosensitivity may play an important excitatory role in SMC mechano-electrical feedback. However, a note of caution is required. We set the resting potential hyperpolarized to allow for full NaV1.5 availability, but this system limits our ability to determine whether sub-threshold events by either channel can result in firing, as may happen in excitable cells. In a set of preliminary studies, we investigated the potential influence of G615E-NaV1.5 on mechano-electrical feedback in GI SMCs by computational modeling. In silico modeling simulated NaV1.5 voltage- and current-clamp in vitro behavior, and the resulting SMC electrical activity and cytoplasmic Ca2+ concentrations were affected by mechanical forces for WT but not G615E-NaV1.5 .We are ultimately interested in understanding how Nafeedback . These ctivation –13 and stivation . Therefo studies ,28, we fV) [V1.1 mechanosensitivity leads to “mechanical braking” of neuronal excitability [in vivo and to replicate these for studies in vitro. It is unclear how the current in vitro mechanostimulation protocols correlate with in vivo physiology. To fully understand mechano-electrical coupling we will need to integrate mechanosensitivity of NaV1.5 and other mechanosensitive ion channels into cell models and to place these models into physiologically relevant mechanical contexts.In addition to mechanosensitive voltage-gated sodium channels, cells have other mechanosensitive voltage-gated ion channels, such as potassium (KV)  and mechV) . The efftability . FurtherV1.5 missense mutation G615E disrupts NaV1.5 mechanosensitivity without a significant impact on voltage-dependent function, which may have important consequences for mechano-electrical coupling in myocytes. This raises the possibility of directly targeting NaV1.5 mechanosensitivity in disease with a drug such as ranolazine [V1.5 peak current but can inhibit mechanosensitivity [In summary, the disease-associated Nanolazine ,22, whicsitivity ,31.


We propose two generalized Haldane models on laser-coupling optical lattices. Laser-assisted nearest neighbour tunnelings generate artificial staggered magnetic flux, facilitating the realization of topological nontrivial band structures. As generalizations of Haldane model, these models support topological insulator and semimetal phases featuring high Chern numbers. We show simple rules for computing Chern numbers of our models and display the phase diagrams. Moreover, numerical calculations of energy spectra are in perfect agreement with our theoretical expectations. Our models may serve as two new family members for generalizing Haldane model on optical lattices. In lattice systems, the topologically nontrivial band structures are currently attracting a great deal of interest3. The band topology is characterized by a topological invariant-Chern number-taking4 integer values and signaling topological order. Nontrivial topological order has experimental consequences, such as quantized Hall conductivity5 and the existence of edge states3. In two-dimensional lattice systems, one of the simplest models supporting topological bands was proposed by Haldane6. This model features real nearest-neighbor (NN) hopping and complex next-nearest-neighbor (NNN) hopping on a honeycomb lattice. Haldane showed that this two-band model is topologically equivalent to integer quantum Hall state.Topologically ordered phases of matter have been a hot topic in condensed matter physics since the discovery of the integer quantum Hall effect10, topological superconductor13 and topological semimetal19 have been widely explored. To simulate these fascinating quantum phenomena, ultracold atoms in optical lattice is an ideal playground, for its versatile control of interactions which are hard to adjust in traditional solid state systems20. Several methods have been proposed to realize topological nontrivial band structures with cold atoms, such as lattice shaking23, rotation24, laser coupling28, or using synthetic dimensionality29. Recently, Haldane model has been realized experimentally through lattice shaking30. Measurement of Chern number of Hofstadter bands has been successfully performed31. The quantized conductance in neutral matter system has also been observed32. Besides, there are many other developments on Chern number detection34, models design that support nontrivial topology37 and so on. Recently, some three-band tight-binding models have been proposed40, because of their high Chern numbers and easy access to topological semimetal phases.In recent years, topological insulatorIn the present paper, we propose two simple lattice models that support topological insulator and semimetal phases featuring high Chern numbers. Our tight-binding model is defined on laser-coupling optical lattice, on which complex NN tunnelings are laser assisted. The three-band model is defined on state-dependent lattice whereas the four-band model is on state-independent lattice, both pierced by effective staggered magnetic fields. As generalizations of Haldane model, these models support topological nontrivial energy bands. Chern numbers are computed in a simple way and rich phase diagrams are exhibited. Moreover, the topological edge states show time-reversal (TR) symmetry for a special case, although TR symmetry breaking in the bulk is required for a nonzero Chern number.i of three sublattices. Normal NNN tunnelings of amplitude t take place within the same triangular sublattices. Laser-assisted NN tunnelings43 of amplitude λa(b) are accompanied by recoil momentum p  of the laser coupling processes behave like onsite potentials. To eliminate the explicit spatial dependence of our Hamiltonian, we perform a unitary transformation36In our three-band model, fermionic ultracold atoms are trapped on honeycomb-like lattice. Atoms of three different hyperfine states are located on inequivalent sites, labeled by A, B and C Fig. . In the m p Fig. , generatThus giving a transformed Hamiltonian of real NN tunnelings and complex NNN tunnelingslux Fig. . The preui is the NNN vector and νi is the NN vector.ψn,k . The topological order of each band is characterized by the Chern numberz component of curl Given the momentum-space Hamiltonian (4), one easily knows there are three energy bands featured by Bloch wave functions x, y, z} of three bands. Setting t as unit, high Chern number phases a(b) becomes large, it reduces to an effective two-band model with Chern numbers ϕ affect the nontrivial scale of detuning Δ, allowing for the largest value |Δ| = 9 when it approaches ±2π/3. The coupling strength λa(b) has no influence on Chern numbers, but it turns out to modify the energy gaps and lead to topological semimetal phases as shown in the following numerical calculations.We are more interested in two symmetric cases and show their phase diagrams. For the isotropic case ϕ. For the isotropic case of phase diagram Fig. b with fixed Δa. Specially, there is a symmetry kx by −i∂x and integrating over x, one can find effective edge Hamiltonian ϕ is presented in Fig. ϕ, two edge states show up between the two energy gaps. Here the upper gap is indirect zero, signaling topological semimetal phase. Comparing Fig. λa(b) modifies the energy gap and results in topological insulator-semimetal phase transitions.The defining characteristic of topological nontrivial band structure is the existence of gapless edge states. We numerically calculate the energy spectra for 1D ribbons with zigzag and armchair edges, which is periodic in x and y direction respectively. The displayed edge states within the bulk energy gaps address different topological nontrivial phases. To demonstrate previous analytical results, we show two kinds of topological phase transitions driven by resonance detuning Δ or artificial magnetic flux H = HT + HL + HV:HT is the NNN hopping term on the honeycomb lattice  and . We find topological insulator phases characterized by one or two pair of edge states when resonance detuning ϕ = π/2, the two nontrivial intervals are  and . The two topological insulator phases become semimetal phases at the same detuning  at the two Dirac points. We also show the analytical phase diagrams of two symmetric cases, verified by numerical calculations of energy spectra. For the four-band model, the Peierls phase depend on the sublattice indices instead of internal states. Different topological nontrivial phases characterized by one pair or two pairs of edge states, can be easily transformed to each other by changing the resonance detuning δ and artificial magnetic flux ϕ. The effects of laser coupling on topological band structures are discussed. Compared with Haldane model, topological properties of our models are much richer, featuring high Chern number and easy control of topological insulator-semimetal phase transitions. For the staggered artificial magnetic flux, our models can be viewed as two new family members for generalizing Haldane model on optical lattice.In conclusion, we have proposed and studied two generalized Haldane models on laser-coupling optical lattice. Laser-assisted NN tunnelings generate artificial staggered magnetic flux, facilitating the realization of topological nontrivial band structures. For the three-band model, we show a simple rule for computing Chern numbers, which are obtained from the values of energy Hk in equation (En(k) the energy of n-th band. If wave function is analytic in the whole Brillouin zone (BZ), the contour integral in equation  and (8) on 1D ribbons of width 25 and 30, respectively. The symmetric and non-symmetric energy spectra correspond to different boundary conditions of armchair and zigzag edges, respectively.


The full width at half maximum (fwhm) is 103 nm, the internal quantum efficiency (IQE) exceeds 88%, and the external quantum efficiency (EQE) is 69%. According to Rietveld refinement analysis and density functional theory (DFT) calculations, Bi3+ ions randomly occupy all La sites in orthorhombic La4GeO8. Importantly, the oxygen-vacancy-induced electronic localization around the Bi3+ ions is the main reason for the highly efficient orangish-red luminescence. These results provide a new perspective and insight from the local electron structure for designing inorganic phosphor materials that realize the unique luminescence performance of Bi3+ ions.Phosphor-converted white-light-emitting diodes (pc-WLED) have been extensively employed as solid-state lighting sources, which have a very important role in people’s daily lives. However, due to the scarcity of the red component, it is difficult to realize warm white light efficiently. Hence, red-emitting phosphors are urgently required for improving the illumination quality. In this work, we develop a novel orangish-red La Phosphorescent substances that give white-light-emitting diodes a more natural reddish hue can be made by tuning the electron structure of their activator ions. Jun Lin of China’s Changchun Institute of Applied Chemistry, Chinese Academy of Sciences and colleagues combined germanium, lanthanum and bismuth oxides to manufacture a reddish phosphorescent substance that, when added to blue and green phosphors on an LED chip, produced more natural-looking light compared to conventional phosphor-converted white LEDs. LEDs are constantly being improved as an energy-efficient light source, but the colour of the light they emit can be unnaturally white or blue. Fabricating high quality red-emitting phosphors to change this has been a challenge. The researchers developed a substance with a unique reddish photoluminescence caused by electrons localizing around bismuth ions. Their approach offers a new perspective for exploring luminescence in inorganic materials. Conventional WLEDs are composed of a blue LED chip and yellow YAG:Ce3+ phosphor; however, they emit cold white light because the emission spectra do not cover the red region5. Therefore, red phosphor is very important for producing warm white luminescence and improving the luminous efficiency. Many previous works have explored the use of red-emitting phosphor to enhance pc-WLED lighting quality. Eu3+-doped inorganic phosphor materials are the most frequently investigated red phosphor materials due to the typical 4f–4f partial spin and forbidden transition7. However, Eu3+ is rarely utilized in warm pc-WLEDs because its excitation spectra do not fit well with near-ultraviolet (n-UV) and blue light8. The most widely commercially available red phosphors are Eu2+-doped nitride phosphors12, such as CaAlSiN3:Eu2+ and Sr2Si5N8:Eu2+. Although these phosphors realize high quantum efficiency, harsh synthesis conditions (high temperature and high pressure) and deep-red emission limit their large-scale application  in the production of warm white light. In addition, Zhang et al. reported a narrow-band red-emitting SrLiAl3N4:Eu2+ phosphor that was obtained via facile atmospheric pressure synthesis, which could easily compensate the red component for YAG:Ce3+ and potentially serve as an alternative phosphor for n-UV pc-WLEDs. However, the spectral overlap still remains a large problem13. To date, linear red-emitting Mn4+-doped fluoride phosphors have attracted substantial attention, as their quantum yield can exceed 98% under blue light irradiation15. However, they have two serious drawbacks: low thermal stability and massive HF acid use. Hence, exploiting high-quality red-emitting phosphor materials remains challenging.Phosphor-converted white-light-emitting diodes (pc-WLED) have become the next-generation solid-state lighting source in both indoor and outdoor lighting areas3+-activated phosphors have been extensively investigated due to their unique luminescence performance16. Their excitation spectra are located in the n-UV area; thus, spectral overlap can be efficiently avoided. However, Bi3+ ions typically emit blue and green light and rarely emit red light, except in a suitable matrix, such as ScVO4 and ZnWO420. Bi3+ contains a naked 6s6p energy level; hence, the surrounding coordination environment is very sensitive22. The luminescence performance of Bi3+ can be easily influenced by tuning the surrounding electron structure. Recently, it was demonstrated that the formation of a vacant defect could contribute to the spectral adjustment. For instance, Zhang et al.23 reported a giant enhancement of Bi3+ luminescence that was realized by generating an oxygen vacancy, which is a new strategy for exploring novel Bi3+-doped phosphor materials.Recently, Bi4GeO8:Bi3+ (denoted as LGO:Bi3+) phosphor. Under 397 nm n-UV excitation, LGO:Bi3+ displays an orangish-red emission for which the peak is at 600 nm, fwhm = 103 nm, IQE = 88.3%, and EQE = 69%. Via experimental and theoretical studies, we demonstrate that the unique photoluminescence performance is caused by oxygen-vacancy-induced electron localization around the Bi3+ ions. This finding provides a new insight into the design of novel luminescence materials by changing the local electron structures of activator ions. The fabricated pc-WLED devices realize a high color rendering index (CRI = 95.1) and a low correlated color temperature (CCT = 5323 K), thereby indicating that LGO:Bi3+ is a superb red-emitting candidate in the field of solid-state lighting.In this work, we develop a novel and high-quality red-emitting La3+ phosphor is evaluated in detail through diffuse reflectance (DR) spectra. The DR spectra of the LGO matrix show only one prominent band, which is centered at 280 nm , in addition to the prominent band in the region of 232–321 nm, a shoulder band appears at ~400 nm. The former is mainly attributed to the matrix absorption of LGO and the 1S0→1P1 transition of Bi3+, while the latter originates from the 1S0→3P1 transition of Bi3+,24. The optical bandgap value can be obtained via linear extrapolation based on the DR spectra according to the following equations:25A represents the absorption constant, R is the reflectance coefficient (%), F(R) is the absorption coefficient, Eg is the optical bandgap value, and hv represents the photon energy. The bandgap values for the LGO matrix and LGO:0.007Bi3+ are 4.89 and 4.95 eV, respectively . This emission can be attributed to the characteristic 3P1→1S0 transition of Bi3+. In the PL spectrum of LGO:Bi3+, an emission redshift of ~50 nm and a broader fwhm compared to those of YAG:Ce3+ (maximum at 550 nm and fwhm = 87.5 nm) are observed, thereby demonstrating that LGO:Bi3+ could cover more of the red component in PL spectra. Simultaneously, LGO:Bi3+ shows no reabsorption between the PLE and PL spectra, the IQE value can reach 88.3%, and EQE = 69%. The calculated Commission Internationale de l’Eclairage (CIE) diagrams for LGO:0.007Bi3+ (λex = 397 nm) and YAG:Ce3+ (λex = 460 nm) are located in the orangish-red region  and yellow region   is consistent with the RT spectra (298 K)  Fig. , respect) Figure  and S1b,7 Figure -S1f. The27. In Fig. xBi3+ (x = 0.01 and 0.03) samples and standard α-Bi2O3 powder are plotted. All samples show the typical Bi3+ peaks at ~159 and 164.6 eV, which are assigned to Bi 4f7/2 and Bi 4f5/2, respectively. In addition, when the LGO:Bi samples are treated under a N2/H2 (90%/10%) reducing atmosphere, no luminescence is observed. PL spectra that are treated in a different environment are shown in Figure 3+ ions.Since the valence of Bi has a critical influence on its luminescence properties, it is necessary to define its valence in the LGO matrix29. Fig. 3+. Although its emission intensity gradually declines as the temperature increases from 298 K to 573 K, it maintains 78.4% emission intensity at 398 K of the original intensity at 298 K. The quenching temperature, which is denoted as T50 , is 488 K. The quenching process is ascribed to the thermally excited nonradiative transition. Furthermore, the emission peak position and shape show almost no shift  could be indexed with the standard La4GeO8 (PDF No. 40–1185), thereby demonstrating the formation of a pure phase . All the as-prepared samples crystalize in orthorhombic unit cells with space group P1. The refined lattice parameters for LGO are a = 7.6642(8) Å, b = 5.8470(9) Å, c = 18.2897(4) Å, and V = 819.629(3) Å3 and the detailed structural information is listed in Table c and cell volume V as functions of the Bi3+ ion concentration x. The lattice parameters gradually decrease as x increases, which results from the smaller ionic radius of Bi3+  compared to La3+ . Furthermore, the lattice parameters a and b gradually increase with x, thereby indicating that the La lattices may be locally distorted with the incorporation of Bi3+  system. Figure b-axis direction, LGO exhibits a highly symmetric structure, which consists of three types of [LaOn] polyhedra  and two types of [GeO4] tetrahedra. The six crystallographic La sites can be sorted into three categories: La1–La3. The La1 site is connected with six O atoms to form a distorted octahedron, while the La2 and La3 sites are coordinated with seven O atoms to form decahedra. The O atoms in LGO can also be divided into three types: Oi-Oiii. The structural information that is obtained via DFT calculations well agrees with the Rietveld refinement results. The calculated average La–O bond lengths are summarized in Fig. Density functional theory (DFT) calculations are used to further investigate the electron properties and structural configuration of the LGO:xBi3+  is determined from FT-IR spectra . The peak splitting degree at ~400 cm−1 also differs between the two samples (marked by a pink dashed rectangle). These variations in the Raman spectra demonstrate that the local lattice coordination environment in the LGO matrix changes slightly with Bi3+ ion incorporation and may result in the formation of lattice defects. XPS analysis is an effective method for elucidating the presence of vacancies. With the doping of Bi3+ into the LGO matrix, the 3d orbital of La and the Ge binding energy remain unchanged  maps around the Bi atoms at the La1–La3 sites , 2.08 eV (600 nm), and 2.24 eV (550 nm). The crystal splitting field energy (Dq) is expressed as follows:34Z is the charge of the anion, e is the charge of one electron, r is the radius of the d wave function, and R is the average La–O bond length. As the bond length decreases, the crystal field splitting increases. On the basis of Fig. 3+ at the La1, La2, and La3 sites, respectively. The oxygen-vacancy-induced electron localization around the Bi atoms is the crucial factor for generating orangish-red emission in the LGO:Bi3+ phosphor.To further investigate the influence of oxygen vacancies on the Bites Fig.  without tra Fig.  and life3+ phosphor in a pc-WLED device, we fabricated a pc-WLED device by using LGO:0.007Bi3+, commercial green Ba3Si6O12N2:Eu2+ phosphor, blue BAM:Eu2+ phosphor, and a 400 nm n-UV LED chip. For comparison, the pc-WLED is fabricated similarly by using commercial YAG:Ce3+ phosphor with a 460 nm LED chip. The electroluminescence (EL) spectra are presented in Fig. 3+ covers more of the red component in the visible light region than YAG:Ce3+. With a 3 V, 20 mA current driving, the CCT (correlated color temperature) and CRI (color rendering index) of the as-fabricated pc-WLEDs for LGO:0.007Bi3+ are 5323 K and 95.2, respectively, which are superior compared to the commercial YAG:Ce3+ phosphor (6015 K and 72.2). The luminous efficiency of the as-fabricated pc-WLEDs for LGO:0.007Bi3+ is 6.4 lm/W, which should be optimized via proper process treatment. Moreover, the CIE color coordinates of the former  correspond to a more suitable white-emitting position than those of the latter . The fabricated pc-WLED of LGO:Bi3+ exhibits much warmer white light than that of YAG:Ce3+  and the fwhm = 103 nm. Its IQE could reach 88.3%, EQE = 69%. Rietveld refinement confirms that the as-prepared samples crystalize in orthorhombic unit cells with space group P1, and its lattice parameters are a = 7.6642(8) Å, b = 5.8470(9) Å, c = 18.2897(4) Å, and V = 819.629(3) Å3. The XRD spectra and DFT calculation results demonstrate that Bi3+ ions randomly occupy La1–La3 sites in LGO, which will emit deep-red (1.93 eV), orange (2.08 eV), and yellow (2.24 eV) light when substituting at La1, La2, and La3 sites, respectively. Interestingly, the unique orangish-red luminescence behavior should be ascribed to the electron localization surrounding Bi3+ ions with the presence of adjacent O vacant defects through ELF maps analysis. The fabricated n-UV-based pc-WLED with a hybrid of LGO:Bi3+ phosphor, blue BAM:Eu2+ and green Ba3Si6O12N2:Eu2+ phosphor achieves high CRI (95.2) and low CCT (5323 K), indicating a superb candidate in lighting field. The concept of focusing on the electron localization properties surrounding activators ions offers a new perspective and insight for exploring unique luminescence behavior in inorganic luminescence materials.In summary, we have successfully exploited an orangish-red-emitting LGO:Bix4-GeO8:xBi3+ (x = 0‒0.03), which is denoted as LGO:xBi3+, were prepared via a conventional high-temperature solid-state approach. The raw materials of La2O3 (99.99%), GeO2 (99.999%) and Bi2O3 (99.99%) were all purchased from Aladdin. First, stoichiometric raw materials were weighed, put into agate, and ground together for 1 h. The obtained mixtures were transferred into corundum crucibles and annealed in a horizontal tube furnace at 1100‒1300 °C for 4 h in air. After naturally cooling to room temperature, the annealed samples were ground again. In addition, Lax4-GeO8:xBi3+ (x = 0‒0.03) samples were prepared under an N2/H2 (90%/10%) reduced atmosphere with the same other conditions for comparison.Samples of La3+ phosphor, blue BAM:Eu2+ phosphor, green Ba3Si6O12N2:Eu2+ phosphor, and a 400 nm LED chip. In a typical fabrication process, the LGO:0.007Bi3+, BAM:Eu2+ and Ba3Si6O12N2:Eu2+ phosphors were evenly blended with silicone resins A and B (A:B = 1:1) in the agate mortar and the resulting mixture was coated on a 400 nm LED chip. The packaged devices were cured in an oven at 120℃ for 12 h to form the resulting pc-WLED devices. The commercially available yellow YAG:Ce3+ phosphor and a 460 nm LED chip were also fabricated to pc-WLED via the same method for comparison.pc-WLED devices were fabricated by using the as-prepared orangish-red LGO:0.007BiPowder X-ray diffraction (XRD) were collected on a D8 Focus diffractometer with Ni-filtered Cu-Kα . Rietveld refinements were performed with General Structure Analysis System (GSAS) software based on XRD data. Photoluminescence excitation (PLE) and photoluminescence emission (PL) spectra were collected by a fluorescence spectrometer  whose excitation source is a 450 W xenon lamp. Diffuse reflectance (DR) spectra were collected with using a UV–vis-NIR spectrophotometer (Hitachi U-4100). Photoluminescence decay curves were obtained with using a Lecroy Wave Runner 6100 Digital Osilloscope (1 GHz) (Contimuum Sunlite OPO), the excitation was a tunable laser (pulse width = 4 ns and gate = 50 ns). Fourier-transform infrared spectra (FT-IR) were performed on spectrophotometer  by using KBr pellet technique. Raman spectra were obtained on Raman spectrometer (JYT6400) using a 512 nm laser. The photoluminescence quantum yield (QY) was obtained on an absolute PL quantum yield measurement system . The electroluminescence performances of pc-WLED devices were measured analyzer system (tarspec SSP6612.) by using an integrating sphere. All the above measurements were performed at room temperature. Temperature-dependent PL spectra (10–300 K and 298–573 K) were recorded on a fluorescence spectrophotometer (Edinburgh Instruments FLSP-920) with a temperature controller.s25p65d16s2), Ge (4s24p2), O (2s22p4) and Bi (6s26p3) electrons were treated as the valence electrons. The exchange and correlation functional were described via the Perdew–Burke–Ernzerhof (PBE) generalized gradient approximation. To investigate the electronic properties and obtain an accurate description of the density of states, we employed the Heyd–Scuseria–Ernzerhof (HSE06) method. A plane-wave cutoff energy of 400 eV was applied in our calculations and 4×4×2 Monkhorst–Pack k grids were used during the optimization. The iterative process was considered to have converged when the force on the atom was less than 0.01 eV Å−1 and the energy change was <10–5 eV per atom.The first-principle density functional theory (DFT) calculations were performed with the Vienna ab initio simulation package (VASP) code. The electron–ion interaction was treated with the projector augmented wave (PAW) method. La (5supplemental materials