The aim of this study was to describe data on epidemiology, ventilatory management, and outcome of acute respiratory distress syndrome (ARDS) in immunocompromised patients.We performed a post hoc analysis on the cohort of immunocompromised patients enrolled in the Large Observational Study to Understand the Global Impact of Severe Acute Respiratory Failure (LUNG SAFE) study. The LUNG SAFE study was an international, prospective study including hypoxemic patients in 459 ICUs from 50 countries across 5 continents.p < 0.0001), despite similar severity of ARDS. Decisions regarding limiting life-sustaining measures were significantly more frequent in immunocompromised patients . Use of noninvasive ventilation (NIV) as first-line treatment was higher in immunocompromised patients , and immunodeficiency remained independently associated with the use of NIV after adjustment for confounders. Forty-eight percent of the patients treated with NIV were intubated, and their mortality was not different from that of the patients invasively ventilated ab initio.Of 2813 patients with ARDS, 584 (20.8%) were immunocompromised, 38.9% of whom had an unspecified cause. Pneumonia, nonpulmonary sepsis, and noncardiogenic shock were their most common risk factors for ARDS. Hospital mortality was higher in immunocompromised than in immunocompetent patients  contains supplementary material, which is available to authorized users. In recent decades, significant advances in the management of immunocompromised patients have led to improved survival rates –3. Hence2/FiO2) ≤ 300 mmHg while receiving IMV or NIV with positive end-expiratory pressure (PEEP) ≥ 5 cmH2O and (2) new radiological pulmonary parenchymal abnormalities. In patients with AHRF, a more detailed set of data was collected to determine whether they met the Berlin definition criteria for ARDS. Data on comorbidities, etiology of AHRF, and risk factors for ARDS were recorded. Data on arterial blood gases, ventilatory support, use of adjunctive therapies , severity of ARDS, and other organ involvement by modified nonpulmonary Sequential Organ Failure Assessment (SOFA) score [LUNG-SAFE was an international, multicenter, prospective observational cohort study conducted in a 459 ICUs worldwide. During 4 consecutive weeks in the winter of 2014 (February–March 2014 in the Northern Hemisphere and June–August 2014 in the Southern Hemisphere), participating ICUs enrolled patients undergoing IMV or NIV. Participating ICUs obtained ethics committee approval and either patient consent or waiver of consent as per local guidelines. National coordinators, site investigators, and endorsing societies are listed in Additional file A) score  were colA) score .We defined “immunocompromised” patients as all patients with at least one of the following conditions listed in the LUNG SAFE CRF: (1) immunosuppression , (2) active hematologic malignancy , and (3) active neoplasm . Patients without these conditions were classified as “controls.” For the purposes of this analysis, the study population was restricted to the subset of patients fulfilling ARDS criteria on day 1 or 2 following the onset of AHRF.NIV encompassed all forms of NIV modes and interfaces (including continuous positive airway pressure). ARDS severity was assessed from the first to the second day from ARDS onset, according to the Berlin definition criteria: mild (PaO2/FiO2 201–300 mmHg), moderate (PaO2/FiO2 101–200 mmHg), and severe (PaO2/FiO2 ≤ 100 mmHg). Changes in ARDS severity were evaluated in patients staying in the ICU for at least 2 days, and they were classified into four categories: (1) no change, (2) worsening (shift to a more severe category), (3) improvement (shift to a less severe category), and (4) resolution. Duration of invasive ventilation was computed as the number of days that the patient required IMV up to day 28. Survival was evaluated at ICU and hospital discharge or at day 90, whichever event occurred first.In regard to management, patients were subdivided in three ventilation subgroups: (1) IMV, defined as patients invasively ventilated from day 1, independently of the type of support received after the eventual extubation; (2) NIV, defined as patients treated exclusively with NIV from day 1 to study exit ; and (3) NIV failure, defined as patients initially treated with NIV and subsequently intubated during the study period. The term t test or the Wilcoxon rank-sum test  for continuous variables and the χ2 or Fisher’s exact test (according sample size) for proportions. We used analysis of variance or the Kruskal-Wallis test (as appropriate) and the χ2 test (or Fisher’s exact test) to assess differences among the NIV, NIV failure, and IMV groups. The Bonferroni correction was applied to determine significance in the setting of multiple comparisons.Continuous variables were expressed as mean (SD) or median (IQR), and categorical variables were presented as count and percent. No assumptions were made for missing data, which were rare . To asseTo evaluate factors associated with the use of NIV, we applied a multivariable logistic regression model, and the independent predictors  were identified through a stepwise regression approach. This approach combines forward and backward selection methods (combined with a significance level of 0.05 for both entry and retention) in an iterative procedure to select predictors in the final multivariable model. This approach was also applied to identify factors associated with hospital mortality in immunocompromised patients. In this case, the stepwise approach also evaluated as possible predictors ventilator setting variables measured at ARDS onset.Survival analysis was performed according to the Kaplan-Meier method. We assumed that patients discharged alive from the hospital before 90 days were alive on day 90. The log-rank test was used to compare survival curves among groups.p values were two-sided, and values less than 0.05 were considered significant. Statistical analyses were carried out with R version 3.3.3  and SAS version 9.4 software .All p = 0.0163) and had a lower body mass index (BMI) . They had a lower prevalence of chronic obstructive pulmonary disease, diabetes mellitus, and heart failure (New York Heart Association classes III–IV) and a higher incidence of pneumonia, pulmonary vasculitis, and noncardiogenic shock. Among immunocompromised patients, 28.6% had mild, 46.4% moderate, and 25.0% severe ARDS, and the most common risk factors for ARDS were pneumonia (70.5%), nonpulmonary sepsis (16.1%), and noncardiogenic shock (10.3%). Nonpulmonary SOFA score at day 1 and ARDS severity were similar between immunocompromised and controls. Additional file p = 0.0045). Comorbidities and PaO2/FiO2 were not different among the subgroups. There was a marked difference in the mean nonpulmonary SOFA score, which was significantly higher in IMV than in NIV  and NIV failure subgroups .A total of 459 ICUs from 50 countries enrolled patients in the LUNG SAFE study. Among 12,906 mechanically ventilated patients, 4499 had AHRF, and of these, 2813 fulfilled the Berlin criteria for ARDS on day 1 or 2. Among ARDS patients, 584 (20.8%) were immunocompromised . The proportion of patients remaining on NIV from day 1 to study exit (NIV subgroup) was similar, whereas the incidence of NIV failure was significantly higher in immunocompromised patients .Figure p = 0.0005). Other factors associated with NIV are shown in Additional file A multivariable logistic regression model revealed that, adjusting on confounders, immunodeficiency was independently associated with the use of NIV  , whereas there was no difference in duration of mechanical ventilation and changes in ARDS severity. Survival curves for hospital (or 90-day) mortality are shown in Fig. p = 0.0078) and NIV failure  subgroups. Of the NIV patients who died, 68% had a limitation of life-sustaining measures. The incidence of NIV failure was 48%. ICU and hospital mortality of patients with NIV failure were significantly higher than those of patients managed exclusively with NIV , whereas they did not differ from those of IMV patients. Survival curves for hospital (or 90-day) mortality of immunocompromised patients stratified by ARDS severity and by ventilation subgroups are shown in Additional files p = 0.0032), higher peak inspiratory pressure level , lower PaO2/FiO2 ratio , lower degree of improvement in PaO2/FiO2 ratio between day 1 and day 2 of ARDS , and lower BMI  . The results of this analysis are reported in Additional file p < 0.0001) and had lower hospital mortality  and fewer limitations of life-sustaining measures .Additional file The main findings of this analysis can be summarized as follows: immunosuppression is frequent in ARDS patients; causes of ARDS in immunocompromised patients are mainly related to infection; immunocompromised patients are more likely to receive NIV as first-line ventilatory treatment; and the outcome of ARDS is worse and limitation of life-sustaining measures is more frequent in immunocompromised patients. Among the 2813 ARDS patients included in the LUNG SAFE study, one-fifth were immunocompromised, and 62.7% of them had an active malignancy. In line with recent literature , 21, a dImmunocompromised subjects had a significantly higher ICU and hospital mortality, despite similar ARDS severity and nonpulmonary SOFA score. Another subanalysis of the same database confirmed that active neoplasm, hematologic malignancies, and immunosuppression are independently associated with increased mortality . In addiHospital mortality of our patients was lower than that (64%) reported by Azoulay et al. in a large retrospective analysis of 1004 patients with cancer and ARDS admitted to the ICU over a period of 21 years . In thatThe actual advantage of ICU admission of immunocompromised patients remains debated , 29, 30.Two important questions on the optimal ventilatory management of AHRF in immunocompromised patients remain unanswered. First, is NIV the optimal first-line ventilatory support? Two randomized controlled trials conducted almost 20 years ago showed that NIV, compared with standard oxygen therapy, significantly reduces the rate of intubation and mortality , 17, butab initio with IMV. However, two factors may limit the relevance of this observation: the relatively low number of patients in the NIV failure subgroup and the lack of information on the actual duration  of the NIV period before intubation, which would be important to know in the light of literature data showing that delaying endotracheal intubation after a prolonged NIV trial may negatively impact patient survival [The impact of NIV failure on patients’ outcomes is the second important issue. We observed a significantly higher incidence of NIV failure in immunocompromised patients than in controls, in keeping with the observation of Thille that a diagnosis of active cancer is independently associated with NIV failure . In our survival , 20. In survival . Taken tThe present study has several limitations. First, it is a post hoc analysis of a prospective multicenter observational trial, and unknown confounders associated with the subgroup analysis may bias the results. Although the data were prospectively collected from a high number of centers from 50 countries, different approaches to clinical decisions from different centers  may have influenced the outcomes. Second, the criteria used to define the immunocompromised cohort were quite heterogeneous. It was impossible to stratify the patients according to the prognosis of baseline disease and to the severity of immune deficiency. Indeed, causes of immunosuppression, other than malignancies, were not specified in nearly 40% of the whole cohort of immunocompromised patients. Moreover, in patients with cancer, no information was available on the type of cancer, its staging, and the nature and timing of anticancer treatments. This lack of information, related to the LUNG SAFE original CRF, should be considered a major limitation because the outcome of immunocompromised patients is strictly dependent on the type of underlying diseases and associated therapeutic approach. All these factors may limit the generalizability of our findings. Third, to limit the burden on investigators, data were collected once daily only, and information on the actual hours of duration of ventilatory treatments was not available. This is particularly relevant for patients treated with NIV because the precise duration of NIV before the eventual intubation might be important to understand the impact of NIV failure on outcome . Fourth,ab initio with IMV. These data should be considered in light of the nonspecific criteria used to define the immunocompromised population and the potentially heterogeneous approaches to clinical decision making in the participating centers.Immunocompromised patients represent an important proportion of ARDS patients in the ICU. Compared with immunocompetent subjects, they had higher mortality, regardless of ARDS severity, and a higher frequency of limitation of life-sustaining measures. Nonetheless, nearly half of these patients survive to hospital discharge. They were more likely to receive NIV as the first ventilator strategy, and those who did not require invasive ventilation had a lower mortality. Mortality of immunocompromised patients who failed NIV was not different from that of patients treated Additional file 1:List of LUNG SAFE investigators. Names and affiliations of the LUNG SAFE investigators. (PDF 172 kb)Additional file 2:Table S1. Patient characteristics of immunocompromised patients according to the type of ventilator support. This table shows patient characteristics, including comorbidities, ARDS risk factors, and illness severity at ARDS onset of immunocompromised patients according to the type of ventilator support. (PDF 74 kb)Additional file 3:Table S2. Factors associated with the use of noninvasive ventilation. Multivariate logistic regression model describing the factors associated with the use of noninvasive ventilation. (PDF 49 kb)Additional file 4:Table S3. Ventilator settings during the first day of ARDS in the immunocompetent (Control) and immunocompromised (Study) groups. This table shows ventilator settings during the first day of ARDS in the immunocompetent (Control) and immunocompromised (Study) groups. (PDF 50 kb)Additional file 5:Table S4. Adjunctive measures/therapies during at least one day during follow-up in immunocompetent and immunocompromised patients. This table shows the proportions of adjunctive measures/therapies during at least one day during follow-up in immunocompetent and immunocompromised patients. (PDF 97 kb)Additional file 6:Table S6. Ventilator settings during the first day of ARDS in immunocompetent (Control) and immunocompromised (Study) patients, stratified by the type of ventilatory support . (PDF 60 kb)Additional file 7:Figure S1. Kaplan-Meier curve for hospital survival in immunocompromised patients according to ARDS severity. Kaplan-Meier curve for hospital survival in immunocompromised patients according to ARDS severity. Mortality is defined as mortality at hospital discharge or at 90 days after onset of acute hypoxemic respiratory failure, whichever event occurred first. We assumed that patients discharged alive from the hospital before 90 days were alive on day 90. Severity of ARDS was evaluated at the day of onset according to the Berlin definition. Note: The number of patients reported in the bottom of figure is referred to as the end of the corresponding day. (PDF 402 kb)Additional file 8:Figure S2. This figure shows a Kaplan-Meier curve for hospital survival of immunocompromised patients according to the ventilation subgroup. This figure shows a Kaplan-Meier curve for hospital survival of immunocompromised patients according to the ventilation subgroup. Mortality is defined as mortality at hospital discharge or at 90 days after onset of acute hypoxemic respiratory failure, whichever event occurred first. We assumed that patients discharged alive from the hospital before 90 days were alive on day 90. Type of ventilator support: IMV Patients invasively ventilated from day 1, independently of the type of support received after the eventual extubation; NIV Patients treated exclusively with noninvasive ventilation, from day 1 to study exit, independently of outcome; NIV failure Patients initially treated with noninvasive ventilation and subsequently intubated during the study period. Note: The number of patients reported in the bottom of the figure is referred to as the end of the corresponding day. (PDF 396 kb)Additional file 9:Table S5. Factors associated with hospital mortality in immunocompromised patients. Multivariate logistic regression model describing the factors associated with hospital mortality in immunocompromised patients. (PDF 49 kb)Additional file 10:Table S9. The most important factors leading to death in the ICU in immunocompetent and immunocompromised patients. (PDF 44 kb)Additional file 11:Table S7. Patient characteristics and clinical endpoints of immunocompetent patients, according to the type of ventilatory support. (PDF 88 kb)Additional file 12:Table S8. Patients’ characteristics and clinical endpoints of immunocompromised (study) patients, according to the cause of immunosuppression . (PDF 79 kb)


Hydrologic data has traditionally been collected with permanent installations of sophisticated and accurate but expensive monitoring equipment at limited numbers of sites. Consequently, observation frequency and costs are high, but spatial coverage of the data is limited. Citizen Hydrology can possibly overcome these challenges by leveraging easily scaled mobile technology and local residents to collect hydrologic data at many sites. However, understanding of how decreased observational frequency impacts the accuracy of key streamflow statistics such as minimum flow, maximum flow, and runoff is limited. To evaluate this impact, we randomly selected 50 active United States Geological Survey streamflow gauges in California. We used 7 years of historical 15-min flow data from 2008 to 2014 to develop minimum flow, maximum flow, and runoff values for each gauge. To mimic lower frequency Citizen Hydrology observations, we developed a bootstrap randomized subsampling with replacement procedure. We calculated the same statistics, and their respective distributions, from 50 subsample iterations with four different subsampling frequencies ranging from daily to monthly. Minimum flows were estimated within 10% for half of the subsample iterations at 39 (daily) and 23 (monthly) of the 50 sites. However, maximum flows were estimated within 10% at only 7 (daily) and 0 (monthly) sites. Runoff volumes were estimated within 10% for half of the iterations at 44 (daily) and 12 (monthly) sites. Watershed flashiness most strongly impacted accuracy of minimum flow, maximum flow, and runoff estimates from subsampled data. Depending on the questions being asked, lower frequency Citizen Hydrology observations can provide useful hydrologic information. Natural resource managers rely on timely and accurate data to make management decisions. Though water resources for human purposes is one of the most fundamental ecosystem services  has the potential to overcome these limitations. Fienen and Lowry  demonstrWe were interested in how decreased observation frequency associated with Citizen Hydrology observations affects the ability to accurately characterize critical streamflow metrics . Based on our review of the literature using search terms of streamflow, citizen science/hydrology, subsampling, and sample frequency, we could not identify other previous works addressing this particular theme. While Moss and Tasker  used subAn immediate application of this research is to inform monitoring plans for a Citizen Hydrology campaign in Nepal called SmartPhones4Water-Nepal . The initial objective of S4W-Nepal is to further constrain the water budget in the Kathmandu Valley using underutilized sources of information, including water level and streamflow data collected by Citizen Hydrologists. At streamflow monitoring locations, low-cost staff gauges will be installed, and water level data will be collected by local residents with smartphones using an open source Android data collection platform called Open Data Kit (ODK) Collect  stream gauges in California. The three hypotheses we further evaluate are: (1) decreased observational frequency will negatively impact accuracy of flow and runoff estimates, (2) the nature of this impact will differ depending on the parameter in question, and (3) there will be correlations between accuracy of flow and runoff estimates and latitude, watershed area, Richards-Baker Flashiness Index (R-B Index), and storage ratio . The following analysis assumed (1) subsampled water level observations were as precise and accurate as continuous USGS records and (2) an equally accurate stage-discharge curve was available for converting water levels to flows. While not addressed in this paper, these simplifying assumptions highlight two important areas where further research is required if Citizen Hydrology is to help fill the globally widening hydrologic data gap.We compiled an inventory of the 403 streamflow gauging stations (gauges or sites) in the state of California operated and maintained by the USGS with 15-min water level and flow data from January 1st 2008 to December 31st 2014. From this inventory, 50 streamflow gauges were randomly selected. For these 50 gauges, we compiled 15-min records and station metadata including the name, location, and elevation of the gauging station. Figure https://github.com/jcdavids/CAFlowSubsample. This procedure was then repeated for 50 iterations to provide additional information about the distributions of the resulting statistics. The following is a description of the subsampling process.To mimic Citizen Hydrology observations at a lower observation frequency than the continuous record, we developed a bootstrap randomized subsampling with replacement procedure to generate randomized subsample datasets from each gauge record. The subsample datasets were randomly selected from the continuous record at average subsample intervals of once a day, every three days, weekly, and monthly. The subsampling procedure was similar to that used by Jones et al.  to assesqy is a vector  containing records of flow rate for gauging station y from records 1 to r; r is the total number of records in the 15-min time series for each station. Now suppose that we randomly sample from qy based on the formulaqssy,i is the subsample flow vector containing all subsampled records for gauging station y and iteration i. Because we require the subsample to be a random selection with on average even spacing between subsamples, we define the records that should be used for the subsamples used to develop qssy,i with the formularssy,i is the subsample record vector containing the randomly selected records used to develop the subsample flow vector qssy,i. S is the average subsample interval (an even integer) and n is the subsample number ranging from 0 to N. The value of N is given by the formulaSuppose the original 15-min time series data set is given by the formular/S. For example, if r/S was 83.94, then the combined functions would return 83. RIn is a random integer ranging from −S/2 to S/2. Offsetting n ensures that each subsample will be somewhere within the range of S centered about S was set to 96 (daily), 288 (three days), 672 (weekly), and 2922 (monthly). Per the minimum recommended number of bootstrap samples by Efron and Tibshirani  to assess the resulting distributions for minimum flow, maximum flow, and runoff volume.The functions int and floor select the nearest integer below bshirani , 50 itery sites times i subsamples, or 50 times 50) for each of the four subsample intervals S, for a total of 10,000 subsamples. The average size of each resulting subsample was 2571, 857, 367, and 84 records for daily, three day, weekly, and monthly subsampling intervals, respectively. A sample result of the subsampling procedure is presented in section Example subsampled hydrographs for the Truckee River near Farad (SiteID 10346000).To summarize the subsampling process: first, we developed subsample record vectors using Eq. We compiled the 50 original 15-min data sets and the 10,000 subsamples into Microsoft Access SQL databases. SQL queries were developed to compute normalized statistical comparisons (see section Flow ratios) for the 15-min records and subsampled data. In all cases, the flow ratios were aggregated over the entire 7-year period (period) from the beginning of 2008 to the end of 2014. For purposes of comparison and normalization, the actual period minimum flow, maximum flow, and runoff volume for each station was determined from the 15-min data. As previously stated, each individual subsample observation was assumed to have the same flow measurement accuracy as the original 15-min observations.y) and iteration (i)  and actual minimum flow from 15-min record  expressed as a fraction  was used for comparison purposes. The actual minimum is placed in the numerator so that the minimum flow ratio ranges from 0 to 1.A normalized minimum flow ratio between minimum flow obtained from subsampled data for each gauging station (y) and iteration (i)  and actual maximum flow from 15-min record  expressed as a fraction  was used for comparison purposes. Maximum flow ratio ranges from 0 to 1.A normalized maximum flow ratio between maximum flow obtained from subsampled data for each gauging station (y) and iteration (i)  and actual runoff from 15-min record  expressed as a fraction  was used for comparison purposes. Runoff ratio ranges from 0 to infinity.A normalized runoff ratio between runoff calculated from subsampled data for each gauging station  latitude, (2) watershed area, (3) the R-B index, and (4) storage ratio. The first three variables were chosen to explore possible geographic, spatial scale, and temporal/magnitude-based dependencies, respectively. Storage ratio was selected because of the intuitive relationship between the “flattening” of the hydrograph discussed by Vörösmarty and Sahagian  and the The R-B Index is a unitless value used to quantify the flashiness of a watershed  hypothesis in Table The subsampling selections and resulting hydrographs for daily, three day, weekly, and monthly subsample intervals are shown on Fig. Each hydrograph can be constructed by (1) selecting a horizontal gridline representing a subsample iteration, and then (2) moving vertically from each light blue dot on the selected subsample iteration gridline until the 15-min hydrograph is reached. The random distribution of the roughly 1500, 500, 200, and 50 light blue dots, respectively, illustrates that the subsampling method described in section Subsampling procedure is providing good subsample randomization.Table For minimum flow ratio with a daily subsample interval, 39 and 42 of the 50 sites had a 50% chance that subsampled minimum flows were within ±10 and ±20% of the actual minimum, respectively. For the monthly subsample interval, 23 and 25 of the 50 sites had a 50% chance that subsampled minimum flows were within ±10 and ±20% of the actual minimum, respectively.For maximum flow ratio with a daily subsample interval, only seven of the 50 sites had subsampled maximum flow within ±10 and ±20% of the actual maximum. None of the 50 sites had monthly subsampled maximum flows within ±10%, and only two were within ±20% of actual maximum flows.For runoff ratio with a daily subsample interval, 44 and 49 of the 50 sites had a 50% chance that subsampled minimum flows were within ±10 and ±20% of the actual runoff, respectively. For the monthly subsample interval, 12 and 22 of the 50 sites had a 50% chance that subsampled runoff values were within ±10 and ±20% of actual runoff, respectively.Results for minimum flows are shown in Figs A histogram of minimum flow ratios for daily, three day, weekly, and monthly subsample intervals Fig.  shows noResults for maximum flows are shown in Figs n = 617 or roughly 25%). In contrast, the monthly subsample distribution was more heavily weighted to the left, with 0.0 to 0.05, and 0.05 to 0.1 containing the highest number of site-subsamples (n = 915 or roughly 37%).Figure Results for the runoff (volume) are shown in Figs There was a systematic negative bias in the runoff estimates, as evidenced by the greater number of sites below 1 than above 1 for all subsample intervals. Runoff was underestimated for 54, 54, 55, and 61% of site-subsamples for daily, three day, weekly, and monthly subsample intervals, respectively. This indicates that the negative bias was stronger as the subsample frequency decreased. This trend is also illustrated by the median being consistently below the 1 runoff ratio line in Fig. Figure r values between average flow ratios  and (1) latitude, (2) watershed area, (3) R-B Index, and (4) storage ratio. Pearson’s r values were tested for significance with a two-tailed p-value hypothesis test . Values shown in dark red had mathematical dependencies between variables  subsampled water level observations are as precise and accurate as continuous observations and (2) an equally accurate stage-discharge curve is available for converting observed water levels to flows, this analysis indicates that lower frequency observations of stream stage and flow can be useful, and could play a role in hydrologic data generation. The utility of lower frequency data depends largely on what the ultimate use(s) of the data are. Table  subsamplOne limitation of our approach was the assumption that citizen science spot measurements of water level or stage could be converted to flow with the same accuracy as 15-min continuous USGS records. Much of the challenge of streamflow monitoring lies precisely in the conversion from stage to flow, or the development of the stage-discharge rating curve Braca . For exaEstimates of minimum flow discussed in section Minimum flow results, as compared to maximum flow and runoff , were the least sensitive to changes in subsample intervals. Because minimum flows tend to persist for longer timescales, they were estimated within 10% for half of the subsample iterations at 39 (daily) and 23 (monthly) of the 50 sites. There were statistically significant correlations between subsampled average minimum flow ratios and latitude and R-B Index. Precipitation in California has a positive correlation with latitude. We suggest that the observed negative correlation between latitude was due to north-to-south trends in precipitation, resulting in fewer ephemeral streams and more variable minimum flows as latitude increases. Subsampled measurements are most likely to characterize minimum flows for ephemeral streams, or streams that normally go dry for at least certain parts of the year. Streams that run dry also typically have a higher flashiness index.Because maximum flows occur only briefly, it is unlikely that reliable maximum flow estimates (section Maximum flow results) will be obtained from subsampled measurements with average observation intervals of daily or greater. For example, maximum flows were estimated within 10% for half of the subsample iterations at only 7 (daily) and 0 (monthly) sites. This is consistent with Cheviron et al.  who foun3 s−1, or storage ratios greater than one, runoff was estimated within 20% (except for one site) and 10%, respectively for half of the subsample iterations. There were statistically significant correlations between subsampled runoff residuals and latitude and watershed area . Short period runoff events in flashy ephemeral streams often contribute significant percentages of total runoff. It is more likely that lower frequency measurements will produce less accurate runoff results, because critical portions of the hydrograph can be completely missed as the observation frequency increases.Runoff volumes were estimated within 10% for half of the iterations at 44 (daily) and 12 (monthly) of the 50 sites. The systematic negative bias in runoff estimates that increased as the subsample frequency decreased is congruent with the findings of Coynel et al. . Data asWhile the subsampling procedure used in this paper produced somewhat regularly spaced readings, actual Citizen Hydrology observations will likely consist of an irregular mixture of observation frequencies. Thoughtfully varied observation frequencies, however, are a potential strength of Citizen Hydrology. We envision that, at a minimum, monitoring frequencies could be varied based on (1) typical seasonal hydrologic patterns and (2) individual rainfall-runoff events. In Nepal, for example, where our field work is being completed, it rains for roughly 4 months during the monsoon season (June–September), and is relatively dry for the remaining 8 months. Hydrographs during the monsoon season are quite dynamic, and therefore more frequent observations are desired. During the dry period, the hydrograph mainly undergoes a long recession, so less observations are needed, especially towards the end of the recession prior to the next monsoon. Additionally, depending on rainfall-runoff response timescales, observation frequencies could be altered depending on rainfall duration and intensity, or more simply by if it is raining or not. Therefore, future work should explore how variable observation frequencies, or adaptive monitoring, could lower uncertainty in Citizen Hydrology data.We suggest that data assimilation (briefly mentioned in section Runoff), or a systematic combination of modeling and observations, could be promising methodology for adding value to, and improving accuracy of, Citizen Hydrology observations. For example, higher frequency observations of rainfall collected by a permanently installed sensor could be combined with lower frequency observations of stream stage and flow performed by Citizen Hydrologists. Then, in the context of a rainfall-runoff model, these data could be combined to help “fill in the gaps” of the hydrograph. Data assimilation has the possibility to improve minimum flow, maximum flow, and runoff estimates based on lower frequency observations, and should be the focus of future Citizen Hydrology research.The results of this research are most meaningful if the watersheds chosen for subsampling from the “data rich” region(s) are similar to those of the “data poor” region(s) targeted for applications of Citizen Hydrology. For our purpose of designing a Citizen Hydrology monitoring campaign in Nepal, we specifically chose stream gauges from California for subsampling because of (1) the abundance of high quality stream gauging stations and (2) the topographic and climate similarities with Nepal. For example, both California and Nepal have well-defined 4 to 5 month long wet periods when the majority of precipitation occurs , followed by prolonged dry periods. During the wet periods, both California and Nepal have significant precipitation events that occur due to the strong winter Pacific jet stream  of the Bagmati River watershed in the Kathmandu Valley. Precipitation patterns and amounts for the Kathmandu Valley are similar to those in Northern California . There are 31 watersheds with a latitude above 36 included in this study ranging in size from 1 to 31,313 km2. The highest R-B Index observed for these 31 sites was 0.66 for SiteID 11181000. For daily observation frequencies, out of a total of 1550 site-subsamples (i.e. 31 sites times 50 subsamples), only 28 site-subsamples had runoff errors greater than 10%, and only one site-subsample exceeded 20%; the average runoff error was 1.9%. With the assumptions previously stated at the end of the section Background and introduction in mind (i.e. regarding water level observation accuracy and stage-discharge curve availability), these results give us reasonable confidence that runoff estimates based on daily Citizen Hydrology observations should be within 10% of actual runoff, if not better.As a sample “data poor” region application, we are using Citizen Hydrology observations to estimate runoff in several sub-watersheds , similar to those that could be produced by Citizen Hydrology, on the accuracy of basic streamflow statistics like minimum flow, maximum flow, and runoff. To answer this question, we performed a subsampling analysis on 7 years of streamflow data from 50 USGS gauging stations in California. Depending on the questions being asked, and the characteristics of the watershed(s) in question, lower frequency observations, such as those produced from Citizen Hydrology, can provide useful hydrologic information. In general, as watershed flashiness decreases and storage ratio increases, the reliability of minimum flow, maximum flow, and runoff estimates obtained from low frequency observations increases. Also, as latitude increases, which for California is a reasonable proxy for precipitation, the reliability of runoff estimates based on low frequency observations increases. Interestingly, watershed size seems to play a less prominent role than latitude , R-B Index, and storage ratio in determining reliability of low frequency observation based runoff estimates.Supplementary MaterialSupplementary MaterialSupplementary MaterialSupplementary Material


These data demonstrate that progesterone mediates a phenotypic change in BAT, which contributes to the gestational metabolic environment, and thus overall fetal size.Pregnancy is associated with profound maternal metabolic changes, necessary for the growth and development of the fetus, mediated by reproductive signals acting on metabolic organs. However, the role of brown adipose tissue (BAT) in regulating gestational metabolism is unknown. We show that BAT phenotype is lost in murine pregnancy, while there is a gain of white adipose tissue (WAT)-like features. This is characterised by reduced thermogenic capacity and mitochondrial content, accompanied by increased levels of markers of WAT and lipid accumulation. Surgical ablation of BAT prior to conception caused maternal and fetal hyperlipidemia, and consequently larger fetuses. We show that BAT phenotype is altered from day 5 of gestation, implicating early pregnancy factors, which was confirmed by reduced expression of BAT markers in progesterone challenged oophorectomised mice. Moreover,  Furthermore, altered metabolism as a result of maternal obesity is associated with an increased risk of gestational diabetes and pre-eclampsia7, and predisposes the infant to metabolic disease in later life10.Pregnancy is associated with a gradual metabolic shift from normal maternal serum concentrations of triglycerides, free fatty acids, and glucose at the time of conception to hyperlipidemia and insulin resistance in the third trimester. These changes support the growth and development of the fetus. The importance of maternal-fetal energy transfer is exemplified by several pathophysiological conditions. Under-nutrition or obesity during pregnancy can increase the risk of babies being born with intrauterine growth restriction or large for gestational age11. However, little is known about the prevalence and function of brown adipose tissue (BAT) during pregnancy. Adult humans possess classical brown adipocytes and beige cells of white adipocyte origin, both of which are thermogenic and can contribute to whole body energy metabolism  results in lipid accumulation, whereas towards term, in conjunction with placental hormones, there is insulin resistance and lipid catabolismIn this report, we examine the impact of pregnancy on BAT, the role of BAT in regulating the maternal and fetal metabolic environment, as well as alterations in BAT phenotype mediated by early pregnancy signals.Ucp1, Dio2, Zic1, and Cidea, accompanied by significant increases in white adipose tissue (WAT) markers Lep and Adrp. Gene expression of the β3-adrenergic receptor was unchanged  on pregnancy, a period associated with profound metabolic change, we first identified a point during gestation to study BAT biology that follows significant alterations in serum lipid concentrations. Longitudinal sampling of sera over the course of pregnancy identified gestational day (GD) 14 as a point that follows significant increases in serum cholesterol, HDL, and triglyceride concentrations  of female mice prior to conception, to enable assessment of the relative contribution of BAT to gestational metabolism. Gestational days 14 and 18 were chosen as time points of interest to capture the impact of BAT ablation prior to, and at the end of, the period of rapid fetal growth and development Fig. . AblatioThe impact of BAT ablation on fetal parameters was evident at GD18, as the average fetal and placental weight increased by 7.8% and 12.3% respectively Fig. , whereasThe mouse fetus undergoes a rapid growth phase between GD14 and parturition, which coincides with our observation that at GD18, the fetuses are heavier from iBATx mothers. This may be as a result of altered fetal or maternal lipid homeostasis and fuel availability. Biochemical analysis of FFA concentrations in the serum of fetuses at GD18 from iBATx mothers revealed a significant increase of 117% Fig. , whereasSrebp1c and Acc1 in the fetal liver -mediated RER value. The levels of mitochondrial markers were also reduced at GD14 and, when combined with reduced UCP1 expression and increased lipid concentrations, could be responsible for the abrogation of BAT function during pregnancy. These results demonstrate that BAT undergoes adaptive changes characterised by a loss of classical thermogenic phenotype and function accompanied by whitening during pregnancy. Similarly, whitening of BAT with a loss of BAT phenotype and function has been observed in diet-induced obese mice23.To ascertain if the diminished BAT phenotype resulted in decreased BAT function, we assessed the capacity of β3-adrenergic agonist-stimulated thermogenesis at GD14 using indirect calorimetry, avoiding the muscular contribution to O24, was surgically ablated prior to conception. Days 14 and 18 of gestation were chosen to study the impact of BAT ablation, as they lie before and towards the end of the period of rapid fetal growth26. Removal of BAT had no effect on maternal body weight. This is not surprising, as mice lacking UCP1 housed below thermoneutrality do not become obese and O2 consumption remains unchanged29. However, the iBATx group had significantly higher levels of serum cholesterol and hepatic triglyceride concentrations. This is consistent with the view that gestationally adapted BAT contributes to whole body lipid metabolism during pregnancy.Evidence for a functional role of gestationally modified BAT was provided by experiments where iBAT, which represents approximately 60% of total BAT33. We therefore studied the consequences of iBAT ablation on the fetal compartment. Fetal and placental weight were increased at GD18 in the iBATx group as a result of a decrease of a factor or function associated with brown fat during pregnancy that contributes to the regulation of fetal growth. There are striking similarities between maternal obesity, which is associated with hypertriglyceridemia and an increased risk for babies to be born large-for-gestational age35, and our findings in iBAT ablated mice that have raised maternal hepatic triglyceride levels and heavier GD18 fetuses. This emphasises the importance of BAT in regulating systemic maternal lipid levels during a critical period of fetal development.It is well established that the metabolic status of the mother can affect the developing fetus36. We hypothesised that the increased FFA levels originated from the maternal serum, as gene expression analysis of lipid homeostasis targets in the fetal liver were not increased and, in some cases, were reduced, possibly as a measure to rebalance the elevated fetal serum FFA levels. The probable source of the increased fetal serum FFA is hydrolysed triglycerides from the placenta, as the expression of placental lipases was significantly increased in GD18 iBATx mice. This may be a homeostatic response to reduce the elevated levels of GD14 maternal triglycerides  and allowed free access to food and water. They were housed in stable conditions . Mice were acclimatised to the animal house for 1 week prior to experiments.For pregnancy experiments, 7–8 week old female mice were mated with males. Virgin mice were used as controls  0). GD1 was considered to be the day the copulatory plug was identified. For all animal studies, mice were sacrificed after a 4 hour fast, and maternal and fetal serum and tissues were collected and snap-frozen immediately or fixed in 10% neutral buffered formalin for 24 hours.2 consumption and CO2 production was measured in each cage sequentially every 12 minutes, and retrospectively corrected for body weight without pups. Respiratory exchange ratio was calculated according to manufacturer’s software.Indirect calorimetry was carried out using the LabMaster cage system (TSE). GD0 and GD14 mice were terminally anaesthetised by intraperitoneal injection of 90 mg/kg pentobarbital . Once fully sedated, they were placed in the metabolic cages and allowed to acclimatise for 20 minutes, to establish a constant metabolic baseline. Norepinephrine  was then administered by subcutaneous injection at the nape. The O7–8 week old female mice were anaesthetised via isoflurane vapour  and the interscapular BAT (iBAT) was surgically removed. Sham-operated mice underwent the same procedure without the removal of iBAT. Mice were single-housed for 2 weeks during the recovery period, and then paired with males until visual confirmation of a plug, following which they were monitored until sacrifice at GD14 or GD18 of pregnancy, along with GD0 controls.7–8 week old female mice were anaesthetised via isoflurane vapour and underwent bilateral oophorectomy. After a two-week recovery period, the mice were anaesthetised and 14-day release osmotic minipumps  were implanted subcutaneously in the dorsal region. Osmotic pumps contained progesterone , prolactin  or vehicle only (20% (w/v) 2-hydroxypropyl-beta-cyclodextrin, Sigma Aldrich). After 2 weeks, animals were sacrificed as described above.48. Sections were incubated overnight at 4 °C with rabbit anti-mouse primary antibody to UCP1 diluted 1:10,000 in phosphate buffered saline. The UCP1 antibody was a kind gift from Barbara Cannon.Fixed iBAT sections were embedded in paraffin and sectioned at 5 µm onto poly-L-lysine coated slides. Sections were stained with haematoxylin and eosin for histological analysis. Immunohistochemical analysis was performed as previously describedSerum and tissue cholesterol, LDL-cholesterol, HDL-cholesterol, TG, FFA and total proteins were measured using an Unicel DxC 800 autoanalyzer (Beckman-Coulter) using dedicated kits from Beckman-Coulter, excluding FFA which were measured using a kit from Wako Diagnostics (Neuss). Liver and brown adipose tissue were extracted in a 0.125 M potassium phosphate buffer and normalised to the total protein content.49. Cells were seeded and grown in growth media consisting of DMEM/10% NCS with  20 nM insulin  and 1nM triiodothyronine (Sigma-Aldrich), until 70% confluent. The adipocytes were differentiated for 48 hours in the above media supplemented with 500 µM isobutylmethylxanthine, 2 µM dexamethasone, 50 µM indomethacin and 1 µM rosiglitazone (Sigma-Aldrich). On the morning of an experiment the media was replaced with growth media.iBAT was removed from 10 day old C57BL/6 pups and pooled. Primary adipocytes were isolated and differentiated as previously describedUcp1 was undetectable in several of the cultures, and thus the results of the hormonal treatments were calculated as relative to the NE treated group, as previously described43 , and cDNA synthesised using the Superscript II kit (Thermo Fisher Scientific), according to manufacturer’s instructions. Semi-quantitative real-time PCR with SYBR Green JumpStart Taq ReadyMix (Sigma-Aldrich) was used to detect and amplify target cDNA. Relative gene expression was calculated using the ∆∆ Ct method. Genes of interest were normalised to the housekeeping gene Cyclophilin B. See Supplementary Experimental Procedures online for a table of primers and sequences. Basal expression of Frozen iBAT samples were homogenised in Qiazol (Qiagen) and incubated for 5 minutes at room temperature, after which chloroform (Sigma-Aldrich) was added. Following centrifugation, total DNA was precipitated from the interphase and the phenol phase with 100% ethanol. After further centrifugation, the resulting DNA pellet was washed in 0.1M sodium citrate, followed by 75% (v/v) ethanol. The DNA pellet was air-dried, resuspended in 8mM sodium hydroxide, and neutralised by the addition of HEPES buffer and 100 mM EDTA, and stored at −20 °C until use. Relative mitochondrial content was assessed by comparing the Ct of mitochondrial Cox2 relative to nuclear β-actin, as described above.Data are presented as mean ± standard error of the mean (SEM). Statistical analysis was performed using Origin Pro software (OriginLab). An unpaired Student’s t-test was used for comparison between two groups. One-way ANOVA was used when comparing more than two groups, followed by Tukey’s post-hoc analysis. A p-value of <0.05 was considered significant. Data was assessed for normality by carrying out standard distribution plots. Where there were outliers, sensitivity analysis was performed and analysis repeated with the outlier(s) removed.All experiments were performed in accordance with the Animals (Scientific Procedures) Act 1986 Amendment Regulations 2012 and approved by King’s College London’s Animal Welfare and Ethical Review Body and the Home Office.Supplementary Information


Hybrid palm oil, which contains higher levels of oleic acid and lower saturated fatty acids in comparison with African palm oil, has been proposed to be somehow equivalent to extra virgin olive oil. However, the biological effects of its consumption are poorly described. Here we have explored the effects of its overconsumption on lipid metabolism in a non-human primate model, the common marmoset. Dietary supplementation of marmoset with hyperlipidic diet containing hybrid palm oil for 3 months did not modify plasma lipids levels, but increased glucose levels as compared to the supplementation with African palm oil. Liver volume was unexpectedly found to be more increased in marmosets consuming hybrid palm oil than in those consuming African palm oil. Hepatic total lipid content and circulating transaminases were dramatically increased in animals consuming hybrid palm oil, as well as an increased degree of fibrosis. Analysis of liver miRNAs showed a selective modulation of certain miRNAs by hybrid palm oil, some of which were predicted to target genes involved in cell adhesion molecules and peroxisomal pathways. Our data suggest that consumption of hybrid palm oil should be monitored carefully, as its overconsumption compared to that of African palm oil could involve important alterations to hepatic metabolism. After it has been proved that partially hydrogenated fat–which is rich in trans isomers– was harmful to health, its use was forbidden in different countries. African Palm oil (AP)  has been the vegetable oil most widely used by the food industry as a trans-fat substitute even though it is rich in saturated fatty acids. However, recently, Chen et al. showed that for each kilogram of palm oil consumed annually per capita, the mortality due to ischemic cardiomyopathy increases the equivalent to 68 for 100,000 deaths in developed countries1. Even if metabolic consequences of AP consumption are still controversial3, its consumption remains, a source of major concern within health policies regarding the reduction of cardiovascular and metabolic diseases risk.In the last decades, the increasing consumption of processed and industrialized food with low nutritional quality has correlated with an increment of chronic diseases prevalence, such as obesity and cardiovascular diseasesElaeis guineensis) and South American species, Elaeis oleifera has been exploited, with the objective of developing varieties as productive as African oil palm, together with resistance to diseases and pest, reduced height and improved nutritional value, specifically, increased monounsaturated oleic acid and reduced saturated fatty acids levels5. EMBRAPA  has developed the first Brazilian interspecific hybrid cultivar between Elaeis oleifera and Elaeis guineensis, which has all these characteristics, i.e. high productivity and improved nutritional value6. In this context, hybrid palm oil (HP) has been proposed as a promising new lipid source for industrialized products and a substitute for AP that could bring benefits to human health.Interspecific hybridization between African oil palm 8. This apparent healthier profile of fatty acids composition of HP was tested and compared to that of extra virgin olive oil (EVOO) in dietary supplementation10. When consumed for three months, both oils increased the degree of unsaturation of erythrocyte membrane lipids9. Moreover, dietary supplementation with HP showed similar effects on plasma lipids composition compared to that of EVOO (no differences between both groups)10. Researchers have suggested that the observed beneficial effects of HP consumption support the concept of HP as a “tropical equivalent to olive oil”.Previous studies on the chemical composition of HP suggest that the ripening status influences the amount of antioxidant phenolic content in the fruit14. Furthermore, the metabolic consequences of AP consumption are still controversial. Its effects can be favorable or unfavorable when compared with other types of fat sources, according to the evaluated parameters3. According to Wilson et al.15, these controversial metabolic effects of AP can be justified by the high presence of tocopherols and tocotrienols in their fat. These tocols can promote an antithrombotic state by reducing platelet aggregation and modulating prostanoid synthesis. But once the fat is refined, bleached and deodorized, depending on the degree of processing, the antithrombotic properties are inhibited15. Moreover, although no detrimental side effects of monounsaturated fatty acids (MUFA) rich diets were reported, there is still no consensus regarding monounsaturated fatty acids consumption recommendations16. Indeed, some large studies also suggest that substitution of saturated fatty acids by MUFAs can increase the risk of ischemic heart disease13. In this context, whether the consumption of HP–enriched in MUFAs–should be promoted due to its putative beneficial effects is still unclear.On the other hand, recent studies suggest that there is no clear evidence to conclude that dietary saturated fat is associated with an increased risk of CVD17. Indeed, fatty acids have been shown to influence miRNAs expression in different metabolic tissues including intestine18, adipose tissue19 and liver20. Whether saturated or MUFAs influence liver miRNA expression is poorly characterized21. Moreover, whether HP can influence miRNA expression is completely unknown.miRNAs are posttranscriptional regulators of gene expression. Increasing evidence suggests that miRNAs can be modulated by dietvs. that of AP, rich in saturated fatty acids on lipid metabolism, and to identify liver miRNAs that could be modulated by these dietary fatty acids. For this purpose, we used the common marmosets, a New World primate species that has great phylogenetic similarity with humans, being omnivorous animals, with diversified eating habits and easy adaptation to experimental diets. Nonhuman primates have also a lipid metabolism that is similar to ours and are recognized as a good animal model for dyslipidemia and metabolic disorders studies22. The experimental design is shown at Fig. The main objective of this study was to evaluate the effects of overconsumption of HP, source of unsaturated fatty acids–obtained from the interspecific hybrid grown in EMPRAPA/Brazil–, First, we decided to study the detailed lipid composition of the experimental diets Table  in orderNext, we decided to determine whether there were differences on food intake due to the experimental diets. Animals consumed normally the prepared diets during the intervention period, both during normocaloric and hyperlipidic phases. No apparent differences in food intake were observed. Despite the consumption of hyperlipidic diets, no significant changes in body weight during the nutritional intervention were observed, either when analyzed by sex or computing both sexes together . Tissue analysis of the fatty acid synthase (FASN) gene showed an increased expression of the mRNA in animals consuming HP  or higher availability of palmitic acid (C16:0) as a substrate for the enzymatic reaction (p < 0.0001), suggesting that the increased SCD1 activity in the liver of HP group might be involved in the process of hepatic lipid accumulation with potential to promote NAFLD Table .25. Epigenetic factors, such as DNA methylation, histone modifications or microRNAs (miRNAs), have been involved in liver disease27. Over the last decade, miRNAs have emerged as critical regulators of lipid metabolism . In order to gain insight into possible molecular mechanisms modulated by HP that could be leading to the observed hepatic lipid accumulation, we next evaluated hepatic miRNAs. A selection of miRNAs from marmoset, which are conserved in humans , was first screened in both male  because of its high saturated FA content, specifically palmitic acid (C16:0 ≈ 44%), and its use in foods and food products has been somewhat discouraged. On the other hand, although controversies persist concerning the association between the intake of dietary saturated fatty acids and CVD risk31, several studies have shown the beneficial role of diets with a relatively high MUFA content on CVD risk factors, obesity and diabetes. These beneficial MUFA are mainly provided by the Mediterranean diet and, specifically, by olive oil, oleic acid-rich oils32. In this context, higher degree of FA unsaturation is therefore a desirable characteristic to alter in palm oil. Hybrids between E. guineensis × E. oleifera excited much interest, because of the shorter size, more resistance to several diseases, higher desaturation of palm oil and, in particular, putative healthier oil, constituting an alternative approach to increase oleic acid content in palm oil5. Seeds of high yielding interspecific hybrids palm oils (HP) are produced in Brazil by EMBRAPA/Dendê do Pará S.A.12 (MANICORÉ hybrids), using wild E. oleifera palms indigenous to the Coari and Manicoré municipalities in the Amazon river basin.Dietary FAs play significant roles in the cause and prevention of cardiovascular disease. Palm oil may be an unhealthy fat, mainly extracted from vs 42.1 ± 0.01 in original oils, data not shown) together with a lower palmitic acid content . Furthermore, as expected, a high-fat diet manufactured from the HP showed higher contents of monounsaturated and lower of saturated fatty acids compared to AP. However, the amount of the polyunsaturated or essential fatty acid, linoleic acid, does not differ significantly  and similar saturated fatty acid content (33.5% ± 0.5), while the percentage of others fatty acids does not undergo significant changes. Moreover, a recent positive effect of HP –grown in Colombia– on human plasma lipids has been observed, where after three months of 25 ml per day HP consumption, the total cholesterol levels decreased by 6.3%, whereas LDL-C concentration decreased by more than 13%10. This is the only study found in the literature that shows similar effects on plasma lipids composition compared to EVOO10. On the other hand, our results provide evidence that a high-fat diet richer in HP –grown in Brazil– does not result on a healthier liver lipid content profile in marmoset.In the present study, our results of fatty composition revealed significant differences between the HP and AP produced in Brazil, where HP presents a higher level of oleic acid  might not induce appreciable changes in circulating lipids levels. In the context of a high fat diet, it has been previously described that some positive effects related to dietary fatty acids can be lost under high-fat intake35, such as in our study. Indeed, animal studies when fed high fat diet have shown that palmitic acid or palm oil enriched diet can induce obesity, hepatic steatosis or changes in the microbiota37. In humans, only one study shows that overfeeding SFAs from palm oil increases hepatic fat compared to that of n-6 PUFAs (sunflower oil)38. Thus, whether this HP grown in Colombia, when consumed in large quantities in the diet also produce liver damage in humans, as observed here for marmosets, has not been evaluated.The increased liver volume of animals consuming HP diet prompted us to search for other markers of liver damage. The AST/ALT ratio is an index used in clinical practice as a non-invasive method of determining the progression of various liver diseases, especially NAFLDet al., suggested that hybridization seemed to modify the biosynthesis of fatty acids rather than their assembly in the triacylglycerols8. In fact, they found that the sn-2 position of triacylglycerols in HP was mainly esterified with oleic acid (≈10% increment compared to that of AP)8. This apparent healthier profile of fatty acids composition of HP was tested and compared to that of EVOO in dietary supplementation10. Although more direct experiments are required in the HPs produced in Brazil, it is possible that the HP grown in Colombia, due to its higher proportion of unsaturated FA chains in position 2 of the TAG molecule8, could be quickly oxidized, resulting in a significant impact on the circulating lipid levels when in dietary supplementation.There is evidence that the metabolic effects of dietary TAG depend not only on their FA composition but also on the stereochemical configuration of specific FA in the TAG molecule. In Colombia, Mozzon et al.39, saturated fatty acids  exert lipotoxic activity in liver cells, inducing caspase activity and, therefore, apoptosis. To overcome this, liver cells upregulate the activity of SCD, because MUFAs  can be stored in cells without the toxic effects observed with saturated fatty acids. The authors suggest that the accumulation of fat in liver is a mechanism of adaptation to avoid liver cells death39. Hepatic SCD1 activity, has been shown to correlate with non-alcoholic fatty liver disease (NAFLD)40. In the present study, we observed increased 16:1/16:0 ratio in HP diet indicating a higher activity of the SCD1 enzyme, suggesting that, high-fat diet richer in HP –grown in Brazil– contributes to hepatic fat accumulation with potential to promote NAFLD. In fact, Puri and colleagues41 have previously performed lipidomic analysis on human healthy livers, and those of steatosis and non-alcoholic steatohepatitis patients. From their data we can obtain the SCD ratio for healthy (0.053), steatosis (0.076) or steatohepatitis (0.062) liver patients, which are within the same range of our findings. Yamada and colleagues42 analysed also similar groups and found a clear difference between healthy (0.10) and non-alcoholic steatohepatitis (0.13) patients. They also correlated these data with different histopathological analysis and showed a significant correlation between these ratios and the progression degree of NAFLD42. On the other hand, the increased glucose levels observed in the HP group, might be indicative of hyperinsulinemia, and insulin can also upregulate the activity and expression of SCD43. Overall, our data suggest that HP diet, by mechanisms still unknown, alters liver function when compared to that of AP supplementation. Whether or not this HP given in a normolipidemic diet also alters liver function, is a subject for future investigations in this experimental model.The finding in this study of increased total lipid in liver tissue from animals consuming HP diet, motivated us to study the ratio of C18:1/C18:0 and C16:1/C16:0 and therefore, SCD1 activity was indirectly estimated. The enzyme SCD1, which is the isoform predominantly expressed in the liver and adipose tissues, plays an important role in the synthesis of monounsaturated long-chain FAs from saturated fatty acyl-CoAs. According to Li 44; (b) Cell adhesion molecules (CAMs), adhesion of leukocytes to endothelial cells via cell adhesion molecules is thought to be pivotal in the initiation of atherosclerosis45, and it has been described that certain miRNAs play important roles in lipid uptake and adhesion molecule expression in oxLDL-effected macrophages/DCs46; and (c) Peroxisome, it is well known the role that peroxisomes play in cellular lipid metabolism, being involved in key pathways such as fatty acid beta-oxidation, etherphospholipid biosynthesis and fatty acid alpha-oxidation47. These pathways could be mediating an inflammatory response to the observed hepatic damage  in an attempt to recover normal liver function. Interestingly, putative gene targets to some of these miRNAs were also related to glucose metabolism or glucose transport, such as Sirtuin 1 (Sirt1) or Sortilin 1 (Sort1), respectively, and several other insulin signalling/receptor related genes including: Vamp2, Hnf4a, Cacna1c, Stx1a, Cacna1d, Snap25, Grb2, Fgf23, Fgf1, or Igf1r. Future experiments will determine whether the expression levels of the putative target genes are modulated by the HP diet.In order to identify liver miRNAs that could be modulated by these high-fat diets richer in HP and AP, we evaluated hepatic miRNAs in this experimental model. To the best of our knowledge, no specific studies have been conducted either on the qualitative liver expression of miRNAs, or on their modulation by these hyperlipidic diets. miRNAs are powerful post-translational regulators that mediate gene regulation by providing the means of fine tuning entire gene networks. In this manner, the modulation of a small number of miRNAs allows the regulation of many genes involved in different pathways. The characterization of the miRNAs modulated by the Hybrid palm diet vs. the African palm diet, has allowed us to identify 10 miRNAs whose expression is consistently altered under the HP diet on marmosets of both sexes. Analysing the putative gene targets to these miRNAs and studying the pathways in which these genes are involved by gene ontology, five pathways have risen that could be involved and/or mediating the effects observed on the experimental animals under the HP diet. It is worth to highlight three of these pathways due to their relationship to lipid metabolism: (a) Nicotinic acetylcholine receptor signaling pathway, nicotinic acetylcholine receptors (nAChRs) are known to interact with anti-inflammatory pathways and have been implicated in control of appetite and body weight, as well as lipid and energy metabolism48. The inability to obtain liver biopsies  at time 0 of the intervention, and the inability to obtain enough biopsied tissue for additional histological analysis, still the gold standard method for diagnosing liver disease despite its limitations49, has prevented a more thorough and detailed characterization of the effects induced by the tested diets. The strength of our study relies on the use of marmoset as a non-human primate model and a strict control of the fed diet. Nonhuman primates have a lipid metabolism close to ours and provide a very good model for metabolic disorders22. The control of the fed diet reduces the variability inherent to free will subjects, giving strength to the statistical analysis. Moreover, diet is known to change not only classically used biochemical parameters, but also novel biomarkers such as miRNAs50.Some limitations of the present study should be noted though. For instance, the limited number of subjects has reduced the possibility (if any) to find any differences attributed to sex, given the case that these differences exist. Indeed, sex differences may also influence lipid metabolismIn conclusion, our study provides evidence that HP supplementation in the diet of the common marmoset can induce liver abnormalities not easily detected by classical biochemical parameters (i.e. plasma lipid levels). Our data suggest that overconsumption of HP should be monitored carefully, as its consumption, compared to that of AP, might rise important concerns related to hepatic metabolism. Further studies will be needed to discern the differences of HP supplementation with the variety used in other previous studies.Callithrix jacchus, ten males and ten females) were used in this study. The animals were housed in couples in species-specific cages of stainless steel with internal sliding wall (squeeze-back system), according to the established guidelines in the Guide for the Care and Use for Laboratory Animals51.Twenty common marmosets  and water ad libitum, as well as low-fat fruits and vegetables offered daily as environmental enrichment, to break the food monotony, following the recommendations of the National Council for Animal Experimentation ControlThe marmoset experiments were approved by the Ethics committee for the use of experimental animals of the Health Care Center of the Federal University of Rio de Janeiro, under reference number INJC014. All experiments were performed in accordance with relevant guidelines and regulations.53 as showed in Table E. guineensis and E. oleifera interspecific hybrid) were kindly provided by AGROPALMA .To perform the study, primates were divided into 2 groups  subjected to two different diets: African Palm (AP) and Hybrid Palm (HP) groups. The dietary intervention consisted of a 3-month metabolic insult with hyperlipidic  and normoglicid diet, with a high glycemic index, that differed only in the lipid source of the diet: HP vs. AP. Experimental diets were made by Prag Soluções , following the recommendations of the European Association of Zoos and AquariaBody mass was measured at each blood collection, once the animals were sedated. Biometrics were also performed at this moment to determine the measure of body length , tail , abdominal perimeter  and thoracic perimeter (measured around the axillary region) with anthropometric tape Fig. .At the beginning of the study we characterized the animals running hematological and immunological analysis, as well as determining hepatic and renal status. Also, blood samples were taken before dietary intervention  and after 1, 2 and 3 months within the nutritional interventions  and blood was collected by puncture in the femoral vein (0.8 to 1.0 mL) and separated in aliquots with different anticoagulants: in tubes containing heparin (20 IU/ml blood) or EDTA , as required to run the different intended analyzes. Samples with heparin were sent under refrigeration to Laborlife Clinical Analyzes (Rio de Janeiro) to determine biochemical parameters such as: total cholesterol and its fractions, triglycerides, and glycaemia by means of colorimetric chemical kinetic assays with an automated clinical analyzer (Metrolab 2300). The samples collected in EDTA were immediately centrifuged at 3,000 rpm for 15 minutes at 4 °C. Plasma and resulting erythrocytes were frozen at −80 °C for further plasma lipoprotein analysis by fast protein liquid chromatography (FPLC) and assessment of erythrocyte fatty acid profile by gas chromatography, respectively  and xylazine (1.5 mg/kg), both intramuscularly. The hepatic tissue was collected by laparotomy wedge biopsy. Samples were immediately frozen in liquid nitrogen and stored at −80 °C for further analysis  from animals fed AP or HP were fixed in formalin, paraffin embedded, and sections were stained with hematoxylin and eosin (H&E) and Pico Sirius Red. Images were captured with a camera DP72 coupled in a microscope BX51, both Olympus. Three hepatic biopsies of each group were processed.54, the internal standard C13:0 being added for quantification of the methyl esters.The fatty acid profiles of the diets and hepatic tissue were determined by liquid-gas chromatography according to the AOCS Ce2b-11 method55. The alkaline direct methylation method (Ce 2b-11), described by the American Oil Chemists’ Society56, was used for transesterification of the oils extracted from the diets, as well as for the extraction, purification, and transesterification of the liver samples. Tridecanoic acid (13:0)  was added to each methylation tube as internal standard. Fatty acid methyl esters were separated and quantified using the Agilent Technologies 7890A CG System with a flame ionization detector and a 100 m × 0.25 mm capillary column with 0.2 µm inner diameter . Hydrogen was used as carrier gas. The fatty acid methyl esters were identified based on purified standards , and their quantification was performed as a function of the corresponding peak areas compared to that of the internal standard.The lipids of the experimental diets were extracted and purified using the Bligh and Dyer methodCT scans were performed on all animals prior to and at the end of the dietary intervention in an Optima PET/CT560 equipment, located at the Nuclear Medicine Service of the Hospital Universitário Clementino Fraga Filho- Rio de Janeiro, UFRJ. The settings for CT scans were 120 kVa, 80 mA and slices of 0.65 mm. The volume of the livers was calculated using OsiriX Lite v.7.0.4 software, where each tomographic slice was manually segmented, and its volume was calculated later. A 3D image of the animal bone structure was processed for a better view of the location of the region of interest.55, and the results are expressed as percentage.Total fat in liver samples was determined by the gravimetric method after lipid extraction. Lipid extraction was performed as described by Bligh and Dyer2-EDTA and 0.02% NaN3, pH 7.4, at a flow rate of 0.3 ml/min. Fractions of 0.4 ml were collected and cholesterol and triglyceride concentrations in each fraction were determined enzymatically .Equal volumes of plasma from every marmoset in each group were pooled and 220 μl of pooled plasma were subjected to gel filtration by fast protein liquid chromatography (FPLC) using a Superose 6 HR 10/30 column (Pharmacia). The samples were eluted with 150 mM NaCl, 10 mM Tris-HCl, 2 mM Na57), we compared the marmoset miRNA sequences with the assessed human miRNAs, including in our screening the sequences presenting 100% similarity with human miRNAs, and sequences with up to two mismatches outside the seed sequence (nucleotides 2–8) and/or differing in up to two nucleotides in length. The number of marmoset miRNAs selected was 370, to which we added miRNAs to be used as normalizing miRNAs.We selected the miRNAs presenting the highest sequence similarity with human miRNAs. Using data related to miRNAs sequence provided by The Marmoset Genome Sequencing and Analysis Consortium . RNA was quantified using a NanoDrop-1000 Spectrophotometer and purity was determined by calculating the 260/280 nm and 260/230 nm ratios. RNA integrity was assessed using the Agilent 2100 Bioanalyzer. For the selected (see above) miRNA screening, cDNA was synthetized from total RNA (1000 ng) using miScript® II RT Kit (Qiagen), following the manufacturer’s protocol. miRNA quantification was performed by real time PCR (qRT-PCR) using the miScript SYBR®Green qPCR Master Mix (Qiagen) on a 7900HT fast real time PCR system (Applied Biosystems) using a 384-well plate format. Reactions were performed in a final volume of 10 µl following these cycling conditions: 15 min at 95 °C for one cycle, then 40 cycles at 94 °C for 15 s and 58 °C for 30 s. The dissociation curve was analyzed at 95 °C for 15 s, followed by one cycle with increasing temperature starting at 60 °C and ending at 95 °C. RNU6 and RNU1A1 were used as reference small RNAs for normalization. miRNA relative expression analysis was performed using the GenEx software .Validation of selected miRNAs from the primary screening were performed by Real-time RT PCR using a second different pair of primers. Reactions were performed using cDNA obtained as indicated previously in 384-well plates, using miScript SYBR green Mastermix (Qiagen) and gene expression determined using a 7900HT Fast Real-time PCR System (Applied Biosystems) as described above. Specific oligos for Callitrix miRNAs were used and values were normalized by the housekeeping small RNAs RNU1A1 and RNU6.58 algorithm v.3.3, setting a minimum alignment score of 150 as a threshold for a valid mRNA-miRNA interaction. For functional analysis of these predicted targets, marmoset genes were mapped to their corresponding human orthologous, and enriched pathways and gene ontology (GO) biological processes were identified using the Genecodis enrichement analysis tool59.Marmoset transcripts 3′UTR sequences were obtained from Ensemble database (Dataset C_jacchus3.2.1), ending up with a set of 30642 sequences. After the selection of differentially expressed microRNAs, putative targets for these miRNAs were determined using the miRandat test was performed to establish statistical significances between experimental groups with GraphPad Prism program, version 7. The level of significance was set at 5% (p < 0.05).Results are expressed as mean ± error standard of the mean (EPM). Kolmogorov-Smirnov test was used to test the normality of the data. Those data that were not parametric were log transformed. Supplementary Information


The fresh pod weight of pw was only about 40% of that seen in the wild-type (WT) Zhonghua16, while the hull and seed filling of the mutant both also developed at earlier stages. Pods from both pw and WT lines were sampled 20, 40, and 60 days after flowering (DAF) and used for RNA-Seq analysis; the results revealed highly differentially expressed lignin metabolic pathway genes at all three stages, but especially at DAF 20 and DAF 40. At the same time, expression of genes related to auxin signal transduction was found to be significantly repressed during the pw early pod developmental stage. A genome-wide comparative analysis of expression profiles revealed 260 differentially expressed genes (DEGs) across all three stages, and two candidate genes, c26901_g1 (CAD) and c37339_g1 (ACS), responsible for pod width were identified by integrating expression patterns and function annotation of the common DEGs within the three stages. Taken together, the information provided in this study illuminates the processes underlying peanut pod development, and will facilitate further identification of causal genes and the development of improved peanut varieties with higher yields.Pod size is the major yield component and a key target trait that is selected for in peanut breeding. However, although numerous quantitative trait loci (QTLs) for peanut pod size have been described, the molecular mechanisms underlying the development of this characteristic remain elusive. A peanut mutant with a narrower pod was developed in this study using ethyl methanesulfonate (EMS) mutagenesis and designated as the “pod width” mutant line ( Arachis hypogaea L.) is one of the most important oil crops in the world. Thus, as the demand for oil is ever-increasing, there is an urgent need to breed new peanut varieties with high yields, a characteristic that is dependent on pod size. Previous research has shown that pod size is mainly determined by quantitative trait loci (QTL), and several of these have been identified , p-hydroxyphenyl (H), and syringyl (S) subunits  monolignols can be incorporated into lignin polymers to varying extents in specific tissues and certain species  results in elevated levels of H lignin in plants  eliminates, or reduces, the number of lignin S units content  biosynthesis  mutant with a narrower pod (seed width) (pw) than the Zhonghua16 wild-type (WT), and performed transcriptome profiling of both lines at three key pod developmental stages. The RNA-Seq data presented in this paper enhances our understanding of the transcriptome dynamics of peanut pod development and reveals the genes that are responsible for determining pod size.A limited number of studies are available that provide information on the developmental process and regulation of peanut pods. Given the fact that these plants possess the unique characteristics of “aerial flowers and subterranean fruit,” the genes responsible for pod development are likely distinct from those in model plants such as pw mutant used in this study was isolated from an EMS-induced population that originates from the Chinese elite cultivar Zhonghua 16, a WT that possesses both high yield and oil content characteristics. Both WT and pw (M6–M7 generations) lines were planted in the same areas  between 2014 and 2016, and pod samples for further experiments were collected 20, 40, and 60 days after flowering (DAF), in 2015 from 12 individual plants. We then selected 12 representative pods from both the WT and pw mutant for each biological library construction ; total pod samples from both lines were sliced, immediately frozen in liquid nitrogen, and held at −80°C for subsequent RNA extraction with a Tiangen extraction kit (DP432). Three biological replicates were performed in all cases.The pw mutant ; a total of 20 representative pods from 10 individual plants were selected for each biological experiment from both lines.Pod and seed traits were investigated by applying previously published standard procedures  that ended with 100% TBA, before being infiltrated with saturated paraffin-TBA mixtures for 24 h and embedded in paraffin for 48 h. Blocks were then polymerized completely at 4°C, and semi-thin sections between 5 and 8 μm in thickness were cut with a microtome blade KD-P , stained with safranin O/fast green, and visualized with a Nikon ECLIPSE TI-SR microscope . Image J software was explored to measure cell size of the parenchymal cell.pw and WT developmental stages via sequential extraction with three different solvents, comprising sodium phosphate  at 37°C, 70% ethanol at 80°C (repeated more than three times), and acetone at room temperature. Lignin content was then measured by the acetyl bromide method; in short, between 2 and 5 mg of cell wall extract was dissolved in a 2.5 mL acetyl bromide:acetic acid  solution at room temperature for 24 h. This mixture was then transferred to a 10 mL volumetric flask, and 0.35 mL of 0.5 M hydroxylamine hydrochloride was added and the flask was filled with acetic acid to volume. Absorbance was then measured at 280 nm . A 3-μg sample of total RNA from each pod sample was then used to enrich messenger RNA and to construct complementary DNA libraries. High-quality reads  were acquired by cutting adaptor sequences and removing low-quality reads with ambiguous nucleotides; all sequence data was uploaded into the BioProject database hosted by the National Center for Biotechnology Information (NCBI) under the BioSample accessions SAMN07437075 and SAMN07437076 (BioProject ID: PRJNA396814). Transcripts were then assembled using Trinity software  was detected for pod samples at DAF 20, DAF 40, and DAF 60, and three repeats of each reaction for individual genes were performed. The relative expression of each gene among different samples was calculated by using the 2−ΔΔCt method and normalized by using the internal reference actin gene. Thermal cycle parameters were 95°C for 30 s, followed by 40 cycles of 95°C for 10 s, and between 50 and 56°C for 25 s in a 20 μl volume.Reverse transcriptions were performed using an Invitrogen SuperScript Reagent Kit. We performed qRT-PCR using a Bio-Rad IQ5 qRT-PCR detection system  and the SYBR® Premix ExTaq® (TAKARA). Gene expression of both lines  in each experiment denoted in corresponding figure legends. Values were considered statistically significant at the P < 0.05 level and very significant at the P < 0.01 level; gene expression was scaled using the FPKM Z-score based on a mean value of three biological replicates in a heatmap.All statistical analyses reported in this paper comprised Student's pw and WT, we compared dry weight, length, pod and seed widths, and hull weight. Results show that both dry weight and width of both pods and seeds were significantly lower (P < 0.05) than those of WT after harvest  at DAF 50, an increase that continued until DAF 60 and DAF 70 , lignin staining was similar in the pw and WT lines . Thus, 17 genes with FPKM ≥ 2 were selected for confirmation  and c37339_g1 (ACS) were selected, both of which were significantly up-regulated in the pw mutant (Table c26901_g1 gene declined from 64.65 (DAF 20) to 36.88 (DAF 40) and fell further to 7.32 (DAF 60), while in the pw, the FPKM value this gene decreased from 104.13 (DAF 20) to 68.07 (DAF 40) and then fell further to 43.04 (DAF 60). At the same time, the expression level of the c37339_g1 gene in the WT decreased from 2.84 (DAF 20) to zero (DAF 40 and DAF 60), while the expression level this gene in the pw line decreased from 7.86 (DAF 20) to 2.43 (DAF 40) and then fell further to 0.85 (DAF 60). Specifically, the gene C26901_g1 encodes a CAD which is thought to be the key enzyme in lignin monomer synthesis; CAD genes in maize, Medicago truncatula, soybean, poplar, Arabidopsis, wheat, and pine have been shown to function in plant lignification and cell wall organization  and 5′-deoxy-5′-methylthioadenosine (MTA). This is important because ethylene is known to be involved in the lignification of plant secondary cell walls  were selected as candidates for controlling the pw mutation phenotype. The results of this study elucidated the peanut pod developmental process and will be useful in the future as a resource for the supply of genes and as a theoretical basis for developing improved peanut varieties with high yields.Our current understanding of the role of the hull in peanuts is that this structure determines the size and shape of seed . Subsequent quantification of cell wall components and histological staining experiments revealed that the lignin content varied at these two stages between the two lines; we also found that the seed enlarged along with the hull in the pw mutant, whereas the hull developed before the seed in the WT. These results clearly show that hull lignification and seed development conform to distinct patterns, phenomena that have also been observed in different peanut varieties with large and small pods (unpublished data). These hull lignification differences might be due to differences in lignin metabolic pathway expression; a faster seed developmental process in the pw line might be due, for example, to the accumulation of carbohydrates and proteins. We determined that the key modulator of both lignin and carbohydrate metabolism is related to higher regulatory networks, including TFs and hormone signaling pathways.Peanut hull size is one factor that influences seed size and shape. We therefore hypothesized that the narrower pod phenotype might result from the changes induced in the cell wall or to hormone signaling pathways. Initial observations of the p-hydroxycinnamyl alcohol monomers on plant secondary cell walls  in the pw mutant, and that PAL, CCR, and to some extent 4CL, CAD, HCT, and CCoAOMT, exhibited higher levels of expression at DAF 60 in the WT as compared to the pw line  have also been shown to be important genetic regulators of lignification in Arabidopsis fiber cell walls  that had extremely low levels of expression at DAF 20 and DAF 40 and higher levels of expression at a late stage (DAF 60) in the pw mutant. We identified one 1-aminocyclopropane-1-carboxylate synthase (ACS) gene involved in ethylene synthesis that had differences in expression during all three developmental stages.Plant hormones and TFs play essential roles in pod and seed development. The RNA-Seq data presented in this study shows that hormone signaling pathway genes in the 0 Figure . Express0 Figure , but the0 Figure . Based opw and WT and can therefore be selected as important candidates controlling the development of peanut pods. We hypothesize that the ethylene and auxin signaling pathways interact cooperatively with one another, thereby modulating the expression of genes of lignin synthesis and those involved in C metabolism to influence the lignification process and the accumulation of carbohydrates and proteins during the early developmental stages. At the same time, enhanced C/N metabolism and lignification repress the auxin signaling pathway in the pw mutant (Figure pw mutant.The results presented here show that the auxin, ethylene, C/N metabolism, and lignin synthesis pathways all vary between t Figure . FurtherConceived and designed the experiments: LW, YL, LY, HJ, and BoL. Performed the experiments: LW and BeL. Analyzed the data: LW. Contributed reagents/materials/analysis tools: WG, XD, YL, LY, HJ, XR, YC, and AC. Wrote the paper: LW and BoL. All authors have read and approved the manuscript.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.