Data was obtained over a 5-year study period (2009–2013) from two freshwater marsh sites located in Everglades National Park that differ in hydrology. At the short-hydroperiod site  and the long-hydroperiod site  fluctuations in precipitation patterns occurred with changes in ENSO phase, suggesting that extreme ENSO phases alter Everglades hydrology which is known to have a substantial influence on ecosystem carbon dynamics. Variations in both ENSO phase and annual net CO2 exchange rates co-occurred with changes in wet and dry season length and intensity. Combined with site-specific seasonality in CO2 exchanges rates, El Niño and La Niña phases magnified season intensity and CO2 exchange rates at both sites. At TS, net CO2 uptake rates were higher in the dry season, whereas SRS had greater rates of carbon sequestration during the wet season. As La Niña phases were concurrent with drought years and extended dry seasons, TS became a greater sink for CO2 on an annual basis (−11 to −110 g CO2 m−2 yr−1) compared to El Niño and neutral years (−5 to −43.5 g CO2 m−2 yr−1). SRS was a small source for CO2 annually (1.81 to 80 g CO2 m−2 yr−1) except in one exceptionally wet year that was associated with an El Niño phase (−16 g CO2 m−2 yr−1). Considering that future climate predictions suggest a higher frequency and intensity in El Niño and La Niña phases, these results indicate that changes in extreme ENSO phases will significantly alter CO2 dynamics in the Florida Everglades.This research examines the relationships between El Niño Southern Oscillation (ENSO), water level, precipitation patterns and carbon dioxide (CO Teleconnections from the El Niño Southern Oscillation (ENSO) are known to strongly affect climate patterns across North America 2) exchange rates have been linked to changes in surface hydrology in short-term studies 2 exchange rates respond to changes in surface hydrology 2 exchange. As a result, inter- and intra-annual fluctuations in CO2 exchange rates in the Everglades region may be significantly influenced by El Niño and La Niña phases.In the Florida Everglades, changes in the long-term hydrologic cycle have been linked to extreme ENSO phases (El Niño and La Niña phases) 2 and other greenhouse gases are expected to alter the frequency of El Niño and La Niña phases Increased atmospheric concentrations of CO2 dynamics in the Everglades. Because El Niño and La Niña phases are expected to alter the frequency and intensity of precipitation and temperature regimes, it is unknown how, when, and with what magnitude ecosystem CO2 exchange rates will respond to these fluctuations. However, this information is key to develop a prognostic understanding of how these ecosystems will behave in the future.Surface hydrology is managed differently among watersheds within Everglades National Park, which provides a unique opportunity to examine ecosystem function with differing hydroperiods, while still experiencing similar climate. Schedlbauer et al. 2 exchange rates .The goal of this research is to understand the relationship between extreme ENSO phases and intra- and inter-annual fluctuations in CO We hypothesize that El Niño and La Niña will amplify the site-specific seasonal responses in CO2 fluxes. At the short-hydroperiod site  it has been shown that enhanced net carbon uptake rates are associated with the dry season, while at the long-hydroperiod site  greater net carbon uptake rates are associated with wet season conditions 2 dynamics in the respective processes of uptake and efflux. For example, longer and wetter wet seasons will increase the capacity to uptake carbon at long hydroperiod sites, whereas longer and hotter dry seasons will increase the capacity for carbon uptake at short hydroperiod sites. In this study we used the eddy covariance method to estimate whole ecosystem exchanges in CO2, and a combination of linear, non-linear and time series modeling techniques to statistically address these hypotheses.The Florida Everglades are classified as subtropical wetlands with a year-long growing season and distinct wet and dry seasons that define annual variation http://fcelter.fiu.edu/research/sites/; The study sites are two oligotrophic freshwater marsh ecosystems that are within the Florida Coastal Everglades (FCE) long-term ecological research (LTER) program in Everglades National Park  and Muhlenbergia capillaris (Lam.) Cladium jamaicense, Eleocharis sp. and Panicum sp.) dominate ridge areas. Sloughs are dominated by short-statured (0.70 m), submerged species . Periphyton exists at both sites on submerged structures and as floating mats at SRS  and water vapor molar density , and a paired sonic anemometer  was employed to measure sonic temperature  and 3-dimensional wind speed . These paired sensors were 0.09 m apart and installed at 3.30 and 3.24 m above ground level (a.g.l.) at TS and SRS, respectively. Data were logged at 10 Hz on a datalogger  and stored on 2 GB CompactFlash cards. Both IRGAs were calibrated monthly using a trace gas standard for CO2 in air (+1.0%), dry N2 gas and a portable dewpoint generator . Footprint analyses air; °C) and relative humidity   mounted within an aspirated shield , and barometric pressure  . The Tair/Rh sensors were installed at the same height a.g.l. as the IRGA and CSAT.At each site, open-path infrared gas analyzers  were used to measure CO−2 s−1) , incident solar radiation  , and net radiation  . Precipitation measurements were made with tipping bucket rain gages (mm) . Soil volumetric water content  was calculated from equations developed for peat and marl soils using the methodology of Veldkamp & O'Brien s; °C) was measured at 5 cm, 10 cm, and 20 cm depths at two locations within each site using insulated thermocouples . When inundated at SRS, water temperature,  was measured using two pairs of insulated thermocouples , each pair located at a fixed height 5 cm above the soil surface and another attached to shielded floats that held the thermocouples 5 cm below the water surface. At TS, Tw was measured using insulated thermocouples  located at a fixed height 2 cm below the water surface. Water level (m) at both sites was recorded every half-hour with a water level logger .At each site, additional meteorological data was measured at 15-sec, and collected as 30-min averages through a multiplexer (AM16/32A Campbell Scientific Inc.) with another datalogger (CR10X Campbell Scientific Inc.). This included photosynthetically active radiation  w) was first estimated mean to streamline using a 2-d rotation in a Cartesian coordinate framework −2 s−1) was then estimated using the covariance of the turbulent fluctuations of the vertical rate of change of mean molar density of CO2 (c′), and the vertical scalar flux divergence (w′), where the turbulent fluctuations are the instantaneous deviation (at 10 Hz) from the mean block average (term I) over 30 min, and the storage flux (term II):−1 µmol C mol−1) of the molar density of CO2 measured at a fixed plane above the plant canopy (Z), z is the vertical dimension, and the overbar is the averaging period, in this case is 30-min. NEE was then divided by the molar volume of air, V, (m3 mol−1) to convert the units from density to molar fraction, i.e., µmol CO2 m−2 s−1, such that:R is the ideal gas constant (0.082 L atm K−1 mol−1), P is atmospheric pressure (1.10325 atm), and kT is the actual air temperature, estimated by:q is the molar fraction of water vapor calculated by unit conversion of vρ. Micrometeorological convention is used here, where negative NEE values indicate ecosystem uptake of CO2.Net ecosystem exchange (NEE) of COH; W m−2) was determined from the covariance of the turbulent fluctuations of w and sT, and w and q  and pC is the specific heat of air at constant pressure (J kg−1 °C−1). Corrections for the effect of water vapor on the speed of sound were applied Sensible heat  was calculated from the covariance of the turbulent fluctuations of w and vρ and averaged over 30-min,λ is the heat of vaporization (J g−1), and airM and wM are the molecular weights of air (28.965 g mol−1) and water (18.01 g mol−1), respectively. Corrections for thermal and pressure related expansion and/or contraction, and water dilution were applied Similarly, latent energy , LE (<−100 or >800 Wm−2), and NEE  values, stationarity criteria, and integral turbulent statistics n on a monthly basis. When R2 values were less than 70%, annual relationships between Rn and H or LE were used to gap fill data in that month.10 Hz raw flux data were processed with EdiRe , and when PAR was <10 W m-2, NEE data was gap-filled using an Arrhenius approach :α is the apparent quantum efficiency , and maxP is the maximum ecosystem CO2 uptake rate (µmol CO2 m−2 s−1).0R is the base respiration rate when air temperature is 0°C and b is an empirical coefficient. In ecoR is an estimated model parameter, whereas ecoR measurements are the dependent variable in n (with replacement) from the original dataset of size n for each estimated gap-filling model (Eq.6 and Eq.7) on a monthly or annual basis where appropriate were used NEE and ecoR data (Eq. 8).http://ameriflux.ornl.gov).Missing half hourly NEE data were gap-filled using separate functions for day and night. When PAR was ≥10 W mt denotes the tth daily value in the time series. Similar to methods used by Nuttle H and LE .As an indication of season intensity, the seasonal mean Palmer Drought Severity Index was used PDSI;  [41]. PDhttp://www.esrl.noaa.gov/psd/data/climateindices). The ONI is the running 3-month mean sea surface temperature (SST) anomaly from a 30-year mean SST for the Niño 3.4 region . Cold (warm) phases are defined as 5 consecutive months at or below (above) the -0.5° (+0.5°) anomaly.Long-term weather data were obtained from the nearest weather station, NCDC Royal Palm Ranger Station (25°23′N/80°36′W), where NOAA surface meteorological data was available from 1964 to 2013. The Oceanic Niño Index (ONI) was used to define extreme ENSO phases and was retrieved from the National Oceanic and Atmosphere Administrations Earth Physical System Research Laboratory  of order q, and if necessary, differencing of degree dtY) using its one period previous value (tY-1) and its associated error (εt-1):α1 is the first-order AR coefficient, θ is the first-order MA coefficient, εt is the current period error, and εt-1 is its one period previous error. “Memory” can be added to the model by adding lags and associated MA and/or AR components, and potential covariates can also be added as predictor variables, which may also have lagged components.An intervention time series approach was used To facilitate the inclusion of independent categorical variables for ENSO, El Niño and La Niña phases were coded as indicator variables, where a value 0 or 1 specified the absence or presence, respectively, of a categorical effect. We then determined if there were teleconnection lags between ENSO phase and precipitation and temperature.In developing time series models, first, all data series were tested for stationarity via the augmented Dickey-Fuller unit root test Because of the presence of autocorrelation in the explanatory series, input series were pre-whitened eco to light and temperature, respectively, non-linear To examine changes in the response of NEE and R2 dynamics  and a set of explanatory variables over a 5-year time series of daily data (2009 to 2013). These variables included: water level, season, ENSO phase , daily precipitation, drought condition, and average air temperature. The combined effect of ENSO phase and season (e.g. El Niño × wet season and El Niño × dry season) on CO2 fluxes were included in time series models as predictors with indicator variables. Indicator variables were also developed to identify sections of each season that directly followed an ENSO phase (post-La Niña and post-El Niño). Beckage et al. 2 exchange rates, indicator variables were used to identify the day of a precipitation event (Rain Day), the day after precipitation, and the quantity of precipitation . The indicator for the day after a precipitation event identified the first rain free day following a day with precipitation. Finally, drought conditions were defined as those days where PDSI<-2 and verified the drought extent with the National Oceanic and Atmospheric Administration's Drought Monitor (http://droughtmonitor.unl.edu/DataArchive.aspx).An intervention time series approach was used to identify and model the relationship between CO Monitor . Drought2 exchange rates in the Everglades freshwater ecosystems In addition, previous studies identified water level as one of the most important drivers for COecoNEE, GEE, R, and the Water Index) using pre-whitened explanatory series as predictor variables. Cross-correlation coefficient plots identified relationships at various lags, and autocorrelation plots were used to verify that the residuals had characteristics of random error. Model selection was based on minimum AIC, removing the least significant parameters. For each dependent variable, a single model form was selected with common predictor variables to aid site comparisons. Non-significant parameters remained in the model only if the parameter was significant at one site and it did not affect the final model of the other site. Multicollinearity between explanatory variables was also explored to ensure models did not contain input series that were highly correlated.As in the models of monthly weather data, all daily time series were tested for stationarity and non-stationary series were made stationary by differencing The long term ONI data ranged from 2.5 to −2.05 and indicated that the neutral phase occurred over about half (46%) of the period 1964–2013, while the El Niño and La Niña phases occurred 26 and 27% of this long-term study period, respectively. Neutral phases ranged in length from ∼1 month to>12 months and just 6 years contained no neutral phase . There were 14 different El Niño and 14 La Niña events, and the average ONI index was ∼1.1, −1, and −0.02 for all El Niño, La Niña and neutral phases, respectively. Although it is common for an El Niño event to be separated from a La Niña event by a short neutral phase (14 occurrences), a neutral phase occurring between consecutive El Niño (1 occurrence) or La Niña phases (4 occurrences) were less frequent. The majority (∼52%) of wet season months (May to October) were associated with a neutral phase while just ∼25% and ∼23% of wet season months occurred during El Niño and La Niña phases, respectively. Dry season months (September to April) were associated with the neutral phase ∼41% of the time, while El Niño and La Niña phases occurred 27 and 32% of time. Time series analysis of long-term monthly precipitation and minimum and maximum daily temperatures versus ENSO phase showed that rain increased the month after the start of El Niño phases  and declined the month following the start of La Niña (p = 0.7719), though not significantly ; S1 Fig.Throughout the study (2009–2013), 3 short La Niña phases , and an El Niño phase (2009–2010) occurred with shifts in precipitation patterns that resulted in both wetter than average (2010) and drier than average years , which corresponded to changes in season intensity. The ratio of GEE to Reco suggests that CO2 exchange rates at both sites were most similar in 2010  occurred during the La Niña phase at TS followed by the neutral phase. At SRS the greatest CO2 uptake rates were during the El Niño and neutral phase compared to seasons associated with a La Niña phase  and the dry season of 2013   .eco was often higher than wet season Reco, increasing CO2 release at SRS when the dry season was extended , GEE surpassed Reco and the site became a larger sink for CO2  was greatest during La Niña and directly following La Niña phases compared to rates during neutral and El Niño phases, although at higher PAR values (>1500 µmol m−2 s−1) curves for La Niña and the neutral phase converged (eco) were consistently higher at TS, for all ENSO phases as compared to SRS (−2 s−1).In addition to site differences, ENSO phase and season altered photosynthetic capacity and ecosystem respiration ; Fig. 6.onverged . Seasonad to SRS . At bothd to SRS ;. Overaleco differed between ENSO phases, and Reco showed distinct seasonal patterns in temperature sensitivity  but statistically significant autocorrelation remained in pre-whitened series; however, this sensitivity resulted from the large number of observations available and was judged to be biologically insignificant eratures , lagged −1; p<0.0001) had significant positive relationships with NEE, showing that as the change in water level and the quantity of daily rainfall increased, net CO2 uptake decreased  compared to neutral and El Niño phases at TS (p = 0.0039); however, post-La Niña phases in the dry season at SRS were associated with greater NEE . Moreover, there was a significant increase in NEE at TS (higher net CO2 uptake) the day after rain. At TS where hydroperiods were shorter, the effect of rain and post-La Niña phase during the dry season were significantly stronger than at SRS  were significantly linked to increased Reco ] and AR [AR(1)] component at both sites p<0.0001;. The dayModels for the water index had a significant 1-day lagged MA [MA(1)] and AR [AR(1)] component at both sites  in Everglades freshwater marsh ecosystems.The goal of this research was to understand the relationship between ENSO phases and CO The relationships between ENSO extremes, precipitation and hydrology in the Everglades region suggest that El Niño and La Niña phases could be important for C dynamics 2 dynamics, demonstrating that in addition to climate change and water management, ENSO is an additional source of variation in C cycling in Everglades freshwater ecosystems.Interannual variability in precipitation can be large in the Everglades region Abnormal precipitation and water level patterns over the study period coincided with El Niño and La Niña phases. Changes in ENSO phase have been associated with shifts in the position of the midlatitude jet, which is important for patterns in frontal precipitation. In the Everglades where frontal precipitation is the main source of dry season rainfall 2 exchange rates.Results found by Beckage et al. 2 exchange rates. Hydroperiods alter ecosystem production by interfering with exposed leaf area 2 fixation within the water column 2 exchange rates during and directly following El Niño and La Niña phases. The lag in El Niño and La Niña phase effect on CO2 exchange rates is the result of the effect of extreme ENSO phases on water levels during transition periods. El Niño (La Niña) phases increase (decrease) surface water levels during seasonal transitions and, major drainages often contain no water during transitions in a La Niña phase 2 exchange rates between ENSO and post-ENSO phases at TS and SRS.Hydroperiods have shaped soil conditions and species composition at each site in ways that have led to different seasonal patterns in CO2 exchange rates were sensitive to El Niño and La Niña phase in both the wet and dry seasons, while at SRS the effect of El Niño and La Niña phase on the wet season was not as strong as the effect in the dry season. La Niña phases resulted in higher photosynthetic capacity and greater seasonal net carbon uptake rates in both seasons at TS compared to neutral and El Niño phases. While La Niña increased photosynthetic capacity at SRS, the increase in Reco reduced net exchange rates compared to neutral and El Niño phases. As a result of changes in GEE relative to Reco, El Niño led to greater net CO2 uptake at SRS during the dry season compared to La Niña and neutral phases. As water levels and precipitation increased, NEE and GEE also increased at both sites (less CO2 and net CO2 uptake). At TS where hydroperiods were shorter, the effect of rain and the post-La Niña phase during the dry season were significantly stronger than at SRS  or dryer (-) than normal, which magnifies CO2 exchange rates at TS and SRS.The sites differed in their response to ENSO phases. At TS, COn at SRS . Rain waeco, and GEE. At TS where mean GEE surpassed Reco during the dry season, an increase in dry season length and intensity amplified the site's net CO2 uptake rates. Annually, SRS was usually a small source of CO2, although when wet season conditions intensified during El Niño, net CO2 uptake increased. The effect of ENSO phase also differed by site, showing that longer hydroperiods mute the effect of climate fluctuations on CO2 exchange rates, and as water levels decline the system becomes more vulnerable to climate. The ecosystem's sensitivity to climate fluctuations has important implications for water management and climate change 2 exchange rates.The results presented here show that the length and intensity of the wet and dry season varied annually with climate patterns. Considering the site-specific response to season, these results support our hypothesis that variations in season length would explain interannual fluctuations in NEE , Reco, a2 exchange rates. Shifts in water management and land use change  could also significantly alter both hydrology and CO2 dynamics in the Everglades region, making it important to develop a baseline understanding of how hydroperiod drives changes in CO2 dynamics and how climate alters hydrology. With water managers striving to adjust hydroperiods closer to natural values, in the future we might expect water levels in TS to increase, offsetting changes in climate by maintaining current patterns in hydrology. Alternatively, we might anticipate higher water levels to increase hydroperiods, making the system less sensitive to climate change altogether. With longer hydroperiods, SRS will likely remain less sensitive to changes in climate and land management. As a result, the two sites will likely behave more similarly in the future as SRS remains neutral or a very small source of CO2 to the atmosphere and TS becomes more neutral.In sub-tropical ecosystems, phenology is less sensitive to temperature and photoperiod, and more tuned to seasonal shifts in precipitation 2 dynamics Patterns observed in ENSO phases and the co-occurrence of extreme wet and dry seasons suggest changes in climate patterns can significantly alter ecosystem function. Equatorial Pacific SST during the past half century show a clear warming trend that is consistent with global warming 2 exchange rates showing that extreme ENSO phases magnify the site-specific seasonal response in freshwater marsh ecosystems. Although a longer time series is required to verify that this relationship is persistent, this research provides initial insights into an important driver of seasonal and inter-annual variation in CO2 exchange rates in Everglades ecosystems.This research shows that the length and intensity of the wet and dry season vary annually with ENSO phase in the subtropical Everglades region where dry season precipitation is dependent on frontal systems. Changes in season length and intensity are also correlated with COS1 FigARIMA model versus observed data of (a) precipitation, (b) maximum temperature and minimum temperature. Long-term weather data were obtained from NCDC Royal Palm Ranger Station (25°23′N/80°36′W), where NOAA surface meteorological data was available from 1964 to 2013.(TIF)Click here for additional data file.S2 Figeco at TS and SRS.The ratio of GEE to R Seasonal patterns in the ratio of GEE to Reco at (a) TS and (b) SRS shows that there is no clear pattern in wet season CO2 uptake rates and the (c) annual ratio of GEE to Reco were most similar during El Niño and neutral phases at TS and SRS.(TIF)Click here for additional data file.S3 FigSeasonal NEE at TS and SRS. At TS patterns in (a) wet and (b) dry season NEE shows that net CO2 uptake was greatest in years associated with La Niña phases (2011 and 2012). At SRS (a) wet and (b) dry season NEE suggests that the greatest net CO2 uptake occurred in years associated with El Niño (2010) and neutral phases (2013).(TIF)Click here for additional data file.S4 Figeco, (c) GEE, and (d) the water index for TS.ARIMA model versus observed data of (a) NEE, (b) R An intervention time series approach was used to identify and model the relationship between CO2 dynamics  and a set of explanatory variables over a 5-year time series of daily data (2009 to 2013).(TIF)Click here for additional data file.S5 Figeco, (c) GEE, and (d) the water index for SRS.ARIMA model versus observed data of (a) NEE, (b) R An intervention time series approach was used to identify and model the relationship between CO2 dynamics  and a set of explanatory variables over a 5-year time series of daily data (2009 to 2013).(TIF)Click here for additional data file.S6 FigThe relationship between season length and NEE at TS and SRS. NEE had a negative relationship with dry season length at (a) TS and a positive relationship with dry season length at (b) SRS. Annual NEE was positively correlated with wet season length at (c) TS and negatively correlated with wet season length at (d) SRS.(TIF)Click here for additional data file.


Background. Hepatocellular carcinoma (HCC) is a highly aggressive malignancy. Traditional Chinese Medicine (TCM), with the characteristics of syndrome differentiation, plays an important role in the comprehensive treatment of HCC. This study aims to develop a nonnegative matrix factorization- (NMF-) based feature selection approach (NMFBFS) to identify potential clinical symptoms for HCC patient stratification. Methods. The NMFBFS approach consisted of three major steps. Firstly, statistics-based preliminary feature screening was designed to detect and remove irrelevant symptoms. Secondly, NMF was employed to infer redundant symptoms. Based on NMF-derived basis matrix, we defined a novel similarity measurement of intersymptoms. Finally, we converted each group of redundant symptoms to a new single feature so that the dimension was further reduced. Results. Based on a clinical dataset consisting of 407 patient samples of HCC with 57 symptoms, NMFBFS approach detected 8 irrelevant symptoms and then identified 16 redundant symptoms within 6 groups. Finally, an optimal feature subset with 39 clinical features was generated after compressing the redundant symptoms by groups. The validation of classification performance shows that these 39 features obviously improve the prediction accuracy of HCC patients. Conclusions. Compared with other methods, NMFBFS has obvious advantages in identifying important clinical features of HCC. Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related death worldwide and the leading cause of death in patients with the complication of cirrhosis , 2. The As one of the most popular complementary and alternative medicine modalities, Traditional Chinese Medicine (TCM) plays an active role in treatment of malignant tumors including HCC in Chinese and some East Asian countries , 8. UnliIn this study, a nonnegative matrix factorization- (NMF- ) based fIn this work, the questionnaire survey dataset of HCC includes 407 samples within two years, and each patient was observed on 57 clinical symptoms . Each paRefinement of Feature Set. Our original dataset consists of 407 HCC patient samples  can be obtained. samples . The firSimplification of Clinical Staging. The clinical staging of HCC patients in our original dataset was marked with collections “IA,” “IB,” “IIA,” “IIB,” “IIIA,” and “IIIB.” For identifying the symptom signatures related to three clinical stages, all the samples would be relabeled as three classes. Here, we remarked class label “1” for the samples labeled “IA” and “IB.” In a similar way, class label “2” is used for “IIA” and “IIB” and “3” is for “IIIA” and “IIIB.” Finally, all the 407 clinical samples can be distributed in three categories: 82 samples in phase I, 195 in phase II, and 130 in phase III. The details of the refined dataset were described in Feature selection can be organized into three categories, depending on how they interact with the construction of model. Filter methods employ a criterion to evaluate each feature individually and are independent of the model . Among tNonnegative matrix factorization (NMF) aims to obtain a linear representation of multivariate data under nonnegativity constraints. These constraints lead to a part-based representation because only additive, not subtractive, combinations of the original data are allowed . In geneX be n × p nonnegative matrix; that is, each element xij ≥ 0 in X. Nonnegative matrix factorization (NMF) consists in finding an approximation basis matrix W and the mixture coefficient matrix H are n × r and r × p nonnegative matrices, respectively, where r > 0 and r ≪ min. The objective behind the small value of r is to summarize and split the information contained in X into r factors . The matrix H has the same number of samples but much smaller number of features rather than matrix X. Therefore, the metafeature expression patterns in H usually provide a robust clustering of samples j. is jth row of H. ‖[H]j.‖22 and ‖[H]j.‖12 define the l2-norm and l1-norm of [H]j.. The algorithm proposed by Lee is a well-established method to solve the optimization of NMF T, we determined that jth clinical sample is placed into kth cluster if max(Hj) = Hj(k), where k ∈ {1,2, 3}. Hence, we used matrix H to group all the samples into 3 clusters, which correspond to 3 bases (metafeature). DR.The matrix matrix X . TherefoW, each column also corresponds to a metafeature or basis  j [i relates to certain basis j if wij is the largest entry in row i of matrix W. From  clinical phase-specific important symptoms via NMF. Moreover, the partition of 49 clinical symptoms shown in  nausea is observed as a common adverse effect in HCC patients in phase I [ ascites, anorexia, fever, and jaundice often occurred in phase II [yellow complexion” and “yellow skin and eye” shown in  jaundice. For phase III, pain is the most obvious characteristic in HCC patients [pain in shoulder and back,” “chest pain,” and “distending pain in hypochondrium.” Moreover, fatigue and weakness were also common in HCC patients [In matrix asis see . Entry wbasis) j . Therefo phase I . The symphase II , 45–48. patients . Togetheθ = 0.95  were identified as basis 2 related features, and they are most possibly belonging to phase I  are potentially related to basis 3, the frequency of positive for each is over 50%, and the mean value of positive for these three symptoms is 1.77, which further indicate that they might be related to some patients whose conditions are very serious. Although the symptoms V46, V42, and V25 were not identified as redundant symptoms with given threshold (0.95), their compressed patterns in matrix W in W facilitates evaluating the difference among symptoms, and matrix H can validate the high degree of correlation between class labels of samples and basis indexes. After inferring the redundant symptoms with given threshold, we combined each symptoms' group together and converted it into a new feature (named mixed feature). Finally, we obtained 39 clinical features (FS1) of HCC as the optimal feature subset, which consisted of two parts: 33 original symptom features (FS2) and 6 new mixed features (FS3)  . Based o1, FS2, and OFS on the training set (120 representative samples). FS1 and FS2 were generated by feature selection with the threshold θ (0.95). OFS denoted 49 original symptom features in the dataset DR. 2 was much better than OFS; however, it was still worse than FS1 because the new mixed features also have important contributions to classification.For evaluating the potential of NMFBFS-inferred optimal feature subset, we firstly tested the classification accuracy of three candidate feature subsets FSR packages were applied for mRMR and Elastic Net based feature selection, respectively. Supplementary Figure S1 represents the ReliefF-based feature ranking. Supplementary Figure S2 represents the Elastic Net (λ = 0.5) solution paths for feature selection. We selected Top 20 features and Top 40 features as two candidate feature subsets for each method to evaluate their classification performances: FSRF20 and FSRF40 generated from ReliefF; FSMR20 and FSMR40 inferred from mRMR; FSEN20 and FSEN40 inferred from Elastic Net. 1 on the training set (120 representative samples). The results indicate that NMFBFS-inferred feature subset has the best classification accuracy in training samples.We then compared the performance of our NMFBFS with three well-known feature selection methods  from the rest of the samples and then evaluated the classification accuracy of inferred feature subset by each method . θ determines how many groups of redundant symptoms will be screened out. Here, we further discussed the effects of threshold θ to the optimal feature subsets on the classification performance. θ. From θ will screen redundant symptoms strictly, which leads to less similar symptoms that would be obtained. With a smaller value of θ, much more symptoms can be categorized into the same groups; hence, the original feature space will be sharply reduced by our approach. θ, the size of optimal feature subset was narrowed down but the classification accuracy was also decreased. These results suggest that a bigger value of θ will result in less redundant symptoms and therefore induce a larger size of optimal feature subset; oppositely, smaller θ can provide more redundant symptoms and sharply reduce the feature dimension. An extreme case is that θ equals “0,” which means that we can get one mixed feature for each basis and the size of optimal feature subset is equal to the number of bases. In a word, how to determine the value of θ depends on the size of optimal feature subset and its corresponding classification performance.Finally, the more important thing is that the selection of threshold In this study, we developed the NMFBFS approach to efficiently extract the important clinical symptoms of HCC from clinical observation data. NMFBFS is a two-stage filter method for feature selection as follows. (1) In the first stage, preliminary screening is implemented to detect and remove the irrelevant features; (2) in the second stage, NMF was applied to identify the redundant features by groups which might represent similar distribution patterns. Each redundant symptom group was then transformed into a new mixed feature so that the dimension of dataset was further reduced.The application of NMFBFS on a clinical dataset of HCC proved the effectiveness of this approach. The optimal clinical features derived from NMFBFS approach contained many well-recognized symptoms of HCC patients. Moreover, this study also provides a general computational framework of a novel feature selection approach to efficiently extract the optimal feature subset from a high-dimensional dataset.Supplementary information includes two Figures and one Table.Fig S1: denotes the results of feature ranking with ReliefF.Fig S2: denotes the results of feature selection with Elastic Net.Table S1: represents the frequencies of each symptom feature appearing positive over the samples in all the clinical stages.


There is now ample evidence that blind individuals outperform sighted individuals in various tasks involving the non-visual senses. In line with these results, we recently showed that visual deprivation from birth leads to an increased sensitivity to pain. As many studies have shown that congenitally and late blind individuals show differences in their degree of compensatory plasticity, we here address the question whether late blind individuals also show hypersensitivity to nociceptive stimulation. We therefore compared pain thresholds and responses to supra-threshold nociceptive stimuli in congenitally blind, late blind and normally sighted volunteers. Participants also filled in questionnaires measuring attention and anxiety towards pain in everyday life. Results show that late blind participants have pain thresholds and ratings of supra-threshold heat nociceptive stimuli similar to the normally sighted, whereas congenitally blind participants are hypersensitive to nociceptive thermal stimuli. Furthermore, results of the pain questionnaires did not allow to discriminate late blind from normal sighted participants, whereas congenitally blind individuals had a different pattern of responses. Taken together, these results suggest that enhanced sensitivity to pain following visual deprivation is likely due to neuroplastic changes related to the early loss of vision. In a recent study we showed that congenitally blind individuals have reduced thresholds to heat and cold pain, and rate supra-threshold nociceptive stimuli as more painful compared to normally sighted individuals There is abundant evidence from animal experiments that visual deprivation from birth causes dramatic plastic changes in the structural and functional organization of the visual cortex. The extent of these neuroplastic changes depends more strongly on the onset than on the duration of visual deprivation To investigate whether late blind individuals also show hypersensitivity to nociceptive stimulation, we compared thermal pain thresholds and supra-threshold pain ratings of late blind (LB), congenitally blind (CB) and normally sighted (NS) volunteers. Participants also had to answer questionnaires regarding attention and anxiety towards painful encounters in daily life, since these factors are known to influence pain perception Participants were recruited from our database of congenitally and late blind subjects or by advertisement. Our total study population consisted of 23 CB , 12 LB  and 48 NS  volunteers of whom 18 NS and 18 CB were included in a previous study and their data reused We calculated a blindness duration index (BDI) according to the formula “(age-age onset blindness)/age”. The BDI score can vary from 0 to 1, expressing the relative amount of time a person has been blind, with low scores indicating recent onset of blindness and high scores long duration of blindness. All blind participants suffered from blindness due to peripheral origin . In the LB group, the average onset of blindness was 19.7±14.5 years and the average BDI was 0.6±0.3. Blindness due to diabetic neuropathy was an exclusion criterion. None of the participants suffered from known neurological or psychiatric disorders that might interfere with the experiment's results. Demographic details on the blind participants are provided in We used a 3×3 cm Peltier-based thermotest  to determine thresholds for innocuous and noxious thermal stimuli on the dominant medial forearm. In order to reduce anxiety and fear, participants were familiarized with the thermal stimulation equipment and underwent practice trials prior to data acquisition. All participants were blindfolded after the familiarization period. The baseline temperature of the thermode was set to 32°C and we used a ramp rate of 1°C/s for the warmth and cool thresholds and 3°C/s for heat pain and cold pain thresholds. Stimuli were cued 2 to 5 s prior to onset. Participants had to click on a response key as soon as they detected warmth or cool or felt heat pain or cold pain. Thresholds were measured five times for each type of sensation with an inter-stimulus interval of 10–15 s for innocuous stimuli and 15–20 s for noxious stimuli.We used a CO2 laser stimulator device with a spot diameter of 6 mm  to generate highly accurate and contactless heat stimuli. This device is equipped with a contactless measurement unit with online monitoring of target skin temperature that controls the laser power in a closed-loop. This instant feedback guarantees that the skin is brought and maintained with a high accuracy at the exact target temperature, allowing the stimulation of the thinly myelinated Aδ- and the unmyelinated C-fibers without co-activation of the large myelinated Aβ-fibers At the end of the session, participants filled in the Pain Vigilance and Awareness Questionnaire (PVAQ) adapted for a non-clinical population In order to estimate and account for the influence of demographic variables , we conducted a multiple linear regression analysis that generated the regression models separately for threshold assessments and supra-threshold pain ratings. In each condition we modelled age and gender as independent and thresholds/supra-thresholds as the dependent variables. We obtained new supra-threshold/threshold values for each subject from the residuals of the multiple linear regression modeling. Data are presented as means ± SD.We used Levene's test for assessing equality of variances of the data distributions for noxious and innocuous thermal threshold assessments (factor  =  “group” and dependent variable  =  “threshold”). Then, we conducted two-tailed Student t-tests in order to compare groups for noxious and innocuous thermal thresholds. For supra-threshold ratings, we conducted a 1-way ANOVA with the factor “group” as independent variable and “temperature” as dependent variable, checking for the equal of variances of the data distribution with a Levene test. Post-hoc comparisons were done using two-tailed Student t-tests, correcting for multiple comparisons .To investigate the effects of onset of blindness and blindness duration index on pain perception, we performed Pearson's correlations between these variables and pain thresholds and supra-threshold pain ratings in the LB group.We performed the analysis of the PVAQ and PASS data using a principal component analysis  on the questionnaires' raw scores to make a dimensionality reduction while preserving as much data variability as possible. Thereafter, we performed a Fisher Linear Discriminant Analysis (FLDA) on the resulting PCA factor scores to test whether LB, CB and NS responded differently. The variables were entered using the “all-variables together” method, while the goodness of classification analysis was tested using “leave-one-out” cross-validation and balanced for unequal sample sizes.1 = 2; df2 = 63, p = 0.062; cold pain: F = 2.587, df1 = 2; df2 = 63, p = 0.083; innocuous warmth: F = 2.165, df1 = 2; df2 = 63, p = 0.123; innocuous cool: F = 0.198, df1 = 2; df2 = 63, p = 0.821). Comparisons of pain thresholds . In line with the results of the pain thresholds, LB rated supra-threshold heat nociceptive stimuli  indicated that correlations between PVAQ and PASS sub-factors were sufficiently high for PCA. Two components had eigenvalues over Kaiser's criterion of 1 and in combination explained 72.5% of the variance. Thereafter, we performed a FLDA to classify the participants on the basis of their regression factor scores derived from the PCA analysis. As illustrated in 2 = 0.969, χ2 = 1.3, Dof = 2, p = 0.521). On the other hand, the FLDA allowed us to correctly discriminate CB from NS with an accuracy of 75.3 % . Since the above classification scores were mainly driven by factor II (attention to pain), we infer that CB are more attentive to signals of threat than NS. Inversely, the poor contribution of factor I (anxiety) suggests that CB are not more anxious than NS about pain encounters in daily life. Pearson correlation analysis within LB indicated that the onset of blindness had no effect on either factor I  or factor II . Likewise, BDI scores also did not correlate with either factor I  or factor II .The Kaiser–Meyer–Olkin (KMO) measure verified the sampling adequacy of the PCA analysis we conducted . Bartlett's test of sphericity (χThe purpose of the present study was to investigate if individuals with acquired blindness show thermal hypersensitivity to noxious thermal stimulation as previously reported in congenital blindness Our findings are in line with previous studies indicating that late blind individuals show no compensatory plasticity for auditory Our psychophysical data are further corroborated by the psychometric results that also failed to find differences in attitude and responses to signals of threat in daily life between LB and NS. Indeed, results of the pain questionnaires indicated that LB and NS pay similar attention to environmental threats and react with the same level of anxiety to such threats. In sharp contrast, CB scored higher than NS on attention to pain. This increased awareness of potentially dangerous stimuli could partly explain the increased pain responsiveness in CB since attention is known to exacerbate the experience of pain Previous studies have shown that increased attention to threatening stimuli can be driven by augmented levels of anxiety In conclusion, we show that blindness acquired at the age of six or later does not lead to pain hypersensitivity. Our data therefore suggest that hypersensitivity to noxious stimulation is the result of neuroplastic changes that occur early in development. Whether attention is the chief determinant of the exacerbated sensitivity to pain in congenitally blind individuals, or simply a potentiating factor, needs further investigation.


Nyctalus spp.) under an LED street light than in the dark. LED street lights reduce the anti-predator behaviour of moths, shifting the balance in favour of their predators, aerial hawking bats.The light-emitting diode (LED) street light market is expanding globally, and it is important to understand how LED lights affect wildlife populations. We compared evasive flight responses of moths to bat echolocation calls experimentally under LED-lit and -unlit conditions. Significantly, fewer moths performed ‘powerdive’ flight manoeuvres in response to bat calls (feeding buzz sequences from  The chawildlife ,6.In Britain, many moth species have suffered major population declines . Light p2.2.1pectraSuite . The integration time was set to 4 ms, and the curve represents the average of 20 scans. The end of the fibre with the cosine corrector was positioned 162 cm away, directly below the LEDs  fitted with a panel of 24 LEDs was top-mounted on a 4 m high tripod  using a custom-made aluminium adaptor to conform with the light's mounting specifications. The light was positioned along field-woodland boundaries, directed away from the woodland into the open grassland  and powered by a Honda Eu10i portable generator  positioned on average 81 m away (range=68–90 m). Irradiance measurements of the light were taken in a darkened room using a cosine corrector at the end of a 400 μm diameter UV-visible fibre-optic cable connected to a spectrometer  controlled by a PC running Sthe LEDs .2.2http://www.timeanddate.com/.Defensive behaviour of moths was tested across four randomized treatments, with one treatment per night . Illumin2.3Nyctalusnoctula were recorded at Blagdon Lake  between 6 May and 27 June 2013 using a Pettersson D1000X ultrasound bat detector . It is possible that some calls emitted by the much rarer Nyctalus leisleri were recorded, as the calls of the two species are often impossible to discriminate. Both species emit similar calls with dominant frequencies that are close to the best hearing frequencies of many moths, and both feed to some extent on Lepidoptera [ab Pro . Sequences were trimmed to include the final few search phase calls, as well as the entirety of the approach phase and terminal buzz . Sequence order was randomized and sequences were not repeated on the same night. The output intensity of the speaker was calibrated prior to field experiments . The mean maximum output of the speaker across sequences was 107 dB peSPL at 1 m (range=103–109 dB), equivalent to a source level of 127 dB peSPL at 10 cm, which corresponds closely with the source levels of bat echolocation calls –22 and i2.4Flight behaviour of moths was recorded using a HXR NX70 professional video camera  under infrared lighting . Moths could not be identified to species as they flew too high, fast or unpredictably for capture. However, all were relatively large and probably belonged to the families Geometridae, Noctuidae or Notodontidae. Moths in these families possess ultrasonic hearing  and compVideo recordings were made 4 m from the street light column at 90° to the edge of the woodland. All moths were tested at a height of 2–4 m, within 5 m of the street light column and 4 m (±1 m) horizontally from the speaker. Moths flying less than 3 m or more than 5 m from the speaker were excluded. Call intensities likely to have been heard by moths less than 3 m were more intense than those produced by wild bats and therefore may have unrealistically influenced moth evasive responses. Moths flying more than 5 m from the speaker may not have heard the bat calls. Each playback presentation was initiated once a moth was within 5 m of the speaker and therefore in an appropriate position to hear the recordings. Ultrasonic pulses  and comparable in frequency and intensity to those used in our study) emitted less than 5 m away caused moths to spiral or dive towards the ground, whereas pulses emitted 5–12 m away caused moths to alter the direction of their flight path . These a2.5Moth video recordings were analysed manually by eye ‘frame-by-frame’ and scored in a similar way to . Respons2.6p<0.05) fixed effect term following model simplification using backwards multiple regression .Owing to the difficulty in distinguishing sound-induced directional changes from light-induced flight erraticism, moth flight responses from category (ii) were pooled with category (i) and compared to category (iii) to test the hypothesis that moth powerdive behaviour decreases under LED lights. Pooled data were analysed using log-linear generalized linear mixed models (GLMM) with binomial errors in R , using the package lme4 . Site wa3.n=14; bat n=15; LED n=27; LED-bat n=38) from 16 nights at four different sites were included. For each model, data from 39 moths were omitted from analyses because they were either flying more than 5 m from the street light column; flying less than 3 m from the ultrasonic speaker; flying greater than 5 m from the ultrasonic speaker; or because echolocation calls from wild bats were heard clearly on the bat detector during treatments. A further 18 recordings were excluded as these moths' responses were placed in category (iv) (behaviour unclear). The number of powerdives was significantly lower under ‘LED-bat’ treatments than ‘bat’ treatments  but significantly higher than under ‘LED’ treatments . Only 24% of moths performed powerdives during ‘LED-bat’ treatments compared with 60% during ‘bat’ treatments  and was not significantly different between the two bat-free treatments .Data from 94 moths  as they lack UV, which causes ‘flight-to-light’ behaviour and high levels of mortality ,31.


However, probing such buried interfacial processes is experimentally challenging. Infrared spectroscopy is sensitive to molecule vibrational signatures, yet to approach the interface three stringent requirements have to be met: interface specificity, sub-monolayer molecular detection sensitivity, and electrochemically stable and infrared transparent electrodes. Here we show that transparent graphene gratings electrode provide an attractive platform for vibrational spectroscopy at the electrolyte/electrode interfaces: infrared diffraction from graphene gratings offers enhanced detection sensitivity and interface specificity. We demonstrate the vibrational spectroscopy of methylene group of adsorbed sub-monolayer cetrimonium bromide molecules and reveal a reversible field-induced electrochemical deposition of cetrimonium bromide on the electrode controlled by the bias voltage. Such vibrational spectroscopy with graphene gratings is promising for real time and  Understanding of electrolyte-electrode interfaces is limited due to the lack of suitable probing techniques. Here, the authors present a vibrational spectroscopy based on graphene gratings, which enables sensitive and interface-specific detection of molecular vibrations at electrolyte-electrode interfaces. In this paper, we report a new technique based on graphene gratings that overcomes these difficulties, which enables sensitive and interface-specific detection of molecular vibrations at the electrolyte/electrode interface. An infrared spectroscopy technique that overcomes all these difficulties will be a promising method for sensitive interface detection of molecules at the electrolyte/electrode interface.The electrolyte/electrode interface is of great interest for both fundamental science and applied technology: a rich variety of electrical field-dependent physical1246713410111314Graphene is an attractive electrode for studies of electrolyte/electrode interfaces. It is stable and transparent to infrared light, and is being actively explored for applications in supercapacitors, batteries202 vibration peaks of polymer residue on graphene surface within seconds and the new spectroscopy enhances the relative contrast by ∼50 times compared with conventional absorption spectroscopy. Using the surfactant cetrimonium bromide (CTAB) as a model system, we observe CTAB adsorption on graphene and the reversible deposition/dissolution of CTAB at the graphene electrode with new diffraction vibrational spectroscopy.In this paper, we report a new vibrational spectroscopy technique based on diffraction of monolayer graphene gratings. This method overcomes the difficulties of probing buried interface with infrared, and enables sensitive and interface-specific detection of molecular vibrations spectra at the electrolyte/electrode interface. The advantages are demonstrated by detecting CH−1 and a repetition rate of 150 KHz. The infrared radiation is incident on a graphene grating electrode, which forms an electrochemical cell together with a platinum counter electrode and the aqueous electrolyte , where Ein and Emol are the electrical field of incident light and molecular radiation, respectively, and |Emol|2 is omitted because |Emol|≪|Ein|. The relative intensity change due to light absorption is therefore determined by ΔIt/It≈2·Re(Ein*·Emol)/|Ein|2. For a monolayer of material, |Emol| from molecular vibration is typically <10−3 of Ein  and the periodic modulation of molecular species within the electrical double layer (Emol) (Id/Id≈2·Re(Eg*·Emol)/|Eg|2. Because the diffracted electrical field from the graphene grating (Eg) is about 1% of the incident electrical field Ein  . TherefoEin refs , 25, the2 stretching modes  at the electrolyte/electrode interfaces in an electrochemical cell. The spectra of the diffracted infrared light from the graphene grating are taken with a two-dimensional InSb infrared camera.We demonstrate the capability of this vibrational spectroscopy based on graphene gratings by investigating CHEg from the graphene grating electrode using 12 mM NaCl solution as the electrolyte. −1 as a function of the bias voltage from 1.1 to −0.4 V (black squares), which is normalized by the intensity value at the charge neutral point (CNP) of graphene. The diffraction intensity at 3,000 cm−1 contains contribution mainly from the graphene grating because this frequency is away from any molecular vibration resonances in this system. The bias voltage of the electrochemical cell acts as an electrical gate that controls carrier concentration in graphene. At Vbias=0 V graphene is strongly p-doped in the NaCl solution, and it becomes charge neutral at Vbias=1.0 V. We observe that the diffraction intensity shows a local maximum at the CNP, which decreases upon hole doping to ∼10% of the CNP signal at Vbias=0.5 V. Afterwards, the diffraction signal starts to increase with further hole doping. This non-monotonically change in infrared diffraction from the graphene grating originates from the gate-dependent complex optical conductivity in graphene2021Ig is proportional to (Re(σg))2+(Im(σg))2, where Re(σg) and Im(σg) are the real and imaginary parts of graphene conductivity σg, respectively. σg), Im(σg) and |σg|2 at 3,000 cm−1 as a function of the Fermi energy EF. Near the CNP, Re(σg) from interband transitions dominates the diffraction signal. Re(σg) decreases significantly when 2|EF| equals the incident photon energy due to Pauli blocking, and accounts for the reduced diffraction intensity from carrier doping initially. However, at high doping levels Im(σg) from intraband transitions starts to dominate, which ultimately leads to diffraction intensity increasing at high |EF|. The solid line in First, we examine the diffraction signal −1) from as-prepared graphene electrodes in the 12 mM NaCl solution at different bias voltages. Id(ω)/IdCNP(ω), where IdCNP(ω), the spectrum at CNP, is used as the reference. We observed two sharp resonances at 2,916 and 2,848 cm−1 on top of a broad background signal. The broad background arises from the graphene electrode, which first decreases and then increases with higher hole doping controlled by Vbias. The two resonances at 2,916 and 2,848 cm−1 correspond to the anti-symmetric and symmetric stretching vibrations of the methylene group (-CH2-), respectively. A weak resonance at 2,960 cm−1 corresponding to methyl group (-CH3) is also present. The appearance of CH2 vibration peaks indicates that there are hydrocarbons on top of as-prepared graphene in NaCl solution, presumably due to the residue from the polymer transfer and/or photolithography processes. The diffraction spectra in 2 vibration signals, and their understanding requires quantitative description of diffracted electrical field from both graphene and CH2 vibrations at different bias voltages. The normalized diffraction intensity is described byNext we show the infrared diffraction spectra 15N(CH3)3Br) as a test system. CTAB have been widely used to assist DNA extractionThe vibrational spectroscopy based on graphene grating, with its high sensitivity and interface specificity, enables us to −1 from the graphene grating in 11 mM CTAB solution. Compared with graphene in NaCl solution, the voltage dependence shows a similar behaviour except that the CNP is strongly shifted to Vbias=0.2 V. Apparently CTAB largely cancels the strong p-doping in as-prepared graphene, presumably due to the adsorption of cation CH3(CH2)15N(CH3)3+ ( abbreviated as CTA+) that induces a significant amount of electrons in graphene.We plot in 2 vibration frequencies from the CTAB solution/graphene electrode interface at different bias voltages are shown in Vbias=0.2 V is used as the reference. The spectra are reproducible in many cycles when the bias voltage is varied between −1 and 1.5 V, and there is a finite hysteresis when scanning direction switches. The bias-dependent spectra are quite similar to that in NaCl solution  and 2,944 cm−1 (blue arrows). The evolution of these spectra features as a function of electrical potential and their fittings are shown in When d −1.2 V , the evo−1 is associated with the head group of CH3-(N+) in crystalline CTAB31Vbias reaches −1.5 V, a new electrochemical process is triggered to deposit highly ordered CTAB on the graphene electrode. This deposition process can be monitored in real time with our diffraction spectroscopy. When the bias voltage is further increased to positive, the deposited CTAB systematically modify the diffraction spectra. At the CNP with Vbias=0.2 V, the normalized spectrum shows distinct features rather than a constant. When Vbias is further scanned beyond 1 V, however, the features associated with the deposited CTAB layer become weaker and then disappears, and the spectral shape becomes similar to those in Vbias=−1.5 V in the negative scan, coincidence with the start of CTAB deposition, and a peak around Vbias=1 V in the positive scan, coincidence with the disappearing of CTAB deposition. After the Vbias reaches 1.5 V, most of the deposited CTAB is dissolved, and the graphene electrode recovers to its initial status. When Vbias is scanned from 1.5 to −1.5 V afterwards, the diffraction spectra display the same behaviour as that in Previous studies of CTAB have shown that a new vibrational resonance around 2,943 cmVbias=−1.5 V, it can potentially oxide Br− into Br2 at the electrode. In this case, one may expect the corresponding CTA+ cation reduction and deposition in the platinum counter electrode. However, we observe the deposition of CTAB layers at the graphene electrode. Further theoretical investigation will be required to understand microscopically the unconventional deposition processes taking place at the interfaces.The origin of the observed electrochemical deposition and dissolution of CTAB layers is not clear yet. When the graphene electrode is acting as an anode at in situ monitoring of molecular vibrations at the electrochemical interface. In this proof-of-principle study we used CH2 vibrations as an example, but the technique can be readily applied to other functional groups at different infrared frequency ranges. The diffraction spectroscopy enhances the relative contrast by ∼50 times compared with conventional absorption spectroscopy. Currently we demonstrate a sub-monolayer detection sensitivity, which is mainly limited by our laser fluctuation. With improved laser stability and/or balanced detection, we envision that the technique can probe as low as about 1% of a monolayer coverage and provides a powerful tool to investigate fundamental processes at the electrolyte/electrode interfaces in active electrochemical cells.We demonstrated a vibration spectroscopy at the electrolyte/electrode interfaces using graphene grating electrodes. Its high detection sensitivity and interface specificity enables fast and In this experiment, graphene is grown on Cu foil by chemical vapour deposition method−1 is collimated and sent to graphene at normal incidence. The first-order diffraction light is around 25.9° to the normal. The diffraction light is sent to a liquid nitrogen-cooled InSb camera with128 × 128 pixel arrays. The wavelength is then calibrated by absorption of thin film CTAB with its Fourier transform infrared spectrum. The detailed spectroscopy design is shown in The infrared radiation is generated by a femtosecond laser source. Specifically, an amplified femtosecond laser system  delivers laser pulses at 1,026 nm with a pulse duration of 260 fs and a repetition rate of 150 KHz. The laser amplifier pumps a broadly tunable optical parametric amplifier  covering wavelengths from 600 to 2,200 nm. The mid-infrared wavelengths are generated by mixing the pump laser  and the optical parametric amplifier output through difference frequency generation. The output infrared beam at around 3,000 cmHow to cite this article: Bie, Y.Q. et al. Vibrational Spectroscopy at Electrolyte/Electrode Interfaces with Graphene Gratings. Nat. Commun. 6:7593 doi: 10.1038/ncomms8593 (2015).Supplementary Figures 1-6, Supplementary Table 1, Supplementary Notes 1-3 and Supplementary References


Stanene is one of most important of 2D materials due to its potential to demonstrate room temperature topological effects due to opening of spin-orbit gap. In this pursuit we report synthesis and investigation of optical properties of stanene up to few layers, a two-dimensional hexagonal structural analogue of graphene. Atomic scale morphological and elemental characterization using HRTEM equipped with SAED and EDAX detectors confirm the presence of hexagonal lattice of Sn atoms. The position of Raman peak along with the inter-planar ‘d’ spacing obtained from SAED for prepared samples are in good agreement with that obtained from first principles calculations and confirm that the sheets are not (111) α-Sn sheets. Further, the optical signature calculated using density functional theory at ~191 nm and ~233 nm for low buckled stanene are in qualitative agreement with the measured UV-Vis absorption spectrum. AFM measurements suggest interlayer spacing of ~0.33 nm in good agreement with that reported for epitaxial stanene sheets. No traces of oxygen were observed in the EDAX spectrum suggesting the absence of any oxidized phases. This is also confirmed by Raman measurements by comparing with oxidized stanene sheets. Two dimensional 2D) layered materials have recently gained renewed interest due to their exotic electronic properties along with high specific surface area. The prospects of exploiting these properties in sensing, catalysis, energy storage, protective coatings and electrochromism have witnessed a paradigm shift towards the exploration of these sophisticated 2D materials. The exemplary performance of grapheneD layered346Here we report the synthesis of few-layer stanene (FLS) using ultra-fast laser-material interactions. FLS is analogous to few-layer graphene and can be visualized by replacing carbon atoms by tin on a graphene lattice. Structural characterization performed using high resolution transmission electron microscopy (HRTEM) equipped with energy dispersive X-ray analysis (EDAX) and selected area electron diffraction (SAED) detectors confirm the presence of hexagonal lattice of Sn atoms. EDAX and comparative Raman studies that oxide phases are absent and rules out the possibility of (111) α-Sn sheets. Height profile measured using atomic force microscopy (AFM) suggests interlayer separation of ~3.3 Å and in good agreement with that of recently reported epitaxial stanene. Further the UV-Vis spectrum and Raman spectrum are in good agreement with the optical spectra and phonon frequencies calculated using first principles techniques. The structural characterization along with optical signature suggests the synthesis of free standing stanene sheets.Free standing stanene sheets were synthesized by impinging pulses from a tunable Ti:Saphire ultra-fast femto second laser (140 femto-second pulse width and 80 MHz repetition rate) on to a target in liquid medium. This interaction of femo-second laser pulse due to inverse Bremsstrahlung multiphoton absorption process1 ~ 0.25 nm and d2 ~ 0.16 nm. These ‘d’ spacing neither agree with that of standard β-tin2 samplesThe samples were characterized for structure, symmetry and chemical composition using HRTEM, equipped with SAED and EDAX detectors. HRTEM image of a representative sample in 2O2. Similar results are observed in the case of graphene* transitions due to presence of oxygen atoms.The samples were characterized optically using UV-Vis absorption and Raman spectroscopy. The as prepared stanene samples show strong absorption peak ~196 nm (6.325 eV) and two shoulders at ~225 and ~250 nm as seen in z orbital. This suggests that most of the low energy optical transitions occur due to the transitions between the π and π* states. The σ states lie farther down below the π states about 3 eV below the Fermi level. The optical properties of the material can be described in terms of complex dielectric function ε = ε1 + iε2 and the absorption coefficient is directly proportional to imaginary part of the complex dielectric function (ε2). ε2 is obtained using first principles DFT calculations within the generalized gradient approach. Stanene displays pronounced asymmetric peak in the UV region at photon energy of ~6.325 eV. The peak positions at ~191 nm and ~233 nm in the calculated ε2 shows a good similarity with the experimental data.The electronic states near the Dirac point are dominated by π states with major contribution coming from the out of plane p−1 (−1–110 cm−1 is understood to arise from out of plane vibrations of the Sn atoms as suggested by selective dynamics of atoms using first principles techniques. It is important to note that no negative phonon frequencies were found at gamma point suggesting the stability of the structure. These Raman frequencies do not correspond to any gamma phonon frequencies reported for (111) α-Sn thin flims, β-tin, SnO or SnO2−1. This shift is understood to occur due to change in oxidation state of the Sn atoms. The appearance of additional vibrational modes at ~288 cm−1 suggest that large does of high intensity laser beam oxidizes stanene layers forming domains of SnOx in stanene layers. The peaks ~288 cm−1 are in good agreement with the Eu mode observed in the IR spectrum of crystalline SnO2Raman studies were performed using 532 nm continuous wave, 100 mW Nd:YAG fiber coupled laser show the presence of a very strong peak ~149 cm−1 . This isIn a nutshell we have synthesized stanene up to few layers using ultrafast laser materials interactions. Structural characterizations using HRTEM equipped with EDAX and SAED detectors confirm the hexagonal symmetry of Sn lattice. First principles calculations along with ‘d’ spacing obtained from SAED and XRD measurements corroborate that stanene occurs in low buckled structural form. Optical signature for stanene with buckling height of 0.85 Å obtained using DFT calculations is in very good agreement with that of the UV-Vis absorption spectrum. Absence of oxygen peak in the SAED spectrum suggests absence of any oxidized phase of the material. Comparative Raman spectrum studies of FLS with oxidized samples, Raman peaks reported for (111) α-Sn thin films and the agreement of the frequency in FLS with that of the gamma phonon calculated for bi-layered stanene provide supporting evidence for synthesis of stanene up to few layers.The samples were prepared using tunable Ti: Saphire ultra-fast femto second laser (140 femto-second pulse width and 80 MHz repetition rate) (Coherent) on to a target in hexane. The laser beam was focused using a biconvex lens of focal length 20 cm. The synthesized samples were characterized structurally using HRTEM, SAED, EDAX,  AFM (Agilent 5500). Optical characterizations were performed using Raman (Witec Alpha RAS 300) and UV-Vis absorption (Shimadzu UV-2600) spectrometry.First principles were performed using Density functional theory within the generalized gradient approximation (GGA) as implemented in the Vienna ab-initio simulations package (VASP). The structure was first optimized using high energy cutoff of 500 eV. The reciprocal space was sampled using K-Point mesh of 23 × 23 × 1 centered at gamma point. Projector augmented wave (PAW) potentials with exchange correlation as parameterized by Perdew-Burke-Ernzerhof. The unit cell was constructed such that a 2 nm vacuum space was available only along the z direction to avoid any interactions between the two cells in the direction normal to the layers.How to cite this article: Saxena, S. et al. Stanene: Atomically Thick Free-standing Layer of 2D Hexagonal Tin. Sci. Rep.6, 31073; doi: 10.1038/srep31073 (2016).


A simple optical lens plays an important role for exploring the microscopic world in science and technology by refracting light with tailored spatially varying refractive indices. Recent advancements in nanotechnology enable novel lenses, such as, superlens and hyperlens, with sub-wavelength resolution capabilities by specially designed materials’ refractive indices with meta-materials and transformation optics. However, these artificially nano- or micro-engineered lenses usually suffer high losses from metals and are highly demanding in fabrication. Here, we experimentally demonstrate, for the first time, a nonlinear dielectric magnifying lens using negative refraction by degenerate four-wave mixing in a plano-concave glass slide, obtaining magnified images. Moreover, we transform a nonlinear flat lens into a magnifying lens by introducing transformation optics into the nonlinear regime, achieving an all-optical controllable lensing effect through nonlinear wave mixing, which may have many potential applications in microscopy and imaging science. A traditional optical lens refracts light with designed spatially varying refractive indices to form images; such images can be magnified or demagnified according to the law of geometrical optics on both surfaces of the lens by linear refraction, e.g. plano-convex lens, plano-concave lens. These images formed by optical lenses have a limited resolution due to the well-known diffraction limit, caused by lack of detections of the near-field evanescent waves at the far field2468910111314To address this problem, alternative approaches have been proposed in nonlinear optics to achieve the nonlinear version of negative refraction using phase conjugation, time reversal and four wave mixing (4 WM)1718202122232420Here we experimentally demonstrate a new type of dielectric magnifying lens based on nonlinear negative refraction by degenerate four-wave mixing with a thin glass slide. A multi-color imaging scheme is realized at the millimeter scale by converting the original infrared beams into the negatively refracted visible ones, spatial refractive index of the lens is carefully designed to ensure the magnification. By doing so, we surprisingly turn a demagnifying plano-concave lens in linear optics into a magnifying one in nonlinear optics. Moreover, inspired by the transformation optics, we successfully transform a non-magnified nonlinear flat lens into a magnifying one by controlling the divergence of pumping beams, effectively creating a magnifying lens controlled by another optical beam for the first time. This new imaging theme may offer a new platform for novel microscopy applications.χ(3) can internally mix an intense normal-incident pump beam at frequency ω1 with an angled-incident probe beam at frequency ω2, generating a 4 WM wave at frequency ω3 = 2ω1 − ω2, which is negatively refracted with respect to the probe’s incidence20k3 = 2k1 − k2 during 4 WM in order to ensure efficient wavelength conversion. This phase matching condition can be further translated to a Snell-like angle dependence law and create an effective negative refractive index ne as  around 2.8 × 10−22 m2/V2θ2 is set to 7.4°, close to the phase matching condition inside BK-7 glass material in order to ensure nonlinear wave conversion about 10−5 efficiency. In a non-collinear configuration, a USAF resolution card is placed on the probe’s path with a distance u away from the lens, while the images formed with the 4 WM beams around 578 nm wavelength can be captured by a CCD camera. u, showing good agreement between experimental measurements and the theory. It is also worth mentioning that the rainbow colors in the images are resulted from multicolor 4 WM processes, which are enabled by the slight phase mismatching inside the nonlinear glass due to finite spectrum spreading of the incoming beams and the glass slide’s thickness .


Compared to controls, the levels of TNF-α, vWF, and sEPCR were significantly increased in the experimental group at 12 hours and 24 hours and the NO level was significantly decreased. After 12 hours, the aortic endothelial apoptosis index and Bax mRNA expression in aortic endothelial cells had increased in the experimental group, but Bcl-2 mRNA levels had decreased. All these changes appeared at both 12 h and 24 hours. The results indicated that vascular endothelial injury and apoptosis markers were elevated in SAP. Endothelial injury and increased apoptosis in the experimental group were related to the increased expression of TNF-α.We explored mechanisms of vascular endothelial injury that lead to systemic multiple organ failure by detecting the soluble endothelial protein C receptor (sEPCR), von Willebrand factor (vWF), serum nitric oxide (NO), and tumor necrosis factor alpha (TNF- Severe acute pancreatitis (SAP) progresses rapidly, often induces systemic multiple organ failure, and has a mortality rate between 10 and 30%. Scientists originally thought that increased trypsin activity triggered acute pancreatitis and hypothesized that SAP developed when pancreatic enzymes were released into the circulatory system , furtherα (TNF-α), von Willebrand factor (vWF), soluble endothelial protein C receptor (sEPCR), serum nitric oxide (NO), and Bcl-2 mRNA and Bax mRNA in the blood of a rat model of SAP.The specific mechanisms of circulation disturbance in SAP remain unclear. In order to delineate these mechanisms, we measured the amount of pathological injury to the pancreas and the levels of tumor necrosis factor-The study protocol was approved by the Animal Care Committee of West China Hospital of Sichuan University. A total of 48 two- to three-month-old Sprague-Dawley (S-D) rats that weighed between 180 and 200 g were randomly divided into a control group and an experimental group. After surgery, 12 rats in each group were sacrificed at 12 hours and 12 were sacrificed at 24 hours, making four groups of 12 animals each.The rats were housed six to a cage at room temperature on a 12 : 12 light/dark cycle with free access to food and water for five days before the study. Food, but not water, was withdrawn 12 hours before the study started.The rats were anesthetized with 2% ketamine 80 mg/kg) injected into the intraperitoneal cavity. SAP was produced in the experimental group by two injections of an 8% solution of L-arginine   given one hour apart [0 mg/kg iTwelve rats from each group were sacrificed under anesthesia 12 h and 24 h after the injections. Blood samples were taken and centrifuged for 10 min at 3,000 rpm. The serum was preserved at −80°C. The aorta was removed and preserved at −70°C after being frozen in liquid nitrogen for PCR analysis. Part of the aorta was fixed with 4% formalin and embedded in paraffin sections for a TUNEL test.α. A reverse transcriptase polymerase chain reaction study (RT-PCR) was performed according to the manufacturer's instructions. Aortic endothelial apoptosis was detected with a TA200 In Situ Apoptosis Detection Kit .Tissue extracted from the head of the pancreas was fixed with a 40 g/L formalin solution, paraffin-embedded, sliced, and stained with hematoxylin and eosin (HE). A pathologist examined the tissue and gave it a pathology score according to the Kusske standard  based on in vitro studies are expressed as mean ± SD. After a homogeneity test of variance, a comparison between the means of two samples was made using two t-tests of group design.Results fromPancreatic tissue from the control group revealed no edema or inflammatory cell infiltration at any time point . In the α were significantly increased in the experimental group compared to the control group  (P < 0.05), and this discrepancy was also observed at 24 h  . These l at 24 h .P < 0.05) and remained at this level at 24 h  (P < 0.05) and significantly more Bax mRNA expression  than the control group at 12 h, and this was also true at 24 h. The Bcl-2/Bax mRNA ratio was lower in the experimental group than the control group (P < 0.05) at both 12 and 24 h  , Table 2and 24 h .Despite a great deal of basic science research, the exact mechanism of SAP remains unknown. Treatment is mostly ineffective, and the SAP mortality rate remains high at 10–30%. Many researchers attribute multiorgan failure in SAP to a combination of primary pancreatic enzyme activation and pancreatic autodigestion, followed by secondary immune-mediated damage and the breakdown of vascular barriers. Vascular endothelial cell injury and functional changes may be the core mechanism of circulatory disturbances in SAP; but whether vascular endothelial injury actually exists in SAP and whether this injury triggers multiorgan failure have not been extensively researched.α expression.We chose a classic rat SAP model to observe changes in the serological markers of arterial endothelial injury and endothelial apoptosis. We also explored whether the mechanism of vascular endothelial injury and apoptosis in SAP is associated with increased TNF-Rising sEPCR and vWF concentrations in plasma are a sign of vascular endothelial cell injury. vWF has strong sensitivity but lacks specificity because levels fluctuate with mild stimulation , 11. sEPP < 0.05). This suggests that SAP injured the vascular endothelial cells and the degree of injury rose as the condition progressed. We think that both vWF and sEPCR can be used to determine the severity of SAP and formulate a probable prognosis.The vWF and sEPCR levels in our experimental group were significantly higher than the levels in the control group at both 12 h and 24 h  and SAP. TNF-α is a cytokine produced by mononuclear macrophages that has a very wide range of biological activity. TNF receptors exist on the vascular endothelial surface and research according to [α has a direct cytotoxic effect on vascular endothelial cells [In recent years, research has focused on the relationship between cell factors, such as tumor necrosis factor-rding to  suggestsal cells .α concentration in our experimental group was significantly higher than that of the control group (P < 0.05), and as the TNF-α concentration rose so did the rats' vascular endothelial injury factor. Both concentrations continued to rise and reached their highest levels at 24 h.At 12 h, the serum TNF-α played an important role in arterial endothelial injury in our SAP rat model. In this study, we were not sure about the causality between the tumor necrosis factor and vascular endothelial injury; previous researches have confirmed the tumor necrosis factor through the four aspects of the mechanism damage of vascular endothelium. Four factors may contribute to the mechanism. First, TNF-α can cause direct damage [α can activate the lysosomal enzyme complex and increase cell surface protease expression, which further damages endothelial cells [α can significantly promote platelet growth and release. A high platelet count signals injury in the body, and this signal attracts monocytes that transform into macrophages to clean up cellular debris. Large amounts of TNF-α are released in the process, creating more injury that attracts more monocytes and damages the vascular endothelium in a repetitive loop. Fourth, the increased expression of TNF-α increases the permeability of vascular endothelial cells, resulting in vascular endothelial dysfunction [We concluded that TNF-t damage . Second,al cells , 26. Thifunction , 28.P < 0.05). Bcl-2 mRNA expression had decreased significantly and Bax mRNA expression had increased significantly 24 h after SAP was induced. The Bcl-2/Bax mRNA ratio was lower in the experimental group than in the control group at 12 h and continued to drop until 24 h (P < 0.05).Bcl-2 family proteins are the regulators of apoptosis; this family of interacting partners includes inhibitors and inducers of cell death. Together they regulate and mediate the process by which mitochondria contribute to cell death known as the intrinsic apoptosis pathway . Bcl-2 aα. In our study, we did not determine the aetiological relation between endothelial cell apoptosis and TNF-α; previous researches demonstrated that TNF-α induces endothelial cell apoptosis by calcium overload, lipid peroxidation, and the function of the redox imbalance [These results suggest that aortic endothelial cell apoptosis in rats with SAP is associated with alterations in the Bcl-2/Bax mRNA expression ratio, which is supported by other researches . Endothembalance –36. In ambalance .α.Vascular endothelial injury markers and Bax markers of vascular endothelial apoptosis increased in SAP, but Bcl-2 markers of vascular endothelial apoptosis decreased. This indicates that multiple organ failure in SAP is associated with direct damage to the pancreas as well as circulatory disturbances, and endothelial injury and increased apoptosis are associated with the increased expression of TNF-


Fmax) and raw EEM measurements indicated that several raw fluorescence measurements at target excitation-emission wavelength region could provide similar DOM information to massive EEM measurements combined with PARAFAC. Regression analysis between DOC concentration and raw EEM measurements suggested that some regions in raw EEM could be used as surrogates for labor-intensive routine indicators. SOM can be used to visualize the occurrence of pollution. Relationship between DOC concentration and PARAFAC components analyzed with SOM suggested that PARAFAC component 2 might be the major part of bulk DOC and could be recognized as a proxy indicator to predict the DOC concentration.High content of organic matter in the downstream of watersheds underscored the severity of non-point source (NPS) pollution. The major objectives of this study were to characterize and quantify dissolved organic matter (DOM) in watersheds affected by NPS pollution, and to apply self-organizing map (SOM) and parallel factor analysis (PARAFAC) to assess fluorescence properties as proxy indicators for NPS pollution and labor-intensive routine water quality indicators. Water from upstreams and downstreams was sampled to measure dissolved organic carbon (DOC) concentrations and excitation-emission matrix (EEM). Five fluorescence components were modeled with PARAFAC. The regression analysis between PARAFAC intensities ( The agricultural and rural NPS pollution mainly includes: (1) nutrient elements such as nitrogen and phosphorus caused by high rates of fertilization, which lead to eutrophication in ambient waterse.g. chemical oxygen demand (COD)), create toxicity in aquatic ecosystems (e.g. pesticides), introduce emerging organic contaminants  such as hormones and antibiotic resistance genes derived from manure fertilization)Agricultural and rural non-point source (NPS) pollution is mainly caused by the release of fertilizers, pesticides and other additives applied in agricultural landse.g. combination of PARAFAC and SOM)15DOM is a kind of mixture which is so far still poorly defined. DOM can be classified into two categories according to origin: (1) allochthonous DOM which is terrestrially derived and dominated by humic substances; (2) autochthonous DOM which is microbially derived and dominated by non-humic organic matter9EEM has been considered as a competitive analytical tool applied to examine water quality in natural and engineering aquatic systems. In water supply systems, EEM was used as an assessment approach for water quality from groundwater systems181926The objectives of this study were to (1) characterize and quantify DOM in a watershed affected by NPS pollution, (2) assess fluorescence properties with SOM analysis as proxy indicators of NPS pollution, and (3) assess the accuracy and reliability of capturing DOM components by monitoring raw fluorescence at a small number of target wavelengths rather than massive EEM measurements.λex/λem = 250/440 nm and a shoulder at λex/λem = 330/440 nm. Fluorescence in this region is referred to as peak A (humic-like) based on Coble9et al.λex/λem = 230/300 nm, whose shape was different from component 1. It overlaps with the region of peak B (tyrosine-like) based on Coble9et al.λex/λem = 290/490 nm. Fluorescence of component 3 had a similar location to peak C (humic-like) based on Coble9et al.1 (tryptophan-like) with the peak at λex/λem = 280/330 nm and a shoulder at λex/λem = 235/330 nm. The majority of component 4 located in Region IV is considered as soluble microbial product (SMP)-like by Chen, et al.λex/λem = 265/480 nm. Fluorescence in this region is referred to as peak A (humic-like) based on Coble9et al.9et al.PARAFAC is considered as a robust analytical tool to discriminate DOM compositions from massive data of EEMs20et al.9910291 and T2 91030et al.According to the methods for DOM fractionation developed by Coble9An approach introduced in 1980s for data mining, called SOMSample distribution on SOM map is illustrated in 637394041To combine the sample distribution , the hite.g. peak B and T) are the best indicators for pollution181, T2 and C to examine whether peak-picking could be considered as a better indication for NPS pollution than SOM analysis. Previous studies on monitoring pollution in surface waters and drinking water supply concluded that protein-like fluorescence peaks (Fmax) for component 1–5 and fluorescence intensities of each ex-em pair from original EEMs. The left panels of 2), with the highest values (red region) indicating strongest correlations near PARAFAC component peaks (white crosses), and the relative low values (blue region) indicating poor correlations far away from PARAFAC component peaks. The right panels of Fmax from PARAFAC is equivalent (the intercept is zero) to fluorescence intensity from original EEM measurements.To validate fluorescence components from PARAFAC as a proxy indicator for NPS pollution, the relationship between PARAFAC scores and EEM measurements was explored. Correlation between fluorescence intensities of PARAFAC component peaks and raw EEM measurements was analyzed to examine the effectiveness of fluorescence results as indicators for NPS pollution. 2) and the regression coefficient (m) are both closer to 1.0 (the intercept is zero) means more accurate and reliable prediction of fluorescence phenomenon in original EEM measurements using PARAFAC scores as proxy indicators. Additionally, the phenomenon that the reddest region is closer to the white cross in the left panels of 2 and m equivalent to 1.0 are both located at the same point, viz, the white cross, is the best and ideal scenario for the prediction using PARAFAC model. For component 1 in 2 and m at the peak point (λex/λem = 250/440 nm) and shoulder point (λex/λem = 330/440 nm) are both close to 1.0, indicating the position of component 1 peak is a good indicator for fluorescence DOM composition. For component 1 in the right panel, the region around the point that m is equivalent to 1.0 is a gentle slope, with a larger distance between two contour lines, meaning that little deviation in the fluorescence position during measurements would not significantly diminish the accuracy and reliability of prediction using PARAFAC scores as proxy indicators. However, for component 2 in the right panel, the region around the point that m is equivalent to 1.0 is a steep slope, with a small distance between two contour lines, meaning that the prediction using PARAFAC scores as proxy indicators is sensitive to the wavelength positions of EEM measurements. For component 3, the R2 and m near the peak point (λex/λem = 290/490 nm) are both close to 1.0, and the region encompassing the peak has a gentle slope. Accordingly, it is a good scenario to predict PARAFAC component 3 using raw EEM. For component 4, the R2 and m at the peak point (λex/λem = 280/330 nm) and shoulder point (λex/λem = 235/330 nm) are both close to 1.0. However, the regions around the peak and shoulder are steep slopes, meaning that the prediction is sensitive to the wavelength regions of EEMs. For component 5, the R2 and m near the peak (λex/λem = 265/480 nm) are also both close to 1.0, and the slope around the peak is relatively gentle. Accordingly, it is a relatively reliable for the prediction of PARAFAC component 5. From the results above, we can infer that conducting a small number of fluorescence measurements at the target excitation-emission wavelength pairs without PARAFAC analysis could still provide relatively accurate and reliable fluorescence DOM information similar to massive measurement combined with PARAFAC.In To verify the hypothesis that several raw EEMs could be used as surrogates for labor-intensive water quality indicators, relationship between bulk DOC concentration and raw EEM was explored. Linear correlation between DOC concentration and raw EEM measurements was analyzed to inspect the effectiveness of effortless EEM measurement as surrogate indicators to predict routine and labor-intensive water quality indicators such as DOC concentration .2 > 0.8) between DOC concentration and a region of fluorescence ex-em pairs  was located within excitation 230 to 285 nm and emission 305 to 455 nm of EEM region. This region includes peak B, which was associated with tyrosine-like, and PARAFAC component 2. In the last section discussing reliability evaluation of Raw EEM measurements surrogate for PARAFAC EEMs, there is a strong correlation between raw EEMs and scores of PARAFAC component 2 in the region encompassing the peak of component 2. Accordingly, we can infer that there might be a significant correlation between DOC concentrations and scores of PARAFAC component 2.In em pairs . The mose.g. ultraviolet absorbance at 254 nm (UVA254)) and fluorescence values from EEM spectrum (e.g. FDOM370/460) have been shown to be reliable predictors of DOC concentration4344254 and DOC in this study  . SUVA254, in general, is proportional to the aromaticity of DOC (the amount of chromophore or aromatic carbon per unit of DOC) and has also been widely considered as a surrogate for indicating DBP precursors8254 value for DOC indicates that few conjugated double bonds and aromatic carbon existed per unit DOC. In addition, using one fixed fluorescence peak value (e.g. FDOM370/460) will bring bias to the prediction of DOC concentration, because the best wavelength location for fluorescence peak value to predict DOC will vary with different conditions (e.g. DOM source). In this dataset, the best DOC prediction location falls on λex/λem = 265/310 nm, both of which emission and excitation wavelengths were shifted towards shorter wavelengths away from FDOM location. Therefore, fluorescence peaks used to predict DOC concentration or other water quality indicators are DOM source dependent and should not be fixed to several single EEM locations.Using optical properties as surrogates for labor-intensive routine water quality indicators has been studied for many years10is study  did not is study . Here, R 455 nm) , Fig. S2 455 nm) . MoreoveAs mentioned above, 5 fluorescence components were obtained from PARAFAC. We further inspected the relationship between DOC concentration and PARAFAC components with SOM approach.−1) are a response of high PARAFAC component 1 scores (>0.474 Raman unit (RU)), high PARAFAC component 2 scores (>0.523 RU), high PARAFAC component 3 scores (>0.282 RU), high PARAFAC component 4 scores (>0.380 RU), and high PARAFAC component 5 scores (>0.380 RU), collectively (2 = 0.87). Incorporation of all the five components into the model resulted in a better fit (R2 = 0.91) (2 = 0.19) between component 1 and DOC concentration.The component planes for each variable of the SOM output are illustrated in ectively . Regress = 0.91) , suggeste.g. paddy fields), which could be a source of aromatic protein in DOM in samples. The aromatic protein is also known as a kind of DBP precursorsThe strongest relationship between DOC concentration and PARAFAC component 2 indicated that aromatic protein associated with peak B (tyrosine-like) contributed the greatest part to the bulk DOC. Since aromatic protein is autochthonous  DOM, it can be inferred that anthropogenic practice such as agricultural and rural NPS pollution contributed high content of autochthonous DOM. NPS pollution from agricultural lands via runoff or seepage contained soluble microbial products formed in the biochemical processes in agricultural fields  in Quanchengwu Village Luniao Town Yuhang District, Hangzhou, Zhejiang. The annual average temperature was 17.5 °C, with a summer average temperature of 16.2 °C and a winter average temperature of 3.8 °C. The annual rainfall is 1454 mm and annual average relative humidity is 70.3%. This watershed is the origin of East Tiaoxi River. The water of the watershed originated from the hills within it, with a good closure, thus the watershed was a proper site to study the effect of NPS pollution.To assess the effects of NPS pollution on water quality, samples were collected from six sites in the upstream of river and from four sites in the downstream of river over the whole year of 2014 . The sam−1. Fluorescence EEMs were measured on filtered samples with an F-4500 fluorescence spectrophotometer  with a 5-nm band pass and 0.050-s integration time. Fluorescence intensity was measured at excitation wavelengths of 230 to 450 nm at 5-nm intervals and emission wavelengths of 300 to 600 at 5-nm intervals on room temperature samples (25 °C) in a 1-cm quartz cell. Inner filter corrections were applied to EEMs with ultraviolet absorbance at 254 nm (UVA254) greater than 0.03 (1-cm cuvette) as described by Gu and KennyDOC concentration was determined with a MultiN/C2100TOC/TN analyzer of analytikjenaAG with a detection limit of 0.05 mg LTo visualize the cluster of sample distribution and the relationships between DOM bulk indicators and PARAFAC components, the SOM approach was performed with MATLAB (Version 7.00) software. The SOM is a competitive artificial neural networks based on unsupervised learning50To decompose the fluorescence signal into underlying individual fluorescence composition information, the PARAFAC analysis was performed with MATLAB (Version 7.00) software. PARAFAC analysis is a competitive technique for modeling and visualizing complicated multi-variate data20PARAFAC model was derived for all samples using DOMFluor Toolbox for MATLAB with non-negativity constraints applied on all modes. The majority of Raman scatter was removed by subtracting the pure water spectrum from the sample spectrum. The first and second order scatter peaks were cut from EEM spectra and replaced with zeros. Two different split half analyses were run to inspect whether the model was validated. Tucker congruence coefficientsFmax) of each component obtained via PARAFAC. To each ex-em wavelength pair, we can get a 36 × 1 vector of raw EEM fluorescence intensities. This 36 × 1 vector was regressed against the 36 × 1 vector of DOC concentrations and 36 × 1 vector of PARAFAC scores of each component. Thus, regression coefficients (m) and determination coefficients (R2) were obtained as a function of wavelength, which can be plotted as contour graphs.To evaluate the potential for estimating DOC concentrations and PARAFAC scores from raw EEMs, the original measured EEM data were regressed against the DOC concentrations the maximum fluorescence .