The test of “Porter hypothesis” shows that environmental regulation has not encouraged firms to innovate more. We further used the 2013 China Social Survey (CSS) data to verify the human capital mechanism of PM2.5 affecting firm innovation at micro level. The results show that PM 2.5 deteriorates the healthy human capital in a firm, which reduces the innovation output. This article helps to understand the relationship between air pollution and firms’ innovation and to develop appropriate policies.The potentially serious economic consequences of China’s severe air pollution problem cannot be overlooked, especially the impact on corporate innovation, which is a core driver guiding firms towards efficient and high-quality development. This paper explores the direct effect and mechanism of PM2.5 on firms’ innovation output through the identification strategy of instrument variable. Based on the data of Listed Companies in China from 2003 to 2016, we used thermal inversion as the instrument variable for PM2.5 for estimation. The results show that each 1 ug/m Innovation is an important engine for enterprises to achieve long-term development and is an inexhaustible driving force for economic growth. However, China’s air pollution problem, especially PM2.5 pollution, is serious. More than half of China’s cities exceeded air pollution concentrations, and the number of days with PM2.5 as the primary pollutant accounted for 78.8% of the days with severe and heavy pollution. It is estimated that the annual number of deaths attributable to PM2.5 pollution in China has increased to 971,000 by 2017 In the context of such severe pollution in China [3 increase in the annual average PM2.5 concentration in the region, the total number of patent applications and the total number of invention patent applications of listed companies decreases by about 7.0% on average. At the same time, we further control the environmental protection policies and the number of regional environmental regulations, as well as other environmental factors such as pollutant emissions. The results show that PM2.5 does not have a significant impact on the innovation output of individual firms through environmental regulation, and the “Porter hypothesis” of PM2.5 and firm innovation does not hold.In this paper, thermal inversions are used as the instrumental variable of PM2.5 to analyze the effect and mechanism of PM2.5 on micro-firm innovation. In terms of impact effect, we take the innovation output of Chinese listed companies in 2003–2016 as the analysis object. The results of IV estimation model show that PM2.5 leads to the reduction in firm innovation output. Specifically, for every 1 ug/mIn terms of the mechanism of action, we used the 2013 Chinese Social Survey (CSS) data to verify the human capital mechanism from the individual micro level. IV estimates indicate that regional PM2.5 can significantly worsen the health of individual laborers, leading to a decline in labor productivity, lower job satisfaction, and subjective well-being, and increase their negative emotions at work. In general, PM2.5 significantly worsens the human capital of firms, which may lead to the reduction in innovation output of firms. This verifies that the mechanism of the human capital of PM2.5 affects firm innovation. At the same time, we show that there is an inverted U-shaped relationship between PM2.5 and firm innovation output, and analyze the heterogeneity of PM2.5 on firm innovation output from the perspectives of PM2.5 pollution levels, capital intensity, and labor productivity. The results also confirm the existence of the human capital mechanism, and the environmental regulation mechanism is not significant.This paper has the following contributions: First of all, this paper expands the research on the influence of PM2.5 on micro-firms to the field of firm innovation. Studies on air pollution on inventory level , but resThe rest of this paper is organized as follows: Air pollution has a wide-ranging impact on various social and economic actors. Since the effect of air pollution on individual residents is considered relatively direct and easy to observe ,10, a laThe early research on the impact of air pollution on firm subject was mainly indirectly identified through the explicit variable of “environmental regulation”, and the research conclusions did not obtain a more consistent consensus. Some scholars believe that the increase in air pollution is mainly caused by the expansion of industrial production scale or renewable energy consumption scales , and larIn recent years, some scholars have tried to estimate the direct impact of air pollution on firms by designing and constructing some sophisticated identification strategies. For example, some scholars used the closure of a large oil refinery as a quasi-natural experiment to study the impact of air pollution on the labor supply of firms . The resAlthough scholars have begun to study the impact of air pollution on the labor supply, productivity, and inventory of micro-firms through the design of more accurate identification strategies, the research on the theme of firm innovation needs further development. Existing domestic and international literature on environmental pollution and firm innovation is carried out around environmental regulation or the “Porter hypothesis” ,40,41, aIn view of the above problems, this paper focuses on the direct impact and mechanism of PM2.5 on micro-individual firm innovation. Based on the existing literature, this paper focuses on the use of instrumental variables to build a more accurate identification strategy. PM2.5 is related to regional economic and social activities on the one hand, and local natural climatic conditions on the other. It can generally be considered that the long-term stable natural climatic conditions in a region do not have an impact on regional economic or social activities. Therefore, if we can find a natural climate variable that directly affects the formation of PM2.5 in the region as an instrumental variable, the effect of PM2.5 on the innovation of micro-individual firms is more accurately identified. Thermal Inversions may be such a naturally occurring meteorological phenomenon. Under normal circumstances, the temperature in the troposphere decreases with the increase in altitude. This kind of atmospheric stratification is prone to convective movement, and the pollutants in the near-surface layer can be dissipated to high altitude or even far away, thus reducing the degree of urban air pollution. However, under certain weather conditions, the atmospheric structure experiences an abnormal phenomenon where the temperature increases with height, resulting in stable atmosphere. The occurrence of inversion temperature is not conducive to the rising movement of air, so that low-level water vapor and pollutants cannot be diffused out to high altitude, which causes fog formation and accumulation of pollutants. The occurrence of inversion directly leads to an increase in air pollution, but at the same time, inversion is a naturally occurring meteorological phenomenon, its formation not affected by regional economic or social factors, so many economists use it as an instrumental variable for regional air pollution ,56,57.In this paper, the firm’s innovation output is the micro-firm level data, the PM2.5 is the city-level data, and the main control variables are the relevant factors at the firm level. For factors outside the firm level, a fixed-effects model is used for processing, including the control of regional and temporal fixed effects. This paper sets the following quantitative analysis model:The explanatory variable The main goal of this paper is to analyze the direct effect of PM2.5 on firm innovation. The core strategy chooses inversion as the instrumental variable of regional PM2.5 and uses two-stage least squares (2SLS) for regression analysis. At the same time, in order to examine the impact of environmental regulation on firm innovation, in the regression of instrumental variables, group regression is further carried out according to the implementation of regional environmental regulation and innovation policy to test the original estimation results.On the basis of examining the impact of environmental regulation on firm innovation, this paper focuses on the possible mechanism of PM2.5 affecting firm innovation from the perspective of human capital. This paper chooses to use the micro survey data of individual residents to test the impact of PM2.5 on labor health human capital and labor loss, and construct a quantitative analysis model as shown below:Equation (2) is an analytical model at the individual level. Among them, the explanatory variable This paper selects firm patent data as a proxy indicator for firm innovation. There are many variables that reflect firm innovation, such as R&D expenditures and personnel inputs that reflect innovation process inputs ,59, and There are many sources of data for PM2.5, such as environmental monitoring stations ,62,63, sIt can be said that the various PM2.5 data sources have their own advantages. The object of this paper is that firm innovation is a micro-data at the individual firm level. Therefore, this paper chooses spatial remote sensing information that is more advantageous in space as the source of PM2.5 data in this paper. In terms of time, this paper considers the time frequency of the listed company’s annual report data, and selects the annual urban PM2.5 average concentration, which also avoids the problem of the spatial remote sensing information monitoring time interval. Specifically, this paper selects PM2.5 data from the Center for International Earth Science Information Network (CIESIN)/Columbia University, which is hosted by the National Aeronautics and Space Administration (NASA). CIESIN developed and produced a global PM2.5 continuous monitoring algorithm system by using space remote sensing satellites and multi-angle imaging spectrometers to scientifically solve the problem of converting spatial remote sensing information into PM2.5. They provide PM2.5 concentration surface data with a spatial resolution of 0.5° × 0.625°  with a grid length ranging from 70 degrees north latitude to 60 degrees south latitude. On this basis, this paper further uses ArcGIS software to extract Chinese data from global datasets and uses the method of Inverse Distance Weighting (IDW) ,69,70, mIn order to better match the PM2.5 data, this paper also obtains the Thermal Inversions data from the space remote sensing information released by the National Aeronautics and Space Administration (NASA). NASA divides the Earth into a grid with a latitude and longitude interval of 0.5° × 0.625°, and from 1980 onwards has continuously reported the temperature of 42 different sea levels every six hours. In this paper, we obtain the three sea level temperature data closest to the ground. First, we average the sea level temperature of each grid every day and count the first layer temperature lower than the second layer as one inversion temperature, then accumulate the total inversion days of each natural year of each grid. Finally, we determine the number and weight of grids covered by each prefecture-level city in China according to the IDW method, and finally calculate the annual number of inversion days in each prefecture-level city. In order to test the robustness, this paper also calculates the case where the first layer temperature is lower than the third layer.The goal of this paper is to accurately estimate the direct impact of PM2.5 on the firms’ innovation output. On the one hand, it is necessary to eliminate the influence of environmental regulation on firm innovation as much as possible. On the other hand, it is necessary to explore the direct mechanism of PM2.5 affecting the innovation output of firms.In terms of eliminating the impact of environmental regulations, this paper uses policy changes to reflect the impact of environmental regulations. Although existing descriptions of environmental regulations are abundant, such as pollutant discharge , energy The data description statistics of each research variable in this paper are shown in In the regression, we focus on the innovation of patents and examine the impact of regional PM2.5 on firm innovation from two aspects. First, we examine the impact of PM2.5 on the number of innovative firms, including the total number of patent applications applied for and the total number of invention patent applications filed. Second, we further investigate the impact of PM2.5 on the total number of other types of patent applications, including utility model patents and design patents. The regression is analyzed according to Equation (1) using a two-way fixed-effect model and an instrumental variable model.3 increase in the annual average PM2.5 concentration in the region, the total number of patent applications and the total number of invention patent applications of listed firms falls by about 7% on average, that is, the PM2.5 significantly reduces the firm’s innovation output, and the “Porter hypothesis” is not established. The validity of the instrumental variables passed the tests. Similarly, the analysis of patent application data also confirms that there are serious endogenous problems between PM2.5 and firm innovation. The estimation of instrumental variables in this paper may be more accurate.3 increase in the annual PM2.5 concentration in the region, the number of utility model patents and grants of listed firms is reduced by about 6%, and the number of applications and licenses for design patents decreases by approximately 2.6% and 3.7%, respectively. Compared with the estimation results in In This paper further uses the instrumental variable method to test the multi-faceted robustness of the baseline estimates. First, to control the influence of regional and industry-level factors that may change over time, we examined the fixed-effect control of regions, time, and industry. Second, to control for selection bias due to different sample sizes, we examined the estimates of the sample sizes of different firms. Third, to control for the possible effect of cities heterogeneity on the estimation results, we examined the estimates of the characteristics of different cities. Fourth, to control for bias caused by the explanatory variable measures, we replaced the dependent variable measures. Finally, we replaced the ordinary robust standard errors with clustering robust standard errors.This paper further examines the robustness from the regional urban level, the results are shown in The dependent variable in the baseline regression is the total number of patent applications, and this paper further replaces the dependent variable with the share of patents for robustness testing, and the results are shown in Ordinary standard errors are used in the benchmark regression, and this paper further uses clustered robust standard errors for robustness testing, and the results are shown in Many extant literatures suggest that environmental regulation has an impact on firm innovation ,47, and 2 and wastewater). We control the impact of environmental regulations by adding pollutant emissions to the IV baseline regression model.This paper argues that environmental regulation itself is mainly embodied in the form of external policies. This paper sorts out relevant policies related to environmental regulation in the country and cities, including the “Ecological Demonstration Area” , “Atmospheric Ten Articles”, and “Two Control Zones”. Since these environmental policies are based on the scope of the city, in the specific estimation, we put the interaction items of these policy dummy variables and urban fixed effects into the baseline model for control. At the same time, this paper also collects the number of laws and regulations related to environmental protection in each city, and adds it directly as a control variable to the IV estimation model. In addition, we also consider the impact of other pollutants in various cities . The results show that the PM2.5 has a significant effect on the innovation output of the non-excellent group, but it is not significant for the firms with superior classification. This shows that only when the PM2.5 pollution reaches a certain level (such as when it is at a non-excellent level), its effect on the innovation output of the firm appears, which also verifies the above conclusions about the relationship between PM2.5 and firm innovation.We further examine the heterogeneity impact of PM2.5 on firm innovation, and the results are shown in Secondly, we analyze the impact of PM2.5 on the innovation output of firms with different industrial structure attributes. Regression analysis by grouping according to the level of capital intensity shows that PM2.5 has a significant effect on the innovation output of firms with low capital intensity, but has no obvious effect on firms with high capital intensity. The results of this analysis can also better verify that the mechanism of action of PM2.5 on firm innovation output is mainly human capital mechanism rather than environmental regulation mechanism. On the one hand, industries with high capital intensity are mainly refueling industries and basic industries, including steel smelting, petrochemical, and other industries . Firms iFinally, we observe the impact of PM2.5 on the innovation output of firms with different productivity levels. Based on the level of labor productivity, we conduct group regression analysis. The results show that PM2.5 has a significant effect on the innovation output of firms with low labor productivity, but not on firms with high labor productivity.Different from the traditional research on environmental pollution and firm innovation, this paper focuses on the direct influence effect and mechanism of PM2.5 pollution on individual firm innovation output through the identification strategy of constructing instrument variables.3 increase in the annual average PM2.5 concentration of the city, the total number of patent applications and the total number of invention patent applications of listed companies falls by an average of 7.0%. Based on the existing research conclusions on environmental regulation and firm innovation, we further investigated environmental regulations. Different from the idea that some studies directly internalize environmental regulation into firm specific behaviors (such as pollution investment), we mainly measured environmental regulations in the form of policies, including the “Ecological Demonstration Area”, “Atmospheric Ten Articles”, “Two Control Zones”, and other national-level environmental policies, and the number of environmental regulations in each city. At the same time, we also considered the influence of other pollutants (such as SO2 and wastewater) in various cities. The results show that the effect of PM2.5 remains after controlling for environmental regulation. Although the impact of environmental regulation on firm innovation has been controversial in academic circles [We used the patent data of Chinese listed companies in 2003–2016, and matched the annual PM2.5 concentration data with each firm according to the regional cities, and selected the urban inversion as the instrumental variable of PM2.5 pollution to carry out the regression estimation of the impact effect. The results show that PM2.5 leads to a reduction in the innovation output of firms. Specifically, for every 1 ug/m circles ,44,47,87After verifying that the environmental regulation mechanism is not established, this paper attempted to explore the mechanism of other PM2.5 affecting the innovation output of firms. Taking into account the direct impact of PM2.5 on individual health, we focused on the possible mechanisms of action from the perspective of human capital. The results show that PM2.5 can significantly worsen the human capital of firms, which may lead to the reduction in innovation output of firms, thus verifying the role of human capital mechanism. In addition, we further tested the relationship between PM2.5 and firm innovation output, and found that there is an inverted U-shaped relationship between PM2.5 and firm innovation output. When PM2.5 is at a lower pollution level, PM2.5 has a positive impact on the innovation output of firms, mainly through the mechanism of large-scale development of firms; when the pollution of PM2.5 is higher than a certain level, PM2.5 has a significant negative effect on firm innovation. It mainly acts through the human capital mechanism, and the role of the environmental regulation mechanism is not significant. The results of heterogeneity analysis can also verify the existence of human capital mechanisms.The research in this paper has positive guidance and reference significance for understanding the relationship between PM2.5 and firm innovation and formulating corresponding policies. Of course, there are also some shortcomings in this paper. The indicators of innovation output are not rich enough, the description of environmental regulation may be omitted, human capital mechanism testing is not targeted at sample firms, firms’ production and innovation might take place at different hubs, insufficient exogeneity of instrumental variables, measurement errors due to intra-city pollution differences and other issues need to be resolved by future scholars.


This paper proposes a tandem mass spectrometry (MS/MS) approach in isomer recognition by playing in the “energetic dimension” of the experiment. The chromatographic set up (HPLC) was tuned to minimize the run time, without requiring high efficiency or resolution between the isomers. Then, the MS/MS properties were explored to solve the signal assignment by performing a series of energy resolved experiments in order to optimize the parameters, and by applying an interesting post-processing data elaboration tool (LEDA). The reliability of the new approach was evaluated, determining the accuracy and precision of the quantitative results through analysis of the isomer mixture solutions. Next, the proposed method was applied in a chemical stability study of human plasma samples through the simultaneous addition of a pair of isomers. In the studied case, only one of the isomers suffered of enzymatic hydrolysis; therefore, the influence of the stable isomer on the degradation rate of the other was verified. In order to monitor this process correctly, it must be possible to distinguish each isomer present in the sample, quantify it, and plot its degradation profile. The reported results demonstrated the effectiveness of the LEDA algorithm in separating the isomers, without chromatographic resolution, and monitoring their behavior in human plasma samples. Most medicinal chemistry investigations involve the design, chemical synthesis and development of pharmaceutical agents or bio-active molecules (drugs) suitable for therapeutic use . These sIn regards the topic of the recognition of isomers, our group proposed and developed an MS/MS post-processing mathematical algorithm named LEDA  that allows the recognition of isomer compounds without their chromatographic separation ,24,25,26Thanks to this experience, the logical trend suggests the evaluation of the hydrolytic kinetics of plasma enzyme systems when spiking a pair of isomers in the sample. In detail, we propose the case where only one of the isomers is hydrolyzed; in this way, adding the sample to the mixture of isomers, we can verify the influence of the stable isomer on the degradation rate of the other.To carry out this evaluation, the LEDA approach was applied to the plasma stability experiments on two pairs of isomers of P-glycoprotein (P-gp) inhibitors candidate. These molecules are characterized by a tertiary amine group carrying two polymethylene chains of variable length (between 3 to 6 carbon atoms), linked to different aromatic moieties through two ester bonds . The strThe aim of this work is to propose a single HPLC separation method and to develop a series of MS/MS conditions that allow the recognition of studied isomer pairs when they are present simultaneously in the sample, supporting their chemical stability study.the checking of the achieved chromatographic separation;the exploration of the MS/MS features in the isomers distinction;the evaluation of a mathematical device (LEDA) that allows the conversion of the common MS/MS signals in specific isomer abundances, related to their relative concentration in the sample;the assessment of the LEDA quantitative performances;the application of the optimized conditions of the LEDA approach in plasma stability experiments.The investigation was devoted to monitoring the hydrolytic activity of human plasma enzymes towards selected couples of isomers simultaneously added to the samples. The description of the experimental results of the proposed study is presented in the following steps:The obtained chromatographic profiles showed an appropriate distinction between the internal standard (IS) and the analytes as a result of the different characteristic ion transitions , however, an acceptable separation among the isomers was not reached. The peak parameters  for each analyte were calculated and reported in the The chromatographic profiles of the studied compounds are reported in the N ≈ 5000), mainly due to the short column used. In any case, this set up allowed us to take advantage of a rapid elution gradient, allowing for a brief window to rinse the column and restore the initial condition; as a result, a faster chromatographic run is obtained. Therefore, the primary feature required by chromatography in the presented approach was reached: being simple and rapid. In this way, it was possible to process a large number of samples (about 150–160/day) with the same reliability of common HPLC-MS/MS methods, avoiding the complicated method setup for each pair of isomers. However, to obtain a suitable distinction  between the tested compounds and to ensure their proper monitoring during the degradation experiment, the MS/MS features for each analyte must be explored.The proposed HPLC method achieved a poor efficiency, evaluated as the number of theoretical plates (+ species), with the same m/z value for each pair of isomers , were performed  could be selected. Four of these Pis were related to the bond cleavages of the ester groups, following the general scheme proposed in 1 and/or Pi2 and the corresponding neutral alkyl-alcohol structure. Pi1 and Pi2  show the same structure regardless of the precursor compound, but their maximum abundances are different and characteristic for each isomer , reducing their ability for the recognition of isomers. Finally, two other Pis were observed (Pi5 and Pi6) that can be related to the bond cleavage between the tertiary amine group and the propyl chain linked to the aromatic moiety (Ar1 or Ar2), but their formation yield is also less than 20%. In addition, in this case, the structures of the ions are the same, regardless the considered isomer, but their relative abundances are different. The fragmentation hypothesis and molecular structures of Pi5 and Pi6 are reported in the The MS/MS most intense signals, for all compounds, were obtained by the bonds cleavage 1 and/or 2 with the formation of Pih isomer . On the + species of the selected P-gp inhibitors highlighted common dissociation pathways with the formation of six main Pis that could characterize each isomer. Indeed, these ions show different abundances depending on the precursor isomer; hence, it may be possible to set up a suitable “mathematical device”, able to distinguish the common signals and elaborate the MS/MS data by assigning the proper abundance to the isomer or isomers present in the sample.The fragmentation studies on the [M + H]The proposed mathematical device, LEDA, is a matrix of linear equations that elaborates each MS/MS signal, distinguishes the possible components and assigns the correct abundance to the isomers present. The algorithm is based on the rationale that the MS/MS signal might be represented as the sum of contribution of each isomer present in the sample. As demonstrated in the breakdown curves shown above , each ism is the abundance ratio between the product ion (Pi) vs reference ion (Ri) measured (m) in the sample.(Pi/Ri)x is the characteristic abundance ratio between the Pi vs Ri of pure isomer.(Pi/Ri)x is the concentration (%) of the isomer in the sample.[%]n isomers requires a matrix composed of n linear regression Equation (1) obtained from n different experimental data. In our case, the experimental data used were the abundance ratios of product/reference ions selected during the MS/MS method set-up. In this study, a pair of isomers was spiked in the samples; then, a LEDA matrix composed by two equations (square matrix) could be adequate. However, to verify the reliability of MS/MS signal deconvolution, an overdetermined system of linear equations (six equations) was also evaluated by assembling a LEDA matrix that includes all the Pis revealed during the ERMS experiments. Therefore, two LEDA matrices have been proposed: the first was composed by only two equations ; the second was , a single Equation (1) related to only a product ion ratio (Pi/Ri) could be sufficient. Indeed, by assuming that only the pair of isomers constitutes the MS/MS signal, the concentration of B is calculated as B% = (1 − A)%. However, in this case the possible contribution of signals from unknown isomers (or any co-eluting compound having the same product and reference ions) is neglected. Therefore, to avoid ill-conditioned algorithms, the resolution of the mixtures of LEDAall,  assemble195–221 matrix is shown.The LEDA reliability was evaluated by determining the accuracy and precision of the quantitative results obtained from processing the MS/MS data from the analyzes of the mixture solutions  of each 2 = 0.999), the slope near to 1 indicates an optimal accuracy of recognition of the ELF96 isomer in the analyzed mixtures and the y-intercept close to the origin assures a good isomer distinction also in low relative abundance. Clearly, the minimum amount of isomer detectable in the mixture also depends on the precision of LEDA elaboration, represented by the standard error of the calculated linear function (SE-Lin). Then, to estimate the best accuracy and precision performances of the LEDA process, two mathematical matrices for each pair of isomers were used to elaborate the MS/MS data acquired from the analyzes of the standard mixtures. The comparison with obtained results were reported in The reported validation plot demonstrated how the estimated linear function was representative of the data points  that showed no significant differences was noteworthy. In this case, the evidence suggests that increasing the number of Equation (1) to compile the LEDA matrix does not influence the accuracy and precision of the elaboration. However, the outcome could depend by the low number of isomers involved (only two) or by the “simplicity” of the standard mixtures, which were diluted in neat solvents. To settle this point, both LEDA matrices were applied in plasma stability studies of the selected couple of isomers to also verify the effectiveness of the algorithm in a complex matrix.All the slope values shown in In this section, the parameters  that define the range of applicability and the trueness of the quantitative analysis by the proposed approach are reported and described.2) and the estimated LOD and LOQ values for each analyte are reported in the The analyte determination was carried out by using the verapamil hydrochloride as internal standard (IS) that, with its similar molecular characteristics to the studied compounds, assures reliable and robust quantitative data analysis. The linearity range of the of response of each compound was verified by analyzing, with a proper MS/MS method, its calibration solution  and plotThe standard error of y-intercept (Y-SE) of linear regression, calculated for each calibration curve, was used to establish the limit of detection (LOD) and limit of quantitation (LOQ) of the analytes in the selected quantitation signals . By follThe matrix effects (ME) and recovery (RE) values for IS and each analyte were obtained through the analysis of the sample sets prepared, as described in The collected results about quantitative parameters demonstrated that the proposed HPLC-MS/MS methods are suitable to be applied in the chemical stability study of selected P-gp inhibitors.The present investigation was carried out to evaluate the variation of hydrolytic activity of the human plasma enzymatic system when two potential substrates were simultaneously present. To achieve this aim, we studied the behavior of two couples of isomers which, in a previous study , had bee1/2 and degradation rate or k) of the samples added with the mixture of isomers were calculated by processing the quantitation data obtained from the proposed LEDA matrices and the results, reported in the 19–-221 matrix assembled with two Equation (1), relating of the most intense Pis (Pi1 and Pi2) in the isomers fragmentation, was adequate to represent the system of the studied compounds, ensuring their correct distinction in sample mixtures. Therefore, all the data reported below were obtained by processing the MS/MS data with the LEDA195–221 matrix.The characteristic degradation parameters , reported in 1/2 43 and 32 min.), however, these differences are statistically non-significant .The effectiveness of the LEDA approach was evaluated by comparing t samples . The obtTo verify the efficiency of the LEDA algorithm, the set of plasma samples spiked with an equimolar mixture of isomers was processed using the ‘scan-by-scan’ method, allowing the graphical separation of the isomers present in the chromatographic profiles. In this case, the computation of the LEDA matrix was repeated on each MS/MS data point acquired; consequently, a relative abundance of the Ri signal for each isomer was assigned. By considering a generic chromatographic profile, the performance of the algorithm was reflected in the deconvolution of unresolved peak of Ri signal that delivered its abundance between the isomers (LEDA reconstructed chromatographic profiles). In In In this study, we proposed the evaluation of the activity of hydrolytic human plasma enzymes towards the simultaneous presence of two possible substrate compounds. To achieve this aim, we selected two couples of isomers, one pair was characterized by a substrate isomer (susceptible to hydrolysis), while the other remained stable. The challenge of this application was to monitor the behavior of both isomers when they were present at the same time in the sample by using an HPLC-MS/MS method. Indeed, the MS and MS/MS methods are known to have poor specificity in the recognition of isomers. To overcome these difficulties, a new HPLC-MS/MS approach was proposed, using an interesting post-processing data elaboration (LEDA) to solve possible signal misassignment. Taking advantage of the characteristics of LEDA, the chromatographic set up to analyze the samples was tuned to minimize the run time, without requiring high efficiency or resolution between the analytes. The only aim of the chromatographic column was to avoid or limit the interference of sample matrix in the analyte ionization process (matrix effects). Therefore, we were able to develop an HPLC-MS/MS quantitative method that is sensible, specific and fast.The LEDA “mathematical device”, introduced to process the acquired MS/MS data, was demonstrated to be reliable in recognizing and separating the possible components present in the sample signals. Its effectiveness was tested by processing the HPLC-MS/MS analysis of a series of known mixture solutions, and we introduced the “validation plot” that, through the evaluation of the characteristic parameters, described the accuracy and precision of the quantitative ability of the LEDA elaboration. The obtained results showed an accuracy near to 100%, with precision values ranging between of 1% to 3%. The obtained MEs values (ME 88–100%) demonstrated that the chromatographic setup (short column and fast elution gradient) was required to avoid the matrix interferences, while the REs values (RE 87–99%) indicate a reliable sample preparation, despite only a protein precipitation being carried out. Therefore, the general procedure proposed was found to be adequate in studying the selected isomer compounds without their chromatographic separation and applying and developing the MS/MS features. For this purpose, the LEDA post-processing algorithm was introduced to allow the use of the sole and simple chromatographic method, which led to increased productivity, without losing determination specificity.1/2 e k rate).Finally, we applied the developed HPLC-MS/MS method to the analysis of three series of human plasma samples, spiked with each pure isomer (series 1 and 2), and an equimolar mixture of both isomers (series 3). The obtained results confirmed the stability characteristics of the selected compounds: the substrate isomers were hydrolyzed, while the other isomers kept their concentration constant in the sample solution. Furthermore, the presence of the stable isomer did not appear to affect the degradation of the substrate-isomer , verapamil hydrochloride and ketoprofen  were purchased by Merck . Ketoprofen ethyl ester (KEE) was obtained by Fisher’s reaction from ketoprofen and ethanol. Ultrapure water or mQ water (resistivity 18 MΩ cm) was obtained from Millipore’s Simplicity system . Phosphate buffer solution (PBS) was prepared by being dissolved in mQ water and reported salts at the following concentrations: 8.01 g L−1 of NaCl, 0.2 g L−1 of KCl, 1.78 g L−1 of Na2HPO4 2H2O, and 0.27 g L−1 of KH2PO4. The samples of human plasma were collected from healthy volunteers, pooled and kept at −80 °C until use. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the local Ethics Committee  Institutional Review Board .The P-gp inhibitors selected for this study were obtained as reported in Ref. . AcetoniThe HPLC-MS/MS analysis was carried out using a Varian 1200 L triple quadrupole system , equipped with two Prostar 210 pumps, a Prostar 410 autosampler and an electrospray ion source (ESI). Raw data were collected and processed by Varian Workstation version 6.8 software . A thermostatic oven G-Therm 015  was used to maintain the samples at 37 °C during the degradation test, while the centrifuge Eppendorf 5415D  was employed to centrifuge the plasma samples.−1 and stored at 4 °C. Working solutions of each analyte were freshly prepared by diluting stock solutions up to a concentration of 1.0 µg mL−1 and 0.1 µg mL−1  in mixture of mQ water:acetonitrile 50:50 (v/v). The IS working solution was prepared in acetonitrile at 33 ng mL−1 (IS solution). The quantitative data of each analyte were obtained by building a five-level calibration curve, prepared by adding proper volumes of the working solution (1 or 2) of each analyte to 300 μL of IS solution. The obtained solutions were dried under a gentle nitrogen stream and dissolved in 1.0 mL of 10 mM of formic acid in mQ water:acetonitrile 80:20 (v/v) solution. Final concentrations of calibration levels of each analyte were: 5.0, 10.0, 25.0, 50.0, and 100.0 ng mL−1. With the aim of evaluating the accuracy and precision of the proposed approach, five mixture solutions for each couple of isomers in different proportions were prepared. Mixture 1 had 90% of A isomer and 10% of B isomer, while in mixture 2 the proportion was 75% and 25%; mixture 3, 50% and 50%; mixture 4, 25% and 75%; and mixture 5, 10% and 90%. The sum of isomer concentrations in the five mixture solutions was 100 ng L−1. Each solution was used to prepare the precision and accuracy set samples as follows: 100 μL of mixture solution and 300 μL of IS solution in autosampler vial were added. The obtained solutions were dried under a gentle nitrogen stream and dissolved in 1.0 mL of 10 mM of formic acid in mQ water:acetonitrile 80:20 (v/v) solution. The calibration levels and mixture solutions were analyzed six times by the HPLC-MS/MS system, with the proper conditions. Finally, the spiked solutions, used in the sample preparation of chemical stability study, were separately prepared by diluting the respective stock solutions in mQ water:acetonitrile 80:20 (v/v) solution, to obtain an analyte final concentration of 10 μM. For each couple of isomers, three spiked solutions were prepared: solution of isomer A, solution of isomer B and solution of mixture of both isomers (each at concentration of 10 μM).Stock solutions of analytes and verapamil hydrochloride  were prepared in acetonitrile at 1.0 mg mL−1, employing a binary mobile phases elution gradient. The solvents used were 10 mM formic acid and 5 mM ammonium formate in mQ water:acetonitrile 90:10 (solvent A) and 10 mM formic acid and 5 mM ammonium formate in acetonitrile:mQ water 90:10 (solvent B). The program of elution gradient was set up as follows: initially at 90% solvent A, which was then decreased to 10% in 4.0 min, kept for 2.0 min, returned to initial conditions in 0.1 min and maintained for 2.0 min to a total run time of 8.0 min. The column temperature was maintained at 40 °C and the injection volume was 5 µL. The ESI source operated in positive ion mode by using the following setting: 5kV needle, 42 psi nebulizing gas, 600 V shield, and 20 psi drying gas at 280 °C. The MS analyzes were obtained in an ion scan by acquiring the m/z range from 250 to 750 with 600 ms of scan time. The ERMS experiments were performed to study the fragmentation of [M + H]+ species of each analyte and build its breakdown curves [m/z range from 50 to 650, scan time of 600 ms and by using argon as collision gas. The ERMS experiments were performed by introducing working solution 1 to each analyte  of each ion signal, present in the MS/MS spectra acquired, versus the maximum intensity of precursor ion (Ri) and the CV applied. In this way, each ratio abundance represents the yield of formation of Pi to the precursor ion to the applied CV. The MS/MS analyzes were acquired in multiple reaction monitoring (MRM) by selecting, from the ERMS experiments, the most characteristic ion transitions for each analyte , at a constant flow of 0.25 mL minn curves ,25,26,27 analyte , via syr analyte .In order to evaluate the human plasma chemical stability of studied compounds, a dedicate MRM method was arranged for each pair of isomers with the specific MS/MS transitions of the IS and the monitored isomers, each acquired for 25 ms (dwell time).g). Then, the supernatant was transferred to autosampler vials, dried under a gentle stream of nitrogen and dissolved in 1.0 mL of 10 mM of formic acid in mQ water:acetonitrile 80:20 solution [−1 (depending on the MW of the studied compound), values placed in the middle of calibration curves. The final solutions of blank and all of the sets of samples, prepared as described above, were analyzed using the proper HPLC-MS/MS method.Each sample was prepared by adding 10 µL of proper spiked solution to 100 µL of human plasma matrix in a 1.5 mL microcentrifuge tube. The obtained solution corresponds to 1 μM of analyte that undergo degradation study. Each set of samples was prepared in triplicate and incubated for four different times: 0, 30, 60, and 120 min at 37 °C. Therefore, each stability panel of studied analyte was represented by a batch of 12 samples . After the incubation, 300 µL of IS solution was added to the sample and then centrifuged , calculated between the analyte and the IS quantitation ions signal, versus the nominal concentration of the calibration solution. A linear regression analysis was applied to obtain the best fitting function between the calibration points. Taking into account that both the calibration and sample solutions were prepared with the same procedure, the calibration curve parameters, the LOD and LOQ values for each analyte, referred to the concentration of sample solution actually analyzed by the HPLC-MS/MS methods. In order to obtain reliable LOD and LOQ values, the standard error (SE) of response and slope approach was employed . The estn linear equations (see Equation (1)) to allow the resolution of the mixtures of n isomers. Naturally, to increase the specificity and reliability of isomers speciation, an overdetermined system of linear equations could be assembled; in this study, the LEDA matrix was composed by a number >n of linear equations (see Equation (1)). All calculations for the deconvolution of MS/MS data are processed using an Excel™ macro. The deconvolution was performed by applying the algorithm either to the area abundances, obtained from the integrated peak intensities of each MRM channel, or to individual MS/MS data points of the chromatographic sample profile. In the first approach, the LEDA provides the relative amounts (%) of each know component present in the sample. In the second approach, each MRM signal is deconvoluted ‘scan-by-scan’ and assigned to the present isomers, allowing a graphical separation of the processed chromatographic profiles. The characteristic abundance ratios were calculated by data obtained from the highest level of the calibration curve (100 ng mL−1) of each pure isomer by the HPLC-MS/MS methods described above. The ratios between Pi vs. Ri selected in the MRM methods were calculated and the resulted values were reported in In the proposed HPLC conditions, the chromatographic peaks of analytes were unresolved; therefore, LEDA algorithm application was necessary to elaborate the peak area of each MRM channel of the unresolved isomer signals, separating their components and assigning the correct abundance to the identified isomers. The LEDA algorithm is a matrix composed by The LEDA tool performances were evaluated by processing the MS/MS data obtained from the analysis of the standard mixture samples, prepared in M), the enzymatic degradation rate is described as a first-order kinetics. Therefore, by converting the quantitative results as natural logarithm, the natural logarithm of concentrations can be plotted versus incubation times and, once the slope of the linear function is obtained, will represent the degradation rate constant (k). Then, the half-life (t1/2) of each tested compound can be easily calculated as follows:In general, the presence of an ester group in the structure of a new drug candidate compound leads to the performance of a plasma stability study in order to verify its possible hydrolysis by esterase enzymes. The plasma samples were prepared by spiking a known amount of compound , then being analyzed by a proper method, and finally their stability profiles were plotted. The stability profile of each compound was obtained by monitoring the variation of the analyte concentration at different incubation times in plasma samples. Generally, when the substrate concentration was smaller than the Michaelis–Menten constant  and, after application of the same procedure followed for the studied compounds, its degradation plot (SF34) showed a decrease comparable to that found in literature (t1/2 ≤ 120 min.), confirming the activity of plasma enzymes [For the compounds that showed a value of k rate < 0.006 (ln(μM)/min.), a high t enzymes ,25,27,28In the proposed HPLC-MS/MS method, we have used a mathematical algorithm (LEDA) to solve and separate the MS/MS spectra from the unresolved chromatographic peak of a pair of isomers. The development of LEDA involved an investigation on the energetics of the fragmentation pathway and allowed for the selection of the better product ions for each analyte in terms of both sensitivity of detection and specificity, i.e., the capability to distinguish between isomeric compounds. It is noteworthy that the choice of characteristic product ions and the optimal abundance ratios play an important role for the application of the LEDA approach to a multi-component mixture analysis. The developed method was applied in a human plasma drug stability study of two couples of isomers to verify its effectiveness. The obtained results confirm the ability of the LEDA approach to distinguish the isomers, allowing the evaluation of their behavior in plasma samples. It is worth emphasizing that the investigation was carried out with a conventional HPLC-MS/MS system, without using expensive ultra-efficient chromatography systems or ancillary MS techniques . In the proposed approach, we have explored the potential of MS/MS techniques, introducing the “energetic dimension” of the experiment, which proved to be fruitfully employed to solve the MS problems in the recognition of isomers.


The flows of people and material attributed to international tourism exert a major impact on the global environment. Tourism carbon emissions is the main indicator in this context. However, previous studies focused on estimating the emissions of destinations, ignoring the embodied emissions in tourists’ origins and other areas. This study provides a comprehensive framework of a tourism telecoupling system. Taking China’s international tourism as an example, we estimate the carbon emissions of its tourism telecoupling system based on the Tourism Satellite Account and input–output model. We find that (1) the proposal of a tourism telecoupling system provides a new perspective for analyzing the carbon emissions of a tourism system. The sending system (origins) and indirect spillover system (resource suppliers) have been ignored in previous studies. (2) In the telecoupling system of China’s international tourism, the emission reduction effect of the sending system is significant. (3) The direct spillover system (transit) and indirect spillover system’s spatial transfer effects of environment responsibility are remarkable. (4) There is a large carbon trade implied in international tourism. This study makes us pay attention to the carbon emissions of tourists’ origins and the implied carbon trading in tourism flows. Tourism development is accompanied by large-scale, fast-growing and multiple flows of people, material, capital and service, which exerts major and complex impacts on the social economy and ecological environment from the local to the global ,2. From However, previous work at different levels focused on tourism carbon emissions within a particular area, with little attention paid to tourism-integrated systems, and it especially overlooked that of tourism origins ,10. The Given the above considerations, this paper intends to explore the issues of tourism telecoupling in the dimension of carbon emissions. The specific aims of this study are to (1) construct a theoretical framework of the tourism telecoupling system, (2) establish the models of tourism carbon emissions from the perspective of a telecoupling system, and (3) taking China as the example, explore the synthesis effects of international tourism carbon emissions under this framework. There are two main contributions of this study. (1) First, it builds the theoretical framework of tourism telecoupling system and constructs the calculation model of tourism telecoupling system emissions, which expand the perspective and content of tourism system research. (2) Two important variables in tourism carbon emission system are identified, which are the emission reduction in the origins and the implied carbon trading in tourism flows. These help to improve the understanding of tourism system carbon emissions.2e in 2013, which is four times greater than that previously reported [Previous studies on tourism carbon emissions mainly focused on global, national and regional levels ,22. On treported . The resreported ,24.2e emissions by analyzing tourism energy consumption and tourist behavior in New Zealand. Cadarso et al. estimated the contributions of residents and visitors to the tourism’s carbon footprint in Spain, and Meng et al. quantified the direct and indirect CO2 emissions of tourism of China [On the national level, top–down and bottom–up approaches were the most commonly used . The resof China ,27. In gof China . Some reof China ,33,34,35Moreover, the research on the regional level tends to pay attention to environmental vulnerability. Sun found that the island destination leads to a larger share of carbon emissions outside their geographic territory, and Farreny et al. highlighted the significant effects of transport on tourism carbon emissions in Antarctica; approximately 70% are attributable to cruising and 30% to flying ,36. ThesIn recent decades, increased human interactions over long distances have profoundly impacted global socioeconomic and environmental sustainability . TraditiAs a universal paradigm, the telecoupling system has been implemented in diverse fields, such as international trade ,41, resoThis study intends to discuss the tourism telecoupling system from the perspective of carbon emissions. China’s international tourism is adopted as an example to estimate the carbon emissions of the tourism telecoupling systems, which include the inbound tourism, outbound tourism and the whole sectors of international tourism. There are two reasons for choosing the international tourism of China as the case. First, international tourism, as a global activity, is a typical telecoupled phenomenon. Second, the international tourism of China has a large scale, and its partners are distributed worldwide. In recent years, the scale of the inbound and outbound tourism sectors in China has been among the highest in the world, and there exists a stable two-way tourists flow between China and the 189 international tourism partners. Hence, its representativeness and typicality are both significant.The telecoupling framework provides a new perspective for the discussion of tourism system carbon emission. With the deepening of globalization and regional integration, the tourism industry in a given country is increasingly dependent on regions over distances, involving merchandise trade and personnel mobility. Therefore, the telecoupling under the tourism scenario needs to consider the resource supplier system, especially the island tourism destination, which is dependent a lot on the import of various resources. Therefore, this paper improves the telecoupling framework proposed by Liu et al.  in a tou2 emissions stemming from energy consumption [In the context of tourism carbon emission, there is actually implied carbon trading in international two-way tourism flow. In the view of tourism telecoupling system, the reduction in the sending system carbon emission makes these synthesis effects more complex and more interesting. Therefore, the calculation of tourism telecoupling system carbon emissions contains six parts: the sending system, the receiving system, the direct spillover system and the indirect spillover system, the total carbon emissions of the tourism telecoupling system and the implied carbon trading of tourism. In this study, the carbon emissions are the COsumption ,23,51. TSS). This part includes the CO2 emissions reduction in the sending system, resulting from residents’ departing for travel. Based on the statistics of the International Energy Agency (IEA), the daily CO2 emissions of residents include those of electricity and heat production, manufacturing industries and construction, transport, residential, commercial, and public services. The model of SS is as follows:2 emissions of tourists’ residences, including emissions of electricity and heat production, manufacturing industries and construction, transport, residential, commercial, and public services at residences from fossil fuels, and (1) The sending system carbon emissions (RS). This includes the CO2 emissions generated by tourists at destinations. Studies on the carbon emissions of destinations are relatively mature, and the input–output approach is often used. So, this study adopts an environmentally extended input–output approach used in an interesting research made by Sun et al. [2 emissions databases, thereby tracing the CO2 emissions originating from the consumption of tourist products and services. The model of RS is as follows [N matrix of the carbon emissions intensity, N × N identity matrix, N × N MRIO matrix listing the international trade transactions between countries, N × M matrix of the final demand of M global agents  of N products. N × 1 matrix acting as the final demand block of the MRIO system.(2) The receiving system carbon emissions . The DSS contains the emissions of transportation from the origins to the destinations, which is the key part of tourism emissions [DSS model is as follows:(3) The direct spillover system carbon emissions (missions . Referrimissions . The DSSdistance .ISS). According to the statistics of the International Energy Agency, the energy industry (energy exploration) is the biggest contributor to carbon emissions of global trade. Hence, the ISS in this study is the CO2 emissions originating from fossil fuels exploitation by the resource supplier.(4) The indirect spillover system carbon emissions . The TSS is the actual emissions of a certain tourism system. Hence, the TSS would be the sum of RS, DSS, and ISS minus SS.(5) The carbon emissions of the tourism telecoupling system (ICT). Two-way tourism flows between countries/regions not only include economic transaction but also imply spatial transfer of the environmental responsibility. Suppose two countries A and B; the two-way tourism flows make A and B both the sending system and receiving system. The ICT is the difference value between the change in the tourism carbon emissions of country A (the ARS minus the ASS) and the change in the tourism carbon emissions of country B (the BRS minus the BSS).ARS is that when A is the receiving system, ASS is that when A is the sending system, BRS is that when B is the receiving system, and BSS is that when B is the sending system. When ICT > 0, A has an implied carbon trading surplus with B, and when ICT < 0, A has an implied carbon trading deficit with B. When ICT = 0, A has an implied carbon trading balance with B.(6) The implied carbon trading of tourism  in China were captured and analyzed to ensure information saturation. (4) The distance from the origins to the destinations considers the actual distance of round direct flight route between their capitals. The emission factor per unit distance is from Gössling and Peeters [http://data.IEA.org/, accessed on 10 January 2020), which is consistent with the calculation method of IPCC. (2) The tourism energy consumption coefficient is from a journal article [https://worldmrio.com/eora/, accessed on 10 January 2020). It should be noted that since the maintenance and updating of tourism satellite accounts in various countries around the world are not synchronized, the carbon emissions of China’s international tourism are estimated only in 2015.The data of this study come from multiple sources. The first part is tourism-related data. (1) The data of inbound and outbound tourists of China are from the World Tourism Organization. A spot of missing data is supplemented by the China Tourism Statistical Yearbook. (2) The data of tourism satellite accounts are from the World Tourism Organization, Organization for Economic Co-operation and Development, European Union, governmental reports, and journal articles. (3) The length of stay of inbound tourists is obtained from the Tourism Sample Survey of China, while that of outbound tourists is from online travel notes. More than 3000 notes from two major tourism websites  and Japan (0.43 MtCO2) (2/capita), Canada (0.41 tCO2/capita) and Australia (0.36 tCO2/capita) are more than 20 times higher than those of developing countries, such as the Philippines (0.02 tCO2/capita), Cambodia (0.02 tCO2/capita) and Myanmar (0.02 tCO2/capita).The COame year . Conside3 MtCO2) . On a pe2 in China. On one hand, sending system emissions account for about 20% of that of receiving system. This shows that the estimations of the tourism system emissions in the past literature are too high. On another hand, the carbon emission in destinations is more than five times that of in origins. In other words, people’s carbon emissions from tourism activities are more than five times those from daily activities. Therefore, it is of great importance to encourage low-carbon tourism behavior. Regarding the total CO2 emissions of different origins, the total emissions of Pacific Rim countries are generally higher (2), Japan (1.92 MtCO2) and the Russian Federation (1.46 MtCO2) occupying the top three countries. In per capita terms, the emissions of developed countries are also significantly higher than the others. For example, French emissions (0.72 tCO2/capita) are approximately 77 times higher than those of Myanmar (0.01 tCO2/capita).The total carbon emissions of the receiving system are 17.01 Mt, i.e., inbound tourists produce 17.01 MtCOy higher , with Ko2 emissions of the spillover system (direct spillover system and indirect spillover system) are 63.08 Mt. This part is the largest of the inbound tourism telecoupling system of China, and it is more than triple those of the receiving system (17.01 Mt). The spatial transfer of the environmental responsibility caused by tourism development cannot be ignored. In detail, the carbon emissions of the direct spillover system account for 61.38% (38.72 Mt). Moreover, the CO2 emissions of the direct spillover system  are significantly higher than those of destinations, which is consistent with the previous studies [The total CO studies . On the 2, which are approximately three times higher than the sending system emissions of inbound tourists. This accounts for 3.93% of the Chinese total residential carbon emissions in 2015 [2) are the three largest countries that contributed to the sending system emissions of Chinese outbound tourism. Thailand is the preferred destination for Chinese residents to travel abroad in these years. On a per capita basis, Canada (0.23 tCO2), Australia (0.22 tCO2), United States (0.21 tCO2), Italy (0.20 tCO2) and France (0.19 tCO2) rank in the first five countries because of their longer distance from China and people’s longer stay duration. Meanwhile, those of Singapore, Korea and Japan are below half of them.In terms of outbound tourism, the sending system emissions reached 14.10 Mt in 2015. This shows that the outbound tourists reduce Chinese emissions by 14.10 MtCO in 2015 . Specifi2 in their destinations. This number is 1.53 times those saved in the sending system. Meanwhile, this ratio is 3.20 in Chinese inbound tourism. This shows that under the existing conditions, the energy consumption of Chinese residents’ tourism activities and daily activities is closer. In terms of specific countries/regions, as shown in 2), Korea (2.64 tCO2), Singapore (1.38 tCO2), Vietnam (1.16 tCO2) and the United States (1.14 tCO2) are the five largest in the 189 destinations. The receiving system emissions of Thailand stand head and shoulders above others. The per capita emissions of the receiving system show similar characteristics with those of the sending system.The receiving system emissions reach as high as 21.60 Mt, which means Chinese tourists emitted 21.60 Mt COThe carbon emissions of the spillover system (direct spillover system and indirect spillover system) of China’s outbound tourism are 96.97 Mt, of which the direct spillover system emissions reach 70.68 Mt, accounting for 72.89% and the indirect spillover system emissions are 26.29 Mt, accounting for 27.11%. Similar with that of inbound tourism, the spillover system emissions (both direct spillover system and indirect spillover system) are much larger than those of the sending system and receiving system. Meanwhile, an interesting indicator also reveals the particularity of China as a destination. The ratio of spillover system emissions and the receiving system emissions in China’s outbound tourism is 4.49, while that in China’s inbound tourism is 3.71. This shows that the spatial transfer of the environmental responsibility caused by tourism development of China is lower than that of other destinations as a whole. This result is consistent with the fact that the self-sufficiency of China’s tourism-related industries is relatively high.2 . This result is consistent with the study of Sun et al., in which the carbon footprint of global tourism was discussed [The tourism telecoupling system emissions in this study are the actual emissions of China’s inbound and outbound tourism, which are the sum of the receiving system, the direct spillover system and the indirect spillover system minus the sending system. In 2015, the tourism telecoupling system emissions of China’s international tourism reached 179.24 MtCOiscussed . The senOn a specific country basis, the carbon emissions of the tourism telecoupling system between China and another 189 countries showed a spatial heterogeneity. Geographical distance is the key variable of the telecoupling relationship. According to the geographical distance (GD) between the couple countries and the amount of their tourism telecoupling system emissions (TTSE), the 189 countries are divided into four categories: HL , HS , LL , and LS  . High TTICT) of China’s international tourism is the net value of the change in the tourism carbon emissions of China (the RSChina minus the SSChina) and the change in the tourism carbon emissions of another 189 countries (the RSrelevant countries minus the SSrelevant countries). On the whole, China has a large carbon trading surplus in international tourism, reaching 23.26 CO2 Mt in 2015. This indicates that China exported 23.26 MtCO2 by two-way international tourism, accounting for 0.26% of the total CO2 emissions of fuel combustion in China [The implied carbon trading  the proposal of a tourism telecoupling system provides a new perspective for analyzing the carbon emissions of a tourism system. The sending system (origins) and indirect spillover system (resource suppliers) have been ignored in previous studies. (2) In the telecoupling system of China’s international tourism, the emission reduction effect of the sending system is significant. The sending system emissions accounts for 50.27% of the receiving system(destinations), while this part has been ignored in previous research and practice. (3) The direct spillover system (transit) and indirect spillover system’s spatial transfer effects of environment responsibility are significant. These two systems account for 89.29% of the total carbon emissions of the tourism telecoupling system. (4) There is a large implied carbon trade in international tourism. On the whole, China has a carbon-trading deficit in international tourism in 2015, reaching 23.26 MtCO Zealand . Of courThis study has several theoretical and practical implications. First, the telecoupling system perspective is of benefit to the exploration of tourism systems. The tourism telecoupling system expands the perspective and content of tourism research. It is conducive to the in-depth exploration of the hidden elements in tourism systems by taking it as a continuously developing, globally expanding and dynamic complex system . FurtherHowever, there are also some limitations. Although this study hopes to calculate the carbon emissions of a tourism telecoupling system as accurately as possible, the overall estimate may be slightly larger due to the slight differences in the classification of tourism satellite accounts among countries. Meanwhile, the tourism telecoupling system emissions could be explored in a long-time series. Not only the environment interactions but also the economic, social and cultural issues should be discussed. Furthermore, the exploration of the subsystem level of the tourism telecoupling system, such as agents, causes, and effects, should be performed to improve the effectiveness of policies targeting socioeconomic and environmental sustainability from local to global levels.


The main purpose of this paper is to compare the chromatographic properties of capillary columns prepared with polymers with different backbone structures and to demonstrate the possibility of polymer differentiation via inverse gas chromatography. With the use of addition and metathesis types of polymerization of tricyclononenes, two new stationary phases were prepared. The metathesis polymer contained double bonds in the polymeric backbone while the backbone of the addition polymer was fully saturated and relatively mobile. A comparison of the separation and adsorption properties of new phases with conventional gas chromatography (GC) stationary phases clearly indicated their non-polar characteristics. However, the difference in the polymer structure appeared to have very little effect on the stationary phase separation properties, so other parameters were used for polymer characterization. The thermodynamic parameters of the sorption of alkanes and aromatic compounds in both polymeric stationary phases were also very similar; however, the entropy of sorption for hydrocarbons with seven or more carbon atoms was different for the two polymers. An evaluation of the specific surface energy of the stationary phases also allowed us to discriminate the two stationary phases. The modern practice of analytical gas chromatography is essentially a technique performed using wall-coated (WCOT) or porous layer open-tubular (PLOT) capillary columns. The columns available today afford an unprecedented level of durability, inertness, and ease of use. The invention of fused silica capillary columns in 1979 ,3.General information on GC columns can be found elsewhere in the literature , and accAn alternative technique for the preparation of PLOT columns is the synthesis of the stationary phase directly inside the quartz capillary ,7. The ag is widely reported in the literature . Th. Th21]. The thermostability of the new stationary phases was tested by TGA a and by Both new stationary phases were tested for physical ageing as described in the Materials and Methods. However, no changes in selectivity and efficiency were observed when the columns with AP and MP stationary phases were heated within a few hours at increased temperatures up to 175 °C. These data coincide well with the stability test of the AP and MP membranes used in membrane chemistry, which could not detect physical ageing of these polymers .It was reported in a membrane study  that AP The elution order for the test mixture during the AP and MP stationary phases was similar to the elution order for the non-polar stationary phase b–c: the The polarity of the stationary phase is also a very important parameter for chromatographic separations, and it is frequently characterized by McReynolds constants, which are evaluated from the retention times of five probe analytes: benzene, 1-butanol, 2-pentanone, 1-nitropropane, and pyridine . Both APo and the entropy of sorption ΔSo. They are related to the partitioning coefficient of the analyte K (Henry constant) between the moving and stationary phases according to the equationo is the free energy of sorption; R is the universal gas constant; s and Cm are analyte concentrations in the stationary and mobile phases, respectively; and T is the column temperature, K. The partitioning coefficient K correlates with the retention factor k [m to that of the stationary phase VS in the column, and R and to being the analyte retention time and column hold-up time, respectively. Combining Equations (3) and (4) allows one to obtain the relationship allowing the evaluation of the thermodynamic parameters of sorption from the linear correlation between lnk and 1/T:The thermodynamic parameters of sorption for analyte separation in GC are usually evaluated by means of IGC . The thefactor k ,29:K = ko and entropy ΔSo of sorption for the investigated saturated and aromatic hydrocarbons are shown in o and entropy ΔSo of sorption for the n-alkane carbon number. The slopes of the linear dependences are called the methylene selectivity of the stationary phase and are shown in Corresponding straight-line correlations were observed for all of the polymeric stationary phases studied in the research :(6)sD ofCH2 is the cross-sectional area of a CH2 group (m2) [CH2 = 69.919/(568.02–T)0.5 (Å2), N is the Avogadro number, 6.023 × 1023, γCH2 is the free energy of a CH2 group [CH2 = 35.6 + 0.058 (293–T) (mJ/m2).Here, αoup (m2) : αCH2 = H2 group : γCH2 = sD and, correspondingly, had an equal tendency for non-specific interactions with solutes. These values were 1.5 times higher than the free dispersive energy of the polar PG 20M stationary phase. Low values for PEG 20M can be explained by weak interactions of n-alkanes (used as solutes) with the polar surface PEG 20M. The small increase in the free surface energy of AP and MP over SE-30 and PTMSP may be attributed to the non-polar features of the polymer structure.As one can see from D, cm3/mol) is used:3) of the test molecule. In the first step, the graphical dependence of the free energy of adsorption on ∆G = −RTlnkβ on the molar deformation polarization PD for n-alkanes was plotted. The obtained straight-line dependence is called the alkane line (SP) , which are two to three times lower than those values, e.g., for the adsorption of dichloromethane on the quartz surface  were quite close to the literature values.The specific surface energy of the stationary phases was evaluated based on interactions with polarized analytes, which were aromatic hydrocarbons in this study. The carbon backbone of the MP-based stationary phase provided a high number of double bonds , which cane line  and is cine (SP) . The val.5 kJ/m2 ). These New representatives of microporous polymeric matrixes, addition and metathesis polymers of tricyclononene, were synthesized using different catalytic systems and were investigated with the IGC method to differentiate the isomeric stationary phases using chromatographic data. The backbone of the metathesis polymer had numerous double bonds while the backbone of the addition polymer was saturated. The presence of double bonds made the MP polymeric chains rigid and conformationally less flexible. In contrast, the addition AP polymeric chains seemed to be more mobile. However, the difference in the polymer carbon backbone structures had very little effect on the separation properties of the stationary phase. The elution sequence of the test solutes and McReynolds coefficients were similar for both stationary phases prepared from AP and NP polymers. The evaluation of the thermodynamic parameters of sorption such as the enthalpy and entropy of the sorption of alkanes and aromatic compounds, methylene selectivity, and the compensation temperatures did not reveal significant differences between the stationary phases. The only parameter that was able to differentiate the isomeric polymeric stationary phases was the entropy of sorption for hydrocarbons with nine or more carbon atoms. The evaluation of the specific surface energy for aromatic analytes also allowed the discrimination of the polymers. This high similarity between the AP and MP polymers points to the high-shielding double bonds in the MP polymer in contradiction to the clear difference observed between the polymers and the typical polar stationary phases.


Piaractus mesopotamicus) subjected to fasting (four and fifteen days) and refeeding . Our results showed that different fasting and refeeding periods modulate the expression of the genes mtor, rps27a, eef1a2, and cdkn1a. These alterations can indicate the possible protection of the muscle phenotype, in addition to adaptive responses that prioritize energy and substrate savings over cell division, a process regulated by ccnd1. Our study reveals the potential of meta-analysis for the identification of muscle growth regulators and provides new information on muscle responses to fasting and refeeding in fish that are of economic importance to aquaculture.The regulation of the fish phenotype and muscle growth is influenced by fasting and refeeding periods, which occur in nature and are commonly applied in fish farming. However, the regulators associated with the muscle responses to these manipulations of food availability have not been fully characterized. We aimed to identify novel genes associated with fish skeletal muscle adaptation during fasting and refeeding based on a meta-analysis. Genes related to translational and proliferative machinery were investigated in pacus ( Piaractus mesopotamicus, popularly known as the pacu, is one of the most widely cultivated native species, with production in nine Brazilian states [Fish are the most diverse living vertebrates on the planet, with important ecological and economic roles in many nations . Regardin states . The popn states . In addin states . Recentln states ,6, with n states . The skeletal muscle constitutes more than 60% of the teleost body mass . In addimurf1 and mafbx) and a loss of muscle mass. Then, during refeeding periods, muscle mass recovery occurs through the stimulation of anabolic genes (igf-1 and mtor) [Hippoglossus hippoglossus [Salmo salar [Oncorhynchus mykiss [Paralichthys adspersus [Fasting and refeeding studies conducted in our lab showed changes in the classical pathways that regulate the muscle protein balance in the pacu skeletal muscle, which is well-characterized in the literature ,15,16. Fnd mtor) . These ooglossus , Salmo smo salar , Oncorhys mykiss , and Pardspersus , demonstIn finfish aquaculture, the maintenance of the fish muscle mass underpins the sector’s profitability. Thus, understanding the muscle phenotype regulators is essential in order to improve the management techniques. In this context, mRNA profiles in public databases may favor the exploration of markers involved in maintaining the skeletal muscle phenotype and its growth. Among the available methodologies for the mining of the public data to be analyzed, meta-analysis has been used in several areas of science as a statistical method that compiles, compares, and evaluates data from pre-existing and independent studies ,23. ThisPiaractus mesopotamicus. Therefore, this work aimed to identify, through meta-analysis, novel genes associated with the growth and maintenance of the muscle phenotype. We investigated the expression levels of these genes during fasting and refeeding conditions in the pacu fish, http://www.prisma-statement.org/, accessed on 2 February 2020) [The integrative meta-analysis was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses  . The keyhttp://www.ncbi.nlm.nih.gov/geo/geo2r/, accessed on 1 June 2020) was used, which applies the same script  to all the datasets. To select the differentially expressed genes (DEGs) in this system, adjusted p-values (padj) of <0.05 and a |fold change (FC)| of >1.5 were considered . In this analysis, the lists of up- and downregulated genes were combined using the tool due to the similarity of the biological processes in which these genes take part. The Biological Process, Molecular Function, WikiPathway, and KEGG libraries were considered for the selection of the terms, considering the significance value of p < 0.05 (Fisher’s test) . The databases, text mining, experiments, co-expression, and co-occurrence were considered as active interaction sources. The minimum interaction score required was the most reliable (0.900), and the p-value of the PPI enrichment indicated the statistical significance provided by STRING (<1.0 × 10−16). For a better visualization, the nodes without connections were hidden, and the names of the proteins were retained. Based on this network and with the support of the literature, key genes were selected for the investigation of the muscle of pacus (Piaractus mesopotamicus) subjected to fasting and refeeding conditions through RT-qPCR. Based on the DEGs of each dataset, the enrichment of the pathways was performed using the Fish EnrichR tool (’s test) . Based o’s test) . These c’s test) , had the3 tanks containing 30 pacus. All the fish underwent a 15-day adaptation period with ad libitum food two to three times a day, following the diet previously described in the publications of our research group [The experiments were carried out on animals and their muscle tissue from previous studies ,16 accorAfter this adaptation period, the youngest fish 12.6 g ± 2.24) were separated into the following groups: control , fasting  and refeeding . Meanwhile, the older fish (28.5 g ± 8.8) were divided into the following groups: control , fasting , and refeeding . Fish of different weights were selected to ensure that the investigations would cover different growth periods. In this context, the lengths of the fasting and refeeding periods were selected based on the size of the fish, considering that these nutritional changes would modify the muscle transcriptional signature without triggering the death of the fish before their experimental evaluation. In addition to these aspects, our research group has already obtained significant results by evaluating the classical genes of the skeletal muscle in these periods of fasting and refeeding in pacus  g ± 2.24, suggest® reagent , following the manufacturer’s instructions. To quantify and analyze the purity of the extracted RNA, a NanoVueTM Plus spectrophotometer  was used, which provides these parameters through absorbance at 260 nm (amount of RNA) and 280 nm (amount of protein). To assess the integrity of the extracted RNA, 1% agarose gel electrophoresis and capillary electrophoresis were used with the 2100 Bioanalyzer  were used, which provided an RNA integrity number (RIN) based on 28S and 18S ribosomal RNAs. The samples with a 260/280 ratio of ≥1.8 and RIN of ≥7 were considered for further analysis. To prevent any genomic DNA from contaminating the samples, the extracted RNA was treated with RQ1 RNase-Free DNase . RNA reverse transcription was performed using a GoScriptTM Reverse Transcription System , according to the manufacturer’s guidelines. To investigate the genes selected in the in silico analysis, we extracted the total RNA from pacu muscle samples previously used in other experiments ,16 using® qPCR Master Mix , and the primers for the respective targets , eef1a2 , ccnd1 (Cyclin D1), mtor , and cdkn1a (cyclin-dependent kinase inhibitor 1A) were designed based on the Piaractus mesopotamicus transcriptome obtained by our research group [https://bioinfo.ut.ee/primer3-0.4.0/, accessed on 20 October 2021) and NetPrimer  , in which the expression was normalized based on the ppia mRNA, the expression of which remained constant among all the samples.The expression level of the mRNAs was evaluated by quantitative real-time PCR (qPCR) using the QuantStudio™ 12K Flex Real-Time PCR platform , with two technical and five biological replicates. All the qPCR reactions were performed according to the Minimum Information Guidelines for Publishing Quantitative Real-Time PCR Experiments (MIQE) . The cDN targets  were synch group . The tooCA, USA) . The read, 2ΔΔCt  using Dap < 0.05) .For the gene expression analysis, comparisons were performed between the experimental groups of late juveniles C, F15d, R6h, and R15d, respectively, and between the early juvenile groups C, F4d, and R3d. The Kolmogorov–Smirnov normality test was performed for all the genes evaluated, and one-way ANOVA tests with Tukey’s post-test were applied to the data with a normal distribution. The Kruskal–Wallis test and Dunn post-test were applied to the data that did not have a normal distribution. The data are presented as the mean ± SD, and for all the tests, the statistical significance was set at 5%  and ribosomes (lower region), demonstrating the interaction between these two processes. Furthermore, such genes were responsible for enriching the common cellular processes of proliferation and protein synthesis in the different species subjected to different treatments . rps27a, which connects the two large clusters of the network, and based on their close interactions, the other targets selected were ccnd1, cdkn1a, and eef1a2. The selected key genes were investigated using the fasting and refeeding conditions, which involve proliferation and protein synthesis processes and have great relevance in aquaculture.From this network, key genes were selected by considering their topology and the possible importance of these genes for fish skeletal muscles, according to the literature. This selection involved mtor and eef1a2 expression in the skeletal muscle of the early juveniles in the F4d group compared to the C and R3d groups, while the expression of rps27a was reduced in the R3d group compared to the F4d group. Furthermore, considering the genes associated with the cell cycle, a reduction in the ccnd1 expression was observed in the R3d group compared to both the C and F4d groups. The expression of cdkn1a, meanwhile, was downregulated in the R3d group compared to F4d  compared to the R6h group. Regarding the rps27a ribosomal gene, there was a decrease in its expression in the R15d group compared to the F15d group. On the other hand, the expression of the eukaryotic elongation factor eef1a2 showed no change in its expression between the different groups. Regarding the cell cycle and replication regulators, the expression of the ccnd1 transcripts showed an increase in the F15d group and the R6h group compared to the C and R15d groups. Meanwhile, the expression of cdkn1a was downregulated in both the R6h and R15d groups compared to the C and F15d groups , the d groups .The meta-analysis using the fish skeletal muscle microarrays allowed us to identify the critical connection between ribosomes and the cell cycle, as evidenced by the molecular interaction network. This relationship was also observed by a group of researchers who described the limiting role of the ribosomal machinery in cell proliferation. This association becomes more evident when considering the roles of many ribosomal proteins (RPs) in communicating the disturbance in ribosome biogenesis to the cell cycle . Furtherrps27a and eef1a2, mTOR (the mechanistic target of rapamycin kinase) plays an essential role in ribosomal biogenesis by regulating the translation of RPs and the transcription of rRNAs  [mtor transcript was also evaluated. Based on this analysis, the upregulation of mtor in the F4d compared to the C group may be involved in maintaining the translational capacity, defined as the number of ribosomes present in the tissue [mtor in the R3d group, the levels of which became similar to those of the control group. On the other hand, the lack of alteration in the expression of this gene in the F15d group compared to the control may be related to the more severe catabolic environment compared to the F4d condition. In this group, it is possible that the processes maintaining the survival of the organism, as a whole, overcome the protection of the muscle mass.In addition to the other targets obtained and investigated in this work that are related to ribosomes, such as al RNAs) . Althouge tissue , during e tissue ,36,37. Trps27a was similar when comparing F4d with R3d and F15d with R15d. In all cases, the expression of this gene increased during fasting. Similar to other RPs, RPS27A plays an important structural role in the ribosomes, and its participation in the crosstalk between ribosomal stress and the cell cycle has already been described [rps27a is also abundant in the muscle cells, demonstrating the role of the translation machinery in the tissue and the presence of multiple copies of rDNA in the skeletal muscle [rps27a expression was observed in the R3d and R15d groups compared to F4d and F15d, respectively. These results may be associated with the reduction in stress in the context of food availability in the refeeding groups, as was also observed in the refeeding of Gadus morhua after fasting periods, which promoted the resumption of ribosomal pro-biogenesis conditions in the muscle of this fish [Interestingly, the expression of escribed . Accordiescribed . In addiescribed . Our datl muscle . Furtherhis fish . eef1a2 , another component of the translational machinery transcript, was also evaluated in our study. In its canonical function, eef1a2 acts through the elongation of the polypeptide chains, ensuring that the tRNAs and their respective amino acids correctly couple on the ribosomes [eef1a2 expression, followed by a reduction in the expression of the R3d group. According to some authors, Eef1a2 participates in the maintenance of muscle mass in rats, and its absence triggers atrophy [The expression of ibosomes . In this atrophy ,44. Simi atrophy .mdx mice, eef1a2 overexpression improves Duchenne muscular dystrophy, highlighting the relevance of this gene to tissue maintenance, formation, and morphology [eef1a2 in the conservation of the muscle mass and morphology, the increase in its expression in the F4d group may indicate an attempt to maximize protein synthesis during food restriction. In contrast, the food availability in the R3d group reduced its expression to levels similar to those of the C group. These data, as well as the lack of alteration in eef1a2 in the more extended periods of fasting and refeeding, corroborate the transcriptional modulation of mtor. In the shorter periods of fasting and refeeding, it was still viable for cells to maintain the muscle mass through the utilization of amino acids from muscle catabolism during the short protocol [Furthermore, in rphology . Given tprotocol ,47, someCcnd1 (cyclin D1) is one of the genes that coordinate cell proliferation, connecting nutrient availability in the environment with cell division. The expression of this cyclin varies throughout the cell cycle, with high levels of ccnd1 found in the G1 phase. In the transition from the G1 to S phases, there is a reduction in cyclin expression, which allows for DNA synthesis. During the entire S phase, the ccnd1 levels remain low, thus increasing the cyclin expression in the transition from S to G2. At this point, the cyclin D1 levels only increase due to environmental signals, such as the presence of food [ccnd1 expression was observed in the R3d group compared to the F4d group, while high levels of ccnd1 were identified in the F15d and R6h groups compared to the C and R15d groups, respectively. The increased ccnd1 expression in the F15d group may represent an adaptive measure that saves energy and substrates in the skeletal muscle of fish under fasting conditions. In senile human cells, this was reported to occur due to high levels of cyclin D1, which prevent DNA synthesis and delay cell cycle progression during the G1 phase [ccnd1 can compromise the proliferative processes required for the fish’s muscle growth. However, the progressive reduction in these cyclin levels resulting from refeeding, in both the short (R3d) and long (R15d) periods, may be associated with the nutritional restoration and continuity of the cell cycle, thus allowing muscle growth to resume. It should be noted that the lack of change in ccnd1 expression in the F4d group compared to the C group may be associated with this less severe period of fasting, in which proliferative processes may not be affected. As in the case of the genes associated with the protein synthesis machinery, changes in the nutritional status of cells also modulate the expression of cell cycle regulators . Ccnd1  levels was observed in the R3d group compared to the F4d group and in the R6h and R15d groups compared to the C and F15d groups, respectively. In addition to regulating the cell cycle, Cdkn1a acts on cell survival, with the mRNA of this gene expressed in both the liver and skeletal muscle of rats fasting for 24 and 48 h [ppar in the red muscle of the pacus during fasting, a muscle compartment that is less affected than white muscle [cdkn1a, the literature data partially corroborate our results, demonstrating the important reduction in the cdkn1a levels during refeeding [cdkn1a in the F4d and F15d groups, similar to the levels in the C group, may be associated with muscle adaptation to food restriction, and after food is made available to the muscle fibers, the transcriptional expression of cdkn1a decreases to very low levels. Regarding the final investigated target, a reduction in the and 48 h . Accordiand 48 h . PPARα iand 48 h . In thise muscle . Though efeeding . In thisMicroarray data provide unprecedented opportunities to explore new biological questions. Although our strategy has limitations associated with the low number of datasets available in the databases, the key genes selected in vitro were validated in pacus subjected to fasting and refeeding conditions, reinforcing the findings described here and the prospective power of the meta-analysis.Our meta-analysis was an efficient strategy for the data mining and identification of non-classical genes associated with the maintenance of the skeletal muscle phenotype and growth in fish. Specifically, we demonstrated that ribosomal and cell cycle genes act in these processes in different species and under distinct experimental conditions. Furthermore, the investigation of the selected targets in the pacus demonstrated specific transcriptional modulations in short and extended fasting and refeeding periods, which can be associated with the maintenance of basal protein synthesis and tissue protection, together with energy saving. Together, our results help researchers to understand the adaptations of the skeletal muscle to the manipulation of food availability and are relevant for strategies that employ periods of fasting and refeeding in finfish farming and the natural environment.