Taking the perspective of corporate social responsibility and institutional theory, this research establishes an innovative relationship between variables such as charitable donation, political connection and crisis spillover effect of firms through quantitative analysis using the event study method, regression analysis and the Heckman two-stage model. Taking 8 food safety incidents from 2011 to 2016 as research samples, this paper studies the impact of food safety incidents on the market value of both firms under crisis and their competitive firms, as well as the influence of political connection and charitable donation. Based on the current situation that the product crisis or reputation crisis of a firm will, inevitably, affect the market performance and value of its competitive firms in the same industry, this paper attempts to answer questions such as “what kind of firms are capable of minimizing this negative influence?” “will the political connection of competitive firms exert a positive or negative impact?” and “can actions taken before the crisis, such as charitable donation of competitive firms, help these firms in reducing the harm?” The conclusions are as follows: first, the occurrence of food safety incidents not only has a negative impact on the market value of the crisis firm, but also has a negative spillover effect on the competitive firm; second, charitable donations made by the competitive firm before the crisis demonstrates a positive competitive effect on the competitive firm, and the intensity of such charitable donations is positively correlated with this positive competitive effect; third, the political connection of the competitive firm has no significant impact on the crisis spillover effect. These findings provide enlightenment for the operation and management of firms in the food industry. Taking the perspective of corporate social responsibility and institutional theory, this research establishes an innovative relationship between variables such as charitable donation, political connection and crisis spillover effect of firms through quantitative analysis using the event study method, regression analysis and the Heckman two-stage model. Taking 8 food safety incidents from 2011 to 2016 as research samples, this paper studies the impact of food safety incidents on the market value of both firms under crisis and their competitive firms, as well as the influence of political connection and charitable donation. The conclusions are as follows: first, the occurrence of food safety incidents not only has a negative impact on the market value of the crisis firm, but also has a negative spillover effect on the competitive firm; second, charitable donations made by the competitive firm before the crisis demonstrates a positive competitive effect on the competitive firm, and the intensity of such charitable donations is positively correlated with this positive competitive effect; third, the political connection of the competitive firm has no significant impact on the crisis spillover effect. These findings provide enlightenment for the operation and management of firms in the food industry.As a matter of fact, crisis management of firms is a research subject that is constantly updated and iterated. Researches on individual firms are gradually extended to researches on the industry, and crisis spillover has gradually become a new research field in crisis management of firms. Faced with highly-developed social media networks and closely-connected supply chains, firms can hardly fight any battle alone in the context of industrial crisis. This paper mainly explores the following four major issues as regard to this circumstance. First, how would market value of the firms under crisis change with the occurrence of food safety incidents. Second, with such incidents happening, whether competitive firms will be affected by the crisis spillover effect; and if so, will contagion effect or competition effect take place? Third, how would competitive firms' political connection influence such crisis spillover effect? And finally, how would charitable donations made by competitive firms impact the crisis spillover effect?It is found that the biggest and most harmful food safety incident in American history happened in the well-known fast-food chain Jack-in-the-box and has caused illnesses to more than 700 people and death of 4 children across America, due to excessive addition of Escherichia coli in food. This fast-food chain has not only lost tens of billions of dollars in sales, but also continued to pay the price for such incidents many years later . The eveH1a: Incident of food safety crisis has a negative impact on the market value of the crisis firm.H1b: With the passage of the incident, its negative impact on the market value of the crisis firm generally shows a weakening trend.H1c: The occurrence of food safety incident creates crisis spillover effect on the competitive firms, and the contagion effect is greater than the competition effect.First, the relationship between political connection and crisis spillover is two-sided. On one hand, political connection may relieve the contagion effect of the crisis spillover. When a crisis occurs to the firm, investors may tend to believe that firms with political connection will be protected by the government from being involved in the crisis . SimilarSecond, firms will usually adopt strategies that are in consistency with their own institutional advantages. There is a mutually dependent relationship between politically connected firms and the government. In order to achieve its economic goals, the government will exert pressure on the firms, and in the meanwhile, it will also deregulate or offer certain preferential policies to such firms. In this scenario, consumers' perception of the product quality of the firm will be affected by the deviation of its political connection, which will create a bad impression of the firm. In addition, people tend to have a natural distrust of politically connected firms and believe that such firms are likely to engage in unethical behaviors . The criH2a: Political connection of the competitive firm has no significant impact on the average cumulative abnormal rate of return of the competitive firm.H2b: Political connection of the competitive firm has a positive impact on the average cumulative abnormal rate of return of the competitive firm.H2c: Political connection of the competitive firm has a negative impact on the average cumulative abnormal rate of return of the competitive firm.Charitable donation is an important part of corporate social responsibility, while the demonstration of corporate social responsibility is very likely to weaken the negative spillover effect of the crisis . On one A crisis may be attributed to factors related to moral issues or capability issues. A moral-issue related crisis involves acts of firms that are contrary to the existing moral standards of consumers, such as those with dishonest conducts; while a capability-related crisis involves products offered by the firm which cannot meet the perceived expectations of consumers. If the crisis incident is considered to be a moral crisis, the halo effect formed by the previous actions of the competitive firm representing its corporate social responsibilities will be helpful in separating it from the impact of the crisis. If the crisis is attributed to capability issues, then, charitable donations can effectively alleviate the crisis spillover effect by affecting consumers' perception of the brand or firm under crisis. Some scholars have proposed that consumers' evaluation of the brand or firm under crisis will form a hypothesis-confirming context, and it is based on this background knowledge that consumers will understand the crisis; thus, this hypothesis-confirming context will help reduce the negative evaluation of the brand or the firm . More imCharitable donation is an important act representing corporate social responsibility, which affects consumers' attribution, and in turn, consumers' perception of the brand and the firm. Once a crisis occurs, due to information asymmetry in the market, consumers tend to use their existing rigid knowledge to evaluate relevant competitive firms, which will indirectly affect the spillover effect of the crisis . The criH3a: Charitable donation made by the competitive firm before the crisis will positively influence the average cumulative abnormal rate of return of the competitive firm.H3b: The intensity of the charitable donation made by the competitive firm before the crisis has a significant influence on the average cumulative abnormal rate of return of the competitive firm.H3c: The greater the intensity of the charitable donation made by the competitive firm before the crisis, the greater the positive influence it will have on the average cumulative abnormal rate of return of the competitive firm.This paper takes 8 firms under crisis related to food safety incidents as the research object, while selecting their corresponding competitive firms, respectively, with the same or similar range of business operations, and studies the spillover effect of the food safety incidents based on the stock price changes of both the crisis firms and the competitive firms.After sorting out news reports and government regulatory releases on food safety incidents in the food industry from 2010 to 2016, and excluding food safety incidents of non-listed companies and non-mainland A-share listed companies, eight food safety incidents of listed companies were eventually selected as the research object, with a total of 70 research samples.Three kinds of variables are involved in this study, namely, the core independent variables, dependent variables and control variables. Based on the extant literature, these three types of variables, which are used in subsequent empirical study, are defined as below. According to the extant literature, the event study method typically applies to two main types of events. The first type of events in all samples are of the same nature, but maybe with different dates of occurrence. The second type is a single event, that is, the events and times to be studied in all samples are the same. This paper studies eight food safety events in crisis firms, all of which are associated with the spillover effects of food safety events. So they are typically similar events, but with inconsistent time of occurrence.The estimation period and time window period need to be defined to reflect the influence of events with the changes of stock prices during the studied period. The date of occurrence of the event should be included in the event period, which includes a period of time before and after the event. The stock price some time before the event can capture the responses of the capital market on the eve of the event, which may affect the stock price due to leakage of information and other reasons. The stock price for a period of time after the event can clearly reflect the attitude of the capital market toward such event. The estimation period is defined to estimate the rate of return before the event occurs by using the data of the estimation period. The expected rate of return is subtracted from the actual rate of return after the event to obtain the abnormal rate of return brought by the event. In this study, the event window period is defined as 21 days before and after the event day, i.e., the event window is , and the estimation period is defined as 70 days to 11 days before the event, i.e., the estimation window is .This paper takes 8 A-share listed firms encountering crisis with food safety events as the main research object, and studies the spillover effect of food safety events based on the stock price changes of crisis firms and competitive firms by associating the crisis firms with different numbers of competitive firms within the same or similar business. After excluding issues related to incomplete data and so on, 70 research samples were eventually obtained according to classification based on the similarity of main businesses among the firms.Event study method is a research method used to analyze the impact of specific events on the company's stock price, which offers a means to measure the change of the firm value. In a rational financial market, the impact of an event will be immediately reflected in the stock price, so the impact of the event can often be considered through the change of the stock's return on equity . UsuallyThe market model is the most complex one among the three models, and it is also the most widely used research model in the event study method , 19. Theit is the expected return of stock i on day t; Rmt is the market rate of return on day t;αi and βi are both estimating parameters; ξit is the residual term. In this paper, different indicators were selected to represent the market rate of return according to different indicators actually used by different exchanges for the listed companies. To be specific, for listed companies in Shanghai Stock Exchange, Shanghai composite index rate of return is used as the indicator of the market rate of return; and for listed companies in Shenzhen Stock Exchange, Shenzhen component index rate of return is used as the indicator of the market rate of return.In the equations shown above, RIn the event study method, calculating the abnormal rate of return of stocks is a key step. Abnormal rate of return refers to the difference between the actual rate of return and the expected rate of return, which is shown as follows.it is the abnormal rate of return for stock i on day t of the incident period; Rit is the actual rate of return for stock i on day t of the incident period; and E(Rit) is the expected rate of return for stock i on day t of the incident period.In the above equation, ARi,T1 < t1 ≤ t2 ≤ T2, according to accumulated period of time (shown as follows).In order to more intuitively show the impact of the incidents on stock value, while studying the abnormal rate of return of stocks during the incident period, it is also necessary to calculate the cumulative abnormal rate of return of sample firms CARAccording to the known average abnormal rate of return, the time can be further aggregated to obtain the cumulative average abnormal rate of return of individual stocks in the incident period (shown as follows).In the above equation, CAAR represents the cumulative average abnormal rate of return, and N represents the number of days.Through the above calculation, the cumulative abnormal rate of return and average cumulative abnormal return of the sample in the incident period can be obtained see .t-test to determine whether the average cumulative abnormal rate of return is significantly different from 0. The results are shown in After obtaining the average abnormal rate of return and the cumulative average abnormal rate of return, a statistical test should be carried out to verify whether the incident has an impact on the stock value at a certain significance level. This paper uses P-value represents the level of significance. Through the test of the above statistics, it is found that during the incident period, food safety incidents have a significant impact on the stock prices of both crisis firms and competitive enterprises, and there is a negative contagion effect presenting on the competitive firms. Within  days of the incident period, the normalized average abnormal rate of return is significantly different from 0 at the 90% confidence level; within  days of the incident period, the normalized average abnormal rate of return is significantly different from 0 at the 95% confidence level, and even more significantly different from 0 at the 99% confidence level within the day before and after the incident. Therefore, hypothesis H1a is verified, i.e., food safety crisis of the firm has a negative impact on the market value of the crisis firm. As can be seen from In the above table, mean value represents the standardized average abnormal rate of return, T value represents the standardized average abnormal rate of return, and It can be noticed through the performance of the competitive firms that, within  days of the crisis period, the average abnormal rate of return of the competitive firms after standardization is significantly different from 0 at the 95% confidence level, and is significantly negative. For these competitive firms, the average cumulative abnormal rate of return also shows an obvious negative relationship. Therefore, H1C is supported, i.e., the occurrence of food safety incidents of crisis firms has a spillover effect on their competitive firms, and seen from the overall picture, the contagion effect is greater than the competition effect.The above quantitative research mainly explores our first research question, that is, after the occurrence of food safety incident, whether the crisis firm will bring about a spillover effect on the competitive firms. The results show that the crisis firm causes an obvious contagion effect on the competitive firms; however, with the development of the incident and the measures taken by the competitive firms, this contagion effect weakens in a gradual manner. Unfortunately, the utilization of the event study method can only explore issues such as the market value of competitive firms caused by the food safety incident; while factors affecting the change of market value of competitive firms involve various comprehensive facets. Therefore, based on the above research, our next step is to further study the impact of the characteristics and actions of competitive firms on their market value, i.e., whether the charitable donation of competitive firms before the crisis can regulate the crisis spillover effect; and if yes, how this spillover effect can be regulated, and whether the greater the donation amount is, the stronger this effect will be; in addition, in what direction the political connection of competitive firms will regulate the crisis spillover effect? Based on these two research questions, an empirical study will be carried out as below.In order to study the impact of charitable donations and political connections of competitive firms before the crisis on their average cumulative abnormal rate of return, a regression equation model is built for empirical analysis. The detailed model is shown as follows.i represents the average cumulative abnormal rate of return of competitive firms, where i = 1,10; Pol indicates the political connections of key executives of the competitive firm during the crisis period; if Don represents whether the competitive firm had made charitable donations in the previous year before the crisis occurred; size indicates the size of the competitive firm; Debt represents the asset liability ratio of the competitive firm; CF represents the cash flow of the competitive firm; OC represents the equity concentration of the competitive firm; incident is the incident identifier. βi represents the regression coefficient to be estimated, where i = 1,2,3,…,8; ε is the random residual term.In this equation, PCARThis paper uses EViews 7.0 to carry out ADF test. It is found that the individual stock returns and relevant market returns of the sample firms remain series stationarity, therefore, corresponding correlation regression analysis can be carried out. Before the econometric analysis, descriptive statistics is conducted to observe the sample data.Before regression analysis of the equation, the correlation of each variable must be determined through correlation analysis. Correlation analysis aims to test whether there is a relationship between each variable, and uses correlation coefficient to quantify the strength of this relationship.According to It can be seen from The above regression analysis has verified that charitable donation actions of competitive firms will alleviate the negative effects of crisis spillover. However, the difference made by the positive roles of different levels of donation intensity remains to be explored. In view of this, this study applies the Heckman two-stage model for regression analysis. The application of the Heckman two-stage model mainly involves consideration of the following two aspects. First, this model takes the virtual variable of “donate or not” as the explained variable. When there is donation made by the competitive firm before the crisis, the variable is set to be 1, otherwise it is set to be 0. According to the above, a probit model recorded as model 1 is established to study the impact of donation made by the competitive firm before the crisis on the crisis spillover effect. Second, model 2 or 3 are established on the premise that the competitive firm has made charitable donation, that is, the virtual variable of ifDon is 1; then the variable of real charitable donation intensity “Don” is used as the explanatory variable to examine the specific impact of the intensity of the charity donation made by the competitive firm on the spillover effect.The regression results are shown in In terms of control variables, it can be seen in model 1 that the size, cash flow and equity concentration of the firm are significantly positively correlated with whether the firm donates, indicating that the larger the firm size, the more sufficient the cash flow and the higher the equity concentration, the more likely the firm is to make charitable donations. In models 2 and 3, firm size, cash flow and equity concentration are positively correlated with the average cumulative abnormal rate of return of competitive firms, which is consistent with the results of previous research.Through methods such as event study, regression analysis and Heckman two-stage model, this paper explores the crisis spillover effect of food safety incidents on firms in the food industry, and conducts an in-depth study on the impact of political connections and charitable donations of competitive firms on their average cumulative abnormal rate of return. It is found that the occurrence of food safety incidents will have a negative impact on the market value of crisis firm, but with the passage of the incident, the effect of the negative impact continues to weaken. The main reason is that after the incident, crisis firms will take effective countermeasures, such as clarification, apology, distinction, etc.; in addition to the fact that consumers' attention is limited after all, both consumers and the media will eventually pay less attention to the incident; and with involvement of other influencing factors, the negative effects will be weakened with the passage of time. Furthermore, the occurrence of food safety incidents will also exert an impact on competitive firms inside and outside the industry. The results show that the overall crisis spillover effect of food safety incidents on competitive firms is a negative one, to be exact, a contagion effect. It is noticed from the overall sample that, in the first 3 days of the incident, due to the possible early disclosure of the incident information and the small-scale spread of information related to the incident, consumers will have a sense of distrust of competitive firms in the same industry. However, with the gradual unfolding of the incident, consumers and investors will come to understand that the incident may only be caused by only an individual firm or a small group of firms due to some illegal operation. After such clarification of facts, the negative impact of the incident will be gradually weakened.In addition, our research results show that the political connection of competitive firms before the crisis has no significant effect on their average cumulative abnormal rate of return during the crisis period. In other words, political connections of competitive firms play no significant positive or negative role in influencing average cumulative abnormal rate of return during the crisis period. This may due to the fact that there are too few samples collected in this paper, and the implicit impact of political connection on the yield of the competitive firms is yet to be accurately captured. However, the donation actions made by the competitive firms before the crisis will increase their cumulative abnormal rate of return during the crisis, meaning that the charitable donation actions of competitive firms will cause significant positive effect. The greater the intensity of such charitable donation made by the competitive firms before the crisis, the more positive the impact on their average cumulative abnormal rate of return; and the significance of this impact increases with the extension of the incident window.On the basis of quantitative and empirical research, this paper verifies the existence of crisis spillover effect, discusses the relationship between political connection, charitable donation and crisis spillover effect, and reaches a few practical conclusions. Although only the food industry is explored, this study still provides useful implications for firms in various industries in avoiding negative spillover effect of crisis and striving for positive spillover effect in the future. To be specific, this study provides managerial implications at both industrial and firm levels.At the industrial level, the industry should strengthen the norms of industrial autonomy. Food industry, especially, is an industry with a high incidence of crisis. Once a food safety incident occurs, it will not only put the crisis firm into business meltdown, but also affect a large number of competitive firms in the same industry and bring a significant impact to the whole industry. In detail, trade associations should play an active role in strengthening the autonomy and standardization of the industry, constructing the review mechanism of the industry, and guiding the whole industry to form a high-quality business model, which takes quality as the foundation and services as the guarantee.Seen from the firm level, first of all, firms should lay absolute emphasis on product quality. Quality is the foundation of a firm's survival, and ensuring product quality should be considered the most fundamental and prioritized obligation for a firm. To be specific, firms should establish an effective safety management system to ensure product quality from various aspects such as the sources of raw materials, production, processing, distribution and so on. Second, attention should be paid to information related to industrial crisis. Firms should not develop themselves behind closed doors; instead, attention should always be paid to the dynamics of competitive firms through sensing and collecting all kinds of information related to potential and existing crisis, grasping the public opinion guidance of the media and the public, and formulating and taking effective responses in a timely manner. Third, firms should actively undertake corporate social responsibilities. Our research conclusion shows that actions related to corporate social responsibilities in such form as charitable donation can effectively reduce the negative impact during the crisis. Through actively shouldering social responsibilities, firms are able to establish a decent corporate image, play a positive external role in the crisis, thus, protecting or buffering the damage of the crisis.The limitations of this study are mainly as follows. From the perspective of sample size, only a limited number of incidents of listed companies are used as our samples, which has brought difficulty especially in the observation stage. Also, since most of the incidents studied are individual and independent ones, so the specific performance of systematic incidents in different incident windows can seldom be displayed. Therefore, according to the above-mentioned issues, subsequent research can expand its scope to include not only the domestic A-share market, so as to obtain more samples. In addition, we need to control the types of events in different classifications in order to further study the corresponding disparities.Furthermore, from the perspective of research scope, the sample of competitive firms selected in this paper are firms with the same or similar main businesses in the industry. However, the scope of spillover effect is actually far more than this. Upstream and downstream firms in the same industry and related firms outside the industry will also be implicitly implicated. Future studies can be conducted through expanding the research scope and classifying the type of incidents in order to explore more distinctive scenarios. In addition, the impact of upstream and downstream firms within the industry and relevant firms outside the industry can also be studied. From the perspective of research methods, this paper mainly uses the event study method to test the spillover effect, and then verifies the existence of such spillover effect through constructed statistics, namely, standardized abnormal rate of return. However, due to the immaturity of China's stock market, our research results may also be affected by other factors within the relatively clean event window. For example, there may be a deviation in calculating the cumulative abnormal rate of return of individual stocks. For the sake of robustness, this issue should be further considered in the future. Therefore, other statistical methods are suggested to be utilized in the future.Last but not the least, from the perspective of research content, this paper also studies the impact of such variables as political connection and charitable donation on crisis spillover effect; other factors that may also cause fluctuations in stock prices should be taken into account such as the impact scope and significance of the food safety incidents, the respective industrial status of crisis firms and competitive firms and so on. However, due to the difficulty of obtaining these control variables, these variables are not added in this paper for the sake of time and energy. In the future, more firms should be considered and appropriate indicators should be selected for classification-based researches.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.RX was in charge of the formulation of the general research topic, the construction of the research framework, and the proposing of the theoretical hypothesis. MW generally contributes to the construction of the theoretical framework based on the in-depth accumulation of a large volume of literature reading and analysis and contributes a lot in the data analysis process. LL was in responsible for the overall development of this study, including the selection of research angle, research dimensions, the planning of sample collection, data analysis and proof-reading, and polishing of the whole paper. DW was in charge of data collection, analysis of this study, and responsible for all the procedures taken during data collection. All authors contributed to the article and approved the submitted version.This research is financially supported by the National Natural Science Foundation of China (Grant number: 71773115), the National Natural Science Foundation of China (Grant number: 71973129), and the Major Bidding Project of National Social Science Fund of China (Grant number: 20&ZD124).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Ginkgo biloba displayed extremely potent CES2 inhibition activities and high specificity over CES1 (>1000-fold). Further investigation showed that ginkgetin, bilobetin, sciadopitysin and isoginkgetin potently inhibited CES2-catalyzed hydrolysis of various substrates, including the CES2 substrate-drug irinotecan. Notably, the inhibition potentials of four biflavones against CES2 were more potent than that of loperamide, a marketed anti-diarrhea agent used for alleviating irinotecan-induced intestinal toxicity. Inhibition kinetic analyses demonstrated that ginkgetin, bilobetin, sciadopitysin and isoginkgetin potently inhibited CES2-catalyzed fluorescein diacetate hydrolysis via a reversible and mixed inhibition manner, with Ki values of less than 100 nM. Ensemble docking and molecular dynamics revealed that these biflavones could tightly and stably bind on the catalytic cavity of CES2 via hydrogen bonding and π-π stacking interactions, while the interactions with CES1 were awfully poor. Collectively, this study reports that the biflavones isolated from Ginkgo biloba are potent and highly specific CES2 inhibitors, which offers several promising lead compounds for developing novel anti-diarrhea agent to alleviate irinotecan-induced diarrhea.Human carboxylesterase 2 (CES2), one of the most abundant hydrolases distributed in the small intestine, has been validated as a key therapeutic target to ameliorate the intestinal toxicity caused by irinotecan. This study aims to discover efficacious CES2 inhibitors from natural products and to characterize the inhibition potentials and inhibitory mechanisms of the newly identified CES2 inhibitors. Following high-throughput screening and evaluation of the inhibition potency of more than 100 natural products against CES2, it was found that the biflavones isolated from  Human cntestine . Currentntestine . Howeverntestine . Therefo50 values are at the micromole levels), which motivates us to find more efficacious CES2 inhibitors. Recently, we have carried out high-throughput screening campaign to evaluate the inhibitory potentials of more than one hundred natural products on CES2. Among all tested natural products, four biflavones  isolated from Ginkgo biloba leaves were found with the most potent CES2 inhibition activities  for CES2, a wide variety of natural products  with strong to moderate CES2 inhibition activities were discovered . Howevertivities . It is w effects . Thus, tGinkgo biloba against CES2, as well as to characterize the inhibitory mechanisms of these biflavones against CES2. For these purposes, a set of inhibition assays were used to assess the inhibition potentials and specificity of four biflavones against CES2, inhibition kinetic analyses, ensemble docking and molecular dynamics simulations were used to explore the action mechanisms of these four biflavones against CES2. All these studies will be advantageous for deciphering the interactions of four biflavones with CES2 inhibition, while the key findings present here will be promising to develop novel CES2 inhibitors for ameliorating CES2-associated drug toxicity.This study aimed to assess the inhibition potentials and specificity of four biflavones isolated from Ginkgetin, bilobetin, sciadopitysin and isoginkgetin were ordered from Shanghai Standard Biotech Co., Ltd. . Loperamide (LPA) and fluorescein diacetate (FD) were purchased from TCI . Irinotecan (CPT-11) and corresponding active hydrolytic metabolite SN-38 were obtained from Alfa Aesar  and HEOWNS , respectively. The purities of all tested biflavones were identified by LC-UV and the data indicated that the purities of tested biflavones were higher than 98% totally. The 4-methylumbelliferyl-acetate (4-MUA) was obtained from Sigma Chemical . Recombinant CES1 (Batch No.150006A), recombinant CES2 (Batch No.153015A). the pooled human liver microsomes from 50 individual donors (HLMs) and Pooled human intestinal microsomes  were bought from Bioreclamation IVT  and stored at −80°C. The stock solution of each compound (100 mM) was dissolved with dimethyl sulfoxide , which were stored at 4°C. LC grade acetonitrile , phosphate buffered saline  and ultrapure water (produced by a Millipore water system) were used in following experiments.The incubation system contains following components : 2 μL DMSO/inhibitor, 2 μL HLMs , 194 μL buffer PBS (pH = =7.4). Three concentrations of each inhibitor  were established for preliminary screening. Two control groups were set up at the same time, one was the inhibitor-free system  represented the 100% enzyme activity, the other was the positive inhibitor (LPA). The incubation system pre-incubated at 37°C for 3 min minutes and 2 μL fluorescent substrate FD  was added for fluorescence analysis in fully automated fluorescent microplate reader  .CPT-11, a known CES2 substrate-drug, was also used to ascertain whether the identified four biflavones displayed strong inhibition on CES2-catalyzed CPT-11 hydrolysis. Experiments were performed in Eppendorf Tubes : 2 μL DMSO/inhibitor, 2 μL HLMs/HIMs , and 194 μL PBS buffer (pH = 7.4). Two control groups were set up concurrently, an inhibitor-free  system regarded as 100% enzyme activity, and the other one with the positive inhibitor (LPA). The incubation system pre-incubated at 37°C for 3 min followed by adding 2 μL substrate CPT-11 , after incubated for another 50 min, reactions were terminated with an equal volume of 10% perchloric acid-ice acetonitrile (1:2). The mixture was centrifuged at 20,000 × g, supernatants were collected for follow-up analysis in LC system . ChromatThe incubation system contains following components : 2 μL DMSO/inhibitor, 2 μL recombinant CES1 or recombinant CES2 , 194 μL buffer PBS (pH = 7.4). Two control groups were set up simultaneously, one was no inhibitor system  regarded as 100% enzyme activity, and the other was the positive inhibitor (nevadensin for CES1 and LPA for CES2) . The incKi) and the inhibition modes of four potency inhibitors against CES2-catalyzed FD hydrolysis were explored by utilizing a cluster of kinetic assays for FD hydrolysis at the presence of incremental concentrations of each biflavone (Ki) values on the basis of kinetic data.The inhibition constant (iflavone . The Michttps://swissmodel.expasy.org/) based on the template structure of CES1 (PDB code: 5A7G) with the resolution of 1.48 Å  were selected as the initial conformations for molecular dynamics simulations by using Gromacs (Version 2019.3) software . The for50 and Ki values).All data were shown as mean ± SD of triplicate assays. The GraphPad Prism 8.0 was applied to determine the inhibition constants , which encourages us to further investigate the specificity and inhibitory mechanisms of these four biflavones against CES2.Furthermore, the inhibitory effects of four biflavones on CES2-catalyzed irinotecan hydrolysis were also investigated in HLMs . As listed in ectively ; Table 1n system . Further50 values as low as determined as 0.08, 0.14, 0.10 and 0.07 µM, respectively. Meanwhile, the inhibitory effects of four biflavones against CES1-catalyzed 4-MUA hydrolysis in recombinant CES1 have also been determined, and the results showed that the IC50 values were larger than 100 μM  as the substrate. As shown in n 100 μM . These f50 shift assays were used to identify whether these biflavones were time-dependent inhibitors or reversible inhibitors against CES2  surrounding to the catalytic cavity of CES2 were involved to form hydrogen bonds with this agent, while this agent could also interact with Phe469 and Trp348 via π-π stacking. Isoginkgetin created strong interactions with a panel of residues  in CES2 via forming hydrogen bonds, while this agent also interacted with Trp348 and Phe153 via π-π stacking, and with Asp456 via amide-π stacking. Notably, Trp348 could interact with all tested biflavones via π-π stacking, indicating that this residue may play a crucial role in binding on CES2. In addition, we discussed the relationship of virtual screening scores and activities against 13 representative structures obtained from clustering of molecular dynamics simulations  and bring bleeding or other side effects, when this herb is co-administrated with warfarin, an anticoagulant agent that is metabolized by CYP3A4 and CYP2C9 (Ginkgo biloba extract and its major constitutes are able to modulate the pharmacokinetic properties of co-administrated drugs via modulating the function of drug-metabolizing enzymes. Considering that Ginkgo biloba extract are frequently used over the world and the recommended daily dose is very high (12 g/d), of which total biflavones were about 48.9 mg/d. The metabolic interactions of Ginkgo biloba ingredients with human drug-metabolizing enzymes and the potential risks should be carefully investigated , much attention should be paid when Ginkgo biloba extract or Ginkgo biloba-containing dietary supplements are co-administrated with CES2 substrate-drugs. For example, CES2 can hydrolyze the antitumor drug LY2334737 , as well as the poor oral bioavailability of the biflavones in Ginkgo biloba, it is easily conceivable that the local exposure of these biflavones will be much higher than that in the liver, following oral administration of Ginkgo biloba leaves extract. It should be noted that CES2 inhibitors may affect CPT-11 hydrolysis in the liver, which in turn, reducing the plasma exposure and the in vivo anti-tumor efficacy of SN38. Thus, an ideal CES2 inhibitor is expected to reduce SN-38 formation in the intestinal tract, but does not affect the hydrolysis of CPT-11 in the human liver. In this study, our findings demonstrated that these biflavones displayed very high specificity toward CES2 over CES1. In these cases, the intestinal CES2 is more likely inhibited by these biflavones but CES1 (a known serine hydrolase responsible for lipid metabolism) in the liver is hardly inhibited by these natural products.Taking into account that intestinal CES2 is one of the key targets involving in CPT-11 induced life-threatening diarrhea, via targeting CES2, owing to these compounds display potent CES2 inhibition activities, excellent isoform-specificity, good safety profiles and high local exposure in the gastrointestinal tract. Furthermore, it has been reported that the biflavones isolated from Ginkgo biloba leaves possess a range of bioactivities , which are also beneficial for the patients with irinotecan-induced intestinal toxicity. In near future, to develop more efficacious biflavone-type CES2 inhibitors, the naturally occurring biflavones could be further modified by the medicinal chemists, to improve the CES2 inhibition potency, safety, and drug-likeness properties . It is well-known that the biflavones are abundantly distributed in a range of medicinal plants  . Thus, the key interactions between the C-7 phenolic group in these biflavones and the catalytic cavity of CES2 should be conserved in the development of novel biflavone-type CES2 inhibitors.From the perspective of drug development, biflavones are promising lead compounds for the development of novel anti-diarrhea agents for ameliorating irinotecan-induced intestinal toxicity ’s wort) , while t’s wort) . Thus, tGinkgo biloba displayed potent CES2 inhibition potency and high specificity over CES1 (>1000-fold). These biflavones showed potent inhibition potentials CES2-catalyzed hydrolysis of various substrates, including the CES2 substrate-drug irinotecan, while their inhibition potentials were more potent than that of loperamide . Inhibition kinetic analyses showed that ginkgetin, bilobetin, sciadopitysin and isoginkgetin potently inhibited CES2-meidated FD hydrolysis via a reversible and mixed inhibition manner, with Ki values of less than 100 nM. Furthermore, ensemble docking and molecular dynamics give deeper insights into the molecular mechanisms of these newly identified CES2 inhibitors. All these findings clearly demonstrated that the biflavones isolated from Ginkgo biloba were potent and reversible CES2 inhibitors, which offered several promising lead compounds for the medicinal chemists to develop novel CES2 inhibitors for some specific purposes, such as modulating the pharmacokinetic profiles of CES2-substrate drugs and alleviating irinotecan-induced diarrhea.In summary, this study reported that four biflavones isolated from


The multivalent inhibition potencies for the tacrine clusters were investigated for the inhibition of acetylcholinesterase. Two of the tacrine clusters displayed a small but significant multivalent inhibition potency in which the binding affinity of each of the tacrine binding units increased up to 3.2 times when they are connected to the central scaffold.The synthesis of four tetra-tacrine clusters where the tacrine binding units are attached to a central scaffold  To the best of our knowledge, if we neglect bivalent enzyme inhibitors, multivalent enzyme inhibition potency has only been achieved for a few groups of enzymes including, glycosidasesa,a,a,a,,Multivalent interactions  constitute a widespread recognition phenomenon in living organisms to establish interactions between carbohydrates and proteins, which are essential for the adhesion of viruses and bacteria to the surface of a cell in addition to cell adhesion1) (Alzheimer’s disease (AD) is a multifactorial progressive neurological disorder that represents the most common form of dementia1) , which uvia a heptamethylene linker to obtain bis(7)tacrine (2) (2) triggered an avalanche of reported bivalent AChE inhibitors,When the structure of tacrine complexed with AChE was solved by X-ray crystallography, it was concluded that it binds to CAS in the solid staterine (2)  that is 3a–6a  the synthesis of the tetra-tacrines 3a–6a  the multivalent inhibition potencies of tetra-tacrines 3a–6a against AChE by comparing them with reference compounds 3b–6b.The tetrameric structure of AChE that contains four catalytic subunits3a–6a  as multies 3a–6a , (2) the® 40–63 µm or Aluminium oxide Sigma–Aldrich 58 Å pore size was used for flash column chromatography. NMR spectra were recorded on a Bruker Avance NMR spectrometer; 1H NMR spectra were recorded at 400.13 or 850.13 MHz, 13C NMR spectra were recorded at 100.61 or 213.76 MHz, in CDCl3, MeOD, or DMSO. Chemical shifts are reported in ppm relative to an internal standard of residual chloroform , residual methanol  or residual DMSO . High-resolution mass spectra (HRMS) were recorded from on a Qexactive spectrometer in positive electrospray ionisation (ESI) mode.DMF has dried over 4 Å molecular sieves (oven-dried). All reactions were carried out under an argon atmosphere unless otherwise specified. Microwave reactions were performed in a CEM Discover-SP, max power 300 W. TLC analyses were performed on Merck silica gel 60 F254 plates or Sigma-Aldrich aluminium oxide 60 F254  plates using a UV light for detection. Silica gel NORMASIL 607) , azide 8, 9, 10, or 11 , and copper (II) sulphate pentahydrate (0.3 equiv.) in DMF (3 ml) in a foil-covered round bottom flask was added sodium ascorbate (0.6 equiv.). The mixture was kept stirring at room temperature overnight under Ar atmosphere. The solvent was then removed under reduced pressure and the concentrate was purified by silica gel flash column chromatography.A mixture of propargyl alcohol , azide 8, 9, 10, or 11 , and copper (II) sulphate pentahydrate  in DMF (5 ml) was added sodium ascorbate . The mixture was irradiated in a microwave at 300 W and 115 °C for 45 min. Water (10 ml) was added and the crude mixture was extracted with dichloromethane (3 × 20 ml). The organic phases were combined, dried with MgSO4, and filtered. Evaporation of the solvent by reduced pressure yielded a crude material that was purified by column chromatography.A mixture of the alkyne Electrophorus electricus  was used, conducting minor modifications on Ellman’s protocolMK. The formation of the chromophore was monitored during 125 s at 405 nm and 25 °C.For the assessment of enzymatic inhibition, commercially available acetylcholinesterase from 50 values was accomplished by plotting %I vs. log[I] and adjusting to a second-order equation. Substrate concentration was kept at 121 µM, using 2–4 independent assays, each of them, being run in duplicate.Calculation of IC3a, five different substrate concentrations, ranging from ¼ MK to 4 × MK were used. Cornish-Bowden method3a; for that purpose, two different plots were used: 1/v vs. [I] (Dixon plot) and [S]/v vs. [I]. Mixed inhibition was found for such compound, which means that it binds both, the free enzyme (iaK) and the enzyme-substrate complex (ibK). Kinetic parameters  were obtained through non-linear regression analysis (least squares fit) using the GraphPad Prism 8.01 software and inhibition constants were calculated using the following equations:For the calculation of the kinetic parameters of the free enzyme, and in the presence of SD.Data are expressed as the mean ± 1) as a reference compound for the evaluation of the multivalent inhibition potency of 3a–6a. Instead, for each tetra-tacrine 3a, 4a, 5a, and 6a a mono-tacrine reference compound 3b, 4b, 5b, and 6b, respectively, was prepared to contain the 1,2,3-triazole moiety and the same number of CH2-groups between the tacrine ring and the hydroxyl group as the corresponding tetra-tacrine contains CH2-groups between its tacrine rings and central scaffold. Reference compounds 3b–6b were obtained when propargyl alcohol (7) underwent Cu(I) catalysed alkyne-azide 1,3-dipolar cycloaddition (CuAAc) with azide armed tacrine derivatives 89–11a , which was subjected to propargylation upon treatment with propargyl bromide and sodium hydride to provide 13 (13 was armed with four tacrine inhitopes when it underwent Cu(I) catalysed alkyne-azide 1,3-dipolar cycloaddition with azides 8, 9, 10, and 11 to obtain tetramers 3a, 4a, 5a, and 6a, respectively, with variable length of the linker between the central sugar scaffold and their inhitopes. The formation of 1,4-regiosisomeric triazole moieties in 3a–6a was supported with 13 C-NMR spectroscopy where the carbon atoms in 5-position in the triazole moieties consistently appeared in the range 124.8 to 122.7 ppm, which agrees with reported data for such isomers2-protons  between the triazole moieties and the central sugar scaffold  and 6b (m = 8)] between the tacrine and triazole rings are significantly stronger inhibitors than 3b (m = 2) and 4b (m = 3) with shorter linkers, which indicates that the longer ligands establish more efficient simultaneous interactions with PAS and CAS in the active gorge. The tetra-tacrines 3a–6a also displayed potency in nM concentration range (IC50 = 12.5 nM to IC50 = 232 nM). However, for these tetra-valent inhibitors, there was no clear trend between the linker length between the tacrine and triazole rings as the strongest tetra-tacrine AChE inhibitor 5a  behaves as a 36-fold stronger inhibitor than the weakest tetra-tacrine inhibitor 6a .The potency of tetra-tacrines 50 value of the mono-tacrine with the IC50 value of the corresponding tetra-tacrine, which contains the same number of CH2-groups between the tacrine and triazole rings . The relative inhibition potencies for tetra-tacrines 3a–6a demonstrates that longer linkers between the triazole and tacrine rings have a destructive impact on the inhibition potency, as the rp-values gradually decrease from rp = 12.9 for 3a (m = 2) to rp = 0.03 for 6a (m = 8). The inhibition potencies on valency-corrected basis (rp/n) showed that tetra-tacrines 3a  and 4a  exhibit small but significant multivalent inhibition potencies for AChE. The rp/n-values for 5a  and 6a  on the other hand demonstrate that the mono-tacrines 5b (m = 6) and 6b (m = 8) were 75% and more than 99% less active, respectively, when they are connected to the central multivalent sugar scaffold. From a Cornish-Bowden plot  and ibK = 45.0 ± 5.9 nM (non-competitive inhibition constant)], which implies that it binds to the catalytic site in addition to a second binding site, for instance, PAS on the entrance of the active gorge. Thus, the multivalent inhibitory potency observed for 3a and 3b might be due to the chelation effect in which the length of the linkers in 3a and 4a are of sufficient length to allow simultaneous binding of their tacrine inhitopes to more than one active gorge in the tetrameric AChE enzyme. On the other hand, shorter linkers in the tetra-tacrines imply higher effective concentration nearby the active gorges, and thus a statistical binding effect cannot be excluded as the reason for the observed multivalent inhibition potency observed for tetra-tacrines 3a and 4a. However, as rp/n ˂ 1 for 5a and 6a, in such statistical binding effect scenario, it implies that another effect is involved, which oppose the binding of the inhitopes to the enzyme for example that longer linkers affect the position of the tacrine rings in such a way that they become less accessible for the enzyme.The relative inhibition potency (rp) was obtained by dividing the ICden plot  for 3a, 3a–6a in which the tacrine rings are connected to a central scaffold via linkers of variable lengths. Two of the tetra-tacrines 3a and 4a with the shortest linkers displayed a small but significant multivalent effect in the inhibition of AChE. The observed multivalent inhibition potency is proposed to arise from the chelation or statistical binding effects.We have applied the Cu(I)-catalysed azide–alkyne Huisgen cycloaddition reaction to obtain four tetra-tacrine clusters Click here for additional data file.


Agaronia razetoi) and the bivalves (Tivela tripla), can be used for biomonitoring.The present study investigated the trace metal levels in sediments, enrichment in sediments, and its bioaccumulation in mollusks collected from the intertidal sediments of the coast of Accra, Ghana, which is influenced by anthropogenic activities. The metals  were analyzed in sediments. The metals  were analyzed in the soft tissue of the mollusks collected from the different coastal areas. The results indicate that although Accra is the capital city and the coast receives tons of anthropogenic wastes, trace metal levels were low and there was no enrichment for the metals analyzed except Cu which showed minor to moderate enrichment in the coastal sediments. There was bioaccumulation of the trace metals in the soft tissue of the mollusks, and the results suggest that the mollusks studied, the gastropods ( Trace metal pollution remains an environmental concern around the world. This is because metals are persistent in the environment and can cause toxic effects on organisms including humans –3. MetalMetals in sediment bioaccumulate in benthic invertebrates and present a potential health risk when they are fed on by organisms at higher trophic levels. As a result of the potential risk of metal exposure to organisms, metals are monitored by determining metal levels in sediment, water, and in the tissues of aquatic organisms such as fish, mollusk, and other invertebrates .Generally, benthic macroinvertebrates are used for biomonitoring to assess the extent of environmental pollution. However, the species selected for biomonitoring is very important, and in the present study, we used mollusks because they are abundant with restricted movement to a particular local environment , 13. TheAccra, like most major cities in West Africa, is along the coast of the Gulf of Guinea. Accra is characterized by high population growth, urbanization, informal e-waste recycling, and industrial activities which contribute to the trace metal load along the coast of Accra. Previous studies have investigated metals in fish, bivalves, and gastropods , 16, andThe Accra coast is part of the Gulf of Guinea which is a large marine ecosystem on the Atlantic coast of West Africa. The Accra coast is the coastline of Accra, the capital city of Ghana which has lots of industries and is characterized by rapid urbanization. The coastline is approximately 40 km long, and it has a high population density, which is characterized by various economic activities such as fishing and recreation , 21. The2 along a transect from three different sites at a depth of 15 cm in the intertidal area. The sediments collected from the different sites were placed in ziplock bags and transported to the laboratory for analysis of Fe, Mn, Cu, Zn, and Cr. At each of the coastal areas, mollusks in the coastal sediments were collected by hand from the sites and transported on ice to the laboratory where they were stored at −20°C until analysis. The same mollusk species were not present at all the sites; we, therefore, collected the mollusks that were present at the different sites. We collected 17 and 2 individuals of the gastropods Agaronia razetoi from Sites 1 and 2, respectively, while at Site 3, there was no Agaronia razetoi; therefore, we collected 15 individuals of the bivalve Tivela tripla. We however did not find any mollusk in the sediment at the time of sampling at Site 4.The sediment samples were collected from intertidal sediments of four coastal areas along the Accra coast which are highly populated and influenced by anthropogenic activities such as urbanization and industrialization. These are Sakumono (S1), Teshie (S2), Korle Gonno (S3), and Jamestown (S4) coasts and are referred to as Site 1, Site 2, Site 3, and Site 4, respectively . Sakumonμm mesh sieve. The sediment samples that were sieved and the fraction that was retained on the mesh were then dried at 60°C for 48 h and weighed. The organic matter in sediment was quantified using the loss-on-ignition method where approximately 1 g of ground sediment dried at 60°C was placed in a crucible and heated in the muffle furnace at 550°C for 4 h [The sediment organic matter and silt/clay content were quantified for the sediments collected from the four coastal areas along the coast of Accra . The sil for 4 h .4: HNO3: H2SO4, 1 : 25 : 2.5) was added to 1 g of ground sediment in acid washed beakers and digested under a fume hood. After digestion, the samples were filtered into a 100 ml volumetric flask using a Whatman No. 42 filter paper. Distilled water was then added to the filtrate to make it to the 100 ml mark and analyzed with flame atomic absorption spectrophotometry (AAS) on PerkinElmer PinAAcle 900T.The sediment subsamples from the cores were dried in an oven at 60°C for 48 h in opened ziplock bags placed in acid washed beakers. The sediment was ground, and 10 ml of ternary mixture  was added to the tissues and digested under a fume hood. After digestion, the samples were filtered into a 100 ml volumetric flask using a Whatman No. 42 filter paper. Distilled water was then added to the filtrate to make it to the 100 ml mark and analyzed with flame atomic absorption spectrophotometry (AAS) on PerkinElmer PinAAcle 900T.The soft tissues of mollusks were removed from their shells using stainless steel scalpel blades. The soft tissues were placed in acid washed beakers and dried in an oven at 60°C to a constant weight. After quantification of the dry weight, 10 ml of ternary mixture .The anthropogenic input of metals in sediment was assessed using the enrichment factor. The enrichment factor was quantified by normalizing metal concentrations in samples and background concentration to a reference metal (Fe) concentration .The enrichment factor was quantified by the following equation:To assess the toxicity of trace metals to benthic organisms, we compared the concentration of each metal with the threshold effect levels (TEL). The TEL is a sediment quality guideline (SQG), and metals are considered to be toxic when they exceed the TEL values . We subsR version 3.5.1 [The data were checked for normality using the Shapiro–Wilk test. The datasets that were not normal were transformed or analyzed using the Kruskal–Wallis test. A one-way analysis of variance (ANOVA) was used to evaluate differences in Fe and Zn in sediments from the different sites. A Kruskal–Wallis test was used to analyze the differences in Mn, Cu, and Cr in sediments from the different sites. A one-way analysis of variance (ANOVA) was used to analyze the differences in bioaccumulation and BSAF values of metals in the tissue of mollusks from the different sites. The Pearson correlation was used to analyze the association between trace metals in sediment and sediment organic matter and silt/clay content. All statistical analyses were conducted with on 3.5.1 .P > 0.05) differences in Cu, Mn, and Cr concentrations between the different sites. However, there were significant differences for Zn and Fe. The Zn concentrations in the sediment differed significantly (P < 0.05) between the sites. The Zn concentration was highest at Site 2 and lowest at Site 4. There was a fivefold difference between the highest and lowest concentration. The Fe concentrations in the sediment differed significantly (P < 0.05) between the sites. The Fe concentration was highest at Site 2 and lowest at Site 4. There was a fourfold difference between the highest and lowest concentration. Fe levels were significantly enhanced at Site 2 compared to the other sites. The toxic units ) were less than one for Cu, Zn, and Cr, thus suggesting no toxicity of these metals in the sediment.The average concentrations of metals in the sediments for all the sites are shown in ) showed no enrichment of Mn, Cr, and Zn at all the sites. However, Cu showed minor to moderate enrichment at all the sites except for Site 2 (Teshie coast) where there was no enrichment. There was moderate enrichment at Site 1 and minor enrichments at Sites 3 and 4.The result of the enrichment factors P < 0.05) positive correlation with Zn and Cr. There was a significant positive correlation between Cu and Mn. There was also a significant positive correlation between Zn and Cr. The OM was significantly positively correlated to Fe and Cr.The sediment samples collected from the different sites had low organic matter and silt/clay content. The degree of OM in the coastal sediments from the different sites was in the order of Site 2 > Site 1 > Site 3 > Site 4. The degree of silt/clay content in the coastal sediments from the different sites was in the order of Site 2 > Site 4 > Site 3 > Site 1. The Pearson correlation between metals, organic matter, and silt/clay content was determined . Fe in tP > 0.05) difference in Cu and Cr levels in the tissue of mollusks from the different sites. However, the Zn concentrations in the mollusk tissue differed significantly (P < 0.05) between sites. The highest Zn concentration was recorded in the mollusk from Site 2 (Teshie coast) and the lowest concentration in the mollusk from Site 3 (Jamestown coast). There was a fivefold difference between the highest and lowest concentrations which was significant. The Cr levels in the tissue of mollusks were low irrespective of the levels in the sediment suggesting low bioavailability of Cr to the mollusk. A comparison of the metal levels in the mollusk tissue in the present study to other studies [The average metal concentrations of mollusks sampled in the present study are shown in  studies , 10, 26 P < 0.05) between the sites, and the highest BSAF value was for Site 2 (Teshie coast) and the lowest for Site 1 (Sakumono coast). The BSAF values for Zn differed significantly (P < 0.05) between the sites, and the highest BSAF value was for Site 1 and the lowest for Site 2. The BSAF values for Cr differed significantly (P < 0.05) between the sites. Cr generally had the lowest BSAF values, and the highest value was for Site 1 and the lowest for Site 2. Generally, for the metals studied, there was a trend where BSAF values were generally lower for the mollusk in sediment with higher metal concentrations.The BSAF  values fThe concentrations of trace metals along the coast of Accra in the present study were low. However, the concentrations were within the range reported for other coastal areas along the coast of Ghana , 17. TheAlthough Accra is the capital city of Ghana and the coast receives tons of domestic and industrial wastes, the results of the enrichment factors suggest low anthropogenic input of trace metals. For all the metals studied except Cu, our results suggest that they are mainly from natural sources. This is because the results of the enrichment factors showed no enrichment of Mn, Cr, and Zn at all the sites. However, Cu showed minor to moderate enrichment at all the sites except for Site 2 (Teshie coast) where there was no enrichment. Site 1 (Sakumono coast) which had moderate enrichment of Cu in the sediment is close to Tema, a city in Accra that has a lot of industries and a port, and these may be an anthropogenic source of Cu to the marine environment. The positive correlations between Fe, Zn, and Cr and between Cu and Mn suggest that these metals are transported bonded to Fe or Mn oxides/hydroxides and are from a common anthropogenic and geologic origin , 28. TheAgaronia razetoi and Tivela tripla) studied in the present study may not be a good bioindicator for Cr and Mn pollution. The Cr, Zn, and Cu levels bioaccumulated by the mollusk in the present study compared to other studies around the world indicate that the levels in the tissue are low and within the recommended range [The bioaccumulation of metals in the mollusk was not significantly different between the sites for all the metals except Zn. Previous studies have shown that the difference in metal levels in the tissue of mollusks may be due to the concentration of metals, the environment, metabolism and the uptake routes such as diet, or from the ambient environment . In the ed range , 10.Agaronia razetoi and Tivela tripla). Previous studies have shown that Tivela tripla may be a likely prey of Agaronia razetoi [Agaronia razetoi than Tivela tripla because of biomagnification of metals at higher trophic levels. However, the results of the present study suggest that bioaccumulation of metals was not influenced by the trophic levels but by the metal and the organic matter in the coastal sediment. In this study, the major challenge associated with the analysis of bioaccumulation of metals in mollusks from the various sites was the unavailability of the same species of mollusks in the spatial areas studied. The availability of the same species of mollusks in the various sites studied would have made the comparison stronger.In the present study, bioaccumulation was quantified in two species of mollusks  and the bivalve (Tivela tripla) can be used for biomonitoring of heavy metals.The study shows that the trace metal levels in the coastal sediments of Accra and in the mollusks were below threshold levels. However, heavy metal levels in sediments except Cu tend to be higher in sediments with higher organic matter. The findings of this study also suggest that Cu levels in the sediment have an anthropogenic input. This is because the enrichment factor shows that Cu levels in the coastal sediments of Accra showed minimal to moderate enrichment. However, there was no enrichment for the other metals at all the other sites. There was bioaccumulation of Zn, Cu, and Cr in the mollusks; however, the concentration of Cr in the tissue relative to what is in the sediment suggests low bioavailability to the mollusks. Bioaccumulation of the heavy metals in the mollusk suggests that the gastropod (


Furry animals are an important source of indoor allergens. Diagnosis of allergy to small pets such as guinea‐pigs still relies on animal dander extracts which do not allow to define the primary sensitization source.To identify major guinea‐pig allergens and to evaluate their potential as marker allergens for in vitro IgE‐diagnosis in comparison with dander extracts.n = 29) and a group of patients allergic to cat and dog (n = 30) were recruited for the study. A panel of four guinea‐pig lipocalin allergens was expressed as recombinant proteins in E. coli. Specific IgE were quantified by ImmunoCAP and ELISA.A group of patients allergic to guinea‐pig . Cav p 6 shares 54% sequence identity with Fel d 4 and Can f 6 and was found to be IgE‐cross‐reactive with these allergens. In the group of cat‐ and dog‐allergic patients, 73% had also specific IgE to guinea‐pig dander. However, only 27% of the cat /dog‐allergic patients had specific IgE to any of the non‐cross‐reactive guinea‐pig allergens Cav p 1, Cav p 2 or Cav p 3. The high prevalence of IgE to guinea‐pig dander could be explained by IgE‐cross‐reactivity among serum albumins and certain lipocalins.The availability of specific allergen markers is essential for the assessment of primary sensitization, especially in polysensitized patients. The proposed panel of guinea‐pig allergens Cav p 1, Cav p 2 and Cav p 3 is a first step to component‐resolved IgE‐diagnosis of allergy to small furry pets. Component resolved diagnosis and the availability of specific marker allergens (right) allows the determination of primary sensitization in patients which show multiple sensitizations to animal dander extracts (left). Major and minor cat and dog allergens have been extensively characterized allowing to distinguish genuine IgE‐sensitization from mere IgE‐cross‐reactivity between certain cat and dog allergens.The objective of the present study was to provide specific marker allergens for the diagnosis of allergic sensitization to guinea‐pig, a pet with a high sensitizing power.22.1The first patient cohort consisted of 29 patients  exposed to guinea‐pig dander with a history of inhalant allergy. Of these, 26 were doubtlessly only allergic to guinea‐pigs while 3 had possibly also symptoms to other pets. All were sensitized to guinea‐pig as shown by positive skin prick tests and the presence of specific IgE (sIgE) to guinea‐pig extract. The second patient cohort consisted of 30 patients  with inhalant allergy in presence of cat or dog or cat and dog and having positive skin tests and specific IgE to both cat and dog. These patients had no exposure to guinea pigs. Both cohorts were recruited at the National Unit of Immunology‐Allergology at the Centre Hospitalier de Luxembourg. Skin prick tests solutions were from Stallergenes , and in vitro IgE measurements to guinea‐pig, cat and dog epithelium were performed with ImmunoCAP (ThermoFisher Scientific). Ethical approval was obtained from the National Committee for Medical Research Ethics (No°201001/06 and No 201307/04), and informed consent was obtained for all subjects.2.2E. coli. Cav p 4, guinea‐pig serum albumin, was purchased from Sigma‐Aldrich. Polyclonal antibodies were raised against recombinant Cav p 1 and Cav p 6. Mice were immunized with 50 μg of allergen suspended in Al(OH)3 (Sigma‐Aldrich), and 3 boosts were administered subsequently at 3‐week intervals.Two guinea‐pig lipocalin allergens, Cav p 2 and Cav p 3, have been previously characterized in detail.2.3Specific IgE (sIgE) to recombinant Cav p 1, Cav p 2, Cav p 3, Cav p 6 and to native Cav p 4 , as well as recombinant Fel d 4 and Can f 6, were measured by ELISA as described.2.4sIgE frequencies to individual components in the two cohorts were compared using 2x2 contigency tables and Fishers's exact test using GraphPad Prism 8. The Venn diagram summarizing the sensitization profiles of guinea‐pig‐allergic patients was constructed by using an online tool.33.1IgE‐reactive bands were detected in hair and harderian gland extract from guinea‐pig using sera of guinea‐pig‐allergic patients and the corresponding proteins were purified from hair by ion exchange chromatography Figure . As lipoThe cDNA corresponding to allergen Cav p 1, named Cav p 1.0201, has now been cloned and the sequence clearly classifies it as lipocalin. Sequence identity is 44% to Cav p 2, 46% to Cav p 3 and 29% to Cav p 6 Figure . Other mThe N‐terminal amino acid sequence of the second protein, DEVVRGNFDAEKISG, relates to a newly identified allergen named Cav p 6 by the WHO/IUIS allergen nomenclature subcommittee. It also belongs to the lipocalin family, and it shows a relatively high amino acid sequence identity to a group of lipocalins that have been shown to be cross‐reactive: Fel d 4 (54%), Can f 6 (54%) and Equ c 1 (49%) Figure .10, 1All 4 lipocalin allergens, Cav p 1, Cav p 2, Cav p 3 and Cav p 6, were detected in guinea‐pig hair protein extracts Figure A. The an3.2To assess the diagnostic value of the 4 guinea‐pig allergens, a group of patients allergic to guinea‐pig was screened for the presence of sIgE to the 4 lipocalins. Twenty‐four of 29 (83%) guinea‐pig‐allergic patients had sIgE to rCav p 1 Figure . rCav p A/L), but she is also sensitized to cat (sIgE 4 kUA/L) and dog (sIgE 28 kUA/L). Inhibition with increasing doses of Cav p 6 inhibited IgE‐binding to Fel d 4 up to 83%, binding to Can f 6 up to 21%. The results point to a cross‐sensitization from guinea‐pig to cat and probably to a co‐sensitization to dog, although she does not have a dog.As Cav p 6 shares relevant sequence identity with Fel d 4 and Can f 6, we assessed a potential IgE‐cross‐reactivity to these lipocalins. Sera with sIgE to Cav p 6 were tested for the presence of IgE reacting to Can f 6 and Fel d 4. Thirteen out of 17 (76%) Cav p 6 positive patients were also positive for Fel d 4, and 5 (29%) with Can f 6 Table . All exc3.3In order to assess the specificity, respectively, the cross‐reactivity of guinea‐pig components, a cohort of 30 patients allergic to cat/dog Table  and withA/L. A majority of these sera reacted to the cross‐reactive molecules Cav p 4 and/or Cav p 6: fourteen out of 22 sera were positive for Cav p 4 and/or Cav p 6. Only 7 sera reacted to Cav p 1 (titers 0.6–3.4 kUA/L), and 2 of these were also positive for Cav p 2, resp. Cav p 3. One other patient had specific IgE to Cav p 2, but not Cav p 1. The frequencies of IgE positive sera to Cav p 1, Cav p 2 and Cav p 3 in both patient cohorts are significantly different , indicating that all 3 lipocalins can be considered as markers for primary sensitization to guinea‐pig. The presence of sIgE to Cav p 6 was also more frequent in the guinea‐pig‐allergic cohort, but the difference was less significant (p = .0182).Twenty‐two of the 30 cat and dog sensitized patients (73%) had a positive IgE test to guinea‐pig epithelium, titers between 0.42 and 51.6 kUThe remaining 8 patients were positive to guinea‐pig dander, but negative when analysed for IgE‐reactivity to Cav p 4 and the 4 guinea‐pig lipocalins, pointing possibly to the existence of another cross‐reactive allergen in guinea‐pig dander.A/L, sIgE guinea‐pig 23 kUA/L) and that, according to the inhibition results with Cav p 6, his sensitization to guinea‐pig is a cross‐sensitization from cat and dog.IgE‐cross‐reactivity among Fel d 4, Can f 6 and Cav p 6 lipocalin subfamily members was further assessed by ELISA inhibition using a cat‐ and dog‐allergic patient serum (CD‐31) Figure . IgE‐bin4Animal dander extracts are still widely used for in vivo and in vitro allergy diagnosis. Although they have several drawbacks, such as variable allergen content and the presence of cross‐reactive molecules, they constitute a sensitive screening tool for the detection of sensitization to animals and they are often used as first step in diagnosis.In the present study, we have used four guinea‐pig lipocalins and serum albumin to analyse the IgE‐reactivity profile of a group of guinea‐pig‐allergic patients.E. coli. Another lipocalin, Cav p 6, was identified as allergen present in guinea‐pig hair, cloned and further characterized. Together with the previously identified lipocalins Cav p 2 and Cav p 3, the 2 new allergens were evaluated for their use in component‐resolved diagnosis.A partially characterized 20 kDa protein named Cav p 1 had been purified from hair by Fahlbusch et al. and shown to be a major allergen of the guinea‐pig.Cav p 1 is the most important major guinea‐pig allergen. It is recognized by 83% of guinea‐pig‐exposed and guinea‐pig‐allergic persons. Sequence identity with other known pet lipocalins is below 47%, and Cav p 1 is thus unlikely to present significant IgE‐cross‐reactivity with other known allergens.Cav p 6 on the contrary belongs to the cross‐reactive Fel d 4, Can f 6, Equ c 1 lipocalin subfamily.A/L), to Cav p 1, Cav p 2 and/or Cav p 3, even though anamnesis did not report contact with guinea‐pigs. An explanation could be that these pet‐allergy‐prone individuals had contact with guinea‐pig allergens without knowing it. Other explanations could be that they did not remember previous contacts or that they omitted to report exposure to guinea‐pigs. As animal allergens are ubiquitous, this is certainly a weakness of the study. In an optimal setting, patients would need to be followed longitudinally, with detailed questionnaires and exposure monitoring, in order to assess objective exposure to animal allergens. However, at this point it can also not be excluded that there exists a weak cross‐reactivity between Cav p 1 and other pet allergens, possibly not yet defined lipocalins. Nevertheless, the presence of specific IgE to Cav p 1 is highly indicative of a primary sensitization to guinea‐pig and not to cross‐sensitization to cat and/or dog allergens (p < .0001).In the selected cohort of cat/dog‐allergic patients, 73% were also positive with guinea‐pig dander. Eight of these 22 patients (27%) were positive, although weakly (between 0.6 and 3.4 kUFor 14 cat‐ and dog‐allergic patients, guinea‐pig extract positivity could be explained by IgE‐cross‐reactivity either to Cav p 6 and/or Cav p 4, an assumption supported by a stronger IgE‐positivity in the cat and dog homologous molecules Table . Guinea‐However, eight patients do not have specific IgE to any of the recombinant or native guinea‐pig allergens tested. Although the tested panel is quite extensive, it does probably not include all guinea‐pig allergens as suggested by the results in the primarily guinea‐pig‐allergic cohort, where 10% of the patients could not be diagnosed by using the 4 lipocalins.In conclusion, a panel of 4 allergens was able to detect 90% of guinea‐pig‐allergic patients. Cav p 1 is the most important guinea‐pig allergen detecting by itself primary sensitization to guinea‐pig in 83% of the patients of our cohort. The prevalence of IgE directed to the panel of 3 marker allergens in a cohort of cat/dog‐allergic patients is reduced to 27% compared to 73% when using hair extracts, underlining the urgent need of marker allergens to distinguish polysensitization from cross‐reactivity.Direct and indirect exposure to different pet animals hampers clinical diagnosis of the primary sensitizing source, but also clinical co‐sensitization is difficult to assess without the availability of marker allergens. Molecular components are essential to clarify the sensitization profile of those patients to give an accurate and in‐depth diagnosis and advice.As shown by the analysis of the IgE‐positivity to whole guinea‐pig extract in a selected cohort of cat/dog‐allergic patients, a majority of them do not have a primary sensitization to guinea‐pig allergens. This is a strong argument for the good performance of component‐resolved diagnosis in establishing primary allergy to guinea‐pigs, especially in a context of pet polysensitization. It is also an incentive for the need of further characterisation of other small pet allergens in order to find specific marker allergens for each species and to enable a correct diagnosis in matter of allergies to furry animals.None of the authors has declared a conflict of interest related to this study.CH and KS were involved in all stages of the project, analysed the data and wrote the manuscript; KS and SK performed the experiments; FH and CL recruited patients and participated in data analysis and interpretation; MO contributed to the interpretation of data and revised the final version of the manuscript. All authors critically revised the manuscript.Supplementary MaterialClick here for additional data file.Fig S1Click here for additional data file.Fig S2Click here for additional data file.Table S1‐S2Click here for additional data file.


Recent rapid thinning of West Antarctic ice shelves are believed to be caused by intrusions of warm deep water that induce basal melting and seaward meltwater export. This study uses data from three bottom-mounted mooring arrays to show seasonal variability and local forcing for the currents moving into and out of the Dotson ice shelf cavity. A southward flow of warm, salty water had maximum current velocities along the eastern channel slope, while northward outflows of freshened ice shelf meltwater spread at intermediate depth above the western slope. The inflow correlated with the local ocean surface stress curl. At the western slope, meltwater outflows followed the warm influx along the eastern slope with a ~2–3 month delay. Ocean circulation near Dotson Ice Shelf, affected by sea ice distribution and wind, appears to significantly control the inflow of warm water and subsequent ice shelf melting on seasonal time-scales. The intrusion of relatively warm water is causing the recent rapid thinning of the Dotson ice shelf, West Antarctica. Here, the authors analyzed two-years of mooring data from the Dotson ice shelf front and found that seasonal variability of the ocean circulation and ocean surface stress are the main causes of variability in heat transport. The cause for the recent flow increase is believed to be the thinning of the buttressing ice shelves8. At the Amundsen Sea Embayment (ASE), ice from the West Antarctic Ice Sheet (WAIS) is drained into the ocean through the Pine Island, Thwaites, Haynes, Smith, Pope, and Kohler glaciers. These glaciers had an ice flux of 334 ± 15 Gt yr−1 in 20139, and they have potential to impact sea level rise globally should this flux change significantly. In the 1980s, Pope, Smith, and Kohler Glaciers, located in the western ASE, drained ice about 15% of the total ice mass loss from the ASE, but since 2013 their rate have increased and they now contribute about 23%. In the ASE, warm and salty circumpolar deep water (CDW) can intrude from the deep ocean across the continental shelf under the influence of wind and Earth’s rotation10. This happens in submarine troughs where the seabed is sufficiently deep (>500 m), and in the southern end of these troughs a modified version of CDW  can access deep-draft ice which accelerates the ice shelf melt and the glacier mass loss17.Glacier flow in West Antarctica has been increasing18. Understanding seawater circulation near the ice shelf is essential for determining how changes in oceanic heat transport affect basal melting of ice shelves19. Recently, it has been found that the surface water flowing into the cavity influences Ross and Filcher-Ronne ice shelves melting and their seasonality21. Long-term mooring observation in front of Pine Island Glacier (PIG) have shown that the variation in sea surface heat flux can affect the variability in CDW volume and seawater circulation pattern22. Moreover, the seasonal expansion and contraction of polynya and sea ice melting and formation can affect the seawater density structure, affecting the seawater circulation.The oceanic heat transport in the AS is more significant than the Ross and Weddell Seas adjacent to the giant ice shelves in Antarctica. In the Ross and Weddell Seas, where large cyclonic polar gyres are located north of the continental shelf, only a tiny amount of CDW cooled within the gyre intrusion on the continental shelf−1 between 1994 and 2012, which is 30% faster than the average thickness change in the AS sector (1.94 m yr−1)5. Observations near DIS has been conducted semi-regularly in summertime since 1994 and demonstrate a substantial inter-annual variation connected with the weather systems near the ASE19. Continuous time series, based on moored instrumentation, are rarer. Although there is a clear seasonal variation of the mCDW intrusions further north in the Getz–Dotson trough23, previous records at the ice shelf front25 have been too short to determine any seasonality there. The seasonal variation of atmospheric forcing into the ocean caused by the seasonality of the sea ice distribution will affect the ocean circulation near the ice shelf26. Therefore, confirming the seasonality of the mCDW circulation in front of the ice shelf and identifying the causes is essential for quantifying the influx of oceanic heat into the ice shelf cavity and understanding the thinning process of the ice shelf. In this study, we show that there is a clear seasonal variability of mCDW water flowing into the DIS and how it propagates into the cavity beneath the ice shelf. In addition, temporal and spatial variations in ocean circulation and heat transport to and from the sub-DIS cavity and look into associated forcing mechanisms are investigated.The Dotson Ice Shelf (DIS), located southwest in the ASE, buttresses the Kohler and Smith Glaciers and has thinned by 2.6 m yrIn January 2014, three bottom-moored arrays were deployed from the South Korean icebreaker RVIB Araon along the DIS calving front to measure the variability of temperature, salinity, and currents. These moorings were located near the center of the Dotson–Getz trough (DGT) and on its eastern and western slopes, 2–3 km away from the calving front  and veered westward. Near the western flank, weak south-eastward flows (around 1 cm s−1) were measured at 700 m  does not significantly affect the seasonal variability of the southward flow.The predominantly south-westward flow on the eastern flank Fig.  displaye26. Salinity at mid-depths (above 500 m) varied seasonally near the eastern and central front, as shown by the 34.2 PSU isohaline, rising from ~450 m in early spring to ~300 m in early summer and deepening 450–500 m for the rest of the year. The seawater temperature does not differ significantly between the eastern slope and the center, similar to the salinity. Thus, the baroclinic effect by density gradient seems insignificant in the mid-depth layer. This spatial distribution of salinity suggests that the vertical shear of the southward flow  buoyancy fluxes from sea ice formation and/or atmospheric cooling (less buoyant at the sea surface) creates deep convection, leading to a downward descent of the thermocline and thinning of the mCDW layer at the bottom, while negative buoyancy fluxes  due to surface heating and sea ice melting leads to an upward movement of the thermocline and a increase mCDW volume. In order to investigate the effect of sea ice fluctuations on the change of mCDW and its circulation, local surface buoyancy fluxes were calculated using the heat- and freshwater fluxes from the data-assimilating Southern Ocean State Estimate (SOSE)27. These surface buoyancy fluxes were then compared to the depth of isohalines and meridional velocities  repeats seasonal expansion and contraction, which can affect seawater circulation and spatial distribution of mCDW by causing changes in ocean surface density during sea ice formation and melting. Previous study from PIG23. These can induce barotropic currents. In other to estimate the effect of the barotropic component on the variability of the southward current near the eastern side of DIS front, the OSSC was calculated from sea ice motion and wind using the ice–ocean drag coefficient  with OSSC at 28-day lag  fast sea ice. When a homogeneous wind field blows over an opening in the fast ice, spatial stress gradients 30. The seasonal phase of the maximum meltwater fraction is delayed gradually toward the bottom, with the maximum at 575 m (0.45%) appearing in July. In contrast, the minimum meltwater fraction appeared in early October/September both years for the whole water column.During all seasons, there was a weak southward flow present near the bottom throughout the record at the western side of DIS front Fig. , presumar = 0.9, with a 17-day lag) between the meridional current velocity and the upper layer meltwater fraction 34 that heat content variability in front of DIS can affect the heat transport into the DIS cavity and ice shelf melt.Although it is expected that the seasonal increase in heat transport has implications for the basal melt of the ice shelf, recent results indicate that the barotropic current component, which is a strong contributor to the seasonal variability, can be at least partially blocked at the ice front35, Pine Island Glacier19, Amery ice shelf36, as well as Dotson trough further north than the presently studied system37. A similar circulation pattern has also been noted in comparatively ‘cold’ shelf systems such as Ross ice shelf20 and Filchner-Ronne ice shelf38, although it does not substantially influence the ice shelf melt processes there. It was also shown that high meltwater concentrations correlate with intermittent outflows from the western side of the cavity, which is to be expected but is rarely observed due to the challenging environments for data collection near the calving fronts of these large glaciers.Using two years of mooring data near the calving front of Dotson ice shelf, we identified a substantial inflow of warm and salty water near the seabed at the eastern flank of the deep trough that leads into the ice shelf cavity. We also identified high concentrations of meltwater on the opposite (western) flank. These findings have been observed in other systems that receive warm salty water through deep troughs in the continental shelf, such as Getz ice shelf25, based on summer observation, may have been overestimated. Based on the present data set the average heat transport in summer was 141 MW m−1, 1.27 times greater than the annual average of 111 MW m−1, a number that can be used to scale summertime observations from this region. The seasonal average meltwater flux to the north was at a maximum of 89.2 cm2 s−1 in autumn and 25.7 cm2 s−1 in summer. In contrast, meltwater flux showed negative in winter and spring due to the dominant southward flows, and the annual average was close to zero. This seasonality implies that additional observations are needed to determine how much of the inter-annual variability of heat and meltwater transport seasonality propagates into the ice shelf cavity. The effects of the strong seasonality also have implications for timing of snapshots used to calculate ice shelf thickness evolution and points to the importance of high-resolution remote sensing products when calculating ice shelf mass balance.Both the warm inflow in the east and the meltwater outflow in the west had a clear seasonal variation. Inflow velocity and heat content in the east peaked in summer, with a nearly three-fold increase of the summertime heat transport toward the cavity. There was also an autumn maximum in both meltwater content and outflow velocity in the western side of the ice shelf front, delayed by about 2 months from the inflow peak in the east. The substantial seasonal variability of heat transport and meltwater discharge near the ice shelf calving front, that was observed, suggests that previously evaluated heat- and meltwater transport22 and to Getz Glacier39, where a wintertime maximum in heat content and velocity has been observed. In contrast to the situation at Pine Island Glacier calving front22, where the main cause for vertical migration of the thermocline is local buoyancy flux, short-term variability of heat transport was here caused by local changing stress at the ocean surface due to wind and sea ice conditions. These appear to be similar to the intermittent reductions in heat transport that have been observed at the Getz ice shelf cavity35, except that the forcing in the present locality is mainly local while the Getz ice shelf appears to be predominantly remotely forced and transmitted via topographic waves to the cavity opening.The seasonal variation observed at DIS is in contrast to seasonal variation in the deep troughs leading to Pine Island Glacier40. Previous studies have shown that the wind field and atmospheric circulation affects not only the seawater circulation at the ice shelf front but also the variability of warm water flowing into the DGT near the center of the continental shelf41. Also, local wind forcing in front of PIG has been attributed a significant modulator for the short-term (less than one month) variability of the upper thermocline depth and basal melting there42. Local atmospheric forcing, such as wind, is the most crucial factor in determining the variability of the seawater circulation and oceanic heat transport in the AS. Meanwhile, atmospheric circulation in the AS is much more complex and diverse than in any other sea in Antarctica due to the variability44  in the Amundsen Sea Low (ASL)45. In addition, this variability in ASL was influenced by the Southern Annular Mode (SAM) and El Niño-Southern Oscillation (ENSO) phases in a long-term timescale46.The driving mechanism behind seasonality of the heat transport into the DIS cavity is interpreted to be caused by local OSSC, induced by changes in sea ice and wind distribution, in turn linked to changes of the polynya area and sea ice distributionThrough long-term observations in front of DIS for two years, we found that heat was delivered to the DIS cavity along the eastern flank of the DGT, and glacial meltwater was emitted along the western flank with a time lag of two months. Furthermore, the seasonality of heat transport and meltwater discharge was confirmed due to the variability of OSSC induced by wind and sea ice distribution. The circulation pattern of mCDW in the front of the ice shelf and its seasonal variability by atmospheric conditions demonstrated here allow for quantitative evaluation of the effects of short-term variability in the atmosphere on ocean circulation. This finding implies that the ocean circulation effect by local meteorological conditions in front of the ice shelf plays an essential role in regulating mCDW inflow and the basal melting of the ice shelf. Therefore, understanding the long-term variability of the atmosphere and the subsequent response of the ocean circulation are likely essential for determining the long-term melting trend of the WAIS. These results suggest that further studies are needed, e.g., ice-ocean coupled numerical model study considering the ice shelf.® software and the tidal signal was removed using the t_tide toolbox47.To monitor the temporal and spatial variation of mCDW and its effect on the rapid melting of glaciers in the Amundsen Shelf, we conducted extensive oceanographic surveys 4 times from 2010 to 2016. The temporal variability and properties of mCDW and its circulation in front of the DIS were obtained from the K3–K5 moorings deployed on January 8–9 2014, and recovered on January 19–20, 2016. At each station, the current velocities were observed from dual ADCPs with upward-looking 150 kHz (K4 and K5) or 75 kHz (K3) and downward-looking 300 kHz (K3–K5) RD Instruments (RDI) , and dissolved oxygen (accuracy of 3 μmol kg−1).In addition, the moorings contained 8 (K4 and K5) or 12 (K3) Sea-Bird Electronics (SBE) 37-SM or 37-SMP-ODO MicroCAT sensors (Supplementary Table −3) at each mooring was calculated relative to the freezing point:ρ0 is the reference seawater density (1027 kg m−3); Cp is the specific heat of seawater (3986 J K−1 kg−1); and T and Tf are seawater temperature and seawater freezing point at the surface based on the salinity, respectively.The heat content  from January 2014 to January 2015. In this calculation process, it was assumed that the shear between each layer of the baroclinic component of southward velocity was always constant. The linear regression showed a high coefficient of determination from 0.93 to 0.64  and properties 48:φ) was then calculated from observed in situ temperature and salinity and the end-members of ice and water mass50:χT and χS represent potential temperature and salinity and subscripts melt, CDW, WW, and obs indicate the end-members of ice, CDW, Winter water, and in situ observations at K5. In previous studies, meltwater fraction calculation and end-member selection have used summer observation data50. In this study, an end-member applicable over the entire season was required to calculate the meltwater fraction using mooring data. However, winter water is generated as a result of brine rejection and convection by sea surface cooling and remains until summer above the mCDW layer. Thus, to detect this ‘pure’ winter water, we selected end-member estimated from summer observation already presented. We used the two-year average (2014 and 2016) end member for CDW , WW  and ice  obtained from previous studies in the AS34.The outflow water from DIS was considered a mixture of winter water (WW), mCDW, and ice shelf meltwater based on the assumption that the ice-seawater system was closed. This relationship was expressed by water mass (g is acceleration of gravity (9.8 m s−2) and α at the sea surface  is the thermal expansion coefficient (5.1 × 10−5 K−1)51. QHF is net air–sea heat flux (W m−2) and positive means that the ocean gains heat. ρw and Cp are water density and specific heat of seawater (4190 J kg−1 K−1), respectively. β  is the saline contraction coefficient (7.9 × 10−4)51. QFW is the freshwater flux (m s−1) and negative means that the ocean gains fresh water. S0 is the ocean surface salinity. QHF and QFW are obtained from the data-assimilating Southern Ocean State Estimate (SOSE) for 1/6° from 2013 to 201827.Buoyancy flux is caused by atmospheric heating and formation and melting of sea ice. Buoyancy flux was calculated as52 providing gridded wind data above 10 m at the sea surface with a horizontal resolution of 20 km , 15 km , and 10 km  at 3 h intervals , which uses the Polar Weather Research and Forecasting (WRF) model, the mesoscale model specially adapted for polar regions55 then becomes:CD,ai is drag coefficient between the air and ice; W10 is surface wind; Uw, and Uice are the ocean current and sea ice velocities, respectively; ρa, ρw, and ρice are the densities of air, water, and sea ice, respectively; f is the Coriolis parameter; and h is ice thickness. The rotation matrix R as a function of the angle (θ) between (Uice−Uw) and W10 is given byThe free drift sea ice motion using the Coriolis force, balance of wind, and water stressUice as sea ice velocity can be expressed asα is the wind factor. In an idealized steady ocean, the second and third terms in Eq.  can be estimated, allowing sea ice motion and sea ice velocity in free drift to be defined asNa is the Nansen Number and Ro is the ice’s Rossby Number, given byThe α) and angle (θ) can be written asThe wind factor  laser altimetry  and wind velocity vector at 10 m above the sea level, respectively. The air–ocean drag coefficient (62ρw is the surface water density (1026 kg m−3); Uice is the sea ice velocity (observation)53; and Uw is the water velocity63. Understandably, the water velocity was unknown below ice coverage in the AS. Thus, we estimated a parameterization assuming that a full Ekman spiral developed below the ice and that no other forces than the stress were acting on the water; this gave the surface current velocities as65f is the Coriolis parameter, AZ is the vertical eddy viscosity of 0.1 m2 s−1, and τc) was written asIn the coastal region, the horizontal imbalance of energy from the atmosphere into the ocean can have a large impact on ocean circulation. Therefore, ocean circulation will be strengthened by horizontal variations in the wind field and sea ice condition at the boundary of the polynya. Ocean surface stress is calculated as a combination of sea ice and wind stress asSupplementary InformationPeer Review File