S. strictum 203 were studied. The tests of acute toxicity, immunotoxicity and allergenicity should conclude that the studied proteolytic complex is safe for medical usage. For the intravenous and the intraperitoneal routes of administration, the maximum tolerated dose and the median lethal dose were not determined.The different effects on animals of the thrombolytic protease complex of the new producer  Sarocladium strictum 203 (VKM F-4845D) showed a direct fibrinolytic activity and a plasminogen-activating effect (indirect fibrinolytic activity) [One of the main goals of modern medicine is the diagnosis and treatment of conditions caused by thrombosis. Thrombolytic agents, which can be used in therapy, are proteolytic enzymes with an activity similar to one of haemostatic system proteases . Such acVKM F-484D showed The study of acute toxicity, immunotoxicity and allergenicity is very important to conduct before non-clinical studies. This allows us to draw general conclusions about the safety of the studied substance and to save further time and money. It is important at the early stages of the development of a drug to assess its prospects and the possibility of its further use for the treatment of humans.S. strictum 203.This work aims to study allergenicity, immunotoxicity, and acute toxicity of proteinases of the micromycete S. strictum 203 were supplied by the Department of Microbiology, Moscow State University and deposited in the All-Russian collection of microorganisms (number VKM F-4845D). The strain was maintained in tubes with slant agar. To receive the substance S. strictum 203 was cultured in submerged conditions. A culture of the micromycete was grown on Czapek’s slant medium for 7 days, after that, spores were washed by inoculum media and transferred to the medium for 48–72 h. Part of the biomass (10% v/v) was transferred to the fermentation medium for 96–120 h at a temperature of 28 °C on a rocker at 220 rpm. The medium for enzyme biosynthesis had the following composition (in g/L): sucrose, 40.0; K2HPO4, 4.4; NaNO3, 19.0; KNO3, 2.5; pH 6.5. At the end of the cultivation, the culture liquid was separated from the mycelium by centrifugation . Proteins from the culture fluid were precipitated by acetone (2 v/v) cooled to −20 °C, kept for 1 h at 4 °C, and then filtered. The filtered precipitate was dried in a vacuum desiccator over calcium chloride for 3 days. The received substance was a homogeneous yellowish–white powder without smell, well soluble in water. For the received substance, in vitro the tests were carried out as described earlier [Micromycete The acute toxicity study was carried out on Wistar rats  and C57Bl/6 mice  of both sexes.The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Bioethics Committee of M.V. Lomonosov State University (protocol 16-1 dated 14.05.2021).The allergenicity study was carried out on Wistar rats  of both sexes; CBA mice  of both sexes; CD1 mice  of both sexes; Soviet chinchilla Rabbits (2–2.2 kg) of both sexes.The immunotoxicity study was carried out on (C57Bl/6xDBA)F1 mice  males.All rodents were born in the vivarium of Moscow State University and used for studies in vivo. Animals were kept by groups  under a 12 h light-dark cycle (lights on at 08:00), 30–70% relative humidity and at 20–24 °C. Adult rabbits of either sex weighing 1–1.5 kg were purchased from local market and kept in the vivarium of Moscow State University for one week for acclimatization. Standard food for rabbits and filtered tap water were provided ad libitum.Animal content and all studies involving animals were carried out in accordance with the rules ,8. SubstAllergenicity of substance was studied in four separate allergenicity tests: (1) active cutaneous anaphylaxis in mice; (2) active systemic anaphylaxis test in rats; (3) concanavalin A–induced inflammatory reaction in mice; (4) conjunctive test in rabbits. For carrying out these experiments, Wistar rats of both sexes, CBA mice of both sexes, CD1 mice of both sexes, Soviet chinchilla rabbits of both sexes were used.The division of animals into groups was performed. The first group included 10 CD1 mice  receiving injections of normal saline. The second group included 10 CD1 mice  receiving injections of substance, which was administered at a dose of 24 mg/kg. The third group included 10 CD1 mice  receiving injections of substance, which was administered at a dose of 240 mg/kg.First injection was performed subcutaneously, second and third injections were performed intramuscularly into the quadriceps muscle of the thigh (0.2 mL/kg).An active cutaneous anaphylaxis test was performed on the 14th day after the last substance injection ,10.On the last 14th day, the mice have injected with substance intracutaneously ; the same volume of normal saline was injected intracutaneously to another area of the skin.To assess the reaction, 20 min after the intracutaneous injection, animals were injected by Evans’ blue solution  intravenously. The mean diameter and intensity of the ‘blued’ lesion surrounding the injection sites were assessed 30 min later.Rats were divided equally into three groups of 10 animals  per group and treated as follows: rats of the first group were injected with normal saline subcutaneously (sc) for three days, the second and the third groups were injected with ovalbumin solution (0.012 mg/kg sc) for three days.On the 12th day after the last ovalbumin injection rats of the first group were injected with normal saline intraperitoneal (ip) (1 mL/kg) for four days, the second group was injected with substance solution  for four days, the third group was injected with substance solution  for four days ,12.2 inhalation), and the weights of their small intestine were determined. Then, the small intestine was dried, and the weights were determined. The degree of anaphylaxis development was assessed by the severity of the oedema of the small intestine ).On the 15th day, an hour after the substance injection, all animals were injected with ovalbumin solution (0.036 mg/kg ip). Thirty minutes after ovalbumin injection, the animals were euthanized  receiving injection of normal saline subcutaneously (sc). The second group included 10 mice  receiving injection of substance, which was administered at a dose of 24 mg/kg sc (0.2 mL). The third group included 10 CD1 mice  receiving injection of substance, which was administered at a dose of 240 mg/kg sc (0.2 mL).One hour after substance injection, all animals received Concanavalin A solution  in the pad of the left hind paw. The right hind paw received the same volume of normal saline.2 in-halation). The weights of both paws were determined. The reaction index was calculated by the following formula:Ri—reaction index, expW—the weight of the experimental paw, contW—the weight of the control paw.After 1 h of the Concanavalin A injection, the animals were euthanized (COSoviet chinchilla rabbits (2–2.2 kg) of both sexes were divided equally into two groups of 6 animals  per group and treated as follows: rabbits of the first group were injected with normal saline subcutaneously (sc) for five days, the second group animals were treated with substance  for five days.Fourteen days after the last substance administration, all animals were treated as follows: 1 drop of the substance (100 mg/mL) was dipped in the left eye , 1 drop of the isotonic sodium chloride solution was dipped in the right eye .The state of sclera, cornea, and eyelids was verified. The reaction was determined after 15 min (fast response), 24 and 48 h in accordance with the scale of estimating the ophthalmological indicators .Three following experiments were performed: (1) a delayed-type hypersensitivity test; (2) T-Cell-Dependent Test; (3) phagocytic activity of peritoneal macrophages. For all three tests 90 male (C57Bl/6xDBA)F1 mice were used (30 mice per test).Mice were divided into three groups. The first group included 30  mice receiving injection of normal saline subcutaneously (sc). The second group included 30 mice  receiving injection of substance, which was administered at a dose of 20 mg/kg sc (0.2 mL). The third group included 30 mice  receiving injection of substance, which was administered at a dose of 200 mg/kg sc (0.2 mL).The substance was injected subcutaneously daily for five days (0.2 mL/kg), after which the following tests were performed.To perform a Delayed-Type Hypersensitivity Test 30 mice  from each group) were injected with a solution of sheep red blood cells (SRBC)  subcutaneously at the base of the tail. After 5 days the animals were injected with SRBC  in the pad of the left hind paw. The right hind paw received the same volume of normal saline.2 inhalation), and the weights of their paws were determined. The reaction index was calculated in the same way as was shown above.Finally, 24 h after the injection, the animals were euthanized  from each group) were injected with a SRBC intraperitoneally .2 inhalation), the spleen was evaluated and weighed, the state of the immune system was evaluated, determining the number of the antibody-producing cells (APC) in the spleen by the plaque-forming cell assay, calculating the modulation index (immune response) compared to the control group [After 5 days of the immunization, the mice were euthanized , the peritoneal exudate was isolated. The total number of peritoneal macrophages in the exudate (1 μL) and the number phagocytic cells were counted using a Gorjaev’s chamber.To perform this test 30 mice  from each group) were injected intraperitoneally with 1 mL of ink particles suspension. Ten minutes later the mice were euthanized  and intravenous (iv) administration to C57Bl/6 mice of both sexes and to Wistar rats of both sexes.For the iv administration, 40 mice and 40 rats were used.Mice were divided into 4 groups of 10 animals each. The first group received injection  of substance, which was administered at a dose of 0.551 mg/kg. The second group received injection  of substance, which was administered at a dose of 1.102 mg/kg. The third group received injection  of substance, which was administered at a dose of 2.204 mg/kg. The fourth group received injection  of substance, which was administered at a dose of 4.408 mg/kg.Rats were divided into 4 groups of 10 animals each.The first group received injection  of substance, which was administered at a dose of 0.2755 mg/kg. The second group received injection  of substance, which was administered at a dose of 0.551 mg/kg. The third group received injection  of substance, which was administered at a dose of 1.102 mg/kg. The fourth group received injection  of substance, which was administered at a dose of 2.204 mg/kg.For the ip administration, 40 mice were used. Animals were divided into 4 groups of 10 animals each. The first group received injection  of substance, which was administered at a dose of 0.551 mg/kg. The second group received injection  of substance, which was administered at a dose of 1.102 mg/kg. The third group received injection  of substance, which was administered at a dose of 2.204 mg/kg. The fourth group received injection  of substance, which was administered at a dose of 4.408 mg/kg.After iv and ip injections of substance, the animals were observed for 14 days. After this period, all animals were euthanized, and a diagnostic autopsy was performed.After the administration of the substance, the animals were continuously observed for an hour , then 5 min every hour for eight hours, signs of the manifestation of intoxication were recorded.In the next 13 days, animals were examined daily twice a day.During the study, the following parameters were registered: animal death—daily during the entire observation period; visual inspection of animals (daily); inspection with elements of neurological testing (at least 1 time per week); the weight of animals (at least 1 time per week); consumption of food and water (at least 1 time per week); macroscopic examination of internal organs and histological examination of organs with external pathologies.The parameters of the general state of animals were recorded visually at least once a week.The following parameters were studied: activity in the cell, the presence/absence of tremor, the appearance of animals , the parameters of the individual emotional status , emotional tension in the cell, simple reflexes . The weight of animals was registered at least once a week. Consumption of water and food was registered at least once a week.During the autopsy, we investigated: muscular system, internal surfaces, a skull cavity and a brain surface, neck with organs and tissues, chest, abdominal and pelvic cavities with organs and tissues in them. All deviations from the norm were fully described and documented.p < 0.05 was taken as significant.For performing the statistical analysis Statistica 7.1  was used. Kruskal–Wallis ANOVA on ranks test was used for data analysis. Results are expressed as mean of data ± SEM. In all tests, In the test of active cutaneous anaphylaxis in mice, the mean diameter and intensity of the ‘blued’ lesion surrounding the injection sites were assessed . As a reIn the test of active systemic anaphylaxis in rats, the degree of the development of anaphylaxis was assessed by the severity of the oedema of the small intestine to change the content of the total water in it (method of drying to constant mass) .As a result of this test, the statistically significant differences between the control  and experimental groups  are not identified.Thus, the studied substance does not affect the development of active systemic anaphylaxis in the presented test and does not show allergenic properties.In the test of Concanavalin A-induced inflammatory reaction in mice, the weights of both paws were determined, and the reaction index was calculated .As a result of this test, the statistically significant differences between the control and experimental groups were not identified. The average reaction index had similar values for all three groups.Thus, the studied substance does not affect the intensity of the inflammation reaction and does not show allergenic properties.Cornea ; iris (the degree of inflammation of the iris); conjunctiva . Each of the experimental parameters was assigned a score from 0 to 5, depending on the severity of the trait. At both time points, all rabbits, males and females alike, have the indicators of the state of the cornea, the iris and conjunctiva at the original physiological level.The Draize Test was used for evaluating the irritant action of the substance in the conjunctive test in rabbits. Three-block parameters were considered to estimate the reaction:Thus, the analysis of the results of the conjunctival sample showed that the studied substance does not have an irritating effect.In the humoral immunity in T-Cell-Dependent test in mice, the number of the antibody-producing cells (APC) in the spleen was determined by the plaque-forming cell assay, and the modulation index (immune response) compared to the control group was calculated.The results of this test were expressed as a number of APC per 1 million nucleus-containing cells in the spleen. It has been established that the administration of the substance does not lead to a change in the mass of the cells of the spleen compared with the control animals .The administration of the substance did not lead to stimulation of humoral immune response in doses of 20 and 200 mg/kg.As a result of this test, the amount of APC did not increase in a statistically significant manner, and differences between the control and experimental groups were not identified . Thus, tIn the Delayed-Type Hypersensitivity Test in mice, the reaction index was calculated . The anaIn the Phagocytic Activity of Peritoneal Macrophages test the number of peritoneal macrophages and the number of the ink particle-containing (phagocytic) cells were counted. The phagocytic index was calculated .The test results showed that the studied substance does not significantly change the phagocytic activity of peritoneal macrophages at all doses . Thus, iDuring the entire period of the experiment (14 days), not a single death was recorded in all groups of animals. No clinical symptoms were observed in the control group. In contrast, increased breathing rate and decreased activity were observed in all animals in the experimental groups immediately after administration. In the first minutes after the intravenous administration of the substance, the rats and mice experienced pain in the area of administration for 30–50 min. Thus, we failed to detect the lethal dose of the studied substance. No lethality was observed at intravenous injection at all doses.The intraperitoneal administration introduction of the substance also caused a pain syndrome: the animals were assembled into groups in the corner of the cell, they did not move for 10–15 min, then several animals showed classic indicators of strong abdominal pain for 60–90 min.The general condition of experimental animals was the same as in the control animals during the term of observation (14 days).Animals showed normal activity in the cell, and there was no tremor, the appearance of animals  was normal.Forage and water consumption were within normal limits.The faeces and urine were normal consistency, smell, and colour. The reaction of animals to sound and tactile stimuli was adequate. There were no signs of emotional tension.The weight of animals decreased by the next day after the drug administration by 5–22%, depending on the group, but returned to the initial values a week after the administration. The decrease in body weight was a consequence of stress after the introduction of the substance.During the macroscopic examination, no visible changes in the internal organs were observed.Thus, the studied substance in prophylactic and therapeutic doses and in a dose three times greater than the therapeutic did not show toxic effects.The analysis of the results of the acute toxicity test show that the median lethal dose for the studied substance for the iv and the im administration is more than 4408 mg/kg.S. strictum 203 may be safe for medical usage and very perspective in the biomedical area.Summing up the results of all three used tests, we conclude that the studied substance is safe from the point of view of allergenic properties, immunotoxicity and acute toxicity in the conducted experiments on animals. For the intravenous and the intraperitoneal routes of administration, the maximum tolerated dose and the median lethal dose were not determined. That means the proteolytic complex of


The Global Burden of Disease (GBD) does not produce estimates of heart failure (HF) since this condition is considered the common end to several diseases . This study aims to analyze the interactions between underlying and multiple causes of death related to HF in Brazil and its geographic regions, by sex, from 2006 to 2016.Descriptive study of a historical series of death certificates (DCs) related to deaths that occurred in Brazil between 2006 and 2016, including both sexes and all age groups. To identify HF as the underlying cause of death or as a multiple cause of death, we considered the International Classification of Diseases (ICD) code I50 followed by any digit. We evaluated the deaths and constructed graphs by geographic region to compare with national data.We included 1,074,038 DCs issued between 2006 and 2016 that included code I50 in Parts I or II of the certificate. The frequency of HF as the multiple cause of death in both sexes was nearly three times higher than the frequency of HF as an underlying cause of death; this observation remained consistent over the years. The Southeast region had the highest number of deaths in all years  and approximately double the number in the Northeast region and more than four times the number in the North region. Codes of diseases clinically unrelated to HF, such as diabetes mellitus, chronic obstructive pulmonary disease, and stroke, were mentioned in 3.11, 2.62, and 1.49% of the DCs, respectively.When we consider HF as the underlying cause of death, we observed an important underestimation of its impact on mortality, since when analyzed as a multiple cause of death, HF is present in almost three times more deaths recorded in Brazil from 2006 to 2016. The mentioning of conditions with little association with HF at the time of the death highlights the importance of HF as a complex syndrome with multiple components that must be considered in the analysis of mortality trends for implementation of public health management programs. The number of individuals with cardiovascular diseases (CVDs) increased from 271  million in 1990 to 523 (95% UI 497–550) million in 2019, and the number of CVD deaths increased steadily from 12.1 (95% UI 11.4–12.6) million in 1990 to 18.6 (95% UI 17.1–19.7) million across the 21 world regions analyzed by the Global Burden of Disease (GBD) in 2019. Considering the population growth and aging, the prevalence of CVDs is estimated to increase in several geographic areas. In Brazil, the disability-adjusted life years (DALYs) lost due to ischemic heart disease (IHD), the main component of CVD mortality, ranged from 771.2 (95% UI 679.4–866.3) per 100,000 in Amazonas to 2416.2 (95% UI 2176.7–2686.2) per 100,000 in Rio de Janeiro, a 103.2% difference within the country, with important regional variations ) ) . After dInstituto Brasileiro de Geografia e Estatística, IBGE) from 2000 and 2010, intercensal projections until 2012, and population projections from 2013 onwards.Information about the population residing at each region was also obtained from the DATASUS website,  which ofWe identified the occurrence of HF listed in the DC as the presence of code I50 (followed by any digit) of the 10th revision of the International Statistical Classification of Diseases and Related Health Problems (ICD-10) listed in Parts I or II of the certificate. We excluded those in which the field for information regarding the sex of the decedent was either empty or marked as “ignored.” Since each line in Parts I  and II of the DC may display up to four different ICD-10 codes, we subdivided these lines into four subparts, each with a single code, so that the frequency of each code could be assessed independently.We analyzed the absolute numbers of DCs at a national level, and the absolute numbers of DCs and mortality rates per 100,000 inhabitants in each geographic region (identified by federative units), according to the selection of HF as the underlying cause or it’s mention in any part of DC - as a multiple cause of death. When they are mentioned in more than one line in DC, it was considered just once.All codes cited in Parts I and II of the selected DCs were evaluated and we also performed this analysis by geographic region and built separate graphs for comparison of national data. The codes I50.0, I50.1, and I50.9 were analyzed together and represented by the code I50x.The data were analyzed using Stata (StataCorp LP), and the graphs and tables were built using Excel .Between 2006 and 2016, we identified 1,074,038 DCs with code I50x cited in Parts I and II. Of these, 120 had improper information in the field “sex,” and were not included when analysis by sex were performed.As shown in Fig. Quantifying the number of DCs listing HF as a multiple cause of death in each geographic region and across all regions showed little variability over the years Fig. A. In allFigure Of the 296,715 DCs with HF  cited as the underlying cause of death, 886,442 mentions were found, and the two codes most frequently cited in Parts I and II were directly related to HF (I50.0 and I50.9), listed in 33.5% of the deaths. Table Overall, HF was listed in any line of Parts I and II 1,074,038 times in all DCs, and the codes most frequently mentioned in these cases were those with the term “heart failure”  Table . Of noteFig. In some regions across the world, the age-adjusted incidence of HF has stabilized and appears to be decreasing, but the total number of patients living with HF is increasing, reflecting the chronic course of the disease associated with population aging and growth. Data about the epidemiology of HF are limited, especially in middle-income countries like Brazil, where the prevalence of HF is considered to be growing, and an increasing association of HF with conditions such as rheumatic disease, Chagas disease, hypertension, and others has been reported . A studyAnother Brazilian study analyzing deaths from HF between 2008 and 2012 compared two models redistributing HF deaths to specific causes of death in the age group of 55 years or more. One model, in which the deaths were redistributed based on data from hospital records, redistributed 45.8% of the deaths to HF based on the principal diagnosis at the hospital. The other model (method of multiple causes of death) redistributed the underlying cause of death in records in which HF was cited as the underlying cause of death to hypertensive heart and kidney diseases (25.3%), IHD (22.6%), and diabetes (9.6%) [In our population-based study in adults of both sexes, we also observed that HF was the most frequent diagnosis (33.5% of the cases) when this condition was listed as the underlying cause of death Table . On the Of note, although Chagas’ disease with cardiac involvement is reported to be highly prevalent in Brazil and Latin America, , 9 this , we realized that there was a need to evaluate the frequency of HF listed as a multiple cause of death in different regions of the country.Considering the total number of DCs with HF as the underlying cause of death or as a multiple cause of death, women predominated throughout the period Fig. , particuA study using coarsened exact matching to redistribute deaths records certified to HF  in the US, Mexico, and Brazil  assigned most deaths to IHD , followed by hypertensive heart disease , and diabetes  . AnotherIn the present study, the number of DCs with HF listed as a multiple cause of death was higher in the Southeast region across the entire period, especially in 2016. In contrast, the mortality rate of HF as a multiple cause of death  was higher during the entire period in the South region, with a peak in the year 2011, probably due to the aging of the population and the quality of the information cited in DCs in the South. A study analyzing the observed and predicted standardized mortality related to HF as an underlying cause from 2001 to 2030 in Brazil estimated a progressive reduction in the last years to about 6.1 and 6.2% in men and women, respectively, although the authors stressed that the absolute number of deaths from HF should increase over time due to population growth and aging. They observed higher rates in the South and Southeast regions, which they attributed to greater access to the health care system by patients with chronic diseases and to increased life expectancy in these regions .The evaluation of HF as a multiple cause of death is fundamental since the official data considering only the underlying cause of death substantially underestimates the impact of this condition on mortality data. The report in the DC of conditions at the time of death that have little association with HF, as found in the present study, highlights the importance of HF as a complex syndrome with multiple components that need to be considered in the analysis of mortality trends for implementation of public health management programs.


Using the ecological civilization demonstration zone as a quasi-natural experiment, this study has explored the effect of it on air pollution in China by employing the difference-in-differences model and the spatial difference-in-differences model, and further tested the political promotion tournament in China by employing the binary logit model. The results show that the ecological civilization demonstration zone has basically and effectively reduced air pollution, except for carbon monoxide and ozone. In addition, the spatial spillover effects of the ecological civilization demonstration zone on air pollution are not only basically supported among the treated cities, but also extremely established in the untreated cities neighboring the treated cities. Furthermore, no clear evidence supports the establishment of the political promotion tournament in China, while local cadres tend to cope with the assessment of higher officials passively rather than actively. Overall, this study sheds light on the coordination of economic development and ecological civilization from the perspective of the career concerns of local cadres. As the undesirable by-product of industrialization and urbanization in China, environmental pollution not only hurts the human health of residents, but also threatens the sustainable development of biological systems . In partIn the stage of shifting from high growth to high quality development, ecological civilization becomes a crucial issue in the new era . As one 2.5 non-compliance of prefecture-level and above cities in 2020 has decreased by 28.8% compared with 2015. However, whether and how ECDZ affects air pollution in China still remain as a black box, which leaves an opportunity for this study.Nevertheless, due to inconsistent environmental performance assessment standards across different regions, highly polluting enterprises tend to move from areas with strict control to areas with less control, which reduces the whole effect of environmental regulation and is detrimental to ecological protection . Even soDue to the advantage of identifying the policy effect of quasi-natural experiments in econometric analysis, the difference-in-differences (DID) model has been widely used in policy evaluation . Hence, The marginal contributions of this study can be drawn from two aspects. Theoretically, the current estimates of the effects of air pollution are mainly concentrated in the economic and medical field, while few studies focused on the political field with the consideration of economic growth by establishing a comprehensive framework under the background of the political promotion tournament in China. Hence, this study takes advantage of the opportunity and has great theoretical significance for institutional economics and political economics, especially for the emerging countries with similar situations. In practice, the current discussion on ecological civilization mainly focuses on its measurement and driving forces, while no study has comprehensively explored the environmental impact of ECDZ in China, especially with the consideration of heterogeneous pollutants and spatial spillover effects. Therefore, this study has great practical significance for construction and optimization of ECDZ in China, and also has significant international meanings for other emerging powers.We organize the rest of this study as follows. As a fundamental part of the reform, China’s central authority began to be decentralized to local governments in 1984, and this led to fierce fiscal competition among regional governments with the incentives of economic growth and political promotion . On the However, since economic growth was placed in the primary position and the competition among local cadres was based on the economic performance, local governments tend to generally enforce environmental regulations with low standards to obtain mobility, which leads to extensive development at the expense of environmental pollution . In partAs a philosophical topic and cultural theory, ecological civilization focused on balancing the relationship between humans and nature . HoweverAgainst this background, to promote the construction of ecological civilization, the National Development and Reform Commission and other six ministries issued the “ecological civilization demonstration zone construction program (pilot)” in 2013, and then the first batch of ecological civilization demonstration zones (ECDZ) was officially designated in 2014, followed by the second batch of ECDZ in 2015 . In partTo vividly illustrate two different types of promotion logic, we draw 2.5), coarse particulate matter with a diameter of 10 millionths of a meter or less (PM10), sulfur dioxide (SO2), carbon monoxide (CO), nitrogen dioxide (NO2), and ozone (O3). Second, the biographical and promotional information of local cadres was mainly collected from two official websites, namely xinhuanet.com (accessed on 20 October 2020) and people.com (accessed on 21 October 2020). Third, the list of pilot cities where ECDZ were located was mainly sourced from the website of the National Development and Reform Commission in China. Fourth, the socioeconomic data were collected from the China City Statistic Yearbook and the China Urban Construction Statistic Yearbook. In particular, this study has deleted several cities, namely, the four municipalities directly under the central government , the prefecture level cities with severe data loss and administrative changes , and dozens of national autonomous prefecture cities, and eventually formed panel data including 280 cities from 2014 to 2019 as the studying samples.This study obtained the data from several sources. First, the air pollution data at the city level came from the Chinese Research Data Services (CNRDS) platform, which collects daily data on AQI, which was calculated by the concentrations of six air pollutants, namely, fine particulate matter with a diameter of 2.5 millionths of a meter or less  are adopted to act as the proxy of the dependent variables; DID, a dummy variable, denotes the core explanatory variable, which equals to 1 after the city owns the title of ECDZ, and otherwise 0; in addition, several control variables are introduced into the research framework to control for the characteristics of each city, including the natural logarithmic of per capita gross domestic product (lnPGDP) and its quadratic term ((lnPGDP)^2) to test the establishment of the Environmental Kuznets Curve, population density (PD) measured by the share of total population to municipal area, the share of secondary industry to GDP (SI), the share of third industry to GDP (TI), and financial pressure (FP) measured by the share of the difference between fiscal expenditure and fiscal revenue to GDP.In Panel A, the annual average value of AQI (AQI) and the concentrations of six air pollutants  are employed to act as the proxy of the core explanatory variables; in addition, several control variables are employed to control for the characteristics of each cadre, including age (AGE), education (EDUCATION), gender (GENDER), and tenure (TENURE1 and TENURE5). Specifically, education (EDUCATION) is a dummy variable, which equals to 1 if the cadre has a doctorate degree, and 0 otherwise; gender (GENDER) is also a dummy variable, which equals to 1 if the cadre is a male, and 0 otherwise. Moreover, since the tenure of cadres is no more than 5 years, this study employs two dummy variables, namely TENURE1 and TENURE5, which equal to 1 if it is the first year or the last year of the tenure, and 0 otherwise.In panel B, political promotion (PP), a dummy variable, is adopted to act as the proxy of the dependent variables, which equals to 1 if the cadre is promoted to a higher rank, otherwise 0; economic growth rate with one year lag (EGR) and air pollution reduction  model to evaluate the effects of it on air pollution in China , and conT,T × Dit represents the spatial spillover effects on the treated cities, θ1 represents the coefficient of WT,T × Dit, WNT,T × Dit represents the spatial spillover effects on the untreated samples neighboring the treated cities, θ2 represents the coefficient of WNT,T × Dit, θ3 represents the spatial coefficient of Xit, and the other parameters are consistent with Equation (1).The DID model based on a quasi-natural experiment can to some extent cause the endogeneity problem. However, the spatial spillover effects of air pollution cause the failing at obtaining consistent estimators under the traditional DID model. Fortunately, with the spatial lag terms incorporating into the traditional DID model, the spatial difference-in-differences (SDID) model can control such spatial spillover effects. In particular, considering the advantage of the SLX  model in calculating local spillover effects rather than global spillover effects, this study has constructed the SDID model with the assumption of the SLX model , which iit denotes political promotion at city i in year t, α0 denotes the constant term, πit denotes the independent variables including air pollution reduction , economic growth rate (EGR) and the control variables, α1 is the coefficients of the independent variables, and the other parameters such as γi, μt, and εit are also consistent with Equation (1).Since the dependent variable is discrete  in panel B, the binary logit model is employed to test the establishment of political promotion tournament in China , dividinBefore implementing the regression, two diagnostic tests including the parallel trend test  and the 2.5, PM10, SO2, CO, NO2, and O3, and reported the corresponding results in the columns (1)–(7) of Based on Equation (1), this study has investigated the effects of ECDZ on the seven indicators of air pollution including AQI, PM2.5, PM10, SO2, and NO2. In addition, the coefficient of DID in column (1) is negative but insignificant, which indicates that the reduction effect of ECDZ on AQI is extremely weak in the statistics. However, the coefficients of DID in columns (5) and (7) are significantly positive, which indicates that ECDZ has increased the annual average concentrations of CO and O3. Thus, without the consideration of the spatial spillover effect, the effects of ECDZ on different indicators of air pollution have a clear differentiation; this is because compared with CO and O3, the other four indicators including PM2.5, PM10, SO2, and NO2 directly related to haze pollution, which are more sensitive to the public, and more effort is made to control them. Furthermore, long-term and short-term exposure to ambient ground level NO2 and O3 could play an important role in the phenotypes of cardio-respiratory diseases and inflammatory process in the lungs [3 and the reduced NO2 highlights the complex and difficulty of air pollution reduction in the process of constructing ecological civilization. Therefore, it is not hard to learn why local cadres tend to cope with the assessment of higher officials passively rather than actively.As is shown in he lungs ,28, thus2.5, PM10, SO2, CO, NO2, and O3, and reported the spatial regression results in the columns (1)–(7) of Based on the Equation (2), this study has explored the spatial effects of ECDZ on the seven indicators of air pollution including AQI, PMT,TD in columns (2)–(4), and (6) are significantly negative, the spatial coefficient of it in column (1) is negative but insignificant, while the coefficients of it in columns (5) and (7) are significantly positive, which indicates that ECDZ has indirectly reduced the annual average concentrations of PM2.5, PM10, SO2, and NO2, and increased the annual average concentrations of CO and O3 among the treated cities, while the reduction effect of it on AQI among the treated cities is relatively weak. Moreover, the spatial coefficients of WNT,TD in columns (1)–(4), and (6) are significantly negative, while the spatial coefficients of it in columns (5) and (7) are significantly positive, which indicates that ECDZ has indirectly reduced the annual average value of AQI and the annual average concentrations of PM2.5, PM10, SO2, and NO2, and increased the annual average concentrations of CO and O3 in the untreated cities neighboring the treated cities. Therefore, with the consideration of spatial spillover effects, the impacting trends of ECDZ on the seven indicators of air pollution are basically robust when compared with the benchmark results in As is shown in The effects of ECDZ on air pollution have been verified by adopting the DID and SDID models, while the establishment of the political promotion tournament in China has not been identified, which still needs an empirical test. Based on Equation (3), this study has investigated the effects of economic growth rate and air pollution reduction on political promotion in the treated samples and the untreated samples, and reported the results in As shown in Based on the panel data of 280 Chinese cities from 2014 to 2019, this study has explored the effects of ECDZ on air pollution by adopting the DID and SDID models, and tested the establishment of the political promotion tournament in China by investigating the effects of economic growth rate and air pollution reduction on political promotion by adopting the binary logit model. This study draws the conclusions below.3, ECDZ has to some extent reduced air pollution in China, no matter with or without the consideration of spatial spillover effects, which indicates that the effectiveness of ECDZ on reducing air pollution is basically established for the indicators of air pollution, namely, AQI, PM2.5, PM10, SO2, and NO2, while the controls of CO and O3 have been neglected, which deserves more attention in the future.First, except for CO and O2.5, PM10, SO2, and NO2 among the treated cities and in the untreated cities neighboring the treated cities, but increased the annual average concentrations of CO and O3 among the treated cities and in the untreated cities neighboring the treated cities, which highlights the robustness of the impacting trend.Second, except for the direct effects of ECDZ on air pollution, ECDZ has reduced the annual average values of AQI and the annual average concentrations of PMThird, although the political promotion tournament in China is not supported, the effectiveness of ECDZ has been to some extent proved. In particular, the governance of CO has indeed increased the possibility of political promotion in the treated cities. One possible reason is that the governance of CO is easier to see the effects of in the short-term and in turn becomes the key work of local cadres, which highlights that local cadres tend to cope with the assessment of higher officials passively rather than actively.In order to achieve the win-win of blue sky and economic development and promote the reform of political promotion mechanism simultaneously, several policy implications are provided as follows.First, due to the differentiation of ECDZ on different indicators of air pollution, the traditional “one-size-fits-all” pattern should be changed, and the governance of them should be adapted to local conditions. In addition, to guarantee the effectiveness of environmental governance institutionally, the evaluation of environmental pollution and protection should be incorporated into the political promotion mechanism.Second, due to the existence of spatial spillover effects and the collaborative governance problem caused by administrative segmentation, the joint governance of air pollution should be strengthened by adopting a top-level design. In addition, in order to improve the efficiency of cleaner production and lower the emissions of air pollutants simultaneously, a national exchange market of pollutants quota should be established soon.Third, in order to stimulate the motivation of local cadres and reduce their short-sight behaviors, a lifelong accountability of ecological civilization should be established. In addition, to promote the institutionalization, normalization, and standardization of ECDZ, the institutional joint governance mechanism should cooperate with the joint of the experts in environmental, economic, engineering, and other fields.Since this study has offered a novel perspective on identifying the effects of ECDZ on air pollution and the establishment of political promotion tournament in China, several potential directions still need an in-depth exploration. For instance, due to the limited availability of data, the panel data of this study merely include 6 years, which may to some extent soften the persuasiveness. In addition, political connections may be incorporated into the research framework to test the establishment of rent-seeking . Therefore, with the increase in the possibility of available samples and indicators, this topic still deserves further investigation.


Cancer diseases are currently one of the greatest health challenges in clinical medicine worldwide. Classic methods of treatment often lead to numerous side effects, including the development of multidrug resistance. For this reason, increasing hope is being placed on compounds of natural origin, mainly due to their pleiotropic effect on different types of cells, protective effect on normal cells and toxic effect on cancerous ones. The most studied group are the polyphenolic compounds, which include resveratrol. The effectiveness of polyphenols in the treatment and prevention of many diseases, including cancer of various origins, has become the basis of many scientific studies. The anticancer effect of resveratrol has been demonstrated at all stages of the carcinogenesis process. Additionally, whether administered by itself or in combination with cytostatics, it may play a significant role in the process of reversing multidrug resistance. A review of the effects of resveratrol in in vitro conditions proves that it has a stronger or weaker antiproliferative and proapoptotic effect on the cells of certain neoplasms of the gastrointestinal tract. Despite the differences in the effect of this compound on different types of cancer, a similar tendency can be observed especially regarding the correlation between the concentration of the compound and the incubation time on the one hand and the antitumour effect on the other hand. The information included in this review may prove helpful in planning in vivo and clinical studies in the future. The progress in medicine in terms of prevention programs, diagnostic tests and therapeutic strategies, as well as the awareness of industrialized societies in relation to the importance of physical activity and proper nutrition, have contributed to a significant improvement in the quality of life and the average life expectancy. Unfortunately, this is connected with an increasing incidence of aging-associated diseases, including cancer.Despite the implementation of cancer prevention, the incidence and mortality rates are constantly increasing everywhere in the world ,2. As a ABCB1 gene, encoding the P-glycoprotein (P-gp) protein, through which the drug is transported outside the cell, leading to a reduction in the effective concentration of the drug in the cell . Re. ReVeratoperties ,24,25. T extract . The cispe skins  27,28].,28.VeratSince then, this compound has been extracted from many other naturally occurring plants included in a varied diet, including bilberries, blueberries and peanuts ,30. HoweResveratrol is a phytoalexin, a compound produced by plants in response to the attack of pathogens and as a result of UV radiation and the action of heavy metals ,34. For The antitumour activity of resveratrol was first described in 1997 by Jang et al. ,41,42. DThe main goal of this review is to present the results of in vitro studies on the antiproliferative and proapoptotic effects of resveratrol on human gastrointestinal cancers.Pancreatic cancer is the ninth most common cancer in women and the tenth most common cancer in men ,50. The In the case of pancreatic cancers, the results of numerous in vitro studies indicate the potential antiproliferative effect of resveratrol, depending on the dose and the time of incubation with the compound.In a study on two pancreatic cancer cell lines, PANC-1 and AsPC-1, Xian-Zhong Ding’s team showed that after 48 and 72 h of incubation with a resveratrol solution at various concentrations, there was a statistically significant change in cell viability. These changes were dependent on the time and concentration of the compound tested. The greatest effects were observed at a concentration of 100 µM . HoweverThe studies of Jing Cui’s team also showed an inhibition of cancer cell survival dependent on the concentration and time of the effect of resveratrol. The tests were performed on the PANC-1, BxPC-3 and AsPC-1 pancreatic cancer lines, which were treated with resveratrol in a concentration range from 0 to 200 µM/L at 24, 48 and 72 h . StudiesAnother neoplastic model of pancreatic cancer—the Capan-2 line—was also used in order to study the effect of resveratrol on cell survival. After 24 h of exposure of the cells to resveratrol , a concentration-dependent decrease in cell viability was observed . The stuThe effect of resveratrol on cell survival was also studied by P. Liu’s team. Three pancreatic tumour lines were used in the study: PANC-1, CFPAC-1 and MIA PaCa-2, as well as normal pancreatic cells derived from tissue collected from a patient during surgery. The cells of the lines tested were exposed for 72 h to various concentrations of resveratrol. An increased cell growth inhibition was observed to take place with an increasing compound concentration. Compared with neoplastic cells, normal pancreatic cells showed greater resistance to the cytotoxic effect of resveratrol . The celMost studies confirm that the higher the dose of resveratrol, the stronger its antiproliferative effect on cells of various pancreatic cancer lines. At the same time, such a strong effect is not observed in normal pancreatic cells, which may translate into resveratrol being an effective and safe treatment option in the future.Apoptosis is a complex physiological process that follows a specific pattern that significantly influences the proper functioning of the organism. This process leads to the removal of unnecessary and damaged cells that could potentially pose a threat to the organism  . Due to For pancreatic cancer, resveratrol has been shown to be proapoptotic in many cases. In the studies of Xian-Zhong Ding’s team, the ability of resveratrol to induce apoptosis was examined by using flow cytometry. The pancreatic tumour cell lines PANC-1 and AsPC-1 were subjected to 72 h of incubation with 100 µM/L resveratrol. After this time, the induction of the apoptotic process was 27% and 34% for PANC-1 and AsPC-1, respectively .Similar tests were performed by Jing Cui et al. The cells of three pancreatic cancer lines  were incubated in resveratrol for 48 h at concentrations ranging from 0 to 200 µM/L. The analysis of the results showed that the induction of the apoptotic process was 26.20% for the PANC-1 cell line. A similar value was found for the BxPC-3 line: 22.08%. As for the AsPC-1 line, the percentage increase in apoptotic cells was only observed at a concentration above 150 µM/L . The teaStudies on resveratrol’s ability to induce the apoptotic process performed on the Capan-2 cancer cell line showed that the number of apoptotic cells after 24 h of incubation with 100 µM/L resveratrol solution increased significantly (from 12.92% to 21.31%) compared with cells not treated with the compound .The effect of resveratrol on apoptosis was also demonstrated by P. Liu’s team. Research shows that microRNAs can act as oncogenes or tumour suppressors; hence, they play an important role in the process of cancer initiation and progression . One of The effect of resveratrol on the activation of the executioner caspase-3, which is normally involved in typical apoptosis, was also studied. The tests were carried out on four pancreatic cell lines: PANC-1, MIA PaCa-2, Hs 766T and AsPC-1. The cells were incubated for 48 h with resveratrol (0–40 µM). Caspase-3 activity, which was measured with the help of a fluorometer, was the lowest for the Hs 766T line, while the highest activity was observed for the PANC-1 and the MIA PaCa-2 lines. For the AsPC-1 line, caspase-3 activity was intermediate in relation to the remaining cell lines. These studies did not include a reference to a normal pancreatic cell line .In conclusion, resveratrol is a proapoptotic compound in human pancreatic cancer, and it acts through many different pathways involved in this process, especially at high doses.Despite the observed reduction in the incidence of stomach cancer, it remains one of the most common neoplasms, especially among men . SimilarSeparate reports from recent years indicate that resveratrol is a compound that is also used in the treatment of gastric cancer .Xiaoping et al. analysed in vitro the effect of resveratrol on the survival of human gastric cancer cells as well as its potential mechanism of action. For this purpose, MGC-803 cells were exposed to different concentrations of resveratrol for 24 and 48 h. In this experiment, resveratrol was shown to inhibit cell growth depending on the dose and the duration of the effect. Accordingly, further experiments were performed to evaluate the effect of the compound on the cell cycle. The cells were incubated with resveratrol . The analysis showed that the treatment with resveratrol led to the inhibition of the cycle in the G0/G1 phase . Other sStudies on the effect of resveratrol on the inhibition of cell growth were also carried out on the gastric cancer model SGC-7901 cell line. A 48 h cell incubation with resveratrol (0–200 µM) resulted in an inhibition of cell proliferation proportional to an increasing concentration of the compound . SimilarFurther studies on the effect of resveratrol on neoplastic cells in gastric cancer were carried out on two cancer cell lines: the aforementioned SGC-7901 line and the BGC-823 line, as well as the benign epithelial cell line GES-1. The cell lines were exposed to various resveratrol concentrations ranging from 0 to 400 µM for 24, 48 and 72 h. A significant reduction in viability was observed in all cell lines tested. These changes were dose- and time-dependent .The proliferative activity of gastric cancer cells is significantly inhibited by resveratrol. The effect of the compound’s action is stronger when the concentration is higher and the exposure time is longer.Studies on the effect of resveratrol on the induction of the apoptotic process were carried out on a gastric cancer cell model: the SGC-7901 cell line. In this experiment, cells were incubated for 48 h with the compound in a concentration ranging from 0 to 200 µM. It was shown that the effect of resveratrol results in an increase in the production of ROS (reactive oxygen species), which according to the literature are involved in the apoptotic process . AdditioAs in pancreatic cancer, resveratrol also has a proapoptotic effect in gastric cancer, and the effect observed is stronger when the concentration of the compound is higher. However, there is less documented research on this tumour model.Liver cancer is also one of the most common cancers worldwide. It is diagnosed more often in men than in women . DespiteThe effect of resveratrol on cell viability was also studied in a liver cell model: the HepG2 line. In the experiment, cells were exposed to resveratrol (0–100 µM) for 24 and 48 h. The results of an MTT test showed a concentration- and time-dependent reduction of the viability in the cell line tested . P.-L. KIn conclusion, the tests performed on liver cancer cell models indicate the antiproliferative properties of resveratrol, primarily the inhibition of the cell cycle. The results are dependent on the concentration and time of the incubation with the compound, as well as the tissue origin of the cells tested.The mechanism of apoptotic induction by resveratrol in liver cancer cells in vitro has not been fully understood yet. However, it is known that the effect depends on the type of cancer lines studied, among which there are differences in the response to the effect of the compound.In the case of HepG2 cells, P.-L. Kuo’s team showed that resveratrol inhibits cell growth and induces the apoptotic process. The level of p53 protein in the HepG2 and Hep3B cell lines was analysed after incubation with resveratrol. In the HepG2 cell line, the expression level of this protein increased along with the increase in resveratrol concentration, while in the Hep3B cell line, no significant changes in p53 expression level were observed. These results provide evidence that, in this case, the proapoptotic effect of resveratrol is mediated by the p53 protein . ProteinIn other studies performed on the HepG2 cell line, the most pronounced effect on the induction of apoptosis was observed (by flow cytometry) at a concentration of 50 µM during a 48 h incubation with the compound .The TUNEL method was used to evaluate the effect of resveratrol on the induction of apoptosis in the liver cancer cell lines HepG2, Bel-7402 and SMMC-7721. After 24 h of incubation with 100 µM resveratrol solution, it was observed that resveratrol increased apoptosis in relation to untreated cells. Moreover, it was shown that resveratrol also influences the apoptotic proteins Bcl-2 (antiapoptotic) and Bax (proapoptotic), which leads to a decrease in the Bcl-2/Bax ratio .The results of all the studies described above suggest that resveratrol may play a significant role in the apoptosis of liver cancer cells by participating in the regulation of the expression of cycle-suppressor and proapoptotic proteins.Intestinal cancer is a common cancer characterised by a high mortality rate all over the world . It mostStudies carried out on the intestinal cancer lines HT-29 and WiDr have shown that the effect of resveratrol leads to an increasingly stronger inhibition of cell proliferation as the concentration of the compound increases .In another experiment, the intestinal adenocarcinoma cell lines HCA-17, SW480 and HT-29 were used. The cell lines were exposed to various concentrations of resveratrol. A concentration- and time-dependent inhibition of the number of cells was demonstrated in all the lines tested .Another team’s studies on the HT-29 cell line showed varying resveratrol effects depending on the concentration after 48 h of incubation with the compound. The MTT test showed that lower concentrations of resveratrol (1 and 10 µM) stimulated cell growth, while higher concentrations (25 and 50 µM) inhibited cell proliferation . By exteThe anticancer properties of resveratrol have also been shown in in vitro studies in which the research model was the HCT-116 and Caco-2 human intestinal cancer cell lines. A proliferation inhibition dependent on resveratrol concentration was observed in both cell lines .In another study performed on the intestinal cancer cell lines HCT-116, CO-115 and SW480, after a 24 h incubation with the compound at concentrations ranging from 0 to 50 µM, it was observed that cell viability decreased as the concentration of the compound increased .The effect of resveratrol on the proliferation of neoplastic intestinal cells was also observed in the DLD1 and HCT15 cell lines. Treatment with resveratrol at concentrations ranging from 0 to 40 µM for 24, 48 and 72 h resulted in a dose- and time-dependent cell growth inhibition .In intestinal cancers, resveratrol may act bi-directionally, depending on the concentration. Higher concentrations of the compound result in a significant inhibition of proliferation in particular cell systems, while lower concentrations can stimulate cell growth.Cyclooxygenase 2 (COX-2) overexpression is associated with physiological and pathological processes in the body, including the formation and progression of neoplasms ,99. One In intestinal cancer, resveratrol also has proapoptotic properties. This effect is most pronounced when higher concentrations of the compound are used.Cancer is a complex disease of various aetiologies, and the use of conventional treatments in most cases is associated with numerous side effects. Hence, the current scientific research focuses largely on the search for new therapeutic strategies, especially ones based on compounds of natural origin. High hopes are placed on the possibility of using these compounds in clinical conditions. The most often studied compounds are the polyphenolic compounds, including resveratrol. Due to the diverse range of biological effects, it is considered a promising anticancer agent.Based on the available scientific literature, in this study we have documented the antitumour effect of resveratrol, especially its role in inhibiting cell proliferation and inducing the apoptotic process.In vitro studies conducted over the last few decades show that resveratrol can be antiproliferative  and proapoptotic in various types of cancer.In this work, we focused on the effects of resveratrol in cancers of the gastrointestinal tract .When analysing the scientific reports presented in our study, the effect of resveratrol is strongly dependent on the type of cancer, the concentration of the compound and the duration of its action . In the The effect of resveratrol in stomach cancer (inhibition of cell proliferation) is also specific to certain cells. Changes in cell survival are observed in a wide range of concentrations (0–400 µM). As happens in pancreatic cancer, resveratrol exerts an inhibitory effect on stomach cancer cells in a time-dependent manner.In liver cancer, the effect of resveratrol on cell proliferation also shows a relationship between the concentration and the duration of the effect. However, in this neoplasm, changes are observed at lower concentrations of the compound (0–100 µM).A similar relationship between the concentration and the duration of the action of the compound and cell survival can be observed in intestinal cancer. Studies evaluating the antiproliferative properties of resveratrol showed a notable inhibition of cell proliferation in intestinal cancer at significantly lower concentrations of the compound (0–50 µM).Many of the in vitro studies presented indicate that the antitumor activity of resveratrol may be largely due to the induction of the apoptotic process. Translating the promising results of these in vitro studies into clinical studies is challenging due to the poor pharmacokinetic parameters of the compound—low bioavailability, poor water solubility and fast metabolism —hence, tIn in vitro studies, resveratrol elicits a weaker or stronger response depending on the type of cancer. However, in each case there is a correlation between the concentration and time of the action and the effectiveness of the compound. The promising results of the in vitro studies presented in this paper, as well as in many others, emphasize the need for further experiments, especially in vivo, on the effect of resveratrol to determine the therapeutic doses of the compound and the exact mechanisms of its action in relation to different types of cancer.


Degenerative changes of the spine can cause spinal misalignment, with part of the spine arching beyond normal limits or moving in an incorrect direction, potentially resulting in back pain and significantly limiting a person’s mobility. The most important parameters related to spinal misalignment include pelvic incidence, pelvic tilt, lumbar lordosis, thoracic kyphosis, and cervical lordosis. As a general rule, alignment of the spine for diagnosis and surgical treatment is estimated based on geometrical parameters measured manually by experienced doctors. However, these measurements consume the time and effort of experts to perform repetitive tasks that could be automated, especially with the powerful support of current artificial intelligence techniques. This paper focuses on creation of a decentralized convolutional neural network to precisely measure 12 spinal alignment parameters. Specifically, this method is based on detecting regions of interest with its dimensions that decrease by three orders of magnitude to focus on the necessary region to provide the output as key points. Using these key points, parameters representing spinal alignment are calculated. The quality of the method’s performance, which is the consistency of the measurement results with manual measurement, is validated by 30 test cases and shows 10 of 12 parameters with a correlation coefficient > 0.8, with pelvic tilt having the smallest absolute deviation of 1.156°. Lumbar lordosis is the normal inward curvature of the lumbar spine observed only in human beings. It supports the body, absorbs shock, and ensures stability and flexibility, maintaining the torso within the cone of economy of so-called genuine bipedalism . When thIn data collection procedures, geometrical parameters often are manually measured from X-ray images by experienced surgeons. This can help limiting data collection errors and consequently increasing the reliability of analyses drawn from the dataset. However, it consequently requires a lot of effort and time, especially in research studies that require diverse datasets. To measure an angle parameter, such as a Cobb angle between two vertebrae, the radiologist need to draw tangent lines along the end of vertebrae on X-ray images by a ruler and a pencil, and then measure the angle using a protractor. This procedure highly dependents on the radiologist’s experience and also consumes a lot of time because of the large number of angle parameter between vertebras in one spine. Also, manual measurement of parameters presents a significant challenge in aggregating data for statistical analysis which is mostly performed sequentially by hand.Due to the development of artificial intelligence techniques, especially regarding deep learning, such applications in medical metrology that use vision-based data types have significant potential. One of the strengths of this technique is that it reduces the labor of repetitive work through a combination of the high-volume processing power of computers and the ability of artificial intelligence techniques to generate a set of weighting factors optimized to simulate the experience of experts. In addition, data collection after automatic measurement can be conveniently integrated with statistical tools, especially for large-scale datasets. Aubert et al.  proposedMost research focuses only on localized spinal regions, especially the lumbar region, while there is no comprehensive research on automatic measurement methods to evaluate global spinal alignment. Increased measurement of geometrical parameters of the entire spine could provide information for simultaneous monitoring and diagnosis of spinal sagittal deformities. This paper proposes a method that uses decentralized CNN algorithms to locate required key points and consequently measure the spinal alignment based on 12 parameters distributed throughout the cervical, thoracic, and lumbar spine. The measurement accuracy of the proposed method is validated via a comparison with manual measurements from experienced doctors as standard references.Pelvic incidence (PI) was defined as the angle between a line drawn from the center of the femoral heads (point F) to the midpoint of the sacral superior endplate (line EE’) perpendicular to the sacral superior endplate.Pelvic tilt (PT) was defined as the angle between a line drawn from the center of the femoral heads (point F) to the midpoint of the sacral superior endplate (line EE’) and the vertical line.Sacral slope (SS) was defined as the angle between the sacral superior endplate (line EE’) and the horizontal line.Lumbar lordosis (LL) was defined as the angle between the superior endplate of L1 (line DD’) and the superior endplate of S1 (line EE’).L1 incidence (L1I) was defined as the angle between a line drawn from the center of the femoral heads (point F) to the midpoint of the sacral superior endplate (line EE’) and perpendicular to the L1 superior endplate .T1 incidence (T1I) was defined as the angle between a line drawn from the center of the femoral heads (point F) to the midpoint of the sacral superior endplate (line EE’) and perpendicular to the T1 superior endplate.C2 incidence (C2I) was defined as the angle between a line drawn from the center of the femoral heads (point F) to the midpoint of the sacral superior endplate (line EE’) and perpendicular to the C2 inferior endplate .Thoracic kyphosis (TK) was defined as the angle between the superior endplate of T1 (line CC’) and the superior endplate of L1 (line DD’).C2-C7 lordosis (C27L) was defined as the angle between the inferior endplate of C2 (line AA’) and the inferior endplate of C7 (line BB’).L1 slope (L1S) was defined as the angle between the horizontal line and superior endplates of L1 (line DD’).T1 slope (T1S) was defined as the angle between the horizontal line and superior endplates of T1 (line BB’).C2 slope (C2S) was defined as the angle between the horizontal line and inferior endplates of C2 (line AA’).The angles chosen to characterize spinal alignment are determined according to geometrical relationships between quantities of interests, as shown in Fig. In an approach that differs from determining the lower and superior endplates of typical vertebrae (from C3 to L1), which is based on pairs of upper or lower corners and can be applied for special vertebrae such as C2 and S1, and the representative line AA’ is drawn from the anterior and posterior corner of C2 inferior endplate .After the input image features are extracted by the convolution  and max-However, a highly complex CNN structure can lead to redundancy of untrained weighting factors, which is known as overfitting , and tenL, expressed in Eq.  plots for all angles of the entire sagittal parameters, as shown in Fig. To clarify the interobserver analysis from two experts and compare the variability between manual measurement and the proposed method, the correlation coefficient between values from two experts, between the automated measurement and each manual measurement, and also average of them, were calculated. Besides, the mean absolute error (MAE) and standard deviation (STD) of the absolute error were also considered. This comparison totally considered 360 angles consisting of 12 considered parameters in 30 validation cases as shown in Table About the metric of MAE and STD, it can be noticed that the MAE between two experts is smaller than between the proposed method and experts, including each expert separately and average of them. The reason of this point is that the proposed method’s results are similar to the 1st expert measurement in some cases and, in the other cases, to the 2nd expert measurement. Because the similarity of the proposed method and one of two manual measurements depends on cases, the error between the automated method and each expert, and also average of two experts is larger than error between experts. Therefore, if the error between the automated approach and manual measurement is considered as the minimum value between the error of automated approach and 1st expert, and the error of automated approach and 2nd expert, the MAE and STD between the automated approach and manual measurement, respectively, are 2.995 and 3.003°, which is equal to the MAE and STD between two experts.This paper presents a method using a decentralized CNN to automatically determine spinal alignment by evaluating representative parameters from X-ray images. The proposed method is developed at the hierarchical levels of one model for locating ROIs of the cervical spine, lumbar spine, and femoral heads regions; a second model for positioning the landmarks considered; and a third model for determining the required key points. The advantages of this decentralization are due to the elimination of the effects of areas that are not necessary for detection by narrowing the considered areas at each successive model level based on the geometrical characteristics of the spine. Detection of key points that have geometrical characteristics, such as corners of vertebrae and center circles of femur heads, with small ROIs in the third model shows high accuracy. In addition, the ability to expand the dataset diversity by utilizing vertebral images not included in the image of the entire spine contributes to the quality of this approach.From the perspective of the medical field, this artificial intelligence (AI)-based measurement method possesses highly accurate automated ROI and key point detection rates. Compared to the expert-dependent manual measurement method, this AI-based method has the potential for nearly infinite expansion in its scope of technological applications. The knowledge of spinal sagittal alignment has increased linearly through manual measurement of preoperative and postoperative sagittal parameters in patients requiring surgery. However, the AI-based measurement method will enable an exponential increase in the knowledge of the spinal alignment by rapidly analyzing the chronological and reciprocal change of the spinal alignment of degenerative spondylotic patients as well as the normal population. In addition, the accumulated knowledge will help determine the degree of optimum deformity correction of the spine using a deep mining algorithm to compare patients with good versus bad prognoses.Segmentation of X-ray images or reconstruction of three-dimensional (3D) profiles utilizing the deep learning technique , 11 are There are other research approaches that propose deep learning models designed based on location of key points. After comparing the accuracy of these studies, the proposed method provides a significant advantage. These results compare favorably to those of a state-of-the-art method  for autoHowever, the proposed method has two main limitations. First, utilization of a decentralized CNN requires separate datasets which are contributed for each order and consequently consume time not only for creating the dataset but also for comprehensively training the CNN models. Second, there is a significant obstacle related to the lack of clarity of conventional X-ray images in the ROIs of T1 vertebrae. To visualize deviation of the performance of the proposed method by parameter, the correlations between manual measurements and obtained results of SS, L1S, T1S, and C2S are shown in Fig. These issues might be caused by a characteristic of the shoulder girdle of human, which usually overlaps on the spinal regions in sagittal X-rays. Therefore, visualization of the last vertebrae in the cervical region and the upper half of the thoracic region generally contains significant noise compared to other regions. The noise not only limits the performance of the proposed method but also causes difficulties for manual implementation.About the clinical significance of errors, considering the high correlation and small error in most of parameter cases, except the T1 vertebrae-related parameters, it is considered acceptable, especially with the purpose of this method which is to provide doctors the global deformity of spine for diagnosing and establishing the treatment. Besides, the most significant advantage of this approach comparing to manual measurement is reducing amount of time consumed by detecting features of vertebras by hand, which decreases from around 10 min for one case to less than 1 s.Consequently, the second limitation opens a future research direction on enhancing the visual quality of X-ray images. To deal with this limitation, the future research direction utilizing image-to-image deep learning, which receive the sagittal X-rays of T1 region as the input and provide a de-blurred image with high clarity of vertebrae features for experts and automated method to measure, is now being considered.Based on the time and effort required of an experienced doctor to measure alignment parameters of the spine, development of an automated measurement software that can utilize deep learning and leverage the rapid advancement of big data technologies could free labor from repetitive work and increase productivity because of its ability to process a large amount of data. A fully automatic method was proposed for measuring 12 spinal alignment parameters, PI, PT, SS, L1I, T1I, C2I, LL, TK, C27L, L1S, T1S, and C2S using a designed decentralized CNN algorithm. To validate the performance of the proposed method, the measured results of 30 test cases were compared to standard references created by human experts. The quality of the proposed method, which was evaluated in terms of the correlation coefficient and MAE, was recorded with good results for 10 of 12 parameters, while the gap of accuracy of the two remaining parameters was determined to be an effect of inevitable local noise in the images. In addition, the calculation time was less than 1 s for all cases. Because of difficulties in measuring parameters under in noisy conditions, future research will focus on the reconstruction of images with enhanced visual conditions using image-to-image deep learning techniques.


The compressive strength of MKPC increased with an increase in the glass powder content at the M/P ratio of 1:1, and the addition of glass powder reduced the porosity of the matrix, effectively increased the yield of struvite-K, and affected the morphology of the hydration products. With an increase in the M/P ratio, the struvite-K content decreased, many tiny pores were more prevalent on the surface of the matrix, and the bonding integrity between the MKPC was weakened, thereby reducing the compressive strength of the matrix. At less than 40 wt.% glass powder content, the performance of MKPC improved at an M/P ratio of 1:1. In general, the addition of glass powders improved the mechanical properties of MKPC and reduced the heat of hydration.Waste glass is a bulk solid waste, and its utilization is of great consequence for environmental protection; the application of waste glass to magnesium phosphate cement can also play a prominent role in its recycling. The purpose of this study is to evaluate the effect of glass powder (GP) on the mechanical and working properties of magnesium potassium phosphate cement (MKPC). Moreover, a 40mm × 40mm × 40mm mold was used in this experiment, the workability, setting time, strength, hydration heat release, porosity, and microstructure of the specimens were evaluated. The results indicated that the addition of glass powder prolonged the setting time of MKPC, reduced the workability of the matrix, and effectively lowered the hydration heat of the MKPC. Compared to an M/P ratio (MgO/KH According to a United Nations Environment Programme survey, 1.4 billion tons of waste glass is discarded each year, including flat glass, daily glassware, and glass packaging containers, accounting for more than 80% of glass products . TherefoTo improve the recycling of waste glass, many countries have established recycling policies. The United States has classified glass chips and bottles as environmental pollutants that must be recovered . Italy h43−, H+, and HPO42− plasma, and in a weakly acidic environment, hydration promotes the dissolution of MgO, where the Mg2+ develops a positively charged hydrosol, Mg(H2O)n2+; (2) the hydrosol reacts with the phosphate ions in the system to form hydration products, and the hydration products are connected; (3) the later stage of hydration will form a network structure with MgO as the skeleton and hydration products as the adhesive.In recent years, many researchers have investigated a new type of cement called magnesium phosphate cement (MPC), because of its many advantages, including fast setting and hardening , high ea2O3 can promote the crystallization of Struvite-K, prolong the setting time of the slurry, and reduce the flow rate [Nevertheless, a series of problems are encountered, both in the current research and in engineering applications of MPC, including the high cost of raw materials, rapid setting time, high exothermic hydration of MPC, and the lack of relevant international standards. MPC performance was effectively improved by adding an admixture to the MPC components, thereby reducing the demand for dead-burned MgO. For example, metakaolin effectively improves the MPC compressive strength, tensile bond strength, freeze-thaw resistance, and water resistance ; glass flow rate ; microstlow rate ; and watlow rate . Waste glow rate . HoweverMeanwhile, replacing MgO with an admixture of glass powder reduces the price of the raw materials. In this regard, some scholars have studied the addition of glass powder to magnesium ammonia phosphate cement (MAPC). The results indicate that the alkali component of glass powder (GP) is involved in the MAPC reaction. The addition of GP to MAPC promotes the crystallization process of the hydrate and produces secondary reaction products. When 10 wt.% glass powder is added to a magnesium-to-phosphate (M/P) ratio of 3:1, the matrix’s workability and setting time meet the rapid repair material requirements, and the matrix has a denser microstructure, thereby improving the compressive strength . HoweverIn this study, waste glass was ground into powder and added to MKPC to compensate for its limitation of application as a rapid repair cement. First, glass powder was substituted for a fraction of the dead-burned MgO, and the workability, setting time, and hydration temperature rise of the slurry at various M/P ratios were investigated. Next, the compressive strength of the samples was evaluated, and the porosity of the samples after curing for 28 d was determined by the piezometric porosity method. The type and yield of the hydration products were identified using X-ray diffraction (XRD) and thermogravimetric analysis. Finally, the morphological structure of the hydration products was examined by scanning electron microscopy and energy dispersive spectroscopy.2PO4, referred to as (KDP), analysis reagent, > 99.0%) produced by the Chengdu Jinshan Chemical Plant, China. Borax  produced by the Chengdu Cologne Chemical Plant, China, was the third component. Glass was ground into glass powder from waste glass, and the chemical composition and particle size distribution are shown in The raw materials used in this experiment included dead-burnt MgO . The MgO particle size distribution and chemical composition are shown in 2PO4 mass ratio) of 1:1, 2:1, and 3:1  were studied. The details are presented in In this experiment, the effects of different glass powder contents  on the hydration and hardening properties of MKPC with M/P ratios . The particle sizes of MgO and glass powder were tested in anhydrous ethanol solution and ultra-pure water solution, respectively. The compressive strength of the sample was tested using a microcomputer-controlled electro-hydraulic servo pressure testing machine . The main engine employed a high-performance bi-directional hydraulic cylinder loading with high reliability, zero leakage, a maximum load of 3000 kN, and a loading speed of 2.4 kN/s. The chemical composition of the sample was analyzed by an X-ray fluorescence spectrometer .The setting time was determined according to the Chinese standard method specified by JGJ/T 70-2009  and begaThe pore size distribution, total pore volume, total pore area, and other physical properties of the sample were measured using an Autopore 9500 mercury porosimeter . The maximum pressure was 33,000 lb (228 MPa), and the aperture analysis range was 5.5 nm to ~360 µm.The samples were ground into powders in an agate mortar and then analyzed using a DMAX1400 X-ray diffractometer  made by Rigaku, Tokyo, Japan. The morphology and micro-area composition of the hydration products of the solidified body were tested by a Variable temperature in situ imaging analysis system . The samples are gilded with a small ion sputtering apparatus . Differential thermal analysis of the MKPC-hardened body was performed using a thermal analyzer . The heating rate was 20 °C/min, and the test temperature range was 30–900 °C.In 2O)62+, phosphorrösslerite (MgHPO4·7H2O), newberyite (MgHPO4·3H2O), and Mg2KH(PO4)2·15H2O; the reaction intermediates are formed at high water content and low M/P ratios, (4) the growth acceleration period of the main hydration product struvite-K, (5) the growth deceleration period of the main hydration product struvite-K, and (6) the final hydration stability period. The main exothermic stage occurs during (2), (4), and (5).The hydration reaction of MPC can be divided into six stages : (1) the2+ ions are given by the following formulas:The dissolution of MgO and hydration of Mg4 7H2O), newberyite (MgHPO4 3H2O), and Mg2KH (PO4)2 ·15H2O.Reaction equations for phosphatite (MgHPO2O)62+, phosphate, and free water, but unreacted MgO was still present in the system. This hindered the combination of the three materials in this stage. The addition of glass powder reduced the content of unreacted MgO, minimizing the restriction between raw materials, which prolonged the formation stage of hydration products in (4) and (5), and increased the hydration reaction, leading to the appearance of the second exothermic peak.Based on previous analysis results, the glass powder was added as a filler in the hydration stage. Glass powder weakens the interaction between raw materials due to the large ratio of length to width of the particles and their irregular, angular, and flaky morphology. Friction readily occurs between the particles, which hinders the binding force between the raw material and free water, thus reducing the overall exothermic hydration reaction. Since the heat release of the overall hydration reaction is reduced, the time to reach the peak temperature is prolonged . In the The effect of the glass powder content on the compressive strength of the MKPC is shown in In the mercury intrusion porosimetry (MIP) results shown in 2PO4 content was lower than that of the low M/P ratio sample. Even if an appropriate amount of glass powder could have been added to speed up the reaction, the yield of K-struvite was restricted by the KH2PO4 content. GP can increase the crystallization rate of MPC and form more phases in MPC, including sodium phosphate and calcium phosphate gels [ate gels . Thus, aAs shown in To more intuitively observe the reaction of the GP particles in the MPC hydration stage, we conducted an experiment in which GP fragments with a particle size distribution of 0.1–1 mm were mixed into MPC paste. After 7 d of curing and subsequent SEM-EDS testing, the elemental distribution at the interface between the glass fragments and MPC paste was observed. When the M/P ratio was 1:1, the SEM image a showed The effect of glass powder on the properties of MKPC was investigated, resulting in the following observations.The addition of glass powder prolonged the setting time of MKPC. At an M/P ratio of 2:1, the setting time of MKPC blended with 40 wt.% glass powder was 11% longer than that of the sample without glass powder. The addition of glass powder also reduced the workability of the MKPC pastes. The reduction in workability increased with an increase in the M/P ratio and an increase in the glass powder content.The addition of glass powder can effectively reduce the heat release during the hardening of MKPC. When the GP content and the M/P ratio increased, the peak reduction of the hydration heat of MKPC was significant.When the M/P ratio was 1:1, the addition of glass powder made the MKPC structure dense and improved the compressive strength of the matrix. The analysis of the hydration products indicated that the alkali components in the GP participated in the reaction of MKPC, and the hydration products were closely bound to the MPC matrix. However, with the increase in the M/P ratio, the compressive strength of the MKPC structure decreased with increasing glass powder content. Under high M/P ratios, the increase in the GP content and the excess MgO occupied most of the void space in the MKPC, and some of the hydration products adhered to the surface of the GP, resulting in a decrease in the overall strength of the matrix.


NR) of tofacitinib was significantly slower (59.5%) than that for tofacitinib alone. An in vitro metabolism study showed non-competitive inhibition of tofacitinib metabolism in the liver and intestine by voriconazole. The concentration/apparent inhibition constant (Ki) ratios of voriconazole were greater than two, indicating that the inhibition of tofacitinib metabolism could be due to the inhibition of the CYP3A1/2 and CYP2C11 enzymes by voriconazole. The pharmacokinetics of voriconazole were not affected by the co-administration of tofacitinib. In conclusion, the significantly greater AUC and slower CLNR of tofacitinib after intravenous and oral administration of both drugs were attributable to the non-competitive inhibition of tofacitinib metabolism via CYP3A1/2 and CYP2C11 by voriconazole in rats.Fungal infections are prevalent in patients with immune diseases. Voriconazole, a triazole antifungal drug, inhibits the cytochromes CYP3A4 and CYP2C, and tofacitinib, a Janus kinase inhibitor for the treatment of rheumatoid arthritis, is metabolized by CYP3A4 and CYP2C19 in humans. Here, we investigated their interaction during simultaneous administration of both drugs to rats, either intravenously or orally. The area under the plasma concentration–time curve from time zero to time infinity (AUC) of tofacitinib was significantly greater, by 166% and 171%, respectively, and the time-averaged non-renal clearance (CL Tofacitinib A was devN-demethylation, and comprises about 50% processed by CYP3A4 and 20% by CYP2C19. It is then further metabolized to form glucuronide conjugates  is the concentration of voriconazole as an inhibitor (voriconazole), and Vmax’ is the apparent maximum velocity of tofacitinib (substrate) with voriconazole. The apparent Ki is the concentration of voriconazole to produce a half rate of Vmax for the elimination of tofacitinib. In the case of non-competitive inhibition, the slope of the inverse velocity for the elimination of a substrate against the concentration of an inhibitor decreases as the concentration of substrate increases until the slope lines meet at a point on the x axis; this point where they converge is −Ki ) in the liver and intestine were measured and are shown in I] up to 120 min post-treatment was higher than its hepatic iK (6.52 μM). The [I]/iK ratios in the liver were 3.68, 2.22, 2.56, 2.10, 1.96, and 2.30 at 1, 5, 15, 30, 60, and 120 min post-treatment, respectively , but the values in the intestine were less than the intestinal iK of voriconazole (26.2 μM). The [I]/iK ratios in the liver were greater than two and the ratios in the intestine were less than two (0.158–0.447) up to 240 min  were determined based on the results of previous studies; intravenous and oral doses with a linear relationship between doses and AUC values of tofacitinib  and voriNR of tofacitinib have been reported to be due to changes in tofacitinib metabolism in rats /Ki ratio of voriconazole in the liver and intestine. As mentioned earlier, voriconazole inhibited the hepatic metabolism of tofacitinib when both drugs were administered intravenously. Therefore, the effect of the voriconazole-induced hepatic metabolic inhibition of tofacitinib on the AUC of orally administered tofacitinib does not seem to be minor compared to the effect of the intestinal metabolic inhibition of tofacitinib by voriconazole, since the gastrointestinal first-pass effect of tofacitinib is approximately 46.1% of the oral dose, whereas the hepatic first-pass effect of tofacitinib is 21.3% of the oral dose after oral administration in rats [I]/Ki ratios for the inhibition of tofacitinib metabolism in the liver up to 240 min after the oral administration of both drugs were greater than two, but the [I]/Ki ratios in the intestine were less than two (I] values were obtained from the serosa and not from the enterocytes. Since orally administered drugs are absorbed through enterocytes, a high concentration of a drug during absorption is expected in the enterocytes [I] values had been measured in enterocytes, the [I] values would be high and the [I]/Ki ratios could thus be greater than two in the intestine. Therefore, the significant increase in AUC of tofacitinib (The significantly greater AUC of tofacitinib after the oral administration of both drugs appears to be due to the non-competitive inhibition of tofacitinib metabolism by voriconazole in the liver and intestine . These r in rats . The [I]than two . This coerocytes . In addierocytes ,40. In tacitinib  after thacitinib  via the acitinib . OverallR of tofacitinib was significantly slower in the presence of voriconazole after either intravenous or oral co-administration of both drugs, which is attributable to the significantly greater AUC of tofacitinib, due to the slower hepatic and intestinal metabolism of tofacitinib in both routes of administration (Ae0–24 h values of tofacitinib were comparable in the absence and presence of voriconazole for both intravenous and oral routes, indicating that voriconazole does not inhibit the organic cation transporter for the excretion of tofacitinib. Therefore, the slower CLR of tofacitinib when co-administered with voriconazole is attributable to the greater AUC of tofacitinib, in turn due to the metabolic inhibition of tofacitinib caused by voriconazole.The CLstration . TofacitCmax of tofacitinib were 103% and 16% higher, respectively, when it was combined with ketoconazole, an imidazole antifungal drug, and its AUC and Cmax were 79% and 27% higher, respectively, when co-administered with fluconazole, a triazole antifungal drug [In reports on drug interactions with voriconazole, a mixed inhibition of CYP enzymes in rat hepatic microsomes was reported when it was combined with vonoprazan, a reversible potassium-competitive acid blocker, which is metabolized by CYP2B6, CYP3A4, CYP2C19, and CYP2D6 . In addigal drug . TherefoNR of tofacitinib was significantly slower than that of tofacitinib alone (In summary, after the intravenous administration of tofacitinib and voriconazole to rats, the CLib alone . This coib alone  could be