Regional integration can significantly affect carbon emissions, but scholars have paid more attention to the impact of integration level, ignoring the importance of regional integration expansion. This study attempts to demonstrate whether, in the process of promoting carbon peak and carbon neutrality in China, the transformation of the administrative region’s economy into an integrated economy based on urban agglomeration regional integration expansion affects urban carbon emissions. This study considers the regional integration expansion of the Yangtze River Delta Urban Economic Coordination Committee as a quasi-natural experiment, exploring the carbon emission reduction effect of regional integration with the difference-in-differences model. With the mediating and moderating effect models, this study examines the mechanism of regional integration affecting urban carbon emissions. The results show that regional integration, considering regional integration expansion, can significantly reduce urban carbon emissions. The carbon emission reduction effects of regional integration show significant heterogeneity. For example, there is a significant carbon emission reduction effect of high-hierarchy cities and an insignificant carbon emission reduction effect of general-hierarchy cities. Further research into the driving mechanism finds that deepening collaborative governance, industrial structure optimization, and green technology promotion brought about by regional integration are important mechanisms influencing urban carbon emissions. In addition, the carbon emission reduction effect of regional integration is influenced by the level of urban marketization and development efficiency. Different from the existing studies focusing on the effects of regional integration level, this study assesses the feasibility of promoting urban green development through urban agglomeration regional integration expansion. Based on the relevant empirical research, we propose to better promote high-quality development by strengthening urban agglomeration cooperation, optimizing urban development paths, strengthening innovative development, and improving macro political systems. It also indicates that the relevant policies should be formulated after considering local conditions. For ChiFor the well-functioning government in China, the government-led urban agglomeration expansion is important to promote regional integration development. The Yangtze River Delta is an urban agglomeration with a higher economic development level, deeper integration level, and faster spatial expansion in China. The regional integration development in the Yangtze River Delta has become a national strategy; deepening urban coordination and actively exploring the low-carbon development path are the practical requirements for striving to be the pioneer of carbon peak and carbon neutral development. Therefore, based on the difference-in-differences model, this study takes the Yangtze River Delta as the research area. The Yangtze River Delta Urban Economic Coordination Committee oversees a quasi-natural experiment to explore the carbon emission reduction effect and driving mechanisms of regional integration from multiple perspectives. This study can enrich the existing research in the following aspects. Different from the previous studies exploring the effect of integration level, it considers the reality of regional integration expansion, enriching the perspective of integration research. In terms of research content, it explores the carbon emission reduction effect and drivers of regional integration from multiple perspectives. The research method regards regional integration expansion as a quasi-natural experiment. The difference-in-differences model is used to conduct the study, which improves the credibility of the research results.In the process of factor-driven economic development, factor quality and utilization efficiency directly impact urban development quality. This is also affected by the competitive relationship with other cities. As an important factor influencing urban interaction relationships, regional integration affects the quality of urban development. It does this by optimizing the urban development environment and changing the state of competing factors ,22. FromFrom the theme of this research, urban carbon emission is not only related to economic scale. It is also influenced by urban environmental regulation systems, industrial structures, and development paths, which are also significantly driven by competing relationships with other cities . As showThe environment is a public good with typical externalities. As urban economic linkages strengthen and pollution externalities emerge, strengthening collaboration among spatially adjacent cities is an inevitable path to solving regional environmental problems . Due to Regional integration can reduce urban carbon emissions by promoting collaborative governance. On the one hand, with rapid economic growth driven by regional integration, people’s demand for high-quality environments has increased. Environmental collaborative governance has consequently become an important theme of integration cooperation ,18. EnviIn the industrialization process of China, high carbon emissions are the consequence of an extensive development model with high energy consumption, high pollution, and high emissions. It is caused by the industrial structure directly . In the Resource mismatch in the segmented market is an important mechanism that inhibits the optimization of industrial structures. Regional integration provides the possibility for urban industrial structure optimization. Firstly, regional integration promotes the accelerated concentration of factors in high-efficiency enterprises and cities. If cities cannot adjust their industrial structure, they will be locked in the value chain’s low end, making it difficult to achieve economic growth and forcing cities to accelerate the industrial structure transformation ,32. For Technology promotion is a key factor in promoting high-quality development . The extIn general, regional integration can enhance green technology promotion and drive carbon emission reduction through the following paths. On the one hand, regional integration brings about the improvement of collaborative innovation platforms and market scale expansion by strengthening urban innovation cooperation, developing the technology trading market, enhancing the innovation spillover effect, and strengthening demand-induced innovation . This caDriven by the complex mechanism, costs and benefits in regional integration are also influenced by urban development status. For example, the higher the marketization level, the stronger the ability to gather high-quality factors, which strengthens the positive effects of regional integration. In the process of factor-driven economic development, development efficiency directly influences urban economic growth and carbon emissions. It shows that the more efficient the development and the factor allocation, the lower the carbon emissions. Generally, the marketization level and development efficiency, as characterizations of urban development status and the influential factors of carbon emissions, both regulate the carbon emission effect of regional integration from internal and external perspectives ,26,31. IAs the economic takeoff is significantly later than that of developed countries, the economic rise of developing countries represented by China has brought about rapid growth in global carbon emissions. For example, according to the Global Carbon Project, as the largest developing country, China’s carbon emissions will reach 11.47 billion tons in 2021, twice that of the United States and four times that of the EU. How to effectively reduce the carbon emissions of developing countries has an especially important impact on global carbon emission reduction. Urban agglomeration, an important carrier for China to participate in global competition, is also important for promoting regional integration and narrowing the regional development gap . ConsideThe Yangtze River Delta urban agglomeration is located on the eastern coast of China. With the promotion of multiple subjects such as government, academia, and enterprises, the cooperation mechanism has been improved, driving the administrative economy to transform into an integrated economy . On 5 NoFrom the existing research ,31, regiScientifically defining the regional integration process in the Yangtze River Delta is the key foundation for conducting this research. Urban agglomeration integration has multidimensional complexity . The govThis study focuses on whether regional integration can reduce urban carbon emissions in the Yangtze River Delta. Exploring the regional integration effect, it is not only necessary to focus on the impact of regional integration deepening, but also to consider the reality of regional integration expansion ,30. RegiCar represents urban carbon emissions as the explained variable, D is the explanatory variable, X is the control variable, i and t represent city and time, respectively, ε is the random disturbance term, and μi and ηt are the fixed effects of city and time, respectively.This study aims to scientifically assess the carbon emission effects of regional integration. However, comparing city carbon emissions before and after participation in the Coordination Council may incorporate other factors into integration shocks. Similarly, comparing the carbon emissions of cities within and outside the Coordination Council will lead to the attribution of unobservable individual systematic variation to the effect. This study uses a difference-in-differences model to obtain an unbiased estimate of the carbon emission effect ,33. ThisMed is the mediating variable, and the remaining variables have the same meaning as in Equation (1). The steps of the mediating effect test were shown in The mediating effect models were constructed to identify the mechanisms by which regional integration affects urban carbon emissions ,22.(2)CReg is the moderating variable, and the remaining variables are defined in the same way as in Equation (1).The theoretical analysis suggests that other factors also influence the carbon emission effect of regional integration. Drawing on existing studies , the mod(1)Explained variables. In view of the practice of carbon peak and carbon neutrality in China, this paper uses carbon emissions as the explained variable. The unit for the variable is million tons.(2)i joins the Coordination Council in year t is set to 1 if it does; otherwise, it is set to 0.Explanatory variables. The regional integration in this paper contains the double meaning of integration level deepening and regional integration expansion. Drawing on existing studies ,33, we c(3)GDP); its unit is CNY 1 billion. Government has an important influence on economic development, and government policy regulation cannot be made without the support of fiscal expenditure. We used fiscal expenditure as the proxy variable for local government influence (Fis), which is CNY 1 billion [Ope). Infrastructure influences carbon emissions by driving public facility sharing and reducing trade costs, and transportation plays an important role in regional integration [Tra) is characterized by passenger volume in a million passengers.Control variables. Economic growth is an important source of carbon emissions , and GDP billion . Opening billion . Total iegration ,31. The (4)Cop), based on the number of signed agreements between governments related to environmental governance, uses the measure of centrality of local government cooperation [Str) is measured by the ratio of the added value of the tertiary industry to the secondary industry. Green technology is an important measure to reduce carbon emissions, and scholars also put forward diversified methods to measure technology level [Pat) was measured by the number of green invention patents granted per 10,000 people.i and j represent the city serial numbers, C(i) is the centrality of city i, N is the number of city units, and wij is the actual amount of connection between city i and city j.Mediating variables. This study argues that regional integration impacts carbon emissions through three main paths. Collaborative governance indicator Mark) characterizes the integration and competitiveness features, which are measured using Wang et al. [EFF) characterizes factor allocation efficiency and reduces carbon emissions by reducing energy consumption. This is measured by urban green total factor productivity [Moderating Variables. In the process of factor-driven economic development, the marketization level and urban development efficiency are important factors affecting the carbon emission effect of regional integration. The marketization level  estimates the average effect. However, the benefits of cities joining regional integration may vary because of differences in development levels and macro policies ,39. GiveAs a gradual process of regional integration, the Coordination Council promotes a progressive, deepening trend of government cooperation. It characterizes exploration-integration deepening by holding annual joint meetings with mayors . What isAre the results of benchmark model regressions strongly robust? Based on the differentiation perspectives, the tests in this section verify the robustness of the benchmark model regression results.post−3, post−2, post−1, post0, post1, and post2, were constructed to represent the third year, the second year, and the first year before joining the Coordination Committee, the year of joining the Coordination Committee, and the first year and the second year after joining the Coordination Committee. Then, interaction terms were formed with the dummy variables of the experimental group, namely, D × post−3, D × post−2, D × post−1, D × post0, D × post1, and D × post2. Finally, the explanatory variables in Equation (1) were replaced with the six interaction terms. The results in D × post−3, D × post−2, and D × post−1 are not significant. This indicates no significant gap in the evolution of carbon emissions between the experimental and control groups before joining the Coordination Council. In other words, the data in this study passed the parallel trend test. In addition, the coefficients of D × post0, D × post1, and D × post2 are all significantly negative and the effects show an expanding trend, which further confirms the conclusions in The parallel trend test is a prerequisite for the application of the difference-in-differences model, which requires that the treatment group and the control group have basically the same change trend before the implementation of regional integration . ParalleIMR) to control the endogeneity of local government participation in the Coordination Council to verify the robustness of the benchmark model [The empirical analysis aims to investigate whether regional integration can bring about the carbon emission reduction. The effect assessment based on the difference-in-differences model requires the experimental group selection to be close to random. However, regional integration expansion in the Yangtze River Delta results from local government application and review by the Coordination Council. In fact, the city development level is a key factor in determining whether it can join the Coordination Council. Therefore, government self-selection may result in sample selection bias and affect the regression result robustness in rk model . Column CP), the coefficient of the explanatory variable is still significantly negative in Column (2) in In terms of macro policy, the central government selects typical cities to carry out pilot projects, which is an important initiative to promote low-carbon transition in China. Studies have found that the low-carbon pilot cities can significantly influence urban carbon emissions . During Carbon emissions estimation directly impacts the results, but it is also a current challenge for relevant research. Drawing on relevant studies , the YanIn the process of responding to national environmental protection policies and integrating into urban agglomeration, there are significantly different effects due to differences in location conditions, development levels, and city hierarchy ,24. For Scholars have found that, in the integration development led by developed cities, proximity to developed cities is one of the key factors influencing the effect . The que(1)The manufacturing industry is generally larger in Jiangsu city, and manufacturing is an important source of carbon emissions. When joining the Coordination Council, the rapid agglomeration of manufacturing-related elements increases urban carbon emissions. Jiangsu city benefits from the integrated transportation facilities closely connected to Shanghai, the strong complementarity of industrial innovation in Shanghai, and the superior manufacturing development environment.(2)In contrast to Jiangsu city, the economic growth of Zhejiang city is significantly driven by the service industry. The transfer of manufacturing and other high-energy-consuming industries, together with the concentration of service industries represented by the Internet in the process of regional integration, are important forces driving industrial upgrading. This is coupled with green technology progress driven by the continuous introduction of green development policies. It results in a decrease in carbon emissions under the synergy of structural transformation and technology progress.(3)Anhui achieves economic growth by improving infrastructure and undertaking industrial transfers, and industrial agglomeration may increase carbon emissions. At the same time, the process of receiving green technology spillover and industrial transfer from Jiangsu, Zhejiang, and Shanghai is accompanied by the outward diffusion of high-energy-consuming industries in integrated cities. This then realizes technology progress and production efficiency improvement. In contrast to industrial agglomeration and efficiency improvement, the development orientation of economic growth and the lack of high-end factor agglomeration ability led to a relatively strong effect of the former and an insignificant effect of carbon emissions.This study found that the carbon emission effects of cities in different provinces participating in regional integration differed. There was a significant positive effect in Jiangsu, a significant negative effect in Zhejiang, and insignificant negative effect in Anhui. The main reasons for this disparity are as follows.Differences in economic development levels, technology endowments, and city hierarchy in the factor-driven development process bring about significant gaps in the competitiveness of urban factors. This leads to heterogeneity in the carbon emission effects of regional integration . It has (1)High-hierarchy cities have higher economic development levels, industrial development efficiency, and environmental regulation perfection. These bear the burden of ecological environmental protection demonstrations. In the process of regional integration development, public facilities further reduce city cooperation costs. They support the development’s efficiency improvement and have the technical ability to accelerate green innovation exploration and gather high-quality development elements. In the regional integration, high-hierarchy cities have gathered efficient green elements and accelerated the outward transfer of high-energy-consuming industries, achieving low-carbon development driven by technology and efficiency.(2)Industrial division and industrial transfer in regional integration is an important form of urban cooperation and an important driver of carbon emission pattern evolution . A diffeThe results in This section adopts the mediating effect model of Equations (2)–(4) to quantitatively explore whether regional integration can affect carbon emissions through the paths of collaborative governance, industrial structure optimization, and green technology promotion. This empirically tests the theoretical mechanism.In the process of regional integration in the Yangtze River Delta, it has been necessary to conduct multi-level government consultation. This is done at the annual meeting of the Coordination Council and is the official process for deepening regional environmental governance. The more cooperation agreements there are among cities, the stronger the collaborative governance degree will be. In this regard, based on the keywords of environment, industry, and innovation, which are closely related to carbon emissions, the textual data of intergovernmental cooperation among cities are retrieved from official websites, and the centrality of policy collaboration is calculated with Equation (6) to test whether regional integration can influence carbon emissions by strengthening collaborative governance.For a long time, the efficiency loss caused by the development of low-end and disorderly industries has been one of the direct factors leading to high carbon emissions . The stuBased on Relying on green technology to achieve clean production is an important initiative to coordinate economic growth and green development ,21. ThisBased on the Based on the theoretical analysis, this section explores the moderating effect of urban marketization level and development efficiency on the effect of regional integration on carbon emissions. With the moderating model of Equation (5), There are significant market distortion factors in China . The lowIn the process of factor-driven economic growth, development efficiency reduces carbon emissions by reducing energy consumption and influencing factor agglomeration . This, iRegional integration can effectively reduce urban carbon emissions, which provides theoretical support for China to promote green development relying on urban agglomeration. However, considering the heterogeneity of carbon emission effects of regional integration, relying on regional integration to achieve low-carbon development should not only formulate localized policies but also pay attention to the possible negative effects. Therefore, we propose the following practical issues that deserve further discussion.Scholars have proposed promoting carbon emission reductions by promoting regional market integration, and urban agglomeration integration is an important measure to promote market integration ,19,22. TAlthough regional integration can significantly reduce urban carbon emissions, it is important to pay attention to the heterogeneity. For example, the different effects among different regions and hierarchies show that urban carbon emission management should not be overly dependent on whether they can join the integration region. The key is how to use the positive effect of regional integration to achieve the improvement of development quality, especially to strengthen the cooperation level with developed cities. The focus should be on collaboratively preparing development plans, breaking down administrative boundary barriers, setting unified market access standards, and promoting the factor free flow. In particular, the longer the city is in the Yangtze River Delta Urban Economic Coordination Council, the more significant is the carbon emission reduction effect. It is clear that in relying on urban agglomeration integration to achieve low-carbon development, the focus should be to deepen the cooperation level. In particular, governments should further improve the regional integration benefits by establishing a collaborative and efficient institutional system, and stimulate the impetus for cities to participate in regional cooperation at a deeper level.In the regional integration led by the well-functioning government, scholars have realized the importance of integration for high-quality development and attracting cities to participate in integration to achieve higher quality development ,30. ExpaEconomic growth may lead to an increase in carbon emissions . HoweverFor developing countries during rapid industrialization, the development pattern dominated by high-pollution and high-emission industries is the direct cause of high carbon emissions . In the Innovation is an important initiative to achieve low-carbon development ,31. The In China, the macro policies by the central government, especially the assessment system, have an important influence on local government behavior. Since the reform and opening up, GDP tournaments have led local governments to introduce many high-energy-consuming and high-polluting enterprises in pursuit of political achievements. These have brought serious problems while achieving economic growth . Under tUnder the vertical management system, the central government has a direct impact on the economic development of local governments . For theLocal government is the main body of economic growth and the important undertaker of carbon emission reduction ,12. For Different to previous studies on the regional integration definition ,42, thisFacing the regional integration led by the well-functioning government in China, an increasing number of cities expect to participate in integration to achieve higher-quality development. Expansion has become a common phenomenon of regional integration. Based on city-scale data from 1997 to 2017, this study treats the expansion of the Yangtze River Delta Urban Economic Coordination Council as a quasi-natural experiment. With the difference-in-differences model, we estimate the carbon emission effect of regional integration. It was found that regional integration can significantly reduce urban carbon emissions, which indicates the feasibility of low-carbon development reliant on urban agglomeration integration. The carbon emission effects of regional integration show multiple heterogeneities, for example, the significant negative effect of high-hierarchy cities and the insignificant positive effect of general-hierarchy cities. Driving mechanism tests based on the mediating effect model and the moderating effect model found that regional integration influences urban carbon emissions through collaborative governance, industrial structure optimization, and green technology promotion. However, the carbon emission effect of regional integration is significantly moderated by marketization level and development efficiency. Based on the relevant empirical research, we propose to better promote the high-quality development of China’s economy by strengthening urban agglomeration cooperation, optimizing urban development paths, strengthening innovative development, and improving macro political systems. It should be emphasized that the implementation of relevant policies to promote coordinated development should be formulated after considering local conditions.


Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae, Streptococcus suis, etc.) in swine farms, thereby exacerbating the endemic clinical diseases. We herein, review and discuss various dynamic polymicrobial infections among selected swine pathogens. Traditional biosecurity management strategies through multisite production, parity segregation, batch production, the adoption of all-in all-out production systems, specific vaccination and medication protocols for the prevention and control (or even eradication) of swine diseases are also recommended. After the introduction of the African swine fever (ASF), particularly in Asian countries, new normal management strategies minimizing pig contact by employing automatic feeding systems, artificial intelligence, and robotic farming and reducing the numbers of vaccines are suggested. Re-emergence of existing swine pathogens such as PRRSV or PCV2, or elimination of some pathogens may occur after the ASF-induced depopulation. ASF-associated repopulating strategies are, therefore, essential for the establishment of food security. The “repopulate swine farm” policy and the strict biosecurity management (without the use of ASF vaccines) are, herein, discussed for the sustainable management of small-to-medium pig farms, as these happen to be the most potential sources of an ASF re-occurrence. Finally, the ASF disruption has caused the swine industry to rapidly transform itself. Artificial intelligence and smart farming have gained tremendous attention as promising tools capable of resolving challenges in intensive swine farming and enhancing the farms' productivity and efficiency without compromising the strict biosecurity required during the ongoing ASF era.A few decades ago, porcine respiratory disease complex (PRDC) exerted a major economic impact on the global swine industry, particularly due to the adoption of intensive farming by the latter during the 1980's. Since then, the emerging of porcine reproductive and respiratory syndrome virus (PRRSV) and of porcine circovirus type 2 (PCV2) as major immunosuppressive viruses led to an interaction with other endemic pathogens (e.g.,  Mycoplasma hyopneumoniae (Mhp) and Actinobacillus pleuropneumoniae (App). They can also include secondary pathogens such as Pasteurella multocida (Pm), Bordetella bronchiseptica (Bb), Gläesserella (Haemophilus) parasuis (Gps), and Streptococcus suis (Ss) (Porcine respiratory disease complex (PRDC) is a multifactorial syndrome affecting the respiratory system of pigs in the swine industry worldwide , 2. Enviuis (Ss) , 2. Dynauis (Ss) . In the uis (Ss) . This siSeveral factors can influence on the PRDC prevalence, including a remarkably lower number of the pig population in the affected area, the closing of pig farms due to ASF depopulation, and an improvement of the employed biosecurity program. The worldwide epidemic outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) also affected pig producers, both directly and indirectly –12. As aAsfarviridae. The ASFV genome size of approximately 170–190 kb encodes many proteins  and the highly pathogenic PRRSV infections, the development of a multiplex real-time PCR exploring the presence of these agents in 1,143 specimens taken from various organs of dead pigs during the 2018–2021 period  revealedersified . The SPFersified .During the early period of the PCV2 discovery, a model of a well-known coinfection of PCV2, porcine parvovirus (PPV), and porcine cytomegalovirus was reported in the case of multiple abortions and reproductive failures . CoinfecIn vivo experiments have confirmed that a coinfection of Mycoplasma hyorhinis (Mhr) and PRRSV can cause severe pathological lesions in the pig lung, and that these lesions are more extensive than those caused after an infection with Mhr or PRRSV alone  . VariousAspects related to environmental conditions, population size, management strategies, and pig-specific factors (including age and genetics) have been linked to the impact of the PRDC . The linin vivo models. The use of biofilm reflects the ability of bacteria to survive outside the host body . This system potentially reduces the risk of disease transmission comparing to the traditional continuous production system, in which the younger animals might have exposed to endemic pathogens shedding from the older animals . The AIAanalysis . An effight pigs .Disease outbreaks in pig production normally occur through vertical and horizontal transmissions. In order to control and prevent the opportunity of an outbreak, the design of the pig flow and the pig separation using multisite production and parity segregation is fundamental for the avoidance of disease transmission among different pig populations at risk, and can also improve the pig growth performances as a result . MultisiParity segregation is the method used in order to separate pregnant gilts  and the first parity (P1) sows from second parity sows (P2) and above with respect to differences in their immune status, disease, nutritional management, and their offspring colostrum management. P1 sows and their offspring must be separated from P2+ sows . UltimatLeptospira-Erysipelothrix, and perhaps App vaccines in some endemic farms. Likewise, vaccinations for breeding herds are usually conducted through the mass vaccination of sows on production and comprise of 3–4 times per year administration of a live attenuated CSFV vaccine, an ADV MLV, an FMD-inactivated vaccine, and perhaps an PRRS MLV vaccine. For some PRRSV-stabilized sow herds, the PRRS type 1 inactivated vaccine may be implemented in pregnant sows if the farm is located within a PRRSV-endemic area , inactivated FMD, PRRSV MLV, PCV2, combined PPV-mic area , 87. Addmic area . Howevermic area . Combinamic area , 91.Since the antimicrobial resistance becomes an emerging problem threatening both the animal and the human health, the rational use of antimicrobials has been recommended. Based on data regarding the antimicrobial resistance prevention and control in agriculture and livestock animals, strategy 4 of Thailand's national strategic plan on antimicrobial resistance 2017–2022 has set a goal of a 20% and 30% reduction of antimicrobial consumption for humans and animals, respectively . Recentletc. and should be considered based on a case-by-case basis depending on the disease prevalence, the herd immunity status, and the biosecurity measures. Improvement of biosecurity should be invested not only for PRDC control, but for ASF prevention.In summary, implementation of PRDC control and prevention measures mentioned above requires combination of several biosecurity measures of which the advantage of AIAO and batch production systems, pig and housing separations, quarantine and acclimatization, AIAO and, multisite production systems and parity segregation, and medication and vaccination strategies, In recent years, ASF has triggered a remarkable crisis for pig production worldwide , 96. ASFvia the use of real-time PCR , the Food and Agriculture Organization of the United Nations (FAO) and the World Organization for Animal Health (WOAH) have launched guidelines for the ASF prevention and control in smallholder pig farms in Asia . These gA concern regarding the swill feed use by pig farms in Asia and Africa remains the major ASF prevention issue, as it might be a source of ASFV transmission . In 2020A spatiotemporal analysis was employed in order to describe the ASFV spread throughout Asian countries  from 1 August 2018 to 31 December 2019, and to provide an overview and a comparative analysis. The analysis revealed increased disease outbreak reports from China, Vietnam, and Laos representing two clusters of outbreaks, and further analysis indicated that the wild boar populations, the pig transportation, and the movement of contaminated fomites are probable risk factors for the ASFV spreading across Asia .The Chinese and Vietnamese experiences in fighting the ASF outbreak were used as valuable knowledge for the forming of guidelines for other ASF-free countries and allowed them to prepare their own outbreak dealing plans. Following the identification and the confirmation of an ASF outbreak by veterinary and local authorities, the depopulation and the repopulation policies should be implemented together with a restriction of the human and pig movements . The FAOAfter the ASF introduction into Vietnam, the factors contributing to the overwhelming spread throughout the county include: (i) the poor biosecurity of the small and the small-to-medium commercial farms covering 80–90% of the farms in Vietnam, and (ii) the insufficiency of the veterinary services to deal with the outbreaks . AlthougThe “rebirth” policy for pig farms in China and Vietnam, especially for small-to-medium pig holders, has been a topic of interest, particularly regarding the ASF re-occurrence due to biosecurity limitations. In an attempt to avoid the re-occurrence of ASF, the Chinese government and the related authorities have launched a program of financial support for culling a subsidy of the infected livestock, and have established the criteria for the repopulate farms , along wImportantly, management strategies on how to reduce the human-to-pig and the pig-to-pig contacts should be planned so as to best fit the farm conditions. The use of combined viral and bacterial vaccines that are commercially available (such as PCV2 and Mhp), or the use of combined bacterial vaccines are effective alternatives . IntradeThe ASF-induced farm disruption has caused the swine industry to rapidly transform itself, and to embrace the new era of swine farming. AI and smart farming have gained tremendous attention so as to resolve the challenges faced by intensive swine farming, and to enhance the productivity and the efficiency of the farms without compromising the strict biosecurity required during the ASF epidemic. The application of AI-assisted smart farming also provides essential information that can be utilized for diagnostic purposes, growth monitoring, and effective management –29. For The new normal biosecurity required for ASF-affected areas has introduced many promising tools in the ASF-affected countries. Apart from the external biosecurity management, a checklist for internal biosecurity would routinely ensure the confining of the entry zone, applying quarantine measures in the buffer zone, generating a clean zone, and increasing hygiene and awareness among the farm workers . A list Based on our observations regarding the ASF-survived and repopulate farms, the PRDC-associated problems seem to have disappeared or to have not affected the production performance, possibly due to the fact that most of the pathogens were eradicated after the ASF-induced depopulation together with pathogen reduction from the current strict biosecurity. However, in some farms, clinical signs related to PCV2 are rising after the abandoning of the PCV2 vaccination (so as to avoid human-to-pig contact) during the ASF epidemic . It shouetc.) are implemented based on a case-by-case basis depending on each farm's health status. After the ASF outbreaks, the adoption of new normal management strategies minimizing pig contact to lessen the opportunity of human-to-pig contact and reducing the number of vaccines administered more or less may lead to a re-emergence of existing endemic PRDC-causing pathogens in case of endemic PCV2-infected farms. Vaccination is essential for the endemic pathogens, but increased numbers of pig vaccination may lead to increased risks of ASF transmission. To deliver clear and concise vaccine recommendation, the farm veterinarians should be responsible for the vaccination program based on the farm disease status. Another good scenario after the ASF-induced depopulation is that the concise pig vaccination program and change of biosecurity may help simultaneously eliminate PRDC from the farms. The disruption caused by ASF has forced the swine industry to rapidly embrace a new era of swine farming. AI and smart farming have gained tremendous attention in order to resolve challenges in intensive swine farming. They have managed to enhance the farms' productivity and efficiency, despite the strict biosecurity required during the ASF era.PRDC is a major multifactorial disease affecting the respiratory system of pigs in the swine industry worldwide. Dynamic polymicrobial infections triggered by PRDC pathogens have clinical outcomes that depend on the type of coinfection occurring each time. Traditional PRDC management strategies : RT NRCT Senior Scholar 2022; Grant Number N42A650553.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Stillbirth prevention is a global health priority and a crucial step towards better maternal and newborn health and wellbeing.4The Lancet Global Health, Elizabeth M McClure and colleaguesIn Compared with previous studies,The contribution of PURPOSe to understanding the causes of stillbirths in south Asia calls for a similar large study in sub-Saharan Africa, including stillbirths weighing less than 1000 g. Research to develop improved methods for early identification of placental malperfusion and placental infection is needed. Most importantly, a series of intervention trials addressing the major causes maternal, placental, and fetal causes of stillbirth in south Asia and sub-Saharan Africa are urgently needed.The global burden of stillbirths is a silent disaster that has long-term consequences for women, families, and their communities. Many stillbirths are preventable, and the interventions needed to prevent stillbirths are integrated into efforts to improve the quality of care during pregnancy, childbirth, and the postnatal period. The health-care community need intentional leadership and a greater commitment to efforts to implement and scale-up existing and new solutions to reduce stillbirths within health systems in low-resource settings. Purposefully aiming to reduce stillbirths will ultimately improve maternal and newborn health and wellbeing. By tackling stillbirths, the health-care community will achieve an acceleration toward the Sustainable Development Goals.We declare no competing interests.


Populus genetic diversity and allow enabling molecular breeding to support accurate early selection, increasing the efficiency, and reducing the time and costs of poplar breeding, that, in turn, will improve our capacity to face or prevent the emergence of new diseases or pests.Poplar is one of the most important forest trees because of its high economic value. Thanks to the fast-growing rate, easy vegetative propagation and transformation, and availability of genomic resources, poplar has been considered the model species for forest genetics, genomics, and breeding. Being a field-growing tree, poplar is exposed to environmental threats, including biotic stresses that are becoming more intense and diffused because of global warming. Current poplar farming is mainly based on monocultures of a few elite clones and the expensive and long-term conventional breeding programmes of perennial tree species cannot face current climate-change challenges. Consequently, new tools and methods are necessary to reduce the limits of traditional breeding related to the long generation time and to discover new sources of resistance. Recent advances in genomics, marker-assisted selection, genomic prediction, and genome editing offer powerful tools to efficiently exploit the  Populus, which are traditionally clustered into six sections known as Populus, Tacamahaca, Leucoides, Turanga, Abaso, and Aigeiros . I. Icis- oo et al.  that revIn recent years, genetic studies have allowed to dissect the genetic bases of biotic resistances in poplar and this information has the potential to be exploited for tree breeding, particularly for implementing MAS and, consequently, marker-assisted breeding (MAB). Under certain scenarios, it was shown that MAS can be used to replace or improve truncation selection based on phenotypic values  and has The knowledge acquired in QTL mapping and GWAS shows that, in several cases, a few QTLs or QTNs with large effects underlie the genetic bases of biotic stress resistance . MoreoveThe application of GP, which aims to estimate the breeding value of individuals using only genotypic information , for theR genes (CRY and genes encoding for proteinase inhibitors) [Alternative ways to accelerate the genetic gain in poplar breeding are based on genetic modification. Recently, genetic-engineering applications in poplar have been reviewed and include studies related to the generation of transgenic poplars resistant to diseases by expressing antibacterial and antifungal genes  or insect ibitors) . Even thibitors) . Moreoveibitors) .The development of the new GE technologies based on CRISPR /Cas is revolutionizing the concept of genome modification because allows the introduction of insertions, deletions, or the substitutions of single base or short sequences at specific sites in a target genome in highly efficient, simple, and versatile way ,121,122 WRKY18 and WRKY35, contributed to unravelling their involvement in P. tomentosa resistance to Melampsora [P. tomentosa by activating the biosynthesis of proanthocyanidins [Even if GE in poplar is mainly focused for the improvement of growth in plantations and the use in the pulp and bio-refinery industries ,124, applampsora ; throughyanidins . These eyanidins .Biotic stresses represent major threats in both natural populations and plantations of poplar and forest trees in general. The extent of biotic stresses is intensifying as global warming is rapidly enlarging the distribution and incidence of pests and pathogens towards higher latitudes and their occurrence in new territories ,29. For One of the main issues of poplar breeding is tied to the biology of poplar that, as for other perennials, displays late expression of relevant traits and late development of reproductive structures, characteristics that prolong selection cycles and, consequently, the duration of breeding programmes. In addition, long breeding cycles should be supported by long-term financial investments. Nonetheless, market needs can change over time and the new clones developed by traditional breeding might not guarantee the expected return investments. The usual intensive cultivation of monoclonal plantations of a small number of elite cultivars, strongly adapted to the pedo-climatic conditions of specific territories, is often criticized by environmental associations, more favourable to greater biodiversity as in natural forests, and restrictions have been posed also on the cultivation of hybrid clones in protected areas and bio-conservation sites along rivers. In this condition, selected clones of native species, alone or in mixed clones, could represent a useful alternative to interspecific hybrids in more sustainable poplar plantations. For this reason, it is necessary targeting poplar genetics to guarantee the sustainability of wood productions in the areas where the spread and emergence of pests and pathogens are expected, also because of the higher frequency and incidence of abiotic stress conditions. It is also important to improve the annual rate of genetic gain in poplar breeding programmes using fast and precise applications and exploiting untapped sources of genetic variability, taking advantage of the large poplar genetic variation worldwide and creating large genetic panels that should be shared between research groups from different countries and tested in multiple environments. The molecular characterization of these collections, in combination to common garden phenotypic data, can contribute to the unlocking of this genetic diversity available to breeders and allows the acquisition of new knowledge  to be integrated into breeding and deployment strategies. The development and use of more diverse genotypes and the maintenance of large well-adapted populations will result in a significant increase in frequency of potentially beneficial allelic variants.The annotated poplar genomes and the new genotyping platforms have largely contributed to the mining of the interaction between poplar and pests or pathogens, but the current knowledge is still insufficient to face climate-change challenges. For example, because of the rapid LD decay measured for poplar, many molecular markers are needed to capture the effects of all QTLs. Moreover, strong LD between markers and causative variants that control the traits of interest is desirable to achieve high values of predictive ability in GP. The available SNP arrays, developed using candidate-genomic-region approaches ,69, reprThe understanding of the relationship between changing environment and the spread of poplar diseases, using accurate prediction models of the effects of climate change on poplar plantations, is pivotal to define new breeding objectives based on the enlargement of the genetic backgrounds in parentals for the generation and selection of new clones with large biodiversity and adaptability to changing environments. Finally, the activation of new cooperation projects, involving geneticists, eco-physiologists, wood technologists, and stakeholders, as poplar growers and wood users, would lead to the definition of short- and medium-term objectives for the development of innovative genetic materials with wide variability to cope with future highly uncertain environments and for the definition of cost- and time-effective strategies that meet rapid changes even considering the ecological and economic contexts.


Editorial on the Research TopicPhysiological and biomechanical determinants of swimming performance—volume 2The objective of this Research Topic was to develop and strengthen evidence of training and swimming performance to increase scientific knowledge in the area, considering that understanding the biomechanical, physiological, and neuromuscular determinants of swimming performance is still challenging. This way, 13 manuscripts have been reviewed and approved for this research topic (volume II). We can categorize the 13 manuscripts into three major areas of swimming research: physiology and prescription; biomechanics; performance assessment and prediction. Furthermore, we highlight that 10 of the manuscripts were carried out with the participation of at least two research institutions, often from different countries, which may demonstrate the need for international interchange and exchange of ideas and methodologies across researchers and laboratories.Almeida et al.  assessed 22 male swimmers in an incremental protocol to estimate, among others, the maximal aerobic velocity, then applied intermittent and continuous swimming protocols, both at maximal aerobic velocity. They noted that: (i) intermittent trials training is preferable rather than continuous training for aerobic capacity; and (ii) Massini et al. (The reliability of back-extrapolation in estimatingpeak in different swimming performances at severe intensity domain) estimated Raimundo et al.  suggest that the time constant of the reconstitution of the maximum distance that can be performed above critical speed is not constant during two high-intensity interval sessions with the same recovery intensity.Concerning physiological aspects of swimming training and performance, studies focused on oxygen uptake kinetics, back-extrapolation reliability, and critical speed. Rad et al.  investigated inertial measurement unit (IMU) with a single IMU on the 16 swimmers' sacrum during training sessions, specifically along ten weeks in 25-m all-out front crawl. Five goal metrics from the IMU signals representing the swimmer's performance in the swimming phases  and in the entire lap were estimated. The results showed that the goal metrics for the free-swimming phase and the entire lap predicted the swimmer's progress well. Regarding pressor sensors, Santos et al. (Reliability of using a pressure sensor system to measure in-water force in young competitive swimmers) analyzed the front crawl over 25-m all-out of 15 age-group swimmers with the pressure sensor system (Aquanex System). They concluded that the system seems to be a reliable device for measuring the hand resultant force during front crawl in young swimmers and can be used to monitor the changes over time.The studies that focused on the biomechanics of swimming, in this special volume, can be grouped into three main topics: (i) inertial and pressure systems; (ii) tumble turn; breaststroke pullout; and undulatory underwater speed; and (iii) active drag, propulsion and kinematics. Koster et al. (Implications of the choice of distance-based measures in assessing and investigating tumble turn performance) intended to understand better the implications of choosing a particular distance-based performance measure for assessing and investigating tumble turn performance in freestyle swimming. In this way, 2,813 turns performed by 160 swimmers were analyzed. The results revealed that performance measures with short(er) distances are more sensitive to changes in the adaptation time and reflect the wall contact time better than performance measures with long(er) distances, which in contrast, are more useful if the focus is on the approach speed prior to the turn. David et al.  examined the effect of wall contact time and tuck Index on tumble turn performance and their interrelations by experimentally manipulating both variables. The results underscored the importance of wall contact time and tuck Index of the tumble turn performance, as well as their interrelations with other performance determining variables in this regard, with the importance of individual tuning. Regarding the breaststroke pull-out, McCabe et al. (The Characteristics of the Breaststroke Pullout in Elite Swimming) characterized the underwater breaststroke pullout technique trends and assessed the effectiveness of each technique as utilized by elite male and female swimmers. The study found no difference in performance outcome for each pullout technique, indicating that one's individual preference should guide technique selection. Concerning the undulatory underwater movement, Kuhn and Legerlotz (Ankle joint flexibility affects undulatory underwater swimming speed) investigated the impact of ankle joint flexibility on swimming velocity and kick efficiency during undulatory underwater by comparing kinematics of swimming trials with reduced, normal, and enhanced maximum angles of plantar flexion. Swimming velocity and kick efficiency did not differ between normal and increased plantar flexion. The results suggest that undulatory underwater velocity is affected by impaired plantar flexion.Concerning the tumble turn performance, Lopes et al.  performed a systematic review to update the body of knowledge on active drag in swimming through numerical and experimental methods. Seventy-five studies on active drag in swimming and the methodologies applied to study them were analyzed and kept for synthesis. There were significantly fewer numerical studies than experimental ones. Based on the complexity of active drag, studying this phenomenon must continue to improve swimming performance. About the propulsion, Morais et al. (Understanding the role of propulsion in the prediction of front-crawl swimming velocity and in the relationship between stroke frequency and stroke length) aimed to: (i) determine swimming velocity based on a set of anthropometric, kinematic, and kinetic variables, and; (ii) understand the stroke frequency–stroke length combinations associated with swimming velocity and propulsion in young sprint swimmers. Swimming velocity was predicted by an interaction of anthropometrics, kinematics, and kinetics. Faster velocities in young sprinters of both sexes were achieved by an optimal combination of stroke frequency–stroke length. The propulsion data showed the same trend. The highest propulsion was not necessarily associated with higher velocity achievement.Concerning active drag, Correia et al.  provided a meta-analysis assessing representative variables for the kinematic, arm-stroke efficiency, coordination, and energetic parameters of the 400-m front crawl test. High heterogeneity (>75%) was found among the outcome parameters in the studies on the meta-analysis. The average speeds seem to be the most responsible and influential in the arm-stroke efficiency, coordination, and energetic parameters for improved 400-m front-crawl performance. Finally, Born et al. (Performance development of European swimmers across the Olympic cycle) quantified the performance development of race time and key performance indicators of European swimmers across the last Olympic cycle (from 2016 to 2021) and provided reference values for long-course swimming pool events for both sexes from 50 m to 1,500 m including butterfly, backstroke, breaststroke, freestyle, and individual medley. Individual events from the 2016 and 2021 European swimming championships were included in the analysis. Among the results, clean swimming velocities were faster in 12  and 5  events. For alternating swimming strokes, i.e., backstroke and freestyle, effect sizes indicated improved swimming efficiency with an inverse relationship between reduced stroke rate and increased distance per stroke.Regarding the performance assessment and prediction, considering the 400-m front crawl test as a useful tool to assess aerobic power and capacity, In summary, this issue advances the knowledge in topics of practical importance related to swimming performance. It highlights the contribution of several research areas, including physiology, biomechanics, new technologies and race analysis and, performance progression. In terms of physiology, training planning of continuous or intermittent sessions, oxygen kinetics, and critical speed remain hot topics in swimming research. In biomechanics, analyzing specific parts and components of a race remains critical and will help a substantial performance improvement. New technologies using reliable devices will help improve training quality, and we expect further improvement soon.FSC, PF, AT, TB, and CM wrote and proofread this editorial. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Enamel demineralization, as a type of frequently-occurring dental problem that affects both the health and aesthetics of patients, is a concern for both dental professionals and patients. The main chemical composition of the enamel, hydroxyapatite, is easy to be dissolved under acid attack, resulting in the occurrence of enamel demineralization. Among agents for the preventing or treatment of enamel demineralization, amorphous calcium phosphate (ACP) has gradually become a focus of research. Based on the nonclassical crystallization theory, ACP can induce the formation of enamel-like hydroxyapatite and thereby achieve enamel remineralization. However, ACP has poor stability and tends to turn into hydroxyapatite in an aqueous solution resulting in the loss of remineralization ability. Therefore, ACP needs to be stabilized in an amorphous state before application. Herein, ACP stabilizers, including amelogenin and its analogs, casein phosphopeptides, polymers like chitosan derivatives, carboxymethylated PAMAM and polyelectrolytes, together with their mechanisms for stabilizing ACP are briefly reviewed. Scientific evidence supporting the remineralization ability of these ACP agents are introduced. Limitations of existing research and further prospects of ACP agents for clinical translation are also discussed. Enamel demineralization is one of the most common dental problems which could appear as white spot lesions (WSLs) in the early stage and even progress into cavities if effective interventions are not taken in time . In the Based on the etiology of enamel demineralization, the strategies to prevent or treat enamel demineralization include: 1) Using antibacterial agents such as mouthwash or toothpaste containing antibacterial drugs  which coIn addition to the above strategies, the enamel biomimetic remineralization strategy, which bases on the natural enamel crystallization process , is beinin vitro enamel demineralization model and further confirmed its remineralization ability and potential for clinical transformation in a rat caries model (Amelogenin (Amel) plays an important role in the formation of natural enamel . Amelogees model .Casein phosphopeptides (CPP) are casein extracts from milk which could markedly increase the apparent solubility of calcium phosphate ions by forming ACP . ResearcIn addition to the aforementioned amelogenin and its analogs and CPP, some kinds of polymers can also stabilize calcium and phosphate ions, including chitosan derivatives, poly-amidoamine and polyelectrolytes.2 and K2HPO4 into Pchi solution to construct the Pchi-ACP nano-complex. X-ray diffraction and selective electron diffraction results confirmed the amorphous state of the nano-complex. And the results of scanning electron microscopy and micro-CT proved that the Pchi-ACP nano-complex could realize the remineralization of demineralized enamel  and phosphorylated chitosan (Pchi) could bind calcium ions through chelation reaction of carboxyl groups and calcium ions and then bind phosphate ions to form ACP . The recd enamel .z-axis on the surface of demineralized enamel, and the microhardness of the remineralized enamel is comparable to that of the natural enamel (Poly-amidoamine (PAMAM) was first synthesized by Tomalia in the 1980s . PAMAM cl enamel .-/Ca2+ structure  Mouthwash. Studies used carriers like chitosan  and carb2) Toothpaste and tooth desensitizer. Another form of application of the ACP agents is to make them into pastes. Our group used mesoporous silicon nanoparticles to load ACP agents to achieve the enrichment and storage of ACP and this delivery system can be applied as the filler of toothpaste . CPP-ACP3) Resin product. Particulate forms of ACP have been incorporated into resin products, like adhesives , pit andACP complexes are in the amorphous state of the liquid phase , and ACPMouthwash form of the ACP agent is convenient to use, but the relatively low concentration of ACP and its inability to persist on the enamel surface for long periods lead to the limited effectiveness of ACP agents to enamel remineralization. The paste-like application form could effectively enhance the concentration of ACP and could maintain a high concentration of ACP on the enamel surface during application, but like mouthwash, it still has a short duration of hydroxyapatite formation due to the effect of saliva flushing. ACP agents modified resin products can release ACP on the enamel surface for a long period thus achieving the long-term prevention or treatment of enamel demineralization. However, the effect of ACP agent incorporation on the performance of these products like mechanical performance and biocompatibility needs further exploration. And the long-term stability of the ACP release from these products should be considered in future studies.Herein we summarize the strategies of stabilizing ACP. Calcium and phosphorus ions can be stabilized to the ACP state using a variety of methods, but the preventive and therapeutic effects of these ACP agents on enamel demineralization still await further investigation. There are three main forms of storage and application of ACP agents, namely mouthwash, toothpaste/tooth desensitizer, resin product. However, due to the shortcomings of the above-mentioned forms of ACP agents, more easy-to-use and long-lasting forms of ACP agents remain to be further explored.


Neurodegenerative diseases are fatal disorders of the central nervous system (CNS) which currently lack effective treatments. The application of mesenchymal stem cells (MSCs) represents a new promising approach for treating these incurable disorders. Growing evidence suggest that the therapeutic effects of MSCs are due to the secretion of neurotrophic molecules through extracellular vesicles. The extracellular vesicles produced by MSCs (MSC-EVs) have valuable innate properties deriving from parental cells and could be exploited as cell-free treatments for many neurological diseases. In particular, thanks to their small size, they are able to overcome biological barriers and reach lesion sites inside the CNS. They have a considerable pharmacokinetic and safety profile, avoiding the critical issues related to the fate of cells following transplantation. This review discusses the therapeutic potential of MSC-EVs in the treatment of neurodegenerative diseases, focusing on the strategies to further enhance their beneficial effects such as tracking methods, bioengineering applications, with particular attention to intranasal delivery as a feasible strategy to deliver MSC-EVs directly to the CNS in an effective and minimally invasive way. Current progresses and limiting issues to the extent of the use of MSC-EVs treatment for human neurodegenerative diseases will be also revised. The treatment of most neurological disorders currently represents a therapeutic challenge for the researchers committed to improving and/or extending the quality and lifespan of affected people.Neurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD), and amyotrophic lateral sclerosis (ALS) are characterized by the accumulation of specific proteins within the nervous system accompanied by a progressive loss of neurons in the affected regions ,2. The pThe use of stem cells was considered a potentially suitable strategy for the treatment of neurodegenerative disorders. Stem cells are undifferentiated totipotent or multipotent cells that can be obtained from a variety of adult tissues such as bone marrow, brain, liver, skin, skeletal muscle, gastrointestinal tract, pancreas, eye, blood, fat, and dental pulp . The mesThe efficacy of MSCs in neurological diseases was demonstrated in several preclinical studies ,15. HoweSuch action is achieved through a paracrine mechanism, the release of extracellular vesicles (EVs), which is, indeed, common to almost all cells. EVs are membranous structures derived from the endosomal system or shedding from the plasma membrane ,23,24,25In 2018, the International Society for Extracellular vesicles (ISEV) updated the guidelines for the study of EVs. They are a heterogeneous population whose size may vary typically between 50 nm and 500 nm, but they can be even larger, measuring 1–10 μm. The ISEV recommends the use of appropriate nomenclature for EVs, classifying them by clear, measurable characteristics  thus abandoning terms such as “exosomes” or “microvesicles” that were previously used ,36,37.EVs are present in many biological fluids, including blood, CSF, urine, saliva, and amniotic fluid, as well as in the conditioned medium of cell culture ,39. TheiThanks to their small size, which allows them to pass the blood–brain barrier (BBB) and deliver their cargo  to the brain, they become a powerful therapeutic application tool in neurodegenerative diseases where the BBB represents the main obstacle to reach the injured area of CNS.In this review, we summarize encompassing information about the therapeutic properties of EVs from MSCs to treat debilitating and fatal neurodegenerative disorders.It has been shown that MSC secreted factors are able not only to improve the surrounding environment of the target tissue, but also to exert beneficial effects even in the distal sites, supporting the paracrine hypothesis rather than the local transdifferentiation one ,44. The http://microvesicles.org/) [www.exocarta.org) [MSCs are considered prolific producers of EVs compared to other types of cells ,48 and tes.org/)  or EXOCArta.org)  both accrta.org) ,53. Thanrta.org) , crossinNeurodegenerative diseases are a heterogeneous group of disorders that affect approximately 30 million individuals worldwide with distinct morphological and pathophysiological features. These diseases have a complex multifactorial etiology whose pathogenic mechanisms are currently not fully understood ,56. The Although neurodegenerative diseases present distinct characteristics, common pathways have been identified, through which the neurodegeneration proceeds. Common pathogenic mechanisms underlying many neurodegenerative diseases include abnormal accumulation of insoluble protein aggregates and misfolding , oxidatiCurrently, no neurodegenerative disease is curable, and the available treatments only manage the symptoms or delay the progression of the disease .A large number of neurodegenerative disorders are characterized neuropathologically by intracellular and/or extracellular aggregates of proteinaceous fibrils which are implicated in progressive brain degeneration . For insThe use of MSC-EVs has been reported to act against neuronal damage and synaptic dysfunction , patholoA similar scenario is envisaged with Parkinson’s disease (PD), the second most common chronic neurodegenerative disease in the world , charactThe considerable capabilities of MSC-EVs have been observed in other settings: EVs derived from adipose mesenchymal stem cells (ASCs) showed the ability to promote remyelination after injury and neuroprotection of neurons and motor neuron-like cells, after peroxide treatment in vitro ,81, demoAs previously reported, the regulation of ROS production plays an important role among the pathogenic mechanisms of neurodegenerative diseases. With regard to this, recently, the role of MSC-EVs in reducing oxidative and nitrosative damage has drawn much attention. Antioxidant effects have been observed in models of PD  and in aInflammation associated with chronic neurodegenerative diseases is not typically the trigger itself of such diseases; however, it contributes and sustains their progress due to the contribution of activated microglia and astrocytes in neuronal dysfunction and death . MSC-EVsIn light of this promising evidence, the use of MSC-EVs in the treatment of neurodegenerative diseases currently appears to be a possible innovative strategy for the treatment of incurable diseases.Delivering therapeutic agents efficiently within the CNS represents one of the crucial issues of the therapies for neurodegenerative disorders. The passage through the BBB represents one of the limiting factors in conveying an efficient concentration of therapy in the areas of lesion in the CNS. The latter concern, related to cellular therapies, is strictly linked to the homing and biodistribution of transplanted cells and their vesicular counterpart, as well as to the identification of an efficient method of administration .MSC-EVs have recently been shown to have intrinsic homing capabilities similar to their parental cells  and, dueScientists devoted considerable efforts to identify methods for the labeling and detection of EVs when injected systemically: the use of fluorescent dyes ,91,114, The identification of EVs is fundamental to evaluate their biodistribution in the target organs, which can be predictive of therapeutic responses. Perets and co-workers recently developed a system to track the migration and homing of intranasally administered small EVs derived from bone marrow MSCs in vivo in different brain diseases. The approach combines classical X-ray computed tomography, with gold nanoparticles as labeling agents. They found that MSC-EVs specifically accumulated in pathologically relevant brain regions of murine models , while in healthy controls, they showed a diffuse migration pattern with rapid clearance. Moreover, the accumulation of MSC-EVs correlates with inflammatory signals in pathological brains . The homThe BBB is a selectively permeable membrane regulating the passage of substances between the peripheral vascular circulation and the CNS, thus serving to protect the CNS from harmful substances or overzealous immune responses .The BBB consists of a monolayer of endothelial cells surrounded by capillaries. Endothelial cells interact with and are bound to the basement membrane, in association with astrocytes and pericytes ,120. EndHowever, this cerebral compartmentalization represents the major obstacle in the administration of drugs or other therapeutic substances directed towards injured sites in the CNS. Several strategies are being developed to enhance the quantity and concentration of therapeutic agents delivered to the CNS. Gabathuler describes the “physiological approach” as the most efficient one for obtaining a regular distribution of molecules in the brain through the endothelial cells of the BBB. This approach exploits transcytosis due to the presence of specific receptors expressed on the BBB . EVs froA recent study showed the therapeutic effect of EVs derived from human neural stem cells in an in vitro model of BBB breakdown. Indeed, 5XFAD AD mice present leakage of the BBB, and EVs were shown to be able to initiate and repair the leakage of the BBB, reversing the deficiency of BBB induced by AD .Nevertheless, relatively few researches identify the precise mechanism of the passage of EVs through the BBB. Some evidence has reported that EVs can cross the BBB using a mechanism of endocytosis via brain microvascular endothelial cells (BMECs). An experimental in vitro model showed that small EVs under stroke-like conditions are able to bypass the BBB through BMECs, mainly via the transcellular pathway, rather than via tight junctions involved in the paracellular route, to provide their load to the brain parenchyma .Understanding the mechanisms of the passage of MSC-EVs through the BBB represents a prerequisite for the development of EV-based strategy for the treatment of neurodegenerative diseases using, in particular, the intranasal delivery pathway.Intranasal (i.n.) administration represents an attractive strategy to deliver pharmacological or cellular substances directly into the brain in a non-invasive way. Several advantages of i.n. delivery are reported : the i.nNeurodegenerative diseases are, for the most part, progressive or recurrent and their treatments often require repeated dosages to achieve long-term beneficial effects ,128. TheIn the context of cellular therapies, beneficial effects of injecting MSCs and their vesicular counterpart via the i.n. route have been reported ,131: LosAn immunomodulatory response was also observed in EAE mice treated i.n. with EVs from MSCs with an increase in the frequency of Foxp3+ CD25+ regulatory T cells, accompanied by an amelioration of pathological and clinical changes until the end of treatment . InteresIn a recent study, we compared the effect of small EVs isolated from ASCs (ASC-EVs) using two different routes of administration: intravenous (i.v.) and intranasal, in SOD1(G93A) mice, a mouse model of ALS. We demonstrated that repeated i.v. or i.n. administrations of ASC-EVs improved the motor performance in SOD1 mice compared to controls. In addition, the treatment demonstrated a neuroprotective effect on motoneurons in the lumbar section of the spinal cord of treated mice. Therefore, with the same therapeutic effects, the intranasal route turns out to be a minimally invasive, effective, and direct way to convey EVs inside the CNS, bypassing the obstacle of the BBB .The concentration, dose, and volume of administration are important factors that can affect nasal drug delivery to the brain. However, some restrictions or limitations should be taken into consideration, such as the complex anatomy and physiology of the nasal epithelium .The nasal epithelium proves to be permeable to lipidic small nanoparticles, which can cross the epithelium via transcytosis. Furthermore, stem cells and their EVs also cross the barrier in a similar way  and the Furthermore, the engineering of nanoparticles in general can improve the penetration through the elastic mucin fiber by means of electrostatic changes. Studies by Carlson and co-workers  demonstrThe possibility to translate the bioengineering applications mentioned above to cellular nanoparticles, and in particular to MSC-EVs, could improve their bioavailability through the i.n. route, resulting in a privileged strategy to treat neurodegenerative diseases.Advanced studies have attempted to manipulate and modify the surface or the content of stem cell-derived EVs in order to improve their homing capacity and therapeutic potential for specific purposes. The modifications previously described to improve the efficiency of intranasal administration of MSC-EVs represent only partially the bioengineering applications used to improve the therapeutic action of EVs ,144.Although intranasal delivery appears to be an advantageous route for the application of MSC-EVs, most preclinical and clinical studies currently report the use of systemic administrations. However, systemic delivery causes high dispersion of EVs throughout the body and a very short residence time in the blood. Improving EV stability in the systemic circulation and protecting their content from enzymatic/proteolytic tissue clearance could be a strategy to increase EV concentration, and enhance the number of vesicles that are able to reach target sites before the clearing action of macrophages . As demoThe improvement in the therapeutic action of EVs could be possible through exploiting the targets that are naturally present on EVs  or through their functionalization with engineered approaches.In order to prolong the circulation time of EVs, several chemical modifications have been tested, such as PEGylation: the use of a hydrophilic polymer was shown to improve cell specificity of PEGylated EVs and to prolong their circulation times ,146.These approaches were also used to target neural cells, avoiding accumulation in no specific sites ,147. “ClAn improvement in the therapeutic action of EVs is obtained also by modifying/functionalizing their content: the overexpression of miRNA-21 in EVs from hypoxia-preconditioned MSCs was able to reduce the cognitive deficit and amyloid deposition in AD mice as well as to decrease inflammatory markers . The oveSynergistic approaches can potentiate the therapeutic effect of EVs: Peng and coworkers have developed a self-oriented system for intranasal administration of MSC-EVs in a PD mouse model. They exploited MSC-EVs as a nanocarrier cargo overexpressing miR-133 to promote nerve axons growth and recovering neuronal function. Moreover, EVs were also loaded with superparamagnetic iron oxide nanoparticles (SPIONs), in order to orient them across the membrane barriers and release drugs into the cytoplasm of target cells, together with hydrophobic curcumin to alleviate neuroinflammation and clear α-synuclein aggregates .Combining the intrinsic properties of stem cell-derived EVs with a targeted functional modification could prove to be an effective strategy to treat neurodegenerative disorders.https://beta.clinicaltrials.gov (accessed on 25 January 2023). The majority of those are observational studies focusing on EVs from patient body fluids for diagnostic and prognostic purposes. Promising results have been confirmed in other diseases [Preclinical data on EVs-based therapies, as previously discussed, are very encouraging. MSC-EV therapies proved to be much safer and more versatile than cell therapy, despite few clinical results being unavailable still ,149. Sevdiseases . Worthy Although there are still few clinical studies currently registered, the interest in these therapies appears to be growing and corroborated by the promising results obtained from preclinical studies .The transition from basic or preclinical research to the clinic still has to overcome several gaps at various levels .The complex biological nature of MSC-EVs makes it difficult to identify their mode of action, and we can likely expect that the neuroprotective and immunomodulatory effects will not be limited to a single, unique active molecule. The partial identification of the active components of MSC-EVs complicates the definition of the precise mechanism of action of MSC-EVs .On the other hand, many inconsistencies are largely due to the technical difficulties that impact manufacturing processes   as well The transition towards therapeutic application also requires the identification of an effective therapeutic dose to produce a response in humans. However, results are controversial and remain highly variable from study to study. Several parameters, including the previously discussed route of administration, the homing and biodistribution, as well as dosing and timing of the MSC-derived EVs have to be carefully investigated .The majority of preclinical data are obtained in vitro or from rodents such as mice or rats, representing the starting point to identify the minimal effective dose of MSC-EVs for a specific therapeutic application that could be extrapolated for humans. Comparative studies examining the preclinical literature are reported to identify a possible conversion of the dose of EVs from animal treatment to humans ,159. HowClinical translation would require the use of large animals that could help in pharmacokinetics and formulation studies due to their great similarity to humans. Nevertheless, the use of large experimental models has unaffordable costs, although they would be very suitable, especially for the study of intranasal delivery ,162.In addition, the regulatory requirements, associated with good manufacturing practices (GMP) production and scaling for large distribution, should also be clarified and implemented for several aspects such as quality controls or production variability .In summary, the reported studies represent the proof of concept of the potential of MSC-EVs as a therapeutic opportunity to treat neurodegenerative diseases. The possibility of combining the intrinsic properties of their parental cells with bioengineering modifications could represent a potential improvement for their clinical use. The development of a formulation of EVs that improves their biodistribution and retention in the lesion sites through an appropriate route of administration holds great promise for a facilitated delivery, mainly for CNS diseases. In this regard, the intranasal administration of MSC-EVs represents a flexible treatment, which simplifies the delivery procedure in a direct, efficient way.Although there are still many challenges to be addressed, the clinical translation of MSC-EVs towards a real therapy represents an interesting frontier for the treatment of neurodegenerative diseases. In recent years, the number of ongoing clinical trials that are actively recruiting patients has been constantly expanding, and the successful translation of EV-based therapeutics in the clinic seems to be more realistic and not so distant, thanks to the rapid advances of nanotechnologies and the support of coordinated studies worldwide.


The development of new techniques to rapidly and accurately detect bacteria has drawn continuous attention due to the potential threats posed by bacteria to human health and safety. Recently, a novel strategy based on fluorescent probes has drawn considerable interest for the detection of bacteria due to its high selectivity, fast response, and simple operation. In this review, we summarize the recent progress on fluorescent probes for the specific recognition and discrimination of Gram-negative and Gram-positive bacteria. In particular, we outline current design strategies, such as targeting of the differences in surface components, cell wall components, endogenous enzymes, surface charge, and hydrophobicity of various kinds of bacteria to develop various fluorescent sensors . We also emphasize the application of organic molecules in probe recognition elements. We hope that this review can stimulate this research area in bacterial detection and imaging in the future. The increasing public health problems caused by bacterial infections are the subject of continuous concern. In 2017, the World Health Organization (WHO) released a list of priority pathogens , which aβ-lactamase used to destroy antibiotics +) chloroplatinic (II) complex. The probe binds negatively charged LPS to form LPS-Pt (II) aggregates and quickly distinguish Gram-negative and Gram-positive bacteria within 5 min without washing  (51) with excellent water solubility, showed excellent imaging and antibacterial ability on E. coli. and S. aureus. Compared with probe 51, a mixed-charge AIE polycarbonates probe (52) allowed for the rapid and selective imaging of S. aureus but not E. coli  , which c E. coli .β-lactamase. This kind of method has certain advantages for the detection of super bacteria. In addition, the electrostatic and hydrophobic interaction between the probe and the bacterial surface is currently a hot spot in bacterial detection research. According to the conformation of the probe, this method can distinguish Gram-negative bacteria from Gram-positive bacteria and even roughly judge the types of bacteria according to the ratio of fluorescence signals before and after binding to bacteria. However, the high detection limit of this kind of probe is still subject to some limitations in the field of bacterial detection. At present, the detection requirement of most pathogens in various countries is zero detection, and the detection range of fluorescent probes that only rely on hydrophobic and electrostatic interaction as identifying elements is usually between 104 and 108 CFU/mL. Moreover, the probe is easily affected by cations and surfactants in the environment to be measured. Although the use of highly specific recognition elements, such as antimicrobial peptides and aptamers, to construct probes can effectively detect the presence of pathogens, they are difficult to synthesize and easily degraded, so they cannot be widely used.In this tutorial review, we summarized the recent research progress with respect to construction strategies for bacterial detection using fluorescence probes. We first discussed the strategy of labeling the surface components of bacteria with small molecular organic fluorescence probes in a direct or indirect manner. Although those metabolic fluorescent molecules are highly selective, they are limited by bacterial metabolism. Moreover, they showed some limitations in the detection of bacteria in complex environments. Next, we outlined the fluorescent probes constructed through the binding of specific components on the surface of bacteria, such as the binding of glycopeptide antibiotics in the cell wall and the affinity of boric acid derivatives to carbohydrates. We also mentioned the detection strategy involving the catalytic reaction of specific groups by bacterial endogenous enzymes, such as nitrate reductase, alkaline phosphatase, and A recently proposed strategy based on fluorescent probes has attracted considerable interest for the detection of bacteria due to its high selectivity, fast response, and simple operation. We hope that this review can stimulate this research direction in bacterial detection and imaging in the future.