Marine microplastic pollution (MMP) is becoming one of the most pressing environmental problems facing humanity today. The novel coronavirus epidemic has raised the issue of environmental contamination caused by large-scale improper disposal of medical waste such as disposable masks (DMs). To assess the impact of MMP caused by DMs and to seek solutions for the prevention and control of MMP, this study uses the Driving force-Pressure-State-Impact-Response (DPSIR) framework to establish a causal chain of MMP caused by DMs. The conclusion shows that the novel coronavirus epidemic has led to a surge in the use of DMs, which has brought pressure on resource constraints and environmental pollution at the same time. Improperly DMs enter the environment and eventually transform into MMP, which not only endangers the marine ecological system but also poses potential human health risks as well as economic and social hazards. In addition, further research on environmentally friendly masks (cloth masks and biodegradable masks) is essential to mitigate the environmental damage caused by the large-scale global use of DMs. This study provides a scientific and theoretical basis for the assessment of MMP from discarded DMs, and the findings of this study will provide a reference for the formulation of relevant policies. The emergence of the novel coronavirus COVID-19) disease with strong contagiousness has attracted global attention since December 2019 9 disease,4. SinceDMs are mainly made of polypropylene (PP), which is considered plastic waste after being discarded. Its entry into the environment can cause plastic pollution and release microplastics, severely threatening the aquatic environment and aquatic life once they enter freshwater and coastal areas . The datSeveral studies have focused on the MMP induced by the rapid growth of waste in DMs. Sangkham measured the generation of DMs and medical waste in Asia during the outbreak and discussed the emerging challenges in solid waste management during and after the pandemic . Jung etIn order to fill this gap, this study analyzes and explores the problem of MMP caused by DMs from a multidimensional perspective, including social, economic, and environmental perspectives. The structured approach provided by the DPSIR framework is used to examine, in detail, the interrelations among the five components to better understand the current complex pollution situation of marine microplastics and the potentially significant pollution risks in the future. First, the driving forces of DM usage are discussed. Second, the social and environmental pressures created to meet human demand are examined. Then, the physical, chemical, and quantity states of the DM contaminants and the impact of their resulting impacts on marine ecosystems, human health and socioeconomics are examined. Next, responses to regulate the driving forces, mitigate the pressures and states, and impacts of MMP are reviewed from both upstream and downstream perspectives. Finally, specific recommendations for preventing and controlling MMP from DMs are derived.MMP is a complex environmental problem that requires an adaptive management framework that is action-oriented, integrated with an approach which is interdisciplinary in nature, incorporating both social and ecological systems . The DPSThe DPSIR framework is one of the key conceptual frameworks that are now widely used in marine environmental issues. It has been applied to various marine environmental issues, such as social-ecological systems in coastal environments , social According to the EEA , the comWith the widespread use of the DPSIR framework in environmental studies, the definition of the five components of the framework and the relationships among them have been explained in more detail. (1)The “driving forces” are the most primitive factors that cause environmental change and refer to the social, economic and institutional changes that directly or indirectly trigger environmental stress. It can be defined as “social, demographic, socio-economic development and corresponding changes in lifestyles, as well as overall consumption levels and production patterns” . Rodrigu(2)The “pressures” refer to anthropogenic factors that are directly applied to environmental systems by driving forces and contribute to environmental change, “including emissions of material, physical and biological agents, and the use of resources and space by human activities” . Pressur(3)The “states” indicate both qualitative and quantitative measurements of pollutants or the current state of the environment or environmental parameters that may change due to pressures . It is w(4)The “impacts” refer to the feedback effects of natural system states on human health, socioeconomics, and ecosystems, which often arise from adverse environmental conditions and are the result of a combination of the first three elements . Impacts(5)The “responses” refer to social actions taken by humans to prevent, mitigate, or eliminate negative impacts , which iThe DPSIR framework is used to address the MMP from DMs, following a typical five-step procedure . In thisThe COVID-19 pandemic has severely impacted the health and livelihood of people worldwide. DMs have a great filtering effect on aerosols and droplets, which can prevent respiratory diseases. Wearing DMs effectively reduces the probability of being infected by novel coronavirus , which mTo avoid the crisis of economic downturn and communication blockage brought about by precautionary measures such as lockdowns and travel restrictions, people in different countries choose to wear DMs on a large scale, while maintaining social distancing, keeping good hand hygiene (hand washing), and avoiding public or crowded spaces as alternative measures to keep society functioning properly .Since 2020, health authorities in different countries have issued various recommendations for using face masks to protect against COVID-19, which tend to be dynamically adjusted  32]. Wh. Wh32]. The raw materials of DMs are mostly made of petroleum-based polymer-PP (PP) fiber, which is a non-renewable resource. Therefore, the massive use of DMs can cause a large consumption of natural resources. Furthermore, DMs are used in a wide range of applications and scenarios. They are likely to be directly discarded in municipal solid waste without safe disposal measures or directly in the environment. This creates new challenges for medical waste management, which will worsen especially for developing countries with inadequate waste management . Discarded DMs are found scattered in public places such as parks and streets all over the world . A studyThe DMs are made of melt-blown fabric, non-woven fabric, mask straps, and nose clips. Melt-blown fabric is the core material used to produce DMs, whose main component is PP. The melt-blown cloth consists of ultra-fine fibers with diameters of about 1–5 microns arranged randomly to form numerous tiny pores that trap dust due to the electrostatic effects. When the droplets containing various types of viruses approach the PP melt-blown fabric, they can be adsorbed on the surface of the melt-blown fabric and cannot penetrate. Thus, DM has a strong filtration property, sufficient to reject bacteria, suspended particles, liquid droplets, and aerosols. The inner layer, near the mouth and nose, is made of hot-rolled nonwoven fabric to absorb moisture and ensure that the wearer’s lips are dry and comfortable. The entire mask is designed to fold to improve its fit to the wearer’s face, blocking more than 90% of the 5μm particles. The structure of DMs varies slightly between standards, with disposable surgical masks and N95 masks having a high acceptance rate among health workers and the public . Their sExperimental results show that individual N95 masks and disposable surgical masks have approximately 11 and 4.5 g of PP and other plastic derivatives , respectively ,47. BecaThe use of DMs has reduced the spread of the virus in society while simultaneously causing severe environmental problems in terms of microplastic pollution. During the use of DMs, extremely fine nonwoven fibers tend to break off and form microplastics. Mechanical wear and tear between the fibers is further exacerbated by the repeated use of DMs over time, leading to extra microplastics. In addition, DMs may intercept microplastics in the air during use, thus increasing their microplastic carrying capacity . 21 units of microplastic into the environment [Discarded DMs enter the ocean through improper landfills and dumping treatment. They disintegrate into tiny plastic fragments under a range of physical and chemical effects, such as solar radiation, biological erosion, low-temperature weathering, tidal, wave washing, and so on. As the DMs gradually age and decompose in the environment, the entire DM will become completely microplastic and enter the ocean . Shen etironment . Using kironment . The avaUnlike other plastic products such as plastic bags and bottles, DMs are more likely to be damaged and decomposed in the natural environment, releasing additional microplastic fibers . The melDiscarded DMs entering the environment may not only entangle the bodies of marine wildlife but also release microplastics that are preyed upon by marine life, thus affecting normal growth and reproduction . ExperimMicroplastics have been found in organisms at different levels of the marine food chain ,62. The With the increasing amount of marine organisms caught and seafood supplied by aquaculture year by year, microplastic particles in these marine organisms and aquaculture products may eventually enter the human food chain. The pollution status of the marine environment by microplastic is a potential health problem for humans. It has been shown that harmful microorganisms could attach to plastic debris , increasFoods made from marine organisms, such as fish, are an important food source for humans. MMP raises concerns about global food security, which may eventually lead to the consequences of food shortages . In the To respond to the impact of MMP from DMs, especially the impact on marine ecosystems, the potential impact on human health, and the impact on the economy and society, a series of prevention and control strategies have been developed at different levels. According to the principles of source reduction and process management of MMP prevention and control , the resIn the context of the normalization of epidemic prevention and control, DMs have become an immediate need in people’s daily lives, and their demand cannot be reduced in the short term . The fun(1) Improving public participation in the proper disposal of DMsGuiding the public to properly dispose of DMs is the most effective way to properly prevent the problem of microplastic pollution at the source. Used DMs can greatly reduce the risk of environmental contamination after proper disposal such as disinfection and incineration ,78. At p(2) Strengthen the management of DM wasteDue to the disposable natures of DMs, recycling as a reduction pathway to achieve more limited results, and scientific management based on the disposal process will be sought to reduce the risk of MMP caused by DMs. The COVID-19 pandemic has increased the pressure on municipal waste management, which can lead not only to the spread of the virus but also to environmental problems if not handled properly . TherefoCurrently, several medical waste bins with clearly marked points are placed in public areas such as hospitals and communities to collect discarded DMs. They are packaged in double-layered medical waste bags and disposed of as general medical waste by specific personnel, municipal solid waste workers, and special waste management departments of companies . (3) Using cloth masks or biodegradable masks as an alternativeThe promotion of cloth masks can effectively reduce the consumption of DMs ,45. ClotThe development of biodegradable masks is another solution idea to reduce the risk of the DMs contamination. The advantage of biodegradable materials is their biodegradability through composting, which avoids environmental hazards at the source to some extent. The biodegradable polymer and bio-based material electrospun ensemble can be used as the main material of masks. Experiments revealed that this type of mask reaches 98% particle filtration efficiency during use, and its waste can be biodegraded within four weeks . In addiThe downstream responses mainly focus on recycling and reusing discarded DMs to reduce the risk of MMP. Medical plastic waste generated during the epidemic has a high calorific value compared to other conventional fuels, accounting for nearly 25% of the calorific value of municipal solid waste . Discard(1) Physical-mechanical recyclingImproving the process of mechanical recycling of DMs can produce the recycled product, recycled polyethylene, with better stiffness and strength, which enables physical and mechanical recycling ,91. The (2) Chemical recycling2, CH4, and CO, which suggests an environmentally benign disposal process [Multi-stage pyrolysis was used to catalyze the cracking of discarded DMs to generate H process ,93. Xian process . TherefoDMs, as a new type of microplastic pollutant, can continuously release microplastics into the marine environment through the water systems. However, the rigidity of their demand and single-use nature makes it difficult to establish closed-loop chain management of the entire life cycle such as source reduction, waste process control, and end-of-life recycling. This study is based on the analytical framework of DPSIR, which seeks more effective management measures to curb the MMP prevention and control brought by discarded DMs. The results of the study show the surge in demand for DMs’ uses under the three driving forces of human health risk, social demand, and mask policy promotion in the context of the COVID-19 epidemic, increased the risk of non-renewable resources such as oil tension, plastic waste increase, and microplastic flow into the environment, thus increasing the pressure on both resources and the environment. In addition, the physical-chemical composition of discarded DMs leads to waste ending up in the ocean in the form of large amounts of microplastics. In order to effectively reduce the impact on the marine ecosystem, human health, and socioeconomics caused by MMP from DMs, response mechanisms are proposed for upstream and downstream in a targeted manner.In view of this, countries around the world should guide the public on the proper disposal of DMs and educate the public on the environmental responsibilities that individuals should assume. Secondly, it is necessary to strengthen the management of DM waste and improve the infrastructure to help the public dispose of DMs reasonably. In addition, a system of rewards and penalties for recycling DMs could be developed as a complement to fully incentivize the public to dispose of DMs reasonably. Moreover, eco-friendly alternatives such as cloth masks and biodegradable masks with filtration efficiency that meet basic protection needs and have a longer life cycle can be developed. In addition, further research into the risks of MMP from DMs is essential, and new technologies should be developed to overcome the problems associated with the release of microplastics from DMs.


N = 42 in Experiment 1, N = 41 in Experiment 2) were shown 12 novel objects, asked three questions about each, and did not receive answers to their questions. Children recalled their questions in the first experiment and categorized variants of the novel objects in the second experiment. We found that children have robust working memory for their questions, indicating that these questions may relate to their subsequent thinking. Additionally, children generalize category boundaries more narrowly or broadly depending on the type of question they ask, indicating that children’s questions may reflect an underlying bias in how they think about the world. These findings suggest that future research should examine questions in the absence of answers to understand how inquiry affects children’s cognitive development.Children ask many questions, but do not always receive answers to the questions they ask. We were interested in whether the act of generating questions, in the absence of an answer, is related to children’s later thinking. Two experiments examined whether children retain the questions they ask in working memory, and whether the type of questions asked relate to their categorization. Four to ten-year-old children ( Children are very inquisitive individuals; research has found that children ask an average of 76 information-seeking questions per hour by the time they reach preschool age , and theHow do questions help children learn? One proposal is that children’s questions are information-seeking in nature . That isChildren ask many kinds of questions, such as about visual features, functions or behaviors, locations, people, events, cultural conventions, verbal labels, and causal relations . Some quThe process of generating questions becomes more efficient across development. Preschool and kindergarten-age children often struggle to generate questions spontaneously . As chilThere may also be age-related biases in the information that children seek through their questions. For instance, research has long found that preschool-aged children primarily use feature-based information over other types of information when categorizing and generalizing objects . FurtherIn studies examining what children learn via inquiry, children are generally provided information and/or a direct answer to their question during cognitive tasks. Consequently, it is hard to determine whether children’s questions themselves are related to their thinking and/or inferences. When asking fact-based questions, children likely need to hear the answer to learn something new. For instance, someone would need to tell children that Madison is the capitol of Wisconsin to learn anything from the question “What is the capital of Wisconsin?” However, what about other types of questions? It could be that the act of question asking itself is related to this change and the process of inference making. Another possibility is that questions themselves may not matter at all; simply being provided the information, without having to ask a question, could have yielded the same behavioral response. According to existing theoretical frameworks of epistemic questions as information-seeking behaviors , childreAre there cognitive consequences to children’s question asking in the absence of answers? There is little research to date on the act of generating questions. Most of what we know about children’s question asking comes from observation-based research . In earlIn the current study, we examined whether and how children’s question asking is related to cognition by testing their thinking after a question-asking period. Given that there are a plethora of ways in which questions could impact cognition , we starTo foreshadow the results of Experiment 1, we observed that children had strong working memory for their questions, which suggests that children may use these memories to make inferences during higher-order cognitive processes. Thus, the second way we studied the role of question asking in children’s learning was through examining whether question asking may also have cognitive consequences for higher-order cognition. Specifically, in Experiment 2, we looked at children’s categorization of the items they asked questions about. We chose to look at the relation between children’s questions and categorization because categorization represents how children make inferences about the world , and preWe hypothesized that asking certain questions may relate to how children generalize. For instance, children may ask feature-based questions because they may later tend to generalize based on features. If this is indeed the case, we expected to see a relation between the number of feature-based questions children ask and how broadly or narrowly they generalize category boundaries based on these features. Moreover, we expected to observe a relation between category-based questions  and children’s generalization of category boundaries. Specifically, we predicted that feature-based questions would be related to narrower categorization, and that category-based questions would be related to broader categorization behavior. We tested this hypothesis by looking at how broadly or narrowly children categorized based on the numbers of differing features on novel objects. If we did not find these results, it would provide support for alternative hypotheses. For instance, the nature of the questions that children ask may have no relation to how they generalize category boundaries. There may also be an external, unmeasured factor that impacts both the questions children choose to ask and their later categorization. Taken together, these two experiments provided a first step of examining whether the act of generating questions relates to children’s subsequent learning.We first examined whether question asking would be related to children’s working memory. That is, in the first experiment we asked: Do children retain the questions they ask in their working memory? We chose to start with this lower-level process because working memory for questions could serve as the foundation for how the act of asking questions affects higher-order cognitive processes, such as categorization or problem solving.M = 6 years 11 months, median = 6 years 8 months, range = 4 years 0 months to 10 years 5 months). This age range was chosen because it spans the time in which children begin successfully generating questions to the time when they become adept at using efficient question-asking strategies  provided demographic data about their child and family. Further demographic information is provided in N = 10, and inability to pay attention to the task, N = 4).Effect sizes were gathered from studies on children’s question asking with this age group, which had consistently large effect sizes . A question was coded as a category-based question if it contained elements referring to the novel object’s category membership . Questions could also be coded as being both feature-based and category-based . We also coded for the following question types: function, behavior, ambiguous, location, creation, and social relevance-based questions. However, we did not explore these in Experiment 1 or 2 because they were sparsely asked. Inter-rater reliability across all question categories, including superordinate and subordinate questions was 90%. Examples of all question types are listed in We also coded category-based questions as superordinate and subordinate. Superordinate category-based questions were defined as questions that related solely to category membership . Subordinate category-based questions were defined as questions that explicitly referenced specific feature-based information. For instance, the question “Do all wugs have orange feet?” was coded as subordinate because the feature of “orange feet” was explicitly mentioned. Alternatively, this same question “Do all wugs have orange feet?” could potentially also be interpreted as a superordinate question, because it refers to the broader category of “all wugs” in addition to explicit feature information. However, for the purpose of this study, this question would still be coded as subordinate because it explicitly mentions the feature “orange feet.” We coded children’s questions in this way because it aligns with how feature and category-based information was coded in previous research on children’s question-asking behavior e.g., .Additionally, questions were coded as “correct” if they were recalled mostly word-for-word. Children did not always recall questions completely word-for-word, but nearly always recalled them mostly word-for-word. For instance, if a child asked, “Why does it have purple feet?” but recalled their question as “Why does it have those purple things down there?” while referring to the feet, the question recall was coded as correct. As with the question types, inter-rater reliability was 90% when we coded for correct versus incorrect question recall.SD = 18.3) of the total number of questions they asked. No child remembered less than one-third of the questions they asked (range = 12–36). These results suggest that children attend to and retain the questions that they ask in working memory, possibly serving as foundational representations for later thinking and learning.We were interested in whether children retained the questions they asked in working memory. We hypothesized that children would remember the majority of their questions, as these short-term memories could serve as a foundation for later thinking and learning. To test this hypothesis, we calculated the mean percentage of questions children remembered out of all of the questions asked. We found that children indeed have robust working memory for their own questions: on average, children were able to remember 77.5% (M = 29.98 (out of 36), SD = 8.22, range = 5–36]. Children also asked other types of questions, such as category-based questions; for example, “Is this wug a type of bird?” . On average, children asked more feature-based questions than category-based questions, t(41) = 12.02, p < 0.001, d = 1.85, 95% CI . All 42 children asked feature-based questions, while 25 children asked category-based questions. Thus, we chose to focus on differences in memory for feature and category-based questions for Experiment 1. No single item elicited significantly more feature or category-based questions than the others  and feature-based questions , t(25) = 0.806, p = 0.428, d = 0.16, 95% CI . One might expect children to have better memory for feature-based questions, as children viewed the novel objects when they were asked to recall their questions. However, because children remembered a similarly high proportion of category-based questions, this suggests that category-based questions may relate to children’s thinking and behavior. This is discussed in more detail in the General Discussion.Next, we looked separately at the percentages of category-based and feature-based questions that children remembered. Results revealed that children remembered a similar proportion of category-based questions  the number of feature-based questions they asked between the first and second half of the experiment, with the remaining 37% of children asking the same amount of feature-based questions in each half. Similarly, 61% of children changed the number of category-based questions they asked, with the remaining 39% asking the same amount of category-based questions in each half of the experiment . ChildreWe also separated children’s inquiry behavior based on age, separating them by preschool age (under age 6) and elementary school age (ages 6 and up) because we wanted to determine whether there were developmental differences between these age groups in the types of questions asked. We split children into these age groups based on the age of transition to formal schooling, as children who have entered formal schooling are more likely to have received explicit instruction on question-asking. Indeed, the Next Generation Science Standards and the Common Core State Standards in ELA and Mathematics all include the act of asking questions as an important skill that K-12 students should know and do . Older cM = 7.57, SD = 8.5) than younger children , t(40) = 2.63, p = 0.012, d = 0.86, 95% CI . Older children also asked fewer feature-based questions  compared to younger children , t(40) = −2.97, p = 0.005, d = −0.97, 95% CI . We also conducted these analyses with age in months as a continuous variable, which revealed similar results: there was a positive correlation between age and the total number of category-based questions asked  and a negative correlation between age and the total number of feature-based questions asked . Therefore, it appears that there are developmental differences in the types of questions children choose to ask. These findings are further explained in the Discussion.Analyses splitting children into these age groups found that the average number of category-based questions asked differed significantly based on age: specifically, older children in the sample asked more category-based questions  than the second half , t(40) = 3.34, p = 0.002, d = 0.52, 95% CI .We also examined whether order or item effects were present in children’s working memory for questions. No item effects were found; that is, children’s memory for their questions was similar across all 12 novel objects in the experiment. When examining order effects, paired-samples t-tests revealed that children were more likely to remember the third question they asked compared to the second or first question for a given item see . AdditioM = 2.5, SD = 0.8) compared to the last item  in the experiment, t(41) = 1.58, p = 0.12, d = 0.24, 95% CI . Thus, children also remember their questions even when they do not know that they will be prompted to recall them.Finally, we examined whether children’s knowledge of an upcoming memory test affected their working memory. Children may have retained questions in working memory only because they knew that they would be tested on them. To test for this possibility, we compared performance on the first item, when children did not know that they would be tested, to performance on the last item, when children had likely learned that they would be tested. There was no significant difference in children’s memory for their questions for the first item  and how children categorize novel objects.Experiment 1 showed that children remember the questions they ask. These findings raise the question of whether children’s question asking may also relate to their higher-order cognition. One possibility is that children could draw on memories for their questions to make inferences during higher-order cognitive processes such as categorization. Experiment 2 tested this possibility by examining whether the type of questions children ask related to the manner in which they categorize objects. We hypothesized that children would generalize category boundaries more narrowly or more broadly depending on whether they asked more feature or category-based questions. If children categorize differently based on the types of questions they ask, these results would suggest that question asking may be correlated with how children conceptualize the world.M = 6 years 9 months, median = 6 years 10 months, range = 4 years 5 months to 10 years 2 months). 95.12% of parents (N = 39) provided demographic data about their child and family. Further demographic information is provided in N = 5, inability to pay attention to the task, N = 5, and failure to finish all trials of the study, N = 2).The participants were 41 4–10 year-old children  and 2.76 category-based questions , t(40) = 13.34, p < 0.001, d = 2.08, 95% CI . No single item elicited significantly more feature or category-based questions than the others (N = 21) and the number who included category-based questions (N = 19) did not differ significantly. As with Experiment 1, we found that most children adapted their question-asking behavior slightly over time  than younger children , t(39) = 2.95, p = 0.005, d = 1.15, 95% CI . This age difference was not found for feature-based questions, t(39) = −1.5, p = 0.142, d = 0.52, 95% CI . When we conducted these analyses with age in months as a continuous variable, there was a positive correlation between age and category-based questions asked and a negative correlation between age and feature-based questions asked, but neither were significant. In sum, we saw similar developmental patterns as Experiment 1, with children asking mostly feature-based questions and asking more category-based questions with age.We also conducted age-related analyses as with Experiment 1. We found that the average number of category-based questions asked differed significantly based on age group: specifically, older children (ages 6 and up) asked more category-based questions  and how they categorized. We conducted hierarchical linear regressions with mean last category endorsement as an outcome variable and number of feature and category-based questions for the first and third questions as predictors, and found that the nature of the third question was no more predictive of categorization behavior than the nature of the first question, F = 17.08, p < 0.001, 2η = 0.61. Children endorsed the object that looked most like the target  the most often, followed by the object with two, three, then four featural changes. There was significant variability in which item children endorsed last  and did not observe any item effects. Therefore, there were indeed differences in how children endorse each variant of the target, indicating that the categorization task is a useful measure for determining differences in generalization of category boundaries.Next, we examined the descriptive statistics for the categorization task. It was important to determine that there were clear differences between each variant of the target novel object to ensure that there was diversity in children’s generalization of category boundaries. To investigate whether these differences existed, we conducted a one-way repeated measures ANOVA to determine whether children endorsed objects  that looked more like the target  over objects that did not. We found a significant effect of object similarity to the target, Wilks’ Lambda = 0.392, last See . Pairwislast See . Additior = −0.327, p = 0.037; r = 0.417, p = 0.01; To test our hypothesis that question type is related to generalization of category boundaries, we conducted bivariate Pearson’s correlations between the total number of feature-based or category-based questions asked and the mean last item endorsed during categorization. We found that when children asked more feature-based questions , they categorized more narrowly. That is, they were less likely to endorse items as belonging to the same category as the initial object . Independent samples t-tests revealed no significant differences in categorization behavior based on whether children asked superordinate or subordinate category-based questions. Therefore, these results suggest that when looking at feature-based and category-based questions more generally, question type is related to higher-order cognition, such as how children endorse category membership.Finally, we fit a mixed-effects logistic regression model predicting children’s likelihood of endorsing a given item as a member of the initial category with age (in months) as a fixed factor, and the number of feature-based questions asked, the number of category-based questions asked, and the number of feature changes from the target item as predictors . To contThe central goal of the experiments was to determine whether the act of generating questions relates to children’s subsequent thinking and learning. Specifically, the studies were designed to determine whether (a) children retain the questions they ask in working memory, and (b) whether the act of asking questions is related to how children think about the world . The results revealed that children have a robust working memory for the questions they ask (Experiment 1), and that asking certain types of questions  predicts how near or far they generalize category boundaries (Experiment 2). To our knowledge, this study is the first to investigate whether the act of generating questions, in the absence of an answer, relates to children’s cognition. Thus, the primary theoretical contribution of this work is the knowledge that the act of asking questions is correlated with children’s subsequent thinking and behavior.In Experiment 1, we took the critical first step of examining whether children remember the questions they ask, as these short-term memories could serve as a lower-level foundation for higher-order thinking like categorization. We found that children had strong working memory for these questions—why? There are several possible explanations. First, children may purposefully be trying to attend to and remember the questions they ask so they can ask similar questions again later, with the hopes of eventually getting an answer. Second, children may retain most of their questions in working memory because these questions reflect how they are thinking about the world. That is, children may have good working memory for their questions because these questions are tightly tied to their biases and prior knowledge. For instance, children asked mostly feature-based questions. The fact that they asked mostly feature-based questions could mean that children at this age are biased to think about the world in terms of features, and thus retain feature-based questions in working memory. Indeed, children have stronger memory for information that is in line with their prior knowledge or biases e.g., .Despite this bias, however, children in Experiment 1 remembered an equally high proportion of category-based questions. This further supports the idea that these questions reflect how they think about the world: since children viewed the item again when recalling the questions, they may have recalled more feature-based questions. Instead, they also recalled most questions containing category-based information that was not apparent when looking at the item. Category-based questions may also require more cognitive effort to generate and remember since they are not in line with children’s bias to focus on features. This may explain their strong memory for these questions: previous research indicates that tasks requiring more cognitive effort may support memory for information . HoweverIn addition, we also found that children’s working memory for observations was similar to their memory for questions in Experiment 1. In other words, the children who failed to generate questions and instead made observations also remembered the majority of their observations. This finding suggests that children’s questions may be one of multiple factors that relate to both lower-order working memory and higher-order thinking such as categorization. Children’s observations may be an equally important factor and may also reflect children’s prior knowledge or biases. In short, children who cannot spontaneously generate questions when prompted may rely on their working memory for other sources, such as their self-generated observations, to make inferences during higher-order cognitive processes.Experiment 2 tested the possibility that the act of asking questions relates to inferences made during categorization. We found that children who asked more feature-based questions generalized more narrowly whereas children who asked more category-based questions generalized more broadly. Why did we find that children’s questions related to their categorization behavior? One potential explanation is that children have an underlying bias that leads children to ask the types of questions they do and categorize the way they do. This bias may be age-related in nature. For example, the younger children in this study may be more biased to focus on surface-level, feature-based information. This may lead them to ask questions about feature-related information. Indeed, research has found that children’s categorization behavior is initially based on perceptual similarity, then gradually changes across development to rely on more abstract category-based similarities as children get older . This deA second potential explanation for the results in Experiment 2 is that when children’s questions do not receive answers, they may be answering them mentally. That is, an underlying bias toward certain types of information may bias how children answer their own questions, and these internal answers may then impact their categorization behavior. For example, if a child is particularly interested in how and why objects look the way they do, this may lead them to ask a question such as “Do all wugs have orange feet?.” Then, they may mentally answer their question by deciding that all wugs must have feet that are the same shape and color. Thus, when they see a wug with different feet and are asked “Is this a wug or is this something else?,” they will use this internal answer to categorize all wugs with different feet as part of a different category. If this is true, then children’s question asking may not have a direct impact on their categorization; instead, children’s internally generated answers to their questions may be an intermediate step.As this work was a first step toward studying the act of question asking in the absence of answers, this study also reveals that there is still plenty we do not know about why children ask the questions they do. Specifically, this work was correlational in nature; we do not yet know whether a causal relation exists between children’s questions and their categorization behavior or the direction of this causal relation. Do children’s question themselves truly guide their thinking and behavior? Or do these questions instead reflect a general sense of how children categorize their world? That is, we do not know whether children’s questions change categorization behavior, or whether children’s categorization behavior changes their questions, whether this relation is bidirectional, or whether this relation is only correlational and not causal. Thus, a new direction for future research is to identify the single or multi-step mechanism(s) that explain relations between question asking and categorization. We predict that this relation is bidirectional and multi-step in nature. That is, children’s questions may impact their categorization behavior due to an underlying bias, and their categorization behavior reinforces what information they choose to focus on in subsequent trials.A final possibility is that the impact of children’s questions may vary based on the context in which they are asking the questions. For instance, there is evidence from previous work e.g.,  that theOn a related note, the social context in which children asked questions may have also influenced their inquiry behavior. First, children in both experiments were prompted by an experimenter to ask questions. We may have seen different results in a motivated-questioning paradigm, where children had the freedom to generate questions spontaneously, rather than in our forced-questioning paradigm. For example, children in a motivated-questioning paradigm may have chosen to ask different types of questions they were more interested in gaining information from other than the feature or category-based questions we observed. Second, the social dynamics of this forced-questioning paradigm may have influenced children’s question-asking. In both experiments, the experimenter made children ask questions and proceeded to not answer them. This dynamic is likely not the same context in which children’s questions typically remain unanswered; children often ask questions to parents or teachers who are facing distractions and thus cannot address each question. In this context, children may have perceived the experimenter as a poor conversational partner and wondered why the experimenter did not answer their questions despite appearing engaged with them. Therefore, children may have assumed that the experimenter was a poor informant and thus chose not to challenge them by asking simpler questions such as feature-based questions, rather than exerting more cognitive effort to ask diverse types of questions. Indeed, children adjust their inquiry behavior based on their perceptions of informants’ knowledge . As contA challenge for the field moving forward is to come up with ways to compare cognitive outcomes between questions with and without an answer. In this work, we chose not to include an answered questions condition because there are major theoretical and methodological concerns in doing so; namely, it is impossible to do so in an unconfounded way. For instance, it would be impossible to ensure that all children receive the exact same answers, as the questions asked will differ among children. As previous research suggests, these answers will likely guide children’s questions differently, making it more difficult to isolate the potential impact of questions themselves. Moreover, in a condition where children receive answers, we cannot determine whether the answer alone, the question alone, or an interaction between the question and answer contributed to children’s cognition. Researchers should not directly compare unanswered questions to answered questions until we more clearly understand interactions between the questions that are asked and the answers themselves.In sum, this study expands our knowledge of the role of children’s questions in cognitive development by demonstrating that children’s question asking relates to their working memory and categorization. While we know that children ask questions for information gain, this study shows that question-asking itself, in the absence of information gain, relates to children’s thinking. Indeed, future research should examine questions without answers to determine the mechanisms underlying question-asking, and whether these processes are causal in nature. This approach will allow the field to build a theory that precisely delineates the cognitive changes as a result of asking a question and the cognitive changes due to gaining information.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the University of Wisconsin-Madison Education and Social/Behavioral Sciences Institutional Review Board. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.EL and HV: study conception and design, analysis and interpretation of results, and draft manuscript preparation. EL: data collection. All authors reviewed the results, contributed to the article, and approved the submitted version.This work was supported by the Wisconsin Alumni Research Foundation, the Wisconsin Center for Education Research, and the National Science Foundation under grant #1561531.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.


Although there are several methods for fabricating nanofibrous scaffolds for biomedical applications, electrospinning is probably the most versatile and feasible process. Electrospinning enables the preparation of reproducible, homogeneous fibers from many types of polymers. In addition, implementation of this technique gives the possibility to fabricated polymer-based composite mats embroidered with manifold materials, such as graphene. Flake graphene and its derivatives represent an extremely promising material for imparting new, biomedically relevant properties, functions, and applications. Graphene oxide (GO) and reduced graphene oxide (rGO), among many extraordinary properties, confer antimicrobial properties of the resulting material. Moreover, graphene oxide and reduced graphene oxide promote the desired cellular response. Tissue engineering and regenerative medicine enable advanced treatments to regenerate damaged tissues and organs. This review provides a reliable summary of the recent scientific literature on the fabrication of nanofibers and their further modification with GO/rGO flakes for biomedical applications. The significant development of nanotechnology in the recent decades allows the design and preparation of fibrous nanomaterials with applications in many fields ,2—nanofiSeveral brilliant reviews concerning tissue engineering scaffolds have been published ,16, but Electrospinning is a feasible, low-cost and versatile method that allows the fabrication of nanofibers using different types of materials ,21,22,23Electrospinning is a well-known method of controllable fabrication of nanofibers that is driven by an external voltage ,27. The Individual polymeric fibers are formed and stretched under the influence of the electrostatic field acting between the needle and the collector. The solvent evaporates and the spliced fibers are expanded and then accumulated on the collector . The samGraphene (G), allotrope of sp2 hybridized carbon atoms with unusual electric , thermalThe most popular derivatives of flake graphene are GO and rGO . GO has V−1 s−1) ,48,49 duV−1 s−1) ,52. Prep×103 Ωm) ,54,55. M×103 Ωm) . GO pres×103 Ωm) . For exa×103 Ωm) ,59. A nu×103 Ωm) ,61. Stud×103 Ωm)  concludegraphene ). For exgraphene  and cemegraphene  can imprThe reduction process leading to synthesis of rGO from GO causes removal of most of the oxygen functional groups and can create some defects in the atom-thick structure of carbon atoms . The red2 S m−1)  and hydr2 S m−1) . Nonethe2 S m−1) ,72,73. A2 S m−1) .As this review is focused on the biomedical application of flake graphene, subsequent paragraphs will be devoted exclusively to flake graphene properties closely related with this subject.Escherichia coli (E. coli) [E. coli. Most of the cells lose their cellular integrity after contact with GO/rGO  ,79,80,81E. coli)  took inth GO/rGO . In an iia cells . Smalleropposite . Thus, fspension . In the t needed .A. niger, A. oryzae, F. oxysporu. After a 7-day incubation, growth of all fungi were completely stopped in solutions where rGO concentration was higher than 250 μg/mL [Candida albicans. This eukaryotic fungus is characterized by a cell structure and metabolism hard to suppress by any antimicrobial agent. In this study, the loss of viability increased with incubation time of the analyzed microorganisms. Results revealed that GO has antifungal activity against microorganisms used in this investigation. To summarize, the developed GO exhibited excellent antifungal properties. Sawangphurk and co-workers [Aspergillus oryzae, Aspergillus niger, Fusarium oxysporum. The authors highlighted that graphene (and its derivatives) is of interest due to its high surface area (about 2630 m2 g−1), high electrical conductivity (about 2000 S cm−1), high thermal conductivity (about 4840–5300 W m−1 K−1), and high Young’s modulus (about 10 TPa) what leads to various potential applications. Reduced graphene oxide (rGO) was produced with a modified Hummers method. The graphene was dispersed in agar and poured into sterilized Petri dishes. Agar discs were covered with fungal; next, discs were placed aseptically in the center of agar plates containing rGO nanosheets (0–500 μg mL−1). Experiments were performed for 7 days in five replicates. The average diameters of fungal colonies were determined. The growth inhibition of A. niger, F. oxysporum and A. oryzae was proportional to the concentrations of rGO flakes. The reason that the rGO flakes were effective to inhibit fungi was probably due to the direct contact with the cell walls of the analyzed fungi. Scientists reported [F. oxysporum, A. niger, and A. oryzae were 50, 100, and 100 μg mL−1, respectively. According to the results, the fungitoxicity of rGO against the analyzed pathogenic fungi might support the possibility of using rGO as an antifungal nanomaterial.Antifungal effects of rGO were studied using 50 μg/mL   affecting this phenomenon. All of this can be the result of GO structure and morphology. Researchers use graphite to produce graphene from various sources, thus leading to different properties of the final product. Moreover, there is no single method or strict conditions of GO and rGO synthesis followed by their purification. Thus, GO and rGO flakes studied over the world (even though named the same) should be considered as completely varied materials, characterized with different properties.P. aeruginosa) grown in liquid cultures. Results show that GO and rGO produced 3.8-fold and 2.7-fold higher levels of ROS, respectively, in comparison to the control trial. The presence of reduced GSH and NAC (N-Acetylcysteine) reduced elevated ROS generation. Moreover, when antioxidant is added to the cell culture, about 20% more cells of E. coli survived incubation with GO [All of the above also applies to studies about oxidative stress and its participation in antibacterial activity. This mechanism can be divided into two paths: ROS-dependent and ROS-independent. Oxidative stress caused by ROS  compared to scaffolds deprived of flake graphene, was observed. A key step towards biological assessment of resulting scaffolds was the cell culture including stem cells. The researchers observed significantly more cells adhered to scaffolds enhanced with rGO compared to the other scaffolds. However, the cells were less spread compared to those with GO and without the substances, according to Mahmoudifard and colleagues  were motLiu and his research team  producedStone, Lin and Mequanint  pointed A Taiwanese team of researchers  focused Girao and his team  decided A group of researchers from South China University of Technology  developeIn another paper, Wang and co-authors  producedIn a seminal paper written by Ma and coworkers , the res−5 S cm−1 in the dry state and peaked at about 3 × 10−4 S cm−1 in the hydrated state), electroconductive substrates were obtained that supported neurite growth without the need for external electrostimulation. The presence of GO/rGO did not significantly affect the fiber diameter (about 800 nm) distribution and porosity (70–80%). NG108-15 neuronal cells were able to adhere and grow on each of the fabricated materials but GO-modified materials showed markedly increased proliferation and metabolic activity. The percentage of live cells after 7 days of culture was higher than 95% for all scaffolds.Magaz et al.  conducteStaphylococcus aureus and gram-negative (−) Pseudomonas aeruginosa. Biological analysis showed that high viability and elevated levels of cell proliferation were recorded on the fibrous material designated by the researchers as CsA/PGT2, a nonwoven fabric with 0.25 percent rGO/TEPA composite. Moreover, all of the produced fibrous materials exhibited antimicrobial activity.Cojocaru et al.  started Staphylococcus aureus and E. coli bacteria. The fabricated fibrous materials were left in contact with the bacterial suspension for a limited time. After the necessary procedures, the number of bacteria present in the samples was recorded to determine the antibacterial efficacy of the fabricated materials. It was shown that after 2 h of contact with Ag/rGO-modified fiber materials, 99.55% and 99.46% of Staphylococcus aureus and E. coli bacteria were inactivated, respectively. It is theorized that graphene oxide acts as a nano-sharpener to damage the cell membrane of bacteria and causes bacterial death, and the rate of bacterial death depends on the structure of the cell wall [Pan and co-authors  fabricatell wall ,107.E. coli and Staphylococcus aureus bacteria. The antimicrobial activity was evaluated at time intervals. It was noted that there was a significant decrease in bacteria on PHA/rGO nonwoven fabric and PHA/rGO/Ag nonwoven fabric compared to nonwoven fabric without rGO or rGO/Ag modification. In conclusion, the researchers successfully fabricated PHA, PHA/rGO, and PHA/rGO/Ag nanocoating. The antibacterial properties of the electrospun scaffolds were tested. It was proved that the PHA/rGO/Ag nanofiber showed significant antimicrobial activity against PHA/rGO and PHA.Mukheem and his team  also worEsherichai coli and Staphylococcus aureus.Liu and co-workers  developeEschierichia coli and Staphylococcus aureus. Growth inhibition was noted for both E. coli and S. aureus, so CS/PVA/GO composite nonwovens can be considered as a promising material for skin regeneration applications.A team headed by Yang  has workE. coli bacteria. It was observed that the highest inhibition of colony growth occurred on the GO/Ag modified material. A higher number of bacteria was recorded on the GO-only- modified material than on GO/Ag, but the largest colony was observed on the SF/GT control sample. In conclusion, no antimicrobial effect was observed on the SF/GT material, lower E. coli counts were observed on the SF/GT/GO material. In contrast, the SF/GT/GO/Ag material showed clear antimicrobial properties and toxicity to E. coli. Zhang and his team  generateBased on all the above-mentioned literature and experimental results, it was possible to claim that graphene and derivatives positively affected the cellular response of several types of mammalian cells and exhibited excellent antibacterial and antifungal properties against microorganisms used in the investigations. In the The reason graphene was so effective regarding inhibition of bacteria and fungi was probably due to the direct contact with the cell walls of bacteria and fungi. This physical contact can be further translated to the release of the reactive oxygen-species, triggering graphene derivatives reaction with organic functional groups and polysaccharides on the cell walls of bacteria and fungi.In our opinion, the incorporation of graphene (and derivatives) into the volume of a 3D electrospun fibrous scaffold for regenerative medicine applications is a brilliant idea and the subject of many ongoing studies. Graphene is still a relatively new material, and a lot of research, observation, and clinical trials have to be conducted, but it is likely that its amazing potential will contribute to devising materials that will successfully replace native parts of the living organism.This review presents various concepts for modification of fibrous scaffolds fabricated by electrospinning technique for regenerative medicine and tissue engineering applications. Concepts for obtaining nanofibers from various polymers, which were further modified by GO/rGO addition, are described. It has been reported that studies by many teams of scientists from all over the world clearly indicate that modification of the composition of electrospun scaffolds by the addition of GO/rGO positively affects the cellular response of several types of mammalian cells and imparts antibacterial properties to the fibrous materials. However, some fundamental questions about graphene antimicrobial mechanisms and cytotoxicity of graphene-based nanomaterials, especially during long-term utilization still have to be addressed. The addition of flake graphene to the polymeric matrix of 3D fibrous tissue engineering scaffolds is a promising approach for altering surface properties of resulting materials, triggering desired cellular behavior. To the authors’ best knowledge, fully controllable introduction of flake graphene in regenerative medicine should be preceded by systematic and statistically significant screening of distinct kinds of graphene flakes. This in fact should be treated as an axiom, governing flake graphene fate. Is completely certain that the final effect of modification by GO/rGO depends on many component parts, including oxidation/reduction degree of graphene and its purity, method of graphene production or reducing agent used in the process. Moreover, the size distribution of graphene flakes can vary from a few nanometers to dozens of micrometers. Therefore, rigorous characterization of graphene is needed, before any further implementation is done. This is crucial if any definitive conclusions about graphene-based materials are to be drawn and correlated with antimicrobial properties of resulting materials.In summary, although according to analyzed papers modifications with GO/rGO greatly improve fibrous bioparameters, future studies need to focus on in vivo studies in order to introduce these materials to modern medicine. Furthermore, many of presented papers are focused on including graphene into polymer, and part of them involved modifications on surface polymer fibrous materials—more research needs to be done with both methods of modifications in the same conditions to compare which way of addition graphene is more effective and gives more desirable properties to scaffolds. In addition, antimicrobial and antiviral mechanisms of GO/rGO still require more research to achieve consensus among scientists. Despite those doubts, it is clear that GO/rGO modifications will play a significant role in the future of modified 3D scaffolds and tissue engineering.


Psychiatric emergency visits have been associated to several climate variables. However, the influence of relative humidity has been not well stablished.The analyse the influence of relative humidity on emergency care visits.Daily urgency visits were extracted from electronic medical records of Hospital Universitario La Paz from 1st January 2019 to 31st December 2019. Relative humidity data (%) was obtained from a local climate station. A negative binomial multivariate regression model was performed with relative humidity, weekday and month as covariates.Relative humidity was not associated with number of psychiatric emergency department visits Relative humidity did not influence emergency help seeking for patients suffering from suicidal phenomenaNo significant relationships.


P < 0.01). The findings indicate that the expression levels of BDNF and TrkB in the primary visual cortex of cats decrease with age, suggesting that the change of BDNF-TrkB signal pathway caused by the weakening of brain-derived neurotrophic factor activity may be one of the important reasons for the decline of visual function.To explore the expression of brain-derived neurotrophic factor (BDNF) and specific receptor tyrosine kinase receptor B (TrkB) in the primary visual cortex of young and old cats, especially to reveal the age-related differences in the mediating mechanism of BDNF-TrkB signaling pathway in cats’ visual cortex and their possible effects on synaptic plasticity, Nissl staining was used to display neurons in each layer of cats’ primary visual cortex, and immunohistochemical ABC method was used to label BDNF and TrkB immunopositive cells in each layer of cats’ primary visual cortex. The BDNF and TrkB receptor immunoreactive neurons and non-neurons were observed and photographed. Their density and immunoreactive intensity were measured. Results showed that BDNF and TrkB were widely expressed in all layers of visual cortex in young and old cats. Compared with the young group, the density and intensity of BDNF and TrkB positive cells in each layer of primary visual cortex in the old group decreased significantly ( The research on the neural mechanism of visual aging has always been one of the focuses of brain mechanism research. Brain-derived neurotrophic factor (BDNF) is expressed in the central nervous system and has the most content in mammalian brain. It is mainly distributed in key brain regions such as hippocampus and cortex, of which CA3 region of hippocampus is the most abundant. Tyrosine kinase receptor B (TrkB) is a membrane-bound receptor. BDNF can interact with high affinity receptor TrkB and play an important role in the nervous system. Studies have shown that BDNF and its downstream pathways play a variety of roles in the central nervous system and are related to the development of neurodegenerative diseases such as Alzheimer's disease(AD), Parkinson's syndrome(PD) and Huntington's disease(HD)5. The mechanism of BDNF and its specific receptor TrkB signaling pathway on brain functional activities has been noticed. It is found that BDNF-TrkB signaling pathway is an important regulatory factor involved in the mechanisms of neuronal development, survival, injury repair and synaptic plasticity of the nervous system8. Studies on the expression of BDNF and its receptors in mammalian visual system have been reported, such as the relationship between BDNF and visual deprivation patterns in the process of visual development plasticity; The key role of BDNF in synaptic reorganization of visual cortex; The regulatory effect of BDNF and its receptor TrkB on GABA (gama-aminnobutyric acid) ergic neurons during the critical period of visual development11. However, the expression and possible mechanism of BDNF and TrkB in mammalian primary visual cortex related to visual aging are rarely reported.Visual function is an important way for higher mammals to obtain external information. With the growth of age, visual function will also have a nonpathological declineIn this study, the primary visual cortex of cats was used as the research object because the evolution of cat's visual system is relatively advanced and has better similarity in organizational structure and system function compared with human beings. Therefore, cat is also a more common model animal for visual system related research. Nissl staining methods were used to show layering structure in the primary visual cortex. Immunohistochemical methods were used to compare the expression of BDNF and TrkB in the primary visual cortex of young and old cats, the experimental data results are observed and quantitatively compared to reveal the influencing factors and possible mechanism of visual function decline at the level of visual cortex.The ethics of this study was approved by Wuhu Institute of Technology Academic Committee. All experimental procedures were strictly performed in accordance with the ethical requirements of Wuhu Institute of Technology Academic Committee for the use of laboratory animals.Felis catus) were selected as experimental animals, with an individual weight of 2–3.5 kg . The experimental animals were divided into two groups according to their age: young group and old group. 4 cats (1–3 years old) were selected to form the young group and 4 cats (10–13 years old) were selected to form the old group. The experimental cats were purchased from the animal farm in Jiangning District, Nanjing, China (license No.SX1207). The cats were raised for a week before the experiment. During this period, they were given cage feeding, sufficient food and water and natural photoperiodic environment to eliminate the influence of artificial photoperiodic factors on visual function. The experimental cats underwent strict eye screening to eliminate the impact of eye diseases on the experimental results. Ketamine hydrochloride was injected at the weight of 40 mg / kg until the animals were deeply anesthetized. Cardiac perfusion was immediately performed on the animals . When pale liver was observed, inject 0.1 mol/l PBS  containing 10% formalin and 2.5% glutaraldehyde  to pre fix the tissue. The brain stem was removed by craniotomy and fixed in the above pre fixation solution for 2 h. The primary visual cortex was cut coronally, and the tissue block was transferred into 30% sucrose solution  till the tissue sank to the bottom. Every five continuous coronal frozen slices (50 μm) were separated from each other by an interval of three slices as a set. Every cat included 10 sets of slices. 80 sets of slices were numbered and marked respectively, according to the individuals in the young group and the old group. The five-slice sets were used for Nissl staining, BDNF and TrkB immunohistochemistry labels and negative controls.8 Cats  and Rabbit-anti-human TrkB  primary antibody  at 4 °C, incubated in secondary antibody (goat-anti-rabbit IgG working solution) at room temperature, incubated in SABC(strept avidin–biotin complex) reagents  at room temperature, developed by DAB(Diaminobenzidine) to produce colorimetric end products, dehydrated by gradient  alcohol, transparentized by xylene and then sealed with neutral gum. The only difference in the treatment of the negative control slices was replacing primary antibodies with PBS. All immunohistochemistry kits and the DAB substrate were products of Boster, Wuhan, China.The frozen sections were incubated in 3% H2) and the intensity of immune-reactions . The average absorbance value is equal to the sum integral optical density divided by the unit area, where higher absorbance values indicated stronger immuno-reactions.40 sets of slices were selected from young cats (20 sets) and old cats (20 sets) . Nissl-stained, BDNF and TrkB immune-reactive slices were selected and observed under Olympus bx-51 microscope from the young and old groups. The images were collected by Image-Pro express 6.0 software. All the relevant morphological parameters were quantitatively analyzed. The density of BDNF and TrkB immune-reactive cells . The standard for labeling a BDNF or TrkB immunopositive cell is that the cell body contains obvious immuno-positive matter and a clear nucleus in soma. After collecting images from 20 randomly selected visual fields of each immune reaction slice, the average absorbance value is measured with the help of Image-Pro express 6.0 software, and the mean value is taken as the index indicating the intensity of immuno-reactions. The observer and counter were unaware of the experimental grouping and treatment to ensure data analyses were performed unbiased.Nissl-stained, BDNF and TrkB immuno-reactive slices were placed at low ocular magnification (40 ×) to obtain the effective image. And then adjust the microscope to ocular magnification (100 ×) to acquire the images of the layering structures. And finally adjust the microscope to high ocular magnification (400 ×), select 10 visual fields (50 μm × 50 μm) of each layer (refer to the layering structure shown in Nissl-stained slices in the same group) to count the number of BDNF and TrkB immunopositive cells and calculate their density  method was used for multiple comparisons among means.The collected data were analyzed via SPSS 13.0 software and expressed as means ± The study was carried out in compliance with the ARRIVE guidelinesA six-layer-structure of the primary visual cortex  was shown in the Nissl-stained slices. The hierarchical structure of the primary visual cortex and Nissl-body are clear, the morphology and size of neurons are different, and the cell bodies and processes were dyed light blue, blue or blue purple in each layer  Fig. : A, B.FiThe brown or dark brown somas of BDNF and TrkB immuno-positive cells and their fiber distributions can be seen in each layer of the primary visual cortex of young and old cats  = 32.02, P < 0.01), whereas, those between layer I and IV were significantly different, and those among layer II-III, V and VI were comparable . However, although no differences were found in the densities of BDNF immuno-positive cells in each layer among cats at similar ages  = 1.92, P = 0.13), those in old cats were significantly lower than those in young cats  = 75.43, P < 0.01)  = 48.13, P < 0.01), whereas, those between layer I and IV were significantly different, and those among layer II-III, V and VI were comparable . However, although no differences were found in the densities of TrkB immuno-positive cells in each layer among cats at similar ages  = 1.86, P = 0.14), those in old cats were significantly lower than those in young cats  = 114.22, P < 0.01)  = 58.26, F = 42.14, P < 0.01, Table Compared with young cats, the average absorbance value of BDNF and TrkB immunoreactivity in old cats decreased significantly  in the brain21.The immunolabeling results of the study systematically reflected the age-related expression differences of BDNF and TrkB in neurons and glial cells in cats’ primary visual cortex. BDNF and TrkB were expressed in all layers of cats’ primary visual cortex, and the expression of BDNF and TrkB in the old group was lower than that in the young group. Indeed, in our experiment, while neurons were specifically immunolabeled, some glial cells were also immunolabeled. It has been reported that glial cells not only support neuronal activity, but also participate in the regulation of neural network plasticity. Astrocytes, microglia and oligodendrocytes play complex and important roles in synaptic plasticity. More and more studies have shown that it is closely related to the occurrence of a variety of neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, multiple sclerosis and so on23. Studies indicate that BDNF plays an important role in nutritional support for the survival and functional expression of GABAergic neurons27. Previous reports showed that there was no significant change in the total number of neurons in each layer of cats’ primary visual cortex in the elderly group compared with the young group, and the number of GABA positive cells in cats’ primary visual cortex decreased with age28. Based on these results, we speculate that the decline of visual function in the visual cortex may not be caused by the decrease of the number of neurons, but more likely by the decline of synaptic plasticity of special neurons mediated by neurotrophic factors. The insufficient inhibitory regulation of GABAergic neurons in primary visual cortex may be the result of the declining synaptic connection mediated by BDNF—TrkB signaling pathway. The possible influence mechanism tends to be like this, the insufficient secretion of BDNF and TrkB in elderly individuals can cause the decline of the morphological and functional stability of inhibitory synaptic connections, which will result in a visual recession characterised by the loss of final visual information transmission.This result is consistent with the down-regulation of BDNF aging expression in the serum of patients’ with Alzheimer's disease and the aging changes of BDNF and TrkB in various layers of the lateral geniculate nucleus of catsIn summary, the expression of BDNF and TrkB in cats’ primary visual cortex decreased with age, suggesting that the change of BDNF-TrkB signal pathway  caused by the decline of brain-derived neurotrophic factors’ activity may be one of the important causes of visual recession. We look forward to verification in further morphological and electrophysiological experiments.


Background: This study evaluated the feasibility of reducing the radiation dose in abdominal imaging of urolithiasis with a clinical photon-counting CT (PCCT) by gradually lowering the image quality level (IQL) without compromising the image quality and diagnostic value. Methods: Ninety-eight PCCT examinations using either IQL70 (n = 31), IQL60 (n = 31) or IQL50 (n = 36) were retrospectively included. Parameters for the radiation dose and the quantitative image quality were analyzed. Qualitative image quality, presence of urolithiasis and diagnostic confidence were rated. Results: Lowering the IQL from 70 to 50 led to a significant decrease (22.8%) in the size-specific dose estimate . Simultaneously, lowering the IQL led to a minimal deterioration of the quantitative quality, e.g., image noise increased from 9.13 ± 1.99 (IQL70) to 9.91 ± 1.77 . Radiologists did not notice major changes in the image quality throughout the IQLs. Detection rates of urolithiasis (91.3–100%) did not differ markedly. Diagnostic confidence was high and not influenced by the IQL. Conclusions: Adjusting the PCCT scan protocol by lowering the IQL can significantly reduce the radiation dose without significant impairment of the image quality. The detection rate and diagnostic confidence are not impaired by using an ultra-low-dose PCCT scan protocol. Kidney and ureter stones can cause severe clinical symptoms leading to hospitalization, renal impairment and death. Non-contrast CT scans are usually the preferred cross-sectional imaging method for the detection of calculi occluding the urinary tract. The advantages of non-contrast CT scans are the high sensitivity and specificity for the detection of urolithiasis ,2. A majOverall, the prevalence as well as the incidence of urolithiasis have increased over the past few decades ,4,5. AltVarious techniques to reduce radiation doses have been employed and different low-dose CT scan protocols have been successfully established in clinical routine; e.g., CT protocols with reduced tube charge current, increased pitch factor, additional tin filter or iterative reconstruction algorithms ,12,13,14An entirely new technology designed to reduce the radiation dosage was recently introduced with the first clinical photon-counting CT (PCCT) scanner. These CT scanners make use of a newly developed photon-counting detector (PCD) that consists of a single thick layer of a semiconductor material. Unlike conventional energy-integrating detectors (EID), PCDs convert incoming X-rays directly into an electrical signal and do not require additional conversion steps from X-ray to visible light and from visible light to an electrical signal ,16.An initial study compared the radiation dose parameters as well as the image quality of the novel PCCT and a conventional EID CT when using the clinical scan protocol of each CT scanner for the detection of urolithiasis. It showed that the PCCT required a significantly lower radiation dose (approximately 30%) to provide significantly better image quality, thus leading to an excellent detection rate of kidney and ureter stones as well as to a high diagnostic confidence of the radiologists evaluating the CT scans .The purpose of the present study is to evaluate the feasibility of reducing the radiation dose of the low-dose scan protocol for the detection of urolithiasis by gradually lowering the image quality level (IQL). The impact on image quality, diagnostic performance and diagnostic confidence as well as the influence on measurements of the size of ureter stones of the reduced radiation dose protocols are evaluated.This study was approved by the institutional review board. Patient consent was waived due to the retrospective study design. In total, low-dose CT examinations of 98 consecutive patients with clinical suspicion of urolithiasis were retrospectively included in this study. The CT scans were performed between September 2021 and March 2022. Patients included in this study were not preselected .All CT examinations were performed in a supine position. Scan parameters for all examinations with the PCCT  were as follows: tube voltage 100 kVp, tin filter, detector configuration 144 × 0.4 mm, pitch 0.6 and gantry rotation time 0.5 s.All CT scan protocols compared in this study used automatic tube current modulation techniques, but at different dose levels, which was achieved by adjusting the so-called “image quality level” (IQL) parameter. The IQL is a parameter developed to provide a CT system independent definition of image quality. It represents quality reference milliampere-seconds (mAs)—a previously defined parameter—denoting the effective mAs applied to the patient based on protocol- and patient-specific attenuation information to produce a desired image quality—additionally being corrected according to detector and CT geometry-specific properties. The IQL was initially set at level 70 (N = 31 CT scans), then lowered to level 60 (N = 31 CT scans) and ultimately lowered to level 50 (N = 36 CT scans).The reconstruction parameters were as follows: slice thickness 2 mm, slice increment 1 mm, image matrix 512 × 512, soft tissue kernel (Br36) and quantum iterative reconstruction (QIR) level Q4.The analysis included the following parameters: Computed tomography dose index (CTDI), dose length product (DLP), effective dose  and the size-specific dose estimate (SSDE) . SubgrouRegions of interest (ROI), constant in size (diameter 1 cm) on all images, were drawn in the subcutaneous fat and the paravertebral muscle. Image noise was defined as standard deviation (SD) of the mean density  of each ROI. Signal-to-noise ratio (SNR) was defined as mean density (HU) divided by the SD of each ROI.CT images were evaluated independently by 3 radiologists with at least 2 years of experience. Overall image quality, image noise and image sharpness were rated on a 5-point Likert scale . In addition, the radiologists screened each CT scan for the presence of kidney or ureter stones and rated their diagnostic confidence on a 5-point Likert scale .While evaluating, readers were not aware of the CT scan protocol that was used to acquire the particular CT images. Cases were presented in a randomized order. Ground truth was based on the radiology report and expert consensus reading in discordant cases.In total, the largest axial diameter of 45 ureter stones (15 ureter stones on each IQL) was measured independently by 6 radiologists using the exact same image settings . The interreader reliability was calculated for each IQL.Statistical analysis was performed with established software packages , R: A language and environment for statistical computing, R Foundation for Statistical Computing, Vienna, Austria, RStudio Version 1.4.1106). If not stated otherwise, all data are presented as mean ± SD.W) was calculated to assess the interreader reliability of the qualitative image analysis. Two-way mixed intra-class correlation coefficient was calculated to assess interreader reliability of stone size measurements for the different dose groups. Differences in terms of detection rates were tested with the Fisher’s exact test for statistical significance. p-values < 0.05 were considered indicative of statistical significance.Differences between groups with continuous variables were evaluated using a one-way ANOVA. In case significant differences were found, pair-wise Bonferroni post hoc tests were performed. For ordinal variables and subgroup analysis , non-parametric statistics were used. Kruskal–Wallis test by ranks was used to test for differences between groups, and in case of significant differences, post hoc pair-wise Mann–Whitney test with Bonferroni correction was used. Kendall’s Coefficient of Concordance  were examined using the IQL 70. Likewise, thirty-one patients  were examined using the IQL 60. Thirty-six patients  were examined using the IQL 50. The distribution of the patients in terms of gender and BMI is shown in vol, DLP, ED and SSDE are shown in CTDIp < 0.001). In addition, when considering each individual subgroup, there was a significant reduction in the SSDE between IQL 70 and IQL 50 .In total, lowering the IQL from 70 to 50 led to a significant decrease (22.8%) of the SSDE . The difference in ED between IQL 60 (2.02 ± 0.67 mSv) and IQL 50 , however, is comparatively small. Similar to the SSDE, the greatest difference in ED between IQL 70 and IQL 50 was observed among patients with a BMI > 30 kg/m2 (IQL 70: 3.33 ± 1.36 mSv vs. IQL 50: 2.54 ± 0.76 mSv).In total, the ED decreased by about 18.9% between IQL 70 (2.43 ± 0.99 mSv) and IQL 50  are shown in p = 0.248). The increase of the image noise was not statistically significant. At the same time, the SNR decreased in muscular tissue as well as in subcutaneous fat. The decrease in the SNR in muscular tissue was statistically significant .Lowering the IQL from 70 to 50 led to a minimal increase in the image noise in muscular as well as in adipose tissue  or IQLs 70 and 60 (p = 0.511). The difference in detection rates of IQLs 60 and 50 was statistically significant (p = 0.028).Taking all CT scans using the IQL 70 into account, there were three false negative and three false positive findings among all three radiologists, which translates to a detection rate of 94.7%. The readings of CT scans using the IQL 60 contained six false negative and two false positive findings, leading to a detection rate of 91.3%. When considering the CT scans acquired with the IQL 50, all three radiologists had a detection rate of 100% with no false negative and altogether three false positives. There were no significant differences between detection rates when comparing IQLs 70 and 50 (p = 0.653) between IQL 70 (mean 4.44 ± 0.91), IQL 60 (mean 4.48 ± 0.94) and IQL 50 (mean 4.47 ± 0.83). Likewise, the overall image quality, image noise and image sharpness, as rated by the radiologists, only showed minor differences between IQLs 70, 60 and 50 that were not statistically significant; e.g., the mean rating of the overall image quality was 4.19 ± 0.86 on IQL 70, 4.28 ± 0.94 on IQL 60 and 4.07 ± 1.00 on IQL 50 (p = 0.311). The interreader reliability of the qualitative image analysis was high for all criteria .The diagnostic confidence reported by the radiologists was at a very high level and did not differ significantly  were measured independently by six radiologists (R1–R6) using the exact same image settings  was still markedly higher compared to the other patient groups . When looking at the subgroups including only patients with a BMI < 30 kg/m2, there is, as expected, a steady decrease in the ED from 2.12 ± 0.58 mSv on IQL 70 to 1.65 ± 0.46 mSv on IQL 50 (p = 0.023).Overall, lowering the IQL led to significantly lower radiation doses. However, certain aspects need to be taken into account when considering individual parameters. When looking at the EDs of all patients, it was noticeable that there were only small differences between the individual IQLs; e.g., 0.05 mSv difference between IQL 60 and IQL 50. This is due to the fact that the calculation of the ED does not consider the patients´ body sizes. Although there were no significant differences in terms of BMI between the patient groups, the average BMI of the patients examined with IQL 50 (BMI 28.85 ± 6.19 kg/mp < 0.001). Simultaneously, significant differences in the SSDE were found when comparing each individual subgroup.Due to the differences in mean BMI between the patient groups, the SSDE that additionally considers the body diameter is possibly more reasonable for quantifying the reduction in radiation doses between IQLs. The SSDE decreased steadily and significantly from 4.57 ± 0.84 mGy on IQL 70 to 3.53 ± 0.70 mGy on IQL 50  may even be considered as “ultra-low dose”.The comparison of the radiation doses described in the present study with radiation doses of previous studies is difficult due to differing scan protocols. However, the definitions for the terms “low dose” and “ultra-low dose” described in the literature can possibly serve as a guide. Rob et al., for example, reporting on a systematic review about non-contrast CT scans for the detection of urolithiasis, defined EDs of ≤3.5 mSv as “low dose” and ≤1.9 mSv as “ultra low dose” . Other sThe qualitative as well as the quantitative image quality assessment yielded similar results. Lowering the IQL from 70 to 50 led to a slight increase in the image noise and a slight decrease in the SNR. However, these changes were minimal and in most cases statistically not significant. Likewise, the radiologists evaluating the CT images did not notice any major changes in the image quality throughout the IQLs. The detection rate of kidney and ureter stones did not differ markedly between the IQLs and was highest on the lowest IQL. Diagnostic confidence as rated by the radiologists was high and not influenced by the IQL. Furthermore, lowering the IQL did not influence the measurements of the size of ureter stones. The intra-class correlation coefficients were excellent throughout all IQLs.The present study has certain limitations. Although there were no significant differences, the mean BMI of some patient groups differed markedly, which influenced the applied radiation doses. Forming subgroups and calculating the SSDE can help to overcome this issue. However, this in turn means that the number of patients in some subgroups is relatively low. Thus, no general conclusions about the potential to reduce radiation doses in obese patients can be drawn.In conclusion, adjusting the PCCT scan protocol by lowering the IQL can significantly reduce the applied radiation dose for patients without significant impairment of the image quality. The detection rate as well as diagnostic confidence are not negatively influenced by using an (ultra-) low-dose PCCT scan protocol. The results indicate the potential for further adjustments of the clinical scan protocol aiming at a further reduction of the radiation dose.