To reduce the structural deterioration of mass concrete structures from temperature cracks, and lower energy consumption caused by the traditional mass concrete hydration heat cooling process, this paper reports the preparation of concrete temperature-controlled phase change aggregate (PCA) by a vacuum compaction method using light and high-strength black ceramite and No. 58 fully refined paraffin wax as phase change material (PCM), and the encapsulation technology of the aggregate by using superfine cement and epoxy resin. Further, through laboratory tests, the cylinder compressive strength, thermal stability and mixing breakage rate of the encapsulated PCA were tested, and the differences in mechanical properties such as compressive strength, flexural strength and splitting tensile strength between phase change aggregate concrete (PCAC) and ordinary concrete were studied. A test method was designed to test the heat storage effect of PCA, and the temperature control effect of PCAC was analyzed based on the law of conservation of energy. The research conclusions are as follows: (1) Both superfine cement and epoxy resin shells increase the strength of the aggregate, with the epoxy resin increasing it more than the superfine cement. The thermal stabilization of the PCA is good after encapsulation of superfine cement and epoxy resin. However, PCA encapsulated in superfine cement is more easily crushed than that encapsulated in epoxy resin. (2) Under the condition of water bath heating and semi-insulation, when the water bath temperature reaches 85 °C, the temperature difference between the PCA and the common stone aggregate can be up to 6 °C. Based on the law of energy conservation, the test results will be converted to mass concrete with the same volume of aggregate mixture;, the difference of PCAC and ordinary concrete temperature can be up to 10 °C, so the temperature control effect is significant. (3) The mechanical properties of PCAC with 100% aggregate replacement rate compared to ordinary concrete are reduced to varying degrees, and the performance decline of the epoxy-encapsulated PCA is smaller than that encapsulated with superfine cement; in an actual project, it is possible to improve the concrete grade to make up for this defect. With the development of human society, global resource and energy scarcity have become a challenge that mankind has to face. Civil engineering construction is an area of great resource and energy consumption. Infrastructure construction and maintenance consume resources and energy and cause pollution. It is estimated that about 70% of global greenhouse gas emissions come from infrastructure construction and operation . TherefoMass concrete is widely used in large modern buildings due to its excellent integrity and stability ,4,5,6. HIn recent years, the use of PCM to control the heat of hydration of mass concrete is a new temperature control method. The mechanism of this technique is to reduce the temperature difference between the inside and outside of the mass concrete by absorbing the heat of hydration and slowing down the cooling rate of the mass concrete through the phase change of the material, thus reducing the internal temperature stress of the mass concrete . PCM canThe idea of using lightweight aggregate to adsorb PCM comes from mixing water stored in PCA into concrete to prevent its self-shrinkage ,10,11, aCompared with the embedded pipe method, the use of PCA can better disperse the PCM in every corner of the concrete, and the temperature control effect is more uniform. However, relevant studies have shown that if the PCA adsorbed with the PCM is directly added to the concrete, the mechanical properties of the concrete will be sharply reduced. The main reason is that the phase change material flows out of the aggregate after the phase change ,41, and Phase change temperature control technology has the advantages of a uniform temperature control effect and little influence from construction factors, which provides a new solution for mass concrete hydration heat temperature control. Now, the technology of controlling the hydration heat of mass concrete with PCM is still in its infancy and has not been widely used in practical engineering. Based on the hydration heat control technology of mass concrete, this study prepared a structure-function integrated PCA, carried out its performance test, mixed the PCA into the concrete to study the physical and mechanical properties of the PCAC and finally designed a heat storage test method to study the heat storage effect of the PCAC. It will provide a valuable reference for the popularization and application of PCAC in practical engineering.In this study, lightweight ceramic granules are selected as the aggregate. Light-weight ceramic granules are porous inside, have high air permeability, water permeability and high strength. Its main performance indexes are shown in The PCM used in this study is No. 58 fully refined paraffin, which is a white block, and the purity of the paraffin used is high. The basic performance indexes of paraffin are shown in To avoid the liquid paraffin flowing out of the PCA after the temperature of the concrete increases, it is necessary to conduct a shell-forming encapsulation of the surface of the PCA, and wrap the PCM inside the PCA. In this study, superfine cement and epoxy resin were selected to encapsulate the PCA.This study selected 800 mesh superfine cement and its mechanical performance indicators are shown in In order to obtain the optimal water–cement ratio, superfine cement slurries with water–cement ratios of 0.32, 0.31, 0.30, 0.29 and 0.28 were configured, and the PCA encapsulation test was carried out by using cement slurries with each water–cement ratio. It was found that when the water–cement ratio was less than 0.29, superfine cement can completely coat the aggregate, as is shown in The second packaging material for the phase change aggregate used in this study is an epoxy resin, which is composed of two component raw materials  prepared, stirred, reacted and solidified in a certain ratio. This study used bisphenol A-type epoxy resin, which was material A  and material B (curing agent) in a 2:1 ratio. After multiple indoor adjustments, the model of material A was determined to be WSR6101 (E44). The material is colorless and transparent, and its performance indexes are shown in Methods of viscosity measurement To ensure the encapsulation quality of epoxy resin mixtures on aggregates, the following three necessary conditions must be met: (A) appropriate viscosity, (B) the operational time is longer than 20 min and (C) excellent thermal conductivity. Immerse the PCA into the epoxy resin to evenly adhere to its surface; the epoxy resin shell should completely cover the PCA, with a very smooth surface, uniform thickness and a light-yellow transparent shape. According to 47–2008)  in the ty = 0.0202x + 0.2672, 2R = 0.94, where y is the thermal conductivity, x is the graphite powder content, and R is the correlation coefficient. To increase the thermal conductivity of epoxy resin, a 15% graphite powder mixed with epoxy resin was used to shell the PCA. The aggregate with epoxy resin was wrapped in cement powder to increase the bonding effect between the aggregate and concrete. The indoor shell material display is shown in Shazim et al. ,42 showeSelf-made equipment was used to prepare PCA indoors, as shown in Newly purchased ceramsites are not completely dry. Therefore, it is necessary to dry the ceramsites and remove surface shells from the dried ceramsites through a sieve shaker. Place the paraffin crystals into a vacuum bucket and melt them using a hot water bath, add ceramsites to the molten paraffin and start vacuum adsorption. All ceramsites must be 2 cm below the molten paraffin liquid level. The vacuum adsorption of PCM lasts for 60 min. After the adsorption is completed, remove the vacuum equipment, lift the drain basket and quickly pour the ceramsites that adsorb paraffin into the water-cooled bucket to avoid liquid paraffin flowing out of the ceramsites as much as possible. After water cooling, there is a trace of water on the surface of the PCA, which needs to be dried in a natural environment to evaporate surface moisture.The encapsulation operation of PCA using superfine cement slurry and epoxy resin is similar. The PCA granules are immersed in a freshly mixed superfine cement slurry/epoxy resin, and then thoroughly stirred to ensure that enough encapsulation material adheres to the surface of the PCA. Finally, the PCA granules are dried and formed. If there are still defects in the encapsulation effect, the phase change aggregate can be re-encapsulated. The preparation and performance test flow charts of PCA and PCAC are shown in Conduct cylindrical compress strength tests on ceramsites, unencapsulated PCA, superfine cement-encapsulated PCA and epoxy resin-encapsulated PCA, according to the lightweight aggregates and its test methods according to the Chinese Standard 31–2010) . The cylaf is the compressive strength of the coarse aggregate cylinder, and the unit is MPa. p1 is the pressure value when the pressing depth is 20 mm, the unit is Newton (N); p2 is the mass of the stamping cylinder, the unit is Newton (N), and the mass of the stamping cylinder in this study is 1.85 kg; F is the pressure area . The cylinder pressure strength is the mean value of the three measurements. If the difference between the maximum value and the minimum value of the three measurements is only greater than 15% of the mean value, the sample should be retested.Load at a constant speed of 300~500 N, and record the pressure value when the stamping depth is 20 mm. The cylinder pressure strength of each material is calculated according to the formula:To study the defects caused by the collision of PCA with other coarse aggregates, the PCA granules that have completed the shell-making process and coarse aggregates are directly added to the forced mixer for mixing to simulate the most severe environment of PCA during the mixing process. Mix 100 superfine cement-encapsulated PCA slurry with 20 kg coarse aggregates and place them in a forced mixer for mixing. The mixing time is 240 s, which meets the requirements of the Standard for quality control of concrete 64–2011) . The samThe phase-change temperature and latent heat of paraffin and PCA were measured by differential scanning calorimetry (DSC). DSC is to measure the change of the energy difference between the sample and the reference sample with temperature and time under the control of the setting temperature. A total of four sets of samples were selected for testing in this study, including two sets of paraffin samples and two sets of PCA samples. The four groups of samples are put into the DSC equipment for heating and cooling tests at 80 °C.To investigate the potential flow of PCM through micropores in the shell of PCA after complete melting, a thermal stability test was conducted using a heating-diffusion-exudation cycle method in this study. The following steps were taken during testing:(1)Draw a circle with a diameter of 30 mm in the center of the filter paper, and place the PCA in the drawing area, as shown in (2)Place the PCAs to be tested in the electric blast drying oven and heat then at 100 °C for 24 h. The unencapsulated PCA sample is placed on the top layer of the oven, the PCA encapsulated in superfine cement is in the middle layer and the PCA encapsulated in epoxy resin is placed on the bottom layer, as is shown in (3)Observe the diffusion degree of the PCM on the filter paper, then measure the maximum diameter (4)Calculate the percentage of exudation To investigate the thermal storage effect of PCA and compare it with the temperature control effect of ordinary crushed stone, three parallel experiments were conducted in this study, which were full PCA, half PCA–half stone group and full stone group. To ensure the heating amount and heat dissipation boundary are consistent for the three groups of experiments at the same time, this experiment used self-made water bath heating equipment. Add 500 mL EPC insulation particles at the top of each water bath liquid level to provide top insulation, as shown in Based on the equal volume method, the PCA granules prepared above were used to replace the coarse aggregates in concrete, and a total of four sets of mixed proportions of concrete were designed. They are the experimental control group with a replacement rate of 0% for coarse aggregates (S1), the unencapsulated PCA group with 100% replacement for the coarse aggregates group (W1), the superfine cement-encapsulated PCA group with 100% replacement for coarse aggregates group (C1), and the epoxy resin-encapsulated PCA group with 100% replacement for the coarse aggregate group (H1). The specific mix proportions are shown in According to the Standard for test methods of concrete physical and mechanical properties 81–2019) , experimThe results of the phase-change aggregates cylindrical compressive strength test are shown in The failure mode of the epoxy resin-encapsulated PCA a and supAfter stirring in a forced mixer for 240 s, separate the PCA from the coarse aggregates, observe the fragmentation of PCA shells which were made of superfine cement and epoxy resin and calculate the shell crushing rate. The calculation results are shown in The DSC curves of paraffin melting and crystallization are shown in The initial stage of temperature rise section (stage I): During this stage, the temperature rise curves of the three samples are consistent, and there is no deviation of the temperature curve. Based on the DSC curves of the PCA mentioned above, it can be seen that the initial phase change temperature of the PCM in the PCA is about 50 °C. Therefore, the main reason for the lack of deviation of the temperature curve at this stage is that the melting phase change of the PCM in the PCA has not occurred.Melting phase change zone (section ①): As shown in Phase change temperature rise section (stage II): After passing through the melting phase change zone, the temperature curves of various groups continue to deviate. As the temperature rises, the temperature will continue to increase according to the deviation curve, and the degree of deviation will increase with the increase in temperature.Temperature peak difference zone (section ②): Observe the peak temperature of the full stone group. When the peak temperature of the full stone group reaches 85 °C, stop heating and turn off the heater power. In this zone, it can be clearly observed that the full stone group has the highest peak temperature, followed by the peak temperature of the half stone–half PCA group, and the full PCA group has the lowest peak temperature. The three temperature peaks are 85.37 °C, 83.92 °C and 80.21 °C, respectively, indicating that phase change materials have a significant effect on temperature control. Moreover, the full stone group reached the peak temperature 6 min earlier than that of the full PCA group, which also indicates phase change aggregate has a delay effect in temperature control.Amorphous temperature drop section (stage III): At this stage, the PCM inside the PCA is in a liquid melting state, and there is no significant deviation in the temperature drop curves of the three groups.Crystallization phase change initiation zone (section ③): As the temperature decreases, when the initial crystallization temperature of the PCM inside the PCA is reached, the PCM begins to transform from liquid to solid, and the PCM will release heat. Compared with the DSC curves of phase change aggregates, the initial crystallization temperature of the phase change aggregates is about 53 °C. The results of this experiment show that the initial crystallization phase change zone occurs at around 57 °C.Crystallization phase change section (stage IV): During the cooling stage, the phase change materials in the phase change aggregates gradually begin to crystallize and release heat. With the increase in time, the temperature drop curves of the three sets of tests finally overlap.By summarizing the effect of PCA heat storage performance on temperature control in literature ,35,39,42As the curing age of concrete increases, the compressive strength of both PCAC and ordinary crushed stone concrete increases. However, the reduction percentage between the compressive strength of PCAC and that of ordinary crushed stone concrete varies less with the increase in curing time. For unencapsulated PCAC, the compressive strength of the concrete compared to ordinary crushed stone concrete at 3d, 7d and 28d is reduced by 52.5%, 51.72% and 46.2%. The compressive strength is reduced by 34.4%, 35% and 30.0% for superfine cement-encapsulated PCAC. The compressive strength is reduced by 15.6%, 20.5% and 19.2% for epoxy resin-encapsulated PCAC. In addition, the strength growth rate of ordinary concrete in the early stage is faster than that of PCAC, and there is little difference in the later stage.The mechanical properties of PCAC are summarized in In practical engineering, the defect of strength reduction caused by phase change aggregate can be made up by appropriate improvement of concrete grade, because the cost increase caused by appropriate improvement of concrete grade is far smaller than that caused by concrete cracking due to hydration heat.Considering factors such as site constraints in the laboratory, it is difficult to place mass concrete in the laboratory. Therefore, this paper designs an equivalent water bath heating test, based on the law of conservation of energy, to equivalently replace the heat of PCA in a water bath environment into concrete, so as to investigate the effect of heat storage of phase change aggregates on the temperature control of the hydration reaction of mass concrete.PQ is the heat storage of phase change aggregate, wc is the specific heat capacity of water, wm is the quality of test water, t2 is the water temperature when adding phase change aggregates and t1 is the water temperature when adding crushed stone.The heat storage energy of phase change aggregates can be calculated according to Equation (4):The heat storage capacity of phase change aggregate per unit mass can be calculated according to Equation (5):m can be calculated by Equation (6):The heat storage capacity of mass concrete with a phase change aggregate content of The relationship between the heat storage of phase change aggregates in phase change concrete and temperature is:The specific heat capacity of phase change concrete is:Pm is the quality of phase change aggregate added in the experiment, T2 is phase change concrete temperature, T1 is the temperature of ordinary crushed stone concrete, pcc is the cement specific heat capacity, cn is the ratio of cement mass to concrete mass, psc is the heat capacity of fine aggregate, sn is the ratio of fine aggregate mass to concrete mass, pwc is the specific heat capacity of water, wn is the ratio of water mass to concrete mass, pbc is the specific heat capacity of phase change material and bn is the ratio of phase change material mass to concrete mass.From Equations (4)–(8), it can be found that:Based on the above derivation, the experimental results are converted into the temperature curve of phase change concrete and ordinary crushed stone concrete at the same volume of phase change aggregate. The conversion relationship is based on Equation (8), and the conversion parameters are shown in (1)Both superfine cement and epoxy resin can be used as preferred materials for PCA encapsulation and shell making. When encapsulating PCA with epoxy resin, graphite powder can be added to the epoxy resin slurry to increase its thermal conductivity.(2)Superfine cement shell and epoxy resin shell can increase aggregate strength, with epoxy resin having a better effect than superfine cement. The PCAs encapsulated in superfine cement and in epoxy resin both have good thermal stability.(3)For the concrete at 3d, 7d and 28d, the mechanical properties of concrete mixed with PCA are reduced to varying degrees compared to ordinary crushed stone concrete.(4)When the water bath temperature reaches 85 °C, the temperature difference between the PCA and the common stone aggregate can be up to 6 °C, and based on the law of conservation of energy, the test results will be converted to mass concrete with the same volume of the aggregate mixture, The difference of PCAC and ordinary concrete temperature can be up to 10 °C, and the temperature control effect is significant.This research prepares PCA, and selects different encapsulating materials to encapsulate the prepared PCA. Research was conducted on the mechanical properties of the PCA and the thermal storage effect of PCAC. The conclusions are as follows:


The subjective experience of time can be influenced by various factors including voluntary actions. In our previous study, we found that the subjective time experience of an action outcome can be compressed when an individual performs a continuous action compared to a single action, suggesting that the sense of agency (SoA), the feeling of control over one’s own action outcomes, contributes to the subjective time compression. We hypothesized that enhancing SoA by providing sensory feedback to participants would further compress the subjective time experience.To test the hypothesis, we used a temporal reproduction task where participants reproduced the duration of a previously exposed auditory stimulus by performing different voluntary actions: a combination of single actions with single auditory feedback, continuous action with single auditory feedback, or continuous action with multiple auditory feedback.The results showed that the continuous action conditions, regardless of the type of auditory feedback, led to a compression of the subjective time experience of the reproduced tone, whereas the single action condition did not. Furthermore, a greater degree of subjective time compression during continuous action and a stronger SoA were revealed when enriched with multiple auditory feedback.These results indicate that enriching auditory feedback can increase subjective time compression during voluntary action, which in turn enhances SoA over action outcomes. This suggests the potential for developing new techniques to artificially compress the subjective time experience of daily events. In everyday life, people formulate intentions to act and then move their bodies to produce a desired outcome in the external environment. Such voluntary actions can compress the subjective time experience of their outcome, irrespective of sensory modality, thereby facilitating optimized interactions with the external environment . The intThe internal comparator model  is a welTo increase in the number of matches between predicted and actual action outcomes during a voluntary action may be a possible way to increase the compression of the subjective time experience of the action outcome, leading to an enhanced sense of agency. In a recent study, we investigated how the subjective time experience of action outcomes changes depending on the type of voluntary action. Specifically, we conducted a temporal reproduction task in which participants reproduced the duration of a previously exposed auditory stimulus using different types of voluntary actions . We compEnriching sensory feedback during voluntary actions by increasing the number of feedback change points may provide additional opportunities to generate matches between predicted and actual outcomes. This, in turn, may enhance individuals to predict their action outcomes, compare them to actual outcomes, and accumulate matches. Building on this idea, the current study sought to investigate how the change in sensory feedback provided during a voluntary action affects the compression of subjective time and the sense of agency over the action outcome. We used a temporal reproduction task similar to our previous study  where paM = 29.3 years; SD = 6.7 years) participated in this study and received monetary compensation for their involvement. The sample size was determined through a power analysis using G*Power (f = 0.25) with 80% power and a significance level of 0.05 for a two-way repeated measures within-subjects analysis of variance (ANOVA). All participants had normal or corrected-to-normal vision and were right-handed as determined by the Edinburgh Handedness Inventory  aged 20–39 years . If we found a significant effect of the task condition, we performed subsequent multiple comparisons using Shaffer’s modified sequentially rejective Bonferroni procedure .The temporal reproduction error was tested using a one-sample t-test to compare the values to 1, allowing us to determine whether the reproduced duration was compressed. We then conducted a two-way repeated measures ANOVA with task condition  and duration condition  as repeated measures factors to compare the task conditions. When we found a significant interaction between task and duration condition, we performed subsequent simple main effect analyses. When significant simple main effects of task or duration conditions were found, we conducted multiple comparisons. We corrected rocedure . If the rocedure . To examrocedure . Finallyt-test showed that the temporal reproduction errors in the single-single condition were not significantly different among the duration conditions compared with a value of 1 . On the other hand, in the continuous-single condition, the temporal reproduction errors were significantly higher in the 3-s condition , but not significantly different in the 5-s condition , 7-s condition , and 9-s condition . The temporal reproduction errors in the continuous-multiple condition were significantly higher in the 3-s condition  and 5-s condition , but not significantly different in the 7-s condition  and 9-s condition .The average temporal reproduction error for the single-single, continuous-single, and continuous-multiple conditions are shown in F = 7.77, p < 0.01, ηp2 = 0.29] and duration condition , as well as a significant interaction between the task and duration condition . We then performed subsequent simple main effect analyses and found the expected simple main effects of task condition in the 3-s, 5-s, and 7-s conditions , but not in the 9-s condition . Subsequent multiple comparisons for the significant simple main effect of the task condition in each duration condition revealed that temporal reproduction errors were significantly greater in the continuous-multiple condition compared with the single-single condition in the 3-s, 5-s, and 7-s conditions , significantly greater in the continuous-single condition compared with the single-single condition in the 3-s and 5-s conditions  but not the 7-s condition , significantly greater in the continuous-multiple condition compared with the continuous-single condition in the 3-s condition  but not the 5-s and 7-s conditions . We also found the expected simple main effects of duration condition in the continuous-single and continuous-multiple conditions  but not in the single-single condition . Subsequent multiple comparisons for the significant simple main effect of the duration condition in each task condition revealed that in the continuous-single condition, the temporal reproduction error was significantly greater in the 3-s condition compared with the 7-s and 9-s conditions  and significantly greater in the 5-s condition compared with the 7-s and 9-s conditions , but not significantly different between the 3-s and 5-s conditions  or between the 7-s and 9-s conditions . In the continuous-multiple condition, the temporal reproduction error was significantly greater in the 3-s condition compared with the 5-s, 7-s, and 9-s conditions , significantly greater in the 5-s compared with the 9-s condition , and significantly greater in the 7-s compared with the 9-s condition . However, we found no significant difference between the 5-s and 7-s conditions . The results suggest that the reproduction of the encoded duration was prolonged in both the continuous-single and continuous-multiple conditions, indicating compression of the subjective time of the reproduction tone. Furthermore, the amount of compression was greater in the continuous-multiple condition compared to the continuous-single condition. However, as the duration increased, the amount of compression decreased until it approached an accurate value. This trend was more pronounced in the continuous-multiple condition than in the continuous-single condition. On the other hand, in the single-single condition, the encoded duration was accurately reproduced regardless of the duration, without any significant compression of the subjective time of the reproduction tone.The temporal reproduction errors in t[3] = 3.61, p = 0.04, d = 1.81) and the continuous-multiple condition . A paired t-test revealed that the correlation coefficients were significantly higher in the continuous-multiple condition than in the continuous-single condition . The results suggest that there was a positive relationship between the speed of action and the subjective time compression in both conditions, and that the relationship was stronger in the continuous-multiple condition than in the continuous-single condition.F = 12.29, p < 0.01, ηp2 = 0.39]. Subsequent multiple comparisons revealed that the self-reported ratings of control were significantly higher in the continuous-multiple condition than in the single-single and continuous-single conditions  and significantly higher in the continuous-single condition than in the single-single condition . r = 0.26, p = 0.04). The results suggest that the explicit rating of the sense of agency was higher in the continuous action condition than in the single action condition. Moreover, this trend was more pronounced when the continuous action was accompanied by auditory feedback containing multiple tones. We also observed a positive correlation between the amount of subjective time compression and the explicit ratings of the sense of agency.In summary, our study found that the continuous action led to a compression of the subjective time of the reproduction tone presented as the action outcome, regardless of the type of auditory feedback, while the single action did not. Additionally, we observed that explicit ratings of the sense of agency were stronger for the continuous action than for the single action. We also found that the subjective time compression caused by the continuous action decreased as the encoded duration length increased, leading to more accurate estimations. The speed of the continuous action was positively correlated with the amount of subjective time compression. These trends were more pronounced when the continuous action was accompanied by enriched auditory feedback. Finally, a positive relationship was observed between the subjective time compression and explicit ratings of the sense of agency.Regardless of the sensory modality, voluntary actions can compress the perceived duration of their outcomes, which helps to maintain the experience of control over external events known as the sense of agency. Recent research suggests that when performing a voluntary action, increasing the number of matches between predicted and actual action outcomes can intensify the compression of the subjective time experience of the action outcome and enhance the sense of agency. However, it remains unclear whether increasing the number of opportunities to create such matches between predicted and actual outcomes can further modulate the compression of subjective time experience. Such an investigation could have implications for the development of new techniques for artificially altering our time experiences of daily events involving continuous actions, such as driving.In this study, we aimed to investigate how the subjective experience of time for action outcomes is influenced by the change in sensory feedback provided. To achieve this, we conducted a temporal reproduction task, in which participants were asked to reproduce the duration of an auditory stimulus that they had encountered during different conditions: a combination of single actions with single auditory feedback, continuous action with single auditory feedback, or continuous action with multiple auditory feedback. As a result, consistent with our previous findings , our resMoreover, our study revealed a more significant compression of subjective time experience during the continuous action when enriched with multiple auditory feedback. This could be attributed to the increased richness of the auditory feedback, which may have diverted attention away from the reproduction tone. Prior research suggests that when attentional resources are divided, attention to an event decreases, and the perception of the event’s duration is compressed . Thus, tIt is important to note that our study involved at least two task-relevant factors that may have influenced the results. The first factor concerns the temporal cues provided by the auditory feedback. Enriched auditory feedback during the continuous action not only increases the number of matches between predicted and actual outcomes but also provides more temporal cues during the reproduction task. As a result, participants might have been able to more accurately reproduce the duration during the continuous action with multiple auditory feedback compared to single auditory feedback, regardless of the duration of the action. This effect might have conflicted with the observed increase in the compression of subjective time during the continuous action with multiple auditory feedback. The second task-relevant factor is the well-established finding that subjective time compression induced by action tends to decrease as the encoded duration length increases. This is consistent with previous studies using temporal reproduction tasks , includiThe current study has several methodological limitations that should be taken into account. Firstly, our conclusion that enriching sensory feedback can boost subjective time compression induced by continuous action may only apply to auditory feedback. The sensory modality in which information is presented influences perceived duration . For insExpanding the applicability of the study’s findings to help individuals modulate their daily time experience through voluntary actions is an interesting research direction. For example, enriched sensory feedback has the potential to enhance various experiences, including driving and using automation technology. Routine driving for work can lead to drivers feeling bored and experiencing subjective time dilation. By appropriately enriching sensory feedback, drivers may avoid dilating their time experience and maintain their sense of agency over their drive, leading to a greater sense of responsibility for their actions on the road. Similarly, automation technology can reduce the number of actions required by operators, potentially leading to increased subjective time dilation and decreased sense of responsibility for task outcomes. To ensure the safe and effective use of automation technology, it’s essential for operators to monitor system performance and be ready to intervene when necessary. Providing appropriate sensory feedback can help operators compress their time experience and maintain their sense of agency over their tasks, ultimately leading to a greater sense of responsibility for task outcomes. Future research should focus on developing effective methods for maximizing subjective time compression. The current study suggests that enriching sensory feedback by increasing the number of matches between predicted and actual action outcomes during voluntary action can enhance the sense of agency and compress the subjective time experience of the action outcome. Multimodal sensory feedback, including visual and tactile feedback in addition to auditory feedback, may increase the number of matches and further enhance subjective time compression. However, it’s important to note that subjective time compression may have a limit. Therefore, future studies should investigate changes in subjective time compression and the sense of agency associated with changes in the amount of sensory feedback from multiple modalities during continuous action, to determine potential plateaus in subjective time compression.Our study has demonstrated that enriching auditory feedback can increase subjective time compression during continuous action, which in turn enhances the sense of agency over action outcomes. Importantly, this effect was not caused by a lack of attention toward action outcomes. Our results suggest that increasing the number of matches between predicted and actual outcomes during a voluntary action by providing enriched sensory feedback can effectively compress the subjective time experience of the action outcome. This study is the first to demonstrate the potential of artificially compressing the subjective time experience of daily events through voluntary actions and enriched sensory feedback. Our findings could pave the way for the development of new techniques that allow individuals to control and compress their own time experience. However, further research is necessary to strengthen the validity of our findings and to determine the extent to which these findings can be applied in practical settings.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving humans were approved by the RIKEN Research Ethics Committee. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent for participation in this study was provided by the participants. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.SU developed the study concept and performed the data collection and data analysis. SU and SS performed the drafting of the manuscript. SU and SS contributed to the study design. All authors contributed to the article and approved the submitted version.This study received funding from Toyota Motor Corporation. The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication.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.


MET is a receptor tyrosine kinase (RTK) responsible for initiating signaling pathways involved in development and wound repair. MET activation relies on ligand binding to the extracellular receptor, which prompts dimerization, intracellular phosphorylation, and recruitment of associated signaling proteins. Mutations, which are predominantly observed clinically in the intracellular juxtamembrane and kinase domains, can disrupt typical MET regulatory mechanisms. Understanding how juxtamembrane variants, such as exon 14 skipping (METΔEx14), and rare kinase domain mutations can increase signaling, often leading to cancer, remains a challenge. Here, we perform a parallel deep mutational scan (DMS) of MET intracellular kinase domain in two fusion protein backgrounds: wild type and METΔEx14. Our comparative approach has revealed a critical hydrophobic interaction between a juxtamembrane segment and the kinase αC helix, pointing to differences in regulatory mechanisms between MET and other RTKs. Additionally, we have uncovered a β5 motif that acts as a structural pivot for kinase domain activation in MET and other TAM family of kinases. We also describe a number of previously unknown activating mutations, aiding the effort to annotate driver, passenger, and drug resistance mutations in the MET kinase domain. Receptor tyrosine kinases (RTKs) are transmembrane proteins that play an essential role in the initiation and regulation of signaling pathways . Most RTIn contrast to physiological HGF-regulated activity, MET oncogenic activity arises through a variety of mechanisms such as gene amplification, mutations, gene fusions, and HGF autocrine loops . In MET Here, we use deep mutational scanning (DMS)  to screeTo identify residues that have a direct effect on kinase function, we leveraged the murine Ba/F3 cell line as a selection system. The Ba/F3 cell line has been used as a model to study RTK signaling because it exhibits: (1) undetected expression of endogenous RTKs including Met, (2) addiction to exogenous interleukin-3 (IL-3) for signaling and growth, and (3) dependence on exogenous kinase expression for growth in the absence of IL-3 . IL-3 wiTo perform a DMS in TPR-MET, we generated a site saturation mutagenesis DNA library of the MET KD . Our libAs expected, we observed no selection in the IL-3 control condition and a low correlation was observed across replicates (Pearson’s R=0.30). In this condition, all variants, including premature stop codons, displayed near WT-like fitness –C. In coMET contains a canonical tyrosine kinase domain, sharing structural and conformational hallmarks with other protein kinases . Two hydOur DMS results highlight that the conserved regulatory and catalytic motifs in MET are highly sensitive to mutation –D. CatalOn balance, most solvent exposed and loop regions were more permissive to mutations –D. HowevGiven the importance of juxtamembrane (JM) regions in controlling the activation of many RTKs  and the . Rather, αJM consistently packed against αC helix and maintained the hydrophobic interface across all structures  MET receptor in HEK293 cells. Based on the DMS in Ba/F3 cells, we expected that P1153 mutations would not signal or express poorly in this context. Indeed, relative to the WT MET receptor, P1153L expressed equivalently, but dramatically reduced signals for phosphorylation of the MET activation loop tyrosines, a marker of active MET signaling . To testNext we analyzed the structural environment of P1153. It packs in a hydrophobic cluster with the resistance-associated β3-αC helix “keymaster” position (L1112 in MET)  and F112Exon 14 skipping, which maintains the ECD and transmembrane region while truncating the ICD at the JM, is one of the most common driver mutations observed in MET. The oncWe performed a parallel DMS of the MET KD in the background of an exon 14-skipped ICD (TPR-METΔEx14) . Most reAs an example of the differences between the two backgrounds, we focused on the αJM-αC helix interface. This region displayed relatively high sensitivity to non-hydrophobic substitutions in TPR-MET , but it These results suggest that the correlation between DMS fitness in TPR-MET and acute A-loop phosphorylation in the MET receptor is not absolute. Since KD activation at a single time point was the only parameter we explored with the membrane-associated receptor, other aspects such as sustained signaling, recruitment of specific signaling adapters, or changes in MET regulation may be more consistent with the proliferative readout of the DMS. In addition, the inconsistency in the S1122Q result may be a specific feature of the dimerised, cytoplasmic TPR-MET signaling that is sensitive to interactions with the portion of the JM deleted by exon 14. Collectively, these results highlight that the αJM-αC helix interface is sensitive to mutation and the presence or absence of exon 14 can alter this sensitivity. While the experimental parameters of this screen may limit how translatable some mutations are in a membrane-associated receptor or in non-proliferative conditions, the consistency of most residues between backgrounds provides increased confidence in the atlas of mutational effects on MET activation.To assess the ability of the DMS to classify driver, passenger, and resistance mutations, we first gathered all MET kinase domain mutations reported from clinical observations in cBioPortal  Figure . RelativInterestingly, there are some missense mutations that are strong GOF variants in the DMS that have not been reported clinically, potentially indicative of novel activating mutations. We classify these mutations as “not clinically observed, GOF.” We hypothesized that these variants are more difficult to observe clinically because of the constraints of the genetic code. To test this idea, we calculated the Hamming distance between the WT MET codon and mutant codon for each position within our dataset –E. We foFinally, we assessed the distribution of fitness effects for clinically observed resistance mutations . ResistaOur parallel DMS of the MET kinase domain has revealed how mutations affect the allosteric regulation of MET in two clinically relevant juxtamembrane backgrounds. Auto-regulation via juxtamembrane segments is a common feature of RTKs like the EGF, PDGF, EPH, and IR families . We propConclusively addressing the question of how exon 14 skipping activates MET is difficult in the TPR-fusion system that we have employed here. While soluble, cytosolic oligomerization occurs for MET fusion proteins observed in some tumors, using the TPR-fusion may not accurately model all of the changes found in other types of MET lesions, such as METΔEx14. Therefore, this strategy may underestimate the effects of regulatory mechanisms related to membrane engagement or ICD oligomerization. Nonetheless, the experimental parameters of this study based on cell proliferation allowed us to understand the mutational sensitivities of the kinase domain in a cytoplasmic and constitutively active environment..Analyzing the mutational landscape with the TPR-MET fusion approach employed here has led to multiple new insights, some of which were also validated in the full-length MET receptor. For example, one of the most unexpected regions of mutational sensitivity was P1153 in the β4-β5 loop. Based on the observed proline sequence shift, β4-β5 loop conformational restraints, and conserved hydrophobic network around the β4-β5 loop position, we suggest that this proline residue is an evolutionary dead-end. In ancestral kinases, a proline could be adopted without a significant cost at the corresponding β4-β5 loop position due to this residue being positioned in proline-permissive Ramachandran space. After drift of the surrounding hydrophobic residues, the sequence for these residues has adapted to the unique structural properties of proline, rendering it impossible to substitute for an alternative amino acid. Interestingly, evolutionarily distant kinases that do not have proline at this position exhibit inhibitor resistance mutations at this site . In someIn summary, our parallel DMS of MET and METΔEx14 has built an atlas of variant effects. Moreover, we identified a small number of unique sensitivities in each background, which provides hypotheses about the mechanism of exon 14 skipping in cancer. We also observed a number of strong GOF variants that have not been observed in the clinic. Strikingly, these variants are enriched in 2 and 3 nucleotide changes, suggesting that our DMS will be especially useful in classifying rare driver mutations and that the clinical population has essentially sampled most of the single nucleotide changes. These results comprise a valuable resource for classifying driver, passenger, and resistance mutations for MET and other RTKs.Ba/F3 cells were purchased from DSMZ and maintained in 90% RPMI (Gibco), 10% HI-FBS (Gibco), 1% penicillin/streptomycin (Gibco), and 10ng/ml IL-3 (Fisher), and incubated at 37°C with 5% CO2. Ba/F3 cells were passaged at or below 1.0E6 cells/ml in order to avoid acquired IL-3 resistance, and regularly checked for IL-3 addiction by performing 3x PBS (Gibco) washes and outgrowth in the absence of IL-3 to confirm cell death in the parental, empty cell line.Plat-E cells stably expressing retroviral envelope and packaging plasmids were originally gifted by Dr. Wendell Lim. Plat-E cells were maintained in 90% DMEM+HEPES (Gibco), 10% HI-FBS (Gibco), 1% penicillin/streptomycin (Gibco), 10ug/ml blasticidin, 1ug/ml puromycin, and incubated at 37°C with 5% CO2. Plat-E cells were maintained under blasticidin and puromycin antibiotic pressure unless being transfected.HEK293 cells were maintained in DMEM (Gibco) supplemented with 10% FBS (Gibco) and 1% penicillin/streptomycin (Gibco) at 37°C in 5% CO2.Human MET knockout HeLa cells were purchased from Abcam and maintained in 90% DMEM+HEPES (Gibco), 10% HI-FBS (Gibco), 1% penicillin/streptomycin (Gibco), and incubated at 37°C with 5% CO2Both pUC19 (Cat. 50005) and MSCV (Cat. 68469) were ordered from Addgene. To ensure unique cut sites within the vectors for introduction and shuttling of the variant library, a new multiple cloning site was introduced into each plasmid. Wild type TPR-MET-IRES-mCherry and TPR-METΔEx14-IRES-eGFP genes were cloned into pUC19 as the parental constructs for library generation and all site-directed mutagenesis. The full-length MET, METΔEx14, and RON receptor cDNAs were subcloned into pcDNA3.1 vector by Gibson assembly and incorporated a C-terminal single Flag tag sequence. All mutations were introduced by quick change mutagenesis.The MET kinase domain variant library was designed to span amino acid positions 1059–1345, which contained the full kinase domain (aa 1071–1345) and a portion of the juxtamembrane (aa 1059–1070). The library was synthesized by Twist Bioscience with one mammalian high-usage codon per amino acid to prevent over-representation of specific residues. The library was received in 96-well arrays of lyophilized DNA at 50 ng per well, where each well contained all variants (missense and WT-synonymous) per position of the kinase domain. The lyophilized library was resuspended in 100uL of 1X TE buffer, and 5ng of DNA from each well was amplified with low cycle PCR to increase the starting material using the following the NEB Q5 High-Fidelity recipe per well: 10μL 5X Q5 buffer, 5ng template DNA, 2.5μL 10μM forward primer, 2.5μL 10μM reverse primer, 1μL 10mM dNTPs (2.5μM each), 0.5μL Q5 Polymerase, nuclease free water to a final volume of 50μL. The following thermocycler parameters were then applied: initial denaturation at 98°C for 30s, followed by 10x cycles of denaturation at 98°C for 10s, annealing at 62°C for 30s, extension at 72°C for 1 min, and a final extension at 72°C for 5 min. A 1% agarose, 1X TBE diagnostic gel was run with 2μL of each sample to confirm amplification of all positions, then the samples were PCR cleaned using the Zymo 96-well DNA clean and concentrate kit, eluted in 10μL nuclease free water, pooled together in a low DNA-bind tube, and then DNA cleaned (Zymo) once more to further concentrate the pooled library.The kinase domain variant library was digested with PstI-HF (NEB) and NdeI-HF (NEB) and cleaned up with the Zymo DNA clean and concentrate kit. Next, the two cloning vectors (pUC19_kozak-TPR-METΔEx14-IRES-eGFP and pUC19_kozak-TPR-MET-IRES-mCherry), were digested with PstI-HF(NEB) and NdeI-HF (NEB), phosphatase treated with rSAP (NEB), gel extracted to isolate the backbone, and DNA cleaned (Zymo). The variant library was ligated into each vector with a 1:3 (insert: vector) T4 ligation at 16°C overnight (NEB). Ligations were DNA cleaned (Zymo), eluted in 10uL of nuclease free water, and electroporated into 50μL MegaX 10 beta cells (Invitrogen). Transformations were then recovered in 1mL of SOC for 1hr at 37°C. Post recovery, 10μL cells were collected, serial diluted, plated at varying dilutions  to evaluate transformation efficiencies. The remainder of the transformation then propagated in 50mL LB and Carbyinacillin at 37°C to an OD of 0.5, and then midi-prepped (Zymo).Amino acid variants were successfully synthesized by Twist Biosciences for all positions with the exception of 1194 and 1278. In addition, premature stop codons were not included in the synthesized Twist library. To include these missing positions and early stop control, we generated a “fill-in” library. For positions 1194 and 1278, a forward primer for each amino acid mutation and a single reverse primer was designed for inverse PCR. An early stop codon “fill-in” library was also generated to introduce one stop codon every 33 bases, evenly spaced throughout the gene. This resulted in one stop codon every 11 positions, or 24 total premature stops. Again, a single forward and reverse primer was designed for each stop codon mutation using inverse PCR. Mutations were introduced into wild type pUC19_kozak-TPR-METΔEx14-IRES-eGFP and pUC19_kozak-TPR-MET-IRES-mCherry with the following NEB Q5 High-Fidelity conditions per reaction: 10μL 5X Q5 buffer, 5ng template DNA, 2.5μL 10μM forward primer, 2.5μL 10μM reverse primer, 1μL 10mM dNTPs (2.5μM each), 0.5μL Q5 Polymerase, nuclease free water to a final volume of 50μL. The following thermocycler parameters were then applied: initial denaturation at 98°C for 30s, 10x cycles of denaturation at 98°C for 10s, annealing at 62°C for 30s, extension at 72°C for 4.4 min, and a final extension at 72°C for 10 min. A 1% agarose diagnostic gel was run with 2μL of each reaction to conform amplification. Then all PCR samples were pooled, DNA cleaned (Zymo) and eluted in 50μL nuclease free water, DPN1 digested to remove template (NEB), DNA cleaned again and eluted in 12μL nuclease free water, PNK treated (NEB), and blunt-end ligated at 16°C overnight with T4 ligase. Ligations were DNA cleaned the next morning (Zymo) and electroporated into MegaX 10 beta cells (Invitrogen). Transformations were recovered in 1mL of SOC for 1hr at 37°C, plated at varying dilutions to estimate transformation efficiencies, propagated in 50mL LB and Carbyinacillin at 37°C to an OD of 0.5, and then midi-prepped (Zymo). The Twist-synthesized library was then pooled together with the 1278, 1194, and premature stop “fill-in” libraries at equimolar concentrations to a total of 1ug of DNA.7μg of each pooled library was restriction digested with MluI-HF (NEB) and MfeI-HF (NEB) to cut out variant libraries in the kozak-TPR-MET-IRES-mCherry and kozak-TPR-METΔEx14-IRES-eGFP backgrounds. Digests were gel extracted from pUC19 and DNA cleaned (Zymo). The empty, Puromycin resistant, retroviral expression vector MSCV (addgene) was also cut MluI-HF (NEB) and MfeI-HF (NEB), phosphatase treated with rSAP (NEB), and DNA cleaned. Isolated libraries were then ligated 1:1 (insert to vector) into the MSCV retroviral vector at 16°C overnight with T4 ligase. Ligations were then DNA cleaned (Zymo) and electroporated into ElectroMAX Stbl4 Competent Cells (Thermo Fisher). Transformations were recovered in 1mL of SOC for 1hr at 37°C and after recovery 10μL was serial diluted and plated to estimate transformation efficiencies, while the remainder was plated on bioassay dishes. Colonies were scraped from the bioassay dishes and midi-prepped for transfections (Zymo).The MSCV_kozak-TPR-MET-IRES-mCherry and MSCV_kozak-TPR-METΔEx14-IRES-eGFP variant libraries were transfected into Plat-E cells for retroviral packaging using Lipofectamine3000 (Invitrogen). Two T-175 flasks of Plat-E cells were prepared for each library in the absence of blasticidin and puromycin 24hr prior to transfection such that they would be at 70–80% confluency at the time of transfection. On the day of transfection, each flask of Plat-E cells was gently washed with PBS to remove the culturing media, and replaced with 35mL Opti-MEM. For the transfection, Opti-MEM was brought to room temperature and two pairs of DNA Lo-bind 5mL tubes were prepared following manufacturer’s instructions for Lipofectamine3000 scaled to a T-175 format. A total of 46μg DNA was used to transfect the libraries and package virus in parallel: MSCV_TPR-MET-IRES-eGFP, MSCV_TPR-METΔEx14-IRES-mCherry. Each flask was incubated with the transfection reagents for 5hr at 37°C, 5% CO2; the transfection media was then replaced with 50mL OptiMEM, 5% FBS, 1x GlutaMax, and 2% Sodium Pyruvate (Gibco) for viral packaging. After 48 hr post-transfection, the viral supernatant was harvested, passed through a 0.45 μm filter to remove cell debris, then precipitated overnight with 1:4 Retro-X concentrator (TakaraBio) at 4°C, then pelleted at 380xg for 45 min at 4C, and resuspended in 5mL of sterile, cold PBS and stored at 4°C in 1mL aliquots until transduced into Ba/F3 cells.The concentrated virus was titiered in Ba/F3 cells in a 6-well plate format. Cells were seeded at 1.0E5 cells/ml with 10 ng/ml IL-3 and 8μg/ml polybrene (Sigma-Aldrich). Virus was added to wells at 0, 10x, 20x, 40x dilutions to determine the proper volume for a transduction MOI of 0.1–0.3. Cells were spinfected at 250xg for 60 minutes at room temperature, then incubated for 48 hr. The viral titer was calculated from the percent of fluorescent cells and viral dilution.For the DMS viral transduction, 6 million cells were spinfected at an MOI of 0.1, in triplicate for a total of 3 biological replicates for each library, and incubated post spinfection in a 15cm dish with 30mL Ba/F3 media and 10ng/L IL-3 for 48hr. Infected cells were then selected with 1μg/ml puromycin for a total of 4 days with fluorescence and cell counts tracked each day.After puromycin selection, all three biological replicates for both libraries, TPR-MET and TPR-METΔEx14, were washed free of puromycin and IL-3 with 3x PBS washes. A total of 6 million cells from each replicate was harvested and pelleted at 250xg to serve as the “time point 0” pre-selection sample (T0).To begin selection of each replicate for each library, two sets of 15cm dishes were prepared with 2.0E5 cells/ml in 30mL 90% RPMI, 10% HI-FBS, 1% penicillin/streptomycin. One plate was kept free of IL-3 as the experimental IL-3 withdrawal condition, while the other plate was supplemented with 10ng/mL IL-3 to provide the control condition. Three time points post T0 were collected for each library replicate and condition for a total of four time points . Time points were harvested every 48hr across 7 days; 6 million cells were harvested for each condition and pelleted at 250xg for 5min; 2.0E5 cells/ml were split at every time point and maintained either in IL-3 or IL-3 withdrawal conditions.The gDNA of each time point sample was isolated with the TakaraBio NucleoSpin Blood QuickPure kit the same day the cells were harvested. gDNA was eluted in a 50μl elution buffer using the high concentration and high yield elution manufacturer’s protocol. Immediately after gDNA was isolated, 5μg of gDNA was used for PCR amplification of the target MET KD gene to achieve the proper variant coverage. A 150μl PCR master mix was prepared for each sample using the TakaraBio PrimeStar GXL system according to the following recipe: 30μl 5X PrimeStar GXL buffer, 4.5μl 10μM forward primer , 4.5μl 10μM reverse primer , 5μg gDNA, 12μl 10mM dNTPs (2.5mM each NTP), 6μl GXL polymerase, nuclease free water to a final reaction volume of 150uL. The PCR master mix was split into three PCR tubes with 50μl volumes and amplified with the following thermocycler parameters: initial denaturation at 98°C for 30 s, followed by 24x cycles of denaturation at 98°C for 10 s, annealing at 60°C for 15 s, extension at 68°C for 14 s, and a final extension at 68°C for 1 min.After all time points were selected, harvested, and PCR amplified, the target gene amplicon was isolated from gDNA by gel purification. The entire 150μl PCR reaction for each sample was mixed with 1X NEB Purple Loading Dye (6X stock) and run on a 0.8% agarose, 1X TBE gel, at 100 mA until there was clear ladder and band separation. The target amplicons were gel excised and purified with the Zymo Gel DNA Recovery kit. To remove excess agarose contamination, each sample was then further cleaned using the Zymo DNA Clean and Concentrator-5 kit. Amplicon DNA concentrations were then determined by Qubit dsDNA HS assay (Invitrogen).Libraries were then prepared for deep sequencing using the Nextera XT DNA Library Prep kit in a 96-well plate format (Illumina). Manufacturer’s instructions were followed for each step: tagmentation, indexing and amplification, and clean up. Libraries were indexed using the IDT for Nextera Unique Dual Indexes Set C (Illumina). Then, indexed libraries were quantified using the Agilent TapeStation with HS D5000 screen tape (Agilent) and reagents (Agilent). DNA concentrations were further confirmed with a Qubit dsDNA HS assay. All samples were manually normalized and pooled at 10nM for MET and METΔEx14. The libraries were then paired-end sequenced (SP300) on two lanes of a NovaSeq6000.Demultiplexed paired-end reads were received from the sequencing core and processed further using a snakemake-based pipeline previously developed  according to the manufacturer’s protocols. Cells were harvested 24 hours post transfection, lysed in buffer , 1 mM sodium fluoride and 1 mM sodium vanadate). Clarified lysates were incubated with G1 affinity resin (Genscript) overnight at 4°C. Resin was washed with lysis buffer (without inhibitors) and proteins eluted by addition of Laemmli sample reducing buffer. Proteins were separated by SDS-PAGE on a 4–15% gradient gel (BioRad) and transferred to PVDF membrane (Millipore). Membranes were probed with Flag (2368), MET pY1234/5 (3077) .Human MET knockout HeLa cells were transiently transfected with Lipofectamine3000 (Invitrogen) according to the manufacturer’s protocols in a 6-well plate format. Post transfection (24hr), cells were washed with PBS (Gibco) (3x washes) to remove serum and transfection media, and replaced with DMEM (Gibco) in the absence of any additives. Cells were serum starved for 4 hr, then stimulated with 50 ng/ml HGF (PeproTech) for 15 min at 37°C, then immediately washed with cold PBS (3x washes), and maintained on ice. Cells were then lysed in buffer , 1 mM sodium fluoride, and 1 mM sodium vanadate) on ice. Clarified whole cell lysates were run on a 8–16% SDS-PAGE gel (BioRad) and transferred to a nitrocellulose membrane (BioRad). Membranes were probed with MET pY1234/5 (Cat# 3077), Met (8198), P-p44/42 MAPK Erk1/2 (T202/Y204) (4376), p44/42 MAPK Erk1/2 (4695), and β-Actin (4970) .Ba/F3 cells stably expressing TPR-MET, TPR-METΔEx14, and empty MSCV constructs were seeded at 2.5E4 cells/ml in triplicate in a 94-well, round bottom plate for each time point in the presence and absence of 10ng/ml IL-3. CellTiter-Glo reagent (Promega) was mixed at a 1:1 ratio with cells and luminescence was measured on a Veritas luminometer at 0, 48, and 96 hrs post-seeding. In this study, we use a modified nomenclature, where we refer to TPR-MET as the TPR-fusion of MET with the full-length juxtamembrane sequence and TPR-METΔEx14 as the TPR-fusion lacking exon 14. Cell numbers were determined from a Ba/F3 cell and ATP standard curve generated according to the manufacturer’s instructions. Data are presented as cell viability normalized to the fold change from the 0hr time point.For IL-3 titrations, Ba/F3 cells stably expressing TPR-MET, TPR-METΔEx14, and empty MSCV constructs were 3x PBS washed and 5000 cells were seeded in a 94-well, round bottom plate. IL-3 was added to wells at 0–10ng/ml . CellTiter-Glo reagent (Promega) was mixed at a 1:1 ratio with cells and luminescence was measured on a Veritas luminometer at 0, 24, and 48 hrs after seeding and IL-3 addition.Cell numbers for all proliferation assays were determined from a Ba/F3 and ATP CellTiter-Glo standard curve generated according to the manufacturer’s instructions. Data are presented as cell viability normalized to the fold change from the 0hr time point.Structural visualization, mapping, and analysis was completed using PyMOL unless otherwise stated. All human MET (UniProtKB accession: P08581) kinase domain crystal structures currently available were downloaded from the PDB. All PDB structures were loaded and globally aligned to generate the kinase domain ensemble. Residue distances were calculated from alpha-carbon x,y,z coordinates and computationally analyzed. Raw PDB files were used to categorize structure features: resolution, construct boundaries, conformation, sequence features, mutations, and apo/holo states.To choose representative active and inactive structures for score mapping and visualization, we generated an ensemble of 88 human MET kinase domain structures currently deposited in the PDB, and classified activity states based on alpha-carbon distances between catalytic site residues K1110, E1127, and F1223 , with thCrystal structures of active and inactive human IR , KIT , EPHA3 (PDB 2QO9), RET , MET , and AXL (PDB 5UAB) were obtained through the PDB. RMSD was calculated and plotted with the Bio3D package in R for each kinase using the inactive structure as the reference.For β5 positional comparison and Ramachandran analysis, PDB files were obtained for each kinase, and analyzed with the Bio3D package in R to attain the Phi and Psi angles of each residue. The general and proline contour data was obtained from Cancer-associated missense mutations for the MET kinase domain was obtained from cBioPortal (NCBI ID: NM_000245). Resistance mutations were obtained from literature references .All human RTK protein sequences used in alignments were acquired from UniProt. Unless otherwise stated, alignments were done with MuscleWS using default parameters through JalView, and amino acids were colored according to physicochemical properties, or percent sequence identity where noted.Supplement 1


The idea of harnessing cooperative behaviors for therapeutic benefit involves exploitation of the invasive capabilities of cheats to drive medically beneficial traits into infecting populations of cells. In this study, we employed Pseudomonas aeruginosa quorum sensing cheats to drive antibiotic sensitivity into both in vitro and in vivo resistant populations. We demonstrated the successful invasion of cheats, followed by increased antibiotic effectiveness against cheat-invaded populations, thereby establishing an experimental proof of principle for the potential application of the Trojan strategy in fighting resistant infections.Antimicrobial resistance poses an escalating global threat, rendering traditional drug development approaches increasingly ineffective. Thus, novel alternatives to antibiotic-based therapies are needed. Exploiting pathogen cooperation as a strategy for combating resistant infections has been proposed but lacks experimental validation. Empirical findings demonstrate the successful invasion of cooperating populations by non-cooperating cheats, effectively reducing virulence  Hence, there is a growing interest in developing “evolution-proof” strategies to fight against infections6. The idea of utilizing social cheats as therapeutic agents has been proposed based on theoretical and empirical studies on cooperative virulence and cheat invasion6. However, experimental evidence supporting the effectiveness of cheat therapies is limited.The fight against microbial pathogens is almost completely reliant on the development of new antibiotics. However, the long-term effectiveness of this strategy is threatened due to the evolution of resistance by pathogens9. This cooperative behavior can be targeted directly, similar to conventional drug treatments 6, or it can be exploited by utilizing cheats6. Cheats are cells that do not contribute to the production of public goods but still benefit from cooperation. Consequently, cheats can invade populations by capitalizing on their fitness advantage12. Furthermore, cheat invasion is expected to lead to a reduction of virulence due to reduced cooperation within the population4.Bacterial virulence is a complex phenomenon that frequently relies on cooperative interactions facilitated by the release of public goodsPseudomonas aeruginosa has been demonstrated through in vitro experiments14. Moreover, studies by Harrison et al.15 and Rumbaugh et al.16 demonstrated the invasion potential of P. aeruginosa cheats and subsequent reduction in virulence using animal infection models. Additional evidence of cheat invasion has been shown using animal models of Salmonella enterica serovar Typhimurium and Staphylococcus aureus infections18. Expanding on these findings, cheats in pathogen populations can be further manipulated to become Trojan cheats: strains which act as vehicles for medically beneficial alleles such as antibiotic sensitivity or genes which quench quorum QS signals  cell must overcome significant evolutionary and mechanistic constraints to resist cheating.Why would cells fail to evolve resistance to cheat intervention strategies in the same way that they evolve resistance to antimicrobial treatments? The answer to this question is well-understood from evolutionary theory. The simplest way to avoid exploitation is to refrain from engaging in cooperative actionsP. aeruginosa, defective in QS signaling, to invade and introduce antibiotic sensitivity in populations of resistant cooperators to improve antibiotic effectiveness. First, we demonstrated the invasive ability of a Trojan strain in vitro using head-to-head competition assays in both liquid and viscous media . We then exposed the Trojan invaded populations to antibiotic selection and showed improved antibiotic efficacy. Finally, we provided evidence of a successful application of the Trojan invasion strategy to improve antibiotic efficiency using a murine chronic wound infection model.Here we present a proof of principle for application of cheats as part of a Trojan horse strategy to drive a selected beneficial allele to populations of cooperators. We used cheats of P. aeruginosa PA14 strain was used as the parental strain. The Trojan strain was an isogenic QS signal blind mutant of PA14 generated by insertion mutagenesis in the lasR locus lasR::Gm (PA14ΔlasR)16. A multi-drug resistance plasmid, pAMBL222 was used to obtain antibiotic-resistant variants. To generate resistant PA14 and PA14ΔlasR strains an electroporation protocol was used23. Minimum inhibitory concentrations (MICs) to kill the resulting transformants were 2048 μg/mL, 16384 μg/mL, 1365 μg/mL and 192 μg/mL for streptomycin, carbenicillin, ceftazidime and meropenem, respectively. MICs of sensitive stains were 8 μg/mL, 53 μg/mL, 1 μg/mL and 0.5 μg/mL for streptomycin, carbenicillin, ceftazidime and meropenem, respectively.For all experiments, wild type 2HPO4.3H2O and 1.5 g MgSO4.7H2O, per liter of dH2O]. Then cultures were incubated overnight at 37°C with 200 rpm shaking. Next, the overnight cultures were centrifugated, supernatant was discarded, and the pellet was washed twice in M9 minimal salts media  to remove residual carbon sources from the KB media. Cell density was assessed by measuring absorbance at 600 nm, and each set of cultures were standardized to the same density before the competition assays.To grow cultures, cells were inoculated from frozen stocks into fresh King’s broth media . After 24 h incubation at 37°C, plates were removed and kept at room temperature for an additional 24 h. This allows distinguishing QS cheats from cooperators based on differences in colony morphologies. While cooperators form raised colonies with fuzzy edges, lasR mutant colonies show characteristic colony flattening, sharper edges and surface iridescent sheen25. To change the viscosity and spatial structuring of the media, competition assays were performed in QSM supplemented with 0.5 % agar.To determine the relative fitness of cheats, a defined QS medium (QSM) was used. PA14 and Trojan strains were inoculated in a 10:1 ratio in 50 ml falcon tubes containing 2 ml QSM [1% BSA w/v and 0.1% CAA w/v in M9 solution] as described previouslyTo determine antibiotic survival of populations before or after competitions in QSM, 10 μl samples were spotted in triplicates onto LB agar plates supplemented with varying concentrations of antibiotics. The inoculated plates were incubated at 30°C for 24 h and then at room temperature for an additional 24 h, which made colony counting more reliable by preventing overgrowth. To obtain survival rate data, triplicate counts were averaged, and percentage of survival was calculated for each data point by the formula CFU(antibiotic)/CFU(no-antibiotic)*100.27. For the experiments herein, female Swiss Webster mice were anesthetized using intraperitoneal injection of ketamine and xylazine. The backs of the mice were shaved, and a full-thickness skin excision measuring 1.5 cm in diameter was administered. To cover the wounds, semipermeable polyurethane dressings were applied, followed by the inoculation of bacteria on top of the wounds, beneath the dressings. The initial inoculation of bacteria was performed at a concentration of 105 CFU/mL, with strains inoculated according to experimental group. For co-inoculated mixed infections, a Trojan to PA14R ratio of 1:10 was used for initial inoculation. For delayed-mixed infections, PA14R infection was allowed to establish for 72 hours. At this point, the delayed-mixed infection groups received an additional 106 CFU per 100 μL of Trojan strain, which corresponded to approximately 10% of the wound burden of PA14R at the 72-hour mark post-infection. The remaining groups were sham inoculated with PBS. On day 7 post-infection, the mice were euthanized. The wound beds were harvested and subjected to ex vivo treatment with either a vehicle control (PBS) or an antibiotic solution containing 1.75 mg/mL of streptomycin and 8 mg/mL of carbenicillin. The difference in dosage compared to in vitro experiments is due to the recalcitrant nature of biofilms in wounds, which reduces the efficacy of antibiotics even on genotypically susceptible infections28. Following antibiotic treatment, the wound beds were washed thoroughly to prevent antibiotic carryover, resuspended in PBS, and subsequently homogenized. Serial dilutions were prepared for CFU quantification, which was calculated per gram of tissue. To determine the population compositions, each homogenized tissue sample was plated on Pseudomonas Isolation Agar (PIA) or PIA supplemented with 200 μg/ml ceftazidime, which selects for the presence of the resistance plasmid. All animal experiments were conducted in accordance with protocol 07044 approved by the Animal Care and Use Committee at Texas Tech University Health Sciences Center.The murine chronic wound model utilized in this study has been previously described13. A value of http://www.R-project.org).Relative fitness The data that support the findings of this study are available from the authors on reasonable request, see author contributions for specific data sets.lasR encoded extracellular protease activity is required for optimal growth in QSM24 by comparing growth of antibiotic resistant PA14 (PA14R) and sensitive PA14ΔlasR mutants (Trojan cheats) in QSM without antibiotic selection. After 24h incubation, monocultures of Trojan cheat were significantly lower in density than PA14R monocultures .We confirmed that cultures . We obtaR, they successfully invaded the populations, increasing in relative frequency, even when carrying the costly resistance plasmid . However, unlike our in vitro assays, complete killing of the sensitive populations proved challenging due to the resilient nature of wound extracts. When we treated the wound beds collected from both initially co-inoculated and delayed-mixed infections, we observed a 1–2 log decrease in total bacterial load compared to uninvaded single PA14R infections . In contrast, PA14fections . The redin vivo can induce antibiotic sensitivity in an otherwise resistant infection, highlighting the potential for manipulating antibiotic resistance of a target population through Trojan invasion.These results demonstrate that Trojan invasion in vitro and in vivo populations of PA14R (R (29 when considering the viability of this strategy. These factors include population viscosity, the relative frequency of cheats, population density, the nature of selection, and the mechanism of cheating.We provide experimental evidence supporting the Trojan horse approach as a potential medical intervention strategy. Following the successful invasion of Trojan strains when introduced into both of PA14R , 4 and 5PA14R (R  and 6. Win vivo experiments (lasR cheat invasions of PA1416, but also offer new insights into potential applications of Trojan cheats to combat infections. We demonstrated the feasibility of introducing antibiotic sensitivity to a target population of chronic PA14 infection, leading to increased antibiotic susceptibility in that population (Our findings from the eriments  not onlypulation . Moreove5 CFU/g of tissue30 (While we successfully demonstrated the potential of employing Trojan cheats, it’s important that the total bacterial load after antibiotic treatment remained substantial. Even after reducing a significant portion of the genotypically susceptible Trojan population, the remaining bacterial load exceeded the clinically defined infection threshold for wounds, commonly set at 1031. Using such enhanced cheats would have the added benefit of further reduced virulence, as many cooperative traits are associated with virulence32. To improve the Trojan strain that we used in this work, one could consider using double QS mutants, such as lasR/rhlrR, instead of the single lasR mutant. Furthermore, mutants capable of exploiting both QS and other social traits, like siderophore production15, could be explored. Depending on the specific mechanisms of cooperation and cheating, increasing the extent of cheating could also be considered as a viable option33. Additionally, genome reduction strategies could be applied to enhance overall strain fitness and invasive capabilities34. Since biofilms have been extensively documented to increase antibiotic tolerance36, one can also adopt several approaches to disperse biofilms to improve cheat invasion and antibiotic effectiveness. These approaches include mechanical removal or using molecular dispersal agents like matrix-degrading enzymes, anti-biofilm peptides, and dispersal molecules to disrupt biofilms37.One approach to enhance cheat invasion is through engineering more effective invaders. While single-trait mutants serve as a promising starting point, theoretically, it is possible to create even better invaders that exploit multiple cooperative traits simultaneously39.One major concern in almost all biocontrol methods is the potential restoration of virulence once the target population is invaded by external agents. In the case of antibiotic sensitivity introduction, the primary worry is the Trojan population acquiring resistance through horizontal gene transfer. Preventing this scenario involves implementing fail-safe mechanisms while constructing Trojan cheats. These mechanisms could include synthetic inducible systems for toxin-antitoxin or phage lytic protein expression to trigger programmed self-destruction upon invasion and antibiotic treatmentIn conclusion, this study presents initial experimental evidence demonstrating the potential application of cheats to socially engineer and deliver medically advantageous alleles, such as antibiotic sensitivity, to target populations. The aforementioned technical improvements and the development of robust and secure delivery strategies will facilitate the clinical implementation of Trojan strains as a novel alternative strategy to fight infections.


Poor or suboptimal knowledge of appropriate antibiotic use is a cause for global concern and little is known about Central Asian countries. Therefore, this survey is aimed at evaluating awareness about antibiotic use and resistance among the adult population of Kazakhstan. A cross-sectional study of a random sample was conducted between October 2021 and February 2022 among 727 individuals without medical education and followed the methodology described in the WHO report “Antibiotic Resistance: Multi-country public awareness survey”. Half of the respondents (50.4%) received antibiotic therapy within the last 12 months, 40.1% had no prescription for this and 40.4% received no advice from a medical professional. Nearly two-thirds of respondents (65.3%) never heard about antibiotic resistance and 57.2% believed that it is worth requesting the same antibiotic if it helped to treat a similar condition previously. In general, knowledge about antibiotic use proved to be low in 82.1% of respondents and 91.9% agreed with the statement that a common cold requires antibiotics. There is a need for awareness-raising campaigns to improve the knowledge about antibiotic use and resistance in the population of Kazakhstan. Since the discovery of the first antibiotic (penicillin) by Alexander Fleming in 1928, antibiotics have revolutionized modern medicine by making previously incurable infections and conditions, including pneumonia and other life-threatening bacterial infections, treatable. Today, many routine medical procedures, such as cesarean section, appendix removal, and chemotherapy, rely on effective antibiotics to prevent common infections from becoming fatal. However, decades of misuse of antibiotics and abuse by doctors and patients (to treat mild ailments) and farmers (to promote growth in agriculture and aquaculture) have led to the emergence of antimicrobial/antibiotic resistance (AMR or ABR), which seriously threatens the health of humans, animals, and the environment [Despite a slight reduction in the consumption of antibiotics for systemic use, the irrational use of these drugs remains prevalent in Kazakhstan. This is due to the over-the-counter availability of 27.5% of antibiotics and excessive prescription by medical professionals, where 29.9% of all medications prescribed are antibiotics . AdditioThe excessive prescription of antibiotics can have significant financial implications for individuals and healthcare systems. When antibiotics are over-prescribed, not only can they become less effective due to the development of antibiotic resistance, but they can also cause side effects that lead to further medical expenses. As antibiotic resistance grows, the cost of treating infections increases, as more expensive and effective drugs are required. This can put a strain on health insurance providers and government healthcare programs, which may struggle to cover the cost of these treatments. The bulk of healthcare provided to the population of Kazakhstan is reimbursed from the state-owned social health insurance fund. Out-of-pocket payments contribute to over-the-counter sales and unnecessary prescriptions may affect the financial stability of the poorest population stratum .Being concerned about the growing trend of antibiotic consumption, the WHO developed a global action plan to combat antibiotic resistance which urges all countries to increase public knowledge about antibiotics and antibiotic resistance through effective information and communication campaigns . Thus, iFurthermore, a greater proportion of females (83.2%) disagreed with the notion that it is advisable to use the same antibiotics as a friend or family member who previously treated similar symptoms or disease, compared to males (65.7%). It is noteworthy that while the vast majority of both males and females obtain antibiotics from a medical store or pharmacy, none of the females and 1.8% of males stored antibiotics from a previous time. Lastly, there was a significant difference between genders in seeking advice from healthcare professionals regarding antibiotic use. Specifically, 63.7% of females sought advice from healthcare professionals, while only 46.8% of males did so.Between-group comparisons of individuals aged 24 years and younger and their older counterparts  are of ip = 0.039). Individuals with higher education heard about antibiotic resistance insignificantly more often than those with lower education (37.5% vs. 31.2%).According to The between-group comparisons based on income level are presented in In general, the level of knowledge regarding antibiotic use was found to be inadequate. A total of 12 questions were asked, and respondents’ knowledge was considered low if they answered six or fewer questions correctly, and good if they answered seven or more correctly. The vast majority of study participants (82.1%) had low knowledge about health problems that can be treated with antibiotics, with only 17.9% answering seven or more questions correctly.This survey aimed to evaluate the level of awareness regarding antibiotic use and resistance among the adult population of Kazakhstan. Half of the respondents (50.4%) received antibiotic therapy within the last 12 months, of which 40.1% had no prescription and 40.4% did not receive advice from a medical professional on how to take them. Nearly two-thirds of respondents (65.3%) had never heard of antibiotic resistance, and 57.2% believed that it is acceptable to request the same antibiotic if it had helped to treat the same symptoms previously. In general, knowledge about antibiotic use for specific health conditions was found to be low in 82.1% of the study participants.Our study findings are consistent with reports from other Eastern European countries. A study by Zajmi et al. in Kosovo showed that more than half of the respondents 58.7%) used antibiotics in the past year, and a quarter (25.0%) took them without a physician’s prescription. Notably, 42.5% of respondents believed that viral infections could be effectively treated with antibiotics % used an. In SerbTo mitigate the problem of antibiotic resistance, the sale of antibiotics without a prescription should be prohibited, and self-medication should be discouraged. Nevertheless, it is a common practice in many developing countries to sell antibiotics upon a patient’s request. A qualitative study from India confirmed that pharmacists readily admitted to selling antibiotics over the counter and were generally unaware of the issue of antibiotic resistance . In DamaPoor or suboptimal knowledge of appropriate antibiotic use is another cause for global concern, as it significantly contributes to antibiotic resistance. This includes awareness of the spectrum of diseases that can be treated with antibiotics, the duration of therapy, and understanding when it is appropriate to stop taking them. Akhund et al. conducted an online survey in Pakistan and found that out of 1132 participants, 837 (73.9%) believed that it is possible to stop the course of antibiotics whenever they feel better, and 505 (44.6%) were convinced that frequent and unnecessary use of antibiotics reduces their effectiveness. Notably, 157 (13.9%) of the participants did not adhere to the duration of treatment recommended by a doctor. As many as 467 (41.3%) of the respondents reused antibiotics left over from a previous prescription when experiencing similar symptoms . In our Self-medication with antibiotics is becoming widespread, and many people are convinced that they can use the same antibiotic for a condition with similar symptoms. In this study, more than half of the participants (57.2%) thought it is acceptable to buy the same antibiotic or request it from a doctor if it helped them to get better when the same symptoms were present before. In a study from Romania, 10.34% of respondents took antibiotics following recommendations of a family member or friend, and 22.9% used the same antibiotic prescribed by a doctor at the last visit . In geneA striking finding of this study is that a high proportion of the population (91.9%) is ready to take antibiotics for a common cold. This is probably best explained by the incorrect identification of bacteria as the most common cause of upper respiratory tract infections. The same misconception is shared by the populations of other countries, although to a lesser extent. In Germany, 10.5% of patients asked for antibiotics to treat common colds , and 14%Another commonly shared misbelief is that antibiotics can be used to treat non-bloody diarrhea. According to a systematic review by Carter et al., low- and middle-income countries tend to over-rely on antibiotics in the treatment of childhood diarrhea as they are prescribed in 10–77% of cases . AnotherThere is a need for awareness-raising campaigns to improve knowledge about antibiotic use and resistance in the population of Kazakhstan. This could be done via social media, and the importance of seeking professional advice before initiating antibacterial therapy has to be emphasized. The country’s medical community, in particular the primary healthcare sector, should adopt the best health education strategies, and policymakers have to reinforce their efforts to control the over-the-counter sale of prescription medications. Currently, there is a lack of recognition of antibiotic misuse in Kazakhstan, and therefore it is important to sensitize all stakeholders. The country’s stakeholders can benefit from the strategies proposed by World Antimicrobial Awareness Week, which was designed to enable communication with a focus on effective approaches to prevent and mitigate antimicrobial resistance .The current study has several limitations primarily stemming from its cross-sectional design. One of the main limitations is the inability to establish causal relationships between knowledge and practice of antibiotic use and individual participant characteristics. Originally, our intention was to sample healthy individuals visiting outpatient facilities for routine check-ups or accompanying their diseased relatives. However, we quickly realized that few adults attend healthcare facilities for preventive purposes. As a result, the majority of participants in our study were accompanying persons, leading to observed shifts in gender and age. Therefore, this sample is not representative of the entire population of Kazakhstan and should be considered as a pilot study. Despite these limitations, our study has many strengths. It is based on a reasonably large sample of the adult population enrolled from a typical region of the country. Furthermore, it is the first study to investigate awareness about antibiotic use and resistance among the population of Kazakhstan and can serve as a benchmark for future research.A cross-sectional study was conducted between October 2021 and February 2022, using a random sample of adult residents without medical education in the East Kazakhstan region. This region was selected as a representative area of the country, based on socio-economic indicators, and had an estimated population of 1,349,400 in 2022 . The samWe recruited healthy individuals attending regional outpatient facilities for routine check-ups or as accompanying persons, using a systematic random sampling method. Out of 35 outpatient polyclinic organizations located on the territory of the East Kazakhstan region, 15 were sampled to represent two cities—Ust-Kamenogorsk and Semey (5 facilities from each city) and rural territories. We sampled 5 rural outpatient facilities located in the region’s north-west (Beskaragay district), north-east (Altai district), center (Zharma district), south-west (Ayagoz district), and south-east (Tarbagatay district). A maximum of 15–20 individuals were recruited from each clinic per day. The full WHO questionnaire was administered through face-to-face interviews. Age below 18 years, presence of medical education, and unwillingness to participate in the study served as the exclusion criteria.This survey followed the methodology described in the WHO report “Antibiotic Resistance: Multi-country public awareness survey” . The quep-value of 0.05 and below was considered statistically significant.The study data were analyzed using the Statistical Package for Social Sciences (SPSS) version 20. As a first step of statistical analysis, the type of data distribution was evaluated for continuous variables by the Kolmogorov-Smirnov test. As the distribution of data proved to be different from normal, the continuous variables were presented as the median with interquartile ranges and non-parametric tests were used to compare differences between the groups. Categorical variables were presented as the frequency with percentage and Pearson’s chi-square test was utilized for between-group comparisons. A To facilitate educational level comparisons, we classified educational attainments into two categories: pre-tertiary  and tertiary  degrees). To elucidate the differences between groups based on place of residence, we compared individuals living in urban and suburban areas with individuals living in rural areas. To compare responses from different income groups, we divided all respondents into two groups based on their monthly income levels: a lower income group  and a higher income group . At the time of the survey in 2021, 150,000 Tenge was approximately equivalent to 350 US dollars .This study has been approved by the Ethics Committee of Semey Medical University with registration code 2, dated 28 October 2020.This study sheds light on the level of awareness and behavior regarding antibiotic use and resistance in Kazakhstan, with a focus on gender, age, education, place of residence, and income level. The findings reveal significant differences in the duration since respondents last received antibiotics, prescription patterns, beliefs about antibiotic use, and seeking advice from healthcare professionals between males and females. Moreover, age, education, and income level were associated with differences in antibiotic use and awareness. Our study highlights the importance of increasing awareness and promoting the appropriate use of antibiotics among different population groups in Kazakhstan. Further research and interventions are needed to address the identified differences and improve antibiotic use practices in the country.