Background Antibiotic resistance is a global problem, and the World Health Organization has made this problem one of its priorities for solving. Therefore, a survey was carried out to investigate the knowledge, attitude, awareness, and perceptions of antibiotic resistance among physicians and to assess the correlation between the knowledge of antibiotic resistance and their years of experience in some Palestinian hospitals.Methods This was a cross-sectional study that targeted physicians who are working in different healthcare facilities in Hebron and Bethlehem governorates. We used a questionnaire for data collection. The questionnaire consists of 42 questions to measure the knowledge, attitudes, awareness, and perceptions of antibiotic use and resistance.Results The response rate was 91.33% (137 of 150 physicians completed the questionnaire). The participants' ages ranged from 25 to 56 years, and the majority were males  working in governmental hospitals . Of physicians, 69.3% (n = 95) perceived antibiotic resistance as a very important worldwide problem, while 54.7% (n = 75) perceived a very important problem in the country, 54.0% (n = 74) a very important problem in their hospital, and 59.1% (n = 81) a very important problem in their departments. Methicillin-resistant Staphylococcus aureus was the most known antibiotic-resistant bacteria followed by extended-spectrum beta-lactamases. Only 47 physicians (34.3%) think that antibiotics are not used appropriately in their department. Respondents' physicians showed that the development of antibiotic resistance was due to various factors that include self-medication n= , overuse of antibiotics , and uncompleted treatment . Senior specialists/consultants were found to be more knowledgeable about antibiotic resistance.Conclusion In our survey, physicians showed variable knowledge and perceptions of antibiotic resistance. Introducing educational programs is necessary to improve their understanding and perceptions of antibiotic resistance, as well as their attitude toward antibiotic use. According to the World Health Organization (WHO), antibiotics are the most frequently prescribed drugs worldwide.According to the WHO, more than 50% of medicines are incongruously prescribed, dispensed, and utilized. Furthermore, around half of the patients take their medicines incorrectly.We conducted a cross-sectional study among physicians in the southern part of the West Bank (Hebron and Bethlehem governorates) in March 2021; the study was conducted for two weeks. We included governmental and nongovernmental/private hospitals. Among the selected hospitals, four were governmental  and the other four were nongovernmental/private . These hospitals are located in different cities in the two governorates.A self-administered questionnaire was distributed in the hospitals among physicians of all levels who prescribe antibiotics including general physicians, medical officers/ residents , senior residents/ specialists, senior specialists/ consultants, and also the physicians working at departments of surgery, internal medicine, pediatrics, emergency, intensive care units, obstetrics, and gynecology. One-hundred thirty-seven of 150 physicians in total were found and considered eligible to participate in our study (response rate: 0.91). Physicians who have a medical degree and doing internship training but do not have a license to practice medicine were excluded from the study because they do not often prescribe antibiotics during their training (13 physicians were excluded from the study).We used the questionnaire developed for assessing antimicrobial use in the Korle-Bu Teaching Hospital.The questionnaire is divided into four parts, the first included demographics and general information data . The second part included questions assessing the perceptions on the importance of antibiotic resistance as a problem at the global, national, hospital, and department levels. It used 4-point Likert-scale response options . The questions using the 4-point Likert scale were dichotomized such that correct answers for KAP and preparedness were noted if a participant agreed or strongly agreed  in a positive question or disagreed or strongly disagreed  in a negative question.The third part of the questionnaire acquired information about perceptions on causes of antibiotic resistance, which are , and to what extent each contributes to the development of antibiotic resistance. The fourth and last part included questions on knowledge and attitude on antibiotic-resistant bacteria of public health importance . To assess knowledge of the prevalence of antibiotic-resistant bacteria of public health importance, the last question asked the physicians to choose the correct order of prevalence for MRSA, ESBL, CRE, and VRE.Printed questionnaires (Arabic version) were distributed among physicians for 2 weeks in March 2021. Each questionnaire included a cover letter explaining the purpose of the study and assuring participants that the privacy and confidentially of collected data will be maintained.The entire study data were entered in Microsoft Excel and analyzed using the Statistical Package for Social Sciences Version SPSS 26 . Frequency and percentages among descriptive statistics were used to describe the data. Proportions were calculated for categorical variables; mean and standard deviations were calculated for continuous variables. Descriptive statistics provide summary information about data, for example, the number of respondents who are male or female. Here we collected this information from 137 respondents out of 150 physicians (response rate of 0.91).p-value less than 0.05 were taken as statistically significant.The KAP of antibiotic resistance among physicians scores according to demographic characteristics were compared with independent- samples t-test and one-way analysis of variance as appropriate. Finally, the Pearson correlation (R) test was used to test the relation between the total score of knowledge and mean scores of antibiotics attitude and practice. The strength of the correlation between knowledge, attitude, and practices of antibiotics is described as follows: 0.0–0.19 “very weak,” 0.20–0.39 “weak,” 0.40–0.59 “moderate,” 0.60–0.79 “strong,” and 0.80–1.0 “very strong.” In all cases, theAbout 91% of four different specializations have completed the survey as 137 of 150 physicians filled out the questionnaire. Their age ranged from 25 to 56 years and 116 participants were males and 21 females. The mean age of the respondents was 32.40 ± 3.15 years. Four different specializations of the physicians participated in the study; 22 (16.1%) general physicians, 54 (39.4%) medical officers/residents, 43 (31.4%) senior residents/specialists, and 18 (13.1%) senior specialists/consultants. Moreover, the average years of practice among the study participants were 6.67 ± 2.35 years. Fifty of them (36.5%) attained continuing education/workshops related to antibiotic resistance in the last 3 years, with an average of 14.6 hours per year .n = 130/137) and the lowest knowledge of CRE .Of physicians' respondents, 95 (69.3%) perceived antibiotic resistance as a very important worldwide problem, 75(54.7%) as a very important problem in the country, 74(54.0%) as a very important problem in their hospital, and 81(59.1%) a very important problem in their departments . The knDifferent factors were significantly perceived by the majority of physicians as very important causes of antibiotic resistance  that isp-value was 0.04 that is less than 0.05. We use the post hoc tests (Tukey's) to specify groups that differed to eliminate any ambiguity. It was noticed that the knowledge about antibiotic resistance asserts that the senior specialist/consultant is more knowledgeable. We have also found that there is no statistically significant difference in knowledge on antibiotic resistance related to the variable kind of hospitalsas the p-value is 0.56 that is higher than 0.05.To understand the relationship between the duration of practice and the knowledge about antibiotic resistance and its effect on public health, we used the Pearson correlation measure. Correlation illustrates the direction and strength of a relationship between two variables. For example, the relationship between the duration of practice and the knowledge about antibiotic resistance was determined; there is a statistically significant difference in knowledge about antibiotic resistance related to the variable level of training as theEven though antibiotics have a great advantage in reducing the incidence and spread of infectious diseases worldwide, they are frequently misused, leading to the spread of multiple resistant bacterial strains. As a result, the antibiotics became ineffective and infections persist in the body, increasing the risk of spreading to other people and leading to longer hospital stays higher medical costs, and increased mortality. Undoubtedly, a better understanding of the importance of antibiotic resistance and its healthcare drawbacks can improve physicians' practices in the field of antibiotic prescriptions, and thus alleviate the spread of bacterial-resistant strains.Our study was conducted in Southern West-Bank Hospitals  from a resource-limited setting. Results showed a variation in the knowledge of antibiotic resistance among physicians concerning the cause and the significance of the problem. Our results showed that the knowledge about antibiotic resistance asserts that senior specialist/consultant is more knowledgeable with a high mean of 1.45 followed by general physicians with a mean of 1.35, then senior specialists (1.3) and medical officers/ residents (1.26). These findings are similar to those from another study carried out in a Ghanaian teaching hospital, which showed that senior physicians are more knowledgeable than junior physicians regarding the issue of antibiotic resistance.The majority of questioned physicians perceived antibiotic resistance as a global problem, while half of them considered it a problem in their country and hospital. Approximately 66% perceived it as a problem in their departments. This matches the findings from other studies where respondents agreed that antibiotic resistance is an important problem but less in their departments compared to national and global settings.The causes of antibiotic resistance in our research study were identified and self-medication was the most perceived factor by the physicians causing antibiotic resistance, then the uncompleted antibiotic therapy and overuse of antibiotics in the population, which match the findings of a survey carried out in the Democratic Republic of Congo to investigate knowledge, attitude, and practice among medical doctors and students.p = 0.003). This can be explained by the fact that the lower the years of practice, the closer the physician is to the years of education, and thus the greater amount of information he/she will remember. Moreover, results from a study carried out in Malaysia concluded that a longer duration of practice does not necessarily ensure good knowledge of antibiotic prescribing and antibiotic resistance.We found a weak positive correlation between the duration of practice and knowledge about antibiotic resistancet(135)= 0.574,p = 0.56). To improve antibiotic use and control antibiotic resistance in the hospital, there is a need to increase education on antibiotic resistance among physicians with an emphasis on junior physicians. Results from our study show the importance of increasing activities aiming to raise physicians' awareness regarding antimicrobial resistance and its major consequences on public health. This can be achieved by encouraging them to attain continuing education/workshops related to antibiotic resistance as only 37% of our respondents have ever attended workshops related to that topic. It is also important to focus on junior residents since they showed inadequate knowledge of antibiotic resistance and to incorporate this issue into the clinical education programs of medical schools. Identification of concerns regarding inappropriate antimicrobial prescribing will enable specific initiatives and approaches to improve future antimicrobial use and reduce antimicrobial resistance in Palestine.No difference was found between governmental and private and nongovernmental hospital doctors in their knowledge (There were several limitations in our study, we are not sure if respondents verified their answers with each other, or if they commissioned others to fill out the questionnaire because, like other self-administered questionnaires, our survey was completed by physicians without supervision to ensure privacy, and to allow physicians to fill it within their free time. Moreover, there is a chance that respondents have been biased in their answers to support the hospital they work at, and gave more socially acceptable answers. As a result, and to influence and guide future interventions, more studies related to antibiotic resistance need to be conducted in this area to prove the reliability of the survey in the West Bank.Antibiotic resistance is one of the biggest threats to global health. This study has provided information about prescribing attitudes and practices of physicians working in both governmental and nongovernmental hospitals in South Palestine. Knowledge regarding antibiotic resistance was good among those physicians with junior physicians being the least knowledgeable. Thus, attention has to be focused on conducting lectures and workshops to enrich physicians' knowledge about antibiotic resistance, and as knowledge is the guide of attitudes and practices, this enrichment can lead the community into a better health condition with fewer bacterial-resistant strains threatening our survival. However, for the containment of antimicrobial resistance, it needs different aspects, such as publishing information about antimicrobial resistance rates in Palestine, the development and use of guidelines for antibiotics, and emphasizing the importance of having regular educational activities regarding the appropriate use of antimicrobials.


MET) gene is of importance in lung cancer development, and mutations in this gene are related to poor prognosis. Consequently, it is important to develop new treatment options that specifically target the MET protein. In this systematic review, we aimed to summarize the existing knowledge on the impact of MET on lung cancer development and the effect of currently available medications. Our hope is that the findings of this systematic review will deepen the understanding of other researchers, possibly providing a guiding hand as to what may be most interesting to focus on in future research projects on this subject.Lung cancer is the type of cancer that kills the most people in the world each year. It is difficult to diagnose lung cancer in the early stages and there are only few treatment options available once the cancer has spread. The mesenchymal epithelial transition factor  than those with a lower mutational burden. Conversely, two studies reported no beneficial effect from adjunctive treatment with a MET targeted therapy. Given these findings, there is an urgent need to identify effective therapeutic strategies specifically targeting the MET gene in lung cancer patients.Lung cancer represents the leading cause of annual cancer-related deaths worldwide, accounting for 12.9%. The available treatment options for patients who experience disease progression remain limited. Targeted therapeutic approaches are promising but further understanding of the role of genetic alterations in tumorigenesis is imperative. The  EGFR) and less than 25.0% of patients are tested for alterations in the mesenchymal epithelial transition factor gene (MET) .MET gene alterations varies greatly between the articles included in this systematic review. In a prospective clinical trial by Palmero et al., genetic testing of treatment naïve NSCLC patients was carried out to identify genetic alterations. Next generation sequencing (NGS) of circulating cell free tumor DNA in blood was compared to standard-of-care tissue-based biopsy testing in order to determine the patients’ mutational burden. The results showed no difference between the methods’ ability to identify mutations in NSCLC. Out of 186 tested patients, 11.0% harbored MET exon 14 skipping mutations (METex14) and 22.0% harbored amplification of the MET gene . Tw. TwMET atudy arm . This daMET antibody mixture entitled Sym015 was investigated. The study included 57 patients divided into three different treatment arms, one with KRAS wild type patients with MET amplifications, one with MET amplified NSCLC, and one where the patients harbored METex14 deletions. The outcome measure presented was the ORR and the results showed that the KRAS wild type cohort had 0.0% ORR while the MET amplified and the METex14 deletion cohort both had an ORR of 25.0% [MET gene rather than cohorts of mixed cancer genotypes. This will make future results more easily interpreted and clinically useful.One other study had available preliminary results at the time of writing this systematic review . In this phase I/II study, the monoclonal anti of 25.0% . A vast MET expression varies between the included trials, with some research groups applying IHC, some performing NGS, and some using different methods. A strength of this review is the variation in location of the included trials, leading to more generalizable and widely applicable conclusions. The exclusion criterion of a publication year prior to 2013 might be considered a limitation; however, this also ensured that only current results were included in this systematic review.The most apparent limitation of the articles included in this review is the lack of control groups in most of the phase I trials, leading to difficulties in interpreting the results. Furthermore, some articles have a low number of included patients and overall, the number of participants varies greatly. Since the included studies have a high grade of variation in patient characteristics, such as age, gender, and smoking history, the comparison between the studies’ results need to be interpreted with some caution. As the targeted therapies are tested on different patient categories, with varying cancer genotypes, it is difficult to compare the presented results and draw conclusions on the efficacy of the investigated drugs. On the same note, the definition of the criteria for what is considered aberrant MET positive NSCLC patients but no convincing results in cohorts with mixed MET mutational statuses. The studies on crizotinib lacked control groups for comparison of the outcome measures. Many ongoing trials on pipe-line targeted therapies exist, which are investigating anti-MET agents on clearly defined cohorts of patients with aberrant MET expression. This systematic review summarized the current status of publications on the MET gene’s implications in lung cancer development and the status of existing and up and coming targeted therapy options. More research is needed and should be encouraged to fully understand how, when, and to whom these drugs should be recommended in order to improve patient outcomes.It remains difficult to compare different targeted therapies and to draw conclusions regarding their potential place in the future treatment panorama. The inter and intra study variation in cohort composition and included cancer genotypes is large. Onartuzumab has shown prolonged PFS and OS among


The mechanical properties, water permeability, porosity, and chemical composition of pervious concrete under two curing conditions were investigated and compared. The effects of CO2 curing on the properties of pervious concrete with different paste–aggregate ratios were derived. Through mechanical experiments, it was revealed that early CO2 curing can enhance the mechanical strength of pervious concrete by about 15–18%. Meanwhile, with the increase in the paste–aggregate ratio, the improvement effect induced by early CO2 curing became more significant. The water resistance of carbonated concrete was not significantly reduced. And with the increase in the paste–aggregate ratio, the carbonation degree of pervious concrete was reduced; the differences in porosity and water resistance became less significant when the paste–aggregate ratio exceeded 0.39. Micro-structural analysis shows that the early CO2 curing reduced both total porosity and the volume of micropores with a pore diameter of less than 40 nm, while it increased the volume of pores with a diameter of more than 40 nm. This is also the main reason that the strength of pervious concrete under early CO2 curing is higher than that without CO2 curing. The effect of varying paste–aggregate ratio and curing methods adds to the limited knowledge of the performance of pervious concrete.To improve the comprehensive performance of pervious concrete, the properties of pervious concrete in different paste–aggregate ratios were subjected to both early CO As a green infrastructure solution for urban areas, pervious concrete has received significant attention in recent years as the permeable pavement material used by many cities worldwide ,2. EspecCarbonization curing, as a new green curing technology, can not only improve the performance of cement-based materials, but also achieve the carbon dioxide capture and reduction in the current global greenhouse effect ,10. Thro2 curing, as a sustainable method, has been studied by researchers both domestically and internationally to improve the performance of pervious concrete. Tian et al. [2 curing could improve the compressive strength of pervious concrete, especially at early ages. In addition, CO2 curing could significantly improve the freeze−thaw and leaching resistance of pervious concrete. Macmaster [2 curing can effectively improve the strength of pervious concrete. Chen [2 curing can significantly improve the early compressive strength of pervious concrete. Compared to ordinary concrete, the carbonation degree of cement stone inside pervious concrete is higher, and the internal and external carbonation is more uniform. Fang et al. [2 curing, thereby improving the density of the aggregates. Wu and Xu [COn et al.  believesn et al.  found thacmaster  proposedacmaster  also proacmaster  believeste. Chen  found thg et al.  improvedu and Xu  exploredu and Xu  greatly u and Xu  conducteu and Xu  studied 2 curing can improve the strength of pervious concrete, showing good carbon fixation ability. However, comprehensive reports on the effects of CO2 curing on the properties of pervious concrete in different paste–aggregate ratios are still limited. Based on this, this paper aims to study the influence of paste–aggregate ratio on the strength, permeability, porosity, and composition of pervious concrete with and without early CO2 curing treatment, to analyze how early CO2 curing could influence the performance of pervious concrete in different paste–aggregate ratios. The perpetual fixation of carbon dioxide in pervious concrete contributes to the sustainable development of the construction. Through this study, it is expected to provide more theoretical and technical support for the preparation of high-performance pervious concrete and ecological environment construction while capturing more carbon dioxide at the same time.In summary, early COThe cement was ordinary Portland cement (P·O42.5) created by Hailuo Cement Co., Ltd., in Anqing, China. Its chemical composition and performance parameters are shown in The pervious concrete design method based on the optimal paste–aggregate ratio  is used 2 curing method, respectively.Based on the mix proportions in 2 curing was carried out under accelerated carbonation conditions referring to the Testing Methods for the Long-term Performance and Durability of Ordinary Concrete (GB/T 50082-2009) [The uncarbonated curing method was used to cover the specimens with a layer of thin membrane, to prevent it from coming into contact with carbon dioxide in the air. The early CO82-2009) . During At a curing age of 7 days and 28 days, both carbonated and uncarbonated specimens were taken out of box to determine the compressive strength, according to the Standard Specification for test methods of mechanical properties of ordinary concrete (GB/T 50081-2002)  was usedThe water permeability coefficient of pervious concrete was measured via the constant head method in terms of the Chinese industrial standard Water Permeable Brick (JC/T 945-2005) , as showThe effective porosity of pervious concrete is determined according to Technical Specification for Pervious Concrete Pavements (DB11/T 775-2010) . In addiThe specimens used to test the degree of carbonization were taken out of the curing room at a curing age of 26 days and baked at 60 °C for 48 h. After drying, one side of the specimen was left to draw parallel lines along the length direction at a spacing of 10 mm as the measurement point for the predetermined carbonization depth. The other surfaces are sealed with heated paraffin. During the test, 1% concentration of phenolphthalein absolute ethanol solution was sprayed on the split surface.The pervious concrete specimens cured to 28 days were removed. The paste on the surface of the aggregate was scraped with a blade and then ground with an agate mortar to prepare microscopic phase analysis samples. Both X-ray diffraction (XRD) and Fourier Transform Infrared Spectrum (FTIR) were used to determine the phase composition characteristics of the pervious concrete paste. Additionally, the BET analysis technique  was used2 curing improved the compressive strength by 13~18% and increased the flexural strength by 5~24%. With the increase in the paste–aggregate ratio, the improvement rate by CO2 curing increased first, and then decreased. For flexural strength, the maximum increase rate (23.55%) for early CO2 curing occurs with a paste–aggregate ratio of 0.43. This is because the increase in the proportion of paste contributes to the strength development of the pervious concrete matrix, after early CO2 curing. However, the maximum increase rate of compressive strength (17.45%) was caused by concrete in a paste–aggregate ratio of 0.39. The increase in the paste–aggregate ratio could improve both flexural strength and compressive strength under two curing conditions, but the positive effect of carbonation becomes less significant when the paste–aggregate ratio increases to a certain value. The phenomenon is more obvious in the comprehensive strength increase rate profiles. In summary, early CO2 curing has more significant effects on the development of the flexural strength of pervious concrete, which is consistent with the conclusion in literature that early CO2 curing can improve the mechanical properties of concrete [2 curing accelerates the formation of CaCO3 and increases the average chain length of calcium silicate hydrate, which is an important reason why early CO2 curing can improve the mechanical properties of cement stone. As we all know, flexural strength is an important mechanical property index of pavement structural materials, so this result provides the potential benefits of CO2 curing on performance improvement of pervious concrete.To further compare the effects of paste–aggregate ratio and curing methods on property, the mechanical strength change rate of 28-day carbonated concrete is calculated and summarized in concrete . Early C2 curing for 28 days. With no early CO2 curing treatment, the deformation under ultimate compressive load decreases as the paste–aggregate ratio increases. For example, when the paste–aggregate ratio is 0.30, the corresponding deformation is 4.52 mm. It is about three times that of pervious concrete with a paste–aggregate ratio of 0.45. It can also be seen from 2 curing is larger than that without CO2 curing, which is consistent with the results in 2 curing treatment can improve the compressive toughness of pervious concrete to a certain extent.To characterize the toughness of concrete under different conditions, both compressive and flexural force–displacement curves before ultimate failure are averaged and plotted for analysis. δ, the area enclosed by OAB is T1; the corresponding abscissa of point D is 3δ, and the area enclosed by OACD is T3. Then, the ratio of the area under the load–displacement curve T3 to the area under the load deflection curve corresponding to the initial crack point T1 is defined as the toughness index I3 to measure the flexural toughness of the pervious concrete, as shown in Formula (1),T1 and T3 (N mm) are the areas of OAB and OACD in 2. The difference is mostly sufficient when the paste–aggregate ratio is 0.3. At this ratio, the flexural toughness index of the pervious concrete with early CO2 curing treatment is 2.0, which is reduced by 12% compared to pervious concrete without CO2 curing . With the increase in the paste–aggregate ratio, the differences in the flexural toughness index between two curing methods become less obvious. When the ratio reaches 0.43, the flexural toughness indexes of concrete under two curing methods are quite close to each other. Therefore, the effects of early CO2 curing treatment or flexural toughness is also related to the paste–aggregate ratio.The final indexes for all specimens at 28 days are calculated and presented in 2 curing treatment is slightly smaller than that without CO2 curing. However, like the pattern in The permeability coefficient mainly indicates the difficulty of fluid passing through the void skeleton . Figure 2 curing treatment is slightly lower than that without CO2 curing. This is because a large amount of CaCO3 formed during carbonation process fills the pore size of cement stone, thus refining its pore structure and making the matrix structure more compact [ compact , which i compact , which c2 curing condition. The black colors refer to concrete, and the white gap in the image reflects the pores. As shown in this figure, as the paste–aggregate ratio increases, the bottom pore of the pervious concrete decreases significantly. When the paste–aggregate ratio reaches 0.43, obvious sedimentation of the paste occurs.The water resistance of pervious concrete is largely determined by the porosity characteristics. In this part, the effects of curing methods on macro-surface porosity of concrete in different paste–aggregate ratios are analyzed. 2 curing treatment shows a decreasing trend. The porosity on the top surface is larger than that on the bottom surface. Besides, the micro-surface porosity under early CO2 curing condition is slightly lower than that under no CO2 curing conditions. Therefore, early CO2 curing treatment can to some extent refine the micro-surface property of pervious concrete, particularly when the paste–aggregate ratio is lower than 0.43.Based on the area covered by white gaps in the image, the macro-surface porosity of 28-day pervious concrete in different paste–aggregate ratios is calculated and listed in 2 curing. After spraying the phenolphthalein solution, the entire surface is a red color. Therefore, by comparing the area with and without red color, the carbonation degree of pervious concrete can be derived.The carbonization degree of each pervious concrete specimen is shown in 2 curing for 28 days. As shown in 2 [own in 2 .2 curing condition, the carbonization degree of the pervious concrete gradually decreases with the increase in the paste–aggregate ratio. The possible reason is that as the paste–aggregate ratio increases, the larger amount of slurry filled between the aggregates increases. Meanwhile, when the ratio of paste aggregate is further increased, the excess slurry slips to the bottom of the specimen, and the phenomenon of sedimentation occurs. In other words, the speed of carbon dioxide entering the specimen slows down, resulting in a decrease in the carbonation degree. This phenomenon explains the less significant effects of carbonation on porosity when the paste–aggregate ratios reach 0.39.Summarizing from 2 crystal enrichment site was selected to compare the changes in hydration products under two curing conditions. 2 for 28 days, which mostly manifested as hexagonal layered calcium hydroxide crystals, with a small amount of gel attached to the surface of the crystals. The adhesion between the crystals was relatively weak. Chen’s research also mentioned relevant conclusions [To analyze the mechanism with microscopic perspective, the SEM electron microscope was used to characterize the microstructure morphology of 28-day aggregate surface slurry under two curing methods. In particular, the Ca(OH)clusions .3, as shown by the arrows in the figure, and the structural strength is further improved. 3 in the slurry. 3 crystals appear between the calcium hydroxide crystals, indicating that the structure has been further improved.2 curing treatment for 28 days are shown in 3 of pervious concrete slurry under early CO2 curing condition is much larger than that without CO2 curing, while the main peak of calcium hydroxide crystal is smaller than that without CO2 curing. This is because the early CO2 curing treatment speeds up the reaction between CaO and CO2, generating more CaCO3. However, the C-S-H gel content in pervious concrete slurry under the early carbonation is lower than that without CO2 curing. 3 peak first increases and then decreases. This is because as the paste–aggregate ratio and the slurry content increase, the internal slurry carbonation degree is less significant, which inhibits the formation of CaCO3.The XRD phase compositions of the pervious concrete slurry with and without early CO2 curing treatment for 28 days are shown in −1 is the stretching vibration of OH in Ca(OH)2. According to −1 disappears, indicating that Ca(OH)2 reacts with carbon dioxide. The absorption peaks near 1390 cm−1 and 1220 cm−1 are the antisymmetric stretching vibration and plane bending vibration of CO32− in calcite, which indicates that calcite is formed by the reaction between Ca(OH)2 and carbon oxide. Compared with the IR spectrum of paste cured without CO2, the shift frequency of IR spectrum cured by CO2 is about 20 cm−1. The blue shift of the absorption band indicates that the calcite crystal increases; that is, the CaCO3 content in the specimen increases.The FTIR spectrums of pervious concrete slurry with and without early CO3. The CaCO3 that appeared during the carbonation process was mainly in the form of calcite. When CaCO3 is formed, SiO2 gel is also produced. When it is fully carbonized, due to the complete decomposition of calcium silicate hydrate, there are only characteristic absorption peaks of SiO2 gel and CaCO3 in the IR spectrum [In summary, in the early carbonation process, the hydrated calcium silicate in the hydrated product was gradually decomposed to form CaCOspectrum .P0 is the saturated vapor pressure of the adsorbate at the adsorption temperature; mV is the saturated adsorption capacity of the monolayer; C is the Bet equation constant, its value is exp{(E1 − E2)/RT}, where E1 is the adsorption heat of the first adsorption layer.To analyze the effects on pore structure of pervious concrete at the microscopic perspective, the BET theory is applied to characterize the microscopic pore. The adsorbate in Bet pore distribution analysis adopts nitrogen adsorption, and the temperature of the analysis tank is 195–850 °C, while the heating rate is 10 °C/min. The adsorption and desorption isotherms are shown in P/V(P0 − P) and P0/P are plotted, a straight line is obtained. The slope of the straight line is m = (C − 1)/(mCV), and the intercept on the vertical axis is b = 1/(mCV), where mV, i.e., the saturated adsorption capacity of the single layer can be calculated asIt can be seen from Formula (2) that when the experimental data of physical adsorption mA(nm2), this mA is the surface area occupied by the adsorbent molecule on the surface of the adsorbentAN is Avogadro’s constant (6.02 × 1023); Sg is the specific surface area (m2/g). Emmet and Brownauer [2, so Formula (3) can be simplified to obtain the Bet nitrogen adsorption specific surface area asAssuming that the average cross-sectional area of each adsorbed molecule is rownauer  have pro2 meets the experimental requirements. The specific surface area of pervious concrete slurry cured by early carbonization is 5.25 cm2/g, which is 5.85% higher than that cured without carbonation. This is because with early CO2 curing treatment, CO2 directly reacts with C-S-H gel and the generated CaCO3 mainly exists in the form of calcite; the calcium–silicate ratio of C-S-H gel decreases, and the specific surface area increases after carbonization. The Barrett–Joyner–Halenda model was adopted to further analyze the pore size distribution. The basic principle of the model is introduced in the literature [2 curing treatment will reduce the micropores with a pore size less than 40 nm, and the mesopores with a pore size greater than 40 nm will increase, but the overall pore volume is reduced with early CO2 curing treatment.Through the principles above, the specific surface area of the pervious concrete slurry is measured by calculating the slope and intercept of the straight line, as shown in terature . The res(1)2 curing are higher than those without CO2 curing at all paste–aggregate ratios.With the increase in the paste–aggregate ratio and curing time, the mechanical properties of pervious concrete are improved to some extent under both curing conditions. In addition, the mechanical properties of pervious concrete after early CO(2)2 curing can improve the compressive toughness of pervious concrete but reduces its flexural toughness. However, its influence on flexural toughness decreases with the increase in the paste–aggregate ratio.Early CO(3)The permeability and effective porosity of pervious concrete decrease significantly with the increase in the paste–aggregate ratio. Additionally, pervious concrete cured with early carbonization has poor water permeability and smaller porosity due to its denser structure.(4)2 curing conditions, the degree of carbonization in pervious concrete gradually decreases with the increase in the paste–aggregate ratio.Under early CO(5)2 in pervious concrete directly reacts with C-S-H gel under early CO2 curing treatment, and CaCO3 mainly exists in the form of calcite. With the increase in the paste–aggregate ratio, the formation of CaCO3 is inhibited due to the decrease in carbonation degree in pervious concrete. At the same time, early CO2 curing condition will reduce the pore size by less than 40 nm in pervious concrete and increase the pore size by greater than 40 nm. It is worth mentioning that the overall pore volume is greater in pervious concrete.In the research on micro mechanisms, it was found that COTo improve the comprehensive performance of pervious concrete, this study compared the mechanical properties, water permeability, porosity, and chemical composition of pervious concrete under different paste–aggregate ratios and two curing conditions. Below, we summarize the main conclusions of this paper based on the aforesaid findings:2 curing can help improve the comprehensive performance of pervious concrete, which has guiding significance for the preparation of high-performance pervious concrete and the construction of the ecological environment. In any case, further research is required to quantify and improve the carbonation degree of pervious concrete in different paste–aggregate ratios, which is the focus of our future research content.The results indicate that early CO


The purpose of this work was to develop a substitute material model for the analysis of reinforced concrete structures. This paper presents proposals to solve the problem of limited calculation time, both to perform simulation models and to perform effective numerical or analytical analyses of structural elements in order to achieve results consistent with experimental results. Achieving this aim is conditional upon the determination of the material model parameters, taking into account the type of structure, the system of reinforcement, and the static strength–deformation parameters of the component materials. A universal procedure is proposed for determining the parameters of the substitute material model on the basis of the homogenization function, in which the homogenization coefficient is assumed as being equal to the effective reinforcement ratio of real reinforced concrete structural elements. In addition, the introduction of a new concrete constraint coefficient to this procedure, which corresponds to the proportionality coefficient of biaxial to uniaxial compressive strength, is proposed. On the basis of the conducted comparative analyses, the possibility of using the hypothetical substitute material model for the design of building elements and structures was confirmed. The average values of the obtained results for individual research series did not differ from the experimental results by more than 8.5%, for both the numerical and analytical models. The behavior of the numerical model of a structural system depends strictly on the material model used, which is an integral part of the procedure for modeling the structure of a building. Performing a spatial analysis of the behavior of a structural element or the entire structure of a building requires a complex numerical model. The difficulty of spatial modeling of reinforced concrete elements using high-strength concrete (HSC) and ultra-high-performance concrete (UHPC) lies in the separate modeling of structural materials (concrete and reinforcing steel) with their contact conditions. With a large number of reinforcing bars, each of them must be defined by its interaction with concrete, which increases the number of nodes used in the computational analysis and the analysis time. Due to the difficulties in modeling the properties of complex structural systems with heterogeneous reinforced concrete structures, an approach based on the homogenization of reinforced concrete is used. In one study , the resIn another study , an apprModeling the behavior of structural elements with increased mechanical and strength parameters ,4,5,6 reFurthermore, in , the autIn another study , solutioIn , the autThe authors of  presenteThe authors of  presentew/c) to the water–cement ratio, the silica–cement ratio, cement class, and aggregate characteristics [This study proposes a methodology based on the expected compressive strength of HSC and UHPC concretes. The basic formula for designing the compressive strength of concrete is Feret’s law, which relates the compressive strength of high-strength concrete (HSC)  to the substitute material model, depending on the adopted type of reinforcing steel  [In the substitute material model, it is also possible to take into account the strain rate effect by introducing the generalized dynamic strength coefficient mula (4) :(4)fhcd=cG and tensile deformation ctG are indicated in Gcε and Gctε correspond to the surface area of the energy cG and ctG, and are taken as limit strains of concrete failure in compression and tension. In this way, we can determine the area of potential failure of finite elements in the model as the point of failure in the model. The schematic diagram of a substitute model is presented in The following homogenized model parameters are specified:hP = .The following strain limits describe the homogenized model:hε = ; and t,limε—the strain limit corresponding to the minimum tensile strength (at rupture).The reference model of concrete was described in detail in ,22,23.In In the developed procedure, the compressive strength of concrete was determined on the basis of Formula (5) ,17 usingLtf—the compressive strength of the concrete at the time of t days (MPa); cemf—the strength of the cement as measured on ISO mortar or design value (MPa); kk = 3.0 + (Bρ − sB)/Bρ—the aggregate coefficient, where sB is the number of binders and aggregates lower than 0.2 mm in size per kg in 1 m3 of concrete, and Bρ is the bulk density of all the aggregates in the sample (kg/m3); frk = exp(0.034Sρ)—the reinforcing-fiber coefficient, where Sρ is the percentage ratio of steel-fiber mass to the mass of the cement (%), hereinafter frk = 1.0 for Sρ = 0.0 is taken; szk—the specimen shape and size coefficient (according to [W/(C + 0.22sm)—the total amount of water included in all the mixture elements (including the water in the SP) relative to the amount of cement with 0.22 microsilica fume amount; and sm/C—the ratio of the microsilica fume to cement.rding to ); kt=1−eThe procedure was carried out in such a way as to avoid the need to carry out experimental tests on standardized samples. The flexural tensile strength of concrete was determined on the basis of Formula (6) and In the next step, the value of concrete tensile strength was determined according to Formula (7) in which the influence of the beam height was taken into account :(7)fct=0cE is determined on the basis of Formula (8) [The elastic modulus mula (8) :(8)Ec=22cε at reaching the maximum compressive strength is determined on the basis of Formula (9):The strain cuε for compressive strength is determined on the basis of Formula (10):The ultimate strain ctε) and ultimate (ctuε) tensile strains were determined according to Formulas (11) and (12), respectively.The maximum . The schematic diagram of the substitute material model for analytical calculations is shown in For the application and verification of the substitute material model in analytical calculations, the moment–curvature relationship is presented. The moment–curvature relationship for the cross-section made of substitute material was determined on the basis of the analysis of work phases of a uniform bending cross-section, assuming an elastic–ideally plastic stress distribution with different compressive and tensile strengths of the material, and taking into account the unlimited deformability of the material for tensile strength, as shown in In phase Ia, the resistance of the cross-section is determined by the elastic stresses in the compression zone and the tension zone of the cross-section. In phase Ib, the stresses in the compression zone remain elastic, and the stresses in the tension zone reach the tensile strength of the replacement material. In phase IIa, the stresses in the compressed zone remain elastic, and the strains in the tension zone exceed the value of the tensile strain limit of the replacement material, and then stresses appear in the lower cross-sectional zone equal to the minimum strength of the replacement material. In phase IIb, the compressive stresses reach the compressive strength of the replacement material. In phase IIIa, deformations in the compressed zone exceed the limit deformations of the substitute material for compression, and a zone with a minimum strength of the substitute material is formed. In phase IIIb, the theoretical limit load capacity of the cross-section is reached, which is determined by the rigid–plastic distribution of stresses equal to the minimum strength of the substitute material. In Verifications of the substitute material model in numerical calculations for ordinary concretes are presented in ,8, whileBased on the test results presented in this paper, the parameters of the substitute material were determined in two variants. In variant 1, all parameters of the substitute material were determined on the basis of static and strength parameters being the results of the authors’ research. In variant 2, the parameters of the substitute material were determined based only on information on the materials, i.e., the cement used, the concrete recipe, and the steel grade. Other parameters were determined on the basis of dependencies indicated in this study, results for series B20-B30 are shown in We noticed that the compressive strength of concrete, according to the authors of experimental research  and our Based on the determined parameters of the substitute material model, a numerical analysis was carried out using the Abaqus 6.23 software. Verifications of the numerical substitute model were made on the basis of a comparative analysis of the authors’ own results and the experimental results of Feng et al. . In FiguIn turn, The difference in the displacement point of 47.0 mm was 2.85 kNm for the B20R3-SB series, which was about 1.9% of the breaking force, while for the B20R3-B series, it was 11.1 kNm and 7.5%, respectively. The difference in the displacement point of 52.0 mm was 7.47 kNm for the B30R3-SB series, which was about 4.7% of the breaking force, while for the B30R3-B series it was 12.85 kNm and 8.2%, respectively. It should be emphasized, however, that according to the EC2 standard, the maximum permissible degree of reinforcement of structural elements is 4%. The R3 series analyzed in the article were marked as having 2.9% of reinforcement, while taking into account the upper reinforcement of 3.5%, which is close to the limit value. In most traditionally designed structural members, the economic reinforcement ratio is between 0.8% and 2.0%. In this respect, the convergence of the results according to the described procedure is the highest.(1)Modeling the substitute material model according to the proposed procedure allows for the achievement of numerical results convergent with experimental results for reinforced elements made of UHPC concrete. The average results of displacements in the middle of the beam for the series using the experimental results of the concrete itself differ from the authors’ experimental results by 5.4%, while for the series in which all strength and deformation parameters were determined the results differ by 8.5%.(2)The difference between the results using the analytical substitute model with the experimental results is in the range of 1.9% to 8.2%.(3)In the case of structural elements with a large amount of longitudinal reinforcement, it is recommended to take into account the work of the substitute material in terms of biaxial compression by applying an additional multiplier called the concrete constraint coefficient with a value in accordance with the values in the range (1.16–1.2) adopted in the literature.(4)The highest accuracy of displacement results equal to 1.9% was achieved for the B20R1 series using the results of experimental studies.(5)The highest accuracy of the breaking load results equal to 0.5% was achieved for the B30R1 series using the results of experimental tests.(6)The average displacement results for the R1 and R1-S series reached an accuracy of 5.1%, while for the R3 and R3-S series, it was 11.0%.Based on the tests and analyses carried out, it was found that the procedure for designing a substitute material model allows for achieving satisfactory results in the modeling of structural elements. Results similar to experimental results, and similar approximation to analyses using other analytical or numerical models, confirm the possibility of using homogenization for structural elements made of HSC and UHPC concretes. The following conclusions emerge from the conducted analyses:The presented results of this study confirm the high efficiency of the proposed method for the homogenization of reinforced concrete structures.The modeling methodology based on the homogenization theory can also be applied to structural elements reinforced with the use of nonmetallic reinforcement because it consists of modifying the basic deformation and strength parameters of the concrete model used. The method can also be used to modify other material models for hybrid and composite elements.The model can be used for any configuration of longitudinal and transversal reinforcement, but we must remember that structural elements and the entire building must be designed in accordance with applicable standards, which specify, among others, the minimum amount and shape of individual types of reinforcement.


Cells need to coordinate nutrient availability with their growth and proliferation. In eukaryotic cells, this coordination is mediated by the mechanistic target of the rapamycin complex 1 (mTORC1) pathway. mTORC1 activation is regulated by two GTPase units, the Rag GTPase heterodimer and the Rheb GTPase. The RagA-RagC heterodimer controls the subcellular localization of mTORC1, and its nucleotide loading states are strictly controlled by upstream regulators including amino acid sensors. A critical negative regulator of the Rag GTPase heterodimer is GATOR1. In the absence of amino acids, GATOR1 stimulates GTP hydrolysis by the RagA subunit to turn off mTORC1 signaling. Despite the enzymatic specificity of GATOR1 to RagA, a recent cryo-EM structural model of the human GATOR1-Rag-Ragulator complex reveals an unexpected interface between Depdc5, a subunit of GATOR1, and RagC. Currently, there is no functional characterization of this interface, nor do we know its biological relevance. Here, combining structure-function analysis, enzymatic kinetic measurements, and cell-based signaling assays, we identified a critical electrostatic interaction between Depdc5 and RagC. This interaction is mediated by the positively charged Arg-1407 residue on Depdc5 and a patch of negatively charged residues on the lateral side of RagC. Abrogating this interaction impairs the GAP activity of GATOR1 and cellular response to amino acid withdrawal. Our results reveal how GATOR1 coordinates the nucleotide loading states of the Rag GTPase heterodimer, and thus precisely controls cellular behavior in the absence of amino acids. Cells need to ensure the abundance of nutrients, such as glucose and amino acids in their local environment before they can commit to growing , 3, 4, 5In human cells, mTORC1 activation requires two GTPase units, the Rag GTPase heterodimer and the Rheb GTPase. When amino acid levels are high, the Rag GTPase heterodimer is activated by its upstream regulators, causing the RagA subunit to be bound to GTP and the RagC subunit to be bound to GDP , 23. Undversus GDP). Intrinsically, the two subunits use unique mechanisms of intersubunit communication to coordinate their nucleotide loading states. This system prevents both subunits from binding to the same nucleotide, thus locking the heterodimer in either the active (GTPRagA-RagCGDP) or the inactive (GDPRagA-RagCGTP) state  state , 23, 32.P) state , 36, 37 P) state , 39.GATOR1 is a major negative regulator of the mTORC1 pathway. It is comprised of three subunits, nitrogen permease-like protein 2 (Nprl2), nitrogen permease-like protein 3 (Nprl3), and DEP-domain containing protein 5 (Depdc5) , 34. GATA). By forming this additional contact, GATOR1 pulls RagC away from RagA and breaks the intersubunit communication that locks up the Rag GTPase heterodimer, allowing RagC to bind to GTP  structural model, GATOR1 makes extensive contacts with both RagA and RagC subunits at two interfaces in the GAP mode: Nprl2-Nprl3 contacts RagA and Arg-78 of Nprl2 carries out the enzymatic function , while DFig. 1d to GTP . As a coIn this study, we carried out a structure–function analysis on the auxiliary interface between RagC and Depdc5. We identified a critical electrostatic interaction between the two, mediated by the Arg-1407 residue on Depdc5 and a patch of negatively charged residues on the αG5 helix of RagC. We found that this interaction is essential for GATOR1 to properly carry out its GAP function, with the loss of this interaction resulting in a much lower GAP efficiency. Finally, we showed that Depdc5–RagC interaction is essential for proper mTORC1 response in cells. Our results demonstrate how GATOR1 controls cellular behavior in the absence of amino acids.A). In particular, we observed a strong Coulomb density of a positively-charged arginine residue, Arg-1407 of Depdc5, near a patch of negatively-charged residues, Asp-201 and Asp-205, on the αG5 helix of RagC . These charged residues are within 3 to 5 Å of each other, which allows for electrostatic interactions to form. Moreover, these residues are evolutionarily conserved in charge . This suggests that Arg-1407 of Depdc5 and Asp-201 and Asp-205 of RagC might be candidates that mediate the GATOR1–RagC auxiliary interaction.To investigate the molecular basis underlying the auxiliary interface, we carefully examined a previously resolved cryo-EM structural model (PDB: 7T3C) , in whicFig. 1C), suggesting that the mutations do not affect the integrity of the GATOR1 complex. For the Rag mutants, we expressed them in Escherichia coli following a previously published protocol (C). As a control, we tested their intrinsic hydrolysis rates using a previously established method (D–F), suggesting any defects that we might see in a GATOR1-stimulated assay (see below) are not due to a non-specific disruption of the folding and architecture of the Rag GTPases.We designed a series of point mutants, aiming to specifically disrupt the potential interaction. We either neutralized the charges of these residues by mutating them to an alanine residue  or reversed the charges . For the GATOR1 mutants, after gel-filtration purification, all three subunits co-eluted in the same fraction, as seen on a Coomassie-stained gel C, suggesprotocol . This yiprotocol C. As a cd method , 43. Undd method , D–F, suTo investigate how disrupting the auxiliary interface may affect the stimulatory effect of GATOR1, we first measured and compared the Michaelis-Menten kinetics using wild-type and mutant GATOR1. In order to eliminate the competition from binding to the inhibitory mode, we used the GATOR1[Depdc5(Y775A)] as our “wild-type” background, as this mutant specifically traps the Rag GTPases in the GAP mode by disrupting the inhibitory binding site .GTPRagA-RagC and RagA-RagCGTP, exist in the reaction, and the only one that GATOR1 can bind to and stimulate is GTPRagA-RagC. In this scenario, RagC is not loaded with GTP, so it does not have the optimal nucleotide loading configuration to allow for the formation of the auxiliary interface (kcat) of GATOR1[Depdc5(Y775A)] is 0.016 min−1 and that of GATOR1[Depdc5] is 0.009 min−1 (A and C), which are within two-fold of one another. As we hypothesized that the auxiliary interface plays a minor role in the single turnover hydrolysis case scenario, the slight two-fold defect matches our expectation and further confirms the architectural integrity of the GATOR1 complex carrying the R1407D mutation, as well as the functional integrity of its GAP interface.As a control experiment, we validated our GATOR1 construct using a single turnover reaction. Here, the Rag GTPase heterodimer is singly loaded with GTP, with the other subunit unoccupied. Therefore, only two species, nterface . The onl09 min−1 , A and CGTPRagA-RagCGTP). In this scenario, the auxiliary interface could readily form (kcat) is 0.11 min−1, while the R1407D mutation causes an eight-fold decrease in kcat (B and C). Moreover, the R1407D mutation introduces an 8-fold increase in KM, suggesting a weakened binding (B and C). We calculated the catalytic efficiency (kcat/KM), which represents the effective association rate . When we neutralized the charges by mutating individual aspartic acid residues to an alanine residue, we observed a mild defect in the binding between GATOR1 and RagC, as the kcat/KM value decreases by two- and fourfold for D201A and D205A, respectively . However, when we introduced a double alanine mutant, we observed a further reduction in the kcat/KM value of ∼sevenfold . These results suggest that the two negatively charged aspartic acid residues form partially redundant interactions with the Arg-1407 residue on Depdc5, as individual substitution cannot completely abrogate the interaction to the baseline level.For potential interacting counterparts on RagC, we sought synergistic effects on the Asp-201 and Asp-205 residues. We carried out a similar set of multiple turnover GTP hydrolysis reactions , D and Ekcat/KM) drops by seven- and 22-fold, respectively . Moreover, when we reversed the charges on both aspartic acid residues, we observed an 80-fold decrease in the kcat/KM value . These results suggest that reversing the charges which generate a repulsive force at the auxiliary interface leads to a strong binding defect in the GATOR1-stimulated GTP hydrolysis reaction.Given the nature of the interaction formed between oppositely charged residues, we reasoned that a charge-reversal mutation may induce a more dramatic effect as we change the attraction force to repulsion. Indeed, with a single charge-reversal mutation on RagC (D201R or D205K), the catalytic efficiency (A). We did not see any appreciable difference between the conditions, implying that salt does not affect the intrinsic GTP hydrolysis by the Rag GTPases. We then performed the GATOR1[Depdc5(Y775A)]-stimulated multiple turnover assay in various salt concentrations and observed a steady decrease in kcat/KM values as salt concentration increased (B and C). This suggests a weakened interaction and supports our hypothesis that the Depdc5-RagC interaction is electrostatic.As the Depdc5-RagC interaction is mediated by positively- and negatively-charged residues, we hypothesized that it may be electrostatic. If this were the case, increasing the ionic strength of the reaction buffer would weaken the auxiliary interface. To test this hypothesis, we supplemented the reaction buffer with increasing concentrations of sodium chloride (NaCl) and carried out the stimulated GTP hydrolysis reaction as above. As a negative control, we performed single- and multiple-turnover assays with the Rag GTPases alone at increasing concentrations of NaCl A. We didncreased , B and CA, cell lysates). As shown below, this cell line becomes desensitized to amino acid withdrawal, suggesting a defect in the GATOR1 function. We then stably expressed wild-type or R1407D Depdc5 in the knockout cell line. Expression levels of Depdc5 within the two cell lines are comparable to one another , as well as their interaction with Nprl2 and Nprl3, suggesting the integrity of the GATOR1 complex is maintained .To probe the physiological role of the electrostatic interaction and its relevance in amino acid sensing, we need cell lines in which this interaction is specifically impaired. We first used CRISPR-Cas9 to generate a Depdc5-knockout HEK-293T cell line. Western blotting using the Depdc5 antibody confirms the complete knockout . For wild-type cells, 1 h treatment in amino acid-depleted media caused complete inactivation of mTORC1, as the phosphorylation signal of its downstream target, pThr389 on S6K1, disappeared . In contrast, for Depdc5-knockout cells, this response is blunted - the cells maintained a high level of phosphorylation on Thr389 of S6K1 even in the absence of amino acids , suggesting a negative regulator of the mTORC1 pathway, here GATOR1, failed to carry out its normal function. While expression of wild-type Depdc5 restored the sensitivity to amino acids  and normal cell size response to amino acid withdrawal , expression of Depdc5(R1407D) completely failed to do so . We also probed the response of another mTORC1 substrate, 4EBP, and observed a similar trend based on its laddering pattern: in the absence of amino acids, hyperphosphorylated 4EBP (asterisk) maintains in the cell line expressing Depdc5(R1407D), while disappears in the wild-type cell lines. These results suggest that the electrostatic interaction between Depdc5 and RagC is necessary for GATOR1 to turn off the mTORC1 pathway in the absence of amino acids.With the stable lines in hand, we tested their response to amino acid withdrawal by depleting the amino acids from the culturing media B. For wiF). When we expressed wild-type Rag GTPases in HEK-293T cells, we did not observe any changes in the cellular response to amino acid deprivation . However, when RagC(D201A) or RagC(D205A) was expressed, we observed a slight elevation of the phosphorylation signal in the absence of amino acid , suggesting a partial defect of GATOR1 function in turning off mTORC1 signaling. This defect is relatively mild, which is consistent with the mild reduction in kcat/KM values in stimulated GTP hydrolysis assay in vitro (cf. D). When we expressed the double alanine mutant in cells , we observed a much greater defect, which is also consistent with our previous results (cf. D).To test the synergistic effect between Arg-1407 of Depdc5 and the negatively charged residues on RagC, we performed a similar set of experiments using RagC mutants. In wild-type HEK-293T cells, we transfected cDNA expressing either wild-type RagC or charge-neutralizing mutants (D201A and/or D205A) and tested their response to amino acid withdrawal by monitoring the phosphorylation on Thr389 of S6K1 F. When witro cf. D. When wults cf. D.G). When we expressed single charge-reversal mutants (D201R or D205K), we observed a more severe defect in turning off mTORC1 signaling than the charge-neutralizing mutants . Moreover, the double charge-reversal mutant (D201R/D205K) completely loses sensitivity to amino acid deprivation , as the phosphorylation level of Thr389 of S6K1 remains almost identical to the condition where amino acids are well supplemented. This is fully consistent with the in vitro results of our GTP hydrolysis experiments, suggesting that the negatively charged residues on RagC are necessary for GATOR1 to carry out its normal function during amino acid deprivation.To further test our hypothesis, we introduced charge-reversal RagC mutants in HEK-293T cells G. When wH). Here, we incubated the cells in the absence of amino acids for a prolonged time (∼8 h), and checked the kinetics of a protein marker for autophagy, ULK1. Upon initiation of autophagy, the ULK1 protein level is downregulated by an E3 ligase, NEDD4L , which is consistent with our results above. When probing the protein level of ULK1, we observed that the degradation of ULK1 in the Depdc5(R1407D)-expressing cell line is also severely delayed , suggesting the auxiliary interaction between Depdc5 and RagC is necessary for efficient initiation of autophagy under nutrient-deprived conditions. At late time points, autophagy is eventually initiated in the mutant cell line, suggesting parallel pathways, such as GCN2, may kick in and act independently of mTORC1 , Abcam, Sigma-Aldrich (SA), or Millipore: Rabbit anti-HA: CST 3724; Rabbit anti-pT389-S6K1: CST 9205; Rabbit anti-S6K1: CST 2708; Rabbit anti-4EBP: CST 9452; Rabbit anti-Depdc5: Abcam ab185565; Rabbit anti-Nprl2: CST 37344; Rabbit anti-Nprl3: SA HPA011741; Rabbit anti-ULK1: CST 8054; Rabbit anti-pS757-ULK1: CST 6888; Rabbit anti-RagA: CST 4357; Rabbit anti-Raptor: Millipore 09-217; Goat-anti-rabbit HRP-linked antibody: CST 7074.Chemicals and Flag-M2 affinity gel were purchased from Sigma-Aldrich. 8-Arg10-SUMO-tagged RagA and tagless RagC was transformed into BL21(DE3) E. Coli strain and was grown at 37 °C. When the optical density (OD) of the bacteria culture reached 0.8, protein expression was induced overnight with 0.5 mM IPTG at 18 °C. The next morning, the cell pellets were resuspended in resuspension buffer . The resuspension was passed through a microfluidizer three times and was cleared via centrifugation. The supernatant was applied to a Ni-NTA column (Qiagen) and washed extensively. The eluate was concentrated down and passed through a MonoS column (Cytiva). The Rag-containing fractions were pooled, and the His8-Arg10-SUMO-tag was cleaved by HRV 3C protease (Pierce/ThermoFisher). Following cleavage, the protein was subjected to a second round of MonoS column to remove the cleaved tag. The Rag GTPase heterodimer was then applied to a MonoQ column (Cytiva). The eluate from the salt gradient was stripped by 20 mM EDTA, concentrated, and finally applied to a HiLoad 16/60 Superdex 200 gel-filtration column. Glycerol was added to the final, concentrated product and was flash-frozen and stored at −80 °C.The Rag GTPase heterodimer was purified based on an established protocol . In brie2; 100 μM ATP; 10 mM Na4P2O7; 10 mM Na β-glycerol phosphate; 1% Triton; and EDTA-free protease inhibitor). The insoluble fractions were cleared by centrifugation, and pre-equilibrated Flag-M2 affinity gel (Sigma) was added to the lysate. GATOR1 was immunoprecipitated for 3 h at 4 °C. Flag gel was washed with TLB, followed by TLB supplemented with 300 mM NaCl. GATOR1 was then eluted with 3× Flag peptide. The eluate was concentrated down using a 100 kDa molecular-weight cut-off filter (Millipore) and passed through a HiLoad 16/60 Superdex 200 gel-filtration column to further improve the purity. The GATOR1-containing fractions were pooled, concentrated, flash frozen in the presence of 10% glycerol, and stored at −80 °C.To recombinantly express GATOR1, 293-FreeStyle cells (ThermoFisher) were inoculated at 1 M/ml in SMM 293-TII serum-free culturing media . After 24 h, the cells were transfected using PEI with a mixture of plasmids including Flag-Depdc5, HA-Nprl2, and HA-Nprl3. 36 h post-transfection, cells were harvested and lysed with Triton lysis buffer dimethylammonio)-1-propanesulfonate) (CHAPS)]. GTP hydrolysis kinetics were measured using established protocols  at varying time points. The time points were then run on a thin layer chromatography (TLC) plate to separate the hydrolyzed GDP from the intact GTP. The reaction rates were determined and used to obtain kobsd values, which were fitted against the concentrations of the Rag GTPases to obtain kcat and K½ values. The multiple turnover reactions used a similar setup, except the Rag GTPases were kept constant (2 μM), while the concentration of GTP ranged from 2 μM to 100 μM. The resulting kobsd values were corrected for the turnover number, and fitted against the concentration of GTP to get the kcat and KM values.Unless otherwise specified, all the biochemical assays below were carried out in Assay Buffer [50 mM NaHEPES, pH 7.4; 100 mM potassium acetate; 2 mM MgClrotocols , 41, 43.32P-radioactively labeled GTP. Increasing concentrations of GATOR1 (25 nM–750 nM) were then added to the reaction to stimulate GTP hydrolysis, and the time points were analyzed accordingly. The resulting kobsd values are fitted against the concentration of GATOR1 to get the kcat and KM values.GATOR1-stimulated single-turnover GTP hydrolysis reactions were carried out by first loading 50 nM Rag-Ragulator with a trace amount of 32P-radioactively labeled GTP. Increasing concentrations of GATOR1 (25 nM–750 nM) were then added to the reaction to stimulate GTP hydrolysis and the time points were analyzed accordingly. Similarly, kobsd values were corrected for the turnover number, and fitted against the concentration of GATOR1 to obtain the kcat and KM values.For GATOR1-stimulated multiple-turnover hydrolysis assays, 2 μM of Rag-Regulator was first incubated 100 μM of unlabeled GTP doped with a trace amount of 2; 2 mM DTT; and 0.1% CHAPS. To set up assays in increasing salt concentrations, we supplement the low salt buffer with 50, 150, and 350 mM sodium chloride. Data were acquired and analyzed similarly.The assay to measure the effect of ionic strength was performed using the GATOR1-stimulated multiple-turnover hydrolysis setup, except the buffer conditions were different from the normal composition. For the low salt buffer, we used 50 mM NaHEPES, pH 7.4; 50 mM potassium acetate; 2 mM MgClTo generate Depdc5-knockout HEK-293T cell line, 2 million HEK-293T cells were seeded in a 6-well plate containing 2 ml DMEM with 10% IFS supplemented with 2 mM glutamine, penicillin (100 IU/ml), and streptomycin (100 μg/ml). The next day, the cells were transfected with guides encoded in a pX330-based CRISPR-Cas9 vector and GFP, and sorted by FACS. Single-cell clones were grown out and analyzed with Depdc5 antibody, to select the knockout line. As the CRISPR guide was transiently expressed in a plasmid, the final Depdc5-knockout HEK-293T cell line does not have puromycin resistance.To generate stable cell lines that express wild-type Depdc5 or Depdc5(R1407D), 3 million Depdc5-knockout cells were plated in a 6-well plate containing 2 ml DMEM with 10% IFS supplemented with 2 mM glutamine, penicillin (100 IU/ml), and streptomycin (100 μg/ml). The next day, the cells were infected with lentivirus encoding HA-tagged wild-type Depdc5 or Depdc5(R1407D) and selected using puromycin 36 h post-infection. The resulting cell lines were probed with Depdc5 antibody to confirm the expression of genes of interest.2, 10 mM Na4P2O7, 10 mM Na β-glycerol phosphate, 1% Triton, and EDTA-free protease inhibitor) at specific time points and cleared by centrifugation, before proceeding with the co-immunoprecipitation. Western blots were quantified using LI-COR Odyssey imaging system.Co-immunoprecipitation and cell signaling experiments were performed based on established protocols , 40. BriAll data are included in the manuscript.The authors declare that they have no conflicts of interest with the contents of this article.


Advances in space flight technology will enable the construction of Moon or even Mars bases in the not-too-distant future. Thus, materials will be needed that are suitable for building in microgravity environments. One idea is to use concrete, the most used construction material on Earth, for these challenging tasks. The hardening and the properties of concrete under the boundary conditions prevailing on Earth are well understood, but there is only limited research on concrete produced in microgravity. Hence, a research project called MASON was established, which aims to mix and harden concrete on the ISS and to investigate the properties of the specimens made in microgravity extensively. Since a defined geometry of the specimens would be favorable for these investigations, a special hardware was developed, called the MASON Concrete Mixer (MCM), which allows the production of concrete specimens fulfilling the requirements on the geometry as well as the safety requirements. Subsequently, the development, design, tests, and qualification of the MCM as well as its usage are presented. Continuous advances in space flight technology allow increasingly easier access to space and planetary surfaces3. To explore these surfaces, building materials are required in large quantities in order to build space infrastructure or future Moon or Mars bases6. Accordingly, it is more important than ever to investigate and understand the behavior of building materials under the conditions prevailing in space7. This especially holds for cement-based concrete, the most used construction material on Earth. Although the use of cementitious binders on celestial bodies is rather unlikely due to efforts that would be necessary for the transport, a deep understanding of the effects of microgravity on hardening and mechanical properties of concrete will provide general knowledge for the use of mineral construction materials in space. However, the investigations on the properties of cement-based composite materials hardened under microgravity are fairly limited thus far. Some work has been published on the crystallization of cement under weightlessness, in short duration during parabolic flights12 as well as in long-term14, and only one single experiment on mixtures of cement, sand and liquids: Already in 1994, first investigations were performed onboard Space Shuttle Endeavor (STS-68). However, the “ConCIM”-experiment was only successful to a limited extent, since the single sample that was produced exhibited a poor quality due to a hardware failure17.In the past decades, the material properties of cement paste and solid concrete have been extensively researched within the boundary conditions prevailing on Earth and are thus well understoodMAterial science on Solidification of cONcrete”) project group, the influence of microgravity on the solidification and properties of concrete mixed and hardened in space was studied. For this investigation, an experiment on the International Space Station (ISS) was carried out. Simultaneously, analogous experiments were conducted on Earth to obtain reference samples hardened under Earth gravity. Additionally, concrete specimens using clinostats and random positioning machines (RPM) were produced to achieve insights in the hardening of concrete under simulated microgravity.In the MASON , was designed which meets the requirements for a preferably easy experiment execution and for safety on board the ISS see Fig. . In addiAfter initial feasibility studies, the design of the MCM was developed within 9 months in an elaborate process involving the Institute of Structural Concrete , BIOTESC , Institute for Theoretical Physics  and the German Aerospace Center - Institute of Materials Physics in Space . After the final design of the MCM was found, the experimental and numerical investigations were carried out which were required for the certification of the flight hardware by the ESA safety panel. Once the certification by ESA was obtained, the 64 MCM items were prepared and delivered just in time in autumn 2021 for the upload with SpaceX 24, one year after beginning of the project. In February 2022, Matthias Maurer conducted the experiments on the ISS, where the specimens were stored for planned download on SpaceX 25. Subsequently, the requirements on the MASON hardware, the process of development and qualification, the mixing process on ground and on orbit as well as the specimens obtained using the MCM are presented.19 had to be considered and served as a base for the hardware development.The aim was to obtain a homogeneous concrete specimen with a defined geometry and volume as a baseline for the studies. To produce concrete, the dry cement powder and aggregates must be mixed with water within the weightless environment of the ISS by an astronaut. This environment is very special, as powder, when released, will remain in the air and will be distributed throughout the station by the ventilation system. Cement powder is considered irritating to the skin and the respiratory tract and dangerous when it gets in contact with the eyes. Because of this reason, the cement powder was classified by the NASA mission toxicologist as a "Toxicity Hazard Level 2" (THL 2). The major challenge now was to generate a hardware that fulfills the safety standard of containing this THL 2 substance in all phases of the hardware cycle. It was necessary to integrate 3 independent levels of containment (LOC) into the hardware or produce a design for minimum risk (DFMR). In conclusion, the aspects presented in Table The MCM consists of 18 standard parts and only five custom parts, optimized for the intended use. The design drawings of the custom parts formed the basis for the prototype construction using 3D printing. The design was adapted, tested, improved, and printed again multiple times. The final container shape was later produced by injection molding.The idea was, as already hinted at in the requirements above, to build a container with 3 main parts: The lower part, a cylinder that holds the cement and later the concrete sample, called the mixing chamber  is inserted into a slot on one end. The mixer blade is glued and additionally connected by a bolt (15). After mixing, the mixer blade can be pulled up and stored in the retention chamber to not interfere with the sample. Initial mixing tests have shown that, particularly when sand is added, a high force is required to rotate the tube together with the mixer blade. To counteract this, a special tool  is connected followed by a check valve (12). Filter and thread both contain Luer Lock connectors. The Luer system is used as a uniform standard in medical technology for the connectivity of infusion sets and syringes and was already approved as space hardware for an experiment on behalf of the student laboratories of the German Aerospace Center (“DLR_School_Lab”) within the mission “ISS Horizons”22. The syringe can later be connected to the check valve. The connection is closed with a cap for transport. The entire construction weights approx. 137.5 g and is shown in the final drawing  which are inserted in a slotted hole on the retention chamber and threads on the mixing chamber. In between the walls of the two chambers two O-rings (7) are placed to ensure tightness at the contact surface. To allow compression of the concrete, the retention chamber is equipped with a cylindrical plug, which protrudes into the mixing chamber. This plug will compress the concrete mixture by reducing the volume of the mixing chamber when the mixing and the retention chamber are pressed together. The maximum way of compression is defined by the slotted hole on the retention chamber. By compression of the two chambers pressure will build up. This pressure is released through a hydrophobic filter (5), which on one hand retains the cement powder and on the other hand also the water and liquid basic solution that is generated by adding water to the cement. This filter is additionally enclosed in a PE bag (16) with a volume of approx. 200 ml which acts as a safeguard in case something would escape the hydrophobic filter. The retention chamber gets protruded by the shaft of the mixer. The protrusion is equipped with three shaft seals (9) and two scrapers (8) that allow rotation around the rod axis for mixing the concrete and extraction from the concrete into the retention chamber while at the same time ensuring the leak proofness. This shaft also serves as an inlet pipe for the water or additives. The shaft is a hollow tube made of stainless steel, where a carbon mixer blade23.To mix concrete, water or additive must be provided. This second element is realized with standard medical grades syringes see Fig. , which a24 with a wall thickness of 4 mm was chosen. Polycarbonate in this setup is able to withstand high mechanical forces and is almost impossible to break. The retention chamber performed as well in this configuration, but when liquid cement got in contact with the complex architecture of the upper part of the retention chamber, environmental stress cracks began forming. Following this issue, the material was reconsidered. CoC and Tritan MX73125, a special kind of PET, were tested. Retention chambers produced with CoC were not satisfactory, Tritan MX731 performed well and was chosen. The shaft of the mixer is also part of this DFMR and in this function has to withstand the shear forces during mixing, forces of leverage, when the shaft is moved out and angular forces that could be applied by twisting the MASON crank. At first a wrapped carbon tube was tried, but it sheared already at a force of 1 Nm. It was replaced by a tube made of chemical resistant stainless steel 316L26 with 1.5 mm wall thickness. Also part of the DFMR are the connection of the hydrophilic filter to the mixing shaft and the hydrophobic filter to the retention chamber. The hydrophilic filter is screwed into the mixing tube. For this a thread was cut into the Luer Slip of the hydrophilic filter. The filter was then screwed in place and fixed with instant glue. The gap between the Luer Male and the shaft were potted with a two-component epoxy filler27  approach with the level of containment (LOC) approach Fig. . The mixThe LOC philosophy was applied, where the parts join, or where the container interacts with the environment. The connection of the mixing chamber and retention chamber was sealed by two O-rings, where each O-ring provides one LOC. The hydrophobic filter  and PP using Araldite 201527 was unsatisfactory.At first, some prerequisites had to be clarified. The glues were qualified with the materials they should be used on by a pull test Fig. . Very goIn addition, also the particle size of the cement powder had to be determined. The data sheets usually end with the detection of 0.5 µm particles, but it was unclear if smaller particles exist that might escape the mixer and invalidate the LOC concept with the 0.45 µm filter. Therefore, an airflow was established through a cement filled concrete mixer and the 0.45 µm filter Fig. . DetectiThe MCM itself also had to undergo a variety of other tests, like a pressure leak test at 3.5 bar delta pressure (MDP), and a material compatibility test with the chemicals used. Here it was discovered that abrasion and elevated temperature produced by the hydratization of the concrete do not have an effect on the stability of the container. The cement powder did not have an influence on the container stability as well.18. In an earlier phase of the hardware development, a breaking of the interface between metal tube and the hydrophilic filter was experienced. This was solved by directly screwing the filter into the thread on the metal tube and potting the gap between the metal tube and the female Luer connector on the filter. This connection was especially tested for crew loads in a series of force tests on the hydrophilic filter. Impacts of the crank and bending forces of up to 3 Nm were sustained by the hydrophilic filter.One other thing that needed to be tested were the loads the crew could induce on the concrete mixer, especially to the filter assembly on the mixing tube Fig. . The loaA verification for launch loads was performed as well. Nominal vibration tests were performed at 0 DB upright with combined Launcher Profile Fig. . The harAs all tests were successfully carried out, the assembly of the flight hardware could be started at BIOTESC. In the assembly procedure, each individual step  was described in detail, documented, and checked by a qualified assurance. All parts and connections were inspected during and after assembly and each build MCM was leak tested at 350 mBar for 30 min Fig. , acceptiDuring the development and the qualification of the MCM, tests on the feasibility for concrete specimen production in the intended way were carried out continuously according to the protocol described in “Methods” section. These investigations included test on the demolding of the specimens as well as tests on the properties of the obtained specimens.The concrete samples could be retrieved easily and efficiently from the MCM. The screws were first removed and then the retention and mixing chamber of the MCM could be separated with a little force. A small hole had to be drilled in the bottom of the mixing chamber so that a pressurized air nozzle could be attached there. Due to the slightly conical shape (0.5° inclination) and the smooth surface of the mixing chamber the concrete cylinder was pushed out with the compressed air force easily. During the EST, 64 MCM were used, and all the concrete samples could be retrieved without damage or any problems by this method. The ground test samples were examined in the same way as planned for the microgravity samples to verify the test methods and to show, that the concrete samples produced with the MCM are suitable to obtain scientific results.Since no buoyancy or sedimentation is expected in the fresh concrete in microgravity, the samples produced in space will be examined primarily with regard to the air void and mass distribution as well as the effects on the mechanical properties, especially the compressive strength  in three regions were used  and are now in the hands of the MASON project team for intensive scientific investigations. On ground 223 MCM were used to produce identical concrete reference samples . With EST and ISS experiment a total number of 351 MCM were in use for scientific analysis.Within the MASON project a concrete mixer (MCM) was developed, tested, and qualified for use on the ISS. With this hardware concrete samples can be produced in microgravity by astronauts, which, due to the defined cylindrical shape, are ideally suited for later scientific analyses. The effects of microgravity on the properties of concrete as microstructure, air pore distribution, density, and compressive strength can be determined optimal, thereby providing a major contribution to future constructions in space. The MCM can be used very flexibly also for other two-component binders or construction materials and can be built in batch production fast, cheap and in large quantities. By means of first tentative tests on specimens produced using the MCM on ground, the general feasibility of the hardware in the intended way was proved. Finally, 64 concrete samples in MCMs were sent to the ISS, where the German ESA astronaut Matthias Maurer performed the experiment within the Cosmic Kiss mission in February 2022. On the ISS, the MCM performed well and allowed the execution of the experiment successful in terms of handling and safety. Based on his experience in µg, Matthias Maurer has suggested small improvements for the MCM to further enhance performance for future applications. Complete results of concrete sample properties and the feasibility of simulating microgravity during the solidification process using a clinostat and a random positioning machine will be presented after evaluation of the ISS and ground reference samples. In close communication with the American Penn State University and the designated NASA staff, future applications are already being considered, such as a moon mission.In total, 64 samples were processed by the crew on the ISS, in particular by ESA astronaut Matthias Maurer. Operation and handling, i.e. for storage transfer, involved ESA astronaut Samantha Cristoforetti. The authors affirm that human research participants provided informed consent for publication of the images in Fig. For bonding between Stainless Steel, MABS (Methylacylate-Acrylnitiril-Butadien-Styrol), Polycarbonate, Polypropylene and Tritan MX731 Loctite 4902 was used in combination with the primer Loctite SF770. Both surfaces were cleaned first with Isopropanol, then primed with Loctite SF770. Loctite 4902 was added on one surface and the parts were pressed together for 10 s and cured for 72 h at ambient temperature and humidity.Bonding strength was measured by pulling the bonded pieces apart at a speed of 2 mm/min.The hydrophilic filter was potted onto the stainless steel tube, and the hydrophilic filter to the retention chamber and plastic bag with Araldite 2015. Curing was performed at 40 °C for 16 h at ambient humidity.An airflow of 20 l/min was established through the MCM for 25 min. The exhaust air was led over a pH indicator paper and in addition bubbled into a beaker containing 25 ml 100 mM NaCl solution and phenol red as pH indicator.The MCM was assembled, and the hydrophobic filter was replaced by a cap to close the exhaust port. The MCM was filled with water. The syringes were filled with water and the syringe clamp was applied. A pressure of 3.5 bar was applied to the hardware under test for 15 min. The H/W was inspected for damage and loss of pressure over time.Vibration testing of the Concrete Hardening hardware was performed at the Fachhochschule Nordwestschweiz (FHNW) on a TIRA TV57315 shaker. Launch loads for up to +12 dB were tested for Space X, Soyuz, Progress and Cygnus. After inspection, a pressure test and a vacuum leak test were performed to guarantee integrity of the H/W after vibration testing.Concrete Mixers and syringes were subjected to a vacuum leak test. The samples were weight on an analytical balance (Mettler Toledo AE260) before and after the vacuum leak test. The H/W was inserted into a bell jar. Pressure was reduced to 350mbar for 30 min. H/W was inspected for visible leaks, weight and excluded if more than 20 mg of substance were lost.All items that can get in contact with cement powder were placed inside cement powder for 6 months. The items were cleaned and inspected.Syringes were filled with 10% Air Entraining Agent or 20% Superplasticizer and stored for 6 months. The items were cleaned and inspected and subjected to a pressure leak test.Influence of abrasion was tested by extensive mixing of three regolith samples for 10 min followed by inspection and a pressure test as described above.Starting position of the MCM,Pull up the retention chamber to increase space for mixing,Attach MASON Crank, mix dry ingredients for 1 turn and detach MASON Crank,Attach syringe, inject liquid, and remove syringe,Attach MASON Crank and mix concrete for 2 min by rotating and moving the mixer,Align mixer with the wings of the MCM, wiggle and pull up the mixer tube until blade is visible,Push lower and upper part of the MCM together to reduce space and compress the concrete.The major steps of the mixing procedure are shown in Fig. The whole process takes in average about 5 min crew time for each MCM.Before space flight, the hardware, timing and on orbit scenario were tested on ground in an Experiment Sequence Test (EST) at the User Support Center BIOTESC in Hergiswil, Switzerland.Waveforms were recorded using a digital oscilloscope (Picoscope 5000 series) and Picoscope 6 software. Using custom-written code, the waveforms were normalized and averaged over each pair of opposite receiver-transmitter orientations, then the time-of-flight was determined using a threshold of 0.04 relative to the signal maximum. In Fig. Tomograms were obtained by a General Electrics Nanotom 160 NF with 15 µm voxel size and initially analyzed using Avizo 9 software, providing the shown slices and 3D view in Fig. 31.After successful qualification of the MASON concrete mixer by the ESA safety panel, the design of the MCM was registered for patent approvalFurther information on research design is available in the Supplementary InformationReporting Summary