Also, little is known on how problem’s difficulty in these tools (the shape of CO2 concentration trajectory), as well as the use of these tools as a decision aid influences performance. The primary objective of this paper was to investigate how learning about Earth’s climate via simulation tools is influenced by problem’s surface and structural features, problem’s difficulty, and decision aids. In experiment 1, we tested the influence of problem’s surface and structural features in a simulation called Dynamic Climate Change Simulator (DCCS) on subsequent performance in a paper-and-pencil Climate Stabilization (CS) task (N = 100 across four between-subject conditions). In experiment 2, we tested the effects of problem’s difficulty in DCCS on subsequent performance in the CS task (N = 90 across three between-subject conditions). In experiment 3, we tested the influence of DCCS as a decision aid on subsequent performance in the CS task (N = 60 across two between-subject conditions). Results revealed a significant reduction in people’s misconceptions in the CS task after performing in DCCS compared to when performing in CS task in the absence of DCCS. The decrease in misconceptions in the CS task was similar for both problems’ surface and structural features, showing both structure and surface learning in DCCS. However, the proportion of misconceptions was similar across both simple and difficult problems, indicating the role of cognitive load to hamper learning. Finally, misconceptions were reduced when DCCS was used as a decision aid. Overall, these results highlight the role of simulation tools in alleviating climate misconceptions. We discuss the implication of using simulation tools for climate education and policymaking.Research shows that people’s wait-and-see preferences for actions against climate change are a result of several factors, including cognitive misconceptions. The use of simulation tools could help reduce these misconceptions concerning Earth’s climate. However, it is still unclear whether the learning in these tools is of the problem’s surface features (dimensions of emissions and absorptions and cover-story used) or of the problem’s structural features (how emissions and absorptions cause a change in CO Understanding stocks and flows is a fundamental process in the real world . For exaIt is a well-known phenomenon that people have difficulties in understanding the dynamics of stock-flow problems . Stock-f2) concentration trajectory. Two of the prevalent misconceptions in climate stock-flow problems are the correlation heuristic and violation of mass balance (2 concentration) follows the same path as the inflow (CO2 emissions). This misconception assumes that stabilizing emissions would rapidly stabilize the concentration; and, emission cuts would quickly reduce the concentration and damages from climate change. This reasoning is incorrect because reliance on the correlation heuristic significantly underestimates the time delays existent between reductions in CO2 emissions and their effect on the CO2 concentration  task to test for correlation heuristic and violation of mass balance misconceptions ,b. In th CS task . Overall CS task .2 concentration to a goal level in DCCS by deciding the CO2 emissions and absorptions. Next, in the CS task, participants sketched the CO2 emissions and absorptions corresponding to a CO2 concentration stabilization trajectory. Results revealed that exposure to DCCS before CS task reduced correlation heuristic and violation of mass balance misconceptions.Furthermore, recent research has documented the role that repeated feedback about cause-and-effect relationships plays on human understanding of dynamic systems, particularly for Earth’s climate system . Researc2 concentration under different CO2 concentration scenarios) or the surface features  of the problem in DCCS before attempting the CS task.Although prior research has documented a reduction in correlation heuristic and violation of mass balance due to exposure to simulation tools, little is known on how people improve their stock-flow misconceptions when they interact with these tools. For example, While performing in DCCS, one possibility is that people may learn the problem’s structural features. For example, recent research has shown that structural knowledge helps people reduce their correlation heuristic and violation of mass balance misconceptions in both cases when problems encountered in the CS task are structurally similar or different compared to those presented in DCCS . However, while performing in DCCS, another possibility is that people learn the surface features of the climate problem .In literature, procedural reinstatement principle states that performance would be better at transfer when the problems encountered during transfer are similar to those encountered during training . Also, h2 concentration curve presented: If the shape of the concentration curve is curvilinear, then this curvilinear shape would create more perceived difficulty among participants compared to when the concentration curve is straighter.Moreover, as per the difficulty hypothesis, people’s transfer of learning is improved when they train on difficult problems compared to easy problems . Thus, i2 concentration curve shape may further need additional working memory capacity. Due to the overload of working memory capacity, participants may not be able to learn the stock-flow relationships in DCCS and reduce their correlation heuristic and violation of mass balance misconceptions in the CS task.However, it is also possible that a difficult curve in DCCS may not help reduce correlation heuristic and violation of mass balance misconceptions in the CS task because of the predictions of the cognitive load theory . AccordiFinally, simulation tools could also be used as a side-by-side decision aid that helps people understand relationships between emissions, absorptions, and concentration by a trial-and-error procedure. There is evidence that even in simple descriptive binary-choice decision tasks, when participants are provided with experiential decision aids, they tend to rely on the experience gained in these aids in making descriptive decisions and improve their decision making . When DCThe primary goal of this research is to investigate via lab-based experiments people’s stock-flow misconceptions about climate change and the role that different factors like surface and structural features, problem difficulty, and decision aids play in reducing people’s stock-flow misconceptions. Such research may help policymakers formulate appropriate policies for climate education in schools and colleges that make use of simulation tools to supplement conventional teaching . FurtherIn what follows, we first present the background where we highlight prior research and motivate our hypotheses. Next, we report three experiments where we test how problem’s surface and structural features, problem’s difficulty, and decision aids help reduce misconceptions about climate change. In the first experiment, we present how problems with surface or structural training during DCCS help reduce misconceptions in the CS task. In the second experiment, we investigate how problem difficulty during DCCS training help reduce misconceptions in the CS task. In the final experiment, we study how DCCS as a decision aid helps in lowering correlation heuristic and violation of mass balance misconceptions by allowing participants to test different values of emissions and absorptions in a trial-and-error procedure. We close the paper by discussing our results and highlighting the implications of using simulation tools (like DCCS) in education and policymaking against climate change.Figure 1). In the CS task, participants are asked to sketch CO2 emissions and absorptions that would stabilize the CO2 concentration according to a given scenario by the year 2100 . Participants are given the concentration’s starting value in the year 2000 , and its historic trends and emissions between the years 1900 and 2000. Participants are asked to sketch the CO2 emissions and absorptions shapes that would correspond to the projected scenario of CO2 concentration between 2001 and 2100. Figure 1C shows an example of a participant that relied on correlation heuristic, whereby he inferred that the shapes of the CO2 emissions and concentration should look alike. Moreover, as seen in Figure 1C, the participant commits violation of mass balance in her response as she fails to make emissions equal to absorption when the concentration reaches 2100. This paper uses the CS task with different CO2 concentration trajectories and cover stories to evaluate people’s reliance on correlation heuristic and violation of mass balance misconceptions.Prior research in stock-flow problems concerning Earth’s climate has analyzed reliance on correlation heuristic and violation of mass balance in the CS task   and follow the CO2 concentration trajectory shown in the bottom left panel.Furthermore, recent research has evaluated how repeated feedback in DCCS helps reduce correlation heuristic and violation of mass balance misconceptions . All participants were students from Science, Technology, Engineering, and Mathematics backgrounds . They were randomly assigned to one of the experimental conditions involving DCCS and CS tasks. Participants were paid a flat fee of INR 50 (approximately 0.9 USD) for their participation after they completed the study.Participants were recruited through an email advertisement for a climate study at Indian Institute of Technology Mandi, India. This study was carried out in accordance with the recommendations of Ethics Committee at Indian Institute of Technology Mandi with a written informed consent from all participants. Participation was voluntary and all participants gave written informed consent before starting their study. There were 100 participants in all . Ages ranged from 18 to 26 years : CS-Surface, CS-Structure, DCCS-Surface, and DCCS-Structure. In both DCCS-Surface and DCCS-Structure conditions, participants played 2-rounds of DCCS repeatedly with heterogeneous problems that were either based upon surface features or structural features and were then transferred to the CS task immediately. In the CS-Surface and CS-Structure conditions, participants played an unrelated task for the average time it took to complete 2-rounds in DCCS and they were then transferred to the CS task immediately. Heterogeneity in problems was either based upon surface features or structural features.Participants were randomly assigned to one of four between-subjects conditions . As shown in Figure 3, we used a glucose cover story  and a temperature cover story . In each of these two problems, participants controlled their accumulation trajectory in DCCS along a stabilization trajectory by making inflow and outflow decisions every 5 time periods repeatedly. After finishing two rounds in DCCS, participants were transferred to the CS task where they attempted two problems that were presented in a random order. Both these problems corresponded to Figure 1’s problem, where one of the problems was presented with the climate cover story , while the other problem was presented with the temperature cover story . In both problems, participants needed to sketch the shape of inflow and outflow that corresponded to the accumulation stabilization scenario. The CS-Surface condition contained the same two problems as part of the CS task in the DCCS-Surface condition; however, the CS-Surface condition did not include DCCS training prior to the CS task. In the CS-Surface condition, participants played an unrelated Tetris game before performing in CS tasks for a duration that equaled the time taken to finish 2-rounds of DCCS performance in the DCCS-Surface condition.Surface features refer to the dimensions of emissions, absorptions, and concentration; and, the cover-story used in DCCS. In the DCCS-Surface condition, participants first tackled 2 concentration under different CO2 concentration scenarios in DCCS. In the DCCS-Structure condition, participants first performed in two different climate problems presented randomly in DCCS. Each problem provided a different CO2 stabilization trajectory, where CO2 concentration increased from 765GtC in 2000 to stabilize at 936GtC by 2100 or a year before. In one of these DCCS problems, the stabilization occurred in year 2100 . In the other problem, the stabilization at 936GtC occurred much earlier in years 2070 (DCCS-2070), respectively, and the 936GtC value was maintained till the end year 2100 . In each of the two DCCS problems, participants were asked to control the CO2 concentration to the stabilization trajectory over a 100-year period by making emission and absorption decisions every 5 years, repeatedly. Once participants completed 2-rounds in DCCS, they were transferred to the CS task immediately where participants attempted two problems presented in a random order. One of these two problems were Figure 1’s climate problem , and the other problem was Figure 4B’s climate problem . In both CS problems, participants needed to sketch the shape of CO2 emissions and absorptions that corresponded to the CO2 concentration stabilization scenario. The CS-Structure condition contained the same two problems in the CS task of the DCCS-Structure condition and did not include training in DCCS. In the CS-Structure condition, participants played an unrelated Tetris game before performing the CS task for a duration that equaled the time taken to finish 2-rounds of DCCS performance in the DCCS-Structure condition.Structural features refer to how emissions and absorptions cause a change in COThe CS-2100-Inc, CS-2100-Dec, CS-Temp, and CS-Climate conditions formed the control groups in the experiment. The DCCS-2070, DCCS-2100, DCCS-Temp, and DCCS-Gluc formed the training groups in the experiment. The CS-2100-Inc (DCCS), CS-2100-Dec (DCCS), CS-Temp (DCCS), and CS-Climate (DCCS) formed the test groups in the experiment.2 emissions and CO2 concentration during the period 2000–2100 was greater than or equal to 0.8. A participant committed violation of mass balance for the increasing trajectory stabilizing in 2100 (2070), if CO2 emissions were less than CO2 absorptions before year 2100 (2070) or CO2 emissions were not within ± 0.5GtC of CO2 absorptions in 2100 (2070 and beyond). A participant committed violation of mass balance for the decreasing trajectory stabilizing in 2100, if CO2 emissions were greater than CO2 absorptions before year 2100 or CO2 emissions were not within ± 0.5GtC of CO2 absorptions in 2100. Because of heterogeneity in surface or structural features in DCCS, we expected participants to possess fewer correlation heuristic and violation of mass balance misconceptions in CS conditions following DCCS compared to CS conditions without DCCS exposure. We used an alpha level of 0.05 and a power of 0.80 for our statistical analyses. The dataset for the experiment has been provided as part of Supplementary Data Sheet S1.The dependent variables were the proportion of participants relying on correlation heuristic and the proportion of participants committing violation of mass balance. A participant relied on correlation heuristic when the correlation coefficient between CO2 emissions and absorptions corresponding to the given CO2 concentration trajectory. On completion of the CS task, participants were thanked and paid for their participation.Participants were randomly assigned to different conditions and given instructions about the study. Participants were told about the goal that they had to achieve and they could ask clarification questions, if any, before beginning their experiment. In the DCCS-Surface and DCCS-Structure conditions, participants first performed 2-rounds in DCCS on a desktop computer and then they were transferred to CS tasks, where the CS tasks were given using a pencil-and-paper format. However, in the CS-Surface and CS-Structure conditions, participants first performed an unrelated Tetris task and then they were immediately transferred to CS tasks, which were given using a pencil-and-paper format. In the CS task, participants had to sketch COFigure 5 shows the proportion of participants relying on correlation heuristic in CS tasks and DCCS in the DCCS-Structure and CS-Structure conditions. Furthermore, Table 1 shows the comparison of different conditions and the associated inferential statistics for correlation heuristic reliance. As seen in Table 1, the reliance on correlation heuristic was statistically smaller in CS-2100-Dec (DCCS) condition compared to CS-2100-Dec condition. Likewise, the reliance on correlation heuristic was statistically smaller in CS-2100-Inc (DCCS) condition compared to CS-2100-Inc condition. Furthermore, the reliance was similar in CS-2100-Dec and CS-2100-Inc conditions. Similarly, the reliance on correlation heuristic was similar in CS-2100-Dec (DCCS) task and CS-2100-Inc (DCCS) condition.We compared the correlation heuristic reliance between control groups and test groups in the structure conditions. Figure 6 shows the proportion of participants relying on correlation heuristic in CS tasks and DCCS in the DCCS-Surface and CS-Surface conditions. As seen in Table 1, the reliance on correlation heuristic was statistically smaller in CS-Temp (DCCS) condition compared to CS-Temp condition. Likewise, reliance was statistically smaller in CS-Climate (DCCS) condition compared to CS-Climate condition. Furthermore, the reliance on correlation heuristic was similar in CS-Temp condition and CS-Climate condition. Similarly, reliance on correlation heuristic was similar in CS-Climate (DCCS) condition and CS-Temp (DCCS) condition.Next, we compared the correlation heuristic reliance between the control group and the test group in the surface conditions. Last, we compared the correlation heuristic reliance between control groups and test groups across the surface and structure conditions. The reliance on correlation heuristic was statistically smaller in CS-2100-Inc condition compared to CS-Climate condition. However, the reliance on correlation heuristic was similar in CS-2100-Inc (DCCS) condition compared to CS-Climate (DCCS) condition.Overall, in agreement with our expectations, the proportion of participants relying on correlation heuristic was statistically smaller in DCCS-Structure and DCCS-Surface conditions compared to CS-Structure and CS-Surface conditions, respectively. Also, the correlation heuristic proportions were similar in the CS-2100-Inc (DCCS) and CS-Climate (DCCS) conditions. This latter finding suggested that both the structure and surface features were similar in their ability to reduce people’s correlation heuristic misconceptions.Figure 7 shows the proportion of participants committing violation of mass balance in CS tasks and DCCS in the DCCS-Structure and CS-Structure conditions. Furthermore, Table 2 shows the comparison of different conditions and the associated inferential statistics for mass balance violation. As seen in Table 2, the proportion of violation of mass balance was statistically smaller in the CS-2100-Dec (DCCS) condition compared to the CS-2100-Dec condition. Furthermore, the proportion of violation of mass balance was statistically smaller in CS-2100-Inc (DCCS) condition compared to CS-2100-Inc condition. The proportion of violation of mass balance was similar in CS-2100-Inc (DCCS) condition and CS-2100-Dec (DCCS) condition. Similarly, the proportion of violation of mass balance was similar in CS-2100-Inc condition and CS-2100-Dec condition.We compared the proportion of participants commiting violation of mass balance between control groups and test groups across the structure conditions. Figure 8 shows the proportion of participants committing violation of mass balance in CS tasks and DCCS in the DCCS-Surface and CS-Surface conditions. As seen in Table 2, the proportion of violation of mass balance was statistically smaller in CS-Temp (DCCS) condition compared to CS-Temp condition. Likewise, the proportion of violation of mass balance was statistically smaller in CS-Climate (DCCS) condition compared to CS-Climate condition. Furthermore, the proportion of violation of mass balance was similar in the CS-Temp condition and CS-Climate condition. Similarly, the proportion of violation of mass balance was similar in CS-Climate (DCCS) condition and CS-Temp (DCCS) condition.Next, we compared the violation of mass balance between control groups and test groups across the surface conditions. Last, we compared the violation of mass balance across the surface and structure conditions. The proportion of violation of mass balance was similar in CS-2100-Inc condition compared to CS-Climate condition. Similarly, the proportion of violation of mass balance was similar in CS-2100-Inc (DCCS) condition compared to CS-Climate (DCCS) condition. This latter finding suggested that both the structure and surface features were similar in their ability to reduce people’s violation of mass balance misconceptions.Thus, overall, the experience gained in DCCS helped participants to reduce mass balance violations. Furthermore, the violation of mass balance reduction helped participants to perform better in the following CS task in the DCCS conditions compared to that in the CS conditions in both structure and surface condition.The comparison of the problems in the CS tasks of DCCS condition and CS condition allowed us to measure the effectiveness of the surface or structural heterogeneity in reducing correlation heuristic and violation of mass balance misconceptions. In both the surface and structure conditions, misconceptions related to correlation heuristic and violation of mass balance reduced significantly in the CS tasks following DCCS compared to CS tasks without exposure in DCCS.2 emissions and absorptions affect the CO2 concentration), misconceptions reduce significantly in the CS task post DCCS performance. This finding agrees with recent research that showed that structural knowledge helped people reduce their correlation heuristic and violation of mass balance misconceptions in both cases when problems encountered in the CS task are structurally similar or different compared to those presented in DCCS . In our study, when people attempt to follow different trajectories of CO2 concentration in DCCS, then this exposure to heterogeneous system dynamics likely enables them to learn that the CO2 concentration increases when CO2 emissions are greater than CO2 absorptions, decreases when CO2 emissions are smaller than CO2 absorptions, and stabilizes when CO2 emissions equal CO2 absorptions.First, we found that when we changed the problem’s structural features between DCCS and the following CS task . All participants were from Science, Technology, Engineering, and Mathematics backgrounds . They were randomly assigned to one of the experimental conditions involving DCCS and CS tasks. Participants were paid a flat fee of INR 50 (approximately 0.9 USD) for their participation after they completed the study.Participants were recruited through an email advertisement for a climate-study at Indian Institute of Technology, Mandi, India. This study was carried out in accordance with the recommendations of Ethics Committee at Indian Institute of Technology Mandi with a written informed consent from all participants. Participation was voluntary and all participants gave written informed consent before starting their study. There were 90 participants in all . Ages ranged from 18 to 25 years : DCCS-Difficult, DCCS-Easy and CS. In the DCCS-Difficult and DCCS-Easy conditions, participants first performed 1-round in DCCS and were immediately transferred to the CS task. In the DCCS-Easy and DCCS-Difficult conditions, in DCCS, participants controlled the CO2 concentration to the stabilization trajectory in each round by making inflow and outflow decisions every 5 time periods repeatedly. In the DCCS-Easy condition, the DCCS used Figure 1’s problem. However, in the DCCS-Difficult condition, the DCCS used Figure 9’s problem. The shape of CO2 concentration scenario in Figure 9’s problem was more complex compared to that in Figure 1’s problem . The complexity of the concentration curve made Figure 9’s problem more difficult compared to Figure 1’s problem. After participants finished performing in DCCS, they were transferred to a different problem in the CS task. In the CS condition, however, participants played an unrelated Tetris task for the average time it took to complete 1-round in the DCCS task (in the conditions involving DCCS) and were transferred to the CS task immediately. In CS tasks across all conditions, participants attempted the problem shown in Figure 4B, where they sketched the shape of CO2 emissions and absorptions that corresponded to a decreasing CO2 concentration stabilization trajectory between 2001 and 2100. In this experiment, the CS condition formed the control group, the DCCS-Easy and DCCS-Difficult conditions formed the training groups, and the CS (DCCS-Easy) and CS (DCCS-Difficult) formed the test groups.Participants were randomly assigned to one of the following three between-subjects conditions  or curvilinear (difficult). We found that people could not reduce their correlation heuristic misconceptions after exposure to difficult climate problems in DCCS compared to those who were either not provided DCCS training or were only exposed to easy climate problems in DCCS. Similarly, the same intervention did not reduce the violation of mass balance misconceptions: The committing of violation of mass balance remained the same after DCCS training (among both easy and difficult problems) in the CS task compared to conditions where the CS task was given without exposure in DCCS. The lack of reduction in correlation heuristic and violation of mass balance misconceptions could be attributed to cognitive load theory . The focus of the next experiment is to evaluate how DCCS could be used as a side-by-side decision aid in reducing stock-flow misconceptions.2 concentrations and global temperatures we may need to rely upon decision aids. In simple descriptive binary-choice decision tasks, when participants are provided with experiential decision aids, they tend to rely on the experience gained in these aids in making descriptive decisions and improving their decision making  to assist them in solving complex mathematical problems. Similarly, climate-scientists and climate-policymakers are likely to use decision aids  while formulating future greenhouse gas emission policies. For example, to evaluate the effects of future emission policies on the COn making . The aimSD = 1.5 years). All participants were from Science, Technology, Engineering, and Mathematics backgrounds . They were randomly assigned to one of the conditions involving DCCS and CS tasks. Participants were paid a flat fee of INR 50 (approximately 0.9 USD) for their participation after they completed the study.Participants were recruited through an email advertisement for a climate-study at Indian Institute of Technology Mandi, India. This study was carried out in accordance with the recommendations of Ethics Committee at Indian Institute of Technology Mandi with a written informed consent from all participants. Participation was voluntary and all participants gave written informed consent before starting their study. There were 60 participants in all . Ages ranged from 18 to 26 years : Aid and No-aid. In the Aid condition, participants could use DCCS side-by-side as a decision aid while sketching the CO2 emissions and absorptions in the CS task; however, in the No-aid condition, participants only sketched the CO2 emissions and absorptions in the CS task and they did not use DCCS. In the Aid condition, participants could use DCCS anytime to enter 10-yearly emission and absorption values over a period of 100 years  and simulate the resulting CO2 concentration. The DCCS simulated the entered emissions and absorptions rapidly within 1 to 2 seconds. Participants could then reset DCCS to the year 2000 and simulate a different set of emission and absorption values. In the Aid condition, participants could use DCCS as many times as they wanted to before they sketched the CO2 emissions and absorptions in the CS task. Also, the number of times participants used the DCCS as a decision aid was recorded in the Aid condition. In the No-aid condition, participants were asked to play a Tetris game for an amount time that equaled the time that participants took to use DCCS in the Aid condition. The No-aid condition formed the control group and the Aid condition formed the test group.Participants were randomly assigned to one of two between-subjects conditions .First, we analyzed the number of times DCCS was used as a decision aid in the Aid condition. Results revealed that participants used DCCS between 1 time and 7 times in the Aid condition  = 5.46, p = 0.02, φ = 0.30]. The proportion of participants relying on correlation heuristic in DCCS was close to 0.30. The correlation between the number of times DCCS was used and reliance on correlation heuristic in the CS task was small and insignificant .We compared the correlation heuristic reliance between the CS tasks across the Aid and No-aid conditions. Figure 13 shows the proportion of participants committing violation of mass balance in the Aid and No-aid conditions. Results indicated that violation of mass balance was statistically smaller in the CS task of Aid condition compared to the CS task of No-aid condition . The proportion of participants committing violation of mass balance in DCCS was close to 0.85. The correlation between the number of times DCCS was used and committing of violation of mass balance in the CS task was small and insignificant .We compared the committing of violation of mass balance between the CS tasks across the Aid and No-aid conditions. Overall, in agreement with our expectations, the proportion of participants relying on correlation heuristic and committing violation of mass balance was statistically smaller in Aid condition compared to No-aid condition.Simulations tools may provide effective side-by-side decision aids that enable people to reduce their stock-flow misconceptions. Results revealed that DCCS served as an effective side-by-side decision aid and enabled people to reduce their correlation heuristic and violation of mass balance misconceptions compared to those conditions where DCCS was not present.2 emissions and absorptions influence the trajectory of CO2 concentration  are effective in causing learning of both structural features and surface features in problems. In our experiment, people were not given full-information on the formulations connecting emission, absorption, and concentration . These r2 concentration come closer to the goal are the ones that likely get reinforced over time. While performing the CS task, people retrieve these reinforced instances from memory to make improved decisions. Thus, people likely use their reinforced knowledge acquired in DCCS to draw correct trajectories of emissions and absorptions corresponding to the different concentration curves.Furthermore, our results revealed that the use of complex curve shapes in simulation tools , however, did not help participants to reduce their stock-flow misconceptions. This result could be explained based upon the additional working memory capacity requirements to process complex interaction of different elements like emissions, absorptions, and concentrations . In agre2 accumulation curves would be more likely to help reduce correlation heuristic and violation of mass balance misconceptions.We found that the difficulty hypothesis was unable to account for the findings in the second experiment. One likely reason for this observation could be that the tasks used in our study are different from those that were used for showcasing the difficulty hypothesis . In liteWe also found that the stock-flow misconceptions did not reduce when the concentration curve shape in DCCS was simple compared to when people were not exposed to DCCS at all. Thus, overall, this result disagrees with those reported in the first and second experiment, where performance in DCCS caused people to reduce their stock-flow misconceptions compared to conditions where participants were not exposed to DCCS. One likely reason for the disagreement could be the number of repetitions of DCCS given in the second experiment  compared to other experiment (multiple). Although we can only speculate currently, but, perhaps, more repetitions of DCCS in simple and difficult conditions could lead people to reduce their stock-flow misconceptions. This hypothesis needs to be tested as part of future research.Our findings have important implications for real-world climate education as well as climate policymaking. First, as simulation tools like DCCS likely create both surface and structure learning, they are ideal for educating students from kindergarten to standard 12th about stock-flow problems . Thus, t2 emission and absorption trajectories impacts CO2 concentrations and global temperatures. One expects improved policy analyses with repeated iterations in simulation tools.Third, the use of simulation tools as decision aids should be encouraged for both climate education and policy analyses. Here, simulation tools can be used as a side-by-side decision aid that provides people the ability to test different hypotheses concerning emissions, absorptions, and concentrations. Also, policymakers could use simulation tools like DCCS for climate policy analyses and to evaluate how different CO2 concentration) improve their decision-making in problems with two or more accumulations ? It would be interesting to investigate whether it is people’s conscious or unconscious learning that improves due to the use of simulation tools? And, whether people are really learning something about climate change or just learning to use the DCCS tool to complete the CS task?The current investigation on the use of simulation tools has revealed promising results. However, there are several research questions to pursue as part of research in the immediate future. Although different structural and surface training was taken into account, comparison with homogenous condition was not made. As part of future research, we would like to compare different structural and surface heterogeneous condition with homogeneous conditions. For example, it would be interesting to analyze how heterogeneity in structure, surface, and problem difficulty interacts with people’s science education and other demographic variables. Also, how a group of decision-makers (in contrast to single decision-makers) may improve their correlation heuristic and violation of mass balance misconceptions via simulation tools as well as how these groups show learning of structure, surface, and difficulty? Still, how people who improve their understanding of Earth’s climate in problems with a single accumulation (COPrior research has also reported that a part of the stock-flow misconceptions in the CS task could be because of the format of presentation of material concerning emissions, absorptions, and concentration . As per As part of our future work, we would like to answer some of these open-ended questions by involving complex stock-flow problems that vary in their complexity in terms of the number of stock and flows and nature of stock and flows . Also, hMK was the research lead who designed the experiments and carried out data collection for this work. VD was the principal investigator who served as a constant guiding light for this work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Recent studies suggest that epigenetic regulation of microRNAs might play a role in its development. In this paper we present the application of a complex computational pipeline to perform enrichment analysis of miRNAs in pathways applied to the study of BOS. The analysis considered the full set of miRNAs annotated in miRBase (version 21), and applied a sequence of filtering approaches and statistical analyses to reduce this set and to score the candidate miRNAs according to their potential involvement in BOS development. Dysregulation of two of the selected candidate miRNAs– The number of LTx has increased steadily over the last two decades and currently more than 3,000 lung transplantation procedures are performed yearly worldwide. LTx mainly intends to improve survival. However, long-term survival is hampered by the development of chronic lung allograft dysfunction (occurring in up to 50% of patients in the 5th post-transplant year), whose main clinical manifestation is represented by Bronchiolitis Obliterans Syndrome (BOS) 8]9][8][9]7][[9][8][9][8][9]7]. They coThere are two approaches to the study of the role of miRNAs in diseases. The traditional "omics" approach assesses a wide panel of factors on plasma/tissue samples and identifies those significantly (2-fold to 10-fold) dysregulated in disease samples with respect to controls. In the other, computational algorithms are used to perform a preliminary exploratory analysis to narrow down number of factors to test in the subsequent wet-lab validation phase. These approaches, however, do not always provide information on the specific cellular site of expression, which is far more relevant than absolute expression levels.In this paper, we implemented a pure computational pipeline to perform enrichment analysis of miRNAs in pathways involved in BOS pathogenesis. This enabled us to identify a limited panel of candidate miRNAs with potential involvement in BOS development. Unlike in other approaches, such as GSEA  and miTEmiR-34a and miR-21, two of the resulting candidate miRNAs. Interestingly, hybridization experiments clearly showed, for the first time, dysregulation of these two miRNAs at cellular level in BOS tissue samples. This provided important insights into their role in the pathogenesis of BOS, opening perspectives for further mechanistic and therapeutic studies.Preliminary validation of the selection of miRNAs was attained both through literature analysis during the computational phase; and by subsequent in situ hybridization experiments on https://github.com/sysbio-polito/BOS-miRNA-Enrichment.http://www.genome.jp/kegg/pathway.html). KEGG is unique in its focus on and coverage of yeast, mouse, and human metabolic and signaling pathways, with a set of more than 200 networks for Homo sapiens 23][[23][22][[[23][22]To take into account TF-mediated miRNA regulations, we developed a php script able to process the KEGG Markup Language (KGML) description of a pathway and systematically integrate TF information for each described gene see . TF infoHomo sapiens species representing our initial list of miRNAs was extracted.Our computational selection of candidate miRNAs involved in BOS development started from the full list of miRNAs annotated in miRBase (version 21 published on June 2014) . MiRBaseIn order to perform the miRNA target enrichment analysis, we first retrieved miRNA target information for each miRNA contained in our initial list. miRNA target information was retrieved by automatically mining the TargetHUB repository with a php script see . TargetHIn our study, we analyzed each miRNA considering two different sets of miRNA targets:Targets obtained by querying TargetHUB with results collected from the miRTarBase database referred to as Validated miRNA Targets (VMTs). MiRTarBase is known to collect information related to experimentally validated miRNA targets .Targets obtained by querying TargetHUB for results present in at least one of the five available repositories. These are referred to as Computational miRNA Targets (CMTs) since they contain a large set of computationally-inferred information.VMTs were the main focus of this research and were used as the main source of information to identify the set of miRNAs to be tested in the laboratory. Nevertheless, CMTs were also of interest because they enlarged the scope of the analysis and allowed the formulation of exploratory predictions on miRNAs, to be tested in future experiments. In the remainder of this paper we will use the general term miRNA targets to identify both VMTs and CMTs. The set of scripts used to perform this computational step is available in the “3-Mirna-Processing” folder of the BOS-miRNA-Enrichment repository on GitHub.To identify relevant miRNAs in BOS development we performed enrichment analysis of miRNA gene targets in the selected pathways for each of the 1870 considered miRNAs. This analysis allowed us to rank miRNAs based on their overall enrichment in the selected pathways. This information was used to skim the initial list of miRNAs, identifying a reduced panel with potential higher involvement in the pathogenesis of BOS.Making use of a combination of php and R scripts, we constructed a 2x2 contingency matrix for each miRNA-pathway pair, as shown in To perform a fair analysis, Nm- was computed by merging the list of targets of all considered miRNAs and subtracting the targets of the miRNA under evaluation; and Np- was computed by excluding those genes in the pathway that had never been annotated as a miRNA target in the considered database.For each miRNA-pathway pair we applied a one-tailed Fisher's exact test to compute the enrichment of the miRNA targets in the selected pathway 37]. The. The37].Finally, significance levels computed for each miRNA when paired with each pathway were combined together in order to test the significance of the miRNA enrichment over the joint set of pathways. First, false discovery rate (FDR) was applied to the p-values for each miRNA-pathway pair, in order to correct for multiple-hypothesis testing, thus implementing the correction as previously described in . Second,As an example, Finally, the scored list of miRNAs was filtered according to two different criteria. First we selected the miRNAs identified as being expressed in relevant tissue samples. This step is important to focus on the miRNAs specifically active in the target tissue. To perform this operation, we consulted mimiRNA, a database of miRNA expression across different tissues and cell lines . mimiRNALung tissues;B lymphocytes;Natural killer lymphocytes;Monocytes;Dendritic cells;Granulocytes; andA549 .The second step was to select intragenic miRNA co-expressed by genes or transcription factors in the selected pathways. To perform this operation, we consulted the miRIAD Intragenic Microrna database . Each geThe full list of 349 miRNAs selected along with the tissue or cell in which they are expressed is reported in the miR-34a and miR-21) was performed on formalin-fixed/paraffin-embedded samples of normal human and rat lungs, lung explants of BOS patients and rat models of acute and chronic lung rejection (outbred CD SPF/VAF), and on mesenchymal cells obtained from bronchoalveolar lavage (BAL) of lung recipients. The specimens were collected from the University of Padua and Foundation IRCSS San Matteo/University of Pavia Pathology Units. The study to develop an animal model of obliterans bronchiolitis was approved by the Research Ethics Committee of University of Padua . All procedures used in this study were conformed to the rules and principles of the 2010/63/EU Directive. Lung explants of BOS patients were collected from IRCSS San Matteo/University of Pavia Pathology Units. The study was approved by Pavia Area Ethics Committee of Foundation IRCCS San Matteo . Written informed consents of patients have been obtained before explant. Additional information regarding the Research Ethics Committee are provided in ISH for two candidate miRNAs   with sliμg/mL) for 10' at 37°C using an Abbott Molecular StatSpin ThermoBrite Hybridizer System (Dako hybridizer). Hybridization with 40nM miR-21 probe, 1nM U6 probe and 20nM scramble probe was carried out at 50°C for 60 min. For miR-34a, a less-abundantly expressed miRNA, 100nM labeled probe was used and hybridization was carried out at 37°C for 20h. Furthermore, to prevent miR-34a release and extra-tissutal diffusion during hybridization, an additional miRNA fixation step, using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, was added on tissue sections to anchor miRNAs into the protein matrix  and  and miR- in vivo ,68.miR-21 in homeostasis and disease, its contribution to BOS pathogenesis can be inferred to be at the level of myofibroblast migration and proliferation in the airway lumen, while a possible additional regulatory activity in the mechanism of epithelial-mesenchymal transition cannot be excluded.On the basis of our novel finding and of previous evidence of the role of Overall, this is the first study to systematically analyze and score a large number of miRNAs implicated in BOS. We were able to process all the miRNAs currently annotated in miRBase (version 21) and, by means of a sequence of computational filtering methods, we were able to filter them and then rank them according to their enrichment into the selected pathways.miR-34a and miR-21, other miRNAs have recently been described as being dysregulated in BOS. Two recent papers reported an integrated analysis of miRNA expression in the mouse models of orthotopic tracheal transplantation [miR-21, miR-146, miR-20, miR-302, miR-19, miR-98, let-7a, miR-15a. In comparison with lung recipients without BOS, clear dysregulation of miR-34a, miR-193b, miR-9 and miR-15a, likewise present in the VTM list in In addition to antation . The folmiR-34a and miR-21 dysregulation in fibroblasts obliterating the bronchiolar lumen, we cannot provide mechanistic insights into their role in BOS pathogenesis, and these should be addressed in specifically designed studies.The main limitation of our study is that only two factors identified by our pipeline underwent validation. However, since our wet-lab experiments were intended only to confirm the results obtained with the computational approach, more extensive tissue analysis was beyond the scope of the study. In addition, we chose 2 miRNAs which had never been associated with BO in previous human studies. For these reasons, while we did demonstrate Nevertheless, the data depicted in S1 File(XLSX)Click here for additional data file.S2 File(PDF)Click here for additional data file.S1 TableFor each pathway the table reports: the unique KEGG Identifier, the pathway name, the assigned class based on its relevance, the relevant genes and the related references that identify the pathway as relevant for BOS.(PDF)Click here for additional data file.


Cancer is the second leading cause of death affecting nearly one in two people, and the appearance of new cases is projected to rise by >70% by 2030. To effectively combat the menace of cancer, a variety of strategies have been exploited. Among them, the development of peptide–drug conjugates (PDCs) is considered as an inextricable part of this armamentarium and is continuously explored as a viable approach to target malignant tumors. The general architecture of PDCs consists of three building blocks: the tumor-homing peptide, the cytotoxic agent and the biodegradable connecting linker. The aim of the current review is to provide a spherical perspective on the basic principles governing PDCs, as also the methodology to construct them. We aim to offer basic and integral knowledge on the rational design towards the construction of PDCs through analyzing each building block, as also to highlight the overall progress of this rapidly growing field. Therefore, we focus on several intriguing examples from the recent literature, including important PDCs that have progressed to phase III clinical trials. Last, we address possible difficulties that may emerge during the synthesis of PDCs, as also report ways to overcome them. Cancer is one of the leading causes of death globally behind the heart and circulatory disorders based on statistics of World Health Organization (WHO) . Among aCurrent treatment processes involve a combination of surgical intervention, radiation and chemotherapy. Drugs used for this purpose are inevitably cytotoxic in order to eliminate cancer cells, but they lack selectivity that could be developed through targeting malignant cells . Due to Additionally, chemotherapy with anticancer agents is often hampered by their poor aqueous solubility, low oral bioavailability and metabolic instability. These drawbacks are linked to the unfavorable ADME (absorption distribution metabolism excretion) that are basically described in the following four consecutive axes: 1) Absorption is directly connected with the transportation process of the drug from the site of administration to the systemic circulation . 2) DistTherefore, most conventional cytotoxic agents applied in chemotherapy lack optimum pharmacokinetic properties (ADME) and thus are not very effective to treat malignancies. Moreover, despite the intensive research to construct new cytotoxic drugs, survival rates in most cancers remain low  and clinIt is clear that we do have access to an enormous pool of unspecific cytotoxic agents that can efficiently kill cancer cells. What is currently needed is not to invest so intensively in generating more cytotoxic agents but to re-use and re-cycle available ones and tailor them to be transformed into targeted chemotherapeutics. Along these lines, drug delivery vehicles that can be tailored for different types of cancer and shape personalized therapeutics are continuously gathering attention. Such drug delivery systems are of ultimate importance to effectively surpass these hurdles and eventually improve drug potency.18F-deoxyglucose positron emission tomography (FDG–PET) imaging was developed in order to visualize the phenomenon of increased glucose uptake by cancer cells -Thr-ol), lanreotide  and vapreotide , which bind mainly to the subtype 2 receptor (SSTR2) that is known to be the most frequently overexpressed SSTR [d-Phe-c[Cys-Phe-d-Trp-Lys-Thr-Cys]-Thr-ol) -octreotide. Recently, another similar analog, designated 111In-DTPA-d-Phe-1-Asp0-d-Phe1-octreotide, has been evaluated and presented enhanced tumor accumulation in pancreatic tumor cells and simultaneously lower renal radioactivity -GnRH-III(Dau=Aoa) enzyme stability of the conjugate was decreased while [4Lys(Ac)]-GnRH-III(Dau=Aoa) showed higher stability -GnRH-III(Dau=Aoa) conjugate that almost reached the in vitro biological effects of the conjugates with a cleavable linker. This conjugate showed significant tumor growth inhibition in vivo, not only on subcutaneous implanted but also on orthotopically developed HT-29 colon cancer-bearing mice -GnRH-III), or different drug molecules (-GnRH-III) [8Lys  were attached to the ε-amino group of nRH-III) –153. SomI) [8Lys .The resulted PDC presented a reduced aqueous solubility, thus an oligoethylene glycol linker was inserted between the spacer and the tumor-homing peptide . This PD6-GnRH-I has been exploited by our group to selectively deliver the anticancer agent gemcitabine to the tumor site. We, therefore, designed and synthesized four different bioconjugates consisting of D-Lys6-GnRH-I and the anticancer agent gemcitabine  through different conjugation sites  and using linkers of different lengths (succinyl and glutaryl) as shown in Furthermore, the tumor homing peptide D-Lys6-GnRH-I peptide induces any perturbation on the local microenvironment of the peptide that is responsible for receptor recognition, we utilized 1H 1H 2D-TOCSY NMR [6-GnRH-I allowing to suggest that they will not influence the binding affinity of the targeting peptide unit of these PDC to the GnRH-R. This was further validated since the new conjugates were found to possess higher binding affinity with respect to the parent peptide, with IC50 ranging even up to 1.9 nM for the conjugate 3G. The conjugates were evaluated regarding their antiproliferative effect on prostate cancer cells (DU145 and PC-3) and the PDC GSG showed IC50 values similar to gemcitabine GSG that possessed the highest antiproliferative effect was utilized for further pharmacokinetic studies in mice. These pinpointed that GSG is able to release a high amount of gemcitabine (averaging 500 ng/mL) for a period of over ≈250 min, while administered free gemcitabine was consumed in less than 100 min. At the same time, the levels of the inactive metabolite of gemcitabine (dFdU) were maintained at very low levels for the GSG conjugate in contrast to the direct administration of free gemcitabine. Finally, when injected into mice with xenografted tumors, GSG inhibited the tumor growth more effectively than gemcitabine when using equimolar quantities. Therefore, GSG could pave the way for the construction of other similar bioconjugates in order to effectively enhance the concentration of the cytotoxic drug in the tumor cells and inhibit their uncontrolled growth.In order to evaluate whether the tethering of the cytotoxic agent to the D-LysOCSY NMR . Upon su6-GnRH peptide-targeting-unit tethered through a succinyl linker [OuWe have also designed and synthesized a PDC containing the cytotoxic agent sunitinib and the D-Lysl linker . Sunitinl linker . Therefo8 of D-Lys6-GnRH to form the PDC named SAN1GSC -OH in the presence of HATU or HBTU and DIPEA, it became evident that the aminium moiety of HATU/HBTU could be installed either on the amino (–NH2) group of Lys or on the phenol (–OH) group of Tyr [During the conjugation of 5’- PDC GSG . We hypop of Tyr .6-GnRH and Fmoc-HER2-BP1  with HATU/DIPEA and characterizing the final products by ESIMS and 1H NMR spectroscopy where the same aminium side product was also recorded. Interestingly, when the dipeptide Fmoc-Cys-Tyr-NH2 was reacted with HATU, we found that the side product could be installed both on the phenol group of Tyr as also on the sulfhydryl group of Cys. Therefore, we tested these reaction conditions on the peptide C1B5141–151 subdomain peptide (RCVRSVPSLCG) of protein kinase C (PKC) γ isozymes, which possess 2 cysteines (but no tyrosine or lysine or free N-terminus amine) and bears anticancer properties [Our findings were further verified by reacting other tumor-homing peptides like D-Lysoperties . Again, Along these lines, we suggested a mechanism that this side-product formation is taking place, directly after the formation of the amide bond that occurs from structure (II) to structure (III), as shown in We discovered that the side product, which is difficult to be separated from the expected PDC and therefore results in reduced synthetic yield, could be avoided by using 1 equiv of HATU/HBTU, instead of the classical and established protocols using 1.5 equiv . Taking Currently used chemotherapeutics are in their majority highly toxic, causing severe side effects. Thus, with the aim to enhance their narrow therapeutic index, a wide variety of strategies have been explored. Selective drug delivery via special carriers represents a viable approach to deal with tumors with higher efficacy while using lower doses of the anticancer agent. Specifically, peptide–drug conjugates (PDCs) operate as potent drug delivery carriers and thus have attracted considerable attention over the last decades. The simplicity, versatility and the relatively low cost for the construction of PDCs have rendered them appealing candidates. In the present review, basic and integral knowledge has been accumulated towards the PDCs design through examining every module required to assemble the fully decorated PDC: the peptide, the cytotoxic agent and the linker. We highlighted the overall progress of this field through selective analysis of noteworthy examples in the literature, as also possible synthetic problems that may arise and their solutions. Based on the fact that several PDCs have been selected for clinical trials and presented tumor inhibition with minimum side effects, this field needs to be further refined and explored. Through this review, we made efforts to provide an influential impetus for the construction of new peptide–drug conjugates, which could eventually transform undesired toxic drugs to highly potent formulations for the effective treatment of cancer.


Radiopharmaceuticals are the major and essential component in nuclear medicine and are the most prominent aspect of peaceful applications of nuclear sciences in human being’s daily life. These radioactive materials are formulated in appropriate forms for administration to human being for the diagnosis and the therapy of human diseases. Diagnostic radiopharmaceuticals are the majorly applied radiopharmaceuticals worldwide (>90%), while therapeutic applications are still in the growing process.99Mo/99mTc generators, the most widely used component in the nuclear medicine practice. Since then, many Tc-99m kits for single photon emission computed tomography (SPECT) were prepared and developed in the country together with other diagnostic and therapeutic radiopharmaceuticals in last 3 decades. Recently, positron emission tomography (PET) has been initiated in the country using the most important PET tracer, i.e. 18F-FDG. The advances of radiopharmaceutical sciences in Iran have been recently reported at international levels  or low specific activity (gel moly) Mo-99 loaded on a column shielded in proper lead container to be sent to nuclear medicine centres. The generator systems are then eluted using appropriate aqueous solution and the 99mTcO4- radio-anion is then mixed with appropriate cold kits containing a ligand and some other additives  to obtain an injectable IV solution. Based on the nature of the kit ligands used, the radiopharmaceutical can be used in the diagnosis of various human diseases, such as malignancies, bone problems, heart disorders, liver diseases, etc.Tc-99m is used in more than 80% of diagnostic procedures and is usually prepared in a 131I-NaI oral solution or capsules is routinely used in the detection of thyroid related diseases and metastasis usually in low doses, the radioisotope is produced by the irradiation of natural tellurium target in research reactor. At higher doses this radiopharmaceutical is used for the treatment of hyperthyroid disease and targeted therapy of thyroid malignancies.Other important diagnostic tracer in form of meta-iodobenzyl guanidine (131I-MIBG) used in the diagnosis of neural crest derived tumors, such as neuroblastoma and pheochromocytoma at low doses. At higher dose, this radiopharmaceutical is used for targeted therapy of the neuroendocrine malignancies.Another important I-131 based radiopharmaceutical is the synthetic homologue of neuroamines called I-131 +/K+ ATPase pumps. Since there is no appropriate potassium radioisotope from the physical point of view to be used for human use, Tl-201 thallous chloride has been considered an important radiopharmaceutical for imaging of organs overexpressing this pump at the cellular level such as myocardial as well as some malignant cells. The radionuclide is produced in a medium sized cyclotron (30 meV) located in Karaj  since 1993 and has been used as the gold standard in the myocardial viability test in human.Tl-201 thallous chloride radiopharmaceutical is used in nuclear medicine based on homology to potassium cation incorporating into biological systems specially Na111In-oxinate for the tracing of radiolabeled cells such as stem cells, leucocytes etc. or used as 111In-DTPA for imaging of cerebrospinal fluid in specific diseases (cisternography).Another cyclotron based radiopharmaceutical is Ga-67 citrate, usually used in the detection of infections, inflammations and some malignancies based on homology to ferric cation in the biological systems. In-111 radionuclide is an interesting cyclotron product used hugely in radioimmunoscintigraphy research studies and also in small molecules in form of 81Rb/81mKr generator for pulmonary studies of lung diseases has been also produced since 1996 as a very safe gaseous radiopharmaceutical that could be even used in infants due to ultra-short half-life (13 seconds) Kr-81m radioactive gas.An interesting 18F-FDG) tracer, a miraculously developed tracer in late 1970’s and early 1980’s due to many physical, biochemical and radiochemical properties. The tracer is specifically integrated into GT1+/HK1+ cells of tumors, brain and also heart and since being approved by FDA in 1991 has been used in the detection and follow-up of more than 30 human malignancies as well as cardiac and brain studies. Sodium fluoride-18 (18F-NaF), is another important PET tracer, easily binding to hydroxyapatite part of the skeletal system offering a gold standard of bone imaging in the nuclear medicine. The other important series of PET tracers are developed from the exciting 68Ge/68Ga radioisotope generator providing an in- house gallium-68 production system, letting every single remote PET center around the country to produce in-house radiopharmaceuticals emphasizing the necessity of hospital radiopharmacies. The recent production and evaluation of Ga-68 generator in the country has been reported and initial human studies on prostate cancer using 68Ga-PSMA has been also announced. The other internationally used Ga-68 tracers such as 68Ga-citrate, 68Ga-DOTATATE, 68Ga-DOTATOC has also been developed and are ready to enter the clinical evaluations in 3 existing Iranian PET centers.The most important and recent diagnostic method called PET has been clinically started in the last 4 years in the country, although the research and development on these tracers initiated almost 2 decades ago. The most important PET radiopharmaceutical is F-18 fluorodeoxy-D-glucose , Re-186  and Lu-177  radioisotopes obtained by Tehran Research Reactor and were used in various clinical studies and successful demonstration of bone pain palliation in patients suffering from metastatic irremediable skeletal pains. Soon after in order to back up the supply chain, in case of reactor production discontinuity, the other important bone pain palliative agent; 188Re-HEDP was produced from generator based radioisotope (188W/188Re generator) and successfully went through clinical evaluations.Recently, 90Y-citrate colloid in the management of large joint radiosynovectomy was successfully shown in separate clinical studies in the country and the agent is at the moment available. Y-90 is a generator produced therapeutic beta emitter obtained from long-lived (30 y) generator available in the country. Also 188Re-sulfur colloid is being used for the radiosynovectomy as demonstrated in other clinical evaluations in the country based on its availability via 188W/188Re generator.Another important series of therapeutic radiopharmaceuticals include radiosynovectomy agents used in the significant alleviation of arthritis pains in patients suffering from joint inflammations due to immunologic/pathologic problems. The application of At the moment more than 50 radiopharmaceuticals are available in Iranian Drug List that most of them are produced and used in the nuclear medicine centers at a regular basis and other available per request . The resa.jalilian@iaea.orgDr. Amir Reza Jalilian is currently working as a Professor of Radiopharmacy at Radioisotope Products and Radiation Technology Section, IAEA, Vienna, Austria. He could be reached at the following e-mail address: beikidav@tums.ac.irDr. Davood Beiki is currently working as a Professor of Radiopharmacy at Research Center for Nuclear Medicine, Shariati Hospital, Tehran University of Medical Sciences, Tehran, Iran. He could be reached at the following e-mail address:


Previous studies have shown that cytokinetic abscission at the end of mitosis is executed by the ESCRT machinery in mammalian cells, and that the process is dependent on adhesion-induced integrin signalling via a FAK-PLK1-CEP55-TSG101/Alix-CHMP4B pathway. The present study identified an alternative abscission mechanism driven by mechanical force. In the absence of integrin signals (non-adherent conditions), cytokinesis in non-transformed human fibroblasts proceeds to CEP55 accumulation at the midbody, but after prolonged time (>3 hours) the major midbody components Aurora B, MKLP1 and CEP55 were no longer detected in the area. Upon adhesion to fibronectin, such cells were able to complete abscission without re-appearance of midbody proteins. Live-cell imaging revealed that re-plating on stiff fibronectin matrix (64 KPa) allowed >95% of the cells to complete abscission within 9 hours while the corresponding number was 40% on soft fibronectin matrix (0.5 KPa). The cells re-plated on poly-L-lysine were not able to generate tension and did not divide. Thus, mechanical tension can cause cytokinetic abscission by stretching of the intercellular bridge between the two daughter cells until it eventually ruptures without the involvement of ESCRT complexes. Importantly, regression of the cleavage furrow and formation of bi-nucleated cells did not occur in most of the suspension-treated mitotic cells after re-plating on fibronectin. Septin, which stabilizes the membrane associated with the midbody, was found to remain along the ingressed membrane, suggesting that this filament system maintains the membrane bridge although the midbody had dissolved, thereby preventing regression and allowing tension to act on the narrow intercellular bridge. Proper separation of the cytoplasm between the two emerging daughter cells at the end of mitosis is essential in order to avoid the formation of bi-nucleated cells, since such cells will either die, become tetraploid, or become aneuploid cells in the next mitosis . The higIt is well established that the absence of cell adhesion to extracellular matrix (ECM) causes the cytokinesis process to halt in normal cells . In fibrOur previous studies showed that cytokinesis proceeds in non-adherent human BJ fibroblasts to a stage where CEP55 is present at the midbody and then the process halts . To furtThe behaviour of the three-hour suspended cells after re-plating on fibronectin-coated dishes was analysed by live cell imaging to allow clear monitoring of any cytokinetic abscission events, the formation of bi-nucleated cells, or cell death. Surprisingly, most of the cells divided, although the completion of the process was delayed -coated dishes, as well as on soft (0.5 kPa) and stiff 64 kPa) fibronectin matrices. On PLL, the cells attached but did not spread or migrate and, as expected, abscission did not occur under this condition Figure . The sti kPa fibrTo investigate if abscission by mechanical rupture affected the functionality of the cells, the ability to proceed in the following cell cycle into S phase was tested. Mitotic cells were isolated and cultured in suspension for 3 hours as in the previous experiments, plated on fibronectin-coated coverslip for different time periods, and then incubated with the nucleotide analogue EdU during an additional hour on fibronectin. For comparison, mitotic cells continuously cultured on fibronectin was given EdU at the same time periods. In both culture conditions, almost no cells exhibited EdU-labelled nucleus during the first hour after isolation, but after 12 hours (3 hours suspension culture followed by adhesion for 9 hours) the number of EdU-positive cells gradually increased at a similar rate in cells re-plated on fibronectin and continuously adherent control cells Figure .After the three-hour suspension period followed by re-plating on fibronectin, the morphology of the cells with two closely located nuclei Figure . To avoiSince the septin filament system is known to contribute to the stabilisation of the ingressed plasma membrane , 16, we A similar distribution of septin-7 which persisted after dissolution of the midbody structure was observed also in three other analysed cell lines, i.e. 3T6 mouse fibroblasts, mouse embryo fibroblasts (MEFs), and NMuMG mouse epithelial cells. Under non-adherent culture conditions CEP55 disappeared from the midbody region in each one of these cell lines during a three-hour period, and yet, a septin-7 ring was localized at the ingression site  and mostIn contrast to cancer cells, proliferation of normal cells requires the interaction of cells with extracellular matrix. Integrin-mediated adhesion contributes to the cell cycle progression through G1 to S phase and to the completion of the last phase of cytokinesis , 19. A lThe results of our present study clarifies some of the contradicting observations in previous reports. Separation of mammalian cells by traction force exerted on the intercellular bridge by the migration of the two daughter cells has previously been observed , 24, butThe critical step in midbody maturation that has to be reached in order to prevent regression is presently not known, but our data indicates that the organisation of septin filaments may be a key factor to keep the plasma membrane ingressed without midbody proteins. During the late stages of cytokinesis, the septin distribution was altered from a broad and longitudinal to narrow and perpendicular form relative to the intercellular bridge Figure . This isin vitro, time-lapse recording of live cells is one method which allows clear determination of this issue, but other methods are needed for studies of non-adherent cells or closely associated cells in vitro and in vivo. Our results suggest that the localisation of septin may be a useful marker for the analysis of abscission and regression.Since cleavage furrow regression after failed cytokinesis will result in cells with more than two centrosomes in the following mitosis, it is a potential cause for the formation of aneuploid tumorigenic cells. Cleavage furrow regression has therefore been a topic for studies in numerous reports. However, since the intracellular cytokinetic bridge is narrow, it is often not obvious if two closely located nuclei represent two divided but interacting cells, one bi-nucleated cell with ingressed plasma membrane, or one bi-nucleated cell with regressed plasma membrane. When adherent non-confluent cells are studied 2. In some experiments CDK1 inhibitor RO-3306  and cytochalasin D (Sigma-Aldrich) were used to synchronize the cells at the G2-M phase transition.Human non-transformed fibroblasts , mouse fibroblasts  equipped with a CCD camera  and a cell culture chamber having constant supply of humidified 5% CO©  and ImageJ (http://rsb.info.nih.gov) software.For the adhesive condition, the mitotic cells were cultured on fibronectin-coated glass coverslips, whereas for the non-adhesive condition they were cultured in Pluronic-coated 10 cm bacterial plates  and thereafter the cells were deposited on glass slides by cytospin centrifugation. Subsequently, the cells were fixed by cold methanol at −20° C for 20 minutes and then washed twice in PBS for 5 minutes. After incubation in blocking buffer containing 1% BSA  and 0.1% Tween20  in PBS, the slides were incubated overnight at 4° C with primary antibodies at a 1:50 dilution in blocking buffer. Antibodies directed against the following proteins were used: Aurora B , CEP55 , α-tubulin , MKLP1 , Septin-7 . The slides were then washed with PBS and incubated for 1 hour with a 1:500 dilution in blocking buffer of secondary antibodies, Alexa Fluor 488-conjugated goat anti-rabbit and Alexa Fluor 594-conjugated goat anti-mouse , followed by washing in PBS and mounting with medium containing DAPI . Digital images of the cells were captured using a Nikon fluorescence microscope  equipped with a CCD camera . The digital images were analysed for the presence or absence of immunostained proteins at the midbody and scored using Adobe PhotoshopUpon plating on fibronectin-coated substrate, the isolated round mitotic cells flatten, divide, and migrate away from each other. Successful cell division could be clearly identified when they had moved apart. In the fixed samples, the cells were scored as bi-nucleated when two cell bodies were in contact. Since the migrating cells frequently made transient contacts with each other, live-cell imaging allowed a more accurate analysis.EdU detection was performed according to the protocol . The cells were incubated with 10 μM EdU for 1 hour prior to fixation with 4% formaldehyde and fluorescence microscope imaging to analyse EdU incorporation.t-test. P-values < 0.05 were considered as significant. For all the experiments, 50 randomly selected cells per condition and for each time point were analysed from each of three independent experiments. ***, **, and * represent P-value less than 0.001, 0.01 and 0.05, respectively.The statistical analyses were performed using student’s


Plasmodium falciparum parasitemia by measuring levels of thyroglobulin, ferritin, soluble transferrin receptor, retinol binding protein 4, α-1-acid glycoprotein, C-reactive protein, and histidine-rich protein II. This 7-plex immunoassay technique has potential as a rapid and effective tool for use in large-scale surveys and assessments of nutrition intervention programs in low- and middle-income countries.Deficiencies of vitamin A, iron, and iodine are major public health concerns in many low- and middle-income countries, but information on their status in populations is often lacking due to high costs and logistical challenges associated with assessing micronutrient status. Accurate, user-friendly, and low-cost analytical tools are needed to allow large-scale population surveys on micronutrient status. We present the expansion of a 7-plex protein microarray tool for the simultaneous measurement of up to seven biomarkers with relevance to the assessment of the key micronutrients iron, iodine, and vitamin A, and inflammation and malaria biomarkers: α-1-acid glycoprotein, C-reactive protein, ferritin, retinol binding protein 4, soluble transferrin receptor, thyroglobulin, and histidine-rich protein II. Assay performance was assessed using international reference standards and then verified by comparing the multiplexed and conventional immunoassay results on a training panel of plasma samples collected from US adults. These data were used to assign nominal concentrations to the calibrators of the assay to further improve performance which was then assessed by interrogating plasma samples from a cohort of pregnant women from Niger. The correlation between assays for each biomarker measured from this cohort was typically good, with the exception of thyroglobulin, and the sensitivity ranged from 74% to 93%, and specificity from 81% to 98%. The 7-Plex micronutrient assay has the potential for use as an affordable tool for population surveillance of vitamin A, iron, and iodine deficiencies as well as falciparum malarial parasitemia infectivity and inflammation. The assay is easy-to-use, requires minimal sample volume, and is scalable, rapid, and accurate—needing only a low-cost reader and basic equipment present in most reference laboratory settings and so may be employed by low and middle income countries for micronutrient surveillance to inform on status in key populations. Micronutrient deficiencies including iron, iodine, and vitamin A affect a significant portion of the world’s population. Efforts to assess the prevalence of these deficiencies in vulnerable populations are challenging, partly due to measurement tools that are inadequate for assessing multiple micronutrients in large-scale population surveys. We have developed a 7-plex immunoassay for the simultaneous measurement of seven biomarkers relevant to assessing iodine, iron, and vitamin A status, inflammation and  Plasmodium falciparum . To . To 5]. Summary statistics from the 7-Plex assays and conventional assays from both the training panel and the NiMaNu study specimens are shown in Reference materials measured in the 7-Plex and conventional immunoassays as compared to their certified values are shown in Using a method similar to inter-method harmonization, as described by Wilson, et al. , we modeSpearman correlations between the 7-Plex assays and original study results were high (0.840–0.987) for plasma AGP, CRP, ferritin, RBP and sTfR concentrations, and moderate for Tg (0.753) which compared 7-Plex plasma results to NiMaNu DBS results . HRP2 opA total of 1442 data points for the 7 biomarkers from the 206 NiMaNu samples were generated by the 7-Plex assay. From the initial 7-Plex assay, there were only 21 test results that were out of range (upper or lower) and only 4 samples that failed to meet quality control (QC) criteria of an intra-assay CV of <15% for duplicate sample wells. Only 3 assays were affected by being out of range limits: CRP , ferritin , and sTfR (1 lower). Some samples were out of range for more than one analyte, meaning that retesting a single sample permitted measurement of more than one biomarker that was out of range. Therefore, only 25 from 1442 test results (1.7% of the entire test panel) had to be reassayed.Further comparison of the data sets was made via scatter plots and Bland Altman analyses Figs . Each plOur initial evaluation of the panel showed significant differences in absolute values between results from an R&D Systems sTfR ELISA kit and the 7-Plex sTfR . The NiMFor Tg, the Spearman correlation between the 7-Plex and revised NiMaNu results was originally 0.753 (n = 199). Scatterplots  show ninSensitivity and specificity analysis was used to determine whether the data derived via the 7-Plex could be used correctly to categorize individual samples as deficient/sufficient , positive or negative for malaria parasitemia (HRP2), and positive or negative for acute inflammation (AGP and CRP), using the classification from the NiMaNu study as the gold standard . Using tP. falciparum. The sTfR assay was redesigned because an antibody used in the original design was no longer available. All of the assays were successfully integrated into an immuno-array and then further optimized to allow a single dilution of small sample volume for the simultaneous quantification of seven analytes using a traditional ELISA protocol and equipment, apart from the Q-viewer for plate reading and analysis. In addition, a reference spot was also added to the array in order to simplify the alignment of the data capture grid of the analysis tool with each well of the 96 well plate image. The Q-View software automatically fits calibration curves for all seven analytes using a log 5-parameter logistic model, thus simplifying analysis, as compared to conventional assays which may require the use of additional software to fit assay calibration curves and calculate concentration. Verification experiments demonstrated that the resulting 7-Plex panel met performance requirements for assay precision, linearity, and limits of detection. Good correlation, sensitivity and specificity in comparisons to results of conventional assay results for samples collected from a population at risk for deficiency, inflammation and malaria parasitemia suggest the 7-Plex assay offers a promising alternative to existing methods.This work was designed to expand upon a pre-existing multiplexed assay that we have previously developed by adding additional biomarkers for MN status assessment, and to make improvements to the original assays included in the Quansys Bioscience Q-Plex™ technology . New assIn our previous study, we used the Bio-Rad Liquichek 3 control as the calibrator for our reference curve values ,36. WhilA lack of harmonization between different ELISAs to the same target biomarker is commonly observed and their standardization remains a challenge ,53. Our We noted that the ferritin results differed significantly from the Ramco kit results when the 7-Plex calibrator was referenced to the NIBSC standard, with recovery averaging 12%. The NIBSC standard was intended to qualify the standard concentration but we found that it was poor when used with our assay. The NIBSC ferritin standard is a recombinant protein expressed from a bacterial source and therefore it may be possible that some epitopes are absent or altered in this molecule as compared to human ferritin. We hypothesize that one or both of the monoclonal antibodies used in the 7-Plex sandwich ELISA has reduced avidity to the ferritin standard used. However, an inter-laboratory evaluation of this standard showed generally good high concordance with the standard values . We thenSimilarly, the sTfR results were significantly different in the 7-Plex immunoassay versus the reference method (R&D or VitMin Labs). The challenges with standardization of sTfR immunoassays have been noted before with bias across methods prior to a reference standard being available; the NIBSC standard also uses a recombinant antigen . Even wiA key limitation of our previous study was that the panel used to verify the original 5-plex assay was derived from an adult US cohort, which does not represent the typical populations that the tool is designed to survey in terms of more variable MN status and increased exposure to disease and/or malaria. In this study, we also demonstrate the performance of the 7-Plex array on samples derived from pregnant women in Niger. This represents a key population group where surveillance can play a role in understanding MN status in populations at risk. This panel was ideal for the initial verification of the 7-Plex as the same biomarkers had all been qualified by independent methods, with the exception of seven data points for Tg, from samples that were not analyzed. Overall, the assays on the 7-Plex array had good concordance with the predicate ELISAs when analyzed by Spearman correlation, and scatter plots, and Bland Altman analysis reflect overall agreement in spite of some cases of systematic difference in absolute value. The Bland Altman analyses were key to highlighting that some highly discordant test data points were at either very low or high levels, and so while significantly different, they present the same test outcome . This was further confirmed by applying ROC analyses to determine the optimal cut-off values that still gave high sensitivity and specificity of the 7-Plex assay as compared to the NiMaNu cut off threshold to establish MN status as sufficient or deficient, inflammation status, or malarial infection. When considering all the biomarkers assessed from the NiMaNu panel, only 1.7% had to be retested due to being out of range or being where duplicate test results were variant by >15%, indicating that the combination of broad calibrator ranges and the sample dilution volume permit the detection of most biomarkers in one test run. This limits the need to run multiple repeat assays using different sample dilutions in order to acquire a complete data set saving money, reducing the need for extra sample volumes and the time to process discrepant samples.AGP had the poorest sensitivity in the NiMaNu samples of pregnant women via this analysis and this is largely in part to the few samples that had significantly elevated AGP levels with the majority of samples being tightly clustered at low concentrations. We hypothesize that AGP is prone to more significant variation within small sample dilutions as compared to the other biomarkers due to its relatively high concentration. For example, many conventional ELISA assays for AGP recommend a sample dilution of 1:10000 to prior to measurement of AGP, whereas the 7-Plex plasma sample volume is only diluted by 1:10. In some cases, the values for recovery were significantly different from the original assay while being concordant via Spearman and Bland Altman analysis.While MN deficiencies affect vulnerable population groups globally, LMICs are most significantly affected. Most LMICs attempt to address these deficiencies via supplementation, fortification, and dietary and/or agricultural interventions to improve micronutrient intakes. However, the challenge in assessing MN status at the population level has led to large data gaps in terms of general surveillance. This is due to a variety of reasons but primarily because MN measurements are costly, difficult and complex to perform in-country based upon the current methodologies. Our tool is specifically targeted toward users in LMICs, with the intent of developing a practical and relatively low-cost tool  to enable in-country surveillance of nutritional status across key populations and to measure the performance of ongoing nutritional interventions. We have developed a tool that can be used in a laboratory setting, does not need a technically complex reader, and requires only common laboratory equipment . The protocol follows a standard methodology for performing an ELISA assay and one trained laboratorian can process 10 plates in an 8-hour period .Further validation of the Human Micronutrient 7-Plex assay on other appropriate cohorts is necessary and is presently underway by our team and by other independent groups. We are currently investigating the use of dried blood or plasma spots as a sample type given that an assay only requires 16 μL of sample. We are also in the process of investigating the expansion of the range of assays hosted by the platform in order to assess other key MNs including folate, vitamin B12, and vitamin D.S1 FigConcentrations of each analyte as measured in the 7-Plex (x-axes) plotted against concentrations measured using conventional assays (y-axes) for 72 lithium heparin plasma specimens. Solid line is linear regression. AGP, α-1-acid glycoprotein; CRP, C-reactive protein; RBP, retinol binding protein 4; sTfR, soluble transferrin receptor; Tg, thyroglobulin.(TIF)Click here for additional data file.S2 FigBland-Altman plots showing percent difference between 7-Plex and conventional assay results on the y-axes plotted against average concentration on the x-axes for 72 lithium heparin plasma specimens. Heavy horizontal line and light horizontal lines indicate mean ± 2 standard deviations of percent difference.AGP, α-1-acid glycoprotein; CRP, C-reactive protein; RBP, retinol binding protein 4; sTfR, soluble transferrin receptor; Tg, thyroglobulin.(TIF)Click here for additional data file.S3 FigReceiver operating characteristic (ROC) curves plotting sensitivity (y-axis) against 1-specificity (x-axis) were generated from 7-Plex results of pregnant women participating in NiMaNu study to identify optimal threshold values for each analyte as measured in the 7-Plex to be used in specificity and sensitivity analysis. Optimal threshold values, defined as those resulting in maximal area under the ROC curves (AUC), are reported in (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 TableMedian concentrations and interquartile range (IQR) of sTfR as measured by three immunoassay methods per sample set and comparisons between assays in absolute value (% recovery) and by Spearman correlation.(DOCX)Click here for additional data file.


Sodium-glucose co-transporter 2 (SGLT-2) inhibitors have emerged as a promising drug class for the treatment of diabetic kidney disease. Developed originally as glucose-lowering drugs by enhancing urinary glucose excretion, these drugs also lower many other renal and cardiovascular risk factors such as body weight, blood pressure, albuminuria, and uric acid. Results from the EMPA-REG OUTCOME and CANVAS trials show that these salutary effects translate into a reduction in cardiovascular outcomes and have the potential to delay the progression of kidney function decline. This review summarizes recent studies on the mechanisms and rationale of renoprotective effects.Effects of SGLT-2 inhibitors on the kidney are likely explained by multiple pathways. SGLT-2 inhibitors may improve renal oxygenation and intra-renal inflammation thereby slowing the progression of kidney function decline. Additionally, SGLT-2 inhibitors are associated with a reduction in glomerular hyperfiltration, an effect which is mediated through increased natriuresis and tubuloglomerular feedback and independent of glycemic control. Analogous to diabetic kidney disease, various etiologies of non-diabetic kidney disease are also characterized by single nephron hyperfiltration and elevated albuminuria. This offers the opportunity to reposition SGLT-2 inhibitors from diabetic to non-diabetic kidney disease. Clinical trials are currently ongoing to characterize the efficacy and safety of SGLT-2 inhibitors in patients with diabetic and non-diabetic kidney disease.The glucose-independent hemodynamic mechanisms of SGLT-2 inhibitors provide the possibility to extend the use of SGLT-2 inhibitors to non-diabetic kidney disease. Ongoing dedicated trials have the potential to change clinical practice and outlook of high-risk patients with diabetic (and non-diabetic) kidney disease. The worldwide prevalence of diabetes mellitus will continue to increase in the next decades from 415 million people in 2015 to 642 million in 2040 . ConsequCurrent treatments to prevent or delay kidney (as well as cardiovascular) complications in patients with diabetes focus on lowering blood pressure, HbA1c, body weight, albuminuria, and cholesterol. Targeting these multiple risk factors reduce the risk of cardiovascular disease and kidney function decline , 7. NeveRecently, several strategies have been tested to improve the prognosis of patients with diabetes. One of these strategies was to examine the effects of intensive compared with conventional glucose control on cardiovascular complications. Several large clinical trials in patients with type 2 diabetes showed that aggressive glucose lowering did not result in a reduced risk for macrovascular complications , 9. The The mechanisms for cardiovascular and kidney protection of SGLT-2 inhibitors in patients with diabetes mellitus are incompletely understood. This review summarizes new insights in the potential protective mechanisms of SGLT-2 inhibitors. In the context of recently started renal outcome trials, we will also review the potential benefits of SGLT-2 inhibitors in patients with non-diabetic kidney disease.2 180 gram glucose is filtered each day by the kidney. In these healthy conditions, urinary glucose concentration is absent owing to an effective reabsorption system, consisting of two sodium-glucose co-transporters (SGLT): SGLT-1 and SGLT-2 [18F]fluoro-dapagliflozin . Th. Th69]. An important question to be addressed is of course whether it is safe to use SGLT-2 inhibitors in non-diabetic CKD patients. As described above, the risk of hypoglycemia with SGLT-2 inhibitors in non-diabetic patients is likely low as the degree of glucose-lowering positively correlates with the filtered glucose load which is low in non-diabetic CKD populations. Clinical experience with SGLT-2 inhibitors in non-diabetic conditions is currently limited, but studies in healthy volunteers have shown that SGLT-2 inhibitors can be safely used at high doses, even up to 50-fold the dose used in clinical practice, without inducing hypoglycemia . Thus, tCurrent guidelines recommend to use SGLT-2 inhibitors as adjunct to metformin therapy in patients with type 2 diabetes and established cardiovascular disease . This reVolume depletion is another adverse effect detected in clinical trials. Patients at risk of volume depletion, such as those with gastrointestinal fluid losses or reduced oral intake, are therefore not good candidates to start SGLT-2 inhibitors. In addition, SGLT-2 inhibitors should be withheld during procedures that may reduce renal perfusion such as elective surgery or intravenous contrast procedures in the same way as is done with ACE inhibitors and angiotensin receptor blockers. Along the same line, SGLT-2 inhibitors should not be continued during short-term periods of NSAID use, as the combination of these two drug classes may reduce renal perfusion and may evoke acute kidney injury.The CANVAS program unexpectedly showed that patients treated with canagliflozin had a 2-fold higher risk of lower limb amputations compared with placebo-treated patients. Patients with a history of amputations or with peripheral vascular disease were at highest absolute risk for amputations during the trial, but the relative effect of canagliflozin was similar across subgroups ••. The uSGLT-2 inhibitors are promising. Not only do they improve glycemic control but they also offer substantial protection against progression of cardiovascular and kidney disease in patients with diabetes independent of glucose control. In the last 2 years, several hypotheses emerged that could explain the underlying mechanisms of these protective effects. With respect to the kidney, restoring tubuloglomerular feedback, and attenuating intra-renal hypoxia and inflammation seem plausible mechanisms by which SGLT-2 inhibitors delay the rate of kidney function decline. These glucose-independent effects offer the opportunity to reposition SGLT-2 inhibitors to the non-diabetic CKD population. Ongoing trials will determine whether this novel drug class is indeed an important asset to combat the epidemic of diabetic as well as non-diabetic CKD.