There are concerns about the impact of climate change on Olympic Games, especially endurance events, such as marathons. In recent competitions, many marathon runners dropped out of their races due to extreme heat, and it is expected that more areas will be unable to host the Games due to climate change. Here, we show the feasibility of the Olympic marathon considering the variations in climate factors, socioeconomic conditions, and adaptation measures. The number of current possible host cities will decline by up to 27% worldwide by the late twenty-first century. Dozens of emerging cities, especially in Asia, will not be capable of hosting the marathon under the highest emission scenario. Moving the marathon from August to October and holding the Games in multiple cities in the country are effective measures, and they should be considered if we are to maintain the regional diversity of the Games. Heat stress, which is a stress effect on an organism that results from exposure to excessive ambient temperatures2, has become apparent in humans and is expected to increase further. During 1991–2018, 37% of heat-related deaths in 732 locations across all continents were attributable to anthropogenic climate change3. Population exposure to “deadly heat” (wet bulb temperatures above 32 °C) may increase by a factor of five to ten by 2070–2080 compared with 20204, and the number of excess global temperature-related deaths is projected to be 83 million with the 4.1 °C rise by 21005. While elderly individuals and children are particularly vulnerable to heat stress, individuals competing in outdoor sports may also be at risk6. Tennis matches were suspended during the 2014 Australian Open after temperatures exceeded 43°C7, and 40 of 68 female marathon runners dropped out at a temperature of 33 °C with 73 percent humidity in the 2019 IAAF World Championships in Doha8. High air temperatures and elevated humidity negatively affected the performance of Olympic marathon runners in 1896–20049.The global land temperature in 2011–2020 increased by 1.59 °C from 1850 to 1900 and is projected to increase throughout the twenty-first century10; thus, the feasibility of the marathon in the Games has important implications both for the health of athletes and the culture of modern humanity. Smith et al.11 revealed that few cities outside of Western Europe will be viable to hold the Olympic marathon by 2085 under the Representative Concentration Pathway (RCP) 8.5 based on the wet bulb globe temperature (WBGT)12. DeChano-Cook and Shelley7 pointed out that many historical host cities of the summer and winter Games will no longer be suitable because of the increases in temperature and sea level.The marathon is one of the most symbolic events of the modern Summer Olympic Games , which are the most watched and most expensive event on earth13. Since an increase in the WBGT has a negative impact on the performance of most marathon runners including elite athletes15, and since the WBGT standards suggested by the International Institute for Running Medicine (IIRM)16 and American College of Sports Medicine (ACSM)17 are referred to in the management of various marathons, we adopt the WBGT as an indicator of the feasibility of hosting an Olympic marathon. We utilize the seven GCMs included in the S14 retrospective meteorological forcing dataset (S14FD)18, which was developed based on the Coupled Model Intercomparison Project Phase 5 (CMIP5)19. We reproduce hourly urban climatic characteristics by applying a bias correction using meteorological observation data based on the method of Takakura et al.20. We consider future socioeconomic conditions using the parameters of population22, the gross domestic product (GDP)23, and its growth rate in the 2030–2050 period and the 2070–2090 period, both are 10 years before the evaluation periods, based on the five Shared Socioeconomic Pathways (SSPs)24. We quantify the effects of AMs that have been adopted or considered in recent Games, such as holding the marathon late at night or early in the morning. The main text presents the results for the late twenty-first century (2080–2099), while the results for the mid-twenty-first century (2040–2059) are presented in the Supplementary Information (SI).The purpose of this study is to explain the feasibility of hosting the Olympic marathon more comprehensively, considering the variations in climate change projections, the hourly climatic characteristics of cities, future socioeconomic conditions, and adaptation measures (AMs) that have not been adequately considered in previous studies. We hypothesize that these climatic, socioeconomic, and adaptation factors all have distinctive impacts on the feasibility of hosting an Olympic marathon, and this study quantifies these impacts. We consider the variation in climate change by projecting the future WBGT in August in the mid- and late twenty-first centuries (2040–2059 and 2080–2099) using seven global climate models  and four emission scenarios 11; and (3) the availability of meteorological data for WBGT correction using the method of Takakura et al.20. See the Methods section for the rationale behind the selection criteria above.First, to clarify the impact of climate change alone, we evaluated the number of cities that could host the Olympic marathon in August during the late twenty-first century (2080–2099) under the socioeconomic conditions in 2020, changing only the climatic conditions according to the four RCPs. A total of 70 cities in 25 countries were selected based on (1) socioeconomic conditions as of 2010 , Int’l $ 2005) as of 2010 and a GDP growth rate above 0% as of 2010–2015); (2) an elevation of less than 1,600 m16.WBGT level 1 (good): There is a greater than 90% probability that the WBGT will fall below 18°C for at least three consecutive hours between 7:00 and 21:00 in August. WBGT level 1 corresponds to a low alert level and “good conditions” in the IIRM's manual.WBGT level 2 (caution): Level 1 does not apply, and there is a greater than 90% probability that the WBGT will fall below 22°C for at least 3 consecutive hours between 7:00 and 21:00 in August. WBGT level 2 corresponds to a moderate alert level and “less than ideal conditions” in the IIRM's manual.WBGT level 3 (warning): Levels 1 and 2 do not apply, and there is a greater than 90% probability that the WBGT will fall below 28°C for at least 3 consecutive hours between 7:00 and 21:00 in August. WBGT level 3 corresponds to a high alert level and “potentially dangerous conditions” in the IIRM's manual.WBGT level 4 (cancel): Levels 1, 2, and 3 do not apply. WBGT level 4 corresponds to an extreme alert level and “event cancelled / extreme and dangerous conditions” in the IIRM's manual.As evaluation criteria for cities, the WBGT levels were set as follows based on the four alert levels  in the IIRM Medical Care manual25, which is closely related to the WBGT. However, there would have been few cases where the WBGT exceeded 28 °C in the Berlin Marathon races covered in the study even momentarily, based on the dry bulb temperature range in the races. Therefore, the use of WBGT Level 4 (28 °C) as a threshold for the impossibility of holding a marathon is consistent with the results of the aforementioned research. The 90% criterion is set in reference to previous studies on the feasibility of hosting the summer and winter Olympics27, and the duration (3 h) and timing (between 7:00 and 21:00 in August) is set based on the general competition time of the Olympic marathon, where all Olympic marathons since the 1980 Moscow Olympics were held, except for the men's and women's marathons at the 1988 Seoul and 2000 Sydney Olympics, the women's marathon at the 1996 Atlanta Olympics, and the women's marathon at the 2020 Tokyo Olympics, which was moved up by an hour the day before to start at 6:0033.We determine that cities with WBGT levels of 1 to 3 can host the Olympic marathon, while those with a WBGT level of 4 cannot. It has recently been found that the performance of elite marathon runners improves as the dry bulb temperature risesAs a result, globally, the number of cities that can host the Olympic marathon (WBGT levels 1–3) significantly decreases as the amount of greenhouse gas emissions increases toward the late twenty-first century 2080–2099)  out of the 70 total cities. Under historical (1994–2013) climate conditions, all cities (33 cities) can host the marathon, while the number of cities decreases to 30.3 (92%) under RCP2.6 and to 16.3 (49%) under RCP8.5. The number of cities with a relatively low heat risk (WBGT levels 1–2) decreases from 1 (3%) under historical climate conditions to none under RCP8.5.The number of cities that can host the Olympic marathon decreases significantly as the amount of greenhouse gas emissions increases. Among the viable cities, the number of cities with a relatively low heat risk (WBGT levels 1–2) is very small and will decrease further with climate change. This result may be because many cities in Asia are in the mid-latitude zone of the Northern Hemisphere and are affected by the Asian monsoon, resulting in hot and humid summers.North America: This region contains the second largest number of cities (16 cities). Under historical (1994–2013) climate conditions, all cities can host the marathon. Under RCP8.5, the number of cities decreases to 15.3 (96%), but under the other scenarios, the number of cities does not change at all or remains mostly unchanged. The number of cities with relatively low heat risk (WBGT levels 1–2) decreases from 8 (50%) under historical climate conditions to 4.4 (28%) under RCP2.6 and to 1.0 (6%) under RCP8.5.Latin America and the Caribbean: This region contains the third-smallest number of cities (7 cities). Under historical (1994–2013) climate conditions, all cities can host the marathon. The number of cities does not change at all or only slightly decreases under the four RCP scenarios. The number of cities with a relatively low heat risk (WBGT level 1–2) decreased from 5 (71%) under historical climate conditions to 3.6 (51%) under RCP8.5.Africa: The region contains the second-smallest number of 3 target cities. Under historical (1994–2013) climate conditions, all cities can host the Olympic marathon. The number of cities decreases to 2.6 (87%) under RCP8.5, but under the other scenarios, the number does not change at all or remains mostly unchanged. The number of cities with a relatively low heat risk (WBGT levels 1–2) does not decrease even under RCP8.5, however, the number of cities with WBGT level 1 decreases from 2 (66%) to 1 (33%) under RCP8.5.The number of cities that can host the event (WBGT levels 1–3) only slightly decreases even under the highest emission scenario, while the number of cities with a relatively low heat risk (WBGT levels 1–2) will decrease due to climate change. This finding may be because many cities are in the high latitudes of the Northern Hemisphere (North America) or the Southern Hemisphere  and are thus less likely to be hot and humid in August than Pattern 1 (Asia).Europe: This region contains the third largest number of target cities, 9. Under historical (1994–2013) climate conditions, all cities can host the Olympic marathon, and under all RCP scenarios, the number does not change at all. The number of cities with a relatively low heat risk (WBGT levels 1–2) decreases from 6 (67%) under historical climate conditions to 3.7 (41%) under RCP8.5.Oceania: This region contains the smallest number of target cities, 2. Even under the highest emission scenario, the number of cities that can host the Olympics does not decrease, and all four cities are determined to be safe (WBGT level 1) under all RCP scenarios.The number of cities that can host the Olympic marathon does not decrease even under the highest emission scenario. There were originally many cities with a relatively low heat risk (WBGT levels 1–2), which will be the case under future climate conditions. This finding may be because many cities are in the high latitudes of the Northern Hemisphere (Europe) or in the Southern Hemisphere (Oceania), where the WBGT is unlikely to be high in August.24. The number of cities by country is shown in Supplementary Figs. Next, we estimate the number of cities that can host the Olympic marathon in the late twenty-first century 2080–2099), considering future socioeconomic scenarios (SSPs 1–5) Fig. . Here, a0–2099, cWe selected target cities using the same criteria as in the evaluation of 70 cities for current socioeconomic conditions. The numbers of selected cities for the five SSPs are 94 for SSP1 (sustainability scenario), 120 for SSP2 (middle of the road scenario), 79 for SSP3 , 90 for SSP4 , and 126 for SSP5 (fossil-fueled development scenario).Globally, as in the results under current socioeconomic conditions, the number of cities that can host the Olympic marathon tends to decrease as the amount of greenhouse gas emissions increases, especially in Asia. The number of cities under each emission scenario is the highest for SSP5 and the lowest for SSP3. Under all SSPs, the number of possible host cities increases from the current socioeconomic conditions (2020). For example, under RCP2.6, the number of cities ranges from 72.7 (SSP3) to 122 (SSP5), while under RCP8.5, the number of cities ranges from 57.6 (SSP3) to 99.9 (SSP5).For all SSPs, the number of possible cities under RCP8.5 is approximately 20% lower than that under RCP2.6, and the impact of the emission scenarios is apparent. Dozens of emerging cities, mainly in Asia, that could host the summer Olympics due to economic development under the low emission scenarios will not be able to host the Olympics under RCP8.5, the highest emission scenario.In this section, we evaluate the change in the number of cities that can host the Olympic marathon in August due to AMs in the late twenty-first century (2080–2099) Fig. . The fouUnder RCP2.6, the effects of AMs are limited due to the originally small decrease in the number of host cities  are shown for 19 cities that have hosted and/or are scheduled to host the Games since 1948 Fig. . Note th36. Thus, it is highly likely that equivalent measures will be required in other future host cities if they are assessed as WBGT level 3.If no adaptation measures are taken (NM), the number of cities with a high heat risk (WBGT level 3) will increase from 7 under the past climate to 10 to 12 (out of 19 cities) under the future climate. Even for Tokyo, which is assumed to be WBGT level 3 at the time of the 2020 Tokyo Olympics, the venues for marathons and race walks were relocated to Sapporo, a city more than 800 km away in the north, due to concerns about heatAmong the single AMs, the effect of holding the event in October (AM2: Oct) is the highest. In most cities, the WBGT level will be 1 or 2 even under RCP8.5. On the other hand, in Rio de Janeiro, Sydney, and Brisbane, which are in the Southern Hemisphere, there are no effects of the measure, or rather, the WBGT level increases. Holding the event in multiple cities in a country (AM3: BC) may or may not be effective depending on the cities. Countries with more diverse domestic climates (cooler metropolises) may be advantageous. Notably, this study does not include cities for which sufficient climate data have not been collected, and the actual effect may be greater. The case where all AMs are implemented at the same time (AM4: All) is the most effective, with all cities under RCP2.6 and 4.5 and 17/16 cities under RCP6.0/8.5 having a WBGT level of 1. See the SI for the results for the mid-twenty-first century  and the effectiveness of adaptation measures in addition to the impacts of climate change.The main contribution of this study is that it more comprehensively explains the feasibility of hosting the Olympic marathon in the future than previous studies37 to 206 in Rio de Janeiro in 201638, and since the Munich Olympics in 1972, each of the Games has been held on a different continent from the previous one, which suggests that regional diversity is becoming increasingly important in the Games. If the Games are to be held in various regions of the world in the future, adaptation measures including the four presented in this paper, are worth considering, in addition to mitigating the impacts of climate change by reducing greenhouse gas emissions. In particular, these adaptation measures will be necessary for many cities to be able to host marathons with relatively low heat risk (WBGT levels 1–2).The number of countries participating in the Games has increased from 14 in the first modern Games in Athens in 189639. In North America, July and August are off-season for sports, which may be the reason why most of the recent Games have been held in those months40. The share of broadcasting rights fees from Asia, Europe, Central America, South America, and the Caribbean is increasing39, but it is not clear whether they will be sufficient to offset the possible decline in broadcasting rights fees for North America. In addition, another effective measure for holding the Games in multiple cities in a country (AM3) may increase the cost related to infrastructure, security, and logistics; therefore, careful consideration of cost-effectiveness is necessary.Hosting the Olympics in October (AM2) was found to be an effective adaptation measure, but it requires negotiation with broadcasters in North America (US and Canada), which pays the majority of the International Olympic Committee's broadcasting rights fees42. The above proposal is consistent with the proposal by Muller et al.10 from the viewpoint of the sustainability of the Games .For example, holding the Games in a city with existing infrastructure and experience in hosting large-scale sporting events and/or limiting the number of spectators and people involved in the Games would reduce the costs associated with AM2 and AM343. (2) Although the focus of this study is on athletes, the heat risk to spectators, who make up the majority of those involved, should be considered as well. (3) In this study, the evaluation was based on the WBGT of representative points in each city, and therefore, microclimatic conditions in the city and adaptation measures, such as the construction of heat-shielding pavements, planting of trees, and misting, were not considered. Such evaluations require a large computational resource for modeling and/or detailed measurements in the field; therefore, it is realistic to use these methods for a smaller number of cities. (4) The cities selected in this study may differ from those that can host the Olympic Games in the future due to the lack of relevant data. Factors such as political rights, experience in hosting world championships, the population support for the Games, dispute with the IOC, and existing stadium infrastructure are also important for being selected as a host city44, but it is difficult to predict the situation of such factors in the mid- and late twenty-first centuries. Therefore, we did not include them in the city selection. Additionally, many cities particularly in Africa and Latin America are excluded due to the lack of sufficient meteorological observation data to reproduce the climatic characteristics of cities, even though they have sufficient socioeconomic conditions to be selected. Moreover, from the perspective of sustainability, Olympic Agenda 202045, presented by the IOC as a reform plan for the Olympic Movement, encourages the use of simple temporary facilities rather than new venues, and it allows the games to be held in locations other than the host city . Therefore, it is possible that smaller cities than those targeted in this study will be allowed to host the Olympics in the future. Based on the above, it is necessary to review the conditions for selecting cities in the future, taking into account the improvement in related data and the trend of sustainability improvement in the Olympics. (5) The WBGT, which was used as the index in this study, has several limitations, including the tendency to underestimate heat in environments where sweat evaporation is limited (high humidity and low wind speed), the inability to take into account conditions such as exercise intensity and clothing, and the lack of evidence on the relationship with health risks in sports47. In the future, it is necessary to develop more robust methods for assessing health risks in marathon running and sports in general.Some issues should be considered in the future. (1) The Games include many outdoor events other than the marathon. It is desirable to evaluate the thermal conditions for such events and to consider possible competition programs within 16 days, which is the basic duration of the Games48 and the conditions of the historical host cities, we selected 165 cities in 66 countries based on the following four perspectives: (1) the urban population, (2) national GDP and its growth rates, (3) elevation, and (4) the availability of meteorological observation data to reproduce the climatic characteristics of the city. Regarding socioeconomic conditions, (1) and (2), since the selection of host cities generally starts approximately 10 years before the Games48, this study use data from 2010 (for 2020), the 2030–2050 period (for 2040–2059), and the 2070–2090 period (for 2080–2099), 10 years before the evaluation period. The specific conditions for the four perspectives are described as follows.Referring to the papers that studied the selection of host cities for the Games44. In reality, competitions are rarely all held in a single city, and in most cases, competitions are also held in neighboring cities. Therefore, in this study, the selection is made based on the population of urban agglomerations (“the population contained within the contours of a contiguous territory inhabited at urban density levels without regard for administrative boundaries”21). We use data from 2010 (for 2020), the 2030–2050 period (for 2040–2059), and the 2070–2090 period (for 2080–2099). The data for each period is acquired or calculated using the following methods.21.2010: The annual population of urban agglomerations in 201022.2030–2050: These data were calculated by multiplying the urban population in 2020 by the ratio of the projected national population in 2020 and the average population from 2030 to 2050 in five-year intervals for each SSP22.2070–2090: These data were calculated by multiplying the urban population in 2020 by the ratio of the projected national population in 2020 and the average population from 2070 to 2090 in five-year intervals for each SSPCondition 1: The city has a population of at least 2.5 million. A study of bids for the 1992–2020 Summer Olympic Games pointed out that a city population of 2.5 million or more is an important factor in being selected as a host city49. Therefore, we set the threshold at $300 billion. Regarding GDP, its medium-term growth rate is important for being selected as a host city44, and after the 1992 Summer Olympics in Barcelona, there was no host city with an average national GDP growth rate of less than 0% from 15 to 10 years before the Games. For these reasons, we set a GDP growth rate of above 0% as a condition. We use data from 2010 (GDP for 2020), the 2010–2015 period (GDP growth rate for 2020), the 2030–2050 period (GDP and its growth rate for 2040–2059), and the 2070–2090 period (GDP and its growth rate for 2080–2099)22. For each SSP, the national GDPs in 2010, the average GDP from 2030 to 2050, and the average GDP from 2070 to 2090 are used22.Condition 2: The national GDP is no less than $300 billion  and the GDP growth rate is above 0%. Olympic-related expenditures are often made on a national basis and on a city basis. After the 1992 Summer Olympic Games in Barcelona, no host city had a national GDP of less than $300 billion 10 years before the Games11. Given the negative effects of a high elevation on long-distance running, including marathons that became apparent at the 1968 Mexico City Olympics9, cities with elevations higher than 1,600 m should not be viable for hosting marathon competitions.Condition 3: The elevation of the city is less than 1,600 m20. We used terrestrial weather station data51 and solar radiation data52 corresponding to the weather stations.Condition 4: There is a meteorological station within 20 km of the representative point of the city, with at least 2,500 data points collected from May to October (summer season in the Northern Hemisphere) over multiple years at intervals of 3 h or less. This condition was established to adequately reproduce the site-specific WBGT, referring to the SI of Takakura et al.9. In many cases, the output of GCMs does not provide diurnal variations, and the spatial resolution is coarse , so it is not suitable for this study. Dynamical downscaling of GCM output using regional climate models (RCMs) is a common solution for spatiotemporally detailed calculations, but it is computationally expensive and not suitable for extensive global studies, such as this one. Therefore, we adopt the method of Takakura et al.20, which can provide site-specific hourly resolution WBGT prediction with only relatively simple calculations using GCM outputs, and we can achieve the objective of this study with a reasonable amount of computation.To consider holding a marathon in a particular city for a few hours of the day, it is necessary to calculate the site-specific hourly resolution WBGT. The environmental conditions during Olympic competitions vary even from start to finish during the same race20, bias correction of WBGT calculated from each grid of GCMs was performed using the observation records of WBGT in six cities in Japan. In this study, however, we use the hourly resolution meteorological data of each city to calculate the equivalent WBGT data using the method of Liljegren et al.53 since most of the cities in the world do not have published WBGT observation records. Additionally, we used natural spline interpolation to create 24-h data for cities where data existed at 3-h intervals or less and excluded other cities since many cities do not have a complete set of 24-h observation data. For more explanations of the calculation, see Takakura et al.20 and Supplementary Fig. In Takakura et al.1. The 90th percentile is used to identify the 90% probability of the definition of WBGT levels. In Figs. The feasibility of hosting the Olympic marathon in each city is assessed by comparing the 90th percentile of the WBGT between 7:00 and 21:00 in the RCP/target period (e.g. RCP4.5/2080–2099), averaged over seven GCMs, with the conditions of four WBGT levels defined in “We evaluate the effect of four AMs that have been adopted or considered in recent Games (except AM4) and can be reproduced with the spatiotemporal resolution of this study. However, it has not been confirmed whether these measures were considered heat countermeasures when they were adopted or considered in the past. See Table Supplementary Information.


This AANAT activity enhancement in vivo did not interfere with light-induced AANAT suppression. Systemic or in vitro lipopolysaccharide (LPS) administration markedly increased Stat3 expression and STAT3 phosphorylation, but it did not significantly affect AANAT expression or activity. Simultaneous LPS administration and Stat3 silencing enhanced the aa-nat transcription and AANAT activity to a similar extent as Stat3 inhibition without LPS co-administration. Furthermore, we describe the circadian rhythmicity in Stat3 expression and the phosphorylated form of STAT3 protein in the rat pineal gland. Our data suggest that the higher nocturnal endogenous level of STAT3 in the pineal gland decelerates or hampers the process of NA-induced AANAT activation or affects the AANAT enzyme stability.In rodents, the melatonin production by the pineal gland is controlled through adrenergic signaling from the suprachiasmatic nuclei and regulation of the principal enzyme in its synthesis, arylalkylamine-N-acetyltransferase (AANAT). In the present study, we identified increased isoprenaline-induced  A horizontal line-fit model (H0) and cosinor analysis were used as the alternative hypothesis. The rhythmicity was approved when both tests were significant. Unpaired two-tailed t tests were used for single column comparison, and multiple t-tests with the Sidak–Bonferroni post-hoc test were used for the effect of in vivo LPS administration. The rhythmicity of the p < 0.0001). The cosinor analysis rejected the null hypothesis  and confirmed the significant circadian rhythmicity for the Stat3 (p < 0.0001).To determine whether the Stat3 shows circadian rhythmicity in the rat pineal gland, we performed qPCR to determine changes in mRNA level and immunohistochemistry to reveal changes in the phosphorylated form of its protein. We found that the Stat3 mRNA expressed the circadian rhythm in the pineal gland .The significant rhythmicity was also confirmed for the level of the pSTAT3(y) (p < 0.0001) as expected [p = 0.0177).In search of the role of STAT3 in pineal physiology, we focused on its effect on AANAT, the most intensively studied enzyme in the melatonin synthetic pathway. We prepared organotypic cultures of pineal gland and treated them with the cell-permeable inhibitor of STAT3 signaling WP1066 for 4 h, non-selective β-AR agonist isoprenaline (ISO) for 6 h, or their respective combinations . ISO sigexpected . Our datp < 0.0001; ISO vs. ISO + siRNA: t = 2.187, df = 15, p = 0.0493). The effectiveness of siRNA transfection for the level of STAT3 protein and its phosphorylated forms is shown in As the nocturnal increase in AANAT activity requires a neo-transcription of its gene and a neo-synthesis of its protein ,9, we tep < 0.0001). However, unlike the aa-nat, the Stat3 siRNA did not upregulate ISO-induced Icer mRNA levels (p = 0.2689). p < 0.0001; Control vs. ISO + siRNA: t = 2.722, df = 6, p = 0.0346), suggesting the positive regulatory loop that may exist in the pineal gland between the STAT3 protein and its own gene. The stimulation with ISO did not significantly change the Stat3 expression in this experiment.Besides aa-nat, the inducible cAMP early repressor (Icer) is another NA-induced gene that is significant for pineal physiology. It inhibits the cAMP-stimulated genes and may negatively affect aa-nat transcription . Figure p = 0.0117; CT20 C vs. CT20 WP: t = 5.302, df = 12, p = 0.0002). The results from in vitro experiments suggested that STAT3 does not affect the aa-nat transcription and activity without concurrent stimulation of β-AR signaling. ISO is often used in in vitro conditions to substitute the night-time adrenergic signaling from the SCN. Therefore, to approach the natural conditions, we performed experiments in which the rats were treated with STAT3 inhibitor WP1066 and with AZD1480 to prevent the phosphorylation and activation of STAT3, both during the subjective day (CT4) and subjective night (CT15). The activity of AANAT was measured by radioenzymatic assay in pineal glands collected 5 h later . Our datp = 0.0223; CT20 C vs. CT20 AZD+LP: t = 2.991, df = 10, p = 0.0136; CT20 C vs. CT20 WP+LP: t = 2.569, df = 10, p = 0.0280).In parallel with this experiment, we tested a hypothesis that STAT3 inhibitors, due to their enhancing effect, may interfere with the light-induced AANAT suppression. The 1-min light pulse of intensity 30 lux significantly decreased the AANAT activity, but administration of either inhibitor had no effect , CT15 (p = 0.0002), CT18 (p < 0.0001), and CT21 (p = 0.0001). At CT24 , the Stat3 mRNA level was comparable with the control group. Similarly, as in Stat3 expression of control animals showed circadian rhythmicity ) and ser727 (pSTAT3(s)) in the SCN . Here, wat3 mRNA  and phosat3 mRNA  in the pThe acute systemic LPS administration (1 mg/kg) at both, during the early night, or in the middle of the subjective day induced strong STAT3 phosphorylation on tyrosine A,B, as wp = 0.0045; CT20 vs. LPS: t = 4.408, df = 6, p = 0.0011).Next, we asked whether the LPS-induced STAT3 could be involved in the regulation of AANAT activity. We injected the rats with LPS or with LPS combined with WP1066 at CT15 and harvested their pineal glands 5 h later A,B. Our p < 0.0001; Control vs. ISO+LPS: t = 10.62, df = 33, p < 0.0001; ISO vs. ISO+WP+LPS: t = 2.905, df = 25, p = 0.0074).To isolate the pineal glands from the context of blood circulation and sympathetic innervation, we prepared organotypic cultures and treated them with LPS, WP1066, and ISO or their respective combinations B. Our daaa-nat expression using cultured pinealocytes . Similar results have been obtained for alocytes A. The trTLR4 is a membrane-bound receptor that is well known for recognizing LPS. The molecular weight of TLR4 is approximately 95 kDa. Although the presence of a TLR4 receptor sensitive to LPS has been already found in the pineal gland , we checaa-nat, expression of Icer was upregulated by concurrent stimulation with ISO and LPS , but similarly to a previous experiment, Stat3 siRNA had no additive effect. Stat3 mRNA has been significantly reduced in primary cultures with siRNA, regardless of co-stimulants . LPS induced Stat3 mRNA in cultures, similar to in vivo . Administration of ISO enhanced expression of Stat3 significantly , and co-stimulation with LPS had small additive power .In contrast to Stat3 transcription in the rat pineal gland expresses circadian rhythm with maximum during the subjective night. Furthermore, here we show that also the level of pSTAT3(y) demonstrates circadian rhythmicity. Similarly, the daily rhythm in the Stat3 expression and STAT3 phosphorylation has been shown in the SCN [aa-nat, the Stat3 gene contains a CREB binding site in its promoter [Stat3 expression in these tissues.The present study, similar to our previous study , demonst the SCN ,19,20, bpromoter ,35. Althpromoter ,36 and mStat3 expression and phosphorylation. As the pineal gland’s most fundamental function is melatonin production, we asked whether this endogenous STAT3 could be involved in the regulation of activity and transcription of the rate-limiting enzyme of melatonin synthesis, AANAT. This enzyme is considered to be a major regulatory node on which multiple signaling pathways affecting the melatonin level converge [The majority of reports concerning the STAT3 focus on the regulation of genes involved in cancer and inflammation, and its transcriptional activity is mostly studied in the context of its aberrant upregulation and activation . Howeverconverge ,11,12 anaa-nat transcription. The β-AR signaling in the rodent pineal gland is activated via a multisynaptic pathway from the SCN during the night, and ISO is used in in vitro conditions to substitute this night-time derived adrenergic signaling [We used three systems, organotypic cultures, primary cultures, and in vivo systemic treatment with STAT3 inhibitors. The STAT3 silencing in primary cultures with siRNA and the treatment of organ cultures with the STAT3 inhibitor WP1066 resulted in more than a 60% increase in ISO-induced AANAT activity and more than an 80% upregulation of ISO-induced ignaling . As the aa-nat promoter revealed the presence of regulatory elements other than CREs that can potentially affect its transcription. For example, it contains an activating protein-1 (AP-1) site [c-fos, c-jun, junB, junD, NGFI-A, and Fra-2 [JunB and c-Fos [There are many mechanisms that modulate the transcription and activity of AANAT, which may also interact with STAT3 endogenous activity. Analysis of the -1) site . In the nd Fra-2  and can nd Fra-2 ,41, and nd Fra-2 ,43. STATnd c-Fos ,45, and nd c-Fos ,47,48. Iaa-nat, the NA/cAMP signaling pathway also induces the synthesis of inhibitory transcription factor ICER [Aa-nat gene expression is increased following the Icer gene silencing either in vivo [Icer promoter that has been shown responsible for BDNF-induced Icer transcription in primary hippocampal neurons [Icer in the primary cultures treated with Stat3 siRNA. Although ISO enhanced the Icer mRNA level as expected [Stat3 silencing did not show any effect. We cannot conclude that the STAT3 action is specific to AANAT, but we may suggest that the regulatory role of STAT3 is not universal to all ISO-induced genes in the pineal gland. Furthermore, in cultured pinealocytes and in contrast to the hippocampus [Icer does not seem to be dependent on STAT3. Earlier studies have shown that, besides tor ICER . ICER in in vivo  or in vi in vivo . STAT3 c neurons . We measexpected , the Stapocampus , the expStat3 gene also contains its own consensus binding site [Stat3 mRNA in both control and ISO-stimulated pinealocytes, which proves the autoregulatory loop of Stat3 transcription in pineal glands, as well as the efficiency of our experimental setup.The promoter of the ing site , and thuaa-nat transcription and AANAT activity and, thus, decreases the melatonin level within minutes. As the STAT3 silencing clearly enhances AANAT activity, we tested the hypothesis that the STAT3 inhibition could interfere with light-induced AANAT suppression and rescue, at least partly, the AANAT activity. We have used a 1-min light pulse of intensity 30 lux applied in the late subjective night and showed that the light pulse reliably suppressed the AANAT activity within 20 min. However, the STAT3 silencing had no effect. The AANAT activity from the pineal glands of animals treated with STAT3 inhibitors was suppressed to the same extent as in untreated animals. It is possible that the combination of pulse duration and intensity exceeded the limit for saturation in nocturnal rodents, which the modulatory effects of STAT3 inhibition could not surpass. The 5-h interval between acute administration of inhibitors and the light pulse or tissue harvest has been designed based on in vitro studies [The fundamental property of the pineal gland’s melatonin synthetic pathway is its sensitivity to nocturnal light, which rapidly downregulates the  studies . Since t studies  or short studies . AlthougStat3 transcription for more than 13 h and pSTAT3 levels for longer than 15 h. Number of pSTAT(y) immunopositive cells was slightly, but significantly, higher even 26 h from the LPS administration. In the SCN, the level of LPS-induced pSTAT3 was at its maximum 5 h after injection, while in the pineal gland, the maximum induction had occurred by the first-time measurement, 2 h from the LPS administration. The difference is probably the consequence of faster exposure to the blood circulating LPS in the pineal gland as a circumventricular organ.Our recent work has focused on the LPS-induced changes in the rat circadian system ,19. Withaa-nat transcription [aa-nat transcription and activity, we hypothesized that LPS-induced STAT3 upregulation can play a role in LPS-induced AANAT downregulation. We performed a set of in vivo and in vitro experiments, but our results did not prove the LPS-induced downregulation of aa-nat transcription in vitro and AANAT activity in vivo. LPS alone did not affect Icer transcription but enhanced Stat3 mRNA in cultured pinealocytes, similarly to in vivo, which has proved the effectiveness of our LPS cell treatments. Some reports focused on the effect of LPS and pro-inflammatory cytokines on AANAT and melatonin levels in the pineal gland demonstrated that LPS, TNF-α, or IL-1𝛽 supress the cription ,58,59. Aaa-nat transcription within the interval 0.5–12 h from the administration. Although our experimental setup is in time-course with this study, it is possible that methodical differences, such as culturing conditions or dosing, could differ from our study. In support, a recent study shows that IFN-γ enhances the aa-nat mRNA in the dose 100 ng/mL, but the dose 30 ng/mL has no effect [Icer transcription reflected the presence of LPS more significantly than the aa-nat did, as was similarly observed in macrophages [The principal studies ,59 have o effect . Furthero effect , suggestrophages .aa-nat mRNA level, as well as on night/ISO-induced AANAT activity. These results could implicate that LPS-enhanced STAT3 will lead to the more effective suppression of AANAT, which, however is not the case. The immune-related changes in aa-nat expression and the melatonin synthetic pathway have been recently attributed to the STAT1–NFκB crosstalk [Our data show that STAT3 inhibition has a potentiating effect on the ISO-induced rosstalk . It is prosstalk . Alternarosstalk . It actirosstalk ,66. Exterosstalk ,68,69. T


Genetic architecture and evolution of complex traits and diseases in diverse human populations” presents six review articles and three research studies. Our Research Topic reflects data generation and research performed in Germany, Brazil, United States, Denmark, Estonia, Mexico, United Kingdom and Singapore, spanning countries across the global income spectrum. We present reviews on methodologies to aid complex trait studies in diverse populations, reviews on complex trait studies in locally and globally understudied groups, reviews on studying the evolution and maintenance of genetic variation influencing complex traits using theory and ancient DNA, and new research studies providing resources or insights to understand the genetic architecture of complex traits in Latin America.The research topic “Simonin-Wilmer et al. present an overview of strategies for detecting genotype-phenotype associations across diverse populations. With increased interest and movement towards multi-ethnic association studies that promise to improve detection power and prediction accuracy, this review provides a primer for researchers on assessment and control of confounders related to genetic ancestry to help identify true genotype-phenotype associations. Sticca et al. review the recent methodological developments in detecting identity-by-descent (IBD) segments in the genome. These developments enable IBD detection in large biobank-scale datasets and the authors argue for the need to incorporate IBD-based analyses into genetic studies to improve imputation accuracy, the power to detect rare causal variants, and to gain insights into the demographic history of causal variants.With respect to methodologies that aid complex trait studies in diverse populations, Charleston Chiang discusses his work on Native Hawaiians as a motivating example to review the parameters of ethical community engagement, along with the challenges and opportunities of conducting genetic studies in minority populations. He reviews how the complex peopling of the Hawaiian islands over several millennia has shaped patterns of genetic variation there, and the hypothesis that the high rates of metabolic disease observed among Native Hawaiians and related Polynesian populations may be linked to ancient genetic adaptations. In their article, Sun et al. review studies of obesity in samples with ancestry from East Asia. Due to several 100 generations of partially independent evolution, genetic studies in east Asian samples are well positioned to discover variants that are at low frequencies or are poorly tagged in European samples. Additionally, inconsistencies between GWAS of obesity in European and east Asian samples point to genetic architectures that only partly overlap, suggesting that the environment may be interacting with genetic effects in different ways in different samples.Two articles in our Research Topic reflect on progress conducting complex trait genetics research in groups that have been historically understudied. In his perspective, Koch and Sunyaev review classical and modern population genetic theory on the maintenance, evolution and distribution of genetic variation for complex traits. There is now strong evidence that trait-associated genetic variation is both highly pleiotropic and shaped by natural selection. While population geneticists have been keenly interested in both of these phenomena for decades, the existing theory is not well suited to the rich but complex data that the field now has access to. Koch and Sunyaev highlight more recently developed theory aimed at bridging this gap, as well as the shortcomings of existing statistical tools and challenges for theoreticians going forward. Further, Irving-Pease et al. discuss what particular insights on the evolution of complex traits can be extracted from the analysis of ancient DNA data. The authors analyze how ancient DNA has been used to study the evolution of traits such as height or skin pigmentation, and also assess the potential to predict phenotypes in archaic hominins. The authors review the prospects of using ancient DNA to detect events of polygenic adaptation, how the degraded ancient DNA data can hinder phenotype studies in ancient individuals and point to strategies to improve complex trait studies using ancient DNA.Secolin et al. analyze a region on chromosome 8 associated with obesity. A study from Brazilian individuals of the BIPMed cohort, sampled in Campinas, showed that this region is under positive selection and has a high proportion of Native American ancestry compared to the rest of the genome. The authors analyze this region in individuals collected in the cities of Barretos, Ribeirao Preto and Belo Horizonte and find the same overrepresentation of Native American ancestry. Second, Kaibara et al. find that 48 SNPs associated with genetic generalized epilepsies (GGE) in non-Brazilian cohorts do not retain an association with GGE in Brazilian patients using genotype data from 87 Brazilian patients with GGE and 340 Brazilian controls. However, they find that nine SNPs in the imputed flanking 1 Mb region surrounding the 48 SNPs retain an association suggesting that there are some shared variants that impact the risk for GGE in Brazilians and individuals from other cohorts around the world. Finally, Jimenez-Kaufmann et al. show that the inclusion of more genomes from Mexican individuals with a high proportion of Native American ancestry in reference imputation panels improves genotype imputation accuracy for rare variants in admixed Mexican individuals. This result, along with other observations from this study, suggest that improvements in genotype imputation accuracy in Latin American individuals from particular regions will require local sequencing efforts to include more individuals with a high proportion of Native American ancestry in imputation reference panels.This Research Topic also contains three papers that provide new insights on complex traits in Latin American populations. First, The lofty goal of this research topic was to identify and present state-of-the-art research themes in the genetic architecture and evolution of complex traits and diseases in diverse human populations. The major conceptual and practical research directions and challenges that this Research Topic has helped identify that need to be addressed are 1) Theoretical and conceptual advances to understand trait evolution with clear expectations and assumptions regarding shared and variable genetic architecture across human diversity. 2) The clarification and contextualization of the use of ancestry and populations as sampling and analysis variables in research studies. 3) Methodological advances with transparent assumptions to appropriately analyze complex traits using genetic and environmental data across diverse groups. 4) The construction of local scientific capacity globally to allow for fair and inclusive strategies of data collection, analysis and dissemination of results. 5) Large enough sample sizes across human diversity to allow for meaningful inference. 6) Frameworks incorporating equitable reciprocity for communication with study participants especially those that have been historically marginalized and discriminated against across the globe. In this Research Topic, we present considerations to work towards meeting these challenges. By presenting voices and research done across countries and ethnicities, we show the possibilities of a globally well distributed scientific practice, which we believe is an important precedent for widely representative studies of complex traits and disease.


Combining X-ray micro-tomography with unsupervised machine learning algorithm, we provide a new tool capable to study the internal features of RRP wires and unlock different approaches to enhance their performances. Such tool is ideal to characterize the distribution and morphology of the voids that are generated during the heat treatment necessary to form the Nb3Sn superconducting phase. Two different types of voids can be detected in this type of wires: one inside the copper matrix and the other inside the Nb3Sn sub-elements. The former type can be related to Sn leaking from sub-elements to the copper matrix which leads to poor electro-thermal stability of the whole wire. The second type is detrimental for the electro-mechanical performance of the wires as superconducting wires experience large electromagnetic stresses in high field and high current conditions. We analyze these aspects thoroughly and discuss the potential of the X-ray tomography analysis tool to help modeling and predicting electro-mechanical and electro-thermal behavior of RRP wires and optimize their design.The electro-mechanical and electro-thermal properties of high-performance Restacked-Rod-Process (RRP) Nb These characteristics combined with a high critical current density (Jc) make Nb3Sn the workhorse in superconducting magnets operating in the 10 T to 20 T range. Nb3Sn wire technology is under development since the ‘60s, but only in the last two decades the conductor’s development has made great progress thanks to the research program for the International Thermonuclear Experimental Reactor (ITER)3, presently under construction. Since the ‘90s, the High Energy Physics (HEP) community has taken interest in the development of Nb3Sn wires for post Large Hadron Collider (LHC) accelerators. The effort was led by the European Organization for Nuclear Research (CERN) and the US LHC Accelerator Research Program (LARP)4, and it culminates with the adoption of Nb3Sn wires as baseline for the High Luminosity LHC upgrade (HL-LHC)5, which will include two pairs of dipoles, operating at 11 T, and 24 quadrupoles based on Nb3Sn. Presently, CERN is investigating various design for a new proton-proton collider with an energy of 100 TeV in the center of mass, the so-called Future Circular Collider (FCC)6. The maximum energy of a circular collider is determined by the maximum magnetic field of the dipole magnets and by the radius of the accelerator. Assuming a circumference of 100 km the dipole magnetic field should be about 16 T to achieve the target value of energy. Even though Nb3Sn wire technology has largely improved over the years, FCC requires new steps forward in Nb3Sn technology to achieve 16 T magnetic field in its dipoles7. Key factors are a high non-Cu critical current density, i.e. Jc > 1500 A/mm2 at 16 T and at 4.2 K, tolerance to mechanical loads and good thermal and electrical stabilization in the form of high purity copper (Cu) surrounding the superconducting sub-elements. Recently, four different dipole designs were developed for FCC in the frame of the Horizon 2020 European Circular Energy-Frontier Collider (EuroCirCol) study8: Cosine-theta9, Block-Coil10, Common-Coil11 and Canted Cosine-Theta12. The analyses performed on these designs highlight the large mechanical loads generated by electromagnetic forces on the Nb3Sn wires reaching peak stress of 150–200 MPa at 16 T13. This large stress may lead to the degradation of the electrical transport properties of the brittle Nb3Sn sub-elements and, for this reason, the electro-mechanical properties of Nb3Sn wires are a fundamental factor for the FCC dipoles’ design.The intermetallic compound NbJc in Nb3Sn depends to the applied mechanical loads and its variation has two regimes: one is reversible, i.e. the wire does not present permanent Jc reduction after unloading the applied force, and the other is irreversible, i.e. above a certain applied stress limit, the Jc degradation becomes permanent. The reversible regime has been intensively studied15 whereas the irreversible degradation was less investigated, mostly in relation with the ITER project16 and more recently in the frame work for the EuroCirCol project18 and HL-LHC19. In the latter publication, it has been shown that the temperature selected for the Nb3Sn activation heat treatment is fundamental for the irreversible limit, but, despite the remarkable collection of measurements, the authors were not able to link the phenomenon to the internal properties of the wires and unveil the mechanism behind the degradation. With the present research we offer to the community a new powerful tool to investigate the internal features of Nb3Sn wires at micro-meter level. This will allow to build extremely accurate electro-mechanical models considering one of the key factors in wires degradation, which is the Kirkendall voids22 generated during the activation heat treatment. In fact, the voids degrade the microstructural homogeneity20 and cause local stress concentration, which act as nucleation point for crack’s formation.3Sn wires. RRP is a type of Internal-Tin Nb3Sn wire, and it is considered to be one of the most promising technology for future application in high-field accelerator magnets mainly because of the record Jc achieved23. Five different RRP sample wires have been analyzed at the European Synchrotron Radiation Facility (ESRF) to investigate their internal properties. Synchrotron tomography was successfully used in the past to study and characterize the voids formation of Internal-Tin Nb3Sn wires25. Furthermore, a more recent study focused on bronze route Nb3Sn wires26 was able to correlate the void morphology to the critical current degradation upon mechanical load21 showing that the non-destructive synchrotron X-ray tomography is extremely effective to study Nb3Sn voids. Our analysis tool is the combination of micro-tomographies with an unsupervised machine learning algorithm, which is able to autonomously isolate, reconstruct and analyze the voids. In the studied wires, the tool detected two type of voids: in the high-purity copper matrix and in the Nb3Sn sub-elements. Some of the voids in the Cu-matrix are related to the presence of gas volumes generated during the wire production whereas other are Kirkendall voids27, which are due to the Sn diffusion in the Cu-matrix24. The latter are thus indicators of a degraded electro-thermal stability of the wire. Differently, the voids in the sub-elements are detrimental for electro-mechanical performance of the wire. The accurate 3D geometrical characterization of the wires internal features is a fundamental step along the path of mechanical and electro-thermal enhancement of RRP wires.Therefore, the subject of the present study is the presentation of the tool and its first analysis of voids in Restacked-Rod-Process (RRP) Nb3Sn samples used in the analysis. The second part is devoted to X-ray tomography performed at the ESRF. The last section is dedicated to the detection and analyses of the voids and their implications.The paper first discusses the main properties of the RRP Nb3Sn RRP wires. Two types of wire configurations were investigated: 108/127 and 132/169. These numbers define the RRPs configuration as number of superconducting sub-elements over the total of restacked elements. Each design was used for a different study goal. 108/127 wires have been used for the 11 T dipoles28 and for the Low-β quadrupole magnet (MQXF) of the HL-LHC upgrade29, at a diameter of 0.7 mm and 0.85 mm, respectively. Two different versions of this design were analyzed, both 0.85 mm in diameter: S20 is Ta doped wire with normal Sn content, which correspond to a Nb:Sn molar ratio of 3.4 and S21 is Ti doped with a reduced Sn content, i.e. Nb:Sn molar ratio of 3.6. The different Nb:Sn molar ratios allowed us to study the correlation between Nb content, Sn leakage and presence of voids in the copper matrix. 132/169 wires were developed in the framework of the European Coordination for Accelerator Research and Development (EuCARD)30 and LARP31, and they have been used for studies related to FCC. The three samples are reduced Sn and Ti doped wires with different diameters. The different diameters, and consequently different sub-elements sizes, allowed us to investigate how the distribution of voids in the sub-elements is affected by the dimension of the sub-element itself. Samples characteristics and heat treatments are listed in Table In this study, the emphasis is on the voids present in Nb32. Furthermore the use of narrow bandwidth radiation helped us to improve the contrast between Nb3Sn and Cu voids33 and prevented beam hardening effects, which is the phenomenon that occurs when polychromatic X-ray beam passes through an object, resulting in selective attenuation of lower energy photons34. X-ray micro-tomography allows detecting and defining internal structure of a composite in a non-destructive manner without influencing the sample. The sample is exposed transversally to the X-ray beam and an image of the transmitted beam is recorded with a detector placed behind the rotating sample holder. In our case the sample was rotated of 360° in 0.012° steps, i.e. 30,000 projections images acquired per sample. These projections, once stacked into a three-dimensional (3D) X-ray absorption map, provide a 3D reconstruction of the sample volume with its internal characteristics. The photon energy was 89 keV and the detector had 2560 × 2160 pixels resolution with an equivalent spatial sampling of about 0.7 μm/pixel . The final output of the measurements is a set of two-dimensional (2D) images of the measured Nb3Sn samples where each image corresponds to a cross-section of the wire, see Fig. X-ray micro-tomography has been performed at the beamline ID19 of the ESRF in Grenoble, France. Synchrotron micro-tomography is an exceptionally powerful tool to study the porosity of materials on µm scale. X-ray synchrotron radiation was already proven to be suited to study the internal features of superconducting wires in several studies21. The MATLAB tool analyses the 2D tomography’s cross-sections one by one. To detect the voids, each pixel is compared with a color threshold that is calibrated on the slice average brightness. The voids are saved as binary maps were 1 is a void pixel and 0 is everything else. In Fig. The tomography post-process was begun exploiting a dedicate algorithm written in MATLAB and already tested ink-means method35, an unsupervised classification algorithm developed for clustering. The underlying idea of clustering is to divide a given set of data into a specific number of groups based on certain patterns or similarities present in the data; in our case the groups  were defined based on the pixels brightness of the tomography. k-means algorithm divides the given set of data into k disjoint clusters equivalent to the groups. The analysis of an image, i.e. one slice of the tomography, can be summarized as follows: in the initial step a k number of centers of brightness, ck, are generated based on the picture brightness scale. The initial centers are arbitrarily generated by the algorithm, the operator can control the number of time the k-means algorithm will be run with different initial ck, the maximum number of iteration per run, and the relative tolerance used to declare the convergence36. For every pixel of the image, the Euclidean distance on the brightness scale d, between the centers and pixel brightness, p, is calculated as:The separation of the wire components in the tomography was done using the well-established nk denotes the number of observations in the kth cluster37. The process is repeated until the tolerance is satisfied, i.e. there are no significant changes in the centers positions.Then, the pixels are assigned to the nearest center based on the calculated brightness distance. When all pixels have been assigned, a new set of centers is generated using Eq. , in whick-means algorithm can be found in38. k-means algorithm was implemented using the python scikit-learn library39. After applying the k-means algorithm, further processing was done in order to obtain as a final result three separated binary images: Nb3Sn sub-elements, Cu matrix and voids. Eventually, copper voids were achieved subtracting the sub-elements from the voids’ maps.As a last step the clusters of pixels are saved. More on The 3D reconstruction of the voids for the wire S20, differentiated by type, is shown in Fig. From the direct observation of S20 in Fig. 41 that normal Sn content RRP wires have typically lower Residual Resistivity Ratio (RRR) than reduced Sn content wires. Our RRR measurements are in agreement with this result, S20 has RRR = 89 while for S21 has RRR = 274, indicating a higher Cu purity for the latter.As said, the analyzed 108/127 wires have similar design but different Nb:Sn molar ratio. It has been shown in3Sn ring, allowing visual detection of the phenomenon. In Fig. Since low RRR measurement can be an indication of the Sn presence in the Cu matrix, a manual investigation of Nb barriers macroscopic disruptions was performed on the wires’ tomography looking for barrier failures and related Kirkendall voids. Studying the tomography of S20, several barriers’ ruptures were detected. In presence of Nb barrier breakages, the voids extend from the sub-element to the Cu matrix crossing the NbIn general, it is expected that heavily deformed sub-elements are more prone to barrier failure. The 108/127 design has a hexagonal pattern of the sub-elements and, during the drawing process, the sub-elements in the corners are more subject to deformation stress, as can be observed in the cross sections reported in Fig. 42 and41. As a further step in the improvement of our analysis tool, we plan to automatize the identification of the broken barriers by implementing advanced machine-learning and deep-learning technologies, such as object detection43 and semantic segmentation44.Both tomography and SEM–EDX analysis, performed on S21 samples, were not capable to detect Nb barrier ruptures. These results confirm that a Nb:Sn ratio of 3.4 is more prone to Sn diffusion in the Cu matrix at this heat treatment conditions and that a ratio of 3.6 is recommended, in agreement with the results ofThe analysis tool highlights that even with high purity Cu matrix, i.e. high RRR values > 150), all the analyzed samples show presence of Cu voids, see Table 50, all t3Sn phase. As listed in Table 45. These Kirkendall voids, due to the diffusion of Sn in Cu and Nb, grow by coalescence during the heat treatment steps. They reach the final dimensions during the formation of the Nb3Sn phase, where the majority of Sn bond with the Nb leaving in the sub-elements core large voids in a Cu-Sn solution. More on the sub-element voids formation is described in24.The sub-element voids are generated during the wire heat treatment, necessary for the formation of the NbJc, without reducing the RRR. Previous studies showed that heat treatments longer than 50 h with a temperature higher than 670 °C stimulate the Sn diffusion increasing the probability of Sn leakage into the Cu stabilizer46. On the other hand, in case of lower temperature the Sn may not diffuse enough causing worse performances at high fields47. With these considerations, RRP can only exploit a limited temperature window between 640 and 665 °C to optimize the wire properties.S19, S25 and S26 are three 132/169 RRP wires with different diameters. The heat treatments of the wires are tailored on the wire design as well as the sub-elements dimensions. In particular, the final temperature plateau and its duration are optimized depending on sub-elements size. The temperature and duration are empirically defined by the manufacturer to maximize the A15 reaction and the The three samples have different diameters, and because they have the same configuration, this difference is reflected by the sub-element size. For this reason, the average diameter of the sub-elements, measured from the 2D tomography, shows a linear dependence on the wire size, see Table To verify this relation, we used our analysis tool, which provides the voids volumes and their center of mass, allowing to uniquely locate their position in the sample. Sub-elements voids are strongly irregular, therefore, to assign them a geometrical description, we decided to approximate them to simple ellipsoids. In such approximation, the major axis of the ellipsoid is equivalent to the void length whereas the minor axis of the ellipsoid is the void diameter. These dimensions have been calculated using the projections of the voids to the XZ, YZ and XY planes, as shown in Fig. The resulting frequency distributions of sub-elements voids’ lengths and diameters as function of the wires’ dimension is shown in Fig. 48. In this context a simple mechanical model based only on the sub-elements design cannot provide reliable results, because the sub-elements voids cannot be neglected, as they can act as nucleation point for the cracks in Nb3Sn21. Therefore to study and enhance the mechanical properties of RRP wires, a more complex mechanical model, capable to include the Kirkendall voids in the sub-elements, is necessary. Our geometrical characterization of the voids makes possible to overcome this issue providing the effective internal features of RRP wires calculating position, orientation, and dimensions of the voids and if needed of the sub-elements. This capability will unlock new strategies for pushing forward the development of RRP Nb3Sn wires obtaining wires with enhanced tolerance to stress by adjusting, for example, the heat treatment in order to tailor the voids distribution without neglecting the effects on electro-thermal properties and critical current.The difference in voids distribution among the three 132/169 RRP wires shows the complexity of the problem. The three samples have the same design and are in scale for sub-elements dimensions, but because of their differences in internal features and heat treatment, they have different voids distribution, thus they cannot be considered mechanically equivalent, i.e. one wire cannot be used as mechanical model scale for the others3Sn wires. As matter of fact a clear quantitative correlation between voids and mechanical limits was obtained for Bronze route wires21 while for the other types of internal tin wires, such as RRPs, the correlation has yet to be demonstrated. It was shown that varying the dopants (Ti or Ta) and the temperature of the heat treatment final plateau (600–750 °C), the axial irreversible limit of RRP wire changes49. Using the combination of x-ray tomography and unsupervised machine learning algorithm would be possible to completely characterize Nb3Sn wires and quantify the impact of the voids variation on the irreversible limit due to the different heat treatments. The software detects and separates sub-elements, copper matrix and voids, and calculates their position, volume, orientation, and dimensions. Hence, the tool quantifies the wire characteristics paving the way for Finite Element Models (FEM) at sub-elements level, and further optimization of electro-mechanical properties of Nb3Sn wires.The information collected using the presented analysis tool raises new questions on voids formations, sub-elements deformations and barrier failures, which are a clear indication of the potential impact such instrument can have on the comprehension of RRP wires properties. A possible application could be to use the tool to quantify the impact of voids on the electro-mechanical behavior of the wire and, as consequence, to improve the mechanical limits of RRP Nb3Sn RRP wires, it will have a significant impact on different technologies. This algorithm is ideal to characterize the wires whose production process involves a synthesis reaction or sintering heat treatment between precursors, which can cause the generation of voids or deformations of the superconducting filaments. The strength of the unsupervised machine learning is the ability to separate the elements that constitute a wire regardless of the materials involved, while the reconstruction program can easily be adapted to the wires components. In particular, the tool can provide support in the advancement of Powder in Tube (PIT) superconducting wires. In Bi-2212, voids have almost no effect on the mechanical behavior of the wire51, on the other hand bridging or bonding of the filaments was related to the heat treatment process and proven to be partially responsible for low Jc and n-value53. The analysis tool could easily map the filaments bridges allowing to quantify the number of filaments intergrowths and their impact on Jc. Others superconducting wires based on powder metallurgy, as Iron-based54 and MgB2 wires55, could also benefit from tomography analysis by analyzing, for example, the interaction between the metal sheath and the superconducting phase. The technique can also be applied in in-situ MgB2 wires for the investigation of the voids formation, due to volume contraction during the synthesis process, which could be detrimental for Jc and mechanical limits57. Finally, the tool is not limited to study single wires, more complex system can also benefit from its use. The analysis of voids and cracks distribution of Nb3Sn Rutherford cables after load test58 can provide precious information of the most sensible area of the cable and how to improve the cable design. While the study of the deformations of the wires in cable in conduit conductors (CICCs) for fusion application59 can offer new solutions for the wires twisting pattern in order to optimize such conductors.Furthermore, even if the tool was developed to analyze Nb3Sn wires. The combination of X-ray micro-tomography and unsupervised machine learning algorithms is used to analyze two 108/127 wires with different Sn content and three 132/169 wires with different diameters. The unsupervised machine learning algorithm allows to differentiate sub-elements voids from Cu voids providing their 3D reconstruction and geometrical characterization.In this paper we propose a new tool for the study of the internal features of RRP Nb108/127 samples have been used to study the variation of Cu voids depending on the Sn content. In case of normal Sn content, Kirkendall voids were generated by Sn diffusion resulting from ruptures in the Nb barriers. Sn pollution in the Cu matrix results in poor electro-thermal stability of the wire. In addition, the analysis underlies that, unsurprisingly, the barrier failures are more frequent in highly deformed sub-elements. In case of the reduced Sn wire, i.e. Nb:Sn molar ratio of 3.6, Sn contamination was not detected in the Cu matrix. Nevertheless, a large number of Cu voids was observed in all the samples, these voids were generated by defects trapped between the different constituents of the Cu matrix during the production process and they do not have a negative impact on the copper RRR.3Sn wires.The three samples of different diameters but same design (132/169) have been used to study the voids distribution and dimensions. The voids were morphologically similar, and the maximum voids volume scales with the diameter of the sub-elements. On the other hand, the distribution of the voids dimensions presents differences which do not allows to directly extrapolate electro-mechanical properties from similar wires. The analysis demonstrates the importance of the accurate description of a wire’s internal characteristics and the effectiveness of our analysis technique for studying RRP Nb3Sn wires, whose repetitive unit, called sub-element, is made of Nb filaments in a Cu matrix around a Sn core, the whole assembly being surrounded by a Nb barrier and then an outer layer of pure Cu. The RRP wire is then produced stacking and cold drawing several sub-elements in high purity Cu tube  down to the wire final size, see Fig. 60. The superconducting compound is formed during a tailored heat treatment which activates the Nb-Sn reaction. The Nb-Sn compound is superconducting between 18at% Sn and 25at% Sn, reaching the highest Tc and Bc2 when the compound is about 24–24.4%, i.e. Nb3Sn. Nevertheless, stoichiometric ratio is generally avoided in the wires, as could result in a complete reaction of the barrier, with consequent Sn leakage. Typically a Nb:Sn molar ratio from 3.1 to 3.6 is used to guarantee a supply of Sn that is sufficient to fully react all the Nb filaments, but without an excess that would take valuable “real estate” in the wire and would lead to a too fast growth of Nb3Sn from the diffusion barrier Nb and thus beat the purpose of the barrier itself61. It has been shown in42 that a Nb:Sn molar ratio of 3.6 greatly enhances the Residual-Resistivity-Ratio (RRR) of the wire compared to lower Nb:Sn values. The RRR is used as indication of the purity of the Cu matrix. It is defined as the ratio of the Cu matrix resistivity at room temperature to its resistivity above the superconducting transition. In case of superconducting wires for high field magnets, Cu with high RRR is necessary for electro-thermal stability62 and a low RRR can indicate damages of the Nb barrier and Sn leakage into the Cu matrix.RRPs are a type of Internal-Tin NbJc, RRP production process includes a very low Cu content inside the sub-elements causing proximity between the Nb filaments63. In the heat treatment, the volume expansion during the Nb3Sn formation causes the coalescence of the filaments and the formation of a monolithic Nb3Sn ring, corresponding to filament effective diameter (deff) of 30–70 µm. Large deff can generate issues in application that requires low hysteresis loss and high magnetic field quality as in accelerator magnets64.In order to achieve high 3Sn. The amount of Cu present in a wire is defined using the ratio between Cu and non-Cu materials, so called Cu:Non-Cu ratio. This ratio is tailored depending on the wire application. In RRP wires, the Cu included in the sub-elements dissolves in the Sn core so it cannot be considered as assisting the stability, therefore to reach the desired Cu:non-Cu ratio, usually between 1 and 1.562, some of the wire sub-elements must be substituted by high purity Cu hexagons. In this way, an additional number is given for the wire definition, which is the number of the total superconductive sub-elements. For example, a 132/169 wire consists of 169 restacked sub-elements of which 132 are Nb3Sn.The design of RRP wires is defined by the number of restacked sub-elements. Sub-elements are deformed to hexagonal shape and they are stacked using a centered hexagonal grid. As said, high purity Cu is necessary for electro-thermal stability of the wire. The Cu matrix has double role: it acts as low resistance current shunt in case of transition of the superconductor to normal state and it homogenizes the superconductor temperature because its thermal conductivity is orders of magnitude higher than that of Nb3Sn transition. RRR is defined as the ratio between R at room temperature and at 18 K, which is just after the transition to normal state.The RRR sample holder is designed to test eight straight samples per measurement. The samples are mounted in series and they are soldered to the current leads. The voltage-tap pairs are soldered on each sample separated by about 20 mm. The resistance (R) at room temperature is measured injecting in the samples a current of 1 A and measuring the voltage drops trough the taps. For the resistance at cryogenic temperature, the sample holder is mounted on a probe, which is inserted in a cold cryostat and gradually lowered to cool the samples up to liquid helium temperature. A thermometer is placed on the sample holder to monitor the temperature. A current of 10 A was injected through the samples and the voltage across each wire was measured to determine the resistance. The cryostat is then slowly heat up, increasing steadily the temperature. The resistance is measured as function of the temperature from 4.2 K up to 25 K, which is after the NbElectron microscopy is performed using a JEOL JSM-7600F field emission scanning electron microscope (FESEM). Energy-dispersive X-ray spectroscopy (EDS) is performed in the FESEM using an Oxford Instrument X-Max system, which utilizes a large area analytical Silicon Drift EDS Detector (SDD) with PentaFET Precision, at an acceleration voltage of 16 kV.