Accelerated warming in the Arctic, as compared to the rest of the globe, might have profound impacts on mid-latitude weather. Most studies analyzing Arctic links to mid-latitude weather focused on winter, yet recent summers have seen strong reductions in sea-ice extent and snow cover, a weakened equator-to-pole thermal gradient and associated weakening of the mid-latitude circulation. We review the scientific evidence behind three leading hypotheses on the influence of Arctic changes on mid-latitude summer weather: Weakened storm tracks, shifted jet streams, and amplified quasi-stationary waves. We show that interactions between Arctic teleconnections and other remote and regional feedback processes could lead to more persistent hot-dry extremes in the mid-latitudes. The exact nature of these non-linear interactions is not well quantified but they provide potential high-impact risks for society. Accelerated global warming in the Arctic might have profound impacts on mid-latitude weather particularly in winter, although the evidence for an effect also in summer is also growing. Here Coumou et al. show that these interactions could lead to more persistent hot-dry extremes in mid-latitudes. Scientists are generally confident in the thermodynamic drivers of these changes but are less so in dynamic aspects5. Another pronounced signal of anthropogenic global warming is the rapidly increasing near-surface temperatures in the Arctic at a pace two to four times faster than the rest of the globe, known as Arctic amplification (AA)6. The extent to which AA affects the mid-latitude circulation and possibly contribute to the observed increases in weather extremes has been a subject of active debate7.The observed increases in the frequency and intensity of extreme heat and heavy rainfall events since the late 1980s, especially in mid-latitude regions, have been linked to anthropogenic global warming9. The increased heat stored in the Arctic Ocean owing to sea-ice loss is released into the atmosphere in early winter. The associated expansion of the near-surface air increases Arctic geopotential heights and can affect the circumglobal circulation directly as well as via feedbacks between the troposphere and stratosphere involving the stratospheric polar vortex14. Even though the exact pathways through which the Arctic influences the mid-latitude winter circulation are debated, a scientific consensus is emerging that AA has at least some influence on winter weather16.Most studies analyzing the role of AA on mid-latitude weather have focused on the winter season and the linkage with cold spells. The stronger jet stream, the presence of the stratospheric polar vortex, and the post-1990s increase in abnormally cold winters over central Eurasia have drawn a lot of attention to the winter season17. For instance, any increased frequency in circulation regimes conducive to persistent heat extremes would act on top of the thermodynamically driven increase in heat, creating possibilities for very-extreme heatwaves. Many recent high-impact summer heatwaves indeed occurred in that far-tail of the distribution and cannot be explained by the direct thermodynamic effect of greenhouse gas forcing alone , resulting in an estimated 55,000 heat-related deaths, more than 500 wildfires near Moscow and grain-harvest losses of 30%4. A quantitative global analyses showed that the 2010 event was the most-severe heatwave ever recorded worldwide, based on a heatwave index that can be used across different regions20. Intriguingly, all record-setting heatwaves based on this index occurred in the mid-latitudes, indicating that here heatwaves are becoming more intense at a pace that exceeds the global mean20. Extreme summer heat in the Northern Hemisphere mid-latitudes now far exceeds historical frequencies in the twentieth century117. Over the last decade, Europe has seen an exceptionally rapid increase in the chance of extremely hot summers similar to the 2003 extreme21. Other notable high-impact and record-breaking droughts and heatwaves occurred in the USA in 2011 and 201220, leading to billions of dollars in agricultural losses121.Many recent high-impact summer heatwaves occurred in the far-tail of the distribution and are difficult to explain by the direct radiative warming effect of greenhouse gas forcing alone122. Consequently, extremes of heat and dryness often coincide to produce compound extremes that exert disproportionately large societal impacts123. Certain mid-latitude regions identified as hotspots of tight atmosphere–land coupling, such as central North America 124, are especially prone to concurrent heatwaves and droughts.One reason why these extremes cause so much damage is that temperature and precipitation during summer are anti-correlated virtually everywhere across extratropical land125. These kinds of summertime compound events are therefore highly relevant for the dynamical changes described in this study, and recent studies have developed innovative techniques to separate the contributions from dynamics and thermodynamics128. In particular, the projected trend toward a weaker and poleward-shifted jet stream is consistent with projections of a significantly increased risk of compound hot-dry extremes across much of the Northern Hemisphere this century122. This type of climate change would likely exacerbate the separate impacts of extreme heat and dryness, based on the documented stresses that compound heatwaves and droughts exert in causing disease129, vegetation mortality130, wildfires131, and agricultural losses132.Such events are promoted by positive thermodynamic feedbacks that favor depleted soil moisture and enhanced sensible surface heating as the land warms, but they are also strongly regulated by atmospheric dynamics that initiate and sustain anomalous heating and drying13. The additional heat inflates the lower troposphere over the Arctic Ocean and nearby continents, and increase geopotential heights, which could affect circulation patterns further south.Due to declining sea-ice, the Arctic Ocean absorbs more incoming solar radiation from spring to autumn. By early winter, when near-surface air temperatures drop below sea-surface temperatures, this excessive heat is released into the atmosphere14 might reduce the poleward pressure gradient in the troposphere and therefore weaken the storm tracks and westerly jet. However, this notion based on thermal-wind balance and baroclinicity provides little explanation for the recent changes in winter circulation. In winter, the near-surface warming in the Arctic has been pronounced but confined to high latitudes only, i.e., north of 70° N6. Within the mid-latitudes , neither the poleward temperature gradient nor the zonal-mean jet or storm track have seen any significant changes in winter26. Future climate model projections under high-emission scenarios show that both changes in the tropics28 and in the Arctic31 can influence the strength and position of the mid-latitude winter circulation. Enhanced warming projected in the tropical tropopause region (due to enhanced deep convection and latent heating) acts to increase the upper-level poleward temperature gradient, which strengthens the mid-latitude westerlies28. This has become known as the tug-of-war  that is conducive to cold spells in Eurasia and Siberia34.Many recent winters were characterized by extremely warm temperatures in the Arctic and anomalously cold conditions further south, especially over Eurasia. In fact, large areas over central Eurasia have been cooling since 1990tex Fig. 34. In th36. Still, its relative importance compared to other pathways, tropical influences and atmosphere internal variability remains unclear7.There is relatively high confidence that this stratospheric pathway is a real phenomenon and has a role in the warm-Arctic cold-continent pattern, as it is supported by multiple lines of evidence: From empirical analyses, to causal discovery algorithms, and climate model simulationsCompared to winter, summer circulation in the mid-latitude is weaker, more barotropic and the climatological jets are more zonally oriented, which promotes the formation of circumglobal wave trains . It is less influenced by variability in tropical sea-surface temperatures (SST), and more sensitive to land-atmosphere feedbacks involving soil moisture or snow cover.37. Moreover, variations in tropical SST have less influence on mid-latitude circulation in summer compared to winter . The position of abnormally warm SST in the tropics determines where the strongest deep convection takes place associated with shifts in the Walker circulation. The upper-level latent heat release during deep convection can trigger long Rossby waves that propagate poleward and influence mid-latitude weather40. This mechanism is less important in summer than winter for two reasons: First, the El Niño-Southern Oscillation (ENSO), the dominant mode of variability in tropical SST, tends to peak in boreal winter and is much weaker during boreal summer41. Second, the prevailing easterly winds in the tropics in summer limit the ability of Rossby waves to propagate poleward38. This is not to say that the tropics cannot influence summer mid-latitude weather. Among other things, ENSO gives some predictive seasonal forecast skill in summer43 and the varying location and intensity of monsoon systems, notably over South and East Asia, can affect the mid-latitude summer circulation46.In summer, the reduced pole-to-equator temperature gradient (as compared to winter) leads to weaker and more-narrow upper-level westerlies, and the stratospheric polar vortex is absent all together Fig. . The rol47. Boreal snow cover during spring and summer has shrunk dramatically in recent years, even faster than the decline of Arctic sea-ice extent49. Without snow, the surface albedo is lower and thus the land regions absorb more incoming solar radiation. Furthermore, declining snow cover in spring has a delayed drying effect on the soils by mid-summer, favoring enhanced temperatures due to suppressed evaporative cooling51. These thermodynamic processes in conjunction with reductions in early season snow cover can affect regional to hemispheric circulation53.The state of the cryosphere, in terms of sea-ice and snow cover, from late winter to early summer can influence the strength and latitude of the summer time jet54. Waveguides produce zonally oriented chains of perturbations that fluctuate at relatively low frequency ranging from weeks to months, creating a teleconnection pattern. Early research found that atmospheric disturbances near the jet core are refracted toward the core, meaning that the jet acts as a waveguide55. The energy of the trapped disturbances does not disperse strongly and therefore can propagate much further and possibly become circumglobal. Such CGWT can generate heatwaves and severe weather outbreaks  due to their longer lifetime than synoptic disturbances57.The waveguide effect, i.e., the trapping and focusing effects of the seasonal jet streams on low-frequency tropospheric waves, has an important role in how a changing mid-latitude circulation might promote stagnant weather patterns59. However, the zonal orientation of the climatological jet does not mean that the overall flow is less wavy in summer compared to winter: The total waviness in geopotential height fields on sub-synoptic to sub-seasonal time scales is as pronounced in summer as in winter, if not more62. In winter, Rossby waves are typically oriented along a meridional path arcing from the tropics into the mid-latitudes, or from the mid-latitudes into the tropics . Due to the zonal orientation in summer, any local heating anomaly can generate a sequence of waves of similar wavelength downstream of the jet, forming a stagnant wave packet that affects weather conditions far away. Recent research has shown that when synoptic-scale waves (wavenumbers 6–8) are trapped in a (near) circumglobal waveguide, wave-resonance can greatly increase their amplitude66. The wave-resonance mechanism can lead to highly persistent and anomalous weather conditions around the hemisphere and studies have linked it to several recent high-impact summer extremes, including heatwaves and floods66. Though there is a solid theoretical basis underlying wave-resonances, their exact significance in the real-world in causing extreme weather events is debated17.The climatological jets in summer have less northward tilt compared to winter and therefore waveguides are also oriented west-to-east and have the potential to become circumglobal Fig. . Also, t67 is likely to be affected by AA. The warm, snow-free land surface and the cold Arctic Ocean create a strong thermal contrast along the Arctic seaboard around 70° N, generating strong westerlies here. These sub-polar westerlies, together with the pronounced sub-tropical jet, form the distinct summer feature of double jets , the mid-latitude summer circulation has indeed weakened in conjunction with a reduction in the poleward temperature gradient in the lower troposphere. This weakening has been detected in the westerly jet , the total kinetic energy of synoptic storm systems (by about 15%) and the number of strong cyclones76. Idealized modeling experiments support storm track weakening when sea-ice is reduced but also indicate that sea-ice changes by itself can explain only part of the observed weakening77. Modeling studies indicate that the effects of historic sea-ice reductions can explain up to one-third of the magnitude of the observed anomalies, with an additional role for changes in SSTs78. Thus, other factors including natural variability likely had a role in the recently observed summer circulation changes, but a substantial share of it is likely attributable to AA.There is modeling evidence indicating that these observed trends are at least partly attributable to AA. CMIP5 coupled model simulations of the twentieth century show that the observed changes in the zonal-mean temperature gradient in summer (characterized by AA and enhanced high-latitude land warming) are likely attributable to anthropogenic forcing ("likely" according to IPCC lexicon)80. The changes at the end of the century in the high-emission scenario are comparable to the observed changes over the past decades26. This suggests that either the models underestimate the future changes  or that a substantial part of the observed trend is associated with multi-decadal natural variability81. For the North American sector, the amount of AA in models by the end of century is negatively correlated with the changes in jet speed and wave phase speed in summer28. Models robustly project a weakening of summer storm tracks by the end of the century  and those can, in a complex, non-linear system, lead to very different outcomes87. The inter-model spread of the poleward shift therefore tends to be larger than the signal itself86.Idealized dry atmospheric model simulations indeed indicate a southward shifted jet coming from AA by itself88. For the Northern Hemisphere, some evidence of a poleward shift of jet streams has been identified in reanalysis products89 and satellite observations of clouds91 and is most significant in winter. This is thus largely in agreement with model projections pointing at the important role of the tropics in shifts in the jet position.Observed jet shifts in the Northern Hemisphere are generally small compared to those in the Southern Hemisphere, but still generally indicate a poleward migration63. In a more complex atmosphere of an aquaplanet model , quasi-stationary synoptic-scale wave trains are enhanced when the meridional temperature gradient is reduced and the westerly winds weaken92. Thus, as the background flow becomes weaker, the same heating source in the lower troposphere can trigger a stronger stationary wave response especially for synoptic-scale waves. The increased moisture content in a warmer atmosphere, and the tendency for increased latent heat release in the tropics and over warm ocean currents in higher latitudes, can provide further heating to perturb more or stronger CGWTs.Limited evidence from theory, observations and some model simulations suggests that AA may amplify synoptic-scale, quasi-stationary waves embedded in the summer jet Fig. . Theory 94. Figure 62  could modulate the CGWT and vice versa. However, the summer CGWT can also be triggered by heating sources associated with the Indian monsoon44.Some observational evidence suggests that the quasi-stationary component of mid-latitude summer circulation has become wavier since 1979, in particular over the North American sector see Box . Using t95. Still, future model projections of mid-latitude quasi-stationary short-wave patterns are generally inconsistent with recent observations. Future CMIP5 projections under high-emission scenarios show an overall decrease in blocking both for winter and summer96. The reasons behind these divergent findings are not well understood and may result from competing effects from the tropical monsoons44, changes in land-sea thermal contrast46, model biases in representing summer time Rossby waves39 and the use of different diagnostics to quantify waviness (see Box 3).The few modeling studies with historically observed low sea-ice concentration show a stationary wave-train response in summer emerging from AA135; (ii) a hemisphere-wide pattern with a prominent zonal-mean component like the Arctic Oscillation or the Annular Modes137; and (iii) the trapping and focusing effects of the seasonal jet streams on low-frequency tropospheric waves39, known as the waveguide effect54. The figure below shows schematic representations of these three types of teleconnection patterns.Remote climate effects, known as atmospheric teleconnections, consist of three main types: (i) regional long-wave pattern confined in a longitudinal sector of the globe, like the Pacific-North America pattern and the North Atlantic Oscillation138.The summer North Atlantic Oscillation (SNAO) can be regarded as the counterpart of the more robust winter NAO. The centers of action of the SNAO exhibit a more northerly location, have a smaller geographical extent and a weaker dipole pattern compared to the winter NAO. Panel a schematically shows the surface pressure patterns of the positive SNAO phase, i.e., when pressure differences are strong, together with the position of the warmer/drier and colder/wetter regions. Generally, the negative phase shows a reverse pattern. Like the winter NAO, the impact of the SNAO on climate extremes such as heavy rainfall and flooding is profound, especially for Europe. Post-2007 summers have seen increasingly robust negative SNAO associated with a persistent anticyclonic anomaly over Greenland and a cyclonic anomaly over Northwest Europe. This pattern caused rapid melting of the Greenland ice sheet and brought unusually wet summers to Northwest Europe, including the massive flooding of U.K. in summer 2012. Future projections of climate models suggest an increasingly positive SNAO in warmer climates137. Winter and summer NAM patterns present both a diverse geopotential height field, mean meridional circulation and eddy structure. The summer NAM has a smaller latitudinal extent and has a stronger link to surface air temperature over Eurasia. The positive phase of the summer NAM is associated with negative geopotential height anomalies over Greenland and the Arctic Ocean and an annual band of positive anomalies comprised between 40° and 60° N and particularly extended over Eurasia, as schematically shown in panel b. When the summer NAM is strongly positive, the storm tracks follow the Arctic front137. Anomalously positive summer NAM phases are associated with double jets favoring blocking between the polar and the subtropical jets. During a positive summer NAM phase, surface temperatures over Eurasia show a dipole pattern with warmer conditions over Europe and colder conditions over East Asia70.The Northern Hemisphere Annular Mode (NAM) is an internally driven atmospheric mode maintained by both stationary and transient waves. The NAM is defined as the first EOF of monthly 500 hPa geopotential height fields and coincides with the definition of the Arctic Oscillation during the winter months19. Wave-resonance periods have therefore been linked to both persistent heatwaves and severe flooding events64.The energy of waves trapped in a waveguide is not dispersed as broadly as in teleconnections of type (i) and therefore it can propagate farther before being dissipated. In summer, when an efficiently trapping waveguide becomes  circumglobal, then resonant interactions between free and forced waves  might lead to wave-amplification and persistent, high-amplitude waves. Waveguide formation is favored during double-jet regimes and thus linked to strongly positive NAM phases. Panel c shows a schematic representation of a wave-resonance event with an amplified wave 7. The orange and green areas represent regions of positive and negative upper-level meridional winds. Such events are associated with alternating hot-dry and cold-wet conditions, following the ridges and troughs. Such a situation is prone to blocking weather systems and deepened troughs, and their relative longevity is key to making severe weather extremes139.Finally, blocking itself is also distinctly different in summer compared to winter. Upstream latent heat release has been identified as an important contributor to persistent blocking and this mechanism is especially important in summerFigure Box 2: Panel a shows a schematic of the positive SNAO indicating the anomalously low (blue) and high (red) sea level pressure regions together with cold/wet and dry/warm regions. Panel b shows a schematic of the 500 hPa geopotential height configuration during the positive phase of the NAM. Rainy clouds mark the position of the storm tracks during a strongly positive NAM phase. Panel c shows a schematic of an amplified wave 7. Green and orange regions show the position of pronounced northward and southward wind anomalies. 141. These metrics have the advantage of being intuitive and readily visualized from geopotential height contours, but they have been criticized for lacking a firm physical basis. The existence of wave trains usually lasts 2–4 weeks and they do not necessarily have a preferred phase position38. Averaging over time may thus cancel out any signal. Further, often the diagnostic approach to detect extratropical jet’s waviness involves Fourier decomposition of zonal wavenumbers65 which could lead to spurious wave signals. In the extreme case: Fourier decomposition of a delta function will create amplitudes in a range of zonal wavenumbers even if the circulation feature is extremely local, i.e., it does not involve a wave “train”63. Likewise, a zonal wavenumber 5 along 40° N  or 65° N (polar jet) depicts two very different wavelengths. Dynamically based waviness metrics such as effective diffusivity of potential vorticity142 and finite-amplitude wave activity144 are derived from first-order mass and circulation conservation principles and satisfy exact budget closure equations. These measures provide a theoretical basis for quantitatively relating changes in zonal wind speed to accompanying changes in wave amplitude and eddy fluxes, which is straightforwardly verifiable under idealized conditions. Such approaches are being applied in climatological studies of circulation trends and extreme weather events related to amplified flow patterns but their derivation is more technical and their application is more involved than geometric methods.Although the concept of wavy versus zonal circulation patterns is straightforward, quantifying these opposite states has proven challenging. Approaches can be roughly separated into geometric and dynamic methods. The former focuses on the geometry of the circulation to characterize the departure of the flow from zonality in terms of wave amplitude, sinuosity, or circularity63. Such flow regimes are characterized by sharper sub-tropical jets  which can act as waveguides69. Moreover, in summer, recent AA is characterized by enhanced land warming over the high latitudes (~70° N) and much less warming over the nearby Arctic Ocean, in stark contrast to winter warming patterns. This enhanced high-latitude land warming is likely related to a combination of the smaller heat capacity of land compared to ocean, as well as late-spring to early-summer reduction of snow cover. Thus, while the overall equator-to-pole temperature gradient reduces, the thermal gradient actually increases at the land-ocean boundary around the Arctic circle. This situation favors the formation of the Arctic front jet at ~70° N in addition to the sub-tropical jet which is normally present. Such double-jet regimes have become more frequent in recent years due to high-latitude land warming, something which is partly attributable to anthropogenic greenhouse gas forcing76. Double jets favor waveguide formation and wave-resonance but the evidence for an increase in frequency or persistence of waveguides is limited69.Basic theory suggests that double-jet flow regimes are to become slightly more common with pronounced AAThe described dynamical mechanisms do not operate in isolation but instead interact with each other, with other teleconnections, and regionally with land-atmosphere feedbacks. The exact nature of these interactions is still speculative at this stage but positive feedbacks are certainly possible which would lead to tail risks.98. The emergence of such positive geopotential height anomalies in mid-to-high latitudes induces hot and dry air through subsidence and easterly wind anomalies of continental origin60. Likewise, drying soils in subtropical regions like the Mediterranean (a robust projection in future climate) can favor the formation of a heat low, i.e., low pressures at the surface due to upflow associated with intense surface heating99. A Mediterranean near-surface heat low brings easterly winds to central and western Europe, obstructing the normal westerlies100. Weakened or diverted westerlies can reduce rainfall leading to further soil drying. Due to such feedback mechanisms between soil moisture, snow cover changes, and continental-scale circulation, summer weather in western Europe and interior North America is likely to become more continental as seen in models101 and possibly also in observations102. The effectiveness of this mechanism depends on the delicate balance between land-atmosphere feedbacks and the strength (and thus its long-term weakening) of the storm tracks.Weakened storm tracks favor the buildup of hot and dry conditions over the continents. This can strengthen the land-ocean thermal contrast which, combined with the enhanced wave response to thermal forcing when the background flow is weak, could lead to amplified quasi-stationary waves. The projected weakening of storm tracks and westerlies can interact with soil moisture and snow cover changes, potentially regionally exacerbating zonal-mean circulation changes. Enhanced terrestrial heating in mid- to high-latitudes associated with reductions in snow cover can promote anomalous ridging in the upper troposphere, as suggested by both observational and modeling studies104, something which is projected for future warmer climates as well. This slowdown results in anomalously cold SSTs over the northern Atlantic which can trigger a quasi-stationary Rossby wave response favoring blocking high-pressure systems over western Europe105. So, just like AA, a slowdown of the AMOC leads to weakening westerlies in summer over the Atlantic sector, favoring persistent hot-dry extremes over Europe106. Recent observational studies indeed indicate that weather persistence in Europe and some other mid-latitude regions has increased in boreal summer102.Recent studies indicate that the Atlantic Meridional Overturning Circulation  has seen an unprecedented slowdown in recent decades107. On sub-seasonal time scales, extreme monsoonal rainfall over eastern or western Himalayan foothills is often linked to southward intruding mid-latitude troughs, possibly part of the CGWT108. The monsoon is also a source of diabatic heating that can generate the CGWT44. Thus, wave trains originating from higher latitudes can modulate the intensity of the monsoon, and the monsoon’s strength in turn reinforces the downstream propagation of the wave train45. The projected increase in Indian summer monsoon rainfall is thus expected to strengthen the CGWT.The Northern Hemisphere summer circulation is connected to the tropics primarily via two-way interactions with monsoon systems. Anomalously low snow cover over Eurasia during late winter to early-spring can increases the land-ocean temperature gradient due to snow-albedo and soil moisture effects which strengthens the Indian summer monsoon rainfall26. Second, late-spring to early-summer snow cover extent, which influences summer flow regimes, has dramatically declined109; and finally, summer circulation is less affected by tropical ENSO forcing, and thus potentially more sensitive to Arctic warming44.Several arguments support the AA influence on summer circulation as compared to winter. First, in the mid-latitudes, the equator-to-pole near-surface temperature gradient has seen a pronounced reduction in summer, but not in winterReviewing the literature, we conclude that there is robust evidence that AA causes a weakening of storm tracks in summer. The mechanism is straightforward: Cyclone genesis is directly related to the lower troposphere temperature gradient which weakens with AA. Further support comes from observed trends and historic and future climate modeling experiments. Still, multi-model attribution analyses are needed to quantify the exact role of past and future AA on the weakening of storm tracks.63) but not necessarily to a more-stationary flow. Both observations and climate models suggest that a weaker westerly flow is associated with a more wavy flow pattern, especially in summer, but this does not necessarily imply a cause-effect relationship61  seasonal forecasts, and extreme weather.111. For example, the change in European summer circulation could be described by combining a set of storylines that are based on the response to remote drivers from the Arctic, the tropics, AMOC, and regional soil-moisture changes. This way, the high-end risk  can be identified and studied without assigning a specific probability to it110.Idealized atmosphere models are useful to study individual teleconnections and their drivers. However, a central challenge is to quantify the interactions between Arctic teleconnections and other teleconnections and regional processes, requiring state-of-the-art weather or climate models. Teleconnections are generally state-dependent  and non-stationary. While often the mean circulation response to a certain perturbation  is analyzed, it will be important to quantify the response in probabilistic terms: The change in frequency of certain high-impact circulation regimes such as amplified and persistent quasi-stationary waves. An increased frequency in such rare regimes will have little influence on the mean circulation but can have large societal impacts. To do so will require large ensembles and well-coordinated experiments with a range of different models. To disentangle the influence of different teleconnections, a so-called storyline approach can be insightful as well. This avoids quantifying probabilities associated with dynamical changes altogether and, instead, creates a set of physically plausible scenarios  of future changes112 and ocean-atmosphere feedbacks in the presence of sea-ice113, novel machine learning approaches should be used to better integrate information from observations in climate models. In particular, causal discovery algorithms can identify causal pathways in the atmosphere and quantify their relative importance from observations alone114. They can thus be used to do process-based model validations and quantify how well models represent certain causal pathways in the atmosphere. This can give direct insight into the underlying physical reasons behind model bias and thus provides concrete targets for model improvements.To understand and overcome model biases, e.g., in the representation of summer Rossby waves115. Likewise, tree-ring analyses suggest that enhanced jet stream waviness during summers in recent decades has been unprecedented over the post-1725 period116.Finally, high-resolution paleo-climate records over the Holocene period can provide further insights into the circulation response to temperature gradient changes and put recent trends into a long-term perspective. Paleo proxies typically measure some form of biological activity and are therefore best suited to analyze the summer/growing season. The mid-Holocene provides a possible paleo analog with enhanced high-latitude warming and, interestingly, this period was also characterized by enhanced drought conditions in the mid-latitudes117. Though the uncertainties are large, changes in atmosphere dynamics have the potential to cause rapid transitions at a regional scale leading to surprises for society. In summer synergistic effects between thermodynamic and dynamic drivers of extreme weather could act in the same direction to cause very-extreme extremes17. Recent summers have seen such anomalous weather (Box 118.Future impacts from extreme weather are likely to be most pronounced in summer, as most ecological activity and agricultural production takes place in this season119.The current literature provides robust evidence that AA influences mid-latitude summer circulation substantially by weakening the storm tracks. The uncertainties to do with other dynamical aspects and with how dynamical changes ultimately affect regional weather conditions are admittedly large. Nevertheless, we identified several possible feedback mechanisms for how storm track weakening can lead to persistent and therefore extreme weather in the mid-latitudes. Several studies suggest that Northern Hemisphere summer weather is indeed already becoming more persistentIn summary, this review shows that AA is likely to have substantial impacts on mid-latitude summer circulation. The societal impacts can be severe due to tail risks arising from radiatively forced mean summer warming combined with local and remote processes that favor more persistent summer weather. A coordinated research agenda focusing on summer circulation, its drivers and extremes is needed to resolve the key knowledge gaps.


Autonomous Sensory Meridian Response (ASMR) is a sensory-emotional experience in which specific stimuli (ASMR “triggers”) elicit tingling sensations on the scalp, neck, and shoulders; these sensations are accompanied by a positive affective state. In the current research, functional magnetic resonance imaging (fMRI) was used in order to delineate the neural substrates of these responses.A total of 17 individuals with ASMR and 17 age- and sex-matched control participants underwent fMRI scanning while watching six 4-minute videos. Three of the videos were designed to elicit ASMR tingling and three videos were not.The results demonstrated that ASMR videos have a distinct effect on the neural activity of individuals with ASMR. The contrast of ASMR participants’ responses to ASMR videos showed greater activity in the cingulate gyrus as well as in cortical regions related to audition, movement, and vision. This activity was not observed in control participants. The contrast of ASMR and control participants’ responses to ASMR-eliciting videos detected greater activity in right cingulate gyrus, right paracentral lobule, and bilateral thalamus in ASMR participants; control participants showed greater activity in the lingula and culmen of the cerebellum.Together, these results highlight the fact that ASMR videos elicit activity in brain areas related to sensation, emotion, and attention in individuals with ASMR, but not in matched control participants. Individuals with Autonomous Sensory Meridian Response (ASMR) report experiencing tingling sensations on their scalp, neck, and shoulders—sometimes radiating down the back and limbs—during the perception of specific auditory or audiovisual stimuli . These “Previous research has used neuroimaging techniques to identify potential differences between the brains of individuals with ASMR and matched controls. Using resting-state fMRI, during the ASMR experience itself .The purpose of the current study is to identify brain areas associated with the ASMR experience. Doing so will provide insights into the neurophysiological underpinnings of this phenomenon, thus allowing researchers to see ing ASMR . Lochte The current research builds upon the research of Lochte et al. by testing a larger number of participants and including a control group of ASMR-insensitive individuals. Individuals with ASMR and age- and sex-matched control participants viewed three popular YouTube.com videos designed to elicit ASMR tingles, as well as three control videos  of similar complexity. This design allowed us to corroborate the results of Mage = 22.71; SDage = 4.74) and 17 age (±3 years) and sex-matched control participants . This sample size was based on published calculations for neuroimaging studies . All participants received a remuneration of $50 CAD (approximately $40 USD).After providing informed, written consent and undergoing MR safety screening, participants were brought into the MRI suite. All scans began with an initial localizer image and shimming, followed by a 7-min structural MRI scan. Participants then completed six fMRI runs. Each run lasted 5 min and 15 s and consisted of a 1-min presentation of a fixation cross, a 4-min presentation of a video, and a 15-s presentation of a fixation cross. The sound for the videos was presented via speakers in the MRI suite; all participants reported that they were able to clearly hear the six videos.The six videos presented during this study included three videos designed to elicit ASMR tingles (hereafter “ASMR-related videos”) and three videos that were not likely to elicit ASMR (hereafter “control videos”). The ASMR-related videos all involved whispering and viewing a socially emotional scene . The control videos involved normal-volume speech and were on the topics of studying suggestions, gardening tips, and make-up application instructions. The order of the three ASMR-related videos and the three control videos was randomized across participants; however, the fMRI runs were presented such that participants did not view two ASMR-related or two control videos in a row .After completing the experimental session, participants indicated whether they experienced ASMR during the viewing of any of the six fMRI runs and, if so, the intensity  of the resulting ASMR tingles. Importantly, participants were not told in advance which videos were expected to elicit ASMR.Data were acquired using a three-Tesla Siemens TRIO MRI scanner . Anatomical data were acquired with high-resolution T1-weighted gradient-echo images using a magnetization-prepared rapid-gradient-echo sequence with the following parameters: slice thickness = one mm; distance factor = 50%; TR = 1,900 ms; TE = 2.99 ms; in-plane resolution = 1.0 × 1.0; matrix = 256 × 256; and field of view (FOV) = 250 mm.Functional MRI data acquisition consisted of a 5 min 15 s scan using a whole-brain echo-planar imaging sequence with the following parameters: slick thickness = three mm; distance factor = 0%; TR = 3,000 ms; TE = 30 ms; flip angle = 90°; matrix = 64 × 64; and FOV = 240 mm.The post-scan ratings of ASMR-related videos by the ASMR participants indicated that some individuals did not experience tingles in response to some of the videos. However, all ASMR participants did indicate experiencing tingles in response to at least two of the three ASMR-related videos. Therefore, for each participant, we analyzed the data from two out of the three fMRI runs involving the presentation of ASMR-related videos. If a participant reported experiencing tingles in response to all three ASMR-related videos, we selected the two fMRI runs linked with the highest tingle intensity ratings for that participant. No participants experienced ASMR-related tingles to any of the control videos. The control-video fMRI runs were yoked to the particular ASMR-related video fMRI runs used for the analyses. For example, if an ASMR participant rated ASMR videos 1 and 3 as eliciting the most intense tingles, then control videos 1 and 3 were used for that participant’s within-subjects contrast.For the control participants, we analyzed the data from the two runs used for the ASMR participant to whom they were matched. For example, if an ASMR participant reported experiencing high-intensity tingles in response to ASMR-related videos 2 and 3, then the fMRI runs for ASMR-related videos 2 and 3 were used when analyzing the data for the control participant who was age- and sex-matched with that ASMR participant.F-test interaction was obtained followed by contrasts of interest; specifically (11-1-10000) to address the within-subjects effect of the ASMR-related video compared to the control video in ASMR participants, (000011-1-1) to address the within-subjects effect of the ASMR-related video compared to the control video in control participants, and (1100-1-100) to address the between groups difference of ASMR participants compared to control participants while watching ASMR-related videos. Beta values were extracted and plotted for the contrasts of interest. The within-subjects results were displayed at p < 0.01 and corrected for multiple comparisons using a cluster threshold estimator. The interaction and between-subjects results were displayed at p < 0.05 and a cluster threshold of 10 voxels was applied. Clusters were converted to volumes-of-interest, and peak intensity coordinates, t-value, p-value, and voxel counts were given for each cluster. The peak intensity coordinates were entered in Talairach-Client software  which produced the anatomical labels and Brodmann Areas (BA), if applicable.Imaging data were preprocessed and analyzed in BrainVoyager QX software . Preprocessing of functional data included slice scan time correction, 3D motion correction, linear trend removal, and temporal high pass filtering. The functional data were coregistered to the anatomical data, and were warped to standardized Talairach space. The time-series data were dummy-coded to account for multiple runs per participant. The stimulation protocol was created to represent the timing of the video presentation and baseline. Individual data were analyzed with a single subject general linear model (GLM) analysis. These were entered into a multi-study multi-subject GLM and compiled for group analysis. A random effects ANCOVA was run. The The statistical test for the interaction effect revealed significant activity in bilateral superior parietal (BA 7), in the right paracentral lobule (BA 5) and cerebellum, and in the left cingulate gyrus (BA 32), precuneus (BA 7), inferior and medial frontal gyri (BA 9), and precentral gyrus (BA 6). The results are displayed in Results of the within-groups contrasts are listed in An additional contrast was performed to compare the responses of ASMR and control participants during the viewing of ASMR-related videos. The coordinate and statistical details for the significant results are listed in The current results demonstrate that ASMR has both emotional and sensorimotor characteristics. ASMR-related videos elicited activity in numerous brain areas in individuals with ASMR including medial prefrontal brain areas, the precentral gyrus, left superior temporal gyrus, and left cuneus. The same statistical contrast (ASMR videos > control videos) only elicited a decrease in activity in the right cuneus in control participants. Additionally, a comparison of responses to the ASMR videos showed greater activity in ASMR than control participants in the thalamus, left precuneus, cingulate gyrus, and medial sensorimotor regions. These data show some consistency with the previous neuroimaging study of The results of the current study highlight the sensorimotor nature of ASMR. Individuals with ASMR demonstrated greater activity in both the precentral  and postcentral gyri  during the viewing of ASMR-related videos than when watching videos that did not elicit tingling sensations. Additionally, ASMR participants showed greater activity in the paracentral lobule—the continuation of the precentral and postcentral gyri on the medial aspect of the brain—during the perception of ASMR-related videos than did control participants. Together, these data demonstrate that the tingling sensations elicited by ASMR triggers are not psychosomatic; rather, they are associated with increased activity in the sensorimotor regions of the cortex.The enhanced activity in the visual and auditory cortices may be linked with the affective nature of the ASMR videos. Previous neuroimaging research has reported increased activity in visual areas during the perception of emotional stimuli . Given tAn additional potential explanation for the observed activity in the auditory cortex relates to the acoustic qualities of the ASMR videos themselves. The observed activity in the dorsal anterior cingulate gyrus is more difficult to classify, as this region is involved with a number of cognitive and affective functions. The significant clusters in the current study included Brodmann areas 24 and 32. These regions are part of the cognitive/attentional region of the anterior cingulate typically involved in attentional control . This acThe contrast comparing neural responses of ASMR and control participants during the viewing of ASMR-related videos also detected bilateral activity in the thalamus. Given that the thalamus serves as a “sensory relay station” for visual, auditory, and somatosensory information —the sensThe atypical thalamic activity noted in the current research is consistent with a resting-state fMRI study comparing ASMR and control participants which showed inter-group differences in the functional connectivity of the thalamus . It alsoA particularly surprising result from the current study was the lack of activity in reward centers such as the nucleus accumbens. This region was active in Although the current research provides novel information about the neural responses of individuals with and without ASMR to videos designed to elicit ASMR, its shortcomings also highlight avenues for future research. Survey studies of individuals with ASMR note that whispering, tapping, and binaural sounds are common triggering stimuli . Thus, ior an emotional phenomenon. Instead, the data suggest that ASMR involves sensory, motoric, affective, and attentional components.The results of the current study highlight the complexity of the ASMR experience. When viewing ASMR-eliciting videos, individuals with ASMR showed increases in neural activity in regions of the cortex related to attention, audition, emotion, and movement. This activity was not observed in control participants. When responses of ASMR and control participants viewing ASMR videos were compared, individuals with ASMR showed greater activity in the thalamus, anterior cingulate cortex, precuneus, and medial sensorimotor regions. Together, these analysis demonstrate that ASMR is not simply a sensory 10.7717/peerj.7122/supp-1Supplemental Information 1Click here for additional data file.


Ischemia/reperfusion (I/R) injury is associated with primary percutaneous coronary intervention (PPCI). The current study was performed to compare the effect of tirofiban and recombinant human pro-urokinase (rh-proUK) on the improvement of coronary slow blood after PPCI.Sixty-five ST elevation myocardial infarction (STEMI) patients treated with rh-proUK and an equal number treated with tirofiban after PPCI were employed in the current study. The clinicopathological information regarding the biochemical parameters, thrombolysis in myocardial infarction (TIMI) grade, hemodynamics parameters, thrombus core (TS), sum-STR, left ventricular ejection fraction (LVEF), blood routine parameters, high-sensitivity C-reactive protein (CRP) level, uric acid, hepatorenal function, electrocardiogram (ECG), and echocardiography before and after the interventions were collected. The differences in those parameters between the 2 groups then compared with assess the treatment effect and side effects associated with the both therapies.P = .03), the proportion of TIMI myocardial perfusion grade level III (P = .04), the changes in thrombus score (P < .001) in rh-proUK group were significantly higher than those in tirofiban group while the corrected TIMI Frame Count (CTFC) (P = .02), the incidence of slow flow (P = .02), the thrombus score post-intervention (P < .001), the stent length (P = .02), and the number of receiving administration of sodium nitroprusside (P = .01) were significantly lower than those in tirofiban group. Moreover, the levels of CK (P < .001), CK-MB (P = .01), and NT-proBNP 24-hour post-intervention (P < .02) were significantly lower in rh-proUK group than those in tirofiban group and the sum-STR right after the intervention (P < .03) of rh-proUK group was significantly higher than that of tirofiban group. No significant difference was detected between the 2 therapies regarding major adverse cardiac events (MACE).The results showed that the TIMI level post-intervention (The findings outlined in the current study showed that the improvement effect of rh-proUK on blood flow condition was stronger right after the intervention and the therapy had a similar safety when compared with tirofiban during a 30-day follow-up. The dysfunction of endothelial tissues during atherosclerosis contributes to the initiation of inflammation in vessels, which leads to the fracture of atherosclerotic plaques and ulcer. Subsequently, platelets are activated due to the endothelial dysfunction and induces the formation of thrombus as well as the obstruction of coronary arteries. Primary percutaneous coronary intervention (PPCI) is the mostly adopted strategy to handle STEMI. The intervention has the advantage to re-establish blood flow of the obstructed sites in a relatively short period and has been proposed as the first-line therapy for coronary ischemia. However, PPCI can itself lead to reperfusion injuries characterized by coronary slow flow/no flow, which causes secondary damages to patients. Thus, how to attenuate side effects associated with the application of PPCI has become a hot subject in the clinical treatment of STEMI.Characterized by the sudden attack and the high mortality, ST elevation myocardial infarction (STEMI) has been conceived as one of the major death-related factors in developing countries. The mechanism driving the formation of slow flow/no flow is complicated and one the of the major factor contributing to the symptom is the aggregation of small thrombus in coronaries induced by the adhesion of platelets. The key role platelet in the coronary micro-cycle implies that the targeted regulation of the platelet amount may represent a promising strategy for the management of coronary slow flow/no flow after the treatment of PPCI.Coronary slow flow/no flow is termed as the absent of blood flow in vessels even after the treatment of PPCI or thrombolysis. Therefore, it is well recognized that the application of antagonists of specific glycoprotein antigen can play an anti-platelet function via the inhibition of IIb/IIIa. As the only clinically available specific antagonist of IIb/IIIa, tirofiban has the advantages of short biological half-life and high specificity. The administration can block thrombus formation and attenuate coronary vessel obstruction by inhibiting the aggregation of platelets, which ameliorates coronary micro-cycle conditions. Except for the application of anti-platelet drugs, the utilization of recombinant human pro-urokinase (rh-proUK) is also being gradually recommended for the management of slow flow in recent years. Previous study demonstrated that the load of infarction related artery (IRA) in STEMI patients was relatively high, which was a major risk factor for coronary slow flow/no flow after PPCI treatment. However, the injection of rh-proUK can dissolve small thrombus that contributes to the formation slow flow/no flow. Given the regulation effect of rh-proUK on coronary blood flow, rh-proUK also has the potential to handle slow flow/no flow associated with PPCI administration. Nevertheless, compared with the studies focusing on the treatment outcomes of tirofiban, very few studies have paid attention to the treatment effect of rh-proUK on coronary micro-cycle after PPCI. Thus, the comparison of the treatment outcomes on PPCI-induced coronary slow flow/no flow between the 2 therapies might provide novel information on the clinical improvement of PPCI.The aggregation of platelet is driven by the interaction between surface specific glycoprotein antigens IIb/IIIa.To elucidate the differences between the 2 therapies on coronary micro-cycle after PPCI, the treatment outcomes of 65 STEMI patients receiving PPCI and tirofiban treatment and an equal number of patients treated with rh-proUK were collected in the current study. Then the effect of tirofiban and rh-proUK on the biochemical parameters, sum of ST-segment resolution (sum-STR), hemodynamics parameters, and major adverse cardiac events (MACE) was compared with demonstrates which therapy might have better effect in improving coronary micro-cycle in STEMI patients receiving PPCI.22.1; patients received PPCI treatments within 12 hours after the admissions; patients were treated with tirofiban or rh-proUK after PPCI; patients had no anaphylactic reaction after tirofiban or rh-proUK treatments; the patients received no intravenous thrombolysis treatments before PPCI; patients with Killip level higher than III or cardiac shock. Based on the above criteria, the history of information of 130 STEMI patients were collected in the current study from Oct 2016 to Nov 2017 in the Tianjin Union Medical Center. Among the cohort, 65 cases were treated with tirofiban and the left cases were treated with rh-proUK. All the patients possessed detailed clinicopathologic features and signed Informed Consents for the utilization of the information. The study was approved by the ethic committee of the Tianjin Union Medical Center for the data collection of the patients . All the works were undertaken following the provisions of the Declaration of Helsinki.The study was a retrospective study using case information and patients employed in the current study were selected based on the following criteria: STEMI symptoms were diagnosed based on Guidelines for the diagnosis and treatments of STEMI published by Chinese Society of Cardiology of Chinese Medical Association2.2All the patients were orally administrated with 300 mg aspirin and 300 mg clopidogrel/180 mg ticagrelor, and intravenously injected with 5000 IU heparin upon admission. Coronary angiogram (CAG) was performed to determine the number of pathological branches before the interventions. For patients treated with rh-proUK, 5 mg drug was intravascularly injected during the intervention and another administration of 5 mg rh-proUK was given to the patients after the insertion of the stent. If the symptoms of slow flow still exited, the patient was administrated with 5 mg rh-proUK for the last time. For patients treated with tirofiban, 5 μg/kg was slowly injected via coronary arteries during the intervention. After the insertion of stent, another administration of 5 μg/kg tirofiban was given to the patients via coronary arteries. For patients still showed symptoms of slow flow, 3 μg/kg tirofiban was injected via coronary arteries. Twenty four hours after the intervention, all the patients were administrated with 100 mg aspirin and 75 mg clopidogrel/90 mg ticagrelor as well as 5000 IU heparin for 5 to 7 days. All the patients were followed up for 30 days after the discharge to record MACE.2.3 sum-STR, left ventricular ejection fraction (LVEF), the blood routine parameters, high-sensitivity C-reactive protein (CRP) level, uric acid, hepatorenal function, electrocardiogram (ECG), and echocardiography were collected both before and after the interventions, and compared with determine the differences in the treatment effect and side effects of the 2 therapies.To compare the treatment effect and side effects associated with the 2 therapies, the information regarding TIMI grade, hemodynamics parameters, thrombus core (TS),2.4t test was used to compare the difference between groups of continuous data. Chi-square and Fisher exact tests were used to determine if differences existed between different groups of categorical indices. P < .05 was considered as statistical significance.Statistical analyses were conducted using SPSS version 19.0 software . Continuous data were expressed as the mean ± standard deviation (SD). Categorical data were represented by frequency distribution or case number. The Student 33.1The analyses of the baseline information between the 2 groups showed that no significant difference was detected regarding age, sex, body mass index (BMI), Killip score, smoking history, drinking history, and other indicators before the surgeries. The detailed information was shown in Table 3.2As shown in Table 3.3P = .13) and CK-MB (P = .18), no significant difference was detected between the 2 groups. The sum-STR right after the intervention of rh-proUK group was also significantly higher than that of tirofiban group  to as low as 3% to 4%. However, the efficacy of PPCI is counteracted with some severe side effects such as slow flow/no flow induced by I/R injuries. Slow flow/no flow is one of the risk factors influencing the prognosis of STEMI patients. To improve the treatment outcomes of PPCI, some agents such as tirofiban and rh-proUK are being applied to ameliorate the coronary blood flow after PPCI.PPCI can effectively dredge the infarcted coronary arteries and has been selected as the first-line treatment strategy for the clinical management of STEMI. the author showed that the application of rh-proUK significantly lowered the slow blood incidence when compared with patients solely treated with PPCI, indicating that the application of rh-proUK had the potential to ameliorate blood flow condition after PPCI. Thus, in the current study, we compared the treatment effect of rh-proUK and tirofiban on slow flow/no flow associated with PPCI. The results showed that the application of rh-proUK significantly decreased the incidence of slow flow after PPCI when compared with tirofiban group. Moreover, the thrombus score in patients treated with rh-proUK was also significantly lower than that in tirofiban group, which might be resulted from the stronger thrombolysis effect of rh-proUK against thrombus already existing in coronaries. Regarding biochemical parameters, the peak levels of CK and CK-MB in rh-proUK group were significantly lower than those in tirofiban group while the peak time point showed no significant difference between the 2 groups. Both indicators are well-recognized indicators for myocardial function in clinic. Moreover, the level of NT-proBNP which represents the myocardial damage degree was also lower in proUK group than that in tirofiban group. Taken together, the data evidently indicated the stronger protection effect of rh-proUK on injuries associated with PPCI treatment than tirofiban.Compared with the studies based on tirofiban, few attention has been paid to the effect of rh-proUK on coronary blood flow after PPCI. In the study of Guo et al, Combined with the larger change in LVEF in rh-proUK group, it was evident that rh-proUK could also improve the myocardial function much stronger than that of tirofiban. Additionally, the incidence of MACE occurring in rh-proUK group was also lower than that in tirofiban group, the results were representative of the safety of the application of rh-proUK in clinic. The current study also performed a 30-day follow-up after all the patients discharged from the hospital and the results showed that no significant difference was detected regarding parameters of biochemistry, myocardial function, and MACE between the 2 groups, further confirming the promising application potential of rh-proUK in ameliorating coronary blood flow after PPCI. The current study also showed that the stronger effect of rh-proUK on heart was more frequently detected right after the intervention, but with the time lapsing, the difference in the treatment effect between the 2 drugs gradually descended, which might be due to the stronger effect of rh-proUK in improving micro-cycle by dissolving small clots instead of antagonizing the aggregation of platelets that were utilized by tirofiban.The myocardial function of the patients was also evaluated with ECG and it was found that the change in sum-STR in rh-proUK group was larger than that of tirofiban group. Previous study showed that the recovery of myocardial ischemia injuries was in positive relation to the restoration of sum-STR.Collectively, the current study compared the effect of rh-proUK and tirofiban in protecting STEMI patients against ischemia/reperfusion (I/R) injuries associated with PPCI. Our results showed both drugs could effectively decrease the incidence of slow flow and MCAE as well as improving myocardial function. Moreover, the effect of rh-proUK was stronger in improving most indicators right after the intervention and showed similar safety in application when compared with tirofiban during a 30-day following up, which indicated the promising application potential of rh-proUK in clinical management of slow flow associated with PPCI. However, the sample size of the current study was still small and might result in the bias of some detections. Thus, more comprehensive work with larger sample size are needed in the future to facilitate the promotion of rh-proUK.Data curation: Zhuhua Yao, Wenting Li.Formal analysis: Wenting Li, Lisong Cheng, Mingying Cao, Yongbin Li.Methodology: Lisong Cheng, Mingying Cao, Yongbin Li.Supervision: Zhuhua Yao.Writing – original draft: Zhuhua Yao.Writing – review & editing: Zhihua Pang.


Estrogen receptor-positive breast cancers are treated with tamoxifen, a drug that competitively inhibits the binding of estrogen to its receptor. Resistance to tamoxifen is a major hurdle in effective management of target breast cancer patient population. A number of dynamic changes within the tumor microenvironment, including the phenomenon of epithelial to mesenchymal transition (EMT), determine the response to endocrine therapy. EMT is marked by silencing or suppression of epithelial marker, E-Cadherin and we found significantly down-regulated E-Cadherin, among other epithelial markers, and a significantly up-regulated mesenchymal marker, Twist, among other mesenchymal markers, in a model system that comprised of tamoxifen sensitive MCF-7 cells and their tamoxifen-resistant counterparts, MCF-7-TAM, developed by chronic and escalating exposure of parental cells to tamoxifen. Further, E-cadherin, but not Twist, was differentially expressed in MCF-7-TAM cells because of differential methylation. Treatment with demethylating agent 5-azacytidine increased the expression of E-cadherin thus verifying a role of methylation in its silencing and, moreover, 5-azacytidine treatment also re-sensitized MCF-7-TAM cells to tamoxifen, as evaluated by assays for viability, apoptosis and migration potential. The 5-azacytidine effects were similar to effects of E-cadherin overexpression in MCF-7-TAM cells. This work describes novel mechanism of E-cadherin downregulation in tamoxifen resistant breast cancer cells. Further studies are needed to exploit this information for betterment of breast cancer therapy. Tamoxifen binds to estrogen receptor, but does not activate it2, rendering a good inhibition of estrogen receptor-mediated signaling. Target patients experience the benefits of tamoxifen resistance initially but a large number of patients eventually start showing signs of resistance which remains a major hurdle in the effective management of breast cancer3. Because of the heightened interest in breast cancer, particularly the estrogen receptor-positive subtype, a number of investigations have been carried out to understand the basis of tamoxifen resistance and a number of genes, factors and signaling pathways are now believed to contribute to resistance to tamoxifen8. Even though so many possible mechanisms have been proposed and investigated, the benefits in clinics are minimal.Breast cancer is a cancer with many subtypes and estrogen receptor (ER)-positive breast cancers, which constitute about 70% of all breast cancers, are treated with tamoxifen therapy12. Even the phenomenon of resistance to tamoxifen in estrogen receptor positive breast cancer cells is believed to involve EMT as one of the mechanism17. EMT fundamentally involves a transition from more organized and robust epithelial to a considerably more motile mesenchymal phenotype. The process of EMT is marked by decreased expression of epithelial markers and an enrichment of mesenchymal markers and thus relative expression of these markers serves as a good tool to evaluate EMT. E-cadherin is an important epithelial marker that is down-regulated during the progression of EMT. E-cadherin can convert to N-cadherin and such ‘cadherin switch’ is often seen at the time of cancer progression resulting in aggressive behavior and increased metastases18. Even though E-cadherin is often evaluated with respect to its relative expression in cells undergoing EMT, the mechanistic details of down-regulation of E-cadherin, particularly its epigenetic regulation, if any, and in the context of resistance against tamoxifen are totally unknown.Epithelial to mesenchymal transition (EMT) has been target of research in the fight against drug resistance in breast cancer for several years2 humidified atmosphere at 37 °C within our laboratory. Tamoxifen and 5-azacytidine were purchased from Sigma Chemical Company .MCF-7 (ATCC HTB-22) cells were obtained from Beijing Zhongyuan Limited  and MCF-7-TAM were generated by exposing MCF-7 cells to increasing concentrations of tamoxifen over a period of 6 months starting with a dose equivalent to IC-5 of tamoxifen. Cells were maintained in the culture medium and passaged, as per ATCC’s instructions. All cells were cultured in 5% COWe performed MTS -5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay to evaluate cell viability. The assay was done in 96-well plates and cells were seeded atleast 24 hours prior to the assay. Individual treatments were performed, as indicated. At the end of treatment, absorbance was measured on a Shimadzu spectrophotometer (Japan).Cells were treated with increasing concentrations of tamoxifen, as indicated for individual figures, and harvested, fixed, and labeled exactly according to the instructions provided with the TiterTACS Kit . Colorimetric readings were recorded on a Shimadzu spectrophotometer (Japan).We used the Millipore QCM 5 µm Chemotaxis Assay 24-well-colorimetric kit  which provides a quick and efficient system for quantitative determination of cell migration after cells are subjected to treatments. The Millipore 5 µm QCM Chemotaxis Assay 24-well-colorimetric is performed in a Migration Chamber, based on the Boyden chamber principle. The 5 µm pore size of this assay′s Boyden chambers is appropriate for studying a subset of fibroblast or cancer cell migration. In our experiments, cells were first subjected to drug treatment, trypsinized, counted and seeded in equal numbers on the provided 24-well plates. Assays were performed exactly as suggested by the Instructions. Colorimetric readings were recorded on a Shimadzu spectrophotometer (Japan).19. cDNA synthesis was carried out with SuperScript III reverse transcription  and the quantitative real-time PCR performed using iTaq Universal SYBR Green Supermix . The mRNA expression levels of were quantified by measuring the threshold cycle (Ct).RNA was extracted from cells using Trizol  and quantitative RT-PCR was performed as described earlierE-cadherin, cloned in pCMV6, was obtained from Origene . Transfection of cloned E-cadherin into MCF-7TR cells was done using lipofectamine 2000 reagent , following the procedure suggested by the company.We studied the methylation of individual genes, using MethylDetector Bisulfite conversion kit  which involved heat denaturation in the presence of the bisulfite conversion reagent. All the steps were followed, as described in the Instructions, including PCR.Each experiment was performed a minimum of three times and Student t test  was employed to compare individual test groups with the respective controls. Significant difference was set at 0.05.MCF-7 cells chronically treated with tamoxifen eventually developed resistance against the drug. This resistance was confirmed by cell viability MTS assay. As shown in Fig. Chemotherapeutic drugs mostly function through induction of apoptosis and such action also results in reduced migratory abilities, therefore, we checked the induction of apoptosis by tamoxifen and the resulting affect on migration in our cell line models. As shown in Fig. EMT has been proposed to be a major factor affecting the response of cancer cells to therapy, therefore, we evaluated EMT in our cell line models. We tested three epithelial markers  and three mesenchymal markers . As shown in Fig. We evaluated methylation as a possible mechanism for the down-regulation of E-cadherin and the up-regulation of Twist, the two most differentially expressed EMT markers revealed in the last figure. We found significantly increased methylation of E-cadherin promoter Fig.  in tamoxIn our evaluation of EMT markers shown above, we found down-regulation of E-cadherin. Therefore, we overexpressed E-cadherin in tamoxifen-resistant MCF-7-TAM cells and tried to evaluate if restoration of E-cadherin could reverse the action of tamoxifen. As shown in Fig. In the results described above, we observed increased methylation of E-cadherin in tamoxifen resistant cells. Therefore, we wanted to test if this was mechanistically linked to tamoxifen-mediated effects. We used 5-azacytidine as an agent that demethylates DNA. We chronically treated MCF-7-TAM cells with 1 μM 5-azacytidine for 6 cell passages and found that the expression of E-cadherin was increased after treatment with 5-azacytidine Fig. . As show22. Treatment of breast cancer patients depends upon factors such as presence of estrogen receptor, progesterone receptor or HER2 overexpression. Presence of one or more of these receptors makes it convenient to enroll the patients for targeted therapy. Endocrine therapy, such as tamoxifen, is effective in breast cancer patients with overexpression of estrogen receptor. Tamoxifen functions via binding to estrogen receptor and competitively inhibiting the binding of estrogen to its receptor. It does not affect the levels of circulating estrogen3.Breast cancer remains a major killer among the females23. Now, that does not mean that this is the only mechanism and certainly more mechanism(s) cannot be ruled out, particularly when we look at the relative expression of the most differentially expressed mesenchymal marker Twist. Twist was found to be highly elevated in tamoxifen resistant cells, however, even though the methylation of its promoter was slightly reduces, as would be expected in cells undergoing EMT, the degree of reduced methylation was not significant enough. This would mean that up-regulation of Twist is because of some other mechanism that does not necessarily involves methylation. On the other hand, the levels of methylation were found to be significantly more for E-cadherin, giving confidence in pointing out some epigenetic regulation. This was further confirmed by experiment using 5-azacytidine, which is a demethylating agent24 and, by definition, reduces methylation by removing methyl groups. When we treated cells with 5-azacytidine, not only the suppression of E-cadherin inhibited in tamoxifen-resistant cells, but the resulting attenuation of tamoxifen effects, such as on cellular apoptosis and migration potential, was also observed which clearly gives weightage to the methylation of E-cadherin as a principal mechanism causing its down-regulation in the tamoxifen-resistant cells.In our analyses, we found significantly higher methylation of E-cadherin promoter in tamoxifen-resistant cells. This can clearly be a reason for its observed down-regulation because methylation of gene promoters leads to their silencing27 and the functional implications of these epigenetic events are coming to light now. The epigenetic changes within the tumor microenvironment assume further significance28 where so many cell types mutually interact within a small space with many of them manipulated into serving the needs of cancer cells. Any changes in the expression of factors within the tumor microenvironment are thus bound to have profound effect on cellular signaling and the growth, proliferation, invasion, metastasis and angiogenesis of cancer cells because they happen inside or within a close vicinity of cancer cells. Further, E-cadherin has long been identified as a prominent biomarker with critical role in cancer progression. It has even been called a ‘gatekeeper’ to breast cancer tumorigenesis29 and although there exists plethora of information on its up- or down-regulation under different conditions, the mechanisms remain almost unknown. Thus, keeping in view such gap in our information, our present study provides some novel information on the regulation of E-cadherin, particularly in breast cancers that have turned refractory to endocrine treatment. While we provide in vitro evidence in support of a novel mechanism of regulation of E-cadherin in tamoxifen-resistant breast cancer, we understand that further animal studies are needed to validate these findings so that the information could be of use in future clinical trials.Epigenetic regulation of tumor suppressor genes has been realized for quite some time now31. Moreover, the small clinical study on 22 patients has suggested benefit of this practice even in the determination of response to a chemotherapy31. Further, epigenetics is now understood to play big role in determination of drug sensitivity in breast cancer32, even in the delicate balance between resistance and sensitivity to tamoxifen36. This not only suggests that our work is timely, but also calls for further studies to improve our understanding of the underlying mechanisms, which can lead to much improved targeted therapies for the treatment of estrogen receptor-expressing breast cancers.Even though no specific guidelines were recommended for inclusion of EMT markers in clinical studies until a few years back, this has considerably changed now. New data supports an evaluation of EMT markers, even in relation to novel targets such as circulating tumor cells


Background: Several studies have tried to investigate the role of inflammatory biomarkers in Autism Spectrum Disorder (ASD), and their correlations with clinical phenotypes. Despite the growing research in this topic, existing data are mostly contradictory. Methods: Eighty-five ASD preschoolers were assessed for developmental level, adaptive functioning, gastrointestinal (GI), socio-communicative and psychopathological symptoms. Plasma levels of leptin, resistin, plasminogen activator inhibitor-1 (PAI-1), macrophage chemoattractant protein-1 (CCL2), tumor necrosis factor-alfa (TNF-α), and interleukin-6 (IL-6) were correlated with clinical scores and were compared among different ASD subgroups according to the presence or absence of: (i) GI symptoms, (ii) regressive onset of autism. Results: Proinflammatory cytokines  were lower than those reported in previous studies in children with systemic inflammatory conditions. GI symptoms were not correlated with levels of inflammatory biomarkers except for resistin that was lower in ASD-GI children (p = 0.032). Resistin and PAI-1 levels were significantly higher in the group with “regression plus a developmental delay” onset (Reg+DD group) compared to groups without regression or with regression without a developmental delay . Conclusions: Our results did not highlight the presence of any systemic inflammatory state in ASD subjects neither disentangling children with/without GI symptoms. The Reg + DD group significantly differed from others in some plasmatic values, but these differences failed to discriminate the subgroups as possible distinct ASD endo-phenotypes. To date, the understanding of the underlying molecular mechanisms of some metabolic or neurological diseases and the deepening of knowledge on the role of inflammation in these disorders have radically changed our understanding of their etiology ,2. AlzheRecently, the contribution of immune dysregulation has been described as a common feature of the autism spectrum disorder (ASD), and alterations in circulating cytokine levels have been repeatedly reported ,9. ASD aSome authors have proposed interleukin (IL)-6, tumor necrosis factor-alpha (TNF)-α, and macrophage chemoattractant protein-1 (CCL2) as potentially involved in brain inflammation at least in a subgroup of subjects with ASD . A recenSimilarly, GI problems that frequently occur in ASD subjects seem to be caused by inappropriate immune activation and pro-inflammatory processes of the digestive tract . It has Besides, a particular type of cytokines called adipokines seems to be implicated in the pathogenesis of inflammatory central nervous system (CNS) disorders and ASD  despite Resistin has been implicated in the pathogenesis of several inflammatory CNS disorders  and its Distally regulated by some cytokines , the plasminogen activator inhibitor-1 (PAI-1) seems to directly influence brain functions causing a neuronal dis-connectivity due to abnormal neuronal migration . PAI-1 mThis pilot study aims (i) to investigate the plasmatic levels of several proinflammatory molecules  in preschoolers with ASD; (ii) to explore the correlation between their plasmatic levels and behavioral profiles in preschoolers with ASD to detect possible specific subgroups within the ASD heterogeneity.A total of 85 ASD preschoolers were included in the study and recruited from November 2015 to February 2018 at the ASD Unit of the IRCCS Stella Maris Foundation , a tertiary care university hospital during a clinical trial on the efficacy of probiotic supplementation in ASD preschoolers . In the ASD diagnosis was performed according to Diagnostic and Statistical Manual of Mental Disorders (DSM)-5  criteriaAll children had a comprehensive evaluation including Autism Diagnostic Observation Schedule-2 (ADOS-2) , GriffitThe demographic and clinical characteristics and a complete description of the tools of all participants and in no-verbal vs. verbal groups are reported in To evaluate the presence of GI symptoms we used a modified version of the GI Severity Index (GSI ) splittiplus developmental delay” was defined as a significant lag in the appearance of normal developmental milestones with a later loss of previously acquired skills.Moreover, all preschoolers were divided into regressive or non-regressive (early-onset -EO-ASD-) autism based on the presence/absence of a history of loss of competences such as language or social competences ; childreThis study was carried out according to the standards for good ethical practice and with the guidelines of the Declaration of Helsinki. The study protocol was approved by the Pediatric Ethics Committee of the Tuscany Region . Written informed consent from a parent/guardian of each participant was obtained.A fasting blood sample (3 mL for each child) was collected in Ethylenediamine tetraacetic acid (EDTA) tube to perform the cytokines quantitative analysis. We didn’t use pain patch before the sampling. Each tube was centrifuged for 10 min at 3500 rpm and all the plasma samples were stored at −80 °C until required the bio-humoral investigationsThe cytokines were measured directly in the plasma through specific immunometric tests  using an integrated multi-analyte detection platform  Each sample was analyzed in duplicate. In each one, a sample was analyzed as a quality control. Inter-assay variability was evaluated using two samples at different concentrations and was <10%.Descriptive statistics were computed for selected demographic variables across diagnostic groups. Contingency tables were used to perform the frequency analysis. Since the molecule’s values were not normally distributed, we used log-transformed values with parametric statistic tests and non-parametric tests to compare GI vs. No-GI subjects (Mann-Whitney test) and to compare EO ASD vs. Reg-DD vs. Reg + DD  for all the selected molecules.p value <0.05 were considered significant. StatView software  was used for data analyses. To discriminate different subgroups of ASD children based on biomarker levels, we performed Principal Component Analysis (PCA) using as correlated variables: sex, BMI, age, and cytokine levels . After log transformation and auto scaling  PCA was performed using MetaboAnalystR 1.0.3 . We checked quality control of samples using PCA that allowed us to label the 85 samples as outlier so it was excluded from downstream analysis.Correlation and regression analysis were computed to study the relationship between the molecules and the identified clinical parameters. Findings with p = 0.086). As concerns sex distribution, no differences were found in the prevalence of females in GI versus No-GI groups neither verbal versus non-verbal groups .Thirty children (35%) were in the GI group and 55 (65%) in the No-GI group. Among the 30 GI subjects, 20 children (67%) were in the non-verbal group, whereas among the 55 No-GI, 26 children (47%) were in the non-verbal group. No statistically significant differences were found in the prevalence of GI subjects between verbal and non-verbal groups (p = 0.0016), the Internalizing and Total problem scores of the CBCL , and of the Communication and Daily Living adaptive scores of the VABS .As concerns clinical variables, there were no significant differences between the GI and the No-GI groups, with the exception of the Global Score of the RBS-R . No difference in plasma biomarker levels was found between non-verbal and verbal groups.As concerns proinflammatory cytokines levels, the single and the mean values in the total sample and in each subgroup are reported in p < 0.01 for all). Regarding the onset of autism, the mean values of cytokines were not statistically significant different between EO-ASD and regressive subgroups. Nevertheless, comparing cytokines levels in the EO-ASD subgroup with the two types of regressive preschoolers (with and without DD), resistin and PAI-1 levels were statistically significant higher in the Reg + DD group than in the other two groups, the EO-ASD and the Reg-DD ones  and with RBS-R total scores , and positively correlated with VABS-II Motor Skills . TNF-α and PAI-1 levels negatively correlated with age ; Leptin levels positively correlated with Body Mass Index  and negatively correlated with CBCL1.5-5 Internalizing problems .Finally, after the correlation analysis between each molecule and all the clinical parameters, CCL2 levels negatively correlated with CBCL1.5-5 Internalizing and Total problems  with a low percentage (PC1 = 21.3% and PC2 = 19.0%), indicating that the two and three groups respectively are not partially separated but overlapped .Our study fits within the complexity and the heterogeneity of studies that examine inflammation and immunity dysfunctions in ASD subjects, moving the field forward into the investigation of biological biomarkers to discriminate possible endophenotypes. The narrow age range considered, the detailed clinical characterization with specific and gold-standard tools for ASD evaluation, and an enough large sample represent the strengths of the study.First, we found that the single and the mean values of our cytokines were lower than those expected in subjects with systemic inflammation ,53,54. TSecond, we did not observe significant differences in the levels of circulating cytokines between GI and No-GI ASD children, except for resistin. Notably, there is too scant relevant research on this topic in ASD subjects ,39 to drWe did not find any significant correlations between the basal levels of TNF-α and IL-6 and the autistic features of the total sample, similarly to some investigations ,66 and iThird, we found that the group with regression plus developmental delay prior to the onset of ASD (16.5% of the sample) was significantly different from the rest of the sample as far as the higher plasmatic levels of resistin and PAI-1. We could suggest that Reg + DD children represent a specific subgroup with a definite biological profile and a specific clinical feature. However, using the PCA method, we did not identify the Reg + DD group as a particular cluster of patients, making the individuation of a specific endophenotype unlikely in this sample. Future studies are needed to retest the robustness of these findings before we can consider them as reliable.In addition, we did not identify any significant correlation between the levels of cytokines and the presence or absence of a regression of skills prior to the onset of autism. This result is in accordance with the majority of similar investigations, but in contrast with others where an association, although weak, between regressive autism and TNF-α , or loweWe must consider this study as a pilot investigation with several limitations. Compared to other authors who have measured a series of pro-inflammatory cytokines in ASD subjects , we focuDespite the above-mentioned limitations and the existing controversies within the studies about the role of cytokines in ASD and the extreme variability of their findings, our study finds no evidence of the presence of inflammatory condition in ASD subjects, except for resistin. Our findings do not support the use of anti-inflammatory therapies in ASD children, and paves the way for the search of alternative hypotheses for the etiology of GI symptoms in subjects with ASD. Despite our findings showed a specific plasmatic cytokine profile in ASD children with a history of a regressive way of onset within a previous developmental delay, the specific endophenotype for these subjects has not been identified.The study protocol was approved by the Pediatric Ethics Committee of Tuscany Region (Protocol Number: 126/2014), with written informed consent obtained from a parent/guardian of each participant. The study was conducted following the 1964 Declaration of Helsinki and its later amendments, and the International Conference on Harmonisation Guidelines for Good Clinical Practice.