The Arctic is warming faster than anywhere else on Earth, prompting glacial melt, permafrost thaw, and sea ice decline. These severe consequences induce feedbacks that contribute to amplified warming, affecting weather and climate globally. Aerosols and clouds play a critical role in regulating radiation reaching the Arctic surface. However, the magnitude of their effects is not adequately quantified, especially in the central Arctic where they impact the energy balance over the sea ice. Specifically, aerosols called ice nucleating particles (INPs) remain understudied yet are necessary for cloud ice production and subsequent changes in cloud lifetime, radiative effects, and precipitation. Here, we report observations of INPs in the central Arctic over a full year, spanning the entire sea ice growth and decline cycle. Further, these observations are size-resolved, affording valuable information on INP sources. Our results reveal a strong seasonality of INPs, with lower concentrations in the winter and spring controlled by transport from lower latitudes, to enhanced concentrations of INPs during the summer melt, likely from marine biological production in local open waters. This comprehensive characterization of INPs will ultimately help inform cloud parameterizations in models of all scales. The Arctic is changing faster than anywhere else on Earth. Interactions between clouds and aerosols play a role in these changes. We report how the quantities and origins of aerosols that affect cloud ice formation change over a full sea ice cycle The largest uncertainties stem from the indirect effects of aerosols on clouds, which are poorly represented in models2. This deficiency is especially true for the central Arctic Ocean, in part, due to a dearth of observations and understanding of clouds and aerosols collocated in space and time3. Evaluating aerosol-cloud interactions in this subregion is critical given the heightened sensitivity of the surface energy budget to cloud phase over sea ice.Aerosols are an important component of the atmosphere through their impacts on climate, yet the magnitude of their effects remains unquantified4. Over the sea ice, they impart seasonally-contrasting radiative impacts, warming the surface for most of the year while cooling it for a short period in summer6. Arctic mixed-phase clouds (AMPCs) composed of liquid and ice are persistent7 and common, occurring 41% of the time on average and up to 70% of the time during the spring and autumn transition seasons8. The ability for models to predict the sea ice seasonal cycle largely depends on accurate depictions of AMPCs because of their strong influence on the surface energy budget, but the magnitude of their impact on downwelling radiation inherently depends on the relative amounts of liquid vs. ice9. However, models struggle to represent seasonal variation in AMPCs without significant biases4. Further, inter-model differences in Arctic cloud occurrence throughout the year are driven by inadequate parameterizations, motivating the need for observations of fundamental microphysical processes10.While the enigmatic behavior of clouds causes an incomplete understanding of their role in the Arctic climate system, previous efforts agree on some general qualities. Arctic clouds are most frequent in autumn and least in winter11. At supercooled liquid temperatures (–38 to 0 °C), the primary pathway for cloud ice formation is immersion freezing by ice nucleating particles (INPs)12, wherein aerosols are first immersed in cloud droplets followed by freezing once optimal conditions are met. The dominant types of Arctic INPs have been shown to comprise of mineral and biological materials, emanating from land  and the ocean . At relatively warm temperatures (above −15 °C)—common among central Arctic clouds throughout the year8—the majority of INPs are typically biological19. Pioneering studies by Bigg et al. during the early 1990s alluded to marine biota as the source of central Arctic INPs21. While previous studies characterizing central Arctic INPs are salient, they are also rare, and typically confined to summer, aside from a single study from March18.Cloud formation requires the presence of aerosols22, specifically enabling a comprehensive investigation of atmospheric, oceanographic, and sea ice observations to characterize the sources of INPs. Our findings portray strong seasonality of INPs, with lower concentrations in the winter and spring controlled by transport from lower latitudes, to enhanced concentrations of INPs during the summer, likely from marine biological production in local open waters.Here, we describe a full annual cycle of size-resolved INPs in the central Arctic. To capture the breadth of INP sources and assess their impacts on Arctic clouds and the surface energy budget, a complete annual cycle of observations is essential. The Multidisciplinary drifting Observatory for the Study of Arctic Climate (MOSAiC) expedition offered the opportunity to achieve a holistic characterization of the central Arctic coupled system over the course of a full year (September 2019–October 2020)23 as it drifted from the eastern Arctic Ocean, past the North Pole, and on toward Svalbard, mostly within the central Arctic pack ice  around Polarstern reached a maximum of 6% in early May, while melt pond fraction reached its maximum of 37% in early August. The concentration of sub-ice chlorophyll-a (chl-a)—a proxy for biomass of marine primary producers—was consistent with what has been previously reported for the central Arctic Ocean24, peaking in June–July. The coldest air temperature (down to –42 °C) occurred toward the end of winter, while, following the melt onset in late May, the warmest (up to 3 °C) was observed during the 24-h sunlit summer when air and surface temperature hovered around 0 °C22. The summer experienced relatively calmer wind (4.5 ± 1.8 m s−1 on average) compared to the rest of the year (5.9 ± 2.6 m s−1 on average).As context for our INP observations, MOSAiC measurements presented here were made onboard the research vessel Polarsternce Figs.  and 2. HAirmass trajectory analyses provided a broader perspective for the conditions at Polarstern. Figure 25. The seasonal cycle was shaped by a combination of a relatively consistent presence of cold-temperature INPs (active at <−15 °C) throughout most of the year with a preponderance of warm-temperature INPs (active at ≥−15 °C) during summer. Cold-temperature INPs  peaked during the winter and late summer. The winter peak was likely influenced by dust from continental sources28, as shown by the intermittent transport of continental airmasses from Siberia, Eastern Europe, and Northern Canada . Local wind speed averaged 4.3 m s−1 in June and July, while 3-day airmasses predominantly traveled from over the pack ice (73% of the time) and MIZ (20% of the time) within the typical well-mixed summer boundary layer (below 400 m (ref. 28)) at an average height of 158 m. It is important to note that transit periods when the ship was in open water south of the pack ice did not correspond to enhanced INP concentration, suggesting that the larger area of ice-free open ocean (SIC < 15%) further south was not a significant source of the warm-temperature INPs in the summer. This is supported by the fact that marine INPs from open ocean sea spray are typically low in concentration, unless the waters are subject to shallow shelf regions or collapsing phytoplankton blooms31. Previous work corroborates the fact that the annual variation of sea ice algal presence, derived from chl-a data, is controlled mainly by environmental conditions such as sunlight, and less by latitude32. Additionally, as melt ponds and the MIZ develop, this exposes materials from under-ice and ice-edge algal blooms that manifest during the melt onset and extend into the summer34 and that may become airborne under appropriate wind regimes. As the melt season progresses, these open water sources contain a mixture of ice algal and marine microbial communities that are more densely populated near the surface, while these microorganisms effectively sink in larger areas of open water that has been devoid of ice for longer periods33, and therefore, farther away from potential exchange with the atmosphere. Together, the evidence presented here suggests that: (1) summer INPs were likely from biological productivity in open water sources either locally within the pack ice, from farther north over the pack ice, or from the MIZ; (2) transported airmasses interacted with these surface sources; and (3) winds were strong enough to produce sea spray from localized open water sources within the pack ice36. Future publications will focus on aerosol DNA and fluorescence measurements to corroborate the presence of biological aerosol in the summer.Warm-temperature INPs, which were predominantly proteinaceous  at −15 °C in late August through mid-October and attributed the ice-free ocean as the likely source with smaller contributions from open leads in the pack ice21. Hartmann et al.26 reported summertime concentrations up to 1 × 10−1 l−1 at −15 °C within the MIZ in July and lower concentrations over the pack ice. More recently, Porter et al.44 reported INP concentrations up to 1 l−1 at −15 °C during their Aug/Sep cruise but also observed lower concentrations in the pack ice. In contrast to these three studies, during MOSAiC, INP concentration was highest in the pack ice at 82.1°N in the summer, reaching 2 × 10−2 l−1 at −15 °C. Possible explanations for this contrast could be variability in time of year, airmass transport pathways, ice thickness, local lead and melt pond areal fraction, and primary productivity peak location and date; however, long-term annual studies at multiple locations within the pack ice, MIZ, and ice-free ocean would be required to statistically identify the key limiting factors that control INP concentration. Overall, the observed concentrations are consistent with what has previously been reported in the Arctic but are unique in that: (1) they demonstrate that INPs can be abundant over pack ice with minimal inferred source from the ice-free ocean relative to the pack ice and MIZ regions and (2) they show relationships between INPs and environmental conditions over a full annual cycle.Previous studies report a similar enhancement in summertime Arctic INPs, whereby peak concentration ranged from 2 × 1045. However, this relationship between INP concentration and particle size was not observed during MOSAiC 47, coarse sea spray is present in the autumn prior to freeze up, thus, it is plausible that the low INP concentration observed here (84.8–86.2°N) originated from sea spray, episodically generated by high winds over local leads present in the pack ice  in nature, either existing in that size range  or internally mixed with sea salt50.At the beginning of the study, autumn INPs were coarse ≥3 µm), although total aerosol in this size range was not particularly abundant  and total aerosol, probably due to preferential gravitational settling of larger aerosols during transport over longer distances47 or primary biological organic aerosol52. The highest fraction of INPs to total aerosol in the ~1–3 µm size range was 3–7 in 104 total particles  in winter implies that cold-temperature mineral INPs that are relatively small, like those observed here, could serve an important role in cloud ice formation. However, the fact that the surface is predominantly frozen for most of winter limits the potential local sources of INPs. Additionally, the highly stratified boundary layer, characterized by high variance in sub-cloud equivalent potential temperature, limits the vertical movement of INPs. Given that there are seasonal episodic transport pathways in the lower troposphere from lower latitudes in winter55, it is most plausible that winter INPs affecting clouds have remote sources. The INPs measured near the surface are likely transported aloft and transported downward by mixing and/or precipitation and sublimation.The coldest and highest clouds were present in winter, as observed previously56) offers the potential for more interaction between clouds and the near-surface environment where coarse, biological or organic sea spray INPs active at cold temperatures16 could be produced from more localized leads, melt ponds, or the MIZ. Indeed, high ice fractions in low-level clouds are observed at these times of year. Previous work has indicated that cloud formation in the spring is promoted by strong evaporation from newly-opened water, increasing cloud presence and downward longwave radiative flux, contributing to even more sea ice melt57.Spring and autumn clouds were still relatively cold and somewhat elevated above the surface, but enhanced below-cloud mixing , including onboard the German research icebreaker Polarstern and from Met City, which was an installation on the ice in the CO between 300–600 m from Polarstern22. GPS devices operated on Polarstern and at Met City measured latitude and longitude at hourly and 1-min resolution, respectively. Arctic Mapping Tools67 for MATLAB were used for plotting the maps for the positioning data .The MOSAiC international drift expedition took place in the central Arctic pack ice, leaving Tromsø, Norway on 20 September 2019 and returning to Bremerhaven, Germany on 12 October 2020. The expedition was broken down into legs, some of which required transit of Polarstern back to land for exchange of personnel, fuel, and provisions (transits are noted in figures in the main text). Dates for legs 1, 2, 3, 4, and 5 are 20 Sep–13 Dec 2019, 13 Dec 2019–24 Feb 2020, 24 Feb–04 Jun 2020, 04 Jun–12 Aug 2020, and 12 Aug–12 Oct 2020, respectively68 from the U.S. Department of Energy’s (DOE) Atmospheric Radiation Measurement (ARM) Program, which was positioned on the bow of Polarstern, ~18 m above the sea ice surface69. Air temperature was also measured at 2 m above the sea ice using a Vaisala PTU307 sensor package situated on a 10-m meteorological tower at Met City70 (not shown but used for as a cross check for the temperature measured on Polarstern). Surface skin temperature was measured at a 1-min resolution using an Apogee SI-4H1 infrared thermometer mounted at 2 m on the Met City tower and pointed down at the surface. Winds were measured at a 1-min resolution using a sonic anemometer mounted at a nominal height of 10-m on the Met City tower22. For the May–June period when Met City was not installed, these measurements were obtained from an identical sonic anemometer mounted at a nominal height of 3 m on an Atmospheric Surface Flux Station that was left in place of the Met City installation71. U (eastward) and V (northward) wind vectors were calculated to obtain daily averages using Eqs. 72. Equivalent potential temperature (θe) was calculated following Eqs. (22) and (43) in ref. 73 for the radiosonde profiles. From that, daily mean vertical θe variance between the launch platform and the daily mean cloud base height as retrieved from ceilometer (see details below) were employed as a proxy for sub-cloud mixing. Likewise, daily mean temperature at the estimated cloud base was extracted from the soundings.Radiosondes measuring profiles of pressure, temperature, humidity, and winds were launched every 6 h from the helicopter deck on the stern of Polarstern up to ~20 km76. The first or lowest cloud base height (out of three possible for multilayer cloud scenarios) was used as that is the most relevant for interactions with aerosols detected near the surface. Cloud fraction over time was calculated using the first cloud base height data from the ceilometer. Each 15-s interval was classified as 0 (no cloud base detected) or 1 (first cloud base detected) and monthly mean cloud fractions were calculated from there. Cloud ice water path (IWP) at a 1-min resolution was retrieved via a multi-sensor cloud property retrieval method77. The fractional occurrence of cloud ice was calculated based on when IWP > 0 for data below 3 km, then taking the total minutes flagged for ice divided by the total minutes where clouds were detected below 3 km per day.Cloud fraction and base height were estimated using 15-s data from a ceilometer  operated on the upper deck of Polarstern as a part of the second ARM Mobile Facility (AMF2)https://github.com/rich-iannone/SplitR)79. Reanalysis data from the National Centers for Environmental Prediction were used as the meteorological fields in HYSPLIT simulations . Trajectories were initiated from 30 m AMSL and from the latitude and longitude of Polarstern at 00:00 daily. The initiation height of 12 m above the AOS inlet was used to estimate larger-scale airmass movements outside of the possible wind shear interference from Polarstern’s decks. An initiation height of 100 m AMSL was also used to demonstrate that possible disturbances from the underlying surface did not change the main conclusions and to validate the results from the trajectories ending at 30 m. Trajectories were calculated 3 days back in time to account for average atmospheric aerosol residence times for supermicron aerosol 53, as these aerosol sizes were the most proficient INPs observed during MOSAiC. Additionally, 5- and 7-day trajectories were calculated to account for possible longer residence times of smaller particles. Recent work utilized 7-day trajectories for coarse mode aerosol source apportionment in Svalbard46. It is important to note that the determination of the length of back trajectories is a compromise between typical aerosol lifetimes and the uncertainty in calculation that increases with time. Additionally, particle sphericity impacts the rate of gravitational settling80 but neither particle sphericity or gravitational settling are accounted for here, as the relevant measurements to determine particle sphericity were not made and HYSPLIT is a passive air parcel model. Figure Airmass backward trajectories were calculated using HYSPLIT, the HYbrid Single Particle Lagrangian Integrated Trajectory model with the SplitR package for RStudio  were obtained from the National Snow and Ice Data Center81 and were derived from the Multi-sensor Analyzed Sea Ice Extent Advanced Microwave Scanning Radiometer 2 MASIE-AMSR2 (MASAM2) daily 4-km SIC product that is a blend of two other daily sea ice data products: ice coverage from the product at a 4-km grid cell size and ice concentration from the AMSR2 at a 10-km grid cell size82. MASAM2 was used to meet a need for greater accuracy and higher resolution in ice concentration fields. Daily SIC averaged within a 1-degree grid box around Polarstern and within a 0.1-degree grid box around airmass backward trajectory endpoints were computed and shown in Figs. Daily SIC data  thermal infrared satellite imagery85. Most samples were analyzed at the Alfred Wegener Institute (AWI) after the campaign had ended. During leg 3, one subsample per sampling event was measured on board within 3 days after sampling, while a second subsample was analyzed at AWI (5–9 months after sampling). No systematic difference between the replicates could be detected, indicating no significant degradation of chl-a took place during storage and transport.Samples for the determination of surface chlorophyll (chl-a) were taken from 11-m water depth via Polarstern’s underway system. Depending on chl-a levels, 2–4 l of seawater were filtered onto glass microfiber filters  in duplicates or triplicates and frozen at −80 °C until further analyses. Samples were extracted in 90% acetone over night at 4 °C and subsequently analyzed on a fluorometer , including an acidification step (1 M HCl) to determine phaeopigments following ref. 87. Submicron aerosol size distributions from 60 nm to 1 µm were measured at a 10-s resolution using an ultra-high sensitivity aerosol spectrometer  also located in the AOS89. The UHSAS measurements were converted to aerodynamic diameter (Da), assuming optical diameter is equivalent to the true diameter (Dtrue), using the following equation and density of 1.5 g cm−3 based on estimates from previous studies based in pristine and polluted locations91:94. The whole air inlet of the Swiss container is built after the Global Atmosphere Watch recommendations95. The AOS inlet contained a back-pressure purge system that was applied to minimize sampling of ship emissions, such that during obvious periods of ship emission influences, the purge system would cause the inlet to stop sampling while all instruments remained operational96. However, not all periods were detected and purged. Thus, developing and applying a pollution mask to omit aerosol data affected by local contamination  was essential. The basic principle of the pollution detection algorithm is based on the time derivative of the particle number concentration. Local pollution was characterized by strong fluctuations (and thus high time derivatives) in particle number concentration. If this time derivative exceeds a certain threshold, the data were flagged as polluted. The basic principle of this method was developed and used for the 2018 Microbiology-Ocean-Cloud-Coupling in the High Arctic campaign on the Swedish ice breaker Oden97.Aerosol number concentrations from 10 nm to 10 µm at a 1-s resolution were measured using a condensation particle counter  located in the AOS99 through the AOS inlet, in parallel with the CPC and UHSAS. The DRUM collected daily integrated samples at 29–34 l min−1 (average flow rate of 31.6 ± 1.6 l min−1) from 0.15 to >12 µm in diameter  on sterilized perfluoroalkoxy substrate strips coated with petrolatum—a material containing very few artifacts that interfere with INP analysis. It is important to note that the AOS inlet has a high transmission efficiency for particles from 10 nm to 4 µm but has large uncertainties in transmission efficiency above 4 µm due to low ambient aerosol signal in that size range, thus it is possible the DRUM samples produced lower INP concentration estimates in that size range as well92. Sample start times ranged from 10:25–15:15 UTC (average time of 14:30 UTC) and collected continuously for 24 h. Aerosols were “smeared” onto the sample strips as the discs in each stage rotated slowly over time (5 mm of aerosol loading) followed by quick 2 mm rotation, affording a sample blank in between each daily sample. Weekly flow and pressure checks were conducted throughout the year to ensure obstructions were not present in the orifices. Samples were preserved and shipped frozen (≤–20 °C) until analysis at CSU.Size-resolved aerosols were collected from 23 October 2019 to 1 October 2020 using the Colorado State University (CSU) 4-stage Davis Rotating-drum Unit for Monitoring cascading impactor 100. For preparation, aerosol samples were added to 2 ml of 0.1-μm filtered deionized water and mixed for at least 20 min at 200–500 rpm in a laminar flow clean hood to resuspend particles into a suspension. After preparation, 100 × 2.5-µl aliquots were created on a 3-inch diameter copper plate and covered to prevent contamination. The plate was cooled at ~1–10 °C min−1 from room temperature until all drops froze on the plate or until the cold plate limit of ~ –29 °C. Drop freezing was detected and recorded through monitoring software to provide the freezing temperature and cooling rate for each drop. Each sample was tested three times .The CSU cold plate is an established method used to measure immersion mode INPs101. However, this recent work analyzed deposition-mode INPs, which likely do not overlap with the properties of immersion-mode INPs, as other previous studies have demonstrated how pollution aerosol, in general, is not a sufficient source of immersion INPs106.For MOSAiC, a subset of the 1280 DRUM samples was processed and analyzed using the cold plate. Approximately every third day’s sample set was chosen—a “sample set” corresponds to all four samples from each of the size ranges, per 24-h period, equating to analysis of 388 samples. However, this time resolution varied as samples that were not under the AOS inlet purge were selected. The purging reduced possible contamination from Polarstern, as recent work has shown that ship pollution potentially introduces INPs at low temperatures 107. The filter units were open-faced, secured outside to the AOS railing and shielded from precipitation and blowing snow. Vacuum line tubing connected the filter unit to the flow meter followed by vacuum pump, both of which were housed inside the AOS container. Average flow rate was 21.9 l min−1. Samples were started at 14:15 UTC on average and collected continuously for 72 h. Following collection, filters were removed from the single-use units, placed in sterile Petri dishes, and preserved and shipped frozen (≤ −20 °C) until analysis at CSU.Total aerosol samples were collected on top of the AOS container near the inlet from 23 October 2019 to 1 October 2020 using single-use Nalgene™ Sterile Analytical Filter Units that were prepared by replacing their cellulose nitrate filters with 0.2-μm polycarbonate filters backed with 10-μm polycarbonate filters (each are 47 mm diameter Whatman® Nuclepore™ Track-Etched Membranes), both pre-cleaned at CSU113, a technique which is comparable to the cold plate method114. Filters were placed in sterile centrifuge tubes, 8 ml of 0.1 µm-filtered deionized water added, and particles re-suspended by tumbling end-over-end on a rotator for 20 min. From these suspensions, 32 50-μl aliquots were distributed into sterile, 96-well PCR trays in a laminar flow cabinet. Serial dilutions were applied to cover INP concentrations over the full temperature range. Plates were then placed into the blocks of the IS, the device covered with a plexiglass window, and the headspace purged with filtered N2. The device was cooled at 0.33 °C min−1 until ~ –29 °C using a recirculating low temperature bath, and the freezing of wells recorded every 0.5 °C via a CCD camera system. Thermal treatments and peroxide digestions provide valuable insights into INP composition. Heat treatments were performed on approximately one third of the IS filter samples by heating 2 ml of suspension to 95 °C for 20 min to denature heat-labile INPs, such as proteins115. Hydrogen peroxide (H2O2) digestions were performed on a further 2 ml of suspension to remove all bio-organic material. This procedure, and the neutralization of remnant H2O2 to prevent freezing point depression112. The samples were then re-analyzed to assess the reduction caused by the decomposition of heat-labile and all organic INPs, respectively.Samples were processed using the CSU Ice Spectrometer (IS)116:f is the proportion of droplets frozen, Vdrop is the volume of each drop, Vsuspension is the volume of the suspension, and Vair is the volume of air per sample. See Supplementary Fig. 117. Treatment results for select temperatures throughout the MOSAiC expedition are shown in Supplementary Fig. 119.For both techniques, the fraction of drops frozen at each temperature interval and the known total volume of air sampled were used to calculate INP concentration with a universally-used equationSupplementary InformationPeer Review File


If combined with a prior information on the paleowater stable isotope composition, further insights can be obtained on the temperature and the source of the paleowater. Clumped isotope composition (Δ47) of carbonates is an independent measurement of temperature, and if combined with the conventional stable isotopes, can provide information on the paleowater stable isotope composition. On the example of Provalata Cave (N. Macedonia), we apply for the first time, both conventional and clumped stable isotope analysis, and identify two different isotope alteration trends, reflecting two distinct hydrothermal events: an older, hotter one, where isotope alteration was likely related to isotope diffusion, lowering the δ18O values of the carbonate; and a younger one, related to the cave formation by low-temperature CO2-rich thermal waters, with dissolution-reprecipitation as the alteration mechanism, causing decrease in δ18O values, and unexpected increase in δ13C values. The findings are further corroborated by additional insight from optical petrography and cathodoluminescence microscopy, as well as fluid inclusion analysis of secondary calcite crystals related to the cave forming phase.Alteration of conventional carbonate stable isotopes (δ Hydrothermal caves are formed by thermal waters that have generally higher CO2 concentrations, and dissolve carbonate bedrock as they cool down along their rising flow path due to inverse relationship of calcite solubility and temperature4. At shallower levels of the aquifer, decrease in pressure facilitates CO2 degassing, leading to supersaturation and precipitation of calcite5. They are generally strongly structure controlled and develop a characteristic suite of cave morphologies and deposits7.Hydrothermal caves are one of the main genetic type of caves, a sub-type of hypogene caves, whose origin is related to deep-seated processes (source of acidity and recharge to the soluble rock formation is from depth), contrary to the most commonly found caves whose origin is connected to surface-related processes 9, most of the known hydrothermal caves are fossil, and became accessible only after being intercepted by surface erosion10. Identification of characteristic morphologies has been the most common approach to identify the hydrothermal origin of caves11, however equifinality of some morphological features  makes this approach not resolutive. Study of secondary minerals has been proven to be a more reliable approach, with microthermometry and stable isotope composition used to confirm hydrothermal origin and identify the sources of fluids as well as their formation temperature16.While some active thermal caves are accessible and offer possibility to study some of the related processes occurring in shallower conditions14, unless their relationship to the cave origin can be unambiguously demonstrated4. Likewise, the absence of hydrothermal minerals does not exclude a hydrothermal origin of a cave, as they might not precipitate at all, and later modification in shallower settings can modify and overprint the primary hydrothermal features. Isotope alteration of bedrock, an approach developed for and most commonly used in hydrothermal ore-related studies19, has also been successfully applied to cave wall bedrock. Recent studies, based on alteration of conventional carbonate stable isotope compositions  in cave walls, have demonstrated a hypogene origin in the presence or absence of related cave minerals21. Furthermore, it has been found that alteration halos do not develop in epigene caves22 and this approach can be used to confirm or dismiss hypogene-related cave origin23. The most commonly identified alteration in cave walls is lowering of the bedrock δ18O values, as a result of the interaction with low-δ18O high-temperature meteoric waters, as well as lowering of bedrock δ13C values due to interaction with dissolved inorganic carbon (DIC) with lower δ13C values22.Although secondary minerals might have been deposited from thermal waters, their hydrothermal origin is not a proof of the primary hydrothermal origin of the cave24 allow an independent estimate of the mineral formation temperature, that, if combined with the conventional oxygen isotope composition, can be used to reconstruct the paleofluid oxygen composition25. Carbonate clumped isotope thermometry has found wide applications, such as in reconstructing metamorphic and exhumation histories of marble formations27, sediment diagenesis29, as well as speleothem studies32. The application of clumped isotope thermometry to subaerially formed speleothems has proven challenging, due to disequilibrium effects related to various controls , leading to overestimation of formation temperatures34. However, subaqueous speleothems, especially slowly precipitating ones, were found most likely to have precipitated in equilibrium, thus providing more reliable results37.Recent developments in carbonate clumped isotope thermometry22 by applying both clumped and conventional stable isotope analyses on carbonates. On the example of Provalata Cave (N. Macedonia), we show for the first time, that the characteristics of two distinct hydrothermal events causing different cave wall isotope alterations can be identified, the second one responsible for cave formation. We support our findings by additional insight obtained by optical petrography and cathodoluminescence microscopy, as well as fluid inclusion analysis of secondary calcite minerals, and reconstruct the subsequent evolution of the cave.Here we expand on the cave wall isotope alteration approach38 , that was active at ca. 1.6 Ma, left abundant gypsum deposits formed as a replacement of carbonate bedrock and calcite spar40. The calcite spar is found as coatings, up to 0.5 m thick, that cap the hydrothermal carbonic phase.Hypogene karst in Mariovo (N. Macedonia) is found at several localities, at the intersection of low topography and major faults, and is developed mainly in calcite and dolomite marble, that are part of NNW-SSE oriented nappes along the eastern part the Pelagonian massif41 (C2 and C3) that cover cave wall bedrock and calcite coatings, and two additional samples cut from calcite coatings covering the cave wall (PROV03 and PR20). Furthermore, we collected samples along the NNW-SSE nappes to better characterize the primary isotopic composition of the calcite marble. Details of the sampled material are given in the Supplementary Information.From Provalata Cave we collected two wall-drilled coresThe two cores, C2 and C3, have length of ~ 30 and ~ 6 cm, with calcite marble representing ~ 15 and ~ 4 cm, respectively. In addition to primary grey-colored marble, two distinct types of color alteration are visible: a narrow rim (< 2 cm) with intense white discoloration at the contact of the cave wall and the overlying calcite coating, and a dispersed pale grey discoloration in the inner part Fig. . The thi18Occ) and carbon (δ13Ccc) isotope values that are within the range of values obtained for samples along the NNW-SSE nappes . The white narrow rim shows change in both δ13Ccc and δ18Occ, with Δδ18O of − 6.7‰ to − 8.3‰ and Δδ13C of + 3.3‰ to + 3.2‰, for C2 and C3, respectively, with values trending towards the isotopic composition of the overlying calcite coatings , reflecting predominantly organic-derived carbon45  and very high δ13Ccc (mean + 7.3‰), with a relatively small range of values , reflecting highest apparent temperatures (mean 206 ± 22 °C)  closure temperatures, and FT ages from the eastern part of the Pelagonian massif relate them to Late Cretaceous-Paleocene thrusting49. The altered sections have increased Δ47 values showing two distinct clumped isotope compositions, that reflect mean apparent temperatures of 147 ± 23 °C in the pale grey section and 54 ± 13 °C in the white rim (Table 13Ccc), imply that the two alterations were related to two distinct hydrothermal events.The clumped isotope compositions follow a similar pattern. The grey calcite marble has the lowest Δ47 values, reflecting apparent temperatures of 11–34 °C  of the fluid in equilibrium with calcite for all of the identified sections, using the calcite-water oxygen equilibrium equation31, the measured δ18Occ and the clumped isotope-based apparent temperature . The unaltered calcite marble has δ18Ow values that are within the range for metamorphic fluids50. However, as the apparent temperatures of the unaltered marble represent equilibrium blocking temperatures, their calculated δ18Ow values are not considered as representative. The pale grey section has lower δ18Ow values, which are within the range for magmatic fluids50. The secondary calcite minerals have the lowest values, reflecting largely meteoric water composition, while the narrow rim has intermediate δ18Ow values. The apparent temperatures for the pale grey section are within the range of temperatures (100 to 200 °C) of the ore mineralization at the nearby Carlin-type-like Allchar deposit51  character of this section as also indicated by the isotopic composition. The similar luminescence of the small calcite crystals in the narrow rim to the one of the overlying calcite coatings suggests deposition from the same (or similar) fluid. This is also supported by the white rim δ13Ccc and δ18Occ values, as they are shifted towards the composition of the overlying calcite coating. A continuous transition between blue, violet and orange cathodoluminescence colors can occur due to the relative and absolute intensity of the intrinsic and orange luminescence55. However, in the absence of additional elemental analyses, it is not possible to gain more insight from the cathodoluminescence on the conditions under which calcite precipitation or alteration occurred.The blue luminescence in the grey marble reflects intrinsic luminescence57. The exchange might also first proceed by dissolution-reprecipitation until chemical equilibrium is achieved and then by diffusion58. Rate of diffusion of carbon and oxygen in minerals is strongly temperature-controlled57, but in the presence of water, oxygen diffusion is greatly increased, while carbon diffusion is largely unaffected61. The alteration of oxygen isotopes with no change in carbon isotopes in the pale grey section, as well as the variation in cathodoluminescence towards the crystal surface, suggest hydrothermal alteration by diffusion. Experimental studies on oxygen isotope diffusion in calcite62 suggest that the water effect to oxygen diffusion in calcite is primarily a surface process, facilitating oxygen exchange between calcite surface and fluids. However, while water enhances oxygen isotope diffusion at the calcite crystal surface, to alter the clumped isotope composition, reordering of C-O bonds requires bulk mineral increase in oxygen diffusivity63, which is achieved only at higher temperatures and longer times47. This implies that dissolution-reprecipitation is the more likely process to explain the alteration of the clumped isotope composition at lower temperatures. Additionally, a combination of increased diffusivity, dissolution-reprecipitation, as well as pressure-induced structural changes can increase the reordering rates62. Although our stable isotope data is not detailed enough to investigate variation along a single crystal, the luminescence halo seen along boundaries of some crystal grains, and no apparent sign of dissolution-reprecipitation along the crystals, support the oxygen diffusion interpretation. Thus, the variation of δ18Occ and Δ47 in the pale grey section most likely reflects the degree of isotopic exchange that occurred between the hydrothermal fluid and the calcite crystals, with alteration progressing further in smaller size crystals and crystals with defects, while creating a halo in larger crystals. The lowest apparent temperature in the pale grey section (~ 110 °C) is found in the sample with the lowest δ18Occ values, that likely reflects the temperature of the hydrothermal fluid.Carbon and oxygen isotopes in calcite can exchange with fluids by either dissolution-reprecipitation, when mineral and fluid are far from chemical equilibrium, or by diffusion, when mineral and fluid are at or close to chemical equilibrium18Occ − δ13Ccc, Δ47 − δ13Ccc, and Δ47 − δ18Occ space  clearly point to dissolution-reprecipitation as the alteration mechanism, where previously formed porosity is filled with secondary calcite that continues further from the cave wall as calcite coating. Thus, the stable isotope composition along the white rim reflects the frequency of secondary calcite minerals, i.e., the linear trends seen in δace Fig.  reflect 18Ow values ranging between − 13.9‰ and − 11.6‰ and δ2Hw values between − 87.0‰ and − 76.0‰  inclusions, mostly as large size inclusions in palisade crystals, but also as clouds of small ‘thorn’ shaped inclusions  of 0.37 to 0.50 and 4He/20Ne values of 1.5 to 2.4. The three-component  helium mixing model , the calcite-water oxygen isotope fractionation was calculated , indicating that the δ18Ow values are a result of mixing of two fluids: one cold (< 10 °C) with low δ18Ow (< − 13‰) and another hot (> 35 °C) with higher δ18Ow (> − 9.5‰). The cold one clearly represents a relatively shallow, meteoric water, while the hot one indicates a deep-seated origin. Magmatic waters have δ18Ow in the range of + 6‰ to + 10‰ and metamorphic waters in the range of + 5‰ to + 25‰50, thus based on the δ18Ow/T slope seen in the samples, and considering magmatic or metamorphic origin, the estimated temperatures of the hot component are between 100 and 200 °C, respectively.The calcite coatings in Provalata Cave show small variation in their δope Fig. . For the18Occ − δ13Ccc space cannot be explained by calcite precipitation due to mixing of two fluids. First, the two fluids will need to be highly saturated in calcite, as mixing of two fluids saturated in calcite at different equilibrium concentrations of Ca2+ produces a mixture with lower calcite saturation due to the curvature of the calcite equilibrium line, that will lead to calcite dissolution 72. Second, the very small change observed in the δ13Ccc values would require two fluids of such contrasting origin, such as meteoric and deep-seated, to have similar carbon isotopic composition, which is highly unlikely. Furthermore, while mixing of two fluids with similar isotopic composition and contrasting temperatures can produce correlation in δ18Occ − δ13Ccc space, it is unlikely that two fluids will have similar isotopic composition, indicating similar origin, but have contrasting temperature73. Thus, it is more reasonable to assume calcite precipitation from one fluid under a temperature effect, then due to mixing of two fluids73. This implies that the water mixing occurred prior to calcite precipitation, and likely contributed to the cave passage formation by mixing corrosion.While the water composition is clearly a mixture of two components, the positive correlation in δ13Ccc for a DIC dominated by a meteoric (soil) sourced carbon. The δ13Ccc depends on the δ13CDIC, T and pH . For such high δ13Ccc value (+ 7.9‰) at low temperature (12 °C), if the calcite precipitates in isotopic equilibrium from fluid with pH > 6, both δ13CDIC and δ13C of the CO2 (δ13Cext) should be higher than 0‰, suggesting a deep-seated origin of the CO2. At pH above 7 the δ13CDIC should be >  + 5.4‰, and δ13Cext >  + 7.5‰, with more than 41% of the carbon in DIC coming from carbonate dissolution (see Supplementary Information). However, in such case the alkalinity will be low , the deep-seated CO2 must have even higher δ13C, suggesting that the combination of higher pH with such high δ13CDIC values is not likely. Thus, it is most likely that the onset of calcite deposition at C2 was from low temperature, slightly acidic fluid, where the water was of meteoric origin, but the CO2 was mostly of deep-seated origin. As the subsequent mixing with deep-seated hotter fluid occurred prior to calcite precipitation, this supports the interpretation46 that meteoric and deep-seated fluids converged at depth along a fault structure with dominantly metamorphic CO2 flux.The calcite that marks the onset of deposition at C2 core has very high δ13Ccc values can be modeled in terms of change in temperature with or without CO2 degassing75. The modeled curves show characteristic slopes of T-δ13Ccc for a range of H2CO3 fractions (fH2CO3) in DIC , or a combination. These samples show also larger shift in δ18Ow (< 2‰). While increase in pH has no direct effect on oxygen isotopes, concurrent increase in pH and δ18Ow can occur by increased water–rock interaction. However, this is unlikely as the low temperature setting does not favor such large water–rock oxygen isotope exchange.Since calcite precipitation is accompanied with temperature change in our samples Figs. , 2, 6, t13CDIC of the deep component is a possible explanation , but increase in mantle-derived fluids cannot explain the observed increase in δ18Ow, and noble gas fluid inclusion data show minor and relatively constant mantle He component. Variation in the δ18Ow of the shallow component is very likely, and such increase in values can be due to climate variations . Although the effect of δ18Ow change in the shallow component can be seen in the mixture δ18Ow, the δ13CDIC change in the shallow component  will be overprinted by the large fraction of deep-derived CO2  is seen in the mixture δ18Ow but generally masked in the mixture δ13CDIC by the high amount of deep-sourced CO2. Thus, the offset from the positive δ18Occ - δ13Ccc covariation in Provalata calcite reflects a phase of lowering of deep CO2 contribution.In other words, climatically controlled variation in the isotopic composition of the cold end-member . The second alteration , is related to the cave-forming phase, when convectional features developed along high-angle fractures of a NW–SE oriented fault zone39, related to Late Miocene-Quaternary extension49. Cave passages formed by dissolution and enlargement of fractures during cooling of rising low-temperature (< 35 °C) thermal waters (of mixed meteoric and deep-seated origin), rich in metamorphic CO2, likely enhanced by mixing corrosion. As dissolution progressed perpendicular to cave walls, porosity was increased in the first few centimeters. Cooling continued, but the system shifted to shallower settings, forcing CO2 degassing and calcite precipitation, first filling up pore spaces, and progressing into thick calcite coatings. The thermal evolution and δ13Ccc values of the coatings suggest that after the initial cooling, another heat pulse followed, and variation in pCO2 controlled the variation in δ13Ccc. The calcite deposition took place between 2.5 Ma and 1.6 Ma and is likely connected to the youngest volcanic activity of the Kožuf-Voras system (3.0–1.8 Ma), focused in its southwestern part54, close to the study site  of magmatic or mixed magmatic-meteoric origin Fig. . It was We show that by combined use of conventional and clumped stable isotope analysis of carbonates in the study of cave wall alteration by thermal waters we can better constrain the character of the isotopic alteration, as well as unambiguously identify different alteration events.18Occ values likely due to diffusion-controlled isotope exchange with fluid at or close to chemical equilibrium with the rock, at higher temperature (~ 110 °C). The second one was related to cave formation by fluid flow along a fracture, i.e., progressed perpendicularly inward from the fracture , and proceeded with dissolution-reprecipitation-controlled alteration by lower-temperature fluids (< 35 °C) that transitioned from being calcite-undersaturated (dissolution) to calcite-saturated (precipitation).On the example of Provalata Cave we demonstrate the primary isotopic character of the unaltered calcite marble, and identify two distinct hydrothermal phases causing different alterations. The older one was pervasive with flow along smaller structures and crystal boundaries, causing lowering of δ18Occ and either unchanged or lowered δ13Ccc, our findings show a rare example of isotope alteration with lowered δ18Occ and significantly increased δ13Ccc. We expect that such case is not a unique one and can be expected at sites with high metamorphic CO2 flux. We also show a non-linear thermal evolution of the hydrothermal cave system, suggesting that simple cooling, as usually assumed for such systems, is likely simplifying the evolution, especially for karst systems related to volcanic areas.While most of the examples of cave wall isotope alteration reported in the literature show lowering of δConventional and clumped stable isotope analyses of carbonates were carried out at the Isotope Climatology and Environmental Research Centre (ICER), Institute for Nuclear Research (ATOMKI), Debrecen.PLUS XP isotope ratio mass spectrometer (IRMS)78. Conventional stable isotope results are expressed as δ18O and δ13C values relative to Vienna Pee-Dee Belemnite (VPDB). The precision of the measurements is ≤ 0.08‰ for δ13C and ≤ 0.1‰ for δ18O.Conventional carbon and oxygen stable isotope analyses were carried out on hand-drilled carbonate powders with an automated GASBENCH II sample preparation device (phosphoric acid digestion at 72 °C) attached to a Thermo Finnigan Delta80. Data evaluation was done on the Easotope application81, with the CO2 clumped ETH PBL replicate analyses method and IUPAC parameters85. Δ47 results are given in the I-CDES90 scale80, and apparent temperatures in °C were calculated based on the Δ47-temperature calibration from Anderson et al.86, with temperature uncertainties propagated from the 1σ standard error (SE) of the Δ47 value. Simultaneously during clumped isotope analysis, on the same samples, conventional carbonate stable isotope composition was also determined.Clumped isotope analysis was done on a Thermo Scientific 253 Plus IRMS, after phosphoric acid digestion at 70 °C using a Thermo Scientific Kiel IV automatic carbonate device. Each carbonate sample measurement consisted of at least 10 replicate analyses of 100–120 μg aliquots that were measured alongside carbonate standard samples  with assigned valuesDouble-polished sections for petrographic observations were prepared employing low-speed precision sawing and polishing, which minimize thermal and mechanical stresses on the samples. Fluid inclusion observations of secondary calcite were carried out on a transmitted-light microscope (Nikon Eclipse E 400 POL) at the Institute of Geology, University of Innsbruck and petrographic observations of calcite marble from cave walls were carried out on a transmitted-light microscope (Olympus BX53 equipped with Olympus DP23 camera) at ICER, ATOMKI, Debrecen. Additionally, cathodoluminescence studies were carried out at the Karst Research Institute ZRC SAZU, Postojna, on a Technosyn® cold cathodoluminescence luminoscope (CITL CL8200 MK4) with a 14 kV electron beam and an electron current of 350–400 μA, mounted on a Nikon Eclipse E600 biological petrographic microscope. The thin sections, examined in transmitted light and cathodoluminescent light, were photographed using a Nikon Coolpix 990 digital camera.3He, 4He, 20Ne, 22Ne) was determined at ICER, ATOMKI.Fluid inclusion analyses were carried out at three laboratories. Water stable isotope composition was determined at the Institute of Geology, University of Innsbruck, and at the Institute for Geological and Geochemical Research (IGGR), Budapest. Noble gas isotope composition  and pyrolyzed into H2 and CO at 1400 °C. The evolved gases were separated in a chromatographic column and analyzed using a Thermo Fisher Delta V Advantage IRMS. Detailed description of the method can be found in Dublyansky and Spötl88. The remaining samples were analyzed at the IGGR in Budapest. Sample chips of about 0.5–1 g were crushed under vacuum in stainless steel tubes, after which the released water was purified by vacuum distillation and introduced into a Los Gatos liquid water isotope analyzer (LWIA-24d). Details of the method are described in Demény et al.89. Results are expressed as δ2H and δ18O relative to Vienna Standard Mean Ocean Water (VSMOW). Analytical precision is 0.5‰ for δ18O and better than 1.5‰ (Innsbruck) or 2‰ (IGGR) for δ2H.At Innsbruck sample PR20 was crushed in a heated crushing cell90 and Molnár et al.91.For noble gas isotope analyses, 1.3 to 2.3 g of calcite samples were loaded into stainless-steel holders with a magnetic ball and baked at ~ 60 °C for 10–12 h in vacuum before the measurements. Gas was extracted by single-step crushing (~ 100 strokes) at room temperature (22 °C). The relatively low number of strokes was applied for each measurement in order to avoid significant contribution of an in-situ component. Helium isotope abundances and ratios were determined by a HELIX- SFT mass spectrometer, whereas a VG-5400 mass spectrometer was used for neon. The analytical procedures are described in more detail in Papp et al.3, doped with a 229Th, 233U, and 236U mixed spike, and cleaned and separated U and Th in anion exchange resin column chemistry following the methods of Asmerom et al.92. Measurements were made on a Thermo-Neptune multicollector inductively coupled plasma mass spectrometer at the Department of Earth and Planetary Sciences, University of New Mexico. U and Th were measured separately as static runs where all isotopes are measured in faraday cups except for 230Th and 234U, which were measured in a secondary electron multiplier. Efficiency between the cups and multiplier were determined using an in-house Th standard and the U standard NBL-112. Decay constants and a detailed description of the uranium-series methods are in Cheng et al.93. The measured δ234U value for standard NBL-112 (CRM-112a) is -38.5‰, where δ234U = [(234UA/238UA) – 1] × 1000, and A refers to activity.Three small pieces of densely crystalline calcite coating (core C2) were dissolved in 15 N HNO13Ccc values in terms of calcite precipitation due to change in temperature with or without CO2 degassing, we used the equation of Zheng75 with a modification, that instead of considering two extreme cases of HCO3-dominant or H2CO3-dominant fluid, for which only fractionation of calcite-HCO3 or calcite-H2CO3 is used, respectively, we select a set of fractions of H2CO3 in the DIC of the fluid (where DIC = fH2CO3 + (1-f) × HCO3), that itself reflects fluid pH at a given T. For a selected δ13CDIC, range of temperatures and fH2CO3, first we calculate the δ13C of the CO2 in equilibrium with the fluid (δ13CCO2) and then we calculate δ13Ccc values using the Rayleigh model equation of Zheng75.To model the δ13Ccc as found for the onset of calcite deposition in C2 core, first we calculate the fractions of H2CO3 and HCO3 in PHREEQC Version 3 software94 for a range of pH values, with a fixed temperature and calcium and DIC concentrations determined by forcing charge balance to the solution at fixed calcite saturation index. Then we calculate the δ13CCO2 and δ13CDIC, and using the modeled chemical composition for dissolution of only calcite or calcite and dolomite, we determine the isotopic composition of the carbon from CO2 not accounted for by dissolution of carbonate rocks .To model the fluid composition from which calcite can precipitate in isotopic equilibrium recording temperature and δDetailed explanation of the calculations is given in the Supplementary Information.Consent was obtained from the identifiable person in Fig. S4b (Marjan Temovski) to publish image in an online open access publication.Supplementary Information.


The fine regulation of actin polymerization is essential to control cell motility and architecture and to perform essential cellular functions. Formins are key regulators of actin filament assembly, known to processively elongate filament barbed ends and increase their polymerization rate. Different models have been extrapolated to describe the molecular mechanism governing the processive motion of formin FH2 domains at polymerizing barbed ends. Using negative stain electron microscopy, we directly identified for the first time two conformations of the mDia1 formin FH2 domains in interaction with the barbed ends of actin filaments. These conformations agree with the speculated open and closed conformations of the “stair-stepping” model. We observed the FH2 dimers to be in the open conformation for 79% of the data, interacting with the two terminal actin subunits of the barbed end while they interact with three actin subunits in the closed conformation. In addition, we identified and characterized the structure of single FH2 dimers encircling the core of actin filaments, and reveal their ability to spontaneously depart from barbed ends. The actin cytoskeleton is essential to ensure cell motility, to regulate cell shape and architecture, and to control membrane reshaping during cell division . A largeMost formins gather several functional domains: a RhoGTPase-binding domain (GBD), a diaphanous inhibitory domain (DID), a dimerization domain (DD), two central formin homology domains (FH1 and FH2), and a diaphanous autoinhibitory domain (DAD). FH1 and FH2 are ubiquitous and are key to ensuring the main functions attributed to formins. The FH2 domains dimerize in a head-to-tail manner and encircle filament barbed ends. A “post” domain is located at the C-terminal end of each FH2 domain, while a “knob,” a flexible “linker,” and a “lasso” domain are located toward its N-terminus. The “lasso” domain of a given FH2 interacts with the “post” domain of its counterpart to induce FH2 dimerization. The “linker” domain is flexible and was shown to be either unstructured or able to adopt different secondary structures such as α-helices or β sheets . ResidueThe molecular mechanism governing the tracking of the actin barbed ends by the FH2 domains remains elusive. Two antagonistic models have emerged to describe formin processivity at barbed ends, based on indirect evidence from x-ray crystallography of formin–actin complexes in nonnative states  have proposed an alternative model, named “stepping-second” because the translocation of the FH2 dimer, the “stepping,” is supposedly triggered by the incorporation of a novel actin subunit. In that model, the FH2 domains remain in the direct vicinity of the three terminal actin subunits at the barbed end, encircling them continuously. The terminal actin subunits are in rapid equilibrium between a closed conformation, with a helical twist near 180°, and an open conformation, with a more canonical 167° helical twist. These conformational changes are accompanied by FH2–actin contact modulations and by the stretching of the FH2 linkers. Upon addition of a new actin monomer, the canonical 167° helical twist would be consolidated, thereby straining durably the FH2 linkers. The translocation of one FH2 domain toward the barbed end would relax this strain. After translocation, the barbedend helical twist would again be in rapid equilibrium between 167° and 180°.The primary purpose of our investigations was to discriminate between these two models by using electron microscopy (EM) to directly observe the conformations of the formin mDia1 FH2 dimer at the filament barbed end. To achieve this, we have optimized the sample preparation to enhance the density of single short filaments  and adapted the image processing workflow to the processing of our filamentous ends. Indeed, only a few reports describe the structure of proteins at either the barbed or the pointed ends of actin filaments, and they involve proteins that stably cap the ends and thus neutralize the actin filament dynamics  and 3 thMaterials and Methods. Sonication was carried out on 1 μM rabbit α-skeletal preformed actin filaments in F-buffer at pH 7.8 with 50 mM KCl in order to shorten them before exposing them to formin mDia1 . Sonication has been reported to induce the depolymerization of actin filaments (F-actin), in addition to their severing  performed on globular proteins, our objects of interest are circumscribed to the ends of elongated asymmetrical assemblies whose upstream bodies extend beyond the periphery of the extracted boxes. Raw boxed particles and an example of filamentous ends displaying an additional density are presented in From the resulting 2D analysis, three distinct groups can be identified. From the first round of classification, most of the generated classes correspond to bare actin filament ends . These cTo characterize the possible different conformations in 3D, we have performed multireference 3D classifications and reconstructions merging the two sets of particles displaying additional density at actin filament ends within the 2D analysis . After sample cryo-fixation, we collected data using a 200 kV field emission gun (FEG) Glacios microscope. Typical images are displayed in Supplemental Figure 5. From 3405 images, 12,112 ends were manually picked . Given that the signal-to-noise ratio and the density of ends are lower for cryo-EM images, the final resolution obtained from these data was unfortunately not better than what we obtained with negative stain images. Nonetheless, in 2D classes, additional densities can be pinpointed at the ends of filaments . Owing to the limited number of ends observed, we identified primarily one conformation of formin bound to the barbed ends. From 3D reconstruction and classification, one main structure was generated . Actin subunits as well as formin FH2 domains were docked within this structure. The FH2 dimers connect only the two terminal actin subunits of the barbed end with a free and accessible post domain, a recognizable feature of the open conformation.To ensure that formins were also visible at the barbed ends of actin filaments in a more physiological condition and were not artifacts from staining and drying by negative stain EM, we carried out cryo-EM experiments. To enhance the density of filaments on the cryo-grids, we used C-flat grids coated with a layer of graphene as described by Materials and Methods and Supplemental Figure 7). Four classes were obtained displaying three different orientations of the leading FH2 domain .Nonetheless, we reconstructed a 30 Å resolution structure where the FH2 domains as well as the actin subunits can be pinpointed and on which an FH2 dimer can be docked encircling an actin filament. The low resolution of the reconstruction is sufficient to show that, alongside actin filaments, the relative arrangement of the FH2 domains and actin is comparable to the ones observed at the actin barbed ends. Our observations thus suggest that FH2 dimers can diffuse along actin filaments by interacting with specific binding sites. We estimated that we could identify on average ∼1 formin per 4 μm of actin filament and that ∼8% of the observed filaments had a formin along their core  for an mDia1-bearing barbed end in the open state than for a bare barbed end. We hypothesize that the available actin-binding interface on the leading FH2 domain could provide a first docking intermediate for actin monomers that would help their orientation relative to the barbed end, resulting in a higher on-rate.We also reveal that, in the open state, the orientation of the leading FH2 relative to the long axis of the filament fluctuates . SeveralWe propose a “flapping” model where the angle between an FH2 domain and actin varies from 108° to 135° . We desiet al.Our observation of formin mDia1 along the core of actin filaments  formins et al.The flapping of the FH2 domains of mDia1 that we described earlier could provide an explanation for this observation. In the extreme open conformation, the addition of actin subunits at the barbed end could leave the FH2 dimer “lagging” behind and no longer translocating synchronously with the elongating barbed end . Lagging formins would then start diffusing along the actin core, in both directions, as described by Bombardier et al., 2015); the formin-bearing barbed end could bind to the pointed end of another filament ; finally, a formin could access the core of the filament via the filament pointed end.Other scenarios could be proposed to account for the presence of formins along the core of actin filaments. We can imagine the following: a formin FH2 dimer could open up and encircle the core of a filament; the formin could be displaced from the barbed end by the arrival of another formin (playing the role of the capping protein in the mechanism proposed by Bombardier n = 50) of actin filaments with a formin within their core also display a formin at their barbed ends. This is significantly less than for the global filament population, where 77 ± 0.4%  of barbed ends are decorated with formins. This does not exclude the other scenarios, but it supports the lagging scenario as a likely mechanism putting formins along the core of the filament.In the lagging scenario, the formin going to the core of the filament leaves behind a bare barbed end that will have a limited time to bind another formin before the sample is fixated for observation. The lagging scenario is thus the only one predicting that filaments with a formin along their core should be less likely to also exhibit a formin at their barbed end, compared with the global filament population. To test this prediction, we randomly picked 38 micrographs in which we found 100 filaments with an observable end and with a formin along their core. Of these 100 filament ends, only 24 had a formin. Considering that half of the 100 ends were barbed ends, we can estimate that 48 ± 7%  on ice.Actin was purified from rabbit muscle as detailed previously  followed by steric exclusion chromatography (HiLoad 16/60 Superdex 200) as developed in Recombinant mouse mDia1(SNAP-FH1FH2-DAD-6xHis) formin  was expressed in 2, 0.2 mM EGTA (Tetra[acetoxymethyl Ester]), 0.2 mM ATP, 1 mM DTT, 50 mM KCl) to reach steady state. A total volume of 500 μl containing the polymerized F-actin diluted to 1 µM was sonicated in F-buffer. The sonication was operated with a sonicator Vibra-Cell 75041  equipped with a 3-mm-diameter probe. Ten pulsations of 0.1 s separated by a 3.9 or a 4.9 s rest period were used for the sonication. Sonicated solution (200–300 μl) was then inserted into quartz cuvettes and analyzed for fluorescence signal for at least 20 min using a spectrofluorimeter . The average time from the end of the sonication process to the pyrene fluorescence measurement was 30 s. As a control, the fluorescence of 1 µM G-actin and 1 µM actin at steady state (without sonication) was also assayed.Actin  was polymerized at room temperature for 1 h in F-Buffer  = CG(t) iG + CF(t) iF, where CG is the G-actin concentration, CF is the F-actin concentration, iG is the fluorescence intensity of 1 µM G-actin, and iF is the fluorescence intensity of 1 µM F-actin in the same conditions . At any time, CG(t) + CF(t) is constant and equal to the total concentration of actin in the sample. iG is directly determined by measuring the signal from a solution of 1 µM G-actin in G-buffer. iF is determined by measuring the signal from a solution of 1 µM actin at steady state and considering that the steady-state concentration of G-actin is 0.15 µM.To deduce the concentration of G-actin from the pyrene fluorescence intensity in our samples, we proceeded as follows. At any time g before use to remove any aggregates. Actin (5 μM) was polymerized at room temperature for 1 h in F-Buffer . To generate a high density of short actin filaments, 500 μl of polymerized 1 µM F-actin was sonicated in F-buffer. The sonication was operated with a sonicator Vibra-Cell 75041  equipped with a 3-mm-diameter probe. Ten pulsations of 0.1 s separated by 3.9 s rest periods were used for the sonication. Formin mDia1(SNAP-FH1FH2-DAD-His) was added in the F-actin sonicated mixture directly after the last pulsation, through a Hamilton syringe equipped with a 0.13-mm-internal-diameter needle. A formin concentration of 100 nM was used for negative stain EM experiments, and a formin concentration of 125 nM was used for cryo-EM experiments.G-actin stock solution was ultracentrifuged for 45 min at 80,000 × Twenty seconds after formin addition, 4 μl of sonicated F-actin/formin mixture was collected with a Hamilton syringe. The collected volume was directly applied to an EM grid.For negative stain EM experiments, the solution was adsorbed 30 s on a freshly glow-discharged 300 mesh carbon-coated copper grid. Excess volume was blotted off with a filter paper (Whatman no. 1). Uranyl formiate 1% (4 μl) was transiently added before being blotted off the grid. A volume of 4 μl of rinsing uranyl formiate 1% was deposited on the grid for 30 s before drying with a paper filter (Whatman no. 1).As shown in Supplemental Figure 1B, short filaments of a few hundred nanometers were generated. On average, 13 ends were obtained per image of ∼1 µm² (average over 1632 images). In parallel, we assessed whether generating a density of filaments by using spectrin–actin seeds would sufficiently enhance the density of short filaments at short times. However, the resulting density of short actin filaments remained low and the presence of spectrin–actin seeds that were not generating filaments induced an additional background signal that was deleterious to SPA.For cryo-EM experiments, C-Flat R 2/2 grids were covered by graphene oxide sheets by depositing 0.2 mg/ml aqueous graphene oxide drops. The grids were used after an overnight drying step as already described (The sonicated actin-F/formin mixture (4 μl) was incubated for 30 s on these freshly glow-discharged grids. The excess volume was blotted off with a filter paper (Whatman no. 1) for 3 s before the grids were plunge-frozen in liquid ethane for vitrification. These operations were carried out using an automated plunge freezing apparatus (Leica EM GP) operated at 80% humidity . A total of 4223 movies were acquired.Cryo-EM data were collected using a Glacios 200 kV (Thermo Fisher) transmission electron microscope equipped with an FEG operating at a 200 kV acceleration voltage. Images were captured using a Falcon 3 direct detection camera at 60,000× magnification and 1–3 μm underfocus. The corresponding pixel size was 2.5 Å per pixel. Each image acquisition was performed in dose-fractionated mode with 60 frames over 2.2 s exposition time for a total dose of 60 eThe actin filament length density observed per image was estimated to extrapolate the actin filament length imaged within the data. The actin filament length in our images was then compared with the density of formins identified along the actin filament bodies to assess the density of formin per actin filament length. The actin filament length was measured manually in 10 images and compared with the measurements resulting from a semiautomated approach on the same images . An error of ∼5% was estimated between the two approaches. The semiautomated approach was applied to 32 images with a mean length of actin filament of ∼4 μm per image. This measurement extrapolated to all the images used for the picking of formins along actin filaments (632 images) led to a total length of actin filament imaged of ∼2566 μm. This length compared with the 717 particles clearly identified as formins encircling the actin filament body led to an estimation of ∼0.3 formin per micrometer of actin filament.2 (2.13 Å/pixel). From 632 of these micrographs, 2013 formin-like patterns were handpicked along the actin filament cores.From the 1632 micrographs obtained by negative staining EM, 20,919 actin filament ends were handpicked using EMAN2  softwareA protocol was adapted to the specificity of our data. Dedicated scripts were generated within SPIDER  with the reference was also evaluated. In this step, either the raw particle or its mirror particle was kept by selecting the one with the highest alignment score. On this occasion, all the references have been manually oriented in a given direction. This step allows the use of a more focused asymmetric mask around the reference averages and brings together a larger number of particles in the same classes, thus improving the associated signal-to-noise ratios. Given that the number of formin-capped ends that could be identified in the data set will be a minority, this step appears to be important to strengthen their signal.In the seventh and last SPIDER analysis step, a principal components analysis was performed to roughly distinguish between formin-bound and bare actin filaments. The more homogeneous data sets considered to be formin bound were then subjected to a second round of 2D SPIDER analysis to benefit this time from the preponderant signal of the formins in the new particle subselection.These aligned data sets were subjected to classical 3D analysis under RELION  v3 in orThe 2D classification focused on the first FH2 domain orientation variability among the actin filament ends bound by formin in the “open state” was performed with SPIDER. From 3804 particles, four classes were generated with, respectively, 956 particles (25%), 1092 particles (29%), 651 particles (17%), and 1104 particles (29%). Angle measurements associated with the first FH2 domain orientation in each of the four classes were carried out using ImageJ. Before each measurement, the class average observed through its focused mask was thresholded to distinguish background versus protein density. The edge delimiting background and protein density was calculated with the “Find Edges” ImageJ function. This delimitation was refined with the “Skeletonize” ImageJ function. After that, the angle formed by the main axis of the actin filament and the relative orientation of the first FH2 domain was measured.Movies acquired by cryo-EM were processed by the MotionCor2 program to correct electron beam–induced sample motion. Contrast transferred functions (CTF) from motion-corrected micrographs were estimated using the program CTFFIND4. Of 4233 corrected micrographs, 3405 were retained after eliminating images showing too much drift (broken membrane near the imaged region), aggregates of graphene oxide sheets or proteins, or poor confidence of CTF fitting estimation.2 (2.5 Å/pixel). From this data set and using RELION 2D classification, at least 1548 particles could be identified as actin filament ends bound by a formin dimer.A total of 12,112 actin filament ends were handpicked within EMAN2 software and extracted with a square box size of 154 pixelsStructural models of a formin-bound barbed end in its “open state” or his “closed state” and of a formin encircling the actin filament core were generated within UCSF Chimera based on FH2-actin atomic structure (PDB: 1Y64) and actin filament atomic structure (PDB: 5OOE). These low-pass-filtered (50 Å) structural models were used as multireference models for RELION 3D classifications performed on the data sets previously determined by 2D analysis. For each data subset identified after RELION 3D classification, a crude cylinder displaying the adapted dimensions could be used as reference to generate a coherent 3D reconstruction.After RELION 3D classifications, 3694 particles corresponding to formin-bound filament ends in their “open state” were selected and subjected to a 3D refinement, leading to a final 3D envelope at a resolution of 26 Å.After RELION 3D classification, 977 particles corresponding to formin-bound filament ends in their “closed state” were selected and subjected to a 3D refinement, leading to a final 3D envelope at a resolution of 27 Å.From 2013 particles corresponding to a formin dimer encircling an actin filament core, 717 particles were selected after RELION 2D classification and subjected to a 3D refinement, leading to a final 3D envelope at a resolution of 30 Å.For cryo-EM analysis, 2373 particles were selected through RELION 3D classification, leading to a final 3D envelope at a resolution of 30 Å.In each 3D envelope generated, the corresponding atomic model was fitted using the UCSF Chimera function “Fit in map” by allowing sequential fitting of independent domain groups. Spurious noise from EM densities was hidden with the “hide dust” command in UCSF Chimera to facilitate readability.Click here for additional data file.


Roughly 1.5 million people in the United States suffer from aortic valve stenosis (AVS), whereas an estimated 250,000 patients with severe AVS are symptomatic and require clinical intervention. The fact that both prevalence and severity of AVS increase with age (>65 years) implies an underlying mechanism of age-related disease progression.Recent studies by Wu et al−/−) and ADAMTS13 (AD13−/−) and several unique techniques such as noninvasive in vivo imaging integrated with tissue histology to examine the effect of excess valve endothelial cell–released vWF on leaflet platelet adhesion, aortic valve morphology, valve interstitial cell transformation, valve hemodynamics, and load-dependent changes of the left ventricle.Due to the anatomic proximity of AVD and AS and the current understanding of the role of UL-vWF–platelet interaction in AVD, Ozawa et alJACC: Basic to Translational Science, Ozawa et al−/−/AD13−/− mice. Interestingly, a histology study revealed the Mac-2–positive plaques were present in the aortic sinuses and extended into the base of the leaflets in the LDLR−/−/AD13−/−. However, the valve thickening was mainly caused by the increased matrix and cellularity. The matrix expansion was likely caused by collagen and active matrix-remodeling as assessed by trichome and MMP9 stains. The cellular proliferation was likely caused by myofibroblasts transformation as shown by α-smooth cell actin stains. In addition, valve calcification by calcium staining and osteogenic transformation by in situ hybridizations of osteopontin stain were only found in the LDLR−/−AD13−/− mice. To explore the contribution of the adhered platelets, they evaluated the phosphorylated SMAD2 and β-catenin status of the TGFβ1 signaling pathway. The intensity and extent of expression for both phosphorylated SMAD2 and β-catenin were greater in LDLR−/−AD13−/− mice than in other control groups, suggesting the potential mechanism of valve interstitial cell transformation and proliferation.In their elegant study in this issue of In summary, Ozawa et alA couple of clinical implications are raised by this study. First, this study demonstrated that AVS and AVD likely share similar pathophysiology because both AVS and AVD can be reproduced using the same animal model. Second, vWF and platelets are likely the missing links to AVS disease initiation and progression. Lindner et alThe author has reported that he has no relationships relevant to the contents of this paper to disclose.


Glioma is among the cancers with the highest mortality and morbidity. The complex biology and acquired chemoresistance create a continuous need for novel, effective therapies. In a recent report by Wang et al, an elegant bioinformatic analysis revealed potential functions and underlying chemopreventive mechanisms involving G protein inhibitory α subunit 2 (Gαi2), which activates NF-κB, a master transcription factor in the regulation of glioma. Given that NF-κB takes part in positive regulatory feedback loops during glioma development, Gαi2 is an attractive candidate for targeted therapies. This discovery unlocks new avenues towards understanding the biology of gliomagenesis as well as the discovery of novel targeted antiglioma agents. High grade gliomas are among the most aggressive brain malignancies with a significantly low median survival of less than 15 months. With >300.000 new cases and >250.000 deaths, gliomas are considered as one of the deadliest types of cancer per case. The standard therapeutic protocols include surgical resection with concurrent radiotherapy and/or adjuvant chemotherapy. The latest WHO classification includes several molecular markers that reveal a complex biology which explain the acquired chemoresistance. Indeed, there is a continuous need for novel, effective therapies against this devastating disease Heterotrimeric G-proteins  have been established as essential component of intracellular signal transduction with various roles in physiology and pathology. The different roles of each subunit as well as the discrete combination of trimers can lead to distinct physiological outcomes. G-protein coupled signaling is a universal cellular mechanism that mediates responses to a wide array of signaling molecules . G-proteins are primarily diversed based on the Gα subunit. Inhibitory α subunits, such as Gαi2 lead to cAMP depletion, through the inhibition of adenylic cyclase enzyme in vitro experiments and in ex vivo xenografts.In this issue, the role of Gαi2 subunit in glioma has been report by Wang et al., published in International Journal of Biological Sciences The underlying mechanism proposed by the authors involved the activity of NF-κB, a master transcription factor in the regulation of glioma Taken together, available data support that the activation/expression of Gαi2 may contribute towards gliomagenesis through a possible feedback mechanism that involves the activation of NF-κB and Sp1 (Figure Given the nature of central nervous system malignancies and the low median survival of glioma, there is a continuous need for new therapeutic approaches. The influential study by Wang et al. puts Gαi2 in the map of available targets for glioma and suggest its involvement in feedback loops that drive gliomagenesis Figure  5. This


Prolonged dexamethasone (DEX) administration causes skeletal muscle atrophy through induction of both oxidative stress and mitochondrial dysfunction. Lipoxin A4 (LXA4) is a recognized antioxidant but its effect against DEX-induced muscle atrophy has not been studied yet. This study aimed to assess the potential ameliorating effect of LXA4 on DEX-induced muscle atrophy and investigate the possible involvement of the mitochondrial dynamics pathway and the redox state in this effect. Forty male rats were divided into four groups; normal control, LXA4-treated, DEX-treated, and LXA4 plus DEX-treated. At the end of the experiment, LXA4 counteracted the effect of DEX on different parameters including muscle weight, muscle strength, serum creatine kinase activity, malondialdehyde and protein carbonyl contents, Na/K-ATPase and citrate synthase activities, mitochondrial transmembrane potential, mitochondrial transcription factor (TFAM), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), and nuclear factor erythroid 2-related factor 2 (Nrf2). These findings signify the promising therapeutic effect of LXA4 against DEX-induced skeletal muscle atrophy and indicate the possible involvement of LXA4-induced mitochondrial activation in addition to its well-known antioxidant effects. Skeletal muscle atrophy is a pathological condition characterized by loss of muscle mass and decreased muscle strength leading to weakness, fatigue, and impairment of the quality of life . Many diMoreover, high doses and/or prolonged administration of dexamethasone (DEX) induces muscle atrophy and increases morbidity and mortality, which may limit its clinical use . MitochoSeveral compounds have been studied for their protective effects against DEX-induced muscle atrophy, such as growth factors, androgens, taurine, glutamine, and beta-adrenergic agonists , but nonLipoxin A4 (LXA4) is an endogenous lipid mediator synthesized by the action of lipoxygenase enzyme on arachidonic acid. It has been found to improve mitochondrial dysfunction and reduce oxidative stress in gastric ulcer models . HoweverNormal mitochondrial function depends mainly on mitochondrial biogenesis and dynamics. Peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) is the critical regulator of mitochondrial biogenesis. It modulates mitochondrial dynamics via the regulation of gene expression of mitochondrial DNA that encodes intramitochondrial proteins . DexametDexamethasone (ampules) was obtained from the Amriya Pharmaceutical Industries, Egypt. Lipoxin A4 , as well as the components of the phosphate buffer saline and mitochondrial isolation buffer, was purchased from Sigma-Aldrich Co, St. Louis, MO, USA.Forty male Wistar rats weighing 170–200 g were obtained from the official animal supplier of the Faculty of Medicine, Tanta University. The study was performed at the Pharmacology Department, Faculty of Medicine, Tanta University in January 2021. The animals were kept in well-ventilated cages (5 rats per cage) under strict hygienic measures, at 25 ± 2 °C room temperature with 12/12 h light/dark cycles, and they were allowed free access to commercial rodent chow  and tap water. The rats were acclimatized for 2 weeks before the start of the experiment. All procedures were performed during the same time of the day. The rats were randomly divided into four groups  and were treated once daily for 14 days. Group I  received saline subcutaneously in the back of the rat and intraperitoneally in the same doses that the drugs were given to groups II and III . Group IThe four limb hanging test assessed the ability of the rat to produce sustained tension in the limb muscles. The rat was allowed to grasp aluminum mesh with its four paws; then, the mesh was inverted, and the hang period started with all four paws of the rat grasping the mesh and stopped by falling off the animal or reaching a maximum of 120 s holding the wire. Three trials were allowed for each animal with 5 min inter-trial intervals . The resAt the end of the experiment, the overnight fasting rat was weighed to calculate the percentage of change in its body weight. The rat was then euthanized by diethyl ether, and its blood was collected in sterile tubes by cardiac puncture and centrifuged for 10 min at 3000 rpm. Serum creatine kinase (CK) activity was assayed using the MAK116 assay kit . Gastrocnemius muscles were quickly collected for further analyses.Gastrocnemius muscles were dissected, weighed, and cut into pieces. One piece was immersed in neutral buffered formalin 10%, fixed, and stained with hematoxylin and eosin to be examined for morphological changes. The second piece was used for nuclear extraction using the Nuclear/Cytosol Fractionation Kit 266‐25) . The third piece was homogenized in cold phosphate-buffered saline (pH 7.4), then centrifuged at 3000 rpm for 20 min for the assay of cytosolic biomarkers, and the fourth piece was immediately washed with phosphate-buffered saline and homogenized in 1 ml of mitochondrial isolation buffer  using a glass grinding tube on ice for 20 times. The resultant homogenate was centrifuged at 1500 rpm for 10 min at 4 °C, and the supernatant was collected and centrifuged again at 10,000 rpm for 15 min. The precipitate obtained represented the mitochondria fraction of gastrocnemius muscle according to the method described by Ding et al. [  and protein carbonyl (PCC) contents in muscle tissue homogenates. Malondialdehyde was assayed using a commercial kit supplied by Biodiagnostic, Giza, Egypt , whereasThe Na/K-ATPase activity was assayed using a commercial kit MBS8243226) supplied by MyBiosource, Inc., San Diego, USA. Briefly, 0.1 g from gastrocnemius muscle was homogenized with 1 ml of the assay buffer on ice and centrifuged at 8000 rpm for 10 min at 4 °C. The supernatant was taken into a new centrifuge tube and kept on ice for detection of Na/K-ATPase activity on a plate reader at a wavelength of 660 nm [243226 suIn a freshly prepared mitochondrial extract, CS activity was measured using 5,5′‐dithiobis (2‐nitrobenzoic acid) using a spectrophotometer (absorbance at 412 nm) overtime at 30 °C in the presence of acetyl co‐enzyme A and oxaloacetate . The enzThe ΔΨm was measured as described by Madi et al.  and GuhaAn enzyme-linked immunosorbent assay was used to estimate mitochondrial transcription factor A (TFAM) (MBS942857) and PGC-1α (MBS762203) levels in muscle nuclear extracts using commercial kits according to the manufacturers’ instructions . Color development was monitored by absorbance at 450 nm using a microplate reader .Nrf2 mRNA expression was measured in relation to the housekeeping gene, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The primers were designed using Primer3 software (http://bioinfo.ut.ee/primer3/) as follows: rat Nrf2 forward primer (5′-TAGCAGAGCCCAGTGGCGGT-3′) and reverse primer (5′-TGCTCTGGGGATGCTCGGCT-3′) (GenBank Accession No. NM_031789.2); rat GAPDH forward primer (5′‐GGTGAAGTTCGGAGTCAACGGA‐3′) and reverse primer (5′‐GAGGGATCTCGCTCCTGGAAGA‐3′) (GenBank Accession No. NM_017008) [The frozen muscle tissues were processed, and the total RNA was extracted using the Qiagen RNeasy Total RNA isolation kit 74,104) according to the manufacturer’s guidelines . This was followed by the synthesis of the first strand using the SuperScript III First‐Strand Synthesis System for RT‐PCR kit  . The PCR reactions were performed using Power SYBR Green PCR Master Mix  . The 04 accord_017008)  for the Specimens from gastrocnemius muscles were fixed in 10% formal saline for 24 h, dehydrated in ascending series of ethyl alcohol, and embedded in paraffin. Five μm histological sections were cut, stained with hematoxylin and eosin , and exaOver 200 myofibers per one gastrocnemius muscle were measured manually, and the mean of the cross-sectional area (CSA) was utilized using ImageJ software  in a blinded manner to calculate the average CSA for each group .P values < 0.05 were considered statistically significant.Statistical tests were performed using the SPSS software program . The data were expressed as mean ± standard deviation (SD). Statistical comparisons between different groups were conducted using the one-way analysis of variance (ANOVA) test followed by LSD post hoc test for multiple comparisons. By the end of the experiment, the body weight of the control and LXA4 groups increased by 16.66% and 16.49%, respectively, compared to their initial body weight. Compared to the control group, DEX administration significantly decreased the body weight by about 18.59% of the initial body weight. Concomitant administration of LXA4 with DEX significantly attenuated the decrease in body weight in comparison with the DEX group as it decreased by about 8.14% of the initial body weight. These findings suggested that LXA4 treatment significantly decreased muscle atrophy Fig. .Fig. 1LXAdministration of DEX for 14 days significantly decreased the hang time and holding impulse as compared with the normal control group, while concomitant administration of LXA4 with DEX significantly increased them as compared with the DEX group. Administration of LXA4 for 14 days did not affect the hang time or the holding impulse significantly as compared with the control group. These findings indicated that LXA4 treatment may preserve normal muscle function Fig. , c.Serum CK activity significantly increased in the DEX group as compared with the normal control group. Concomitant administration of LXA4 with DEX significantly decreased it as compared with the DEX group, which indicated a reduction of muscle damage by LXA4 treatment. LXA4 administration alone did not affect serum CK activity significantly Fig. .Dexamethasone administration significantly increased both MDA and PCC levels in gastrocnemius muscles as compared with the normal control group. Concomitant administration of LXA4 with DEX significantly decreased them as compared with the DEX group. Administration of LXA4 did not lead to any significant changes in MDA or PCC levels as compared with the control group Table .Table 1EThere were significant decreases in Na/K-ATPase activity in gastrocnemius muscles of the DEX group as compared with the normal control group, while the combination of LXA4 with DEX significantly increased its activity as compared with the DEX group Fig. .Fig. 2LXNrf2 in gastrocnemius muscles of the DEX group as compared with the control group. The combination of LXA4 with DEX significantly increased them as compared with the DEX group and relative gene expression of Nrf2 reached a normal level. In addition, LXA4 significantly increased ΔΨm, TFAM level, and PGC-1α levels in gastrocnemius muscles of group II as compared with group I (Fig. There was impairment of mitochondrial functions as indicated by significant decreases in CS activity, ΔΨm, TFAM level, PGC-1α level, and relative gene expression of Microscopic examination of hematoxylin and eosin-stained sections from gastrocnemius muscles showed the normal structure of muscle fibers in the form of polygonal fibers with peripheral nuclei in groups I and II Fig. , b. MuscThe present study exhibited that LXA4 administration for 14 days significantly mitigated the DEX-induced muscle atrophy in rats as shown by biochemical and histological findings. Noteworthy, this is the first study to investigate the protective effect of LXA4 against DEX-induced muscle atrophy in rats and elucidate new mechanisms involved in the effect of LXA4 on mitochondrial dynamics that has not been well identified yet.The results of the present experiment showed that DEX-treated rats had a significantly increased percentage of loss of body weight and decreased muscle strength as indicated by the shortening of the hang time and reduction of the holding impulse compared with the normal control rats. These findings agree with the results of Huang et al.  who repoConcomitant administration of LXA4 and DEX significantly reduced the muscles’ atrophic changes and improved their strength as indicated by the reduction of body weight loss and serum CK activity as well as the increase of myofibers’ CSA, hang time, and holding impulse compared with DEX group.The possible mechanisms of the LXA4-induced protective effect may be suggested in light of the results of this study. Administration of LXA4 reduced the elevated muscles’ MDA and PCC levels, indicating that the DEX-induced oxidative damage of the muscles’ lipids and proteins was significantly attenuated by LXA4.Dexamethasone has been reported to induce oxidative stress and mitochondrial dysfunction . MitochoDexamethasone-induced mitochondrial dysfunction was demonstrated in this study by the decrease in CS activity and ΔΨm compared with the normal control group. This could be explained by the overproduction of mitochondrial ROS, which impaired enzyme functions in the respiratory chain and reduced mitochondrial biogenesis .On the other hand, LXA4 improved mitochondrial functions as indicated by the significant increase in Na/K-ATPase activity, CS activity, and ΔΨm compared with the DEX group. This improvement may be due to the activation of the PGC-1α/Nrf2/TFAM pathway, which was inhibited by DEX administration. PGC-1α is a master regulator of mitochondrial biogenesis, which activates many transcription factors that bind to the promoters of nuclear-encoded mitochondrial genes . It’s weIt could be concluded that LXA4 displayed a protective effect against DEX-induced muscle atrophy in rats, possibly through the improvement of mitochondrial functions and activation of the PGC-1α/ Nrf2/TFAM pathway in addition to its antioxidant activity. So, LXA4 may be considered a promising therapeutic agent for DEX-induced muscle atrophy. Future studies are needed to investigate the therapeutic effects of LXA4 regarding other types of muscle atrophy and assess the efficacy of concomitant administration of LXA4 and DEX in patients on long-term corticosteroid therapy.