The experiments were performed at temperatures of 40–70 °C and pressures of 10–25 MPa in a batch system with ultrasonication for 60 min. Transmission electron microscopy (TEM) images revealed liposome products with spherical morphologies and diameters of <100 nm. Dynamic light scattering (DLS) analysis indicated that the curcumin-loaded liposome nanosuspension exhibited good stability. Changing the operating conditions influenced the amount of liposome-encapsulated curcumin; as the operating temperature or pressure increased, the diameter of the liposome products and the amount of liposome-encapsulated curcumin increased and decreased, respectively. Herein, we described an innovative and practical organic-solvent-free method for generating liposomes from phospholipids.Curcumin-loaded liposomes were prepared using a supercritical carbon dioxide (SCCO Curcuma longa L. (turmeric) is a plant that is native to tropical South Asia; it is widely cultivated in subtropical and tropical zones. This rhizomatous herbaceous perennial plant is generally used in production of a natural colorant, namely curcumin. This chemical compound, as the main active substance of turmeric, has a low-molecular-weight polyphenol that is responsible for the color of turmeric (bright yellow). Curcumin is utilized in the pharmaceutical field owing to its anti-inflammatory, antimicrobial, antibacterial, antifungal, and anticarcinogenic properties [operties . Severaloperties ,3,4. Howoperties ,6,7. Theoperties ,8,9.Liposomes are spherical vesicles comprising one or more phospholipid bilayers that are surrounded by an aqueous compartment. Liposomes are involved in various therapeutic (hydrophobic and hydrophilic) diagnostic agents that can protect the encapsulated agent from metabolic processes and provide a higher drug payload per particle. Owing to their unique properties, liposomes are employed in drug delivery systems. Typically, sphere-shaped vesicles have sizes ranging from 25 nm to several micrometers. Based on the lamellae number and size, liposomes are grouped as follows: multivesicular vesicles (MVVs), large multilamellar vesicles (LMVs), oligolamellar vesicles (OLVs), giant unilamellar vesicles (GUVs), large unilamellar vesicles (LUVs), medium-sized unilamellar vesicles (MUVs), and small unilamellar vesicles (SUVs). The sizes for MVVs, LMVs, OLVs, GUVs, and LUVs range from a few hundred nanometers to several micrometers, whereas the sizes for MUVs and SUVs range from 100 to 500 nm and 20 to 100 nm, respectively .2)–water mixture, operated in an ultrasound environment, was employed as a medium to generate organic-solvent-free liposomes. Curcumin was simultaneously encapsulated with the formed liposomes. Carbon dioxide is generally recognized as a safe (GRAS) solvent, and most solvents are employed under supercritical conditions. The critical point of CO2 is relatively low , which may prevent thermal deterioration during the process. When SCCO2 is employed as a medium for liposome generation, it may favor amphiphilic aggregation into amphiphilic nanoforms owing to its unique properties [2 possesses low interfacial tension, high diffusivity, low viscosity, and tunable density, which cannot be easily achieved by other solvents (organic solvents). CO2 is not only readily available, inexpensive, nonflammable, tasteless, odorless, inert, and environmentally friendly, but can also be easily separated from the product under ordinary conditions; hence, it does not result in product contamination.The conventional method of liposome production has tremendous limitations; it requires an organic solvent, the process is a complex, multistage process, the process is costly due to the high energy consumption, and the formed liposomes mostly exhibit poor stability ,13. In toperties . SCCO2 p2–water mixture as a medium for liposome generation to decrease the liposome product size. Furthermore, this study aimed to utilize the SCCO2–water mixture system in an ultrasound environment to generate smaller liposomes and simultaneously encapsulate curcumin with the formed liposomes.There are several methods to decrease liposome size, including homogenization, extrusion, and sonication. Batch and probe-type sonication are easy and common methods to decrease liposome size . In the 2 conditions at 50 °C and 10 MPa with 45 kHz ultrasound irradiation. At each amount of loaded hydrogenated soy phosphatidylcholine, the collected aqueous solution products exhibited similar colors (opaque–transparent). In principle, liposomes can be formed as spherical phospholipid liquid crystalline phases via the dispersion of phospholipids in a liquid water medium by shaking while supplying sufficient energy. Hence, liposomes were generated when hydrogenated soy phosphatidylcholine and distilled water were loaded into the SUS vessel under pressurized CO2, and then ultrasound irradiation was applied to provide sufficient energy input. This phospholipid arranged itself into bilayer vesicles and multilayer structures comprising lipid bilayers. The thickness and length of bilayer vesicles may increase owing to an ultrasound irradiation process [2 may simultaneously infiltrate and dissolve in a liquid water medium, resulting in a dissociation equilibrium forming carbonic acid, and the dehydration reaction might occur on the remaining CO2 molecule [2-derived molecules. When CO2 depressurization was performed to release pressure from 10 MPa to ambient pressure, CO2 molecules quickly flowed out of the phosphatidylcholine, yielding highly dispersed liposomes. As a result, as shown in  process ,17,18. Cmolecule ,20. This2 conditions at 50 °C and 10 MPa with 45 kHz ultrasound irradiation. As depicted in this figure, the morphology of the liposome generation products was spherical or sphere-like at each concentration of loaded hydrogenated soy phosphatidylcholine. This morphology was due to liposomes comprising spherical or circular bundle structures as the main structures; however, they can also comprise concave or oblate circular bundle structures [In addition to generation of small and large unilamellar liposome products, in this study, liposomes with multivesicular and multilamellar structures were also generated. Transmission electron microscopy (TEM) was used to characterize the morphology and lamellarity of the liposome products. The TEM technique is widely utilized for the characterization of nanodelivery systems. ructures ,22. Fredructures . They al2.Photographs of the collected aqueous solution products and their TEM images revealed that the various amounts of loaded hydrogenated soy phosphatidylcholine did not shift the physical properties of the liposome generation products, i.e., shape and size. The size of the liposome generation product was determined by measuring the solution products using a wet laser diffraction sizing (DLS) device, which is the most commonly used tool to measure emulsion system particles between 3 nm and 3 µm . Notably2 conditions at 50 °C and various operating pressures with 45 kHz ultrasound irradiation. The aqueous suspension product had a light transparent yellow color at each operating pressure. As mentioned above, liposomes are spherical vesicles that can be generated by the interactions of phospholipids and liquid water because phospholipid molecules possess naturally amphiphilic features, where the phospholipid lipophilic side generally faces internally and the hydrophilic side faces externally to the liquid water medium. Therefore, liposomes may integrate both hydrophilic and hydrophobic bioactive compounds, including curcumin. Therefore, liposomes were spontaneously generated from hydrogenated soy phosphatidylcholine when loaded and dispersed in the liquid water medium. Simultaneously, the generated liposomes may interact and encapsulate curcumin when hydrogenated soy phosphatidylcholine and curcumin are loaded and dispersed in the same liquid water medium. This incorporation may occur on the polar head group of a lipid bilayer through hydrogen bonding [ bonding ,27,28. A2 molecules in a liquid water medium may promote phospholipid dispersion to generate a nanosuspension, where, at the same operating temperature, increasing operating pressures enhance the dissolution of CO2 molecules in the liquid water medium. CO2 molecules interfered with phospholipid molecules, resulting in enhanced dispersion, leading to the generation of nanosuspensions with smaller particle sizes (<100 nm). Hence, the dissolution of CO2 molecules in the liquid water medium was expected to enhance the dispersion of hydrogenated soy phosphatidylcholine, resulting in the generation of nanosuspension particles with diameters smaller than 100 nm. At the same time, the application of ultrasound irradiation in the SCCO2–liquid water system may also have enhanced CO2 molecule dissolution because the acoustic vibration of ultrasound irradiation can impose CO2 molecule mass transfer into liquid water [These aqueous suspensions were investigated by DLS to measure the sizes of the liposomes containing curcumin; based on these findings, the particle size distribution of in the aqueous suspension products seemed to possess a single peak diameter under each experimental condition. This shows that the nanosuspension particle size was in the wide range of 40–800 nm; the smallest particle size (around 40 nm) was observed when the experiment was performed at 10 MPa. It is well known that the dissolution of COid water ,30,31. A2 molecules in the liquid water medium, resulting in the uniform dispersion of hydrogenated soy phosphatidylcholine with smaller diameters, the aggregation of generated liposomes might also have occurred during CO2 depressurization at the end of each experiment [2, and reported that CO2 depressurization at a slow rate (around 25 bar/min) may better control the size and size distribution of liposomes [2 depressurization rate was not observed in each experiment. In addition to the CO2 depressurization rate, the size of the particle products might also have been influenced by the presence of curcumin in the liquid water medium. Under CO2 conditions, Zhao et al. performed experiments on carotenoid encapsulation in liposomes using lutein as a carotenoid model compound [2 and ultrasound irradiation conditions. Hence, as exhibited in However, as shown in periment . Shashidiposomes . Howevercompound . They reTo determine the total amount of liposome-encapsulated curcumin, the collected aqueous solution products were injected into and analyzed using a high-performance liquid chromatography (HPLC) system. Prior to HPLC analysis, liposome-encapsulated curcumin was separated from the collected aqueous products. In addition to the upper part of the supernatant being analyzed using HPLC, this part was also characterized by using a TEM device to observe the morphology and lamellarity of the generated liposome products containing curcumin. 2 molecules in the liquid water medium. This can interfere with phospholipid molecules more intensely, resulting in enhanced dispersion and the generation of a nanosuspension with small particle sizes. However, as shown in 2 technique. In addition to the operating pressure, changing the operating temperature also impacted the amount of liposome-encapsulated curcumin following preparation by the SCCOOne of the important steps in liposome generation processes involving the encapsulation of compounds with liposomes is to observe their characteristic features. This was used to confirm the desired liposomal products. The physical parameters of the liposome products  are summarized in Similarly to the diameter size of liposomes, the zeta potential of liposomes is another important parameter that should be considered in liposome generation after the liposome preparation is complete. The zeta potential is the potential difference between the bulk dispersion medium in which the particles are dispersed and the slipping plane of the fluid attached to the dispersed particles. This parameter often reflects the liposome surface charge, which can be used to assist in the prediction of the liposome product’s stability. In other words, the zeta potential can be applied to represent the interaction between the biological environment and the stability of the colloidal dispersion. When the value of the zeta potential is low or near zero, there is no electrostatic repulsion between the liposome products. Accordingly, liposome product flocculation related to the instability of the suspension is generally due to the nature of the attractive force. Conversely, if the liposome suspension possesses a large positive or negative zeta potential, the aggregation of liposome products may not occur because they repel each other. A colloidal dispersion system is generally considered to be stable when its zeta potential value is in the range of 30 mV (either positive or negative) ,42. In tSimilarly to other colloidal dispersion systems, liposomes may also undergo aggregation, phase changes, and fusion upon storage. Here, the liposome products containing the curcumin compound were stored in an incubator (37 °C) and a refrigerator (5 °C) for several days to monitor the liposome storage stability, because a stable formulation may physically maintain the quantity of encapsulated materials, including curcumin ,43. Next21H20O6, 036-04922) were obtained from Nikko Chemicals Co., Ltd., Tokyo, Japan and Wako Pure Chemical Industries, Ltd., Osaka, Japan, respectively. Ethyl acetate  and acetonitrile  were purchased from Wako Pure Chemical Industries, Ltd., Osaka, Japan. Phosphotungstic acid  was purchased from Nacalai Tesque, Inc. . Carbon dioxide  was obtained from Tomoe Shokai Co., Ltd., Tokyo, Japan.Hydrogenated soy phosphatidylcholine (S–10 PLUS) phospholipid and curcumin , ultrasonic equipment , an acrylic chamber assembled with an electric heater, an SUS vessel , and a back pressure regulator  as the main parts. Prior to entering the SUS vessel, CO2 was passed through a coil preheater , which was placed in an acrylic chamber containing liquid water as the heat transfer medium. The preheater was equipped with a K-type thermocouple to determine the experimental temperature. An analog pressure gauge  was also attached between the SUS vessel and the BPR via a 1/16 inch stainless steel tube (SUS–316) to inspect the experimental pressure. The experiments were performed as follows. First, hydrogenated soy phosphatidylcholine (30–60 mg) was loaded and sealed in the SUS vessel with distilled water (60 mL). The sealed SUS vessel was then assembled in the SCCO2–ultrasound apparatus, immersed in an acrylic chamber, and heated to 40–70 °C. To achieve the SCCO2 conditions, CO2 was pumped using a high-pressure pump at 10–25 MPa. Subsequently, it was ultrasonically irradiated at 45 kHz with an input power of 600 W. After 60 min, the pressure of the SCCO2–ultrasound apparatus was depressurized and the liposomal dispersion product was collected. Pressure depressurization was performed as follows. The SUS vessel was removed from the acrylic chamber and vertically positioned for approximately 15 min. Subsequently, the pressure was gradually released from the top of the SUS vessel via the BPR device. The same method was applied when the SCCO2–ultrasound apparatus was employed to generate curcumin-encapsulating liposomes. Each experiment was conducted in duplicate or triplicate, and the data represent the mean values from the results. The effects of the wall thickness of the SUS vessel on the liposome products were not investigated.The amount of encapsulated curcumin was analyzed using reversed-phase HPLC with a Jasco system comprising a column heater , degasser , pump , and UV–vis detector . HPLC was performed and the chromatogram was recorded using a PC driven with Jasco-Borwin (Ver 1.5) software via the LC–Net II/ADC controller . Using an Inertsil ODS-3 column , the HPLC analysis system was operated as follows: flow rate: 1.0 mL/min; acetonitrile/water: 90/10 (eluent); UV detector: 420 nm; temperature: 40 °C. The size distribution of the liposome products was measured using DLS . The zeta potential was measured in zeta potential measurement mode using a ZetasizerNano ZS instrument . The liposomal dispersion product was diluted 10 times with distilled water and introduced into the measuring device with 173° scattering at 25 °C . High-re2 in an ultrasound environment has been demonstrated for curcumin encapsulation using liposomes, certain challenges remain, mainly in relation to the SCCO2–ultrasound system being designed for small-scale processes without an agitation tool that may improve the encapsulation efficiency. Nevertheless, this SCCO2–ultrasound system is a powerful technique for generating organic-solvent-free phospholipid-based drug-loaded liposomes for industrial applications and may provide information for the generation of liposomes from phospholipids.Curcumin encapsulation using liposomes generated from hydrogenated soy phosphatidylcholine in an ultrasound environment under pressurized carbon dioxide was successfully performed at operating temperatures of 40–70 °C and pressures of 10–25 MPa. Experiments were performed in a batch system with 60 min of ultrasonication. TEM images showed that spherical liposomes with diameters of less than 100 nm were formed. DLS analysis indicated that the curcumin-loaded liposome nanosuspension exhibited good stability. The changing operating conditions seemed to affect the amount of liposome-encapsulated curcumin, where increasing the operating temperature or pressure increased the diameter of the liposome products and decreased the amount of liposome-encapsulated curcumin. Although the application of SCCO


By producing a first-of-its-kind, decadal-scale wildfire plume rise climatology in the Western U.S. and Canada, we identify trends toward enhanced plume top heights, aerosol loading aloft, and near-surface smoke injection throughout the American West. Positive and significant plume trends suggest a growing impact of Western US wildfires on air quality at the local to continental scales and support the notion that wildfires may have an increasing impact on regional climate. Overlap of identified trends with regions of increasing wildfire emissions and burn severity suggests a link to climate driven trends toward enhanced wildfire activity. Further, time series of plume activity point to a possible acceleration of trends over recent years, such that the future impacts to air quality and regional climate may exceed those suggested by a linear fit to the multi-decadal data. These findings have significant implications for human health and exacerbate concern for the climate–wildfire connection. Aerosol particulate matter with an aerodynamic diameter less than 2.5-microns (PM2.5) has been linked to enhanced population level mortality3 and increased instances of respiratory illness7. Relative to PM2.5 from non-fire sources , mounting evidence suggests that wildfire emitted PM2.5 may be more toxic8, therefore posing a greater risk to human health. In terms of climate, aerosol loading from wildfires has a direct radiative impact in the form of absorption and scattering of incoming solar radiation by black carbon10 and organic aerosols11, respectively. Additionally, wildfire emitted aerosols generate indirect radiative effects by altering cloud microphysics13.Aerosols emitted by wildfires16, annual fire area burned at high-severity14, the number of large wildfires16, wildfire emissions of aerosol17, and in some cases high-altitude burning18. Similarly, portions of British Columbia demonstrate increases in wildfire emissions17. This shift in wildfire activity has been, in part, attributed to a changing climate, with strong links drawn to increasing temperatures and aridity21, reduced summertime precipitation22, and a declining snowpack24. Significant increases in extreme fire weather across the Western US over the years 1979–2020 have been attributed to increases in the vapor pressure deficit, decreases in relative humidity, and the dependence of these aforementioned shifts on increasing temperatures21. Wildfire activity in the Klamath Mountains/North Coast and Sierra Nevada regions of California, which are large contributors to wildfire activity at the scale of the Western US, has been linked to warming temperatures and increased atmospheric aridity20. Similarly, increases in high altitude burning across the Sierra Nevada, Middle Rockies, and Southern Rockies have also been attributed to drier conditions and warmer temperatures18. Global climate models indicate 2 °C of warming by the year 2050 would continue to exacerbate wildfire activity25, thereby increasing the air quality and climate impacts of large wildfires. Given the legacy of fire suppression in Western US forests, it is reasonable to expect these impacts to be further amplified26.Over recent decades, Western US wildfire activity has become increasingly intense, with many mountainous regions demonstrating increases in annual fire area burned29. Conversely, injections confined to the PBL pose a greater risk to local air quality as they are less vertically diluted30. Similarly, aerosol injection aloft is associated with a greater potential for climate forcing by wildfire aerosols, given the longer residence times32, reduced attenuation of incoming solar radiation at altitude29, and temperature dependence of radiative emissions from the smoke plume33. Further, the radiative effects of aerosol-cloud interactions are sensitive to the injection altitude of black carbon34. In extreme cases, deep pyro-convection may inject aerosols directly into the stratosphere36, producing a stratospheric aerosol mass that is comparable to a moderate volcanic eruption37. Injection of wildfire emitted aerosols above the tropopause is linked to positive top-of-the-atmosphere radiative forcing at the hemispheric scale38, reductions in surface temperature40, reductions in stratospheric ozone41, and disruptions of atmospheric circulations in the lower stratosphere43.Wildfire plume rise, a result of buoyancy produced by combustion-derived heating, determines the altitude at which wildfire emitted aerosols are injected into the atmosphere, modulating the air quality and climate impacts of a given wildfire. The plume injection height often dictates the transport pathway of wildfire smoke. For smoke injections of equal mass, penetrative injections above the planetary boundary layer (PBL) enhance the potential for air quality impacts at regional to continental distances from the fire due to the efficiency of long-range transport and the potential for subsequent boundary layer entrainment44. While a weak correlation exists between the fire intensity and plume top height45, atmospheric stability acts to limit the buoyant plume rise46 by inducing plume injection/smoke detrainment. As the plume rises, environmental eddies entrain cold air into the plume, diluting the vertical momentum and therefore limiting the plume top height47. The effects of dynamic entrainment, produced by a strong horizontal wind47, result in a bent-over plume structure48. Furthermore, latent heat release offers a means to fuel additional plume rise, highlighting the importance of moist layers and moist convection to plume top height30. As testament to the role of moist convection, the inverted-V sounding, in which a moist layer sits atop a relatively deep and dry surface layer, has been positively linked to pyrocumulonimbus  development and instances of stratospheric aerosol injection33.The heights of wildfire smoke plumes are a complex function of the wildfire heat flux, fire geometry, and the ambient meteorological profile44, climate driven trends toward enhanced wildfire activity24 and related degradation of Western US air quality50 highlight the need for investigation into the evolving state of wildfire aerosol injection and the associated vertical mass distribution. At present, the temporal and spatial limitations of satellite sampling by space-borne sensors—i.e., the Multi-angle Imaging SpectroRadiaometer (MISR) and Cloud-Aerosol Lidar with Orthogonal Polarization (CALIOP)—prohibit a purely observational investigation of this topic. Plume top heights observed by MISR, which is limited to making observations during the late morning, indicate that the fraction of plumes penetrating above the PBL is highly sensitive to the definition of plume top. When the plume injection height is defined as the median MISR retrieved height for all pixels in a given plume, a mere 4–18% of North American plumes penetrate above the PBL52. However, when the plume injection height is defined as the maximum retrieved height for an individual plume, 48% of North American plumes are found to exceed the PBL45. Further, plume penetration above the PBL is complicated by the presence of diurnal cycles in fire intensity and lower atmospheric instability. When applied to global plume activity, the Freitas et al.53 plume rise model depicts a diurnal cycle in which ~ 25% of plumes penetrate above the PBL during morning hours, while the late afternoon maximum in fire intensity55 and enhanced lower atmospheric instability correspond to a value of ~ 53%56. Due to limited plume top observations and uncertainties surrounding plume rise modeling57, even less is known about the vertical distribution of detrained aerosol mass below the plume top. Observations from 20 controlled burns in the US Southeast suggest that the majority of plumes are either vertically well mixed or preferentially inject emissions aloft58. Coupled atmospheric—fire spread modelling, 3 -dimensional plume rise modeling, and detrained particle trajectory modeling suggest a tendency for plumes to deliver a majority of their aerosol mass toward the plume top60, while existing parameterizations for chemical transport modeling range from parabolic and Gaussian distributions to a constant dilution throughout the depth of the PBL57.Despite remaining uncertainties as to the exact governing equations of the wildfire plume rise and its expression in a modeling framework2.5 emissions, allows the first estimate of how the vertical distribution of wildfire aerosol may evolve under climate driven trends toward increasingly intense wildfire activity.Given limitations in previous observational and modeling approaches, investigation into the evolving vertical distribution of wildfire injected aerosol mass requires a plume rise modeling framework that simulates both the plume top height and the vertical mass distribution in a manner supported by the limited observations and relevant theory. Application of such a framework to simulation of one-dimensional wildfire plumes at the regional-decadal scale may provide the sample size necessary for meaningful statistical analyses in the context of model uncertainty. Here we seek to generate a physics-based wildfire plume rise climatology that (i) leverages satellite burned area data, wildfire emissions, and atmospheric modeling outputs to provide regional scale understanding of plume activity beyond the limited satellite sampling, (ii) combines information on plume entrainment and mass conservation to estimate the vertical smoke detrainment profile of individual plumes, and (iii) when linked to wildfire PM53 plume rise model 61 and are considered in the context of PBL heights adopted from the Stochastic Time-Inverted Lagrangian Transport model —weighted average plume top height . Although trends for other ecoregions and the Western US/Canada as a whole are statistically insignificant, this climatology finds the first occurrence of pyroCb development in 6 ecoregions between 2017 and 2020, demonstrating an acceleration in recent years. These ecoregions include the Colorado Plateau, Southern Rockies, Sierra Nevada, Wasatch-Uinta, Mojave Basin and Range, and Northwestern Great Plains. Hinge fits further support the possibility of a recent uptick in pyroCb activity, with hinged trends  of 2.72, 2.15, and 0.43 pyroCb hours/year for the Colorado Plateau, Southern Rockies, and Sierra Nevada, respectively. A comparison of simulated pyroCb activity for 2013 to the observational inventory of pyroCb activity constructed by Peterson et al.65 indicates that this model framework reasonably captured the pyroCb formation above the Pony/Elk and Beaver Creek fires in Idaho, while failing to reflect the observed pyroCb activity above the Rim fire in California. Should pyroCb trends present greater clarity over the coming years, this result would support the growing concerns surrounding pyroCb induced warming of the stratosphere39.We further undertake an examination of trends in the most extreme fire activity, as represented by pyrocumulonimbus (pyroCb) formation, defined by especially tall plumes with a plume top temperature <  − 38 °C (the homogenous freezing temperature for liquid water) being leveraged as a pyroCb proxy21. When limiting timeseries to 2003–2019 (a rather quiet fire year in the Western US), trend analyses further support the presence of positive and significant trends across numerous ecoregions , bolstering confidence in this result given the effect of extreme 2020 values , and those inherent to the current state of plume rise modelingmes Fig. a. While 72.On a plume-by-plume basis, we find a standard deviation of modeled plume top heights from MISR observations of 46.9 m for 425 sets of reasonably collocated plumes (within 30 min and 4 km). Of these 425 sets of collocated plumes, we see that 47.8% of modeled plume tops fall within the ± 500 m observational uncertainty of MISR and that there is little geographic clustering of model uncertainty  of the 425 sets of collocated plumes are found to straddle the PBL, with the modeled plume top above the PBL and the observed plume top within the PBL, or vice versa. In 71% of such cases, the modeled plume top exceeded the PBL, with PBL heights averaging 77.9% of the modeled plume top heights. In terms of the climatological average of the modeled vertical distribution of smoke below plume top, ~ 78% of the PM2.5 flux-weighted average plume tops . Enhanced high-altitude injections of smoke aerosol mass , explicit simulation of individual wildfire plumes using a coupled atmosphere—fire spread model (e.g. WRF-Sfire)84 was not computationally feasible.While uncertainties remain in the model, F2010 was selected for its adherence to a physical basis and its ability to account for latent heat release by cloud microphysical processes85 model output, and wildfire inputs generated through a combination of wildfire PM2.5 emissions estimates61, burned area estimates (MCD64A1)86, and sub-daily temporal downscaling profiles based on satellite active fire detections80.For this study, we supplied the F2010 model with environmental conditions from nested Weather Research and Forecasting (WRF)57, which can ingest WRF input files directly. The WRF simulations generated for this study used a nested domain setup where a 4 km resolution domain covered most of the Western US and was nested within a 12 km resolution domain covering western North America  for surface wind speed, wind direction, temperature, and humidity 61 are provided at daily resolution for four biome types  and were converted to a heat flux dataset using PM2.5 emissions factors (PM2.5 emitted/kg dry matter burned)90 and assuming that the heat release per unit dry matter burned is 18.6 MJ/kg91. This conversion is depicted in Eq. (2.5 flux and emission factor associated with biome type Gridded QFED PM92), with the majority of collocations occurring within ± 1 day93. Given this temporal window, we linked wildfire heat fluxes to burned area polygons using three complementary approaches that account for the temporal uncertainty in wildfire burned area observations and the idea of a directional fire spread. Specifics regarding this process are provided in SI Sect. Heat flux datasets were then overlaid with daily burned area polygons from Moderate Resolution Imaging Spectroradiometer (MODIS) collection 6 to identify probable linkages between wildfire heat flux and satellite observed burned area. Since QFED emissions are based on MODIS observed thermal hotspots (MOD14A1/MYD14A1 product), the literature suggests an expectation of collocated burned area and wildfire heat fluxes within a ± 8 day window . The resultant values were assumed to apply to each hour within the 3-h window, meaning additional plume top/thermodynamic variability within any 3-h downscaling window would strictly be a result of varied meteorological conditions.Temporal downscaling from daily to 3-h emissions and burned area was performed using diurnal cycles of wildfire activity based on gridded (0.25° resolution) and monthly aggregated satellite active fire detections provided by the Global Fire Emissions Database (GFED)To estimate the vertical distribution of smoke detrainment below plume top, thermodynamic F2010 outputs for each plume were subject to vertical level-by-level mass conservation calculations following Eq. , and the total emissions were subjected to linear regression. Statistical significance was addressed via 100,000 bootstrapped repetitions. Each linear regression was reapplied to 100,000 resampled versions of the annually aggregated values, allowing replacement of selected samples, to determine if the regression to the actual data was distinguishable from the scatter produced by resampling.Trend analyses aimed at understanding the evolution of plume top heights and the vertical distribution of wildfire generated aerosol mass, were performed for US level 3 ecoregions95, the “chngpt” package96 was used to fit 2-segmented linear trends to PM2.5 flux-weighted average plume top height, PM2.5 mass injection relative to the PBL, and PM2.5 emissions timeseries. Significant hinge-points were selected using a bootstrapping procedure that identifies the optimal hinge, with an associated p-value based on the quality of model fit. Hinge fitting has previously been used to compute climate normals amidst a changing climate97.Hinge fitting, or change point analysis, was also used to elucidate potential emergent trend accelerations over recent years. Using the statistical analysis software R45.MISR derived plume top heights were used to evaluate the performance of the F2010 model by comparing the distribution of modeled and observed plume top heights. Additionally, a plume-by-plume analysis was performed, which considers the deviation between modelled and observed plume tops in the context of instrument uncertainty. From the archived plumes maintained as part of the MISR Plume Heights Project 2, we found 480 plumes with a quality flag of “good” or “fair” within western North America Fig. b. These On a plume-by-plume scale, we found 425 MISR observed plumes within 30-min and 4 km of a model-derived plume. A cutoff distance of 4 km was used to compromise between the importance of local meteorology to plume top heights and the uncertainty in plume location as a result of using burned area centroids to denote plume locations. In cases when hourly resolution model outputs result in a plume top estimate both before and after the MISR overpass time, modeled plume top heights are temporally interpolated to the timing of the MISR overpass. Analysis of matched sets of plumes focusses on the standard deviation of modeled plume top heights from their observed counterparts and the fraction of modeled plumes falling within the MISR observational uncertainty.Supplementary Information.


Arabidopsis thaliana germinates and grows in diverse lunar regoliths. However, our results show that growth is challenging; the lunar regolith plants were slow to develop and many showed severe stress morphologies. Moreover, all plants grown in lunar soils differentially expressed genes indicating ionic stresses, similar to plant reactions to salt, metal and reactive oxygen species. Therefore, although in situ lunar regoliths can be useful for plant production in lunar habitats, they are not benign substrates. The interaction between plants and lunar regolith will need to be further elucidated, and likely mitigated, to best enable efficient use of lunar regolith for life support within lunar stations.The extent to which plants can enhance human life support on other worlds depends on the ability of plants to thrive in extraterrestrial environments using in-situ resources. Using samples from Apollo 11, 12, and 17, we show that the terrestrial plant  Arabidopsis plants were seeded onto lunar soil samples taken directly from the Apollo 11, 12, and 17 missions. Transcriptomic analyses reveal that plants grown in lunar soil differentially express genes associated with salt, metal, and ROS stress. Since the return to the Moon is envisioned as a dedicated, longer-term commitment to lunar exploration, questions of the lunar environmental impact on biology and biological systems have become a significant part of the lunar exploration agenda. Plants have long been envisioned as part of lunar habitats8 and exploration environments10. However, until the current study, the interactions between lunar materials and terrestrial biology were unaddressed in the era of modern molecular biology, and there had yet to be any experiments where plants were actually grown in the true lunar regolith. Understanding the impact of sustained exposure of terrestrial biology to lunar regolith, and determining the efficacy of lunar regolith as a viable in situ resource, is important to the concept of returning to the Moon for long durations. Therefore, we used plant growth and gene expression  to both tests the fundamental impact of lunar regolith on terrestrial biology, and provide an initial evaluation of regolith as a matrix for plant growth systems in lunar exploration habitats.The return of humans to the Moon during the NASA Artemis program19. Plants also make available potentially essential components to the long-term habitation of space and extraterrestrial surfaces by providing food and oxygen, recycling water, and scrubbing carbon dioxide from human habitats. However, many designs of extraterrestrial plant growth systems largely rely on hydroponic systems21, in part due to the lack of information on the efficacy of using in situ materials, such as planetary regolith, as a growth substrate. Information on plant growth in lunar regolith, therefore, informs fundamental biology interactions within the lunar environment, and helps scale options for the use of plants in lunar life support scenarios.Plants are key components of the biological sciences within and in support of space exploration. As model organisms, plants provide insights into gravity, radiation, and other space-related biological phenomena, and therefore help drive an understanding of the physiological adaptation of terrestrial biology to space exploration environments. There is an expanding wealth of literature documenting this progress, e.g., 22). In addition, lunar samples were intensively isolated in specialized facilities at the Johnson Space Center Lunar Receiving Laboratory24, where the samples were handled in gloveboxes roughly equivalent to current high-level biological containment. The primary goal was to search for pathogens, and those search procedures relied on direct, though largely transient contact between lunar materials and biology. In the specific case of plants, lunar samples were rubbed onto leaf surfaces and sprinkled onto seedlings and growth media25, but plants were never grown in lunar regolith as the support matrix27. This gap in knowledge is addressed in the following experiment.The Apollo Moon landings were a critical point in the science of space exploration, not only in the accomplishment of lunar visits, but also in that terrestrial biology would be in contact with extraterrestrial material, and samples of that material would be intentionally returned to Earth. The first contact between terrestrial biology and lunar regolith was an unprecedented concern for protecting both the astronauts visiting the Moon as well as the entire Earth biosphere from the potentially harmful effects of returned lunar materials. Novel, extraterrestrial pathogenic microorganisms were the main concern, in addition to toxic mineral compositions and abrasive physical characteristics. To protect the returning human explorers and the terrestrial biosphere, the astronauts and attending medical staff were isolated for 2 weeks upon the return of the early Apollo crews , 12 (12070), and 17 (70051) Fig. , which r29. Agglutinate and nanophase Fe abundance both increases with regolith maturity as prolonged surface exposure to the solar wind reduces particle size, the increases the conversion rate of grains of Fe metal to nanophase Fe29. Many regolith simulants such as JSC-1A contain abundant natural volcanic glass, e.g., 29, but this material does not fully replicate agglutinate assemblages or morphologies. Additionally, the overall Fe oxidation state of the lunar samples differs from that of JSC-1A. Although to our knowledge, Fe oxidation state ratios have not been quantified in these samples, lunar regolith ubiquitously contains nanophase metallic Fe  in addition to solely Fe2+ in mafic silicates and oxides34, whereas JSC-1A is known to contain Fe3+-bearing Fe-Ti oxides (namely magnetite and chromite) in addition to some proportion of Fe3+ in its silicates and glass. Lastly, two of the lunar regoliths (10084 and 70051) are derived from high-Ti basaltic bedrock and have TiO2 abundances of 5–7 wt.%, which is considerably higher than that of JSC-1A at 1.85 wt.%, e.g., 29.There area few notable mineralogical and compositional differences between the lunar regoliths and JSC-1A. The lunar samples used here contain 30–52 vol. % agglutinates, which are aggregates containing mineral fragments, nanophase metallic Fe, trapped gases, and glass that form due to micrometeorite impactsTo provide insights into the potential for using lunar regolith to grow plants as part of the Artemis lunar exploration science program and for future sustained lunar habitation, we developed a small-scale system based on 48-well laboratory plates capable of assessing plant growth in small, obtainable amounts of lunar regolith samples returned from the Apollo missions 11, 12, and 17 Fig. , and devGermination readily occurred on all samples between 48 and 60 hours after planting, and all lunar seedlings exhibited normal stems and cotyledons Fig. , indicat39 39, Jasmonate-regulated gene-21 (AT3G55970), and Defensin-like (AT1G13609), plus two genes encoding proteins of unknown function (AT3G28290 and AT3G28300) that are also involved in aluminum toxicity41 and jasmonate signaling43. In addition to the coordinate responses, plants from each of the Apollo samples differentially expressed genes unique to each sample location  of the plants after 20 days. The gene expression data were parsed based on lunar sample site replicates compared to the JSC-1A controls Fig. . All lun39 Fig. . The DEG39 Fig. . The mosion Fig. , suggestThe statistically supported differentiation of transcriptome responses based on lunar sites indicated that plant responses vary based on the lunar regolith source, and supported the overall conclusion that lunar regolith is more stressful than JSC-1A simulant. However, the various growth morphologies demonstrated within the replicates of each lunar regolith sample suggested the potential for a range of success states for lunar plant growth. To investigate the responses of potential growth success states, the transcriptomic data were regrouped based on plant size as one measure of growth success  demonstrated strong stress-response transcriptomes Fig. . Nine ofThe morphologically normal Small and Large plants demonstrated only 130 and 150 DEGs compared to JSC-1A, respectively Fig. , suggest44. Fe-oxide deposits resulting from the exposure of nanophase iron to atmospheric oxygen could inhibit mineral utilization in the context of regolith45, whereas grain size shape and micropores could influence material surface area to foster an elevated release of ions into solution46. An increase in nanophase iron and a decrease in grain size are hallmarks of mature lunar regolith47, such as the Apollo 11 regolith used in this study (10084). Although examples of poorly developing plants were represented in each of Apollo site sources, overall, the plants grown in Apollo 11 regolith struggled the most and displayed the greatest number of differentially expressed genes. The plants grown in Apollo 17 regolith struggled the least, and displayed fewer differentially expressed genes. These data suggest that more mature regolith provides a poorer substrate for plant growth than immature regolith. Thus, although this study demonstrates that plants can use lunar regolith as a primary substrate, further characterization and optimization would be required before regolith can be considered a routine in situ resource, particularly in locations where the regolith is highly mature.These data demonstrated that terrestrial plants are capable of growth in lunar regolith as the primary support matrix. Soils derived from lunar regolith could therefore be used for plant production and experiments on the Moon. However, these data also demonstrated that lunar regolith was not a benign growth substrate. Plants can fail to fully establish in the lunar regolith, resulting in a range of growth states and success. Moreover, plants broadly interpreted lunar soils as highly ionic and as eliciting oxidative stress, which is consonant with the prediction that the cosmic ray and solar wind damage to surface regolith would leave it very reactive to biological systemsArabidopsis thaliana (Arabidopsis) ecotype Columbia-0 (Col-0) (TAIR CS70000)48.All seeds used in the experiment were from the same batch of seeds from The lunar Allocation Analysis Review Board/AARB (formerly CAPTEM), through the Apollo Sample Curator at Johnson Space Center (Ryan Ziegler), provided 4 × 1 g samples of regolith from Apollo 11 , Apollo 12  and Apollo 17  Fig. . An examDrainage holes were drilled in a specific configuration to distinguish groups of JSC-1A and lunar samples and then labeled   by placing the plates in trays of the solution to soak the plugs from below, and then allowed to drain. Next, 900 mg of each regolith sample (or JSC-1A) was added to the designated wells . The replicates were arranged in four, 48-well cell culture plates  such that each plate held a replicate of each Apollo site regolith  and commercial potting soil. All materials were sized to a collection of particles <1 mm; the JSC materials we obtained pre-sieved to that particle size fraction, and the soil was sieved in the laboratory. The plates were configured as described above with rockwool plugs and filters, and watered from below with a 0.125× MS nutrient solution. The success of this approach informed the lunar materials experiment configuration  according to the manufacturer’s guidelines RNA concentration was determined on Qubit® 2.0 Fluorometer, RNA quality was assessed using the Agilent 2100 Bioanalyzer . First, 50 ng of total RNA was used for mRNA isolation using the NEBNext Poly(A) mRNA Magnetic Isolation Module . Then followed by RNA library construction with the NEBNext Ultra II Directional RNA Library Prep Kit  according to the manufacturer’s user guide. Briefly, RNA was fragmented in NEBNext First-Strand Synthesis Buffer via incubation at 94 °C for the desired time. This step was followed by first-strand cDNA synthesis using reverse transcriptase and random hexamer primer. Synthesis of ds-cDNA was performed using the 2nd strand master mix provided in the kit, followed by end-repair and adaptor ligation. At this point, Illumina adaptors were ligated to the sample. Finally, each library (uniquely barcoded) was enriched by 12 cycles of amplification, and purified with Agencourt AMPure beads . 48 barcoded libraries were sized on the Bioanalyzer and quantified with the Qubit® 2.0 Fluorometer. Finally, these 20 individual libraries were pooled in equimolar concentration. RNASeq libraries were constructed at the UF ICBR Gene Expression Core . Sequencing was performed at the ICBR NextGen Sequencing Core . Normalized libraries were submitted to the “Free Adapter Blocking Reagent” protocol  in order to minimize the presence of adaptor-dimers and index hopping rates. The library pool was diluted to 0.8 nM and sequenced on one S4 flow cell lane (2 × 150 cycles) of the Illumina NovaSeq6000. The instrument’s computer utilized the NovaSeq Control Software v1.6. Cluster and SBS consumables were v1.5. The final loading concentration of the library was 120 pM with 1% PhiX spike-in control. One lane generated 2.5–3 billion paired-end reads (~950 Gb) with an average Q30% ≥ 92.5% and Cluster PF = 85.4%. FastQ files were generated using the BCL2fastQ function in the Illumina BaseSpace portal. One NovaSeq S4, 2 × 150 cycles lane resulted in an average of 50 Million demultiplexed, paired-end reads when sequencing a pool of 48 samples.RNA isolation and sequencing were after the approaches described in Paul et al.15. The quality of the RNASeq sequence data was evaluated using FastQC and low-quality bases trimmed from the reads using Trimmomatic53. STAR Aligner54 was used to map high-quality paired-end reads to TAIR10 genome, and Gene expression values were calculated using RSEM55. The edgeR linear regression model56 was used to perform the differential gene analysis. Hierarchical clustering and principal component analysis were conducted to evaluate the association of the samples. The thresholds for calling significantly DEG were set at, FDR of 0.05, a fold change of >2, and the average FPKM (Fragments Per Kilobase of transcript per Million mapped reads) for at least one replicate of each comparison group being higher than 0. DEG lists were analyzed for overlaps using BioVenn57. Processing and Analysis of the RNASeq data were performed at the UF ICBR Bioinformatics Core .RNASeq Bioinformatic approaches have been previously described in Paul et al.n = 16 total) one representative of each lunar site on each plate . The individual plate configuration is illustrated in Fig. n = 4 controls for parity to the lunar samples. However one of the replicates in Apollo 11 did not provide viable RNA, and so for the transcriptome analyses of Apollo 11 treatment n = 3, while the Apollo 12 and Apollo 17 treatments n = 4. The statistical methods employed for the transcriptome bioinformatics are described in the Bioinformatics Methods section. The quantitative growth graphs in Supplementary Fig. n = 4, in Supplementary Fig. n = 3. The numerical values for all points on the Supplementary Fig. The experiment compared plant growth and transcriptomes of JSC-1A lunar simulant to plants grown in regolith from three different Apollo lunar landing sites: Apollo 11, Apollo 12, and Apollo 17; JSC-1A served as the control, the three Apollo sites were the treatments. The plants were grown in sets on four replicated 48-well growth plates, each composed of four samples of JSC-1A controls (Further information on research design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Reporting Summary


Coulometric approaches overcome the limitations of spectrophotometry and are applicable to a wider range of essential oils.Essential oils are widely used in aromatherapy, medicine, and food industries due to a wide spectrum of bioactivity. Their antioxidant properties can be considered as markers of therapeutic effect and quality. Constant–current coulometry with electrogenerated titrants has been successfully applied for these purposes for the first time. Fifteen types of essential oils from various plant materials have been studied. Their composition has been identified by gas chromatography with mass-spectrometric detection (GC-MS). The reactivity of individual antioxidants of essential oils towards electrogenerated titrants (bromine and ferricyanide ions) has been estimated. Total antioxidant parameters, in particular total antioxidant capacity (TAC) and ferric reducing power (FRP) based on the reactions of essential oil antioxidants with electrogenerated bromine and ferricyanide ions, respectively, have been evaluated. Positive correlations ( Plant materials are widely studied objects in different branches of chemistry. One of the intensively developed directions in this field is the investigation of the antioxidant properties and their impact on human health. Various types of plant material-based samples  are in focus to date. Among them, essential oils are of practical interest due to their wide spectrum of biological activity and application area. In nature, essential oils are involved in the plant defense system and signaling processes. For example, plant aroma compounds participate in the protection of plants from microorganisms, insects, and herbivores, in the attraction of the pollinating insects and fruit-distributing animals, in water regulation, and allelopathic interactions .Since ancient times, essential oils have been used in aromatherapy . AntibacEssential oils are highly concentrated extracts from flowers, leaves, stems, fruits, and roots obtained by hydrodistillation, steam or dry distillation, as well as mechanically (for citrus fruits) . The maj•) [+•) [Antioxidant properties of essential oils are usually evaluated by spectrophotometric approaches  characte•) ,11,12,13•) [+•) . The tot•) [+•) ,16, ferr•) [+•) ,13,14. F• [Mentha species [Further development in the field could be focused on the development of electrochemical approaches for the evaluation of essential oils’ antioxidant parameters as far as reactions of antioxidants proceed with the electron transfer that partially mimicks their action in living systems. Moreover, a wide range of electrochemical methods has been developed for the estimation of total antioxidant parameters of plant materials and their extracts ,22,23,24•  and galv•  radicalsSummarizing that mentioned above, development of simple, reliable and cost-effective electrochemical approaches for the evaluation of total antioxidant parameters of essential oils is of practical interest. Constant–current coulometry with electrogenerated titrants has been shown to be a powerful tool for the estimation of total antioxidant parameters of different types of foodstuffs, biological fluids, spice extracts, and phytopharmaceuticals . HalogenThe application of a coulometric approach to the analysis of essential oils will expand its analytical capabilities.No need to prepare a standard solution of the reagents i.e., the standardization stage is excluded;Titration is an absolute that excludes usage of calibration plots ;No effect of sample dilution;Coverage of almost all types of antioxidants based on titrants’ reactivity;Ease of calculation and the possibility of using different standard antioxidants;High sensitivity, reliability, and reproducibility of the measurements;Possibility of automation for routine applications.The aim of the present study is to show applicability of constant–current coulometry with electrogenerated titrants for the essential oils analysis. This type of reagent is favorable vs. radicals or other typical reagents due to the following reasons:• and total phenolic contents has been carried out.Coulometric titrants (electrogenerated bromine and ferricyanide ions) have been used as reactive species. Their reactions with individual antioxidants of essential oils have been studied. The data obtained allow evaluation of the total parameters of essential oils in particular total antioxidant capacity (TAC) and ferric reducing power (FRP). Fifteen types of essential oils from various plant materials have been investigated and their components identification has been performed by GC-MS. The correlation analysis of total antioxidant parameters with antioxidant activity towards DPPHThymol of 99.5% purity , 98% carvacrol, 99% eugenol, 98% syringaldehyde , 99% vanillin and 97% phythol , 99% benzyl alcohol, 96% limonene, 98% α-pinene, 98% β-pinene, 98% D-carvone, 75% camphene, as well as α-fenchene, myrcene, 99.5% L-menthol, and 97% L-borneol  were used as standard antioxidants. Their 10 mM stock solutions were prepared by dissolving an accurately weighed portion in 5.0 mL of ethanol (rectificate). Less concentrated solutions were obtained with an exact dilution prior to measurements. Other reagents were of chemical purity and used as received.The commercially available essential oils of various plant materials  of diffe2 surface area), an auxiliary electrode (platinum wire separated from the anodic compartment of the cell with the semipermeable membrane), and two indicator electrodes (platinum needle electrodes) for the detection of the titration end point. A polarizing potential of 200 mV was applied between the indicator electrodes. The surface of the platinum electrodes was cleaned with nitric acid followed by rinsing with distilled water.Coulometric measurements were carried out on the coulometric analyzer “Exper-006”  supplied with the electrochemical cell consisting of four electrodes: a working electrode (bare platinum wire with 0.5 cm−2 providing their 100% current yield. 0.2 M KBr in 0.1 M sulfuric acid and 0.1 M K4Fe(CN)6 in 2 M sodium hydroxide were used as precursors and supporting electrolytes for the generation of bromine and ferricyanide ions, respectively.Constant–current generation of coulometric titrants was performed at the current density of 5 mA cmCoulometric titration was performed in a 50 mL cell containing 20.0 mL of the precursor in the supporting electrolyte. The generating circuit was switched on until a 40 µA indicator current was achieved. Then, an aliquot portion (10–100 μL) of the antioxidant standard solution or 10-fold diluted essential oil was added to the cell, and the timer was started simultaneously. The titration end-point was detected by achieving the initial value of the indicator current. The timer was stopped, and the generating circuit was turned off. Faraday’s law of electrolysis was used for the calculation of the number of electrons participating in the reaction of individual antioxidants with titrants.The total antioxidant parameters, in particular the total antioxidant capacity and ferric reducing power, were evaluated for the essential oils as the quantity of electricity spent for the titration of the sample and recalculated per 1 mL of the essential oil.v/v) ratio [−1, and finally increased to 280 °C at 20 °C min−1 and kept at 280 °C for 5 min. Helium with a flow rate of 1 mL min−1 was used as a carrier gas. An electron ionization system with an ionization energy of 70 eV was used. Mass spectra were recorded with total ion currents over m/z 20−550 Da. The components identification was performed by comparison of the retention times and retention indices with reported ones [Essential oils were dissolved in diethyl ether in 1:10 (v) ratio  and analv) ratio ,30,31. Tted ones ,33 as weted ones . The NIS• stable radical  was used as a reagent to evaluate the antioxidant activity of essential oils. The standard procedure was applied [• methanol solution and incubated in the dark for 20 min. Then, the absorption was read at 515 nm vs. methanol with the addition of 4 μL of the sample using spectrophotometer PE-5300 . Control DPPH• absorption was measured vs. methanol. Antioxidant activity of the essential oils was expressed as a relative decrease of the DPPH• absorption due to the reaction with the antioxidants of the sample.DPPH applied . Briefly−1) were placed in a 5.0 mL volumetric flask. Then, 2.5 mL of diluted Folin–Ciocalteu reagent (1:10 (v/v))  were added and thoroughly mixed. After 4 min, 2.5 mL of 7.5% Na2CO3 solution were added, mixed and incubated for 1 h. The absorbance of the solution was measured at 765 nm in a 0.5 cm cuvette. The blank solution contained all the reagents excluding essential oil, which was replaced with 0.5 mL of ethanol. Total phenolic content was expressed in eugenol equivalents per 1 mL of essential oil.The Folin–Ciocalteu colorimetric method  with somFive parallel measurements were performed for constant–current coulometry (three replications for chromatography and spectrophotometry). A significance level of 5% was used for the statistical treatment of the experimental data. The results were presented as an average value and a coverage interval. Relative standard deviation (RSD) was used for random error characterization.Correlation analysis was performed using OriginPro 8.1 software .The essential oils under consideration have been studied by GC-MS for the components identification. The number of constituents varied in a range of 30–79 depending on the type of essential oil .m-cymene, limonene, eucalyptol, trans-β-ocimene, γ-terpinene, linalool, α-terpineol, linalyl acetate, α-copaene, β-elemene, trans-β-caryophyllene, caryophyllene oxide I, α-humulene, α- and γ-muurolene. Other terpenoids are more specific to the type of essential oil. The chromatographic data obtained agree well with those reported in [The major components of the essential oils are terpenes and their derivatives as well as phenolic volatile compounds . The most widely distributed terpenes are α-pinene, camphene, β-pinene, myrcene, tricyclene, α-thujene, orted in ,37,38,39Such composition of essential oils suggests the possibility of using constant–current coulometry with electrogenerated titrants to assess their antioxidant properties. Coulometric titration of essential oils antioxidants has been performed for confirmation.The reactivity of individual antioxidants of essential oils toward coulometric titrants (electrogenerated bromine and ferricyanide ions) has been studied. These two reagents are very different in properties.Oxidation of bromide ions at a platinum electrode in acidic medium results in the formation of tribromide anion, molecular bromine and short-living bromine radicals adsorbed at the electrode surface . TherefoElectrogenerated ferricyanide ions show relatively weak oxidative properties. On the other hand, it acts as a one-electron oxidant that prevents possible accompanying steps that complicate the reaction.Reactions with electrogenerated bromine proceed rapidly and quantitatively for most of the antioxidants studied . The number of electrons participating in the reactions calculated using Faraday’s law is consistent with the number of reactive functional groups in their structure. The only exclusions are benzyl alcohol, L-menthol and L-borneol  that do o-quinone with the participation of two electrons as well as the electrophilic addition of bromine to double bond occurs. As for the isopropylmethylphenols (carvacrol and thymol), both oxidation and bromination reactions take place, leading to the formation of several products, in particular thymoquinone and mono- and dibromo derivatives [Eugenol, carvacrol and thymol  interactivatives ,41.Terpenes and their derivatives  interactThus, reactions of all studied compounds except myrcene and limonene proceed with the participation of two electrons that agree well with the number of double bonds in their structure. Similar behavior has been found for limonene, for which four electrons are involved in the reaction with titrant that means bromination on both double bonds. Myrcene also reacts with electrogenerated bromine with four electrons transfer i.e., one of the double bonds is inactive, which is probably caused by sterical effects. Therefore, conjugated double bonds undergo a reaction .Ferricyanide ions react with phenolic antioxidants, only allowing to conclude that phenolic hydroxyl groups are oxidized that agree with data for hydroxybenzoic acids  and flavThe number of electrons participating in the reactions of all antioxidants under consideration with electrogenerated titrants are reproduced in repeated measurements and are consistent with that reported for some of them , which cTotal antioxidant parameters of the essential oils in particular TAC and FRP based on the sample’s titration with electrogenerated bromine and ferricyanide ions, respectively, have been estimated. A screening of 15 types of essential oils has been carried out .The highest values of both parameters have been obtained for clove essential oils, which is caused by the high content of eugenol being the major constituent. Its concentration in clove essential oil is usually at least 50% . The low−1) of the neroli essential oil is caused by the presence of terpenoids, in particular β-pinene, limonene, linalool and linalyl acetate reacting with electrogenerated bromine. Other essential oils that are free of volatile phenolics show significantly less TAC (83–459 C mL−1), which is reflected in their terpene constituents.High TAC  based on spectrophotometric data have been evaluated  and applicability in routine analysis.Coulometric titration with electrogenerated bromine and ferricyanide ions has been suggested for the evaluation of the essential oils’ total antioxidant parameters. Data for the reaction of individual antioxidants (volatile phenolics and terpenes) with coulometric titrants confirm the applicability of the method for the characterization of essential oils TAC and FRP. Screening of 27 samples from 15 types of essential oils has been performed and statistically significant difference between the samples has been observed. The data obtained agree well with the standard spectrophotometric approaches confirming the accuracy of the coulometric methods. Disadvantages of spectrophotometric methods like instability of DPPHThus, coulometric titration has been shown to be an effective and promising tool for the characterization of the essential oils via total antioxidant parameters that enlarge the practical application area of the method.


Punica granatum L.), which contains high levels of health-promoting compounds, has received much attention in recent decades. Fruit storage potential ranges from 3 to 4 months in air and from 4 to 6 months in Controlled Atmospheres (CA) with 3–5% oxygen and 10–15% carbon dioxide. Storage life is limited by decay, chilling injury, weight loss (WL), and husk scald. In particular, husk scald (HS) limits pomegranate long-term storage at favorable temperatures. HS appears as skin browning which expands from stem end towards the blossom end during handling or long-term storage (10–12 weeks) at 6–10 °C. Even though HS symptoms are limited to external appearance, it may still significantly reduce pomegranate fruit marketability. A number of postharvest treatments have been proposed to prevent husk scald, including atmospheric modifications, intermittent warming, coatings, and exposure to 1-MCP. Long-term storage may induce phenolic compounds accumulation, affect organelles membranes, and activate browning enzymes such as polyphenol oxidases (PPO) and peroxidases (POD). Due to oxidation of tannins and phenolics, scalding becomes visible. There is no complete understanding of the etiology and biochemistry of HS. This review discusses the hypothesized mechanism of HS based on recent research, its association to postharvest treatments, and their possible targets.The pomegranate ( Punica granatum L.), is likely native from Iran and grown in areas with a Mediterranean environment [One of the most well-known fruits in the world, the pomegranate  are typically more severe, and so it can also happen at storage temperatures above 10 °C and these scalded portions of the fruit may become prone to fungal deterioration in advanced stages . Table 1Although there is a lack of information on scald disorder, the review’s goal is to address factors that impact pomegranate husk scald development, as well as the potential mode of action involved.Weight loss, microbiological contamination, oxidative stresses, and browning are important variables that might decrease the storage life of fruit and vegetables during cold storage and handling. Lower or mild degrees of stress tend to enhance the levels of cellular antioxidant compounds, whereas severe stress may result in a decrease in such levels and the development of postharvest physiological disorders ,21,22. Ro-dihydroxy phenols , as well as the dehydrogenation of o-dihydroxy phenols into o-quinones. Polyphenol oxidase (PPO) and peroxidase (POD) are the main enzymes that cause browning in plants. PPO is a type of enzyme known as an oxidoreductase. It catalyzes the conversion of monohydroxy phenols  into Peroxidases (POD), which perform mono-electron oxidation on a wide range of composites in the presence of hydrogen peroxide, are additional enzymes which have polyphenol oxidation activity . POD is 48H28O30) is the most abundant polyphenol in pomegranate skin [As illustrated in ate skin , which aate skin . The PG ate skin . By releate skin . Althougate skin .o-dihydroxy phenols detected in the skin affected regions. Although the amount of o-dihydroxy phenols in pomegranate skin is quite little, it has been demonstrated that during storage, husk scald can occur as result of the enzyme-mediated oxidation of phenols present in the skin [It was discovered that in scalded pomegranate fruits, PPO and POD activity increased while total phenolic and total tannin components decreased . Additiothe skin . Scaldedthe skin .Temperature has a considerable impact on the rate of enzymatic browning. Fruit and vegetables do not become brown when stored at low temperature, and PPO’s enzymatic activity ceases at temperatures below 7 °C even though it is not deactivated .2O2 with the decline of CAT activity made the tannins oxidize to browning substances. In the peel of scalded fruit, it was shown membrane instability, increased lipid peroxidation, and enhanced carotenoid production, which may suggest a response to oxidative stress [In addition, CAT activity in pomegranate peel negatively correlated with the browning index while ascorbic acid oxidase (AAO), PPO, and POD favorably correlated with the skin browning index . Accordie stress .Punicalin levels were strongly and negatively connected with husk scald, but punicalagin had no significant correlation to the incidence of scald. This finding raises the possibility that high punicalin levels could prevent the occurrence of HS in pomegranate fruit .The symptoms and occurrence of pomegranate and apple scald are similar, however, current research indicates that the biochemical reasons and control mechanisms of the two disorders are different. Diphenylamine (DPA) treatment as an antioxidant ingredient, for instance, can decrease apple scald, but it has no effect on pomegranate scald ,41. AlthAnthocyanins are water-soluble polyphenolic pigments that are responsible for the appealing red-violet-blue hues of many fruits, especially pomegranate arils and husks . AnthocyThe findings imply that neither the total soluble solids content nor the titratable acidity or husk color are connected to the development of husk disorders .The study on the anatomical changes in HS of pomegranate skin showed cracks in the epidermis and cuticle as well as cell degradation only in the very outer layers of the skin, while the inner layers seemed intact . HS fruiPgr023188 encoding uncharacterized protein upregulated in HS fruits, this gene has 50% similarity to gene BVC80_1667g87 that encodes for glycine rich protein (GRPs). These genes are involved in increasing tolerance during water stress [Pgr000486 and Pgr006284), defense (Pgr011016), oxidative stress (Pgr006284) and Glycosyltransferase (GTs) (Pgr007593) which is crucial for the biosynthesis of secondary metabolites were highly presented in HS skin [Gene r stress . Moreove HS skin . HoweverA possible mechanism of husk scald development is summarized in Losing selective permeability in the presence of oxygen may lead to membrane leakage and interaction between enzymes  which are located in the cytoplasm with substrate . Higher ratio of PPO/PAL activity is associated with oxidation of phenolic compounds to quinones which creates colored brown products . EventuaTo avoid this phenomenon of HS in pomegranate during storage various methods were developed in the past as postharvest treatments without knowing the mode of action of HS development. However, we can infer according to the proposed model herein  that theInvestigating postharvest treatments preventing husk scald disorder, is helpful in order to confirm the possible mechanism of HS presented herein. Numerous methods and strategies for postharvest storage of pomegranate avoiding husk scald will be discussed in this review paper and their possible targets.Adverse environmental conditions can disrupt normal homeostasis leading to cell death in plant cells and consequently physiological disorder may appear . The tolFor instance, the results of a research on seven pomegranate accessions showed that higher antioxidant capacity, total phenolic compound, and punicalin caused lower WL, husk scald incidence and fungi decay in fruit. On the contrary, the amount of punicalagin had not significant correlation with the scald incidence. In addition, anthocyanin content did not show any significant correlation with HS incidence. However, light colored fruit were more sensitive to chilling injury and husk scald, regardless of having high antioxidant activity . FurtherThere was a correlation between catechol content and peel browning in pomegranate fruit harvested in late season, and there was a stable catechol level in less browned peel during storage . In addiResearch by  showed tIn In 2 and O2 concentrations surrounding the commodities, and can often help to avoid CI or delay the onset of symptoms [Controlled atmosphere (CA) and modified atmosphere (MA) storage include altering the storage gas environment, specifically the COsymptoms .2 to CO2 ratio can prevent peel browning [2, without CO2, reduced husk scald and postponed the onset of chilling injury symptoms in Israeli “Wonderful” pomegranate at 2 °C, but husk scald persisted after removal from storage. “Mollar” pomegranates were kept for up to eight weeks at 5 °C and at or above 95% relative humidity (RH) in air and in various combinations of controlled atmospheres, including 10% O2 + 5% CO2, 5% O2 + 5% CO2, 5% O2 + 10% CO2, and 5% O2 + 0% CO2 ethylene-free. Except at 10% O2 + 5% CO2, controlled atmospheric storage decreased WL, the risk of decay, and the severity of husk scald.Even though pomegranates do not have climacteric respiratory systems, a suitable Obrowning . The rat2 content had a stronger effect in preventing the rate and severity of husk scald after 5 months of storage at 6 °C [2 + 15% CO2, has the highest success rate for decreasing decay and husk scald problems of all of these methods, it has been shown to prolong the postharvest life of pomegranates for up to 5 months at 7 °C [2 levels [In contrast to the level previously suggested for the “Wonderful” cv., the “Mollar” cv. appears to be more vulnerable to husk scald than other types. Different cultivars may exhibit varying sensitivity to this disorder . Higher  at 6 °C . The use at 7 °C ,18. Furt at 7 °C . On the 2 levels .Browning reduction may relate to increased membrane integrity and decreased peroxidase (POD) and polyphenol oxidase (PPO) enzyme activity . Enzymatp-coumaric acid (LPCAC) isolated from the neutral lipids fraction of peel tissue lipid extracts in air-stored fruit increased nearly three-fold than CA-stored fruit [It was shown that loss of phospholipid content in pomegranate skin cells that were stored in air was lower than CA-stored fruit. However, lipophilic conjugates of ed fruit . LPCAC aed fruit . Furthered fruit .In general, these results are in agreement with the proposed model of HS development presented herein , where wIt has been shown that modified atmosphere packaging (MAP) can increase storage life by three months or more and prevent water loss, visible shriveling symptoms, husk scald, and rot in ‘Mollar de Elche’ , ‘GaneshMAP started to be used frequently for pomegranate shipping and storage. The effect of MAP pomegranate was studied using unperforated polypropylene (UPP) film of of 25 μm thickness and Perforated polypropylene (PPP) film of 20 μm thickness in 2 °C and 5 °C storage.PPP at 5 °C was the best treatment for maintaining red skin-color of the arils at the end of storage, however, husk-scald development was higher at 5 °C possibly because of an increase in polyphenol oxidase activity. Additionally, only unpackaged control at 5 °C fruits showed a moderate  level of pitting and husk-scald severity index . 2 concentration in MAP has been found effective in maintaining higher antioxidant activity enzymes including CAT, SOD and APX [2 concentration, POD and PPO do not catalyze phenols oxidation [Although there are several studies on the effectiveness of MAP for prevention of HS, there is not any information about antioxidants, phenolic compounds or anthocyanin alteration in the fruit skin during storage under MAP treatment. However, in litchi fruit, MAP reduced skin browning through a reduction in PPO and POD activities . Brownin and APX , on the xidation . Based oxidation  and sincIt is likely that MAP systems may operate by different modes of action against HS, likely through reduction in water stress and the corresponding oxidative stress signals, and simultaneously by reducing oxygen levels that reduces oxidative stress within the cells . −1 fludioxonil was utilized [Pomegranates were preserved at 8 °C for 6 or 12 weeks, and a combination of film wrapping and 600 mgLutilized . The resIntermittent warming is the interruption of low-temperature storage with one or more short periods of warm temperature for various periods of time. It has been shown to be beneficial in reducing CI and improving in keeping quality of several horticultural crops, presumably by allowing the continuation of aspects of ripening that had been halted by the low temperature storage . IntermiRef.  compared2 + 8.0% O2 could delay browning, with the browning index being 0.15 after 120 days of storage. The results showed that the activities of the browning index of peel were correlated positively with ascorbic acid oxidase (AAO), PPO and POD, while correlated negatively with catalase (CAT) activity. The application of intermittent warming and pomegranate fruit storage under the conditions of 5% CO2 + 8% O2 gas component could delay peel browning [The application of intermittent warming for 24 h at 15 ± 0.5 °C every 5 days for pomegranate fruit, stored at 5.0 ± 0.5 °C and 5.0% CObrowning .Although the mode of action of intermittent warming is not clear in HS, it is possible that different mode of actions might be taking place; on the one hand, changes in membrane fluidity and reduced oxidative stress may be favored  or alterFruit and vegetables are coated to extend their shelf life. Coatings can be made of protein, lipid, polysaccharide, resin, or any combination of, as well as originate from both plant and animal sources. During processing, handling, and storage, coating creates a barrier for gases and moisture and also serve as natural delivery systems for postharvest chemicals ,85,86,87It has been reported that 1-MCP can minimize superficial apple and pear skin scalding and improve the storage and shelf life of fruit ,94.Pomegranate is a non-climacteric fruit; studies have shown that some non-climacteric fruits can benefit somewhat from 1-MCP ,96,97,98−1, nor 1-methylcyclopropene at 1 μLL−1, alone or together reduced scald incidence and severity in pomegranates. On the contrary, it has been reported that the peel browning of ‘Dahongpao’ pomegranates was significantly reduced using 0.25, 0.5, and 1 μL/l 1-MCP concentrations, possibly related to a reduction in PPO activity [2 + 5% CO2) and air on “Wonderful” pomegranate. The results showed that 1-MCP can prevent husk scald in pomegranates stored in air storage. The partial control or delay of scald symptoms by 1-MCP shows that the biochemical mechanism(s) causing scald are activated, but not entirely by ethylene [Ref.  comparedactivity . Moreoveactivity  examinedethylene ,102. Furethylene ,103.Even though non-climacteric fruit do not even actually ripen, the low levels of ethylene they release are likely involved in the fruit’s senescence, therefore treating them with 1-MCP may have some advantages. Pomegranates may benefit from lower husk scald and superior aril appearance and taste due to an inhibitory influence on the fruit’s metabolic activity, which leads to senescence. More work has to be conducted to elucidate the role of ethylene in HS development before promoting 1-MCP to control HS development.Polyamines (PAs) are compounds naturally synthesized in plants and have roles in development, ripening and senescence processes. Putrescine (PUT), spermine (SPE), and spermidine (SPD) are common PAs applied externally to increase the shelf-life of fruit . The appPomegranate is an exotic fruit with high valuable nutritional benefits. This fruit even has the potential to be considered a functional or medicinal food that recently has received much attention among consumers. Due to increased consumptions and demand, extending the marketing season, creating the opportunity for the sustainable consumption of fresh pomegranate and/or its availability to the processing industry by prolonging the storage life of pomegranates is crucial. HS, on the other hand, has a significant negative effect on pomegranate marketability. The results of this review paper and the hypothetical model described herein suggest that storage duration more likely triggers a cascade of oxidative changes that eventually cause a wound-like effect within skin cells and leads to skin enzymatic browning. This process of HS development apparently starts with water loss and a sequential oxidative stress mechanism. Further studies are needed to confirm water loss as the triggering mechanism of HS development. It is interesting that all of the successful postharvest treatments proposed so far in the literature to halt HS development can be explained by the proposed model of HS development described herein, either through control of water loss, control of oxidative stress, maintaining membrane integrity or through redirecting of protein synthesis of key proteins/enzymes involved in HS development. Furthermore, studies are needed to determine the specific phenolics  involvedThis review paper on HS development and mode of action can be the basis to revisit known postharvest treatments and to find and develop new techniques that can prevents HS development. For instance, the combination of treatments using a hurdle approach based on mode of actions of individual treatments may allow us to obtain additive or synergistic effects on the reduction in HS incidence. In spite of the fact that there are a number of studies controlling HS in the literature, proposing a model describing scald etiology and its prevention or even prediction seems essential for developing novel approaches to reduce HS.


We aim to contribute to the assessment of the screening performance in Flanders (Belgium) and to identify valuable mammograms for subsequent studies and training.Initially negative prior screening mammograms (sMx) of 210 women with confirmed breast cancer detected by the Flemish screening programme between 2011–2013 were reviewed by a highly experienced radiologist. The review of the prior sMx was performed in three steps: 1) only prior mammograms available; 2) with index sMx (=subsequent positive sMx) present; 3) with index sMx and clinical information present.The radiological review yielded 94 (45%) mammograms ‘without suspicious lesions’, 77 (37%) ‘with minimal signs in at least one breast’, and 39 (19%) ‘with clearly visible tumours’. In univariate analyses, the reclassification of prior sMx was significantly associated with the date of the prior sMx, the need for a third reader for arbitration, image quality and the detector system used , and it was not associated with the interval between screening rounds, age at prior sMx, breast density, or tumour characteristics . In multivariate analyses, the date of the prior sMx (p = 0.001), need for arbitration (p = 0.001) and image quality (p < 0.001) remained significantly associated with the reclassification.This retrospective review reclassified 19% of the sMx as clearly visible tumours. With this, the Flemish screening programme performs in accordance with similar studies. The sMx reviewed in this study, form a valuable set of mammograms for training and further research. Breast cancer screening programmes have substantially increased the number of early detected cancers . However23To improve cancer detection by mammography screening the European guidelines advise quality control using predefined performance indicators and quality assurance including review and training. An important performance indicator is rating interval cancers (breast cancers arising after a negative screening episode and before the next scheduled screening round). Performing a radiological review of prior screening mammograms (sMx) of interval cancers is part of the quality assurance and also an important teaching tool . Screen-31This study comprises a review of confirmed breast cancer cases detected by the Flemish screening programme. The aims were to quantify the proportion of visible tumours on the prior sMx and to gather insight into associated variables that may hinder cancer detection, such as breast density, age, image quality, imaging technique, tumour size, type of tumour, need of arbitration, screening interval, and date of prior sMx. The study also aimed to identify a valuable set of sMx for training and subsequent studies.In the breast cancer screening programme in Flanders, biennial two-view mammographic screening is offered free of charge to women aged 50–69 years. Two radiologists (first and second reader) independently evaluate the screening mammograms, with third reader arbitration if needed.Between 2009–2013, 254,350 women participated in the Flemish Breast Cancer Screening Programme. From this group, cases for review were selected based on the following inclusion criteria: 1) informed consent for use of data in scientific research, 2) participation in minimum two consecutive screening rounds, 3) a screening interval of 16–30 months, 4) the index sMx (latest sMx) in 2011, 2012, or 2013 resulted in a referral for further diagnostic workup confirming and correctly documenting breast cancer, 5) where the prior sMx (previous sMx) was considered negative, 6) where the index and prior sMx were digital and available in the PACS (Picture Archiving and Communication System) at the Centre for Prevention and Early Detection of Cancer. In total, 292 cases met these inclusion criteria. From those a predefined sample size of 210 was selected by standard SPSS algorithms for random selection.The 210 prior sMx were thoroughly reviewed by a single, highly experienced radiologist . The review followed a stepwise procedure: 1) review of prior sMx, in the absence of other images or information, 2) review of prior sMx with index sMx (subsequent positive screening mammogram) present, and 3) review of prior sMx, where index sMx and clinical information on tumour localization and characteristics  from diagnostic follow up were present. All steps were performed per case in succession. The expert radiologist reviewed all prior sMx for the presence of malignancy, the image quality, and breast density. The reviewing radiologist was not informed of the purpose of the study.Possible associations between relevant variables and the intermediate (step 2) or final classification (step 3) were studied in univariate (chi-square) and multivariate analyses (logistic regression). 1) Breast density , 2) age , 3) image quality (good/not good), and 4) imaging technique  were considered as relevant variables with a possible association with non-detection of a visible cancer. Also 5) tumour size (<T2 versus ≥T2), 6) type of tumour (in situ versus invasive), 7) the need of a third reader for arbitration during the original reading process of the prior sMx , 8) the interval between prior and index screening , and 9) the date of screening of prior sMx  were tested. Tertiles were used instead of screening years due to an imbalanced distribution of cases across calendar years see .Because of the limited number of clearly visible tumours in the intermediate and final classification, bootstrap validation with bias correction and accelerated bootstrap interval was performed. Statistical significance was set at p < 0.05.In the multivariate analysis, the group of clearly visible tumours was first compared with the compound group of minimal and no signs, subsequently the group of clearly visible tumours was compared with the no signs group only.The sMx dataset contained images of 102 left, 103 right, and 5 bilateral breast cancers.The results of the expert review are summarized in By reviewing prior sMx alone (step 1), 24 of the sMx (11.4%) were labelled ‘probably malignant’ and might have been referred. The intermediate classification (step 2), prior sMx with index sMx present, identified 25 cases (11.9%) with ‘clearly visible tumours’. The final classification of prior sMx (step 3), including the use of index images and clinical information, revealed 39 ‘clearly visible tumours’ (18.6%).The intermediate classification was significantly associated with the date of prior sMx (p =< 0.001) and the need of arbitration on the prior sMx (p = 0.002). The final classification was significantly associated with the date of the prior sMx (p =< 0.001); the need of arbitration (p = 0.004), also with the image quality (p = 0.004) and the detector system used (CR versus DR) (p = 0.036). See When clearly visible tumours were compared to the compound group of minimal and no signs, the need of arbitration on the prior sMx (p = 0.005) and the date of the prior images (p = 0.044) were independently significantly associated with false negative clearly visible tumours in step 2 . When clearly visible tumours were compared only to the group of no signs, the significance level for the need of arbitration (p = 0.001) and date of priors (p = 0.004) appeared even higher.In step 3, the final classification , the need of arbitration (p = 0.001) and the date of the prior images (p = 0.006) were still independently significantly associated with false negative clearly visible tumours. Furthermore, the image quality was statistically significant (p < 0.001). These conclusions held, whether comparing to the compound group of minimal and no signs or only to the no signs group. See All statistically significant associations were confirmed by bootstrap validation.This review of a substantial set of ‘initially negative’ prior sMx resulted in 39 (19%) being labelled as ‘clearly visible tumours’. This result is in accordance with similar studies 7. It conThe 19% missed tumours cannot automatically be considered ‘screening errors’, for several reasons:the proportion of cases with ‘clearly visible tumours’ based on image review alone was 1/3 lower, at 12%. The availability of clinical information is known to alter the reading outcome 9.Even if we tried to reproduce the conditions of routinely assessing sMx in the screening programme, the radiologist’s attention was presumably triggered by the clustering of challenging image sets, the slightly different protocol form, the specific categorization, and the stepwise assessment for the review 9.The normal response of the human mind to low probability events  can be a substantial contributor to false negative errors in breast cancer screening .Therefore, the clustering of challenging sMx in this study may have affected the reader’s awareness and the results of the review.The image quality was significantly associated with the final categorisation of clearly visible tumours. This confirms the importance of a good image quality and therefore requires special attention .In order to obtain a sufficient number of prior sMx we had to include sMx from the early stages of digital mammography screening in Flanders. The ‘date of screening’ effect may reflect a learning curve for the radiologists involved in the screening programme.In several studies, DR detector systems seem to be superior to CR detector systems, also in clinical screening performance. Often higher sensitivity is found with higher cancer detection rates and less interval cancers, especially in dense breasts 1011.10Since this review was performed by a single – albeit highly experienced – radiologist, the results of this retrospective review could not be corrected for inter-observer variability. This is a major limitation of this study.The radiological review yielded 94 (45%) mammograms ‘without suspicious lesions’, 77 (37%) ‘with minimal signs in at least one breast’, and 39 (19%) ‘with clearly visible tumours’. These results are in line with similar studies.The screening mammograms assessed in this review are valuable for training and subsequent studies.All relevant documentation or data in order to verify the validity of the results presented is available, but not openly. Due to the nature of this research, participants of this study did not agree for their data to be shared publicly.