Rare earth elements (REEs) are important raw materials for green technologies. However, REE mining and production uses techniques that are often not environmentally sustainable. Life cycle assessment (LCA) is a well-recognized method for evaluating the environmental impacts of products and technologies. This article provides an overview of the environmental impacts based on published LCA results of primary REE production. Existing major REE deposits  and currently possible production routes are compared. Alternative minerals, such as eudialyte, are also discussed. The article shows which environmental effects can be minimized by technology optimization and environmental safety strategies. Additionally, some of the environmental impacts discussed, may be difficult to mitigate, as they depend on the mineral type. Activities along the complex process chain of REEs production that have particularly high environmental impacts are identified. In the last five years, at least 27 studies were conducted evaluating environmental impacts of various REE products and supply chains. For example, Schulze et al.2 and Deng and Kendall3 considered REE production from ion-adsorption clays (IACs), Marx et al.4 and Arshi et al.5 compared REE production from various mineral types, Vahidi and Zhao analyzed in particular the separation of REEs through solvent extraction6 and the production of RE metals via molten salt electrolysis,7 and Schreiber et al.1 published a comprehensive review of LCA activities involving primary REE production. Though detailed industry data are normally not available, LCA studies provide sufficient insight in the cause-and-effect chains to allow a basic evaluation of various primary REE supply routes.Ever since the increasing importance of rare earth elements (REEs) for green technologies, such as wind turbines, electric vehicles, and low-energy lighting, there has been a parallel discussion concerning environmental impacts caused by the mining and production of REEs. The growing interest was disclosed in a recent review of life cycle assessment (LCA) studies,8 recommending that the EU Commission introduce an EU-wide supply chain law, the discussion of environmental impacts will gain further momentum. For example, in June 2021, the German Bundestag had already passed a law on corporate due diligence in supply chains.9 The law aims to better protect human rights and the environment in the global economy. These include the prohibition of child labor, protection against slavery and forced labor, adequate wages, the right to form trade unions, and access to food and water. The law also considers risks to the environment, such as the release of hazardous substances or polluted water.Like all material processing, the REE supply chain is also associated with environmental impacts. They are mainly related to the geology of a deposit, mineral type and composition, the methods of extraction, local supply of energy and auxiliary materials, and regulatory conditions that mitigate environmental impacts. Hence, the environmental impacts vary considerably. In the course of the “Legislative Report on Human Rights and Environmental Due Diligence of Businesses” (adopted by the European Parliament in March 2021),10 and Bayan Obo is its largest deposit. The ore from Bayan Obo is processed in Baotou, 150 km away. REOs are also produced in Sichuan and from IACs in several southern provinces. It is difficult to find precise production data because illegal mining produces a significant amount of REOs, which is especially true for IACs.11 Also, for legal mining reliable public data are scarce. Since 2015, there have been serious attempts to consolidate China’s RE industry into six large corporations controlled by the central government or by provincial and municipal governments to establish industrial order, address environmental issues, and internalize pollution costs.12China represents the largest REEs producer worldwide with 140,000 t rare earth oxide (REO) equivalents in 2020 (58%)10 Currently, RE production only takes place up to RE concentrates at Mountain Pass. The final processing occurs in Asia.Following a temporary shutdown of the Mountain Pass production site in 2016 and 2017, the United States is again the world’s second-largest producer with 38,000 t REO in 2020 (16%).10 The Australian production decreased significantly in 2020 compared with 2018 and 2019, both with 21,000 t of REO,10 due to the COVID-19 pandemic. Australian’s mining company Lynas Corporation operates the Mt Weld mine in Western Australia and a processing plant in Gebeng, Malaysia (near the city of Kuantan), where the RE concentrate from Mt Weld is further processed to individual REOs.Australia is currently the fourth-largest producer with 17,000 t REO in 2020 (7%) after Myanmar with 30,000 t REO (12.5%).13 for example, in Kvanefield (Greenland) and Norra Kärr (Sweden),14 only very limited mining activities have occurred thus far. Other potential mining sites outside Europe include Thor, Hoidas, and Strange Lake (Canada), Bear Lodge (United States), Nolans Bore , and Steenkampskraal (South Africa),15 of which only the last two projects have achieved “permitted status” to date.16Although some European deposits are already identified,17 Smelting capacity in Japan and other Western countries is negligible.Not only mining and processing of REOs are concentrated in China but also most of the RE smelting industry (95%). Some smaller capacities exist in Vietnam and Laos (5%).3) and monazite (REPO4), bastnäsite prevails in Sichuan and Mountain Pass, monazite in Mt Weld, and eudialyte (N15 [M(1)]6 [M(2)]3 [M(3)] [M(4)] Z3 [Si24O72] O’4X2 with N=Na, Ca, K, Sr, REE, Ba, Mn, H3O+; M(1)=Ca, Mn, REE, Na, Sr, Fe; M(2) = Fe, Mn, Na, Zr, Ta, Ti, K, Ba, H3O+; M  = Si, Nb, Ti, W, Na; Z = Zr, Ti, Nb; Oʹ = O, OH−, H2O; X = H2O, Cl–, F–, OH–, CO32–, SO42–, SiO44–)18 in Norra Kärr. IACs are in the Chinese southern provinces (CSP) and in Myanmar. In IACs, REOs are adsorbed on the surface of alumino-silicate minerals 4] * REE). The mineral types affect the level and type of environmental pollution as well as the applied technology.Various deposits present different mineral types. While the Bayan Obo ore is a mixture of bastnäsite  follows. In Baotou and Kuantan cracking is done by roasting with sulfuric acid (H2SO4). In Sichuan, the pure bastnäsite is cracked via hydrochloric acid leaching. Eudialyte (Norra Kärr) is cracked by a dry digestion with hydrochloric acid (HCl) .20 The former Molycorp Inc cracking process (at Mountain Pass) was subject to confidentiality, so no technical details were provided. It is assumed that HCl was used for cracking as chlor-alkali electrolysis was available at the site. The REE salts are then separated from remaining gangue and secondary elements by different leaching and precipitation processes. Solvent extraction to separate the REEs follows the same procedure for all process chains. The separated REE chlorides are precipitated as carbonates or oxalates. These are calcined to REO. Reduction to the metal is carried out by molten salt electrolysis , calciothermic (Dy), and metallothermic reduction (Sm).7The general process chains for bastnäsite, monazite, bastnäsite/monazite mixed ore, and eudialyte are similar, even if they start from different minerals  and the very low concentration of ThO2 and U3O8 with 26 ppm and 18 ppm, respectively, containing the radioactive elements 232Th and 238U.24 The activity (decay rate) is 3566 Bq 232Th/g ThO2 and 10,470 Bq 238U/g U3O8.25 Monazite contains up to 200,000 ppm ThO2 and up to 160,000 ppm U3O8.26 However, for the monazite deposit of Mt Weld lower values are given .25 For the mixed bastnäsite/monazite deposit at Bayan Obo, the ThO2 concentration is reported to be 320 ppm,27 400 ppm,26 and 700 ppm25 and the U3O8 concentration is given as 2 ppm.25 For the bastnäsite deposit in Sichuan, 400 ppm ThO2 and 40 ppm U3O8 are reported; for Mountain Pass 200–10,000 ppm ThO2 and 20 ppm U3O8.25 The radioactivity of IACs is generally low28 and is reported to be 50 ppm each for ThO2 and U3O8.25 Other environmental impacts are particularly high when REEs are extracted from IACs, as much wastewater is produced with high concentrations of ammonium sulfate  and heavy metals.16 In general, a lot of tailings, which are mixtures of crushed rocks and processing fluids from mills and concentrators, and waste are produced from REE processing that is partially radioactive and highly hazardous. The activity of the leaching sludge and acidic process slag is much higher than that of the raw ores.29 The waste contains the acids and organic solvents.16 New techniques such as bioleaching and molecular recognition technology instead of solvent extraction could save a lot of energy and waste.16 However, these techniques exist so far only in the laboratory, and it will take years to reach technical maturity at industrial production scales.Pure bastnäsite is easier to beneficiate than mixed ores or pure monazite and, therefore, generates less environmental impacts.31 The energy demand cannot be influenced very much because drilling expenses for blasting and transport within the mine depend on the specific site conditions.Radioactive dust caused by blasting and mine sewage contribute mainly to the environmental impact categories: particulate matter formation (PM) and ionizing radiation (IR). The quantities of blasting agent depend on the stripping rate. The more rock that has to be blasted and removed, the more dust is generated. The amount of dust can be reduced by irrigation systems, as provided in Mountain Pass and Norra Kärr. Mine sewage contains heavy metals that are released into the soil when discharged uncontrolled, such as in China.4 The environmental impacts of flotation result from energy requirements and from the production of flotation chemicals. However, they do not play a major role in assessing the environmental impacts of the overall chain, as they are only added in small quantities (approximately 3 kg/t ore). Flotation tailings (up to 40 t/t REO depending on the flotation process) are discharged into open tailings ponds. Depending on the site-specific construction features of the tailing ponds , heavy metals and inorganic phosphorus-containing compounds may seep into the soil. The leakage rate is high for Baotou deposit because the tailings pond has no liner system or vegetation cover. Because the dam is located about 35 m above the Yellow River, the tailings pond poses increasing toxicity risks to water, soil, and air through leakage, dust formation, and rain erosion.26 The Baotou tailings are contaminated with radioactive thorium with a mean concentration of 5% and high concentrations of dissolved solids, chlorides, sulfates, fluorides, ammonium, boron, manganese, and iron.26 Western sites such as Mountain Pass and Mt Weld are equipped with liner systems and can respond to leakage.32 However, even at these facilities, the tailings ponds have a high potential for environmental damage should accidental releases occur.Crushing, grinding, and (magnetic) sorting require energy  that depends on the hardness of the rock, which is highest for the Bayan Obo deposit, followed by the Mountain Pass, Mt Weld, and Norra Kärr deposits.2SO4 (as used in Baotou and Kuantan) and different reactions with HCl   Figure . The coneactions  and 2),,2SO4 , and their production is associated with considerable environmental pollution that clearly contributes to the environmental impacts of the process chain. They can be reduced by recycling of chemicals , as was done at Mountain Pass. The resulting leaching sludge has varying levels of radioactivity depending on the ore. The limit for classification as radioactive waste (1 Bq/g for 232Th) is often significantly exceeded by the leaching sludge. As already mentioned for the flotation tailings, the environmental impacts of leaching sludge depend on the type of storage. When operated in accordance with regulations, leakage occurs through infiltration. Liner systems can reduce the environmental impacts.After cracking, further gangue and accompanying elements are separated from the aqueous sulfate solutions. The leaching and precipitation processes require large amounts of chemicals  are consumed.23 The requirement of chemicals is given by the share of each REE in the ore. However, demand can be significantly reduced, for example, by HCl recycling4 and reprocessing of wastewater.The separation of the individual REEs is very complex, due to their similar physical and chemical properties. So far, solvent extraction is the most common method for industrial production. Up to hundreds of mixer and settler stages may be assembled to separate all the individual REEs. This process leads to a high demand of chemicals, especially HCl. Compared to inorganic chemical consumption, only small amounts of organic chemicals such as the extractants P204 (2-ethylhexyl phosphoric acid mono 2-ethylhexyl ester) and N235 , lithium fluoride (LiF)), graphite (anode), and tungsten or molybdenum (cathode). Production of electrolytes, electricity consumption, and used cathodes contribute most to the impacts. The amount of REF3 and LiF could be reduced considerably by dust filter and recycling.33 In contrast, the electricity consumption of approx. 8–12 kWh/kg RE metal (current efficiency is 75–80%)7 cannot be reduced significantly because a voltage reduction would lead to a lower energy input into the electrolysis cell and thus to a disturbance of the heat balance. The size of the metal reduction plant plays a central role for the environmental impact. It is decisive whether it is a small backyard plant of 3–4 kA or a large state-owned plant of 30 kA equipped with an exhaust gas scrubbing.33 Lower environmental impacts can be achieved by decarbonized electricity generation, automated process control, exhaust gas cleaning, and recycling of used electrolytes.4 This effect was shown in studies that compared various Chinese electrolysis scenarios.33Besides REO, the main inputs into the molten salt electrolysis are electricity, electrolytes . The amounts of ammonium sulfate reported in the LCA studies vary substantially from 4 kg3 to 80 kg21 per kg REO due to uncertainty in publicly available operating data. The impact category marine eutrophication (EPMarine) is mainly affected by ammonium emissions into soil and water, while the increase of other impact categories , eutrophication potential freshwater (EPFreshwater), human toxicity potential (HTP), particulate matter formation (PM), global warming potential (GWP)) is influenced by the production of ammonium sulfate.21 The high ammonium sulfate concentration of 3500–4000 mg/l found in groundwater35 suggests a high consumption of ammonium sulfate for in situ leaching. On the other hand, this suggests that the leaching agent remains in the soil and subsequently pollutes the groundwater.Large quantities of ammonium sulfate are used as leaching agent during 37 There is also a lack of data on the real environmental impacts of mine wastewater as well as effluents from flotation, exhaust gas scrubbing, leaching, and wastewater neutralization. In addition, radioactive emissions, especially caused by 232Th, are not considered in most studies.1 Some of the emissions could be reduced by preventing leakage of pollutants  into water and soil through appropriate sealing of tailing ponds.32So far, no emissions based on measured data from the facilities have been published in the LCA studies. Rather, general thresholds prescribed by the Chinese Ministry of Ecology and Environment (formerly MEP) or Environmental Protection Agency (EPA) are given in some studies, which neither reflect the specific ore compositions nor the amount of chemicals used.Figure 21 Normalized figures allow the comparison and adding of different environmental impacts by setting each impact in relation to the same impact induced by an average person per year . The result of the normalization step (relative approach) is expressed in person equivalents (PE).in situ leaching with ammonium sulfate cause the very high impact of EPMarine.21 Neodymium production from Norra Kärr eudialyte shows the lowest environmental impact,19 followed by bastnäsite from Mountain Pass, monazite from Mt Weld/Kuantan and bastnäsite/monazite from Bayan Obo/Baotou.4 However, it must be considered that the eudialyte REE chain is based only on laboratory data. The case of REE production from Mountain Pass bastnäsite shows the effectiveness of measures, such as recycling of saline wastewater to save chemicals, cleaner power generation using a natural gas-fired cogeneration plant, and an alternative cracking process without roasting, which was used by the former operator Molycorp Inc. Still, these measures do not reduce all environmental impacts. For example, PM is determined by the geologic properties of the deposit and can only be reduced to a small extent by an irrigation system. Changes in processing procedure are also rather unlikely, as they are determined by the mineral type. In contrast, improvements in process efficiency have an impact. Also, exhaust gas treatment of electrolyzers and modern sludge treatment concepts can add up to a recognizable improvement as assumed for the Mt Weld/Kuantan process chain.4In general, the production of dysprosium has a higher environmental impact than that of neodymium because dysprosium is always present in lower concentrations in the ores. Eudialyte has the highest dysprosium content (0.03%) in comparison to the Bayan Obo ore and the IACs, whose dysprosium content is about 0.0037 percent. The dysprosium production based on IACs induces the highest environmental impacts of all process chains Figure . EspeciaMarine (> 98%). Flotation and magnetic sorting have lower impacts due to their lower energy and chemical requirements as compared with cracking and hydrometallurgical processes. Roasting, solvent extraction, precipitation to REE oxalates and calcination contribute most to GWP and acidification potential (AP) mainly caused by electricity demand required for chemical supply. Roasting also causes AP due to direct process emissions of HF and sulfur dioxide. The share of the precipitation chemicals on the total environmental impacts amounts to 1–20% for the Bayan Obo/Baotou production pathway and even 10–40% for Mt Weld/Kuantan pathway, depending on the individual impact category. Molten salt electrolysis causes impacts of EP, AP, HTP, ETPFreshwater, PM, and GWP.7 GWP is caused by CO2, CF4, and C2F6; HTP, AP and ETPFreshwater by HF and PM by dust particles. Phosphate emissions during cathode production causes freshwater eutrophication potential (EPFreshwater) and HF emissions during REF3 production account for HTP and AP.Mining and crushing are the largest contributors to PM  are the main contributor to EPMarine. Energy consumption is mostly reflected in GWP and AP. The share of transport and infrastructure facilities on the total environmental impacts is negligible  clearly shows the dominant share of chemicals in almost all impact categories Figure . PM is de Figure .232Th and 238U and their decay products. These radioactive elements can enter the environment through air, wastewater and rain leaching. The entire process chain has potential for improvement through emission treatment technologies and also recycling of chemicals  generated during processing. Closing illegal mines and raising environmental standards should be addressed as a high priority to reduce environmental impacts. Reinforcing responsibilities along cross-border process chains, the supply chain laws mentioned in the introduction could probably be helpful in this regard. Supplying methodically stringent data, LCAs are a credible means to promote this development.Many previous LCA studies provide useful insights into REE production and a good understanding of its environmental consequences. Because the majority of REEs are produced in China, data availability poses a major challenge. The LCA studies showed that each REE mineral and deposit is different and requires an individual analysis of the entire processing route. Therefore, despite many efforts, there is still no complete picture of the environmental impacts associated with REE production. In particular, the environmental analysis of illegal REE production, mainly IACs, is still a blind spot due to the lack of data. Here, scenarios and sensitivity analyses are often used. Moreover, especially for metal refining, it is unclear how much of the RE metals/alloys are produced and with what efficiency . However, the LCAs have highlighted the main problems, such as large quantities of chemicals needed to process REEs and the large quantities of tailings generated during beneficiation, extraction and separation that contain the naturally occurring radionuclides 38 The reasons for this are inefficient collection and technological problems. The complex chemical separation of REEs is a big challenge and a chief barrier to widespread recycling activities. In 2018, Apple rolled out a Robot for iPhone dismantling. Processing 100,000 iPhones has the potential to recover 11 kg of REE.39 Dramatically improving the recycling of REEs is an absolute necessity. LCAs are needed to evaluate improvement options.In addition, new primary resources should be identified that have high REE content, as few radioactive associated elements as possible and are located outside sensitive ecosystems as alternative REE production pathways to China. Moreover, research into new or improved processing technologies and reprocessing of industrial waste streams should be pursued. Other, secondary REE resources and increased end-of-life recycling of REE-containing consumer products are additional options. Despite extensive research, mostly on a laboratory scale, only about 1% of REEs are actually recycled.


Bactrocera oleae are analysed using scanning electron microscopy, transmission electron microscopy, and fluorescence microscopy. Based on these analyses and measurements of patch reflectance spectra, we infer that white patches are due to modified air sacs under transparent cuticle. These air sacs show internal arborisations with beads in an empty space, constituting a three-dimensional photonic solid responsible for light scattering. The white patches also show UV-induced blue autofluorescence due to the air sac resilin content. To the best of our knowledge, this research describes a specialized function for air sacs and the first observation of structural color produced by tracheal structures located under transparent cuticles in insects. Sexual dimorphism in the spectral emission also lays a structural basis for further investigations on the biological role of white patches in B. oleae.Here, the ultrastructure and development of the white patches on thorax and head of  Bactrocera oleae. Their results suggest that white coloring is a result of transparent cuticle and modified air sacs within these patches, providing further insight into how structural white emerges in insects.Manuela Rebora et al. use electron and fluorescence microscopy together with reflectance spectra to examine structural white patches present in the olive fruit fly,  Such a diversity of colours can derive from pigments, either embedded in the cuticle or situated under a transparent cuticle3, or can be produced through structural optical systems from selective light reflection4.Insects exhibit a remarkable diversity of colours which represent important visual cues for intra and inter-specific communication being used in sexual recognition or as disruptive or warning coloration against visually oriented predators6 and can arise from interference, diffraction and/or scattering of incident light owing to the presence of nanostructures characterizing insect cuticular surface and its multi-layered structure. Structural colours, sometimes associated with pigments8, have been extensively studied, especially in some insect orders such as Lepidoptera10, Coleoptera11 and Odonata14 which offer remarkable examples in this field.Structural colours are widespread in insects15. White light may be scattered by unpigmented cuticle, setae, scales and surface waxes with specific shapes and size, thus producing structural white. This has been documented in beetles, due to a three-dimensional photonic solid in the scales of Cyphochilus spp.15; in butterflies (Pieridae), due to an array of microscopic beads suspended within the wing scales17 and in Odonata, owing to the presence of wax crystals on the epicuticle21. In particular, beetle’s white scales optimized to produce optical scatter and highly efficient whiteness, brilliance, and opacity with an extremely thin thickness have been extensively studied24.Structural whiteness in insects is less common than colours and requires scattering processes for all visible wavelengthsBactrocera oleae (Rossi) (Diptera: Tephritidae). Among Tephritidae, which are of major economic importance in agriculture, this fly is a key pest in olive crops worldwide and represents a great problem, especially in the Mediterranean basin26. Adults of both sexes bear, on their thorax and head, white patches (yellowish in dead or old specimens), whose biological function is still unknown. Ultrastructural investigations using scanning (SEM) and transmission electron microscopy (TEM), together with observation in light and fluorescence microscope as well as reflection spectra measurements allowed us to shed light on the nature of the structures responsible for light scattering. Among different white patches, located on the thorax and head, we focused our observations mainly on the scutellum, a small triangular plate placed posteriorly in the mesonotum, because it is wider and more easily, both microscopically and experimentally, accessible than the other white areas.In the present study, we analyze, in detail, the mechanism underlining the production of structural white in the olive fruit fly B. oleae shows the following white patches: the scutellum, two post pronotal lobes, two anepisternal areas, two notopleural calli, and two katatergites  (mean ± SD), which is significantly different  from that of the males measuring 2.9 ± 0.8 µm (n = 5) (mean ± SD). There is no significant difference  between the two sexes in the maximal thickness of the arborisations of the air sac which is 15.4 ± 2.4 µm (mean ± SD) in females (n = 6) and 16 ± 2.9 µm (mean ± SD) in males (n = 5).Semithin sections of the scutellum Fig.  reveal iB. oleae, is similar to other white patches present in the fly, and is constituted of a smooth cuticle with reduced or absent microtrichia in comparison with other body areas , decreases to the lower range of wavelengths, and demonstrates multiple low amplitude maxima within 220–320 nm   Fig. . The cutge) Fig. .The reflection from white patches is close to the angle-independent Lambertian reflection. Thus, after observation angle correction the reflection intensities from white spots are 0.246, 0.267, 0.262  and 0.218, 0.228, 0.219  for observation angles 0°, 30°, and 45°, respectively  the cuticle is transparent lacking melanisation; (2) under the epidermis, the fly air sacs assume a peculiar structure with developed arborisations; (3) a highly vacuolated layer is interposed between the cuticle and the air sacs.The present investigation describes in detail the ultrastructure of the white patches visible in the thorax and in the head of 5. Structural whiteness in bird feathers or in insects is usually produced by empty structures made of a continuous biopolymer phase and air: voided spaces contain air, while the other phase is a solid material, such as chitin or beta-keratin4. Among insects, the wing whiteness of the cabbage white butterflies (Pieridae) is produced by microscopic beads suspended within the wing scale17, while the body whiteness of the scarab beetles of the genus Cyphochilus is produced by a random network of interconnecting cuticular filaments inside thin scales showing high scattering efficiency27. Accordingly, the arborisations of the aerial sacs of B. oleae with their epicuticular layer and the cuticular spiny beads randomly oriented could constitute a three-dimensional photonic solid surrounded by air able to produce structural white. Indeed, strong diffuse reflection in white spot areas, which is ~20% of the reflection of fine WS1 standard, might be related to the modified air sacs described here. The involvement of these structures in producing structural white is highly suggested by our experiments measuring the reflected and transmitted light of the thoracic white patches of B. oleae in different conditions: intact, with the air sac removed, intact infiltrated with oil and with the air sac removed infiltrated with oil  beetles23.The peculiar arborisations of the air sacs under the white patches, with numerous cuticular spiny beads randomly distributed and oriented in an empty space with air, have features highly compatible with the production of structural white. Indeed the perception of whiteness is due to scattering from a material containing many disordered surfaces reflecting all wavelengthsoil Fig. . The spen ≈ 1.5629 . If the light scattering takes place on the interface between the cuticle and air, it should be suppressed, when the oil fills the interconnected air voids in the modified air sacs. Light transmittance from the white patches samples infiltrated with oil is, however, lower than the transmittance of the cuticle with air sac removed. Remaining air voids or intracuticular defects/inclusions could be responsible for this reduced transmittance.The immersion oil used to infiltrate the air sacs has a high refractive index, which is similar to the refractive index of insect cuticle, The reflection from white patches in males, which is significantly higher than in females, could be due to the different development  of the electron-translucent vesicular layer in the two sexes observed over the air sac arborisations, but further investigations are necessary to clarify this point.B. oleae, in particular the scutellum, the two post pronotal lobes, the two anepisternal areas, the two notopleural calli, the two katatergites together with the white area in the head, are located just in correspondence of the air sacs, expansions of the tracheae, which in Diptera are located mainly in the thorax and head30  tracheal bundles appear as white spots under transparent cuticle32. The reflected white is not as pronounced as in B. oleae, but it could represent a possible preliminary stage for further specialization in insects. In B. oleae the spiny beads located along the arborisations of the air sacs under the white patches could derive from the papillae (punctuate thickenings) described along the inner surface of insect trachee34 and of fly air sacs35. The tracheal cuticle in insects consists of a thin epicuticle and a conspicuous procuticle and follows regular protrusions of the apical plasma membrane of tracheal epithelial cells from which taenidia derive36. Most probably, taenidia, papillae and spiny beads have same evolutionary origin and the same chemical composition of procuticle and epicuticle, also suggested by our ultrastructural observations. In this regard, it is interesting to remember that a peculiar kind of procuticle with an additional protein with the characters of keratins was suggested in insect tracheal taenidia37.The typical functions of aerial sacs in insects are to increase tracheal respiratory efficiency, to assist flight by lowering the specific gravity, hydrostatic function in aquatic insects, thermoregulation, amplification and resonance for sound production/receptionB. oleae emit fluorescence as highlighted in our observations under fluorescence microscope. In particular, the stronger excitation occurs at 365 nm (UV light) and the emission from 397 nm (blue light).The white patches in the body of 39), including flowers, plants, corals, worms, squid, tardigrades, spiders, stomatopods, insects, fish, reptiles, birds, and can be used as a means of intersexual communication40, prey capture41, camouflage42, or as a protection against UV radiation43. Among insects, some species belonging to the groups of beetles, ants, butterflies, dragonflies and one bee species revealed to emit fluorescence (review in ref. 44). In insects, fluorescence is often due to the presence of pteridines, metabolic products of purines absorbing light around 360 nm and fluorescing in the blue region45. As in B. oleae, in many cases, insect fluorescence is not dispersed uniformly on the body surface, but it is localised in some areas, usually the same areas that have markings colored with natural pigments contrasting with the rest of the body, such as the beetles, Pachyrhynchus gemmatus purpuerus Kraatz (Coleoptera: Curculionidae) or Cicindela maritima44 Dejean (Coleoptera: Carabidae). Indeed, the characteristics of the insect surface on which the fluorophore is present can significantly affect the intensity and distribution of the emitted radiation and of the visual signal in the observer46. In this regard, it is interesting to remember the case of the beetles Celosterna pollinosa sulfurea Buquet (Coleoptera: Cerambycidae) and Phosphorus virescens Oliver (Coleoptera: Cerambycidae), where fluorescence occur in conjunction with three-dimensional photonic structures47. Similarly, the butterfly Papilio nireus L (Lepidopotera: Papilionidae) shows in the scales of its wings a two-dimensionally periodic photonic crystal infused with a highly fluorescent pigment represented by pterin. Such photonic crystals enhance the intensity of the observed fluorescence from the butterfly’s wing46.In nature, autofluorescence has been reported in numerous taxa (see reviews in refs. B. oleae might be the presence of resilin (an autofluorescent protein with an emission in the blue region) in the cuticle of the air sac. The strong argument for this is that the UV-excited autofluorescence in the blue region of the dissected thoracic air sacs is similar to that emitted by the white patches of B. oleae 50 can be clearly seen 51, thus supporting the hypothesis of a possible biological role of the white patches in B. oleae life.Interestingly electrophysiological testing of the spectral sensitivity of B. oleae. Similar to the observations in Drosophila52, olive fruit flies that emerged in winter show a darker body colour, compared with flies that emerged during summer, but this concerns only the melanised portions of the body while the white patches on the thorax and head remain unchanged in summer and winter53.To our knowledge, no attention has been previously paid to the possible biological role of the white patches on the body of B. oleae  are focused mainly on chemical cues, while to the best of our knowledge, no studies have elucidated the behavioural role of visual cues in lek defence, courtship and mating behaviour and further research on this point is required54.Concerning the sexual differences observed in the reflectance spectra of the scutellum , one possible explanation could be a role of white patches as visual cues in intra and intersexual recognition during mating. In this regard, it is important to remember that investigations on the recognition of potential rivals/partners during mating behaviour in Lispe consanguinea Loew (Diptera: Muscidae), and L. tentaculata DeGeer (Diptera: Muscidae), inhabiting the supralittoral zone at the shore of a fresh-water reservoir possess similar visual cues namely reflective concave silvery scales on the head face55. Flies occupy different lek habitats. Males of the first species patrol the bare wet sand on the beach just above the surf. Males of the second species reside on the more textured heaps of algae and stones. Male dancing is usually evenly distributed around the female. Rival males also circle about one another at a distance shorter than 15 mm, but not in close contact. Flies of both species often walk sideward and observe the partner not in front but at the side55. We hypothesize that the frontal and lateral patches in B. oleae might have similar function in the courtship of this species and in representatives of other Tephritidae in general.It has been previously demonstrated that two predatory fly species, Rhagoletis56 and Zonosemata58. Interestingly Salticidae females have palps with a UV-excited bright fluorescence40 similar to that observed in the white patches of B. oleae. Imitation of jumping spiders could be of benefit against spiders, insects and vertebrate fly predators since jumping spiders are difficult to catch and poisonous.Another possible function of the white patches could be linked to predator avoidance. Indeed, morphological and behavioural aposematic mimicry with jumping spiders has been reported in other Tephritidae species belonging to the genus B. oleae could potentially serve other non-visual functions, such as thermoregulation. In beetles of the species Neocicindela perhispida (Broun) (Coleoptera: Cicindelidae), white morphs were capable of foraging for a longer time without overheating, when transferred from their natural white sand habitat to a black sand terrain, compared with the resident black morphs, which escaped the heat burrowing into the sand59.Finally, the white patches of B. oleae. The patches have important implications. First, structural white and associated autofluorescence are described in Diptera. Second, a structural colour not produced by an external cuticle but by an internal structure located under transparent cuticle is described in Insects. In particular, portions of the air sacs with their modified internal structure assume a function that, to the best of our knowledge, is so far undescribed in insects. Third, the identification of these complex structures producing structural white lays the foundation for further investigations aiming to understand the biological role of the white patches on the body of B. oleae and their possible use as visual cues in sex recognition or predatory avoidance. This knowledge, adding information on the biology of this dangerous species, could potentially help to develop methods for its biological control.The data here presented describe in detail the ultrastructure and optical properties of the white patches on the body of the olive fruit fly B. oleae adults emerged from pupae obtained from olives collected in the field around Perugia  during October in 2019 and 2020. Olives were kept in the laboratory in a controlled condition chamber , on a net, in order to collect fallen pupae. Pupae were kept inside net cages (300 mm × 300 mm × 300 mm) under the same controlled conditions until adult emergence. Both females and males, after emerging, were maintained in the cages and provided with water and crystallised sucrose.To describe the development of the white patches, we performed observations under a stereomicroscope. Observations of the scutellum of adults at different ages—(1) adults, which have completed the metamorphosis but were still within the pupa (pharate adults), (2) just emerged adults, (3) adults, six hours after emergence, (4) adults, 24 h after emergence—were performed with a stereomicroscope Leica MZ6 .of B. oleae were obtained from the thorax of anaesthetized insects at different ages . Specimens were fixed for 3 h in 2.5% glutaraldehyde in cacodylate buffer , pH 7.2. The fixed scutelli were repeatedly rinsed in sodium cacodylate buffer and post-fixed for 1 h at 4 °C in 1% osmium tetroxide in sodium cacodylate buffer . The samples were then repeatedly washed in the same buffer, dehydrated in ascending ethanol concentrations and finally embedded in an Epon-Araldite resin mixture (Sigma-Aldrich). Afterwards, semi-thin sections (1 µm thick) were sectioned using a Leica EM UC6 ultra microtome , stained with methylene blue and observed in a Leica DMLB light microscope .Semithin sections of the scutellum 60.To calculate the mean thickness of the vesicular layer (six measurements for each sample) and the maximal length of the arborisations in the two sexes, measurements were made from digital images with the open-source image processing program ImageJObservations of the thoracic white patches of adults at different ages—(1) just emerged adults, (2) three-day-old adults, (3) ten-day-old adults—were performed using a fluorescence microscope Zeiss Axiophot  with an excitation filter 365 nm, chromatic beam splitter FT 395 nm, emission 397 nm, with an excitation filter 450–490 nm, chromatic beam splitter FT 510 nm, emission 520 nm, and with an excitation filter 546 nm, chromatic beam splitter FT 580 nm, emission 590 nm. Live males and females were anaesthetised with carbon dioxide, fixed with tape to a microscope slide and observed in the fluorescence microscope. To analyse the autofluorescence of dissected air sacs located inside the thorax  and compare it to that produced by the intact scutellum (with transparent cuticle), the thorax of a fly has been dissected under a stereomicroscope, placed on a glass slide and observed using a fluorescent microscope with the three excitation filters mentioned above.B. oleae insects  were studied in a scanning electron microscope (SEM) Hitachi S-4800  equipped with a Gatan ALTO 2500 cryo-preparation system . For details of sample preparation and mounting for cryo-SEM, see ref. 61. Pieces of the thorax with white patches were carefully cut out, mechanically clamped to the metal holder and shock-frozen in liquid nitrogen. After transferring the frozen sample to the cryo-chamber of the microscope, it was fractured with a metal blade, cooled at −140 °C, and sputter-coated under frozen conditions with gold-palladium (thickness 10 nm) and examined at 3 kV acceleration voltage and a temperature of −120 °C on the cryo-stage within the microscope. In addition, we prepared fractures of the corresponding sites in dry insects and viewed them using conventional SEM.The shock-frozen samples of the thorax of B. oleae were dissected from anaesthetized insects and fixed for 3 h in 2.5% glutaraldehyde in cacodylate buffer , pH 7.2. The fixed scutelli were repeatedly rinsed in sodium cacodylate buffer and post-fixed for 1 h at 4 °C in 1% osmium tetroxide in sodium cacodylate buffer (Electron Microscopy Sciences). The samples were then repeatedly washed in the same buffer, dehydrated in ascending ethanol concentrations and finally embedded in an Epon-Araldite resin mixture (Sigma-Aldrich). Afterwards, ultra-thin sections were cut using a Leica EM UC6 ultramicrotome , collected on Parlodion  coated copper grids, stained with uranyl acetate and lead citrate  and examined using a Technai G2 Spirit BioTwin Transmission Electron Microscope  equipped with a CCD camera Veleta (EMSIS) 2048 × 2040.The scutellum of 2 pharate adults, 4 just emerged adults , 4 three day old adults  and 8 ten-day-old adults  of B. oleae (ten days old) were illuminated at 45° using a light source  through an optical fibre (200 μm diameter) equipped with a one lens condenser. The condenser was placed 10 mm away from the sample. The reflected light was collected by another condenser, which was placed 35 mm away from the sample, and could collect the light at various observation angles , and it was mounted on an optical fibre (200 μm diameter). The optical fibre was connected to a spectrometer . The spectra were recorded with the software Spectral Suite . Further spectra processing was performed in Matlab 7.10  and includes the dark noise subtraction, smoothing with Savitzky-Golay filter62 of 4th order with 12 nm window, and normalization on the WS1 standard  reflection spectrum (45° illumination/detection)  of adult B. oleae with epi- (reflected light) and trans- (transmitted light) illumination, in different conditions: (1) intact, (2) with the underlining air sac removed, (3) intact, infiltrated with oil and (4) with the underlining air sac removed and infiltrated with oil. In particular, a thoracic white patch was cut from the insect thorax using a razor blade and photographed with epi- and trans-illumination in different conditions. Transmission and reflection spectra were also recorded in different conditions. The air sac, underlining the white patch, was scratched out using a piece of a razor blade. A tiny amount of oil  was applied to the cuticle with and without subcuticular air sacs.We observed the thoracic white patches of t test for independent samples. Three-way ANOVA analysis on reflection measurements was carried out using SigmaPlot 12.5 . For post hoc analysis the Holm-Sidak method was used.The thickness of the vesicular layer and the maximal thickness of the arborisations of the air sac in males and females were compared using a Student’s Further information on research design is available in the Peer Review FileSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Reporting Summary


P = 0.04). After irrigation, those without FBs had more patients experiencing pain reduction (67%) compared to those with retained FBs  and had a greater magnitude of change in pain score . An improvement in ocular pain score ≥ 5 points after irrigation predicted the absence of FBs with a negative predictive value of 100%. Eye irrigation significantly lowered corneal FB retention; if ocular pain decreased considerably, the probability of retained FBs was low, making irrigation-associated pain score reduction a feasible diagnostic method to exclude FB retention without needing specialized ophthalmic examinations.This prospective study aimed at determine whether eye irrigation removes ocular foreign bodies (FBs) and whether ocular pain predicts FBs. Emergency department patients complaining of ocular FBs were enrolled. In the irrigation group (n = 52), pain was evaluated with a visual analog scale before and after irrigation, and the presence of FBs was determined under a slit-lamp. In the nonirrigation group (n = 27), the evaluations were performed upon arrival. The corneal FB retention rate was found significantly lower in the irrigation  than in the nonirrigation groups (13/27, 48%;  In 2010, 1.5% of patients visiting the EDs in the United States received an ophthalmic principal diagnosis, and, between 2000 and 2015, 2–3% of patients discharged from the EDs in Taiwan received diagnoses of eye-related injuries3.Eye-related problems are a common reason for emergency department (ED) visits, and the frequency of such visits is increasing5. These patients probably visited the ED because of persistent ocular FB sensation and concerns that a FB remained inside the eye. However, a recent report from New York City revealed that the time from triage to completion of an ophthalmologic consultation for patients complaining of FB sensation was long: 174–263 min5.Among patients visiting EDs with ophthalmic primary complaints, corneal abrasions and foreign bodies (FBs) on the ocular surface are among the leading diagnoses, and these two diagnoses respectively represent approximately 13–14% and 8–15% of the complaints7. Additionally, although 84% of EDs had slit-lamps available, 30% of ED physicians reported no confidence at all in using the instrument for examination6. Such a lack of confidence could lead to avoidable specialist consultations and thus entail long ED waits. In our hospital, where 24-h emergency eye service is available, 11.8% of all consultations in the ED were sent to ophthalmologists, which ranked first among all consultations8.One reason for the long wait is that, although ophthalmic problems are common, ED physicians are uncomfortable dealing with such problems. In the United Kingdom, two surveys conducted 10 years apart reported that 64–69% of senior house officers in EDs had little or no confidence in managing patients with ophthalmic complaints, despite improvements in their ophthalmic training programs9. In our ED, after excluding open-globe injuries, physicians commonly order eye irrigation for patients complaining of ocular FBs. During the ophthalmologic consultations that follow, many patients report that their ocular pain greatly diminished after irrigation, and among such patients, retained FBs are uncommon.External eye irrigation is a standard protocol in the ED for treating ocular chemical injuriesOn the basis of the above observations, we speculated that external eye irrigation could wash out ocular FBs and reduce ocular pain. Additionally, we assumed that after irrigation, an absence of retained FBs could be predicted from a patient’s considerably improvement in the subjective ocular pain sensation. If these theories tested to be true, eye irrigation could be considered as the first-line treatment and a diagnostic procedure which rendering further examinations with special ophthalmic instruments or techniques unnecessary for this common condition in EDs.This prospective observational cohort study was conducted at a tertiary ED in Chang Gung Memorial Hospital, Linkou, Taiwan. The study was approved by the hospital’s institutional review board (No. 201600962A3C101) and adhered to the tenets of the Declaration of Helsinki. Written informed consent was obtained from all enrolled patients.Patients who visited the ED between November 2016 and July 2017 complaining of a foreign body went into the eye, accompanied by persistent ocular discomfort were initially examined by an ED physician. The onset, duration, and symptoms of the eye discomfort were obtained, and basic ophthalmic and general medical history were documented. If both eyes were affected, each eye was separately evaluated. Patients who had no obvious open-globe injury or protruding intraocular content, had a round-in-center pupil with a prompt light reflex, could count fingers at 1 m, and experienced no considerable subjective decline in visual acuity were enrolled in this study. Patients who were younger than 18 years or could not complete a questionnaire were excluded , similar to the score used for dry eye evaluation, was usedUpon confirmation of the FB being removed from the ocular surface, the patient was discharged, and one drop of chloramphenicol 0.25%  was prescribed for application to the affected eye four times a day if the patient had only minimal ocular surface defects and did not regularly use contact lenses. An ophthalmology clinic follow-up was arranged within 1–3 days. If the epithelial defect area was larger than approximately one-sixth of the corneal area, the FB was present upon examination, or the patient regularly used contact lenses, one drop of levofloxacin 0.5%  to be used four times a day, and tobramycin ointment  to be applied at night were prescribed, and an ophthalmology clinic follow-up was arranged within 24 h. The ophthalmic conditions in the follow-up clinics were collected from medical records, and patients were divided into a nonirrigation group and an irrigation group for comparison, and among the irrigation group, into FB retention and FB absent subgroups for analysis.The estimated sample size was calculated based on the preliminary results from the first 3 months of this study. The rate of finding a retained FB was assumed to be 70% without external eye irrigation, and irrigation was assumed to reduce the rate of retained FBs by 50%. Under the assumption that the irrigation group would be twice the size of the nonirrigation group, the estimated sample size was 46 and 23 for the irrigation and nonirrigation groups, respectively .χ2 test for categorical variables and independent t-test and Mann–Whitney U test for normally and non-normally distributed continuous variables, respectively. VAS scores before and after irrigation were compared using the Wilcoxon Signed Ranks test. Spearman’s Rank correlation coefficients were calculated. Receiver operating characteristic (ROC) curves were constructed using the sensitivity and specificity of the ocular pain scores for predicting the presence or absence of FBs after irrigation, and the area under the ROC curve (AUC) was analyzed. All statistical analyses were performed using SPSS Ver. 25 . A two-sided P of < 0.05 was considered statistically significant.Data are presented as mean ± standard deviations. If both eyes were affected, only the eye with the higher initial pain score was included in the analysis. The normality of the distribution of continuous variables was tested with the Shapiro–Wilk test. Variables were compared using the The study was approved by the Institutional Review Board (No. 201600962A3C101) and adhered to the tenets of the Declaration of Helsinki.P = 0.50).After two patients with an intraocular FB discovered during ophthalmologist examination were excluded, 79 patients were analyzed. The mean ages were 44.8 and 44.0 years in the nonirrigation (n = 27) and irrigation (n = 52) groups, respectively, and the majority of the patients were male Table . The ocuP = 0.04). By contrast, the proportions of eyes with FBs retained in the conjunctival sac were similar in the two groups . In total, FBs were absent from 33% and 46% of eyes in the nonirrigation and irrigation group, respectively  than that in the nonirrigation group . No significant correlation was discovered between the ocular surface injury and ocular pain scores.The ocular surface injury scores for the cornea and conjunctiva were comparable between the groups, except for the conjunctival punctate abrasion score, which was significantly higher in the irrigation group than in the nonirrigation group  returned to the ophthalmology clinic as scheduled. Among these patients, one had a retained subconjunctival sand-like FB without active inflammation. The other eight patients had recovered, and none of the patients had signs of infection. Another 16 patients (30.8%) returned to other clinics in our hospital without revisiting the ophthalmic clinic. We presumed that these patients had recovered well since no ocular complaints were documented and no further ophthalmic clinic appointments were arranged.P < 0.001 and P = 0.008, respectively). Notably, patients in the FB (−) group had a significantly greater change in pain score when compared to the FB (+) group .For the 52 patients in the irrigation group, ocular discomfort was re-evaluated after irrigation. We further divided these patients into a FB (−) (n = 24) and a FB (+) (n = 28) group based on whether a retained FB was found after irrigation  in diagnosing FB retention , 0.56–0.85; ion Fig. . FurtherWhether self-reported factors could be used to predict the retention of FBs in the eye was investigated. When a patient reported still feeling an FB, that the FB was possibly metallic in nature, or that the incident had occurred during work, the relative risks of a retained FB were 1.73 (95% CI 1.13–2.66), 1.53 (95% CI 1.78–3.91), and 1.82 (95% CI 1.15–2.89), respectively  reported that the incident had occurred at work. A majority of respondents  were metal workers or mechanics  indicated that some FBs were probably not embedded deeply in the cornea and could therefore be removed through gentle irrigation. ED nurses experienced in performing eye irrigation for chemical burns can learn this procedure easily with minimal additional training. However, the relatively high incidence of FBs in the conjunctival sac (33% in the irrigation group vs. 26% in the nonirrigation group) suggested that some of the dislodged corneal FBs might have ended up in the fornix. Compared with removal of embedded corneal FBs, removal of conjunctival FBs is associated with a lower risk and required skill level.15. Studies have attempted to quantify subjective eye discomfort in dry eye disease. However, such discomfort is affected by conditions including ocular surface diseases16, inflammation17, neuropathic factors18, and nonocular discomfort19. The purely mechanical stress encountered by patients with ocular FBs differs from the multifactorial dry eye disease. Therefore, instead of using dry eye disease evaluation scales, we used a combination of general pain assessment scales including 11-point Box Scale20, 5-category scale21, and Wong–Baker FACES Pain Rating Scale22 as our ocular discomfort measurement tool. These scales have been widely validated and are the gold standards for pain evaluation23. Nevertheless, the perceived magnitude of discomfort greatly varies between individuals and is affected by factors such as sex and antidepressant use25. In the present study, we initially tried to use the absolute mean pain score to predict FBs. Unsurprisingly, the mean score had a low discriminatory power (AUC = 47.6%). Instead, we used the changes in pain score after irrigation to minimize the interindividual differences and achieved a much higher AUC of 71%. Additionally, a cutoff of 5 points for the change in ocular pain score resulted in a high sensitivity (100%) in detecting retained FBs. Thus, the irrigation-related change in the ocular pain score is a simple and acquirable factor for predicting ocular FB retention.The cornea is densely innervated and among the most sensitive tissues27. In a rural setting with nearly 300 participants, prophylactic chloramphenicol ointment prescribed within 18 h after corneal abrasion resulted in no corneal ulcers28. In addition, after noncomplicated corneal FB removal, only 1.7% of eyes exhibited infectious keratitis after treatment with prophylactic fourth-generation fluoroquinolone eye drops, and the development of an infection could be alarmed by the subjective worsening of ocular symptoms29. In the present study, we prescribed chloramphenicol for noncomplicated cases and levofloxacin drops for large abrasions, patients who wore contact lenses, and FBs retained after irrigation. None of the patients in the irrigation group who returned to the clinic developed infectious complications, and their ocular surface injuries all resolved. The above studies and our results suggest that the chances of developing infection under prophylactic antibiotics in abrasion-related ocular surface injuries are low and that external eye irrigation did not compromise outcomes.Although studies have reported that 14–33% of the FBs removed from corneas exhibited positive cultures, 95% and 90% of the isolated microorganisms were sensitive to chloramphenicol and ciprofloxacin, respectivelyThe present study has several limitations. Two patients who complained of bilateral eye discomfort were included, and although we enrolled only the subjectively more painful eye, the sensation in the excluded eye might have affected the pain score of the study eye. Our use of the changes in each patient’s own pain score as the diagnostic factor could have minimized such an impact, but the results may nevertheless be inapplicable to patients affected bilaterally. Another two patients who underwent irrigation were diagnosed with intraocular FBs. They passed the initial open-globe screenings because their entry wounds were small, noncentralized, and self-sealed. Nonetheless, we used sterile saline for irrigation and cautiously avoided applying pressure to the globe. Because the entry wounds were sealed, the risks of intraocular infection and content protrusion should be low. In addition, the ocular pain score was unchanged after irrigation in these patients, which had warranted ophthalmologist consultation. Finally, the FBs were not confirmed by an ophthalmologist with a slit-lamp both before irrigation because the examination itself might affect the location of the FB, especially if the eyelid were to be everted, and this could alter the outcomes of irrigation.On the basis of our results, we propose a flowchart for patients with complaints of ocular FBs Fig. . After oSupplementary Figure S1.


To assess the efficacy of tasimelteon to improve sleep in Smith–Magenis syndrome (SMS).A 9-week, double-blind, randomized, two-period crossover study was conducted at four US clinical centers. Genetically confirmed patients with SMS, aged 3 to 39, with sleep complaints participated in the study. Patients were assigned to treatment with tasimelteon or placebo in a 4-week crossover study with a 1-week washout between treatments. Eligible patients participated in an open-label study and were followed for >3 months.p = 0.0139), and DDTST50 also improved . Average sleep quality  and actigraphy-based total sleep time  improved significantly, consistent with the primary outcomes. Patients treated for ≥90 days in the open-label study showed persistent efficacy. Adverse events were similar between placebo and tasimelteon.Improvement of sleep quality (DDSQ50) and total sleep time (DDTST50) on the worst 50% of nights were primary endpoints. Secondary measures included actigraphy and behavioral parameters. Over three years, 52 patients were screened, and 25 patients completed the randomized portion of the study. DDSQ50 significantly improved over placebo (0.4, Tasimelteon safely and effectively improved sleep in SMS. RAI1) gene variant [Smith–Magenis syndrome  is a rare genetic disorder that results from an interstitial deletion of 17p11.2 and, in rare cases, from a retinoic acid induced 1  was provided for adult patients and an oral suspension administered by 3 ml or 5 ml syringe (4 mg/ml) was provided for pediatric patients. A weight appropriate dosing schedule was developed for the oral suspension in a previous study and administered as ≤28 kg: 0.7 mg/kg or >28 kg: 20 mg. Treatment was administered as a capsule or oral suspension once daily, one hour before target bedtime (±30 minutes). Placebo and tasimelteon were indistinguishable regardless of formulation.P values were calculated using a mixed effects model that included the fixed, categorical effects of treatment, period, and sequence of treatment.The primary endpoints were the average of 50% worst daily diary sleep quality (DDSQ50) (change from baseline) and the average of the 50% worst daily diary total sleep time (DDTST50). These primary endpoints looked at the worst half of nights for sleep quality (DDSQ50) and total sleep time (DDTST50). These data were collected through a daily diary that the patients’ caregivers completed on provided forms. These endpoints were developed through a preliminary, observational study in which the sleep patterns of SMS patients were observed. During the observational study it was observed that patients had highly variable sleep patterns with nights of good and poor quality sleep and of long and short total sleep time. Thus, to better quantify the effect of tasimelteon in this population, the DDSQ50 and DDTST50 were chosen as primary endpoints. The average of 50% worst nighttime sleep quality (DDSQ50) was calculated as the average of the first half of data, sorted from worst to best, if the total number of data points was even, or the first half +1 of data if the total number of data points was odd. For DDTST50 the same approach was utilized. These endpoints were analyzed in the intent-to-treat (ITT) population by comparing the effect of tasimelteon and placebo. Secondary endpoints that were analyzed included the average daily diary sleep quality (DDSQ), daily diary total sleep time (DDTST), 50% worst daily number of nighttime awakenings, 50% worst latency to sleep, daily number of nighttime awakenings, daily total amount of nighttime sleep, and latency to sleep, all by post sleep questionnaire (PSQ) and actigraphy. Additional secondary endpoints included the average 50% worst daily length of nighttime awakenings, 50% worst daily total amount of nighttime sleep, and daily length of nighttime awakenings measured by actigraphy. Finally, changes in clinical impression were determined as Clinical Global Impression Severity and Change  at each visit, and mixed model analysis was used to analyze the treatment differences using the Aberrant Behavior Checklist (ABC) , 22.Data from the open-label treatment arm and open-label extension are also presented below. The same measures were collected as in the randomization arm and are used to provide additional insight into the persistence of efficacy of tasimelteon.RAI1 or an RAI1 pathogenic variant resulting in haploinsufficiency . Moreover two patients' deletions were larger in size and encompassed deletion of CMT1A  or targeted RAI1 gene sequencing. Participants had a confirmed heterozygous deletion (of chromosome 17p11.2) inclusive of Potential patients were identified from SMS registries operated by Vanda; advocacy groups including PRISMS, Inc.; and through phone and web-based patient outreach campaigns. Patient recruitment for the crossover portion of the study began in 2015 and lasted for approximately 3 years. Figure p = 0.0139). Total sleep time on the worst 50% of nights resulted in a difference of 18.5 minutes increase for tasimelteon compared to placebo .Results from the ITT population were analyzed for all primary and secondary efficacy endpoints. Both primary endpoints, average of 50% worst DDSQ (DDSQ50) and average of 50% worst DDTST (DDTST50), favored tasimelteon over placebo, with DDSQ50 demonstrating a statistically significant improvement over placebo Table . Treatmep = 0.0155) and total sleep time as determined by diary . Further, actigraphy-based measurement of total sleep time also showed improvement . Secondary efficacy endpoints are summarized in Table Secondary endpoints showed both subjective and objective evidence of tasimelteon treatment improving sleep related symptoms. These results complemented and extended the conclusions from the primary endpoints showing improvement in overall sleep quality  patients experienced a treatment emergent adverse events (TEAE) while on placebo, and 6 (24.0%) patients experienced a TEAE while on blinded tasimelteon. Ten (52.6%) patients reported a TEAE while on open-label tasimelteon. One (4.0%) patient on blinded tasimelteon and one (5.3%) patient on open-label tasimelteon each experienced a TEAE that led to a temporary disruption of the study drug; no patients experienced any TEAEs that led to study drug being withdrawn. There were no serious adverse events or deaths during the treatment phase. There were no clinically meaningful changes in mean chemistry, hematology, or urinalysis values over time. In general, the adverse events experienced were common for the study population and similar between placebo and tasimelteon for the treatment phase.p = 0.0139). The second primary endpoint did not reach statistical significance but showed an average improvement of 18.5 minutes of sleep  on the worst 50% of nights (DDTST50). In addition to the improvement demonstrated on the primary endpoints, tasimelteon improved additional subjective and objective measures that characterize both the quality and length of sleep. This is particularly noteworthy given that sleep disturbances in those with SMS have been difficult to improve; in fact, in a study assessing the use of prolonged-release melatonin to improve insomnia in children with an autism spectrum disorder and/or neurodevelopmental disorder, results showed a greater improvement in Total Sleep Time (TST) when SMS patients were excluded from analysis [Tasimelteon provided a significant improvement in sleep in patients with SMS treated for 4 weeks in a double-blind, placebo-controlled, crossover study. This improvement was observed in the primary endpoint of 50% worst nights of sleep quality (DDSQ50) , there was an OLE phase into which patients from the randomization and open-label treatment periods could enter. During the OLE phase, tasimelteon continued to show improved sleep by both objective and subjective measures. Interestingly, while the ABC checklist that quantifies behavioral symptoms in SMS did not show statistically significant improvement in the 4-week randomization phase, a large improvement emerged in the OLE phase when the length of treatment was extended for 3 months or longer. This suggests that sleep improvements take additional time to engender behavioral improvements and is consistent with previous findings .One limitation of this study is the relatively small sample size due to the rarity of the disorder and the difficulty of patients to participate in a controlled study, which limits the types of analyses and evaluations of the efficacy of the drug. Another limitation of the study is the duration of the washout period of one week; however, the washout was limited to one week to reduce the amount of time patients were without treatment. Due to logistics, and difficulty bringing in patients for the serum melatonin measurements, melatonin was not measured in this current study. That is one limitation that could be addressed in future studies either via serum melatonin measurement or via salivary dim light melatonin onset (DLMO) tests.RAI1 due to chromosome 17p11,2 deletion or pathogenic variant in RAI1, causes circadian dysfunction in patients, leading to a myriad of symptoms, most critically, sleep disruption. In this study, we provide evidence that tasimelteon can safely provide persistent improvement of the sleep disturbances associated with SMS and may ultimately improve the quality of life for patients and their families. Additional longitudinal real world studies should be conducted to evaluate the benefits of tasimelteon in improving both sleep and behavioral symptoms of patients with SMS.The results of this study are consistent with the putative mechanism of action of tasimelteon as a circadian regulator in non-24 and jet-lag disorder , 19. TheSupplemental material.


Results showed that egg yolk plasma might have been the main fraction, which led to the poor foaming properties of the contaminated egg white protein compared with egg yolk granules. After hydrolyzation, both foamability and foam stability of investigated systems thereof  increased significantly compared with unhydrolyzed ones. However, phospholipids A2 (PLP) seemed to be more effective on increasing their foaming properties as compared to those systems hydrolyzed by lipase (LP). The schematic diagrams of yolk fractions were proposed to explain the aggregation and dispersed behavior exposed in their changes of structures after hydrolysis, suggesting the aggregated effects of LP on yolk plasma and destructive effects of PLP on yolk granules, which may directly influence their foaming properties.As an excellent foaming agent, egg white protein (EWP) is always contaminated by egg yolk in the industrial processing, therefore, decreasing its foaming properties. The aim of this study was to simulate the industrial EWP (egg white protein with 0.5%  Foam is normally defined as a two-phase colloidal structure that is present in a non-equilibrium state containing a continuous phase  in which gas  is suspended as bubbles or gas cells ,2,3. FoaEgg white protein (EWP) is a classic foaming material in foods such as meringues and cake batters . EWP is 2. It is meaningful to determine the effects of esterase treatments on foaming characteristics of industrial egg white protein (egg white contaminated with a low percentage of egg yolk). Therefore, the objective of this study was to compare the foaming and structural properties of industrial egg white protein  being hydrolyzed by lipase and phospholipase A2, and to determine their positive effects on foaming properties of industrial egg white protein via structural changes. Furthermore, lipase and phospholipase A2 were employed in this article in order to enhance and expand their applications scope in food systems, especially in aerated systems.Foaming and structural properties of egg white protein with or without yolk or yolk fractions (plasma and granules) were investigated before and after hydrolysis by lipase and phospholipase A2  was kindly handsealed by Youpuke Biological Technology Co., Ltd. . Sodium dihydrogen phosphate, di-sodium hydrogen phosphate and sodium azide were purchased from Sigma-Aldrich . Water purified by treatment with a Milli-Q apparatus , with a resistivity not less than 18.2 MΩ cm at 25 °C, was used for the preparation of phosphate buffer. The latter was used as the solvent throughout the experiments with addition of 0.02 wt% sodium azide as a bactericide. All the other chemicals and reagents used in this study were of analytical grade.Fresh hen eggs (less than 5 days after laying) were purchased from a local supermarket in Wuxi, Jiangsu Province, China. Lipase  was purchased from Sinopharm Chemical Reagent Co., Ltd.  and phospholipase AEgg white was manually extracted from the yolk of freshly purchased eggs immediately upon arrival in the lab and then homogenized under magnetic stirring (500 rpm speed) for 2 h, as reported previously . No furtv/v) and adjusted to pH 7.0 with 1 N NaOH. After equilibrating overnight at 4 °C, dispersions were centrifuged 45 min  at 4 °C. Then, egg yolk was set apart into egg yolk fractions including plasma (the supernatant) and granules (the precipitate)  .w/w) and 9:1 (w/w), respectively. Therefore, the yield of plasma and granules corresponded to their preparation in yolk. Lipase (LP) and phospholipase A2 (PLP) were dissolved in 10 mM phosphate buffer (PBS) at pH 8.0 in advance. EP and EG were heated to 45 °C quickly and hydrolyzed by LP and PLP at 100 U/g (yolk basis) and 500 U/g (yolk basis) for 2 h in a water bath (under quiescent conditions) at pH 8.0, where enzyme reaction had been completed (not shown), followed by cooling in ice water. Thus, hydrolysates of EP and EG were obtained and called LEP (EP hydrolyzed by LP), PEP (EP hydrolyzed by LP), LEG (EG hydrolyzed by PLP) and PEG (EG hydrolyzed by PLP). In order to make a comparison, hydrolysates of EY  were hydrolyzed by LP and PLP similarly for further tests.Hydrolysates of egg yolk and its fractions were prepared by enzymatic hydrolysis, which was conducted as described by Xing Fu  using thIn order to compare the foaming properties of egg yolk fractions and their hydrolysates, all yolk (yolk fractions) dispersions prepared in i represents the mass of unwhipped dispersions and mf represents the mass of the resulting whipped dispersions (foams) with the same volume of mi.Foam samples were all transferred to cylindrical containers, and a spatula was used to level the top of the foams in order to achieve uniform and plane surfaces to record the foam volume precisely. The foam capability (FC) could be calculated by Equation (1):1 represents the initial mass of foam and m2 represents the mass of drained liquid.Foam stability (FS) was expressed by the mass of drained fluid from the lamella after the whipped foams were stored for 30 min in room temperature and could be calculated by Equation (2):The particle size distribution (PSD) under different treatments was measured by dynamic light scattering via a Zetasizer Nano-ZS  at 25 °C. The refractive index of proteins and aqueous phase was set at 1.45 and 1.33, respectively. Each sample was measured in triplicate.Zeta-potentials of sample dispersions were measured in the standard folded capillary electrophoresis cells, using a Zetasizer Nano ZS instruments . Before measurement, sample dispersions were diluted to 0.005% protein particle concentration using Milli-Q water. Each individual zeta-potential data point was reported as the average and standard deviation of at least five reported readings made on triplicate samples.w/v) Rhodamine B protein stain (excitation 568 nm) and 0.1 mL of 1.0% (w/v) Nile red oil stain (excitation 488 nm). The samples were observed at room temperature (25 °C ± 1 °C) using × 63 objective [Confocal laser scanning microscopy (CLSM) of the sample dispersions before and after hydrolyzing were imaged via a Zeiss LSM 710 confocal microscope , where sample systems were imaged after mixing with 0.1 mL of 1.0% , as described by others ,22. BrieAll measurements were conducted at least in triplicate, and SPSS version 19.0 was used for statistical analysis of means and standard deviations. Significant differences between samples were determined by one-way analysis of variance (ANOVA) with Duncan’s adjustment performed, and the level of confidence was 95%.Foaming properties of egg white protein influenced by yolk fractions and their hydrolysates are shown in Results of particle size distribution are shown in 2. However, the absolute value of zeta potential for yolk fractions hydrolyzed by LP was higher than that of hydrolyzed by PLP, which may be attributed to the different effects of lipase on yolk structures. The zeta potential of LEG (egg granule hydrolyzed by lipase) approximately reached −35 mV, and this phenomenon may be due to the fact that the higher surface charge will be possessed for yolk granules after hydrolysis by PLP compared to NEG , although different extents will happen for the groups exposed after different hydrolyses [As previously reported by Gao et al. , hydrolydrolyses .The CLSM images of sample dispersions under different treatments  are presented in As can be seen, both lipid and protein components in the image are relatively large in 2 (PEY/PEP/PEG), are shown in 2 may effectively act on the complex structures in yolk (both plasma and granules). However, partially aggregated particles were visible, which might have been caused by the heat treatments during hydrolysis [SEM images of sample dispersions, including natural yolk fractions (NEY/NEP/NEG), yolk fractions hydrolyzed by lipase (LEY/LEP/LEG) and yolk fractions hydrolyzed by phospholipase Adrolysis ,34. As cdrolysis .Based on our results, both yolk plasma and yolk granules had undergone the structural changes after hydrolyzed by LP and PLP. Therefore, the schematic diagrams of yolk fractions hydrolyzed by LP and PLP were proposed in As reported, the main constituents in yolk plasma were livetin and low-density lipoprotein (LDL), which includes phospholipids, cholesterol, apoproteins and lipids ,37. From2) were examined. EWP was taken as a standard. Our findings seem to validate the fact that both lipase and phospholipase A2 can increase the foaming properties of yolk fractions, including higher than EWP itself when combined with EWP. However, two types of enzyme showed different mechanisms on the effects on foaming properties of yolk fractions. Compared with LP, higher foamability and foam stability were obtained when hydrolyzed by PLP, which may be attributed to the physicochemical and structural properties of the hydrolysates. Measurements of structural properties seemed to support the idea that loose structures of yolk fractions were beneficial in increasing their foaming properties. Therefore, the fundamental insights of this study may pave a way for improving the foaming properties of industrial egg white  by hydrolyzing sample dispersions. In another aspect, it also provided an idea to generate new types of food foams by retrofitting yolk fractions.Foaming and structural properties of egg yolk fractions  and their hydrolysates (hydrolyzed by lipase and phospholipase A


Hepatitis-associated aplastic anemia (HAAA) is a rare illness that results in bone marrow failure following hepatitis development. The etiological agent remains unknown in most HAAA cases. However, clinical features of the disease and immunotherapy response indicate that immune-mediated factors play a central role in the pathogenesis of HAAA. Activation of cytotoxic T cells and increase in CD8 cells could exert cytotoxic effects on the myelopoietic cells in the bone marrow.4/μL, and alanine aminotransferase level was 1346 IU/L. A blood test indicated cytomegalovirus infection, and 3 bone marrow examinations revealed progressive HAAA. As the disease progressed to the 3rd, 6th, and 9th week after onset, CD4+ T cells were markedly decreased, CD8+ T cells were markedly increased, and the CD4/CD8 ratio was significantly decreased. The number of B cells and natural killer cells decreased with time, eventually reaching 0.0%.A 15-month-old boy was brought to our hospital with complaints of generalized petechiae and purpura observed a week prior to hospitalization. His liver was palpated 3 cm below the costal margin, platelet count was 0 × 10HAAA.Rabbit antithymocyte globulin and eltrombopag olamine (a thrombopoietin receptor agonist) were administered.The patient's platelet count returned to normal, and bone marrow transplantation was avoided. The peripheral blood lymphocytes (PBLs) improved as the patient's general condition recovered.+ and increasing CD8+ PBLs as the bone marrow hypoplasia progresses. The PBLs return to their normal levels with the recovery from the disease. Our case findings thus support the involvement of immunological abnormality in HAAA.This case demonstrates that HAAA induced by cytomegalovirus infection features decreasing CD4 Among 3916 patients with AA reported to the European registry from 1990 to 2007, HAAA accounted for 1% to 5% of all cases. The incidence is high in places where hepatitis is prevalent, mostly in Asian countries,10 and in areas of low socioeconomic status.,11 The etiological agent remains unknown in most cases of HAAA.,5,11Hepatitis-associated aplastic anemia (HAAA) is a well-recognized clinical syndrome in which bone marrow failure follows the development of hepatitis.,5,12,13 These findings suggest that the underlying pathological mechanism in HAAA may be immune mediated.In some studies of patients with HAAA, several immunological abnormalities that responded favorably to immunosuppressive treatment have been described.Here, we present a pediatric case of AA caused by cytomegalovirus (CMV) hepatitis. To the best of our knowledge, this is the first case in which changes in bone marrow findings as well as the time course of the peripheral blood lymphocytes (PBLs) subset were observed simultaneously with HAAA progression. This case is important because it describes the time course of the immunological mechanism in the development of HAAA and recovery from it.2A previously healthy 15-month-old boy was brought to our hospital with complaints of generalized petechiae and purpura observed a week prior to hospitalization. He was born without any problems and had not developed any health problems. His parents too did not have any health problems. He had no siblings, and he was on track with the national vaccination program in Japan. On his initial visit to our hospital, the patient's vital signs were almost normal. His body temperature was 37.1°C, blood pressure was 98/58 mm Hg, heart rate was 120 beats per minute, and saturation of percutaneous oxygen was 98% without supplemental oxygen. His height was 76.8 cm (standard deviation: −0.37 cm), and his weight was 9.4 kg (standard deviation: −0.52 kg). A physical examination revealed spotted hemorrhage and small purpuras scattered throughout the body. Frequent mucosal bleeding in the mouth was noted. The liver was palpated 3 cm below the costal margin but not the spleen.6 cells; normal range: <2.0 × 101/106 cells) were positive, which suggested primary infection with CMV. His autoimmunology-related test values  were all negative. His abdominal ultrasound showed mild thickening of the gallbladder wall and increased brightness around the portal vein . Bone marrow examination was performed to determine the cause for reduction in white blood cell and platelet count. Bone marrow smear revealed slight hypoplasia, with a nucleated cell count (NCC) of 80,000/μL. The cells were not atypical, and abnormal cell proliferations were not observed. Several small lymphocytes with a very high nuclear–cytoplasmic ratio were observed, and megakaryocytes were small in number and size. Furthermore, poor platelet adhesion was noted . No obvious trilineage dysplasia was observed in peripheral blood and bone marrow smears, and blast cells were not present. The diagnosis at this point was CMV hepatitis and myelosuppression owing to CMV infection with negative AA.His blood test results are shown in Table th percentile; normal range: 145-348 mg/dL), triglyceride levels were 88 mg/dL , and ferritin levels were 234 ng/mL . The patient was diagnosed with CMV hepatitis and phagocytosis in the bone marrow due to CMV infection, with no suspicion of AA. He did not meet the diagnostic criteria for hemophagocytotic lymphohistiocytosis (HLH). After the administration of steroids, the number of platelet transfusions decreased; however, the platelet count did not exceed 20,000, and the liver disorder persisted. The third bone marrow examination was performed 8 weeks after the first one  due to CMV hepatitis.The patient's clinical course is shown in Figure 2 copy/mL . Acyclovir and ganciclovir were administered for CMV infection after the hospital transfer, and the CMV-DNA in the blood consequently disappeared. Before bone marrow transplantation for HAAA, rabbit antithymocyte globulin (rATG) was challenged with a cyclosporine combination , and the platelet count recovered to approximately 10.0 × 104/μL. However, 1 month later, the platelet count decreased to ≤50,000/μL, and blood transfusion was required. Up to this point, the bone marrow smear showed hypoplasia. The bone marrow smear performed 4 months after the first administration of rATG showed moderate hypoplasia and an NCC of 18,200/μL. The megakaryocytes were small and did not indicate platelet production . CMV-DNA quantification value using liver tissue was 7.0 × 10ion Fig. A. The reion Fig. B. No obvrd, 6th, and 9th week after the onset, CD4+ T cells were markedly decreased, CD8+ T cells were markedly increased, and the CD4/CD8 ratio was significantly lowered. B cells and natural killer (NK) cells decreased over time and eventually reached 0.0%. T cell receptor recombination circles, kappa-chain recombination excision circles, and severe combined immunodeficiency mutation screening test showed no abnormalities suggestive of acquired immunodeficiency. Furthermore, human immunodeficiency virus antigen antibody test was negative. As the treatment progressed, the significantly decreased CD4+ T cells returned to their normal values and the significantly increased CD8+ T cells decreased to their normal values. Consequently, the CD4/CD8 ratio was normalized. B cells and NK cells showed an upward trend over time. This posttreatment result was the opposite of what was noted during the progression of HAAA.Table At the most recent evaluation 4 years after commencing eltrombopag olamine administration, the patient was healthy without the need for medication and was being carefully monitored for the development of leukemia.3+ and increase in CD8+ PBLs as the bone marrow hypoplasia progressed. Conversely, the findings normalized with the recovery from HAAA. Second, this HAAA case was most probably induced by CMV infection as per the serum CMV antibody and polymerase chain reaction findings using peripheral blood samples.The patient's clinical course reveals 2 important points. First, we were able to follow the progression of HAAA and recovery from the disease with the aid of bone marrow and PBL subset findings. Hence, we were able to observe the decrease in CD4,17 In our case, the lack of hemolytic anemia in the initial laboratory findings was sufficient to eliminate GCH. In addition, the bone marrow findings ruled out other diagnoses, such as hemophagocytic syndrome, megaloblastic anemia, bone marrow infiltration of malignancy, myelodysplastic syndrome, acute leukemia, and thymoma.,19 Myelodysplastic syndrome was excluded based on the absence of precursor cells, blood cell atypia, and chromosomal abnormalities. Flow cytometry did not detect glycosylphosphatidylinositol-anchored protein-deficient blood cells; therefore, paroxysmal nocturnal hemoglobinuria was ruled out. Chromosomal breakage in PBL was not evaluated; however, physical findings and skin pigmentation unique to Fanconi anemia, such as congenital anemia, were observed.As in the present case, when anemia is present at the onset along with severe thrombocytopenia, diseases other than HAAA, such as infantile giant cell hepatitis (GCH) with autoimmune hemolytic anemia, must be differentiated. GCH is characterized by large and multinucleated hepatocytes in the context of liver inflammation. Infantile GCH is typically associated with autoimmune hemolytic anemia. Recent studies have demonstrated the simultaneous expansion of a liver-infiltrating cytotoxic T lymphocyte clone with the development of HAAA. Moreover, CD8+ Kupffer cells might be important mediators of HAAA. Brown et al reported that patients with HAAA have a decreased CD4/CD8 ratio in peripheral blood, which is associated with the activation of cytotoxic T cells and an increase in the proportion of HLA-DR-positive CD8 cells. It has been reported that the phenomenon in HAAA appears more clearly than in idiopathic AA. Kagan et al demonstrated in vitro that in AA, activated CD8-positive lymphocytes could be cytotoxic to the myelopoietic cells in the bone marrow. In our case, PBL subset analysis was performed during the gradual decrease in NCC in the bone marrow, and eventually the diagnosis of AA was reached. The PBL subset analysis demonstrated a decrease in the number of CD4+ cells and an increase in the number of CD8+ cells, which decreased the CD4+/CD8+ ratio. To the best of our knowledge, this is the first pediatric case describing consecutive bone marrow findings leading to HAAA and a decrease in the CD4+/CD8+ ratio using PBL analysis. Our findings support that the increase in CD8+ cells is responsible for the bone marrow hypoplasia in HAAA. Furthermore, solid evidence could be obtained because the changes in the lymphocyte subset were also monitored during the recovery from HAAA with the treatment. Several other studies have demonstrated that activated cytotoxic lymphocytes produce increased amounts of interferon (IFN)-γ in the bone marrow of patients with AA.,23,24 IFN-γ is a marrow-suppressing cytokine that could induce bone marrow inhibition. Hepatic inflammation in HAAA is linked to analogous findings. In our case, unfortunately, we could not prove this theory because we could not measure inflammatory cytokines over time.The clinical features of the disease and its immunotherapy response indicate that immune-mediated factors play a central role in the pathogenesis of HAAA. Gascon et al documented that deficient NK cell activity was an intrinsic feature of AA and that the recovery of NK cell activity was related to hematopoietic recovery. The results from the present study support their theory. The immunological significance of NK cell activity depletion in HAAA, similar to AA, requires further research.The PBL subset analysis showed that the proportion of NK cells decreased with the onset of HAAA and finally reached 0%. Similar findings have also been reported in idiopathic AA.,29 HAAA cases featuring bone marrow infiltration of macrophages and hemophagocytosis have been reported. Because HLH is known to cause aplastic bone marrow if left untreated for a prolonged duration, HAAA may thus be associated with HLH. AA-associated HLH has rarely been documented, and problems in the diagnostic procedure have been discussed. In the present case, the second bone marrow examination showed conspicuous macrophages. Although this observation does not satisfy the HLH diagnostic criteria, changes in cytokine levels associated with hepatitis resulted in an HLH-like pathology after acute hepatitis. Moreover, increased CTL, IFN-γ, and tumor necrosis factor-α might have led to the onset of AA. On the other hand, Brisse et al reported that CD8+ T cells constitute a defensive factor in virus-associated secondary HLH. In the present case, it cannot be denied that the increase in CD8+ cells with the progress to HAAA might have exerted a protective effect against HLH associated with CMV. We could not clearly explain the cause for the increase in macrophages in the second bone marrow examination. Further accumulation and evaluation of HAAA cases with HLH may lead to an adaptive expansion of HAAA etiology.The association between AA and HLH has been debated, particularly the aspect that HLH may be a cause for secondary AA. However, the etiological agent remains unknown in most AA cases.,5,13,25 In a study in Japan, 61 HAAA cases were observed in 525 children with acquired AA. Viral studies of 49 of the 61 cases revealed single patients infected with HAV, hepatitis C virus, EBV, or CMV. In 214 HAAA cases retrieved from the European registry between 1990 and 2007, no causative virus for hepatitis was identified in 94% of the patients with HAAA, whereas it was a hepatitis virus in 15 cases—HBV in 9 cases and HAV in 6 cases. In our case, CMV infection was strongly inferred from the serological findings of CMV and CMV-DNA in blood and liver tissue. The gold-standard assay for CMV is CMV–polymerase chain reaction, which has a reported sensitivity of 85% and specificity of 95%.,35 In the present case, although cytomegalic inclusion bodies were not detected in the liver, the CMV-DNA was detected in blood and liver tissue. In addition, the CMV-DNA disappeared with the treatment and the liver dysfunction  was reversed, which provides strong evidence that hepatitis was caused by CMV. As identifying the virus causing hepatitis in HAAA may be very difficult, large epidemiological studies that consider regional and racial differences are required to comprehend the relationship between the etiology of hepatitis and HAAA.We also hypothesize that this HAAA case was induced by CMV infection. AA cases associated with hepatitis A virus (HAV), HBV, hepatitis G virus, human parvovirus B19, EBV, and transfusion-transmitted virus or echovirus infections have been reported.The present case report has several limitations, including the lack of chromosomal breakage evaluation, the lack of information on changes in inflammatory cytokine levels over time, and the lack of insight into the immunological significance of the depletion of NK cell activity.+ and increase in CD8+ PBLs in a pediatric patient. These findings provide evidence supporting the role of immunological abnormalities in the onset of HAAA. Furthermore, our findings show that HAAA may be induced by CMV.Our case report describes the process of bone marrow hypoplasia progression based on consecutive test findings detailing the decrease in CD4The authors thank the patient's family for providing consent and granting permission to draft and publish this case report.Conceptualization: Toshihiko KakiuchiData curation: Toshihiko Kakiuchi, Katsuhide Eguchi, Daisuke Koga, Hiroi Eguchi, Masanori Nishi, Motohiro Sonoda, and Masataka IshiharaFormal analysis: Toshihiko KakiuchiInvestigation: Toshihiko Kakiuchi, Katsuhide Eguchi, Daisuke Koga, Hiroi Eguchi, Masanori Nishi, Motohiro Sonoda, and Masataka IshiharaMethodology: Toshihiko KakiuchiProject administration: Muneaki MatsuoSupervision: Muneaki MatsuoValidation: Toshihiko KakiuchiWriting – original draft: Toshihiko Kakiuchi and Daisuke KogaWriting – reviewing & editing: Muneaki Matsuo