Ready to eat fried liver meat balls (LMB) were developed to fight anaemia and vitamin A deficiency and promote cognitive function.Randomised controlled trial consisting of two arms: control group with no supplement and LMB group receiving LMB supplement three times a week for 90 d. Criteria of evaluations included dietary assessment, anthropometric measurements, laboratory investigations and cognitive function by Wechsler test.Kinder Garten and primary school in Urban Giza.Sixty boys and girls aging 3–9 years.P < 0·05) only after the dietary intervention with the LMB supplement for 90 d. The standard scores of verbal and non-verbal cognitive function tests (Δ day 90–day 0) increased significantly (P < 0·05) among the LMB group compared with the respective changes observed among the control group. The increase in height-for-age Z score and blood Hb were good predictors for improvement in cognitive function.The LMB supplement contributed to significant increases in the intakes of high bioavailable Fe and vitamin A in the diets of all children. Initial overall prevalence of mild and moderate anaemia was 43 %, which disappeared completely from all children aging < 72 months and from 88 % of children ≥ 72 months after the 90 d dietary intervention with the LMB. Faecal systemic immune globulin A, urinary hydroxyproline index and urinary iodine excretion increased significantly (LMB supplement is effective sustainable nutritious biotherapeutic food in fighting hidden hunger and promoting the cognitive function. The economic cost of hidden hunger, represented by Fe-deficiency anaemia, Zn deficiency, vitamin A deficiency and iodine amount to a loss of 2·5% of the Indian gross domestic product. The most commonly used strategies to combat Fe deficiency are the provision of Fe supplements as FeSO4, which have limited significant impact on BMI–8, health and nutritional status among adolescents in low- and middle-income countries. Currently, there is general agreement that Fe fortification of low bioavailability diets had limited effect on Fe status,11. Meanwhile, the higher food synergy of animal-sourced foods (ASF) render them superior in raising the low bioavailability of dietary Fe relative to the respective technologically reduced Fe-fortified cereal products. ASF consumption indicator associated negatively with stunting in studies involving 30 000 children in sixty-one countries from five continents. ASF-based supplementation to young children increased weight by ≤ 0·14 kg and the height-for-age Z score by +0·06 and are considered the optimum option for improving nutritional quality of a largely plant-source based diet.. Supplementing the meals of African children with cooked beef improved their growth, cognitive and behavioural outcomes–18. The intake of animal products is usually constrained by their cost, but the edible internal organs, which makes considerable proportion of the carcass weight of chicken and farm animals are highly nutritious at reasonable prices. Chicken liver contains high Fe content, when compared with the Fe levels in beef liver (29·3–66·71 mg/kg)(78). The absence of vitamin A deficiency among South African preschool children is due to the common consumption of 60–75 g of liver per month. World’s frozen chicken liver with average price of 540–560 US$ per metric tons (1000 kg) is far much cost effective than the respective price of 143 US$ for one single package of American fortified maize soyabean flour blend, required for the dietary management of an African malnourished child for a duration of 2–3 months.The sustainable development goals 2030 created in the year 2015 by the United Nation organisation imply that each of the 193 member states should do substantial efforts and undertake actions to reduce the stunting, wasting and anaemia, indicators of hunger by 50 % by the year 2030 The objective of the present study is to develop and formulate a nutrient dense ready to eat chicken liver–beef balls to end hidden hunger among pre- and school aged children, with particular emphasis on improving their growth and cognitive outcomes.Ingredients of the liver meat recipe were purchased from the retail market and prepared in a batch of 110 kg. They included chicken liver, bovine meat, rice flour, together with other vegetables. The chicken liver was heated gently until water drainage ceased. The edible parts of carrots, garlic, onions and tomato were obtained by peeling followed by appropriate washing with tap water. The eight ingredients (Table Preschool children aging 36–71 months were recruited from a child care centre (Kinder Garten) and children aging 73–118 months were recruited from a primary school located in urban Giza governorate.Criteria for inclusion was the absence of gastro-intestinal disorders, no use of antibiotics during the last 3 months prior to the study; the taste of the LMB was acceptable to the child; the mother was willing to complete the dietary assessment sheet and to collect stool and urine samples from her own child at predetermined dates.The dietary intervention trial consisted of randomised controlled trial on sixty children. The children were assigned randomly into two age-matched equal groups: group 1 (36–< 72 months) and group 2 (≥ 72 months). Each group was subdivided into two subgroups: the first served as control and did not receive any supplement; while the second subgroup had a serving of LMB. The serving size was one LMB ball to children < 72 months; while the respective service size was two LMB balls for children ≥ 72 months. Serving was three times a week on Sundays, Tuesdays and Thursdays with a total of forty-five servings throughout the 3 months (90 d) duration of the trial..The age and sex of the child were obtained from the birth certificate; body weight and standing height measurements were taken, while the child was with the light clothes and bare footed. Based on the data on age, sex, body weight and height for each child, the respective three nutrition indices of physical growth: weight-for-age Z score, height-for-age Z score (HAZ) and weight-for-height Z score were computed by the EPIINFO software program. The child whose weight-for-age Z score measures below (–2·0) was underweight for his age. The HAZ index identifies linear growth and the child whose HAZ was below (–2·0) from the WHO child growth standards reference population was considered short for his age or stunted. The weight-for-height Z score measures body mass in relation to body length and the child whose weight-for-height Z score below (–2·0) was thin for his height. Boys 3–10 years = 0·095 ×body = weight + 2·11.The BMR was computed in mega joules (MJ) using the following equationsGirls 3–10 years = 0·085 × body weight + 2·03.The BMR was expressed in kcal from the relation 1 MJ = 4180 kcal.. Calculation of nutrient intake and food group consumption was performed using a software application, specifically developed for processing of dietary data.Three 24-h dietary recalls were collected on non-consecutive days from the mother of each child. The mothers recalled all foods and beverages consumed at home and at the day care centre or school throughout the previous 24 h . All mothers were then asked to estimate the quantities of the mentioned foods and to state the ingredients used for preparing the dishes and their respective quantities. To facilitate the quantity estimates, standardised household measures  were provided by the interviewer and marked with several levels to indicate different contents. Raw ingredients were converted from household measures to weight estimates by taking reference weights, calculated on the basis of the average of weights on a digital weighing scale. A trained dietician instructed parents, checked the data and supervised inconsistencies and completeness of the records and was responsible for coding and computer entry of the consumption data. A raw data set for each child contained records with food codes and day, meal, quantity (g or ml), food item and its food code. The food intake data were converted into weights and the combined data were converted into respective energy and estimated nutrient intakes using the data values from the precise Egyptian food composition table (unpublished data) and other food data bases as needed for expressing the individual daily intake of a nutrient into the respective probability of nutrient adequacy (PA). The percentage of reference for each nutrient was capped at 100 % daily value to avoid overvaluing by single nutrient.To determine the adequacy of daily nutrient intake by a sample individual, the child’s daily nutrient intake was compared with the respective age and sex-specific FAO/WHO essential minerals and vitaminsRatios of estimated daily energy intake (mjoules/ BMR MJ) below 0·70 or above 1·80 were excluded from the calculation and the dietary assessment was repeated.. The test identifies how a child’s performance compares to age or grademates. It identifies specific skill strengths and deficits. A battery of cognitive verbal (three subtests) and performance or practical subtests were used. The verbal subtests included word reading, reading comprehension, oral listening, oral expression such as question answering and story generation, summarisation, attention difficulties and concentration difficulties or comprehension of written and spoken language and word fluency. Skill measures included picture completion; the child was asked to find the missing parts in a picture of familiar objects and people and the test is said to reflect visual alertness as well as visual recognition and identification . Age-based normative data was used and the total raw score from each of the subtests was transferred to the respective standard score for each subtest to obtain a weighted raw score. The composite standard scores were calculated by summing the subtest standard scores.Wechsler Intelligence Scale for 3–5 years (grades Kinder Garten) through 10 years was carried out according to the standard methodology using the detailed instructions. Moisture was determined by heating the samples in an oven at 102°C until a constant weight was attained. The protein level was determined using the Kjeldahl method for determining nitrogen content and multiplying this by 6·25. After acid hydrolysis, the fat was extracted with ether using a Soxhlet apparatus. Ash was obtained by incineration at 500–550°C until the ash was carbon-free. The carbohydrate content was determined by difference. The energy density was calculated by multiplying the protein and total carbohydrate content by 16·736 kJ and adding the result to the fat content multiplied by 37·656 kJ.Chemical analysis of the LMB moisture content, protein, fat and ash was determined according to the Association of Official Agricultural Chemists methodBlood samples were collected at baseline (day 0) and endpoint (day 90) by a finger prick. Blood Hb concentration was measured in 20 µl whole blood by the direct cyanmethemoglobin method (Drabkin’s solution) and a photometer at 540 nm  and at endpoint (day 22), each child was asked to collect stool sample into a container and an aliquot was immediately frozen at –20°C for later immunological assay.The  is the major Ig synthesised in the colonic mucous membrane and plays a major role in preventing adherence of pathogenic microorganisms to mucosal sites, and protects against gastrointestinal infections. The s-IGA was assayed by the Chrom ELISA kit  and was used according to the instruction of the manufacturer. Weighed faecal portions (80–120 mg) were suspended in 10-fold excess (1·5 ml) physiological phosphate buffer, vortexed and centrifuged at 13 000 rpm for 5 min. The supernatant was diluted 1:250 in wash buffer. Standards, controls and stool samples were simultaneously run in the ELISA and the concentration of s-IgA was read at 405 nm in ELISA reader against a standard curve and the results were expressed as mg faecal S-IgA per gram stool.Each child was asked after evacuating morning urine to collect void urine sample, which was labelled and saved frozen for subsequent laboratory investigations.Hydroxyproline (HPRO) is a collagen-related marker as it is present in all fibrillar collagens and tissue breakdown contributing to urinary HPRO. Urinary hydroxyproline index (HPRO-I) is a potential biomarker with high prognostic value before growth spurt with cut-off point of ≥ 2·5 ± 0·9 among normal children. This biomarker assists in making the prospect of catch-up growth rate over short periods of time and in close monitoring of treatment regimens.. Urine aliquots (100 µl) were hydrolysed in 10 × 1·5 cm pyrex tubes with screw caps with equal volume of 12 normal hydrochloric acid by heating at 125°C for 2 h. Upon cooling, the pH of the acid hydrolysate was adjusted to 6·0 by the addition of buffer solution. The standard hydroxyproline, the blank and the urine samples were subjected to colour reaction, which is based on the formation of pyrole derivative with tosylchloramine. The pink colour developed with p–aminobenzaldehyde was read at 560 nm, and the results were expressed as μg total HPRO per ml urine.The determination of total urinary (U-HPRO) is based on the colorimetric reaction of Ehrlich.The classical alkaline picric acid and sodium hydroxide reagent were used.The U-HPRO excretion was expressed as total hydroxyproline/creatinine(mmol/mol). The urinary hydroxyproline index (U-HPRO-I) = U-HPRO creatinine ratio/kg body weight urine aliquots (250 µl) was digested in 10 × 1·5 cm pyrex tubes with screw caps with ammonium persulfate in a heating block at 100° for 60 min. The catalytic properties of the liberated iodine were used for the reduction of ceric ammonium sulphate in the presence of arsenious acid, and the yellow colour was measured at 430 nm. A standard curve prepared from potassium iodate solution (10–80 ppm) was used for calculating the urinary iodine. Quality control for iodine was maintained by including reagent blanks to monitor contamination and estimate detection limits.Measurement of iodine concentrations from casual urine is now widely accepted as the best and most cost-effective way of monitoring iodine deficiency in a community, with an iodide excretion below the cut-off point of 0·79 µM iodine/l (100 µg/l) of urine is considered to be at risk of a marginal iodine supply and therefore of goitre and other iodine deficiency disorders. The determination of urinary iodine by ICCIDD and the results were expressed as urinary thiobarbituric acid creatinine ratio (mg/g).Urinary malondialdehyde excretion is a valid biomarker for the oxidative stress of the body to lipid peroxidation, which is assayed by the thiobarbituric acid reactive species (TBARS). Colorimetric methodDescriptive statistics were obtained for all variables and outcome variables stratified by age groups: < 72 and ≥ 72 months. The Whisker box plots of each variable were classified according to age group and dietary treatment. The boxes indicate 25th to 75th percentiles, with mean relative abundances marked as lines and whiskers indicating the range (minimum/maximum) from the boxes. Statistical tests for the distribution of the variable were calculated and compared according to categories based on the Kruskal–Wallis test for continuous variables. Spearman correlation coefficients were used to measure the correlation.Table Table 2) revealed high statistical significant differences (P < 0·001) between the control and the LMB group in the distribution of the number of anaemic and non-anaemic children.Initially, 43 % of all participating children were suffering from mild or moderate anaemia. Following the intake of the LMB supplement for 90 d, anaemia disappeared completely among children in the age group < 72 months, and among the older children ≥ 72 months its prevalence was reduced down to 12 %; while its prevalence remained almost unchanged among the control group  in the mean concentration of s-Ig A compared with the respective pre-intervention levels and compared with the control group  averaged 3·1 ± 0·1 µg (2·87–3·68) per gram faecal material with no significant differences between the groups. The 90-d dietary intervention with LMB was associated with a significant increase (P < 0·05) among children < 72 (4·08 ±0·17) compared to those aging ≥ 72 months (2·39 ± 0·12). The prevalences of children excreting U-HPRO-I) < 2·5  amounted to 28·2 % and 63% among children < 72 months and ≥ 72 months, respectively. Following the 90-d dietary intervention with LMB, the excretion of U-HPRO-I exceeded the cut-off point of 2·5; while the respective excretions of U-HPRO-I below 2·5 were 66 % and 82·4 % among the control group aging < 72 and ≥ 72 months (Table P < 0·001) between the control and the LMB children in the distribution of numbers excreting U-HPRO-I below 2·5 and ≥ 2·5 compared with the expected based on normal distribution.At baseline, the mean (U-HPRO-I) excretion was significantly higher  compared with the respective pre-intervention values .The mean urinary excretion of thiobarbituric acid was not affected by the intervention with LMB and the differences in concentrations between all groups did not attain significant levels  among the control children (32·8 ± 3·2 mg) compared with the respective intakes of 16·7 mg among the LMB children. The estimated daily intake of Fe increased significantly (P < 0·05) from 4·65 ± 0·25 mg on pre-intervention days up to 5·5 ± 0·23 mg during the intervention days with LMB (days 1–90) days. Similar significant increases were found in the estimated daily vitamin A intake following the dietary intervention with LMB (days 1–90) compared with the pre-intervention intake.Tables P < 0·05) were found in the energy among the control children  compared with the respective intake of 1096 ± 46·5 kcal among the LMB children (Table P < 0·05) than the respective fat % Energy of 34·3 ± 1·1 % E in the diet of the control children. The estimated Fe intake was almost comparable (6·14–6·78 mg/d) among both groups; but 35·5 % of the dietary Fe in the diet of the LMB children was derived from the animal sources; fishes, meats and poultry v. only 18·3 % among the control. On the contrary, the estimated dietary vitamin C supply in the diets of control children averaged 46 ± 2·2 mg; which was double the respective supply of 21·7 ± 3·02 mg among the LMB children. Dietary vitamin E was poor in the diets of all children.With regard to the children aging ≥ 72 months, higher estimated daily intakes (P < 0·05) in the PA of total Fe, Fe from animal sources  and vitamin A. Increases were also observed in the PA of Zn, Se, vitamins B1, B2, B6, folic acid and niacin, but the increase did not attain significant level (P > 0·05).The probability of adequacy (PA) of thirteen nutrients is presented in Tables P < 0·05) with the increases (Δ d90–d0) in absolute body heights, the HAZ and urinary iodine excretion. In children ≥ 72 months, the increase in practical subtests of the Wechsler score correlated with changes in HAZ (Δ d90–d0)  Table .The development of the deep-fried LMB used in the present study was culturally acceptable by the participating children, with shelf-life stability for days, reducing the need for refrigeration and is part of the ASF best food-based strategy to prevent stunting and promote cognitive development.. It is estimated that if 30% of the population are anaemic at the onset, an appropriate theoretical sample size of about twenty-five subjects is needed to be followed for at least 3 months to detect an increase in Hb of 1 g/dl. This protocol was practiced in the present study, and the sample size was thirty children and indeed at the end of the 90 d intervention with LMB, the increases in blood Hb concentration averaged 1·3 ± 0·08 and 1·15 ± 0·09 g/dl among children < 72 months and ≥ 72 months, respectively.Body weight contains an average of 70 ml blood/kg. Hb contains 3·4 mg Fe/g and the normal blood Hb concentration is 115–120 g/l than dietary non-heme Fe. Full fat soy flour, isolated soy protein concentrate and even textured soybean are typical example of food products reducing Fe absorption by binding it to insoluble peptides in the duodenum and the term residual ‘soy factor’ is implicated. Commercial ground beef patties and hamburger available on the Egyptian market contain soyproteinsas ingredient in the range between 10 and 25 %, as detected by immunological blotting technique. A randomised controlled trial showed statistical significant (P < 0·05) inferior % apparent Fe retention among male adolescents consuming the sausage extended with soyabean protein (14·6 ± 0·8 %) compared with the respective % apparent Fe retention of 32·2 ± 1·9 % found among the group consuming Fe from 100 % beef under otherwise identical experimental conditions. The consumers welcome the slight reduction in the price of meat extended with soyabean proteins, being unaware that this is at the expense of inferior qualities. % Fe bioavailability can be assessed precisely from the daily intakes of the food constituents of Fe from fish (F), meat (M) and poultry (P). The intake of high dietary ascorbic acids, such as guava (102 mg/100 g) overcome the inhibitory effects of phytate in cereals and enhanced significantly Fe bioavailability in adolescents boys and girls.Fe is present in food in both inorganic (ferric and ferrous) and organic (mostly heme). Heme Fe constitutes 40 % of the Fe in meat, poultry, chicken liver and fish, whose absorption into the intestinal lumen is 25 % times greater. Vitamin A deficiency impairs the utilisation of Fe for haemoglobin synthesis, in part by trapping Fe in the liver and spleen. Baseline dietary supply of vitamin A in the diets of our participating children was poor, and the intervention with the LMB supplement restored the deficiency and the probability of vitamin A adequacy exceeded 100 %. A South African study carried out on preschool children attributed the adequate vitamin A status of the children to regular intake of 60–75 g of liver once per month. Riboflavin deficiency also impairs Fe absorption by altering the turnover of intestinal cells, associated with a change in Fe utilisation for Hb synthesis. The estimated daily intake of riboflavin was acceptable among our children and was close to 90 % of the FAO/WHO requirement. Both vitamin B12 and folic acid deficiencies can cause anaemia. The intervention with LMB provided a generous supply of vitamin B12 averaging 2·74 and 5·48 µg/day, for children < 72 and ≥ 72 months, respectively, which exceed the respective daily FAO/WHO requirement.Also, vitamin A is needed together with Fe for the most effective treatment of anaemia. Fe supplementation of the anaemic children with high pharmacological dosage of 100 mg ferric glycine for 10 weeks increased the blood Hb concentration and the increase was also associated with statistically significant improvement in total The Wechsler Intelligence Scales for Children-Revised score. Our results are in good agreement with these findings.Mild anaemia (Hb concentration between 110 and 119 g/l) was reported to affect the behaviour and cognitive functions of 9-year-old school children and Kenya with small amounts of meats improved their linear growth, physical activity and increased their cognitive skills, behaviour and initiative. Eight interventional studies across eleven countries indicated that supplementation with ASF to 26 299 children (age 1–16 years) showed consistent positive associations with cognitive development, increased cognitive functions (test and exam scores) by up to 20-fold and fluid intelligence and verbal skills by up to twofold.Another study reported that supplementing the basal diet of children from Bhurkino Faso, Ghana, Malawi. The high-quality proteins found in ASF are typical examples in facilitating specific mechanisms, such as speed of information processing, that are involved in learning tasks such as problem-solving capacity. The bioavailability of other nutrients in ASF such as Fe, Zn, iodine and B vitamins  enhance cognitive development through their impact on structural brain development via enhancement of myelination and synaptic connectivity.Neurological studies showed that over a hundred unique factors have been identified to influence cognitive development in children in order to be the only sustainable long-term approach at the community level, where designing the most effective and practical strategies.The present study clearly demonstrates that Fe and vitamin A deficiencies are risk factors affecting children’s growth and cognitive development. Since venous blood sampling from healthy children is prohibited by law in Egypt, it was not possible to assess the biochemical status of vitamin A status and the dietary assessment was the only option to assess the estimated vitamin A intake. The study also clearly demonstrated the role of ASF against undernutrition conditions that generate low/suboptimal children’s cognitive development. Therefore, dietary intervention using ASF should be articulated, publicised and disseminated sufficiently


Alzheimer’s disease is a neurological disorder characterized by the overproduction and aggregation of amyloid-beta and the phosphorylation and intraneuronal accumulation of tau. These events promote synaptic dysfunction and loss, leading to neurodegeneration and cognitive deficits. Astrocytes are intimately associated with synapses and become activated under pathological conditions, becoming neurotoxic and detrimentally affecting synapses. Although it has been established that reducing neuronal tau expression prevents amyloid-beta-induced toxicity, the role of astrocytic tau in this setting remains understudied. Herein, we performed a series of astrocytic and neuronal primary cultures to evaluate the effects of decreasing astrocytic tau levels on astrocyte-mediated amyloid-beta-induced synaptic degeneration. Our results suggest that the downregulation of tau in astrocytes mitigates the loss of synapses triggered by their exposure to amyloid-beta. Additionally, the absence of tau from astrocytes promotes the upregulation of several synaptoprotective genes, followed by increased production of the neuroprotective factor Pentraxin 3. These results expand our understanding of the contribution of astrocytic tau to the neurodegenerative process induced by amyloid-beta-stimulation and how reducing astrocytic tau could improve astrocyte function by stimulating the expression of synaptoprotective factors. Reducing endogenous astrocytic tau expression could be a potential strategy to prevent synaptic damage in Alzheimer's disease and other neurological conditions. et al. reported how the downregulation of tau in astrocytes mitigates the loss of synapses triggered by their exposure to amyloid-beta. The authors suggested that the absence of tau from astrocytes promotes the upregulation of a series of synaptoprotective genes such as Pentraxin 3.Cisternas   The prevailing hypothesis is that hyperphosphorylation, misfolding, and fibrillization of tau impairs synaptic structures and triggers neuronal death;5 it is widely accepted that tau acquires a toxic gain of function. Alternatively, it has recently been suggested that physiological tau functions could possibly contribute to brain disease by allowing other factors and processes to alter other signalling pathways.6 For instance, Aβ-mediated neurodegeneration and cognitive dysfunction are considered to depend largely on tau.6 It has been reported that reducing tau expression prevents or diminishes Aβ-induced toxicity in cultures of rodent primary neurons.8 Moreover, in vivo studies have revealed that total genetic ablation of endogenous murine tau in a hAβPP mouse model prevents behavioural deficits and synaptic alterations.9–12 The results of these studies indicate that lowering tau levels is a potential therapeutic strategy for Alzheimer’s disease, bypassing the need to determine which forms of tau are most detrimental.Alzheimer’s disease is a neurological disorder characterized by extracellular plaques composed of aggregated forms of the amyloid-beta (Aβ) peptide and intraneuronal neurofibrillary tangles (NFTs), neuropil threads, and dystrophic neurites that contain aggregated forms of the protein tau (‘tau-pathology’).13 Therefore, most tau-related studies, including those evaluating the effect of decreasing tau levels, have been focused on neurons.15 Interestingly, tau is also expressed in astrocytes, although at lower levels than in neurons. Under basal conditions, tau expression is 5 and 10 times lower in astrocytes than in neurons in humans and mice, respectively.16 Under physiological conditions, several astrocytic mechanisms contribute to the regulation of neuronal function, synaptic integrity and plasticity.17 Therefore, alterations in astrocytic function may contribute to synaptic loss in Alzheimer’s disease;18 however, the extent of this contribution is currently unclear. As the expression levels of astrocytic tau are low, its relevance has yet to be studied in detail. Recent studies have revealed that lowering tau levels in 4R tau-expressing iPSC-derived astrocytes improves neuronal survival.19 Thus, we aimed to determine if the deletion of astrocytic tau protects synapses against the toxic effects of Aβ oligomers.Under physiological conditions, tau is mainly expressed by neurons.Our present results suggest that astrocytes could be important mediators of Aβ-induced neurotoxicity and that reducing endogenous astrocytic tau levels ameliorates synaptic loss. Our gene expression analyses suggest that this beneficial effect could partly be due to the upregulation of several astrocytic neuroprotective factors, among which PTX3 showed the most significant increase.ad libitum and housed with light/dark cycles of 12 hrs. P0-P1 wild-type  and tau knockout  male and female pups were used for the cortical astrocyte and neuronal culture experiments, and 6-month-old (6 mo) WT and tau−/− mice were used for the immunofluorescence (IF) studies. Tissues were collected after the animals were euthanized by decapitation under deep anaesthesia. Brains were extracted and prepared as previously described.20All work involving mice was performed following the Institutional Animal Care and Use Committee guidelines and in compliance with the Animal Welfare Act, the Guide for the Care and Use of Laboratory Animals, the Office of Laboratory Animal Welfare and the guidelines of Indiana University School of Medicine. Animals were provided with water and food 21 Briefly, brains were extracted, and cortices were dissected and washed with calcium-and magnesium-free Hank’s balanced salt solution (CMF-HBSS). Cortices were resuspended in 4.5 ml of CMF-HBSS and incubated with 2.5% trypsin and 1% DNAse for 15 min at 37°C, with gentle swirling of the tubes every 5 min. Next, a cell suspension was obtained by carefully pipetting the tissue. The cell suspension was filtered through a 40 μm pore cell strainer and centrifuged at 1000 rpm for 8 min. The cells were counted, and 1 × 106 cells/ml were seeded in 12 well plates containing 18 mm sterile coverslips in 37°C prewarmed glial medium [minimal essential medium (MEM)], 0.6% glucose, 1 ×  penicillin/streptomycin, and 10% fetal bovine serum (FBS) for the IF experiments. Sixty-millimetre cell culture dishes were used for astrocyte conditioned medium (ACM) collection. The cells were maintained by replacing the glial medium every 2 days until the desired confluency was achieved at 11 days.Astrocytes were cultured as previously described.22 Briefly, brains were extracted, and cortices were dissected and washed twice with dissection medium . The cortices were suspended in 4.5 ml of DM and incubated with 2.5% trypsin and 1% DNAse for 15 min. After 2 washes with DM, cortices were washed twice with 37°C prewarmed plating media . Tissue was disaggregated using glass Pasteur pipettes with tips previously rounded by gentle flaming. The resulting suspension was filtered through a cell strainer (40 μm pore). The cells were counted, and 200 000 cells/ml were seeded on 12 well plates containing 1 ml of 37°C prewarmed maintenance medium  and 18 mm coverslips treated overnight with 0.5% poly-L-lysine in borate buffer. Half of the media was replaced with fresh 37°C prewarmed MM 5 hours after seeding. On the third day of culture, cytosine arabinoside (AraC) was added at a final concentration of 3 μM. Neurons were maintained for 14 DIV. Half of the medium was replaced with fresh 37°C prewarmed MM every 2 days.The procedure used for cortical neuronal culture was based on previous work.23 Briefly, 0.3 mg of lyophilized peptide Aβ1–42 (03112 Novex by Life Technologies) was resuspended in 500 μl of 50% acetonitrile/water and relyophilized. The protein powder was dissolved in 200 μl of hexafluoroisopropanol (HFIP), and the suspension was incubated for 15 min at room temperature (RT). Next, 700 μl of cell culture-grade ultrapure water was added, and the suspension was stirred at 500 rpm using a Teflon-coated micro stir bar for 36 h at 22°C in a fume hood. An 18-gauge needle was used to make three holes in the caps of the tubes to allow the evaporation of HFIP.Aβ oligomers were prepared as previously described.in vitro (11 DIV), Aβ oligomers were added at a final concentration of 1 μM and the cells were incubated for a further 24 h. This concentration of Aβ, which is higher than physiological levels,24 has been previously used in astrocyte culture to trigger reactivity without cellular death25–27 The next day, the astrocytes were washed twice with 37°C prewarmed cell culture-grade sterile PBS (1X), and the medium was replaced with glial medium without either phenol red or FBS. The medium was conditioned by astrocytes for 24 h. The next day, the ACM was collected and lyophilized. The protein powder was resuspended in 1.5% of the original lyophilized volume in cell culture-grade sterile PBS (1X). The total protein content of the ACM was determined by the BCA method, and 10 μg of total protein was added to 14 DIV neuronal cultures. The endotoxin content of the ACM was quantified using the Pierce Chromogenic Endotoxin Quantification kit  according to the manufacturer’s instructions. The conditioned media contained under 1 endotoxin unit (EU)/ml, a non-toxic concentration incapable of inducing significant glial activation.28–31 Recombinant human PTX3 (R&D Systems 10292-TS-050) was prepared in MM and applied to neuronal cultures at a final concentration of 1 μg/ml as previously described.32 Heat-inactivated PTX3 (iPTX3) was obtained by heating aliquots of PTX3 for 10 min at 70°C. Neurons were incubated with the ACM for 24 h, fixed in 37°C prewarmed 4% paraformaldehyde/4% glucose in PBS 1X for 15 min, and stained for synaptic markers.After culturing astrocytes for 11 days The mouse tau shRNA sequence (CTGGAGCAGAAATTGTGTATAA) was used for the shRNA experiments. The shRNA-tau sequence was cloned downstream of the GFAP promoter and packaged into an AAV9 vector from Vectorbuilder. A scrambled shRNA sequence (shScr) was used as the transduction control. Primary astrocyte cultures were transduced with 2.44 × 1011 viral particles at 11 DIV for 7 days with the shRNA-tau to downregulate astrocytic tau or shScr.33 Immunofluorescence intensity was manually analyzed with Fiji as previously described.34 For each staining procedure, as a negative control, primary antibodies were omitted to determine background and autofluorescence.Fixed cells on coverslips were washed with 1X PBS and permeabilized with 0.1% Triton X-100 in 1X PBS for 15 min, followed by a single 5 min wash with 1X PBS. Next, non-specific epitopes were blocked with 3% BSA in 1X PBS for 1 h at RT. Primary antibodies were diluted in 1% BSA in 1X PBS and added to the cells. The cells were then incubated overnight (ON) in a humidified chamber at 4°C. The following antibodies were used for neurons: Synapsin-1  and PSD95 . GFAP  and PTX3  were used for astrocytes. The next day, the coverslips were washed 3 times with 1X PBS and the corresponding mouse and rabbit Alexa Fluor 488 and 568 secondary antibodies  were added. The coverslips were then incubated in 1% BSA for 1 h at RT. Next, three 1X PBS washes were performed, and the coverslips were mounted with Vectashield containing DAPI (Vector Laboratories) and sealed with nail polish. The percentage of GFAP-positive cells was determined to assess astrocyte purity. The coverslips were examined using a Nikon A1R Scanning Confocal Microscope coupled with Nikon NIS Elements imaging software. Synaptic cluster quantification was manually performed with Fiji (ImageJ) as previously described.35 For each staining procedure, as a negative control, primary antibodies were omitted to determine background and autofluorescence.Paraffin sections were deparaffinized in xylene, rehydrated in descending grades of ethanol (EtOH) and washed with deionized water. After blocking with 3% BSA in 1X PBS, the sections were incubated ON at 4°C with GFAP  and PTX3  antibodies prepared in 1% BSA 1X PBS. The next day, three 5-minute washes with 1X PBS were performed, followed by incubation for 1 h with the corresponding Alexa Fluor 488 and 568 secondary antibodies in 1% BSA 1X PBS. Finally, brain sections were washed threetimes with 1X PBS, quickly dipped in deionized water and mounted with Vectashield containing DAPI (Vector Laboratories). The sections were examined using the microscope and procedures described for the astrocyte and neuronal cell immunofluorescence experiments. The colocalization analysis was conducted automatically using Fiji with the JACoP plugin; Pearson’s correlation coefficient was used as a colocalization index, as previously described.Astrocyte cultures were washed twice with sterile 1X PBS and scraped from the dish with 100 μl RIPA buffer supplemented with one protease inhibitor cocktail tablet/10 ml (Roche 011836170001). The lysate was resuspended thoroughly and centrifuged at 15 000 RPM at 4°C for 10 min. The supernatant was collected, and the protein concentration was determined using the BCA method. The samples were mixed with loading buffer, and 30 μg of total protein was loaded on a NuPAGE 4–12% Bis-Tris protein gel (Invitrogen) and separated at 70 V. The proteins were dry-transferred into a nitrocellulose membrane. The membranes were blocked for 1 h in 5% dry milk powder reconstituted in TBS-T 0.01%; subsequently, total tau  and β-actin  primary antibodies diluted in TBS-T 0.01% -BSA 3% were added, and the membranes were incubated ON at 4°C. The next day, the antibodies were replaced with the corresponding secondary HRP-conjugated antibodies  in 5% milk, and the membranes were incubated for 1 h at RT. According to the manufacturer's specification, the membranes were washed twice with TBS-T and visualized using SuperSignal West Pico PLUS Chemiluminescent (ThermoFisher) developing solution.Total mRNA was purified from primary astrocyte cultures and multiplexed using the nCounter analysis system  combined with the nCounter Mouse Glial Profiling Panel. Briefly, 100 ng total RNA per sample was loaded and hybridized with probes for 16 h at 65° C, following the manufacturer’s protocol. Only samples with an RNA integrity number (RIN) > 9 were used for the NanoString analysis. Counts for target genes were normalized to the best fitting housekeeping genes as determined by nSolver software to account for variation in RNA content. The background signal was calculated as the mean value of the negative hybridization control probes. The expression data were excluded when the background signals were lower than the average negative control background signal, and probes with <100 reads for six or more samples were removed from the analysis. Downstream analyses and visualizations of gene expression datasets were performed using the NanoString nCounter Advanced Analysis Report software.The Abcam Mouse PTX3 ELISA Kit (ab245713) was used to assess the concentration of PTX3 present in the ACM. The 96-well plate was prepared following the manufacturer’s instructions, using 50 μl of undiluted ACM, obtained as described earlier in the Methods. Finally, the plate was read at 405 nm on a microplate reader, and the absorbances on the different samples were analyzed and plotted.Cell survival was measured using the Cell Titer 96 AQueous One Solution kit according to the manufacturer’s directions . Briefly, ninety-six well tissue culture plates were used to seed astrocytes at a density of 10.000 cells per well. At 11 DIV, astrocytes were treated with 1 μM of Aβ oligomers. After 24 h of incubation, the media was replaced with 100 μl of fresh culture medium per well, and 20 μl of Cell Titer 96 AQueous One Solution containing MTS tetrazolium salt was added. MTS is bio-reduced by cells into a coloured formazan product. After 4 h of incubation at 37°C, the absorbance of the wells was measured at 490 nm using a plate reader. The quantity of formazan product is directly proportional to the number of living cells in the culture.n breakdown for each set of experiments is as follows. Synaptic quantification (n = 4). Ten photographs of each culture per condition were randomly taken across the coverslip. From each, a single neurite was randomly chosen, analyzed and quantified for synaptic markers. An individual n was obtained by averaging the quantifications of each of those 10 neurites. For tau western blot, tau qPCR  (n = 3), established from three independent littermates. For GFAP/PTX3 co-staining in vitro (n = 3). We prepared three technical replicates with each culture. Five pictures per replicate were randomly taken across the coverslip and quantified. We obtained a ‘technical n’ by averaging the data obtained from these five photographs and to obtain the final n, we averaged the corresponding three technical n values. For GFAP/PTX3 co-staining in vivo . The fication , 6 and 8tau qPCR , differeanalysis , astrocyntration , AAV-infia (ACM) , we genein vitro  and D, w in vivo , we analty assay , five inP-value calculations were conducted using one-way ANOVA and two-tailed unpaired Student’s t-tests as stated for each experiment. Alternatively, the Kruskal–Wallis test was used when this criterion was not met. Significance was set at P < 0.05. Statistically significant P values are shown in the figures. Data are presented as the mean ± SEM.Statistical analyses were performed using the GraphPad Prism software. Normal distribution of data was evaluated using a Shapiro–Wilk Normality test; subsequently, statistical comparisons and The NanoString data supporting the conclusions of this article are available at NCBI’s Gene Expression Omnibus (GEO) and are accessible via series accession numbers GSE200231. The uncropped version of the western blots shown in this study can be found in 36 Additionally, it has been proposed that astrocytes could be active mediators of Aβ−induced neurotoxicity by exacerbating tau cleavage by caspase-3.37 Here, we first aimed to determine if the absence of tau in neurons confers protection against the synaptotoxic effects of Aβ-stimulated astrocytes. We performed a series of astrocytic and neuronal primary cultures in which WT astrocytes were first stimulated with Aβ oligomers. The culture medium was then replaced, and fresh medium was applied and conditioned by the Aβ-stimulated astrocytes. Next, this astrocyte conditioned media (ACM) was used to treat WT or tau−/− neurons  of the genes contained in the A2 annotation on a heat map and compared the two astrocyte phenotypes. Of the 12 A2-neuroprotective genes analyzed, the expression levels of PTX3 and Cd109 were increased in tau−/− astrocytes 42–50 were upregulated in tau−/− astrocytes versus WT controls (Ranbp9 and Hdac21)51–53 were downregulated in tau−/− astrocytes versus WT astrocytes  and PTX3 on the cortex of brain sections from 6-month-old tau−/− mice and WT controls. We found increased PTX3 expression in cortices of tau−/− mice compared with that in WT mice  as a control  and subsequently exposed these cells to Aβ oligomers. We used a virus with a scrambled shRNA sequence (shScr) as the control , whereas they acquire a neuroprotective phenotype after middle cerebral arterial occlusion, a model of ischaemic stroke. Recently, the heterogeneity of neuroinflammatory astrocyte subtypes in vivo after LPS challenge has been clarified at single-cell resolution, indicating widespread responses and distinct inflammatory transitions in astrocytes with defined transcriptomic profiles in response to inflammation.41 Furthermore, it has become increasingly clear that the binary astrocyte A1 and A2 division is not fixed and that neurotoxic and neuroprotective designations are not all-encompassing.39–41 Microglia-induced astrocytes can be identified by their upregulation of complement component 3 (C3) and have been found to lose many normal homeostatic functions, such as promoting neuronal survival, neurite outgrowth, and synapse formation. This suggests that ‘A1’ astrocytes are either unable to maintain synapses or are actively disassemble them by releasing multiple complement components that help drive synaptic degeneration. C3+ astrocytes also exert a toxic gain of function by secreting soluble neurotoxin(s) that induce neuronal and oligodendrocyte death, supporting the notion that C3+ astrocytes are involved in the development of neurodegenerative diseases.74 Interestingly, a recently published study demonstrated that the manipulation of tau splicing toward more 4R tau promotes a neurotoxic astrocyte phenotype characterized by the upregulation of C3 levels.19 Conversely, under certain stimuli, astrocytes can upregulate the expression of many neurotrophic factors that promote the survival and growth of neurons and thrombospondins, which promote synapse repair.75 Thus, subtypes of astrocytes might have beneficial or reparative functions. PTX3, a known marker for neuroprotective astrocytes,39 is an acute-phase protein linked to immune responses to inflammation.56 PTX3 increases neuronal stem cell proliferation76 and promotes synaptogenesis in hippocampal neuronal culture.32 PTX3 gene knockout has also been shown to reduce neuronal repair and regeneration following ischaemic brain injury.77 Additionally, PTX3 can bind and tune the complement activation pathway and prevent inflammatory reactions.77–79 Specifically, PTX3 regulates C3-deposition by interacting with and recruiting the negative regulator Factor H.79 This point is of great relevance, taking into consideration novel studies demonstrating that the loss of C3 and C3a receptors, which positively correlated with cognitive decline and Braak staging in human Alzheimer’s disease brains, ameliorates synapse loss and neurodegeneration in Alzheimer’s disease mouse models of amyloidosis and tauopathy.81 Our data suggest that a mechanism involving PTX3 could be responsible of the synaptoprotective effect of astrocytic tau ablation. In the present study, we demonstrated that the addition of recombinant active PTX3 to cellular media was synaptoprotective (−/− astrocytes (−/− astrocytes (Transcriptomic profiling has helped identify the diverse heterogeneity and distinct molecular states of astrocytes in different disease models.otective . Neverthtrocytes . Therefotrocytes , we cannin vivo studies are necessary to validate the observations. Reducing endogenous astrocytic tau expression is a potential strategy for managing the deleterious effects of synaptic damage in Alzheimer’s disease and other neurological conditions.Currently, we do not have a clear understanding of the mechanism by which tau regulates PTX3 levels in astrocytes. Nevertheless, based on our results, we speculate that the physiological function of tau in astrocytes is associated with the control of a signalling pathway responsible for PTX3 expression and that the downregulation of astrocytic tau levels not only promotes the expression of anti-inflammatory and neuroprotective factors, such as PTX3, but also directly or indirectly inhibits the upregulation of known pro-inflammatory and synaptotoxic related pathways. In conclusion, our study expands our understanding of how reducing tau improves astrocyte function by stimulating synaptoprotective factors. Additionally, we provided evidence for the possible role of astrocytic tau in the known neuronal effects of tau reduction on health and disease. Considering that the current study was performed in astrocytic primary cultures stimulated with a supraphysiological concentration of Aβ, further fcac235_Supplementary_DataClick here for additional data file.


However, iron limitation sets an upper limit to primary productivity. Here we report on a considerably dense late summer phytoplankton bloom spanning 9000 km2 in the open ocean of the eastern Weddell Gyre. Over its 2.5 months duration, the bloom accumulated up to 20 g C m−2 of organic matter, which is unusually high for Southern Ocean open waters. We show that, over 1997–2019, this open ocean bloom was likely driven by anomalies in easterly winds that push sea ice southwards and favor the upwelling of Warm Deep Water enriched in hydrothermal iron and, possibly, other iron sources. This recurring open ocean bloom likely facilitates enhanced carbon export and sustains high standing stocks of Antarctic krill, supporting feeding hot spots for marine birds and baleen whales.The Southern Ocean is a major sink of anthropogenic CO This study reports a dense, late summer phytoplankton bloom in the Southern Ocean that accumulated unusually high levels of organic matter and supported feeding hot spots for birds and whales. The authors show that this recurring open ocean bloom is driven by anomalies in easterly winds that push sea ice southwards and favour the upwelling of deep waters enriched in hydrothermal iron. This originates from the unique connection between the Southern Ocean and the lower cell of the global overturning circulation3. The Weddell Gyre is a key region of the Southern Ocean where Circumpolar Deep Water (CDW) travels through the Antarctic Circumpolar Current (ACC) at mid depth4, is upwelled to the surface through wind-driven divergence5 bringing high concentration of nutrients and carbon to the surface, and modified to Warm Deep Water (WDW) on its path through the Weddell Gyre6. Later, it is entrained into the deep ocean as Antarctic Bottom Water (AABW) forming at the southern and southwestern borders of the Weddell Gyre7. A recent carbon budget across the Weddell Gyre8, based on observations of inorganic carbon, suggests that intense primary productivity in the region of the Weddell Gyre, away from continental shelves, can explain the carbon drawdown estimated from the carbon deficit directed out of the Gyre below the ACC 10. The strength of the resulting Weddell Gyre carbon dioxide (CO2) sink to depth, at an estimated ~50 Tg C yr−1, is significant when compared to the global ocean biological carbon pump, estimated at 430 Tg C yr−1 across the 2000 m horizon by Honjo et al.11. Satellite-derived ocean color and oceanographic campaigns have however thus far not been able to verify the level of primary productivity required to explain the proposed strength of the Weddell Gyre CO2 sink8.The biological carbon pump in the Southern Ocean, driven by phytoplankton primary production, plays a disproportionately important role in global climate at recent and millennial time-scalesEuphausia superba), marine birds, seals and both baleen and toothed whales. With over 500 million tons of E. superba thriving across its open waters12, the Southern Ocean ecosystem sustains a rich marine wildlife whose life cycles are linked to the short intensive primary production that takes place in specific areas of the Southern Ocean polar waters.Seasonal phytoplankton primary production sustains the Southern Ocean’s rich food-web, dominated by the Antarctic krill , in the Kong Håkon VII Hav, eastern Weddell Gyre ) and Particulate Organic Carbon (POC), as retrieved from a Chl a fluorescence and a backscattering sensor mounted on the Seaglider and calibrated with in situ Chl a and POC measurements (see Methods), were distributed from the surface to 50 m depth (to approximately the base of the surface Mixed Layer (ML)). A subsurface Chl a and POC maximum was however observed at the base of the ML southwards of 68.32° S  and the POC:Chl a ratio (g:g) in the ML was elevated14 .It is believed that we encountered the bloom as it was terminating given that the phytoplankton cells looked chlorotic under light microscopy, the maximum quantum yield of photosystem II, −3 at the base of the ML at the northern edge of the bloom and a maximum estimated POC concentration of 1080 mg m−3 at the base of the ML at the core of the bloom 18, naturally iron-fertilized open waters such as in the lee of the Kerguelen Plateau (maximum POC concentration of 384 mg m−3 and up to 22.5 g C m−2)19 or artificially fertilized open waters (up to 14.4 g C m−2 at the end of the large diatom bloom that was induced by the European Iron Fertilization Experiment (EIFEX) at the Antarctic Polar Front in the Atlantic sector of the Southern Ocean20).This open ocean phytoplankton bloom accumulated striking levels of organic matter over its ~2.5 months duration, with a maximum measured POC concentration of 327 mg moom Fig. . Given toom Fig. . Such hi21 profiles that have been collected in the open Southern Ocean from September 2014 to December 2020, i.e., more than 9500 profiles. We identified only 34 profiles  where the integrated POC over the upper 100 m amounted to more than 15 g C m−2 and up to 19.7 g C m−2 over the whole open Southern Ocean  are typically high and non-limiting. During our campaign, light and macronutrients were not found to be limiting throughout the open ocean bloom . Therefore, the production of such striking levels of organic matter in the open waters of the Southern Ocean must have involved an important iron source and a relatively low grazing pressure at the onset of the bloom as grazing can dominate phytoplankton loss in the Southern Ocean23. During our campaign, dissolved iron (dFe) was relatively elevated24, and probably not limiting, at the sea surface throughout the larger study area, ranging from 0.65 ± 0.2 nM at Astrid Ridge, 0.4 ± 0.1 nM at Maud Rise and 0.6 ± 0.04 nM at 2 stations studied south of the bloom we report here −2 to be between 3.8 and 33.3 μmol Fe m−2 by using lower and upper bound Fe:C ratios from Fe-replete and Fe-deplete Southern Ocean waters (2.3 to 20 mol:mol 10−6)25. Uncertainties on the lower and upper limit of this range are large as they do not account for the role of grazing or vertical export on POC in the ML23, and as POC is both constituted of autotrophic and heterotrophic carbon. A lower estimate could be obtained by considering that phytoplankton only represents 20 to 30% of the total POC26. With this consideration, the necessary iron to accumulate 5 g C m−2  was calculated to be between 1 and 8.3 μmol Fe m−2. An estimation of satellite-derived net primary productivity and the associated iron demands during the 2.5 months bloom period ranged between 14.8 and 42.6 μmol Fe m−2 (see Methods). Together, these observations and calculations highlight that an important iron source or mechanism was required in order to lead to such high levels of POC accumulation inside the open ocean bloom.We calculated the iron necessary to accumulate POC levels up to 20 g C ma in March 2019 follows the deep bathymetric contours at 3500 and 4000 m depth , was larger than the 23-year long mean Chl a + 1 standard deviation over that area  was not significantly different (p > 0.05) between years with and years without an open ocean bloom, and typically initiated on January 15th ±4 days, ended on March 1st ± 3 days, and lasted 44.5 ± 4 days on average.In order to put the observed bloom in a wider context, we inspected satellite-derived ocean color to detect the presence of the open ocean bloom from September 1997 to December 2019 (see “Methods”). Since the surface area of the bloom as observed from satellite-derived Chl pth Fig. , we defia concentration, obtained by averaging de-seasoned anomalies during time periods when the bloom was present shows a large area of positive Chl a anomaly in the open ocean oriented along a northeast-southwest pathway, extending from 55° S to 30° E to 10° W to 70° S .A composite map of satellite-derived Chl ° S Fig.  and impl° S Fig. . Such wiean Fig. . A weak 28.Furthermore, at times when the open ocean bloom takes place, the stronger easterlies push sea ice south, towards the coast. This leads to a negative anomaly in sea-ice concentration in the open ocean west of Astrid Ridge and the opposite positive anomaly in sea-ice concentration along the coast between Astrid Ridge and the Fimbul Ice Shelf Tongue Fig. . By pushThe timing of the wind and sea ice conditions are important for the open ocean bloom. In this region, zonal winds typically increase from January onward, peaking in May . We found that no significant differences existed in preceding winter (i.e. September) maximum ML depths between years with (mean of 124.9 ± 6.3 m) and years without (mean of 113.1 ± 13.3 m) an open ocean bloom observed/(3He/4He)atmospheric − 1) [%]) is a tracer of primordial helium originating from the Earth’s mantle and elevated δ3He ratios (10–12%) are commonly used to detect plumes downstream of hydrothermal vents30. Two sections of δ3He from the Global Ocean Data Analysis Project (GLODAP-2) across the Weddell Gyre, around 0° and 30° E (see “Methods”), and an additional section of δ3He from a recent expedition along 6° E  support hydrothermalism as a major iron source for the open ocean bloom we describe here  between 500 and 100 m depth in the northern part of the section around 50–55° S 29, even though entrainment anomalies are not linked to bloom anomalies in the present study  (between −0.07 at the surface and 0.4‰ at 1000 m depth) from samples collected by a CTD-rosette cast at the northern edge of the bloom (see Methods), suggesting that sea ice meltwater is similarly not a major source of iron for this open ocean bloom38.Considering other known iron sources, atmospheric deposition of iron plays a minor role at these latitudesChaetoceros dichaeta, which represented 39–67% of the total phytoplankton abundance as evidenced from surface underway samples (intake at 4 m depth) and between 31 and 47% of the total phytoplankton abundance within the upper mixed layer, i.e., down to 40 m depth as evidenced from CTD stations . The second most abundant phytoplankton species in this blooming assemblage was the centric diatom Dactyliosolen antarcticus, but it only accounted for a maximum of 10% of total phytoplankton abundance on any occasion.In addition to high biomass, the taxonomical composition of this open ocean bloom has important biogeochemical and ecological consequences. The bloom was dominated by the centric diatom Chaeotoceros dichaeta is a large (~10 to 50 µm) chain-forming diatom species with prominent spines. It can dominate phytoplankton blooms in the coastal and the sea ice zone around Antarctica, particularly in summer, whereas, in frontal regions, it can be dominant in the fall39. Due to their prominent spines, members of the Chaetoceros genus, including C. dichaeta, easily form rapidly sinking aggregates when undergoing mass mortality and are considered to be important carbon exporters40. The large bloom of C. dichaeta we observed over deep waters in the eastern Weddell Gyre was actively contributing to deep carbon export as shown by the large optical spikes created by particles and phytodetritus (i.e Chl a) below 100 m and throughout the bloom area 42 and efficiently export carbon to the deep sea and the sea floor. A similar event could be shown during the EIFEX fertilization experiment which took place north of the ACC, northeast of the Weddell Gyre, where C. dichaeta, contributed significantly to carbon export 24 days after bloom fertilization20. In this instance, half of the bloom biomass sank below 1000 m, and a phytodetritus layer composed of intact cells of C. dichaeta was observed on the seafloor at > 3000 m depth, suggesting that blooms of C. dichaeta contribute significantly to deep carbon sequestration in the Southern Ocean.Euphausia superba) was mostly observed  in two areas: in the vicinity of the open ocean bloom we describe here  outside the open ocean bloom and throughout our entire survey across the Weddell Gyre. In addition, we observed high numbers of humpback whales foraging in the area of the open ocean bloom , but did not report similarly high local abundance numbers like the ones we observed in the bloom area. As shown in previous studies48, the distribution of flying seabirds and baleen whales reflects the predictable distribution of their main prey, the Antarctic krill. Krill is a key prey item for numerous Antarctic species because of the extraordinary size of their swarms49 and their overall dominance in the Southern Ocean12, making it an easily accessible and profitable prey. It is, therefore, very likely that the large number of flying seabirds and baleen whales we observed in the open ocean bloom area were attracted by the presence of Antarctic krill.In addition to Antarctic krill, we observed very high abundances and densities of Antarctic and snow petrels . This possibly brings new insight into the foraging strategy of Antarctic petrels along the Dronning Maud Land coast and highlights this open ocean bloom as their possible favored feeding ground during the breeding season.Dronning Maud Land hosts a large population of seabirds, and in particular half of the World’s Antarctic petrel population, which represents four to seven million breeding Antarctic petrels54. Therefore, our observations along with the results from previous studies highlight this region as an important foraging area for Antarctic wildlife. Although unpredictable, these re-occurring open ocean blooms are a possible key feature of the Weddell Gyre marine ecosystem.Our study provides further evidence that humpback whales feed in the open waters of the eastern Weddell Gyre. These findings are consistent with two recent acoustic studies showing that humpback whales utilize this area during the typical open ocean bloom period: humpback whale vocalizations were recorded from March to May at Maud Rise and almost throughout the year near Ekström Ice Shelf with peak occurrence in February to May55 or for Antarctic petrels that are raising and fledging chicks in February–March51.The evidence we report here points towards the importance of this open ocean bloom for the eastern Weddell Gyre ecosystem; particularly the characteristics directly observed at sea (i.e. high phytoplankton carbon biomass and abundant krill), its recurrence during favorable atmospheric and oceanographic conditions , the likely additional sources of iron sustaining it , and its seemingly long-term significance for the biological carbon pump and top trophic level consumers . The timing of the wind and sea ice conditions are important for the open ocean bloom which typically happens in late summer, i.e., most often in February. Such a productive bloom late in the summer is significant for example for krill that need to prepare for the long austral winter56 due to poleward intensifying winds and increases in meltwater over the last two decades, which could have large consequences on the fate of this open ocean bloom. Given its seemingly importance for the Weddell Sea, the driving forces and the fate of this key ecosystem feature warrant future studies.The next legitimate question concerns the fate of this open ocean bloom and its associated ecosystem in the context of climate change. A recent study described major changes in the Southern Ocean physical and biogeochemical propertiesN = 5) as well as from a SBE 32 carrousel water sampler connected to a CTD (conductivity-temperature-depth) SBE911+ system which was deployed to calibrate the glider sensors at the glider deployment station . Seawater was sampled to measure the concentrations of chlorophyll a , Particulate Organic Carbon and Nitrogen (POC and PON), the phytoplankton species composition, phytoplankton physiology, and stable oxygen isotopic ratio (δ18O)).Seawater was collected from the ship’s scientific seawater intake at 4 m depth throughout the large phytoplankton bloom (a was measured with reverse-phase HPLC including a Agilent Technologies Microsorb-MV3 C8 column (4.6 × 100 mm), a Waters Alliance HPLC System and a photodiode array detector (2996). HPLC-grade solvents (Merck) and the EMPOWER software were used in the analysis. More details can be found in Tran et al.57.Water samples (1 L) for HPLC analyses were filtered on 25‐mm Whatman 0.7 um GF/F glass fiber filters  under low vacuum pressure and then stored at −80 °C until analysis ashore. Chlorophyll Water samples (1 L) for POC analyses were filtered on pre-combusted 25‐mm Whatman 0.7 um GF/F glass fiber filters  under low vacuum pressure, dried at 60 °C and stored at room temperature in PALL filter slides until analyses ashore. 80 μL of 10% v/v hydrochloric acid was added to each filter to remove any inorganic carbon before the filters were folded into aluminum capsules. POC concentrations were then measured with a C/N analyzer Europa Scientific, ANCA-MS 20-20 15N/13C mass spectrometer at the Tvarminne Zoologica Station, Finland. POC values were corrected for dry filter blanks.58.Samples for phytoplankton species counts by microscopy (190 mL) were filled into 200 mL brown glass bottles and fixed with 25% glutaraldehyde and 20% hexamethylenetetramine-buffered formalin solution at final concentrations of 0.1 and 1%, respectively, and thereafter stored cool and dark until analyses ashore. For analysis, 10–50 mL Utermöhl sedimentation chambers  were used for settling sub-samples for 48 h and Nikon Ti-S inverted light microscope for identification and counting by the Utermöhl methodTM fast repetition rate fluorometer (FRRf) integrated with a FastActTM laboratory system. After collection, samples were dark acclimated at in situ temperatures for 30 min before measurement. The active Chl a measurements consisted of a single turnover protocol with a saturation sequence  and a relaxation sequence . The sequence interval was 100 ms which was repeated 32 times resulting in a total acquisition time of 3.2 s. The power of the excitation LED (λ450 nm) was adjusted between samples to saturate the observed transients following the manufacturer specifications. Each sample underwent a fluorescence light curve which measured the active Chl a fluorescence at 15 light levels between 0–1500 μE m−2 s−1, with an optimized duration per light level consisting of 12 acquisitions per light level, except for the first light level (10 μE m−2 s−1) which consisted of double the number of acquisitions (24). All samples were blank corrected using carefully prepared 0.2 μm filtrates59.Measurements of phytoplankton photophysiology were performed using a Chelsea Scientific Instruments FastOcean18O analyses, samples were collected in 25 mL Wheaton bottles. The caps were sealed with Parafilm® and the bottles were stored in +4 °C and dark conditions until analysis in the laboratory. The analysis of δ18O was done with a Thermo Fisher Scientific Delta V Advantage mass spectrometer with Gasbench II. The δ18O data were standardized to Vienna Standard Mean Ocean Water (VSMOW). The reproducibility of the replicate analyses was ±0.04‰.For δ60 using online pre-concentration methods. All samples were measured in duplicate. The detection limit of Fe was 0.08 nM.For the analyses of Dissolved labile (dFe) and total acid leachable (TaLFe) iron, water samples were collected at 12 stations with 5 L acid-cleaned Teflon-lined GO-FLO bottles deployed on a 500 m aramid rope (6 mm-vpg industri), using a dedicated winch and Teflon coated messengers. The samples were collected at 8 discrete depths . After water collection, the GO-FLO bottles were double bagged and taken to a makeshift clean room, placed on a rack under clean air blowing from a Class-100 laminar flow hood and drained into acid washed 125 mL low-density polyethylene (LDPE) Nalgene bottles. Water for TaLFe determination was collected directly into the LDPE without filtration. For dFe samples, water was filtered through Sartorius filters (0.45 + 0.2 μm pore size filtration) using acid-washed Tygon tubes. The dFe fractions were defined operationally by the 0.2 μm nominal pore size. During filtration, an additional HEPA air-filter cartridge  was connected to the pressure relief air-vent valve of the GO-FLO bottles to ensure that the air in contact with the sample during the filtration was clean. All samples for dFe and TaLFe were acidified to pH 1.7–1.8 with ~3 M double quartz distilled HNO3 (UP HNO3). The acidified water samples were stored (> 1 year) until analysis at Stellenbosch University  as described in Samanta et al.http://norgliders.gfi.uib.no/), profiling continuously between the surface and 1000 m depth and measuring a suite of parameters that includes conductivity , temperature, depth (CTD), dissolved oxygen, Chl a, and optical backscattering by particles as a proxy for POC. The Seaglider Chl a fluorescence and backscattering signals were calibrated against the Chl a and POC concentrations measured during a calibration CTD cast shortly after the Seaglider deployment (2.5 h). The Seaglider Chl a fluorescence and backscattering signals were processed following the glider toolbox described by Gregor et al.61. In situ dark counts were calculated from the 95th percentile between 400 and 600 m and removed from the data. A 3-point rolling mean was subtracted from each profile to de-spike fluorescence and backscattering profiles. The mixed layer depth (ML) for each profile was computed as the depth to which temperature exceeded 0.04 °C of the surface temperature, which was a good indicator of the bottom of the ML based on visual inspection of the profiles and general Temperature and Salinity properties in the area15.An autonomous Seaglider (SG563) was deployed and recovered during the cruise, collecting vertical profiles during 31 dives along a 55 km transect across the bloom region between March 12 and March 15, 2019 Fig. . The glia concentration maps are extracted from the Ocean and Land Colour Instrument (OLCI) Level-2 full resolution Near Real Time (NRT) product, obtained by the EUMETCast broadcast system. The OLCI sensors on board ESA Sentinel-3A and B satellites launched in February 2016 respectively April 2018, and have large swath widths (~1270 km) covering large regions with high temporal resolution . The OC4ME algorithm which uses a polynomial approach of a maximum band ratio algorithm of 4 reflectances at 443, 490 and 510 nm over the 560 nm was extracted directly from OLCI Level 2 products and gives the Chl a pigment concentration in [mg/m3]. The cloud free chlorophyll orbit tiles are binned and averaged on a daily base to obtain one image per day. This leads to a very good resolution, coverage in reasonable timeframe and meets the requirements of monitoring water dynamics also on smaller spatial scales.The Chl https://oceandata.sci.gsfc.nasa.gov/SeaWiFS/) and MODIS  level 3 Chl a data processed with the default chlorophyll algorithm (chlor_a) which employs the standard OC3/OC4 (OCx) band ratio algorithm merged with the color index (CI) in 8-day and 9 km resolution.The analysis of satellite-derived ocean color was complemented using SeaWiFs , with prior processing using the NASA team algorithms62.Daily sea ice concentration (1997–2020) were obtained from the NASA’s Nimbus‐7 Scanning Multichannel Microwave Radiometer (SMMR) and Defense Meteorological Satellite Program (DMSP)‐F13, ‐F17, and ‐F18 Special Sensor Microwave/Imager (SSM/I). Data with a spatial resolution of 25 km were provided by the National Snow and Ice Data Centre, University of Colorado in Boulder, CO  using the mean ratio of Fe:C (4.3 μmol:mol) generated for the Sub-Antarctic Zone of the Southern Atlantic Ocean64.To calculate Net Primary Productivity (NPP) in the area where we detected the bloom, we used a 2.25-degree bounding box around the position of the station where we deployed the glider and calculated the mean monthly NPP from satellite data products . We further converted NPP from all methods into Fe demand  SBE911+ system. For helium analyses, water samples were collected into copper tubes, which were clamped off after sampling and later analyzed at the IUP Bremen noble gas mass spectrometry lab HELIS. Samples were processed initially with an ultra-high vacuum gas extraction system. For analysis of the noble gas isotopes  the extracted gas is transferred into a fully automated mass spectrometric system equipped with a two-stage cryogenic trap system. The system is calibrated using atmospheric air standards on a regular basis and measurements are made for blanks and linearity. For details, see Sültenfuß et al.21 from September 2014 to December 2020. Data were collected and made freely available by the Southern Ocean Carbon and Climate Observations and Modeling (SOCCOM) Project funded by the National Science Foundation, Division of Polar Programs (NSF PLR −1425989 and OPP-1936222), supplemented by NASA, and by the International Argo Program and the NOAA programs that contribute to it . The Argo Program is part of the Global Ocean Observing System. We considered the profiles where the integrated POC in the upper 100 m was above 15 g C m−2. In addition, to avoid comparing our data with episodes of eddy-subduction pump which can send high concentration of organic matter in one short-time process, we removed 15 profiles where the integrated POC in the upper 100 m was above 15 g C m−2 but more than 90% of the POC was found below the surface mixed layer.We used all SOCCOM BGC-Argo floats profiles that measured upper ocean POC concentration in the Southern Ocean3 . We then averaged the obtained mixed layer depths to monthly means.We calculated the mixed layer depth in the area of the bloom between 2002 and 2020 by using all Argo floats that drifted through the area within 4 to 8E and −69 to −67S, i.e., total of 1024 profiles of temperature, salinity and depth. Furthermore, we removed profiles earlier than 2007 due to large seasonal gaps. From all profiles, we calculated the mixed layer depth as the depth where the potential density exceeds its 10 m value by 0.03 km/m68. During the campaign, we used two transducers: one on the drop-keel (3 m from the hull when down) and one hull-mounted. The latter was used in ice-covered areas, with low impact on the detection of krill swarms15. The density of krill was calculated as nautical area scattering coefficient (NASC)69.The abundance of krill was studied with a Simrad EK80 research echosounder with six frequencies. For this study, we focused on the 38 kHz frequency. It was scrutinized with the Large Scale Survey System (LSSS) software version 2.5.02 mouth-opening and 3 × 3 mm diamond shaped mesh (7 mm stretched) from mouth to rear. Towing speed was normally 2.5–3 knots. The velocity of the trawl through water and depth of the trawl were monitored by a depth sensor (SCANMAR) attached to the headline. From each trawl, a subsample of approximately 150 individuals of Euphausia superba was taken, and the length of the individual krill was measured (±1 mm) from the anterior margin of the eye to tip of telson excluding the setae. Sex and maturity stages of E. superba were determined using the classification methods according to Makarov and Denis70.Krill swarms were sampled with a Macroplankton trawl at two locations within the bloom region , during daytime and decent weather conditions . Observations were only recorded during ships transits. Marine mammals were primarily recorded from 270-0° (0° being the bow of the ship), on the port side of the ship. Sea birds were both recorded continuously, counting every bird within 300 m in a sector from the bow (0°) of the ship to 90° in 10 min periods, and with point counts every 30 min, identifying ship’s followers behind the ship. As many birds follow the ship for some time, this means double counting the same individuals happens as birds that are counted during a 10-minute period will be counted again during the next 10-min period if they are still present. This method is therefore not for population estimates, but more suitable for density assessment.52. The utilization distribution maps (or heat maps) were created with the kernelUD function of package adehabitatHR in R software version 4.0.2, using the href (reference bandwidth) smoothing parameter (https://www.rdocumentation.org/packages/adehabitatHR/versions/0.4.19/topics/kernelUD).Breeding adults were captured on theirs nest during late incubation and mid chick rearing (from early January to mid-February) and instrumented with Global Positioning System (GPS) loggers  just before leaving on a foraging trip. The GPS units weighed approx. 20 g (ca. 3% of bird body mass) and were taped to tail feathers. Birds were recaptured upon return to their nest to retrieve the GPS units and download the data. GPSs recorded the locations of the birds along their foraging trip at 5 to 30 min intervals. In total, data were available from 161 tracks collected in January/February 2012, 2013, 2014, 2016 and 2018. Additional details about the procedure are given inhttps://www.eoas.ubc.ca/~rich/map.html in November 2021.Data analyses were conducted with Matlab R2019b. All maps were built on QGIS (v. 3.20.0) and using the m_map package as downloaded from Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Onthis journey through uncharted waters, we revealed an expected initialshrinking of the exposed area of the droplet, followed by an unanticipatedexpansion. We attribute this behavior to an initial fast amorphizationof the interface between the droplet and surface, followed by therecrystallization of Ge expelled from the droplet at the interface.As a major surprise, we discovered the emergence of extensive “spaghetti”-likepatterns, which are rationalized in terms of parallel Ge ripples orientedalong, mainly, [−554] and [−55–4] directions.They emerge during spinodal decomposition when passing the eutectictemperature of the system. Their sides are defined by Ge{111} andGe{11–1} vicinals covered with Pt-modified (√3 ×√3) superstructures. The distance between adjacent ripplesis about 18 nm.We embarked on an  This leadsin the ideal case to one big droplet at the thermal summit and inpractice to the accumulation of few large droplets close to the center.We have recently communicated a compelling example for PtGe on Ge{110}.13 While in situ information on the droplet dynamicsabove the eutectic temperature is readily accessible, there are noreports about the evolution during cooling through the eutectic transition.Only postmortem analysis is employed to show, for instance, by TEMthat crystalline AB-remainders of the eutectic droplets reside onthe pedestals of B precipitated from the droplet during cooling onsubstrate B.8 However, insitu information on the crystallization of eutectic dropletsat the very moment when the system is driven through the eutectictransition during cooling is still lacking. It appears that our currentknowledge of the system PtGe/Ge{110} offers a promising opportunityto gain in situ more insights into the processesthat are active during the spinodal decomposition of eutectic droplets.We start with a microscopic view of a relatively small area aroundthe hottest spot at the surface and thus with a large droplet andconsequently a high local Pt concentration. During cooling down, theposition of the hottest spot hardly changes, and as a result, thethermal gradient-induced motion of droplets is minimal. This is ofimportance as the experiment can be executed only once after depositingPt on the virgin Ge(110) surface. Further away from the hottest spot,the local Pt concentration is lower after a prolonged experiment.This allows the experimental evaluation for both high and local Ptconcentrations on a single substrate under the same experimental conditions.Theemergence of eutectic droplets on solid surfaces and theirtemporal evolution above the eutectic temperature have received properattention.2Pt{001} rhombic crystallite, locatedimmediately next to the center and aligned with its a-axis under an angle of 20° wrt the Ge{110}-[001] direction.Further off-center (a few millimeters), extended orthorhombic Ge2Pt{101} crystals emerge, which are aligned with Ge{110} withits Ge2Pt b-axis parallel to Ge[001] atan almost perfect lattice match. These thin islands are stabilizedby quantum size effects.The mirror side of Ge evolution is the coincident emergence ofa large, compact, Gein situ during cooling through the eutectic temperature.Upon cooling toward the eutectic temperature t, wefind an unanticipated “breathing” of the wetting angleand, consequently, of the exposed area of the droplet, which we traceback to the structural changes at the droplet–substrate interface.In addition, we observe the emergence of “spaghetti”-likestructures upon cooling below the eutectic temperature, which arerationalized in terms of a rippled spreading layer of pure Ge aroundthe original droplet. In this process, a crucial role is taken bya Pt-containing (3 × 3) wetting layer on vicinal (111) Ge facetsthat constitute the ripples developing at the Ge{110} surface. Furthermore,at different surface locations ,differently oriented Ge2Pt crystallites are identified.In this study, we apply PEEM (photo-electronemission microscopy),LEEM (low-energy-electron-microscopy), and μLEED  to monitor the structural changes –10 mbar. In PEEM, thesurface was illuminated with a 100 W mercury discharge lamp (λ= 0.253 μm) incident at 16° from the surface plane. Theabsolute temperature reading is estimated to be correct within ca.25 K and calibrated by making the reasonable assumption that the eutectictemperature at the surface equals that of the bulk (1050 K for GePt).A Ge{110} substrate, 10 × 10 mm2, nominally flat,single-side-polished, n-type Ge(110) crystal , has been degassed for about 24 hat700 K, followed by several cycles of argon ion bombardment and flashannealing by e-beam bombardment at a temperature exceeding 1000 K.Subsequently, Pt is deposited from a resistively heated W wire wrappedwith high-purity (99.995%) Pt (Alfa Aesar). The structure of the cleansurface has been examined using LEED. Like the findings in ref  amounts to RC = 90 μm, and the wetting angle θw equals 20°.12 The volumeof the spherical cap is given byAt the start of the current experiment, thesurface was preparedas described above, followed by a prolonged period (>8 h), at atemperatureof about 50 K above the eutectic temperature. As a result, a largeeutectic cluster is situated in the center at the hottest locationat the surface, and several smaller ones are still on their way tothis center. We follow the lifeline of this object during a gentlecool-down. Initially, as can be observed in the accompanying PEEMmovie,V = 8.2 ×103 μm3. Or, with the atomic volume of1 Ge atom per 22.6 × 10–12 μm3 (bulk Ge), we find 3.6 × 1014 atoms inside the sphericaldroplet, assuming that the atomic volumes of Pt and Ge are identicalin this crude estimate.For the numbers above,we obtain 2Pt crystallitesalso emerge below the eutectic temperature,16 which will be discussed in more detail at the end of the paper.Immediately upon cooling, one observes a decrease of the projectedarea of the droplet, as illustrated in sv, γsl, and γlv being thetension of, respectively, thesubstrate–vapor, the substrate–liquid, and the liquid–vaporinterfaces, while θw denotes the wetting angle. Naively,one would expect that within the small variations of the Ge contentinside the droplet only marginal changes in the interfacial tensionsoccur and thus the wetting angle would stay constant. However, thesedimentation of Ge at the droplet–Ge{110} interface duringthe cooling of the eutectic may well result in an increasing kineticroughness at this interface. This roughness would, according to Wenzel,18 lead to a reduction of the wetting anglew* the wetting angleon the rough surface and r the roughness definedas the real surface divided by the nominal surface, and thus by definition, r > 1. Consequently, this would result in an increaseofthe projected area. Therefore, kinetic roughening must be excludedas the course for the discrepancy we ran into. We do realize thatan increase of γsl would lead to an increased wettingangle and potentially would lift the apparent discrepancy. To advancealong this route, we consider how much material is segregated fromthe spherical droplet segment to the droplet–substrate interface.As illustrated by RC = 90 μm and wettingangle θw = 20° amounts to ∼3000 μm2. One atom in the Ge(110) surface covers 1.13 × 10–7 μm2; in other words, the circularbase plane of the droplet counts roughly 2.6 × 1010 atoms. The deposition of all Ge atoms in the droplet at the basewould therefore result in a growth of 1.4 × 104 Ge(110)monolayers. During the 19 K temperature decrease in about 25 s, inline with the quick drop in exposed area exhibited in sl will be relatively high. Accordingly, we attributethe unanticipated shrinking of the exposed area of the cooling downeutectic droplet to the kinetic amorphization of the growth frontat the droplet–substrate interface.The base area of our spherical droplet segment with the radiusof curvature This scenario offersa natural framework for understanding theobserved evolution of the projected area of the eutectic during laterstages, as illustrated in The behavior of the exposed area as a functionof decreasing temperatureclearly reveals that the two active processes (1) kinetic amorphizationand (2) crystallization at the droplet occur not consecutively butsimultaneously. These processes compete with amorphization dominanceduring fast temperature decay and crystallization dominance at slowtemperature decay rates. In this case, the temperature adjustmentwas controlled in three steps, and each time an initial drop is followed,after some time, by an increasing tendency of the exposed area whenthe temperature decay rate decreases. This behavior is in line withthe scenario outlined above.2 crystallites. This appears to be confirmed, however, with an unanticipatedtwist, as we will discuss in detail further below. In this evaluationscheme, our exemplary spherically capped droplet with RC = 90 μm and θw = 20°, whencooled down from 1100 K through the critical temperature, contains7.4 × 1013 Ge atoms which need to be reincorporatedinto the Ge{110} substrate. If these are equally spread over the areaof FoV, of 150 μm in 20When passing through the eutectictemperature, two events occursimultaneously: (1) a spreading of the material originating from theGe–Pt cluster and (2) a partial crystallization of the formerdroplet remainders. In terms of the expected spinodal decomposition,one would naively conclude that the spreading results mainly fromGe incorporation in the Ge{110} substrate, and the crystallizationat the position of the former droplet would result in PtGeA first impression of the events is provided by the snapshotsin 001, becomes larger. Thevalue of the normal component of the wave vector of the diffractedelectrons versus the parallel component change along [001] is plottedin 26 Also, the presence of a (distorted) hexagonal structure in 27 The disappearance of the (110)specular spot already at low energies reveals that, overall, the (111)facets dominate over (110) areas.Upon increasing the electron energy, the distancebetween thispeak and the specular spot of the distorted hexagonal pattern (higherarrow), referred to as Δ–1(35) (red grid) and find, neglectingminor residual image distortions, an almost perfect mapping of themeasured diffraction peaks. This result is a confirmation for thealready concluded presence of (111) vicinals.Note that in this (110) projection the distancebetween the successive[−110] atom strings is smaller by a factor, cos(35.26) = 0.82,than their distance within the (111) facet. This geometric fact causesthe abovementioned distortion of the diffraction pattern of superstructuresat the facets. A decrease in real space gives rise to an elongationin reciprocal space. This is exactly what we observe, as illustratedin the diffraction pattern taken at 4.1 eV and shown in 25 The presence of a metal-induced reconstruction of (111)-type maywell influence the subtle energy balance in favor of the formationof (111) facets. This may even lead to a preference of reconstructed(111) facets above (100) areas. An example of the latter is the Au-inducedgiant missing row reconstruction of Ge{001} with (√3 ×√3) reconstructed (111) facets.26 The current observations with Pt-induced (3 × 3) structureson large (111) facets reveal a similar mechanism. We suggest thatone-third of the Ge atoms in the topmost layer of one of the Ge sublatticesis replaced by Pt, as indicated by the red dots in The diffraction pattern reveals a (3 × 3) reconstructed hexagonalpattern. This is attributed to a Pt-containing cover layer on the(111) facets. We suggest that one-third of the Ge atoms in the topmostlayer of one of the sublattices is replaced by Pt. These are indicatedby the red dots in two equivalent azimuthal directionswhich are about 60° apart. The reason for this at first sightunexpected result must be searched for in the strong anisotropy ofthe (√3 × √3) structure which is responsible forthe evolution of the ripples in the first place. We suggest that theripples are aligned along the directions most densely packed withPt atoms, that is, along ⟨11–2⟩ rather than along⟨110⟩ on the ripple’s facets. This situationis sketched in 28 For ease ofsurvey, It appears attractive to use the simple andstraightforward modeldepicted in To gain additional information on the step densityalong [−554]cf. 7, wehav22 a plot of the changes in the vertical component of the wave vectorversus the parallel one directly yields the angle between the steppedfacets and the constituting {111} terraces. The result is plottedin Encouraged by this result,we now have a closer look at the stepdensity along “[001]”. For this purpose, we inspectthe μLEED pattern obtained for energies between 1.9 and 17 eV.In contrast to the broadening along [11–2] discussed above,we now obtain unambiguous evidence for the presence of well-definedsplit peak pairs for each energy. Characteristic data obtained at4, 6, 10, and 15 eV are shown in 2Ptcrystallites upon passing through the eutectic temperature, in accordancewith the phase diagram in 16 and their Ge2Ptcomposition and crystalline structure were established beyond anydoubt. Several crystalline shapes were detected, including the so-calledelongated hut clusters. These hut clusters have a {001} top face alignedparallel to Ge{110}. An almost perfect match is detected along the[001] azimuth, and twice the periodicity along the [100] azimuth onGe2Pt{100} equals well three times the periodicity along[−110] on Ge{110}. (110) facets complete the hut clusters.Above, we have discussed in considerabledetail the breakaway ofGe from a huge eutectic GePt droplet during a cool-down through thecritical temperature. As we have shown, complex and large-scale patternformation occurs, leading to ripples with vicinal (111) facets ofpure Ge covered by a (√3 × √3) R30° Pt-containingcover layer. The ripples are oriented along, mainly, [−554],[−55–4], and along [−552] and [−55–2]azimuth directions on the {110} surface. Nothing yet has been saidabout the counterpart, that is, the emergence of Ge2Pt are known. However, it appears thatthe intimate connection of these crystallites with the host substratedepends on the cooling rate. We have conducted μLEED measurementson coagulated clusters at about 1 mm from the center of the surfacein a further attempt to unravel the crystalline structure. Only limitedcoalescence has occurred at these positions, and the passing small(er)clusters are still moving under the influence of the prevailing thermalgradient. As a result, we cannot catch live the ultimate moment ofsolidification upon passing the eutectic temperature, but we can identifyseveral crystallites after the action. A representative example isshown in As noted above, both the composition and the overall crystal structureof the emerging Ge2Pt crystallite, as shown below.The crystalline structure of PtGe2 is orthorhombic with a = 6.179 Å, b = 5.779 Å, c = 2.914 Å, and α = β = γ = 90°.29 Note that b equals the Ge latticeconstant within about 2%, and commensurability is thus obtained alongGe[001] if the contact plane is along the Ge2Pt{101} plane,with the b-axis parallel to Ge[001]. The μLEEDdiffraction pattern obtained on top of the Ge2Pt islandin 2Pt island in 2Pt{101} contact plane. The blue latticeis slightly distorted when compared to the theoretical rectangularversion  are leftovers of former dropletsformed in earlier experiments. The irregularly shaped large featureturns out to be a 2D Ge2Pt island. Tocorrect for the ,influences of the electronic structure of the surfacewe have subtracted the intensity from a representative area of thesubstrate. Such a difference curve is shown in 30 Onthe square root of the energy scale, the intensity maxima would appearat equidistant separations. The number of maxima up to the Bragg peakwould equal the number of layers minus 1.Information on the vertical parametersof the crystallite is accessibleby an intensity analysis of the intensity versus electron energy dataobtained in μLEED on top of the Ge2Pt{101} film amountsto a realistic 1.7 eV. A severe problem emerges because the Braggpeak resides at 37.5 eV, which would indicate an interlayer distanceof only 1 Å. For comparison, we show in 2Pt{101} film along its b-axis. This [001] directionis oriented along the normal to the plane of drawing. The Pt atomsare shown as red circles and the Ge ones as black ones. The distancebetween the Pt planes along the normal to the contact plane amountsto 2.64 Å. The Bragg condition for specular diffraction fromthis structure is then given by λ′ = 5.28 Å, withλ′ being the internal wavelength of the probing electrons.This anticipated interplanar distance of 2.64 Å is in clear contrastwith the obtained value of 1.0 Å, and we thus must search foranother solution. This is found straightforwardly by attributing theleft-hand red peak in 2Pt{101} film, one arrives at the expected interplanardistance of 2.64 Å. The two remaining peaks in n layers at a mutual interplanar distance d, the shape of the Bragg peak is given bykz = π/λ, and λ is the electronwavelength. The calculated profiles P(Δkz) for n = 3 and n = 4 areplotted in blue and red, respectively, in n = 4. From this, we derive that the Ge2Pt{101}island from 31f is the Fermi wavelength, N/V isthe free electron density in Ge2Pt, n isan integer number, and d is the interlayer distancein the Ge2Pt{101} film. The ionization energy for Pt ina Ge surrounding is 40 meV.32 At 1050 K,the degree of ionization then amounts to 0.38, and the Fermi wavelength,λf, equals 10.4 Å. From n = 4.0,that is, the thin film is four layers thick, in agreement with thedata in 2Pt{101} film by quantum size effects is a plausible cause for thedevelopment of extended thin Ge2Pt{101} crystallites uponthe solidification of micron-sized droplets when passing the eutectictemperature. With the known wetting angle of 20° before solidification,13 one can estimate the projected view of a fictivespherical cap as the parent droplet for the flat island in The fit in 2Pt clusters wewere able to unveil, we now concentrate on the solidification of thesingle huge cluster that evolved near the center of the substrateduring cooling through the eutectic temperature.13 The resulting compact rhombic Ge2Pt crystalliteis shown in the PEEM image in 13 The red rhombus accentuates theedges of the rhombus (see further below for more details). The crystallinerhombus is a huge monolith on the atomic scale with a long axis ofca. 75 μm. Its height is estimated at several tens of micronstoo. We have recorded μLEED patterns on top of the rhombus inspite of substantial distortions due to huge field inhomogeneities33 around this tall object. The result is shownin 2Pt{001} unit cell. The pattern deviates markedly from theone obtained for thin extended crystallites evolving from smallerdroplets located at 1 mm or more from the center , and along the a-axis, an almost perfectmatch is obtained for every second Ge2Pt trimer (mismatch2%). This provides solid anchor sites for the crystallite on the substrate,as Ge positions in both crystals are common here facets of pure Ge covered by a (3 × 3) Pt-containing superstructure.The ripples are oriented along, mainly, [−554], [−55–4],and along [−552] and [−55–2] azimuth directionson the {110} surface. Spinodal decomposition of the large dropletin the center resulted in a compact rhombic Ge2Pt crystallitewith a {001}-oriented Ge2Pt–Ge(110) interface, withthe a-axis pinned at 20° from Ge[−110].Upon passing the eutectic temperature, spinodal decomposition occurs,resulting in PtGe2Pt{101}-orientedfilms with the b-axis aligned parallel to Ge[−110].These films remain ultrathin with a thickness of about four layersand are stabilized by quantum size effects.For smaller droplets located at 1 mm or more from the center, andthus lower local Pt content, we find Ge