For two biscarbamates, it was determined that they should be able to pass the BBB by passive transport, while for five biscarbamates, this ability was slightly limited. Fourteen biscarbamates did not exhibit a cytotoxic effect toward liver, kidney and neuronal cells. In conclusion, considering their high BChE selectivity, non-toxicity, ability to chelate biometals and pass the BBB, compounds 2 and 16 were pointed out as the most promising compounds for the treatment of middle and late stages of AD.As butyrylcholinesterase (BChE) plays a role in the progression of symptoms and pathophysiology of Alzheimer’s disease (AD), selective inhibition of BChE over acetylcholinesterase (AChE) can represent a promising pathway in treating AD. The carbamate group was chosen as a pharmacophore because the carbamates currently or previously in use for the treatment of AD displayed significant positive effects on cognitive symptoms. Eighteen biscarbamates with different substituents at the carbamoyl and hydroxyaminoethyl chain were synthesized, and their inhibitory potential toward both cholinesterases and inhibition selectivity were determined. The ability of carbamates to cross the blood–brain barrier (BBB) by passive transport, their cytotoxic profile and their ability to chelate biometals were also evaluated. All biscarbamates displayed a time-dependent inhibition with inhibition rate constants within 10 N-methyl-D-aspartate receptor, changes in the homeostasis of biometals, increased MAO-B enzyme activity and oxidative stress  deviated from linearity  had an about two times lower inhibition potency of BChE than bambuterol. The tested carbamates were divided into six groups according to the substituents on the carbamoyl nitrogen. Within a particular group, the compounds differed in the size of the hydroxyaminoethyl chain attached to the benzene ring. Generally, no clear correlation between the length and/or the size of groups on the carbamoyl nitrogen and inhibition potency of compounds was detected It can be said that a lower inhibition potency can be expected for compounds with an aromatic amine on the hydroxyaminoethyl chain since the lowest inhibition potency, about 2400 times lower than that of compound 16, was determined for compounds 10 and 11 with phenyl as a substituent on the hydroxyaminoethyl chain combined with alkyl groups on carbamoyl nitrogen. But, when more detailed structure-activity analysis was performed, analyzing the influence of different substituents on carbamoyl nitrogen with the aniline on the hydroxyaminoethyl chain, it could be seen that replacement of aliphatic substituents does not affect the inhibition potency of the compounds; the increase in inhibition potency occurred when piperidine was used. That observation is in accordance with the structure-activity analysis of the same class of compounds reported earlier by Wu et al., where it was demonstrated that replacing dimethyl groups with ethylmethyl on the carbamoyl part of molecules did not affect the inhibition potency of the biscarbamates :v0 and vi stand for the enzyme activity in the absence and in the presence of inhibitor at time t.The first-order rate constants When the kobs vs. [CC′] was not linear, indicating the presence of reversible enzyme–inhibitor complex, the maximum first-order inhibition rate constant (kmax) and the dissociation constants of enzyme–inhibitor complex (Ki) were determined from:When dependence of ki constant was determined as the ratio:Then the All kinetic parameters were calculated using the statistical package GraphPadPrism .The rate of spontaneous recovery of activity of cholinesterase inhibited by biscarbamates, i.e., the rate of spontaneous decarbamylation, was determined by monitoring the time course of return of cholinesterase activity.The spontaneous decarbamylation proceeds according to scheme:where the carbamylated enzyme reacts with water to form the free enzyme (EH) and corresponding alcohol (COH).® C18-E, Phenomenex, Torrance, CA, USA). The time immediately after filtration was denoted as “zero” time of the onset of spontaneous decarbamylation. After a designated period of time (up to 8 h), aliquots of incubation mixture were added to a reaction mixture (buffer and 0.3 mM DTNB), and measurement of activity was started by the addition of substrate (ATCh for AChE and PTCh for BChE). An identical incubation mixture was prepared with enzyme and buffer instead of inhibitor to measure the control values of enzyme activity.Before spontaneous decarbamylation measurements, BChE or AChE were incubated with 100 µM biscarbamates (incubation mixture) for 60 min to obtain the 90–100% inhibition of enzyme activity ,49. To akdecarb) was calculated according to the equation:v0 denotes the activity of the enzyme in the absence of inhibitor and vt the activity of the enzyme incubated by the inhibitor in time t. The -kdecarb is represented by the slope of the line [The decarbamylation rate constant .For docking ligands into the enzyme receptors, the Flexible Docking protocol was used . LigandsSer198–C=O distance and alignment were finally chosen as representative poses. The parameters used in in silico protocols were configured as reported in a previously published work [The enzyme structures were prepared starting from the crystal structures of free BChE (PDB ID: 1P0I) . The binhed work .2), copper chloride dihydrate (CuCl2·2H2O) and iron dichloride tetrahydrate (FeCl2·4H2O). All metal salts and biscarbamates were dissolved in methanol. Briefly, a fixed amount of biscarbamate (30 µM) was mixed with a fixed amount of metal salt (60 µM). The absorbance spectrum (200 to 600 nm) of such a mixture was measured in the first minute and 30 and 60 min after mixing. The spectra of metal salt and of biscarbamate were also measured [The ability of carbamates to chelate biometals was tested using metal salts: zinc (II) chloride . The intersection point in the plot corresponds to the mole fraction of metal in the biscarbamate-metal complex [The stoichiometry of the biscarbamate-metal complexes was determined using the mole fraction method ,67. The  complex ,51,66,67The study of metal chelation was performed in 1 cm quartz cuvette  at 25 °C using UV–VIS spectrophotometer . All presentations of the spectrum and stoichiometry of binding were carried out in GraphPad Prism, GraphPad Software.The ability of synthesized biscarbamates to penetrate the BBB was estimated by calculating molecular descriptors important for passive transport ,54 , penicillin/streptomycin (PenStrep), glutamine and non-essential amino acids (NEAA)) were purchased from Sigma-Aldrich, Steinheim, Germany. Cell cultivation was performed according to a previously described protocol .The human Caucasian hepatocyte carcinoma HepG2 (ECACC 85011430), human embryo kidney HEK293 (ECACC 85120602) and human neuroblastoma SH-SY5Y (ECACC 94030304) cell lines were obtained from certified cell-bank European Collection of Authenticated Cell Cultures (ECACC) through Sigma-Aldrich . All cell lines were grown and maintained according to standard protocol ,70,71.® AQueous One Solution Cell Proliferation Assay, Promega, Madison, WI, USA). The procedure followed slightly modified manufacturer protocol described previously [The cytotoxic profiles of tested carbamates were determined by measuring the succinate dehydrogenase mitochondrial activity of cells exposed to them . The comeviously . Data we


Idiopathic generalized epilepsy (IGE) is a common type of epilepsy and despite an increase in the number of available anti-seizure medications, approximately 20–30% of people with IGE continue to experience seizures despite adequate medication trials. Unlike focal epilepsy, resective surgery is not a viable treatment option for IGE; however, neuromodulation may be an effective surgical treatment for people with IGE. Thalamic stimulation through deep brain stimulation (DBS) and responsive neurostimulation (RNS) have been explored for the treatment of generalized and focal epilepsies. Although the data regarding DBS and RNS in IGE is limited to case reports and case series, the results of the published studies have been promising. The current manuscript will review the published literature of DBS and RNS within the centromedian nucleus of the thalamus for the treatment of IGE, as well as highlight an illustrative case. Epilepsy is defined as a disease of the brain characterized by an enduring predisposition to generate seizures. Epilepsy is defined by the presence of two or more unprovoked seizures separated by 24 h, one unprovoked seizure with a high probability for seizure recurrence, and/or the presence of a discrete electroclinical syndrome . EpilepsIn a broad classification, generalized epilepsy is the second most common type of epilepsy after focal epilepsy. The term idiopathic generalized epilepsy (IGE) is used to describe a group of epilepsies with presumed genetic basis, manifesting as generalized-onset seizures, most commonly absence, myoclonic and/or generalized tonic-clonic seizures (GTCS). IGE syndromes share several clinical features including normal cognitive development, absence of gross neurological pathology, age dependency, and responsivity to certain anti-seizure medications (ASMs) . ElectroLennox-Gastaut syndrome (LGS) is an epileptic encephalopathy characterized by multiple seizure types including atypical absence, tonic-clonic, myoclonic, tonic and atonic seizures. Typically, EEG shows GSWDs and on a slow background. Due to its generalized clinical and EEG features, LGS was previously referred to as a “symptomatic generalized epilepsy,” a term that is no longer endorsed by the International League Against Epilepsy. LGS is often due to brain injury that almost always involves, but is not necessarily limited to, the cerebral cortex .Despite an increase in the number of ASMs available to treat epilepsy seizures over the last 20–30 years, up to 20–30% of people with IGE are unable to control their seizures with medications alone . PatientThe first neuromodulation modality approved for the treatment of epilepsy was vagus nerve stimulation (VNS). VNS received FDA approval in 1997 for the treatment of focal-onset seizures , which wNeuromodulation modalities have been utilized in the treatment of drug-resistant IGE. More recently, DBS and RNS targeting the centromedian nucleus (CM) of the thalamus have been explored as surgical therapies for people with drug-resistant IGE. Although VNS has been used in the treatment of drug-resistant IGE with some success , this reAlthough many thalamic nuclei may be involved in the generation of generalized epileptiform discharges and seizures, the CM and ANT appear to be activated during the initial spike-wave pattern in generalized epilepsy .GSWDs and generalized polyspike-and-wave discharges are the typical interictal signature of all IGE syndromes. Since the first description  of GSWDscentrencephalic system that includes the thalamus with the diencephalon and the brainstem  activity during GSWDs . Another electrographic signature of LGS is fast rhythmic waves or paroxysmal fast activity, often associated with tonic seizures . Althougduration . Accordiduration .The CM is considered a “diffuse-projecting” nucleus in the thalamus, with the lateral third of the CM exhibiting reciprocal connections with the premotor, motor, and primary somatosensory cortices . TractogDefining the CM as a stereotactic target is primarily accomplished through indirect targeting based on the anterior commissure-posterior commissure line, as the thalamic nuclei are not visible on conventional imaging and intraoperative electrophysiology is not useful to guide placement. Accordingly, studies have attempted to define both optimal targeting techniques as well as success based on active contact location . Newer iWhereas the treatment of focal seizures with ANT-DBS has received considerable attention and FDA approval , the treThree controlled trials assessing the efficacy of CM-DBS have been performed . The firThere have also been several small uncontrolled studies on the use of CM-DBS, most which focused on patients with LGS. Velasco et al. reported a significant difference in seizure reduction, and specifically >87% reduction in patients with appropriate electrode placement . Son et A recent systematic review of DBS targets in epilepsy reported a mean seizure reduction of 60.8, 73.4, and 67.8%, respectively with stimulation of the ANT, CMT, and hippocampus . The anaReports on the use of CM-DBS for IGE are scarce; with the majority of reported cases being in patients with focal epilepsy, LGS or seizure types suggestive of LGS. There are only 6 reported cases of using CM-DBS in LGS. These are summarized in Unlike DBS, RNS is a technology that operates in a closed-loop circuit, via detection of epileptic discharges that are defined based on the patient’s unique electrographic seizure characteristics. When these preset discharges are recognized and detected by the device, responsive therapy is delivered via electrical current. Therefore, RNS provides individualized therapy that can be modified and adjusted based on the patient’s electrocorticography (ECoG) data and seizures. The publication of the RNS pivotal trial in 2011  resultedIn the first reported case, a 19-year-old female with eyelid myoclonia with absences underwent bilateral CM-RNS implantation . FollowiIn another case, a 16-year-old male childhood absence epilepsy underwent bilateral CM-RNS . After iIn addition to focal epilepsy and IGE, other studies have evaluated different indications for the use of CM-RNS as well. The patient is a 41-year-old right-handed female with a history of drug resistant IGE manifesting as typical absence seizures, myoclonic seizures, GTCS. She had a family history of epilepsy . There were no other epilepsy risk factors. Her seizures began at the age of 4 years old. Over time, she continued to experience frequent GTCS that were often followed by non-convulsive status epilepticus . At the time of her initial evaluation in our comprehensive epilepsy center, she would have at least 1 GTCS per year but often would have 1 GTCS every 2–3 months. From 2018 to 2020, she had 11 GTCS that were consistently followed by 1–2 days of NCSE, requiring hospital admissions. Throughout her epilepsy course, she failed medication trials of levetiracetam, topiramate, perampanel, and ethosuximide.In light of her DRE, she was evaluated for epilepsy surgery. A brain MRI was unremarkable for discrete epileptic lesions. Inpatient video-EEG monitoring revealed generalized, frontally predominant, 3–4 Hz, spike- and polyspike-and-wave discharges as well as bursts of GPFA . Her casInitially, following RNS implantation, she continued to experience frequent GTCS with approximately 1 GTCS every 1–3 months. Six months after RNS implantation, she experienced a cluster of GTCS that was followed by absence status epilepticus –3. DurinTo date, the published literature regarding neuromodulation in IGE is primarily limited to case reports and case series. As such, there are many questions related to patient selection, safety, neuromodulation settings, and outcomes. Although the failure of two appropriately chosen medications at appropriate doses denotes drug resistant epilepsy, should patients with IGE have a trial of valproic acid before proceeding to surgery? Are certain electroclinical syndromes more responsive to neuromodulation, e.g., epilepsy with generalized tonic-clonic seizures alone? What are the optimal DBS and RNS settings for IGE? For RNS, the baseline simulation settings in previously published reports have largely adhered to those established for ANT-DBS ; however1 As case reports and series of CM-RNS in IGE continue to grow and the results of randomized controlled clinical trials become available, RNS may emerge as a viable treatment option for people with drug-resistant IGE.Drug resistance is common and may be present in up to 20–30% of IGE, and unfortunately there is no FDA-approved treatment for IGE. Although historically surgical options for IGE have been limited, there is increasing evidence that neuromodulation may be an effective treatment option for these patients. Case series utilizing DBS for IGE have been reported and more recently, case reports illustrating the utility of RNS in treating IGE have been published. Bilateral CM-RNS offers an exciting new treatment paradigm in which detection of epileptiform activity in the brain is possible and therapy may be tailored specifically for each patient. The initial promising results from bilateral CM-RNS for the treatment of IGE has led to the initiation of a prospective single blind, multi-center, randomized study (NAUTILUS).AZ and MS contributed to conception and design of the review. AZ wrote the case illustration. MS provided supervision and project administration. All authors performed the literature review, contributed to drafting of the manuscript, and read and approved the submitted version.


Adansonia digitata L. (Baobab) fruit pulp (ADFP). The prepared ice cream’s physicochemical and microbiological quality, and rheological, microstructural, and organoleptic properties were investigated. Using ADFP instead of skim milk powder with a partial or complete replacement, five ice cream samples were produced and marked as IB-0, IB-25, IB-50, IB-75, and IB-100. Chemical characteristics were not noticeably impacted except protein and ash, which considerably decreased with increasing ADFP levels. Increasing ADFP in the samples increased titratable acidity and reduced pH. All ice cream samples were microbiologically acceptable with no pathogenic bacteria. By increasing ADFP in the samples, the daily values (%DV) of sodium, potassium, and magnesium were not considerably affected. Calcium reduced from 14.91% in IB-0 to 7.75% in IB-100. All microelements found in the study rose considerably as ADFP increased. Increasing ADFP levels significantly boosted antioxidant levels. The IB-100 sample had the highest total phenolic content (149.29 mg GAE 100 g−1), antioxidant activity (98.12 µmol of TE 100 g−1), total flavonoids (5.96 mg QE 100 g−1), and total flavanols (4.01 mg QE 100 g−1). The inclusion of ADFP had a beneficial effect on the color of the samples. It did not negatively affect the ice cream’s organoleptic acceptability as determined by organoleptic, rheological, and microstructural examinations. Interestingly, when skim milk powder was replaced with 50% and 75%, the hardness, viscosity, and aqueous phase separation were significantly improved. In conclusion, ADPF improves the nutritional value of ice cream and can be used as a natural coloring ingredient without affecting microstructural and rheological properties.Ice cream’s appeal is unrivaled. Nonmilk and milk ingredients in ice cream formulas affect their nutritional value, structure, and organoleptical qualities. Seeking novel dietary ingredients instead of artificial flavoring compounds is vital for improving ice cream taste preference, adding antioxidants, and increasing nutritional value. The current study examines the feasibility of manufacturing a new flavored ice cream with excellent dietary value using  Supply for progress and healthful products is periodically bolstered to meet the populace’s needs. Thus, food with additional bioactive components is developed and labeled by food manufacturers to give fundamental nutrients and health advantages in terms of “functional foods” . Extra nThe production procedures of blending, pasteurizing, homogenizing, cooling, aging, flavoring and coloring, whipping, packing, and hardening are all crucial to the final ice cream product. The base ingredients are milk fat and milk solids not-fat (SNF), sugar, water, stabilizers, emulsifiers, and 0.3% liquid flavors and colors . ProteinAdansonia digitata L. (A. digitata), known as baobab, is a member of the Malvaceae family, and is highly valued for its medicinal and nutritional benefits [A. digitata plant—seeds, leaves, roots, blossoms, fruit pulp, and bark—can be eaten, and used in cooking and baking [A. digitata is rich in beneficial nutrients such as vitamins B1 and B2, and minerals. Seeds are high in nutrients such as protein and fat, and can be used as a food source. The chemical composition of 20 kinds of A. digitata indicated 1250–12.91, 4.75–5.18, 2.24–2.42, and 52.17–56.62% for moisture, ash, protein, and total dietary fibers, respectively [A. digitata fruit pulp (ADFP) primarily consisted of hydroxycinnamic acid glycosides, iridoid glycosides, and phenylethanoid glycosides [A. digitata contained significant amounts of flavonoids and phenolic acids such as catechin, quercitrin, quercetin, epicatechin, luteolin, rutin, kaempferol, gallic, caffeic, chlorogenic, and ellagic acids [The stately tree benefits . It can d baking ,13,14. Td baking . The frud baking . The puld baking . Signifid baking , A. digiectively . The sulycosides . An HPLCic acids . Accordiic acids , profiliA. digitata stem bark, had antimalarial efficacy. There was encouraging evidence that eating baobab could help in curbing cravings and promote weight maintenance [A. digitata extracts significantly mitigated lipid peroxidation, which acted as a protective mechanism against hepatotoxicity. At 200 mg daily, ADFP had a cardioprotective effect against induced oxidative stress and led to mostly typical myocardial structure [A. digitata have powerful antioxidant properties [A. digitata fruit pulp, rendering it a potential source of health-promoting compounds [Ismail et al.  showed tntenance . Hanafy ntenance  discovertructure . The flaoperties  that migoperties . Procyanompounds . Baobab ompounds . Incorpoompounds .Interestingly, to the best of the authors’ knowledge, the literature review mainly highlighted that adding ADFP into ice cream has not been investigated so far. Therefore, the current study examines the feasibility of producing a new ice cream product by incorporating a substantial amount of ADFP. To commercially scale up ice cream, the nutritional, physicochemical, structural, rheological, microbiological, and organoleptic properties of prepared ice cream were investigated.Raw cow milk (3.3% fat) from the herd of the faculty of agriculture and veterinary medicine, Qassim University, Al-Qassim, KSA was obtained. Fresh bulk milk was separated, and cream (55% fat) was collected at the processing unit of the Department of Food Science and Human Nutrition. Skim milk powder (95% solids not fat) was supplied by Arla Foods Company . Sugar, gelatin, and vanilla powders were purchased from the local market at Buraydah, Al-Qassim region, Saudi Arabia (SA). A Sudanese source of baobab fruit was obtained from the local market in Jeddah, SA. To extract the coated seeds and dry pulp, dried fruit was washed, and shells were cracked open before grinding using an electronic grinder . The resultant ADFP was then sieved over a 60-mesh screen to obtain a fine, uniform powder that was immediately stored in dark glass jars at a temperature of 18 ± 1 °C until the formulation of different ice cream mixes.According to Arbuckle , five icStandard AOAC (2005) procedures were used to determine the total solids, crude protein, fat, ash, and available carbohydrates .−1) was determined following standard AOAC methods [The pH values of the different ice cream samples were detected using a pH meter . Titratable acidity  the total bacterial count was enumerated according to the conventional method  was homogenized with 225 mL peptone water; then, serial dilutions were performed in a ratio of 1:9 od  on platg to ISO  on a Baig to ISO .−1 multielement certified standard solution  was used as a stock solution for instrument standardization as reported by AOAC [Mineral constituents  were determined using an atomic absorption flame emission spectrophotometer . In addition, 1000 mg L by AOAC .2CO3 (7.5%). The TPC content was calculated as milligrams of gallic acid equivalent (GAE) per gram on the basis of an OD reading at 765 nm taken 60 min after incubation, and compared to a standard curve derived from a gallic acid (GA) solution (R2 = 0.99) (mg of GAE g−1 100 DW).)The prepared samples of ice cream were analyzed for TPC with the Folin–Ciocalteu reagent following a modified procedure developed by Bettaieb et al. . In summ● assay [−1 100 DW).Antioxidant activity (AOA) was assessed using a DPPH● assay , and spe3 and monitored for 60 min at 420 nm. The data were then expressed as mg quercetin equivalent (Q.E.) per 100 g−1 D.W. According to Kumaran and Karunakaran [3 (2%) was added after 5 min, and the optical density (OD) was measured after 150 min at 440 nm; TFL concentrations are reported in mg QE g−1.According to Mohdaly et al. , the TF unakaran , TFL conThe overrun of different ice cream samples and their melting rate over 120 min at room temperature were measured. To measure the overrun %, the weight of freshly prepared and unfrozen ice cream mix was compared with the weight of the ice cream itself , using tThe melting point and melting resistance of ice cream samples were measured by melting 50 g at 22 ± 1 °C. Drippings from the samples were collected in a beaker after they had been set on a fine wire screen over a glass funnel. The temperature at which the initial decrease occurred was noted. Drainage weight was measured periodically for a total of 120 min. Melting resistance was determined by calculating the percentage of relative melted amount for each time interval .The color of each sample was measured with a chromameter  by applying the CIELAB scale  adjusted with typical white, green, and black tiles. The hue angle (Hᵒ), chroma (C), and color changes (∆E), and browning index (BI), compared with the values of the control, were then calculated according to Lavelli et al.  using co−1 was obtained for measuring viscosity. The time was adjusted to 120 s during the experiment. In a linear distribution, 32 points were collected, and duplicates had less than 10% differences. Trios software version 3.1.0.3538 was fitted to the power law models for analyzing the data.The Discovery Hybrid Rheometer  was used to analyze the rheological properties of ice cream samples with different amounts of baobab fruit bulb (ADFP). The applied method conditions were according to the description of Gouda et al. . BrieflyThe microstructures of ice cream samples were examined at the Electron Microscope Unit, National Research Center, Giza, Egypt. TEM  was used to investigate the microstructure of freeze-dried different ice cream samples. Samples were prepared according to the technique of Caroll et al. , and micThe organoleptic attributes of the ice cream of flavor (45), body and texture (35), melting quality (10), color and appearance (10), and total score (100) were judged by a panel of 15 staff members at the Department of Food Science and Human Nutrition, Faculty of Agriculture, Qassim University, KSA. Various ice cream samples were coded using random 3-digit numbers and served randomly by the panelists. The panelists were asked to fill in the scorecards, and data were collected for statistical analysis .The statistical analysis was carried out using one-way analysis of variance (ANOVA) using SPSS, v. 22 . Data were treated as a complete randomization design, according to Steel et al. . MultiplThe chemical composition of the different ice cream samples  is presented in Staphylococcus aureus were not detected in any ice cream samples or their mixes.The results in −1) and Mg (1.70 to 2.20 mg 120 g−1). An inverse observation was that Ca content significantly decreased from 193.8 to 100.8 mg 120 g−1 with the increase in the levels of ADFP. The increase in the Mg content of the ice cream samples from 1.70 to 2.20 mg 120 g−1 due to the rise in ADFA levels was insignificant. The contents of microelements such as P (0.62 to 1.12 μg 120 g−1), Fe (2.4 to 102.18 μg 120 g−1), and Zn (.06 to 18.62 μg120 g−1) of the ice cream samples were increased significantly with skim milk powder substitution.The mineral contents of ice cream samples are presented in According to Food and Drug Administration (FDA) updates , the nutrients’ daily values (DV) are used to inform consumers of the recommended amounts of nutrients that, whether macro- or micronutrients, should be consumed during the day and not exceeded. The daily value (%DV), on the other hand, is the amount of the contribution or content of the nutrient in one serving. Hence, nutritional information should be labeled on different ice cream formulas with the updated daily value for macro- and micronutrients. The TPC, relative AOA using DPPH, total flavonoids, and total flavonols of all formulated ice cream incorporated ADFP were investigated; data are shown in The highest overrun value was observed for the IB-0 sample (35.61), while the lowest was for the IB-50 (13.71) sample. The melting point was determined via the time required for the first drop of the mixture to decrease to a temperature of 22 °C. The fastest sample dropping during melting was IB-75, which took 35.04 min for the first drop to descend, while the longest time was for the IB-25 sample, where the first drop fell after 44.07 min. The melting resistance of ice cream samples containing ADFP is presented in o and C values increased significantly with the increase in ADFP levels, and the highest values of BI and ∆E were when replacing 100% of the skim milk powder content with ADFP .Color is an essential factor in determining product quality; therefore, it is considered to be a criterion for acceptance by consumers . The res0: zero-rate viscosity, η00: infinite-rate viscosity, K: consistency, n: rate index) are shown in As a function of shear rate, the viscosity of various ice cream samples is represented in 00) compared to that of the IB-0 sample, which contained no ADFP. The more the ADFP amount increased, the greater the viscosity (Pa·s) that was observed in all samples  except for IB-100 with zero skim milk powder. The replacement of 75% skim milk with ADFP (sample: IB-75) significantly increased viscosity by about 35.5% more than that of the IB-0 sample, with no significant difference between IB-50 and IB-75 in infinite rate viscosity. The increase in ADFP with the absence of skim milk powder (IB-100) markedly decreased viscosity by about 23.1% more than that of the IB-0 sample with 100% skim milk powder content.The replacement of skim milk powder with ADFP significantly increased the infinite rate viscosity (ηp > 0.05) differences were observed in K values between IB-75 and IB-100 compared to the IB-0 sample. An increase in rate index was significantly observed in samples with greater ADFP amounts (IB-75 and IB-100) than those of the rest of the samples.The consistency coefficient (K) of the ice creams indicating their relative thickness increased with the presence of ADFP in Samples IB-25 (0.875 Pa·sn) and IB-50 (0.498 Pa·sn), while the opposite trend was observed in the rest of the samples. No significant (The transmission electron micrographs of different ice cream samples are presented in p > 0.05) were found among all ice cream samples regarding body and texture, melting quality or color, and appearance, as reported by the panelists. The difference among samples with only the scores of flavors changed the total scores. The flavor attribute for the IB-100 sample (p ˂ 0.05) significantly had the lowest score among all other samples. The main remarks by the panelists were that the IB-75 and IB-100 samples lacked smoothness, and had grainy and rough structures.The mean scores of the flavor, texture, melting quality, and appearance of the different ice cream samples as affected by ADFP addition are presented in This study is closely related to that reported by Umelo et al.  on tiger5 CFUg−1 or less. Many processing steps after the pasteurization of ice cream render it vulnerable to bacterial contamination, especially from pathogenic microbes that contaminate the product after its heat treatment. The microbiological quality of ice cream depends on many factors, including the microbial standards of the raw materials, the production process, and the hygiene of the working environments [5 CFU/g and pathogenic bacteria were absent. This reflects the hygiene and quality of the used materials, and the preparation and production processes of the ice cream under study. Ice cream is a complex food model, and comprehensive approaches to the entire production process are required to ensure high quality and safety standards.According to most hygiene guidelines , TBC should be 0–10ronments . Coolingronments . The obt−1), potassium (988–1240 mg 100 g−1), calcium (237–295 mg 100 g−1), magnesium (90–110 mg 100 g−1), and phosphorus (124 mg 100 g−1), but poor sources of iron, zinc, and copper. On the other hand, skim milk powder is also a good source of sodium (470 mg 100 g−1), potassium (1490 mg 100 g−1), calcium (1140 mg 100 g−1), magnesium (110 mg 100 g−1), and phosphorus (500 mg 100 g−1), as reported by Arellano et al. [Incorporating ADFP into food can be a crucial source of essential nutrients for food fortification due to its high content of macro- and microelements ,36 resulo et al. . From thInterestingly, a remarkable incremental trend in TPC, AOA, T.F., and TFL in formulated ice cream incorporating BPF was observed. This may have been due to the rich content of bioactive compounds and phytochemicals in BPF, which was increased with increasing ADFP levels. The substitution of skimmed milk powder with BPF in ice cream formulas slightly improved the AOA assessed with DPPH scavenging assays. The antioxidant capacity was probably due to the presence of phenolic compounds and ascorbic acid in ADFP ,63. The The increasing ADFP level increased the melt resistance of the prepared ice cream. This improvement was due to the high foaming stability of ADFP and good formulated structure seen in TEM structural analysis . The comThe lack of creaminess and smoothness came from the extremely low overrun, indicating that little air was included, causing an excessively cold sensation in the mouth. On the other hand, a high overrun amount makes the ice cream frothy, even though some countries set 100% as the legal overrun limit for ice cream . NumerouThe color of ice cream is a crucial sensorial parameter that reflects its consumers’ acceptability. The formulation of an ice cream mixture and the coloring ability of the product’s fruit affect the final product’s color quality . As prevIn this study, zero- and infinite-rate viscosity values, as a relation to the shear rate in ice cream samples with opposite amounts of skim milk powder and ADFP, were near the ranges of 4.49 to 15.88 and 0.13 to 0.18 Pa·s, respectively, as the amount of ADFP increased, except for the IB-100 sample. The effect of increasing viscosity as the ADFP amounts increased was also related to the increase in TA% and decrease in pH values , and theObserved increments in zero-rate viscosity, infinite-rate viscosity, and consistency as a result of the ADFP replacement increase  were fouThe texture properties of food are those perceptions based on the evaluation of its physical characteristics as affected by parameters related to the shape, size, structure, number, and conformation of its constituent structural elements and proteins. All play a crucial role in forming the texture of the final product. The protein–fat matrix in an ice cream structure and a cohesive network are essential to produce a homogeneous fat–liquid emulsion. Thus, the combination of many ingredients in the formulation of ice cream, and the four systems that comprise its structure, each an emulsion and each a foam, ice crystals, air bubbles, fat globules, aggregates, and unfrozen serum, compose its complex structure. Thus, TEM is recommended to observe the ice cream microstructure ,72,73,74As compared to the IB-0 sample, ice cream samples containing ADFP showed apparent differences in electron micrographs . ProteinIn both IB-75 and IB-100, the significant decrease in flavor significantly decreased the total score for these samples. This result was in agreement with Ali and El Zubeir , who fouAdansonia digitata L. fruit pulp (ADFP) on the physicochemical, nutritional, rheological, microstructure, and organoleptic properties of ice creams was investigated. Incorporating ADFP into ice cream improved dietary values regarding macro- and micronutrients. Additionally, antioxidant and mineral content enhancement was also detected, which could enhance the ice cream’s stability. Ice cream with no more than 75% ADFP instead of skim milk powder improved the microstructural and rheological properties. When the ADFP level increased, ice cream acceptability was increased regarding organoleptic properties. Remarkably, data gained from the current study highly recommend such a replacement. ADPF can be safely employed as a natural coloring ingredient in ice cream without affecting its microstructural or rheological properties. It also plays a crucial role in improving the ice cream’s nutritional value.The effect of replacing skim milk powder with


Algal biomass and pigments impacted chromophoric dissolved organic matter concentrations. Despite the effective absorption of astaxanthin esters at wavelengths where incoming irradiance peaks, the cellular energy absorption of snow algae was 95% lower than anticipated from their pigmentation, due to pigment packaging. The energy absorption of glacier ice algae was consequently ~ 5 × higher. On bare ice, snow algae may have locally contributed up to 13% to total biological radiative forcing, despite contributing 44% to total biomass. Our results give new insights into the impact of algal community composition on bare ice energy absorption and biomass accumulation during snow melt.Blooms of pigmented algae darken the surface of glaciers and ice sheets, thereby enhancing solar energy absorption and amplifying ice and snow melt. The impacts of algal pigment and community composition on surface darkening are still poorly understood. Here, we characterise glacier ice and snow algal pigment signatures on snow and bare ice surfaces and study their role in photophysiology and energy absorption on three glaciers in Southeast Greenland. Purpurogallin and astaxanthin esters dominated the glacier ice and snow algal pigment pools (mass ratios to chlorophyll  They contribute to regional nutrient and carbon cycling5 and significantly reduce surface albedo, accelerating the melting of ice9 and snow13. Their most striking adaptation is their dark pigmentation, making them effective absorbers of solar irradiance16. They account on average for 9–13% of surface ice melt on the south-western margin of the Greenland Ice Sheet (GrIS)6 and 13% of snow albedo reduction across the Arctic10.Algal communities thrive on glacier and ice sheet surfaces around the world2 with the filamentous Ancylonema nordenskiöldii  and the mostly unicellular A. alaskanum (formerly Mesotaenium berggrennii17). Glacier ice algae accumulate large amounts of secondary, brown-coloured phenolic pigments, mainly purpurogallin-derivatives, in vacuoles in their cells18. Purpurogallin-derivatives absorb light broadly across the near-UV and visible wavelength range (λmax 338 nm19) and influence the absorption properties of the glacier ice algal cells16. The accumulation of secondary pigments protects the internal photosynthetic apparatus from potential damage by excess irradiance20 and can also be observed in the unicellular snow algae 1. They transition from green-coloured, motile cells to red-coloured non-motile, cysts, rich in carotenoids, as the snowpack ripens and melts during the spring thaw23. These carotenoids are stored in lipid globules and consist mainly of astaxanthin isomers and their fatty-acid ester derivatives, hereafter referred to as astaxanthin esters. These absorb solar irradiance in both the visible  and the UV range 25. Exposure to UV radiation and nitrogen limitation are major factors triggering the production of photoprotective carotenoids in snow algae21. In addition, it has been postulated that secondary pigments may also help to generate meltwater around the cells by dissipating heat from absorbed sunlight26. Since the purpurogallin-derivatives and astaxanthin esters are free of nitrogen and phosphorus, they can be produced even during periods of low nitrogen and phosphorous availability on bare ice18 or snow21.The algal community on bare ice is typically dominated by glacier ice algae28. It has been demonstrated that CDOM can be of ecological relevance by attenuating UV light around algal cells in other aquatic environments30, but no data are available yet from supraglacial bare ice environments.Algal pigments and other organic substances can enter the pool of coloured dissolved organic matter (CDOM) in the environment upon excretion by the algae or leakage from dead cells31. Uncertainties on the light absorption efficiencies of glacier ice15 and snow algae31 have been highlighted as one of the major factors limiting our ability to assess algal-driven surface darkening31. Since the pigmentation of algae influences their light absorption and, hence, their darkening potential16, it is crucial to improve our understanding of the extent of pigment accumulation in glacier ice and snow algae for deciphering their impact on surface darkening. For glacier ice algae, in particular, data on their pigmentation are scarce and only available from the south-western margin of the GrIS19 and it remains unclear to what extent their pigmentation could vary regionally. Furthermore, snow and glacier ice algae are traditionally studied independently, however, community mixing occurs upon melting of seasonal snowpacks, so that snow algae are being washed onto the bare ice where they remain on its surface32. This community mixing on the bare ice has so far been rarely considered32 and could impact glacier surface darkening and snow algal population survival since their cysts can act as inoculum for the next melting season34.The contribution of pigmented algae to the melting of the GrIS is not yet fully understoodHere, we studied the relative abundance, pigmentation, and photophysiology of glacier ice and snow algae along with snow and ice CDOM concentrations on three glaciers on SE-Greenland. The different sampling locations enable us to compare algal communities and their adaptation strategies, which may be closely linked to the different environmental conditions of their respective habitats. We analyse how snow and glacier ice algal pigment signatures manifest in their individual light-absorption properties by combining estimated (pigment-derived) and empirically measured  absorption properties. The absorption coefficients were subsequently used to estimate snow and glacier ice algal contribution to biological radiative forcing on the three studied glaciers.Snow and ice samplings were performed on Mittivakkat (MIT), Bruckner Glacier (BR) and Heim Glacier (HE) in SE-Greenland , between 20 and 27 July 2019 Fig. and S2.FiOn Mittivakkat, a land terminating glacier of the Ammassalik island ice field, triplicate samples were collected along a west–sloping transect extending from above to below a persistent snowpack , sub-samples of 2 ml were preserved with glutaraldehyde  and stored at 4 °C in the dark. Cells were counted by performing 3–6 replicate counts of a full haemocytometer chamber per sample  using an inverted light microscope (Olympus CK2). Photos of randomly selected cells were taken and cell lengths and widths were measured using ImageJ . Cell volumes (μm3) were calculated using cylindrical shapes for glacier ice algae and spherical shapes for snow algae after Hillebrand et al.35. The cellular biovolumes were converted to the carbon equivalent biomass (pg C cell−1) using the biovolume to carbon mass conversion constants (0.109 and 0.991) from Montagnes et al.36 to obtain the total mass of carbon of the algal community per snow or ice meltwater volume (µg C ml−1).To quantify the relative abundance of snow  and glacier ice algae  was measured after at least 30 min of dark acclimation at ~ 5 °C. During rapid light curves, the effective quantum yield of PSII (Y(II)) as a function of increasing light intensities was measured with the pulsed light technique and subsequently converted to relative electron transfer rates (rETR). The photosynthesis-irradiance (PI) model of38 was fitted to the rETR vs. PAR data using the phytotools package39 in R. Instrument settings and calculations can be found in Supplementary Methods.To explore the link between algal relative abundances and pigmentation with photophysiology, we assessed the in vivo efficiency of energy conversion of Photosystem II  of the algal communities by measuring variable chlorophyll-fluorescence directly in the field. This was done by using a Phyto-PAM  fitted with a Phyto-ED detector unit equipped with a blue light detector system (470 nm)41 with some modifications as described in Supplementary Methods. Lipophilic pigments were analysed on a high-pressure liquid chromatography system  and quantified using analytical standards (Supplementary Methods). The peak at 338 nm was associated with purpurogallin extracted from algal cells, which was confirmed by additional absorption tests  in the field. These were then wrapped in aluminium foil and stored in a cryoshipper for transport to the home laboratory. There, filters were freeze-dried (− 110 °C) and a two-phase extraction was used to quantify major lipophilic and hydrophilic pigments following Aigner et al. and Holzinger et al.375 within 10%. Prior to analysis, CDOM samples were acclimated to room temperature and measured on a Shimadzu UV 2700 dual beam spectrophotometer using the 10 cm cuvette set-up at an interval of 3 nm. All measurement blanks were prepared using fresh Milli-Q (18.2 ΜΩ cm). The absorption coefficient a(λ) was calculated with the factor 2.303 being the natural logarithm of 10, λ representing the optical density and l being the cuvette pathlength (m−1):Subsamples of melted ice and snow were filtered through 0.2 µm PES filters into combusted (450 °C) 60 ml amber glass vials, transported back to the laboratory and then stored at 4 °C with short exposure to elevated room temperatures. Elevated storage temperature effect on sample CDOM quality quantity was assessed by comparing features with acidified CDOM samples, showing only effects on a−1; Supplementary Methods) to calculate the mass-specific absorption coefficient.A baseline correction was done relative to the mean absorption values at 750 nm. CDOM absorptions were normalised to dissolved organic carbon concentration  based on their pigment composition according to Eq.  in glacier ice and snow algae, respectively. This approach assumes that algal absorption is a linear combination of the absorption of each pigment42. The molar absorption coefficient for purpurogallin was derived by dividing the absorbance (m−1) of the hydrophilic extracts by the given concentration of the standard in the cuvette (mM m−3). The coefficient for all-trans-astaxanthin was taken from Clementson and Wojtasiewicz43 and was corrected for absorption differences of the cis-isomer. Due to the lack of astaxanthin ester standards, the unesterified astaxanthin coefficients were conservatively corrected for molar mass differences using the astaxanthin-ester composition of red snow algal cysts in Bidigare et al.24. All other absorption coefficients were taken from Bidigare et al.44. In the second approach, we measured the cellular absorption coefficients of algal cells directly. In the field, we filtered algal cells from the snow (MIT2) and the site with the highest glacier ice algal relative abundance (HE1) onto GF/F filters, which were stored in plastic dishes for ~ 24 h at room temperature and then in darkness at − 20 °C until analysis. Optical densities (ODs) of the filters were measured on a Shimadzu UV 2700 dual-beam spectrophotometer equipped with an integrating sphere (2600-ISR). A wetted GF/F filter served as blank to account for scattering and absorption of the filter itself. Algal absorption spectra were corrected for the pathlength amplification effect (ß) according to Bricaud and Stramski45 and the optical density of the cells on the filters (ODs (λ)) was converted to the optical density of particles in suspension (ODsus (λ)):We assessed glacier ice and snow algal absorption using (i) reconstructed “unpackaged” absorption coefficients, which are particularly useful to study the role of individual pigment groups for the total algal absorption, and (ii) “packaged” cellular absorption coefficients. For the first approach, following Bidigare et al.sus (λ) was converted to the absorption coefficient (a(λ); Eq. (l (m−1) derived by the ratio between filtered sample volume and sample area on the filter. Algal absorption coefficients (m−1) were subsequently normalised to cell concentrations per meltwater volume (cells m−3), total algal biovolume per meltwater volume (µm3 m−3) and chl a concentrations per meltwater volume (mM m−3). Since glacier ice algae cells are less robust than snow algal cysts and purpurogallin is hydrophilic, the absorption spectrum of glacier ice algae ; Eq. ) using t2 ml−1) by multiplying their biovolume-specific absorption coefficients (m2 µm−3) with the total algal biovolume per meltwater (µm3 ml−1) for each sampling region. The total algal absorption was subsequently multiplied with the incoming spectral irradiance  obtained from the PVSystems solar irradiance program  for 65°N and 24 July 2019  and horizontal plane (perpendicular to the direction of sunlight). We do not integrate the IRF throughout the day to compare the impact of the different algal cell types and their varying biomass only. The sum of the wavelength-specific IRF yields the total energy generated by algae that is available to melt proximal frozen water. The energy used for photosynthesis (~ 1.7%) was subtracted from the IRF for each scenario. We disregarded the effect of scattering by algal cells following Williamson et al.15.We estimated instantaneous radiative forcing (IRF), which is defined as the energy reemitted by particles as heat to their surroundings. Therefore, we calculated the total algal absorption of glacier ice algae and snow algal populations . To test for significant differences between sites we performed a one-way analysis of variance (ANOVA) and in case of a significant result (using p < 0.05), a Tukey Honest Significant Differences post hoc test was performed. Assumptions of normality and homoscedasticity of residuals were tested using Shapiro–Wilk normality tests and Levene’s test. Data were transformed if the assumptions were not met (log-transformation for count data and sqrt-transformation for right-skewed data). For correlations tests, the Spearman correlation coefficient was generally used and the Pearson correlation coefficient for log-transformed data.Sanguina nivaloides) had an average cell volume of 3963 ± 675 µm3 and cell numbers ranged from 0.04 to 0.55 × 104 cells ml−1, contributing on average 74% to the community biomass (1.2 ± 0.9 µg C ml−1). The bare ice algal community on Mittivakkat (MIT3-6) was dominated by glacier ice algae, A. nordenskiöeldii and A. alaskanum, in almost equal proportions of both species with average cell volumes of 2307 ± 421 and 822 ± 133 µm3, respectively. The total glacier ice algal abundance ranged from 1.9 to 7.5 × 104 cells ml−1 and their biomass from 1.71 to 12.97 µg C ml−1. Overall, total algal abundance increased with decreasing altitude along the transect: glacier ice algae on the bare ice by a factor of 2 (2.7 to 5.5 × 104 cells ml−1 from 415 to 274 m), and snow algae on the snow sites by a factor of 15 (0.03 to 0.5 × 104 cells ml−1 from 451 to 445 m). Notably, snow algal cell volumes increased from 7130 to 20,101 µm3 on the bare ice transect , so snow algae still made up on average 44% of algal biomass on the bare ice. On Heim Glacier, the average glacier ice algal abundance was lower than on the bare ice sites on Mittivakkat (on average 1.6 × 104 ± 1.4 × 104 compared to 4.5 × 104 ± 1.9 × 104 cells ml−1) and snow algae contributed only up to 9% to the community biomass. Even though snow was still present on Bruckner Glacier, glacier ice algae dominated the algal community with numbers ranging from 1.5 to 3.0 × 104 cells ml−1 representing on average 72% of the community biomass. Snow algal abundance only ranged between 0.02 and 0.4 × 104 cells ml−1. While A. alaskanum was the dominant glacier ice algal species on Heim Glacier and Bruckner Glacier, A. nordenskiöldii was dominant on Mittivakkat.We sampled replicated snow and bare ice-dominated sites on three glaciers Fig.  and studv/Fm; 0.22 ± 0.08 vs. 0.35 ± 0.06, p < 0.001, n = 12) and photosynthetic activity  were lower in the community collected from the snow compared to the bare ice  in the snow community compared to the bare ice : rETRmax 44 vs. 72 ± 22 µmol é m−2 s−1; Ek 105 vs. 532 ± 153 µmol PAR m−2 s−1. Moreover, Fv/Fm of the bare ice community on Mittivakkat was significantly higher than that of the Heim Glacier community  . LC-HRMS/MS-screening confirmed the presence of both purpurogallin-carboxylic-acid and purpurogallin-carboxylic-acid-glycopyranoside (Supplementary Methods), with the latter present in higher quantities18. Due to their similar λmax, we summarise both as purpurogallin hereafter. The community pigment pool was primarily dominated by purpurogallin as well as astaxanthin esters. Their concentrations were significantly positively correlated with the biomass of their respective algal hosts . The chl a concentrations were also significantly positively correlated with biomass . Other pigments were only found in low concentrations.The pigment compositions of the algal community are presented as % of the pigment pool and as molar ratios to chlorophyll a ratio was on average 20, 56 and 209 at Bruckner Glacier, the bare ice sites on Mittivakkat (average of MIT3-6) and Heim Glacier (average of HE1 and HE2), respectively. Cellular purpurogallin contents varied from 0.01 to 0.02 ng cell−1 within a 95% CI with an average concentration of 0.023 ng cell−1 and did not significantly differ between sites. The log-transformed purpurogallin content per cell showed a positive correlation with biovolume, though not significantly , while no purpurogallin was present on the snow sites on Mittivakkat (MIT1 + 2) Fig. . The purCis-astaxanthin monoesters were the dominant astaxanthin esters on the snow sites and were less abundant in bare ice snow algal communities, resulting in a lower trans to cis-astaxanthin ester ratio at those three sites. We also found traces of a phenolic compound in the hydrophilic extracts of the snow algal-dominated snow sites on Mittivakkat  and Heim Glacier, despite comparably low snow algal biomass. aCDOM 375 absorption is used as a proxy for CDOM concentration and ranged between 0.08 m−1 on the snow (average of MIT1 + 2) to 1.38 m−1 on the bare ice sites on Mittivakkat  and cis conformations  was lower than the expected pigment-derived absorption (λmax 4.07 × 10−9 m2 cell−1 and 8.71 × 10−13 m2 µm−3), resulting in a 95% lower total absorbed irradiance. Using the literature spectra for glacier ice algae including packaging . Their respective contribution to the total radiative forcing of the algal community was estimated depending on their biomass for each site  controlling the presence of snow algae on snow47 and ultimately also on bare ice upon snow melt. Likewise, glacier ice algae can also be found in surprisingly large numbers on snow32. On Bruckner Glacier, they contributed up to 80% of the total community biomass. Since glacier ice algae are not motile, atmospheric deposition of cells could have transported them onto the snow surface or the local conditions of the snow cover being only ~ 10 cm thick and having a frozen crust may have provided favourable growth conditions for glacier ice algae so that they became dominant over time.We observed widespread algal blooms on bare ice and snow surfaces of three different glaciers in SE-Greenland. Traditionally, glacier ice and snow algae are mostly studied separately because they dominate their respective habitats. However, our data show that community mixing occurs upon snowmelt, so that snow algae still contributed to 44% of the total algal biomass on the bare ice. This is in line with the study by Lutz et al.50 can impact algal cell accumulation already on a metre scale and drive local ecological niche formation. Note that due to the restricted accessibility of Heim and Bruckner Glaciers, the number of samples that could be collected there was limited and lower than on Mittivakkat, which could represent a potential caveat for the inter-site comparisons. Despite the high within-site variability, we could still observe large changes in algal abundance along the snow to bare ice transect on Mittivakkat. There, total algal cell numbers increased with decreasing altitude, reaching the highest average concentrations at 343 m elevation (MIT5). Meltwater drainage downslope51 has been shown to drive algal accumulation downstream on the GrIS54, hence likely accounting at least partly for higher cell numbers at sites of the lowest altitude in our study. An extended growth period due to an earlier snow retreat at sites of lower altitudes could additionally explain the higher algal numbers at the lower altitudes, considering the estimated ~ 13 days delay of snow retreat for an altitude difference of 113 m (MIT3-6)55.Overall, the intra-site variability in total algal abundance was higher  than the inter-site variability of the same glacier and between glaciers, despite the different growth conditions . Therefore, local mobilisation and redeposition processes associated with a dynamic weathering crust and supraglacial meltwater flowsv/Fm, rETRmax and α of the snow community (MIT1 + 2) suggest lower photosynthetic performances including a lower efficiency in utilising light compared to the bare ice community (MIT3 + 4). This may be attributed, at least partly, to the different physiological states of the algal groups: typical snow algae such as Chloromonas nivalis dominate the snow community in its dormant cyst stage during the melt season23 of which the Fv/Fm was shown to decrease throughout the summer56, whereas glacier ice algae have no dormant stage and can actively divide throughout the season57. Thus, during the late summer melt season, the snow communities may not need to invest heavily into their photosynthetic capacity, compared to glacier ice algae remaining in their vegetative stage. In addition, solar exposure at both landscape and micro-niche scales can also impact algal photophysiology. Since Mittivakkat faces more south than Heim Glacier  and lipophilic pigment pools32, our understanding of algal community pigmentation on glaciers where snow and glacier ice algae co-occur is limited.Blooms of glacier ice and snow algae darken the GrIS due to their intracellular accumulation of secondary pigmentsa mass ratios of our snow algal samples were on average higher (56) compared to previously documented ratios, e.g., 34 for cells from Svalbard in Müller et al.22 and 50 in Lutz et al.32 for cells from Mittivakkat. This could be due to seasonal pigment variability or composition of the snow algal community, where other species of snow algae that are less pigmented dilute the astaxanthin ester pool relative to chl a59. Note that we can’t exclude a potential impact on the obtained pigment concentrations by the melting of the ice and snow samples in the dark prior sample preservation. In addition, varying environmental conditions may account for some of the observed differences in pigment concentrations compared to other studies.We show that purpurogallin and astaxanthin esters dominated algal community pigment pools  and each pigment group served as a biomass indicator for its respective algal host. Due to the high intracellular concentrations of astaxanthin esters, snow algae had a proportionally large contribution to community pigment pools, despite their smaller contribution to biomass compared to glacier ice algae. The total astaxanthin ester:chl 61, empirical data for glacier ice algae are scarce19, restricting our understanding of their pigment regulation and consequently their ability to darken the ice surface. Several factors should be considered for assessing the active intracellular up-or down-concentration of the purpurogallin pool, of which we discuss the first three in more detail below: (i) duration of growth period/snow retreat19; (ii) photo-acclimation aiming to ensure efficient light capture while simultaneously avoiding damaging-effects of excessive solar radiation ; (iii) cell size and timing since cell division64; (iv) antimicrobial activity (as shown for benzotropolone derivatives65); and (v) adaptation to low temperature by generating heat for ice and snow melt26. We expected an increasing purpurogallin:chl a ratio with decreasing altitude, assuming that altitude is a proxy of time since snow retreat with a consequent higher exposure to irradiance at lower latitude. However, the highest purpurogallin:chl a ratio was found on Heim Glacier located at only 286 m of altitude, while it was ~ 11 × lower on Bruckner Glacier with the highest altitude of 942 m. Overall, the average cellular purpurogallin concentrations  were comparable to the bulk phenol measurements by Williamson et al.15 reporting an average of 0.041 ng cell−1, underlining that purpurogallin is the major phenolic compound18 and demonstrating similar pigment content between West and East Greenland glacier ice algae. Moreover, the cellular and biovolume normalised purpurogallin content did not show any correlation with photophysiological performances across glaciers, suggesting that there is no direct link between the two under in situ conditions. This matches the observation of Williamson et al.15 that glacier ice algae are photo-inhibited during summer and, hence, a downregulation of the purpurogallin stock would in fact increase their risk of photo-damage. Newly divided, smaller cells may have generally a lower photoprotective pigment content compared to older, larger cells as shown for other Zygnematophycean algae41 or phytoplankton64, which could also apply to glacier ice algae as shown by the increasing trend of cellular purpurogallin concentration and biovolume . Hence, purpurogallin could be an important constituent of the DOM pool on glacier surfaces.Cell leakage of pigments could also impact CDOM signaturesent Fig. , which wa ratios of glacier ice and snow algae outlined above and the different absorption properties of their individual pigments would result in higher energy absorption of snow compared to glacier ice algae. Although astaxanthin esters in trans and cis conformations absorb stronger than purpurogallin at mid-visible wavelengths where incoming irradiance peaks  can be lower compared to the potential absorption of the same amount of pigments in solution73. While all algal cells store the pigments intracellularly and, hence, “package” their pigments, the different strength of this effect in both algal groups is likely related to the generally stronger packaging in larger than smaller cells72 and spherical  compared to cylindrical  cells74. Thus, a combination of these factors likely explains the difference in magnitude and shape (flat versus spiky spectrum) between measured “packaged” and theoretically reconstructed algal absorption spectra75. For snow algae, variability in cellular light absorption can be expected between species, individual cells76 and physiological stages of the same species (green pigmented vegetative cells or red pigmented cysts)12. The absorption spectra obtained in our study are therefore likely most representative of red-pigmented snow algal cysts .We studied the snow and glacier ice algal pigment signatures in order to decipher their role in energy absorption during a late summer melt season. We hypothesised that the contrasting secondary pigments:chl 12 and even though their occurrence on bare ice has been reported54, their impact on bare ice darkening after snow retreat has not yet been studied. We demonstrate that within a mixed bare ice community, glacier ice algae remain the main energy absorbers and snow algae contribute only minimally (on average 4%) to the total radiative forcing driven by algae  but may locally be higher (up to 14%). At the glacier scale, this indicates a spatio-temporal effect of biological albedo reduction synchronous with the retreat of a seasonal snowpack: snow algae are the main albedo reducers on wet snowpacks and once melted, glacier ice algae become the dominant albedo reducer even while snow algal cells remain on the bare ice. Our results highlight the role of community mixing during the snow melting season for algal biomass accumulation and bare ice community energy absorption. Future research using in situ albedo measurements and pre-and post-melt algal biomass will help to elucidate the role of community composition for surface albedo reduction in more detail.Supplementary Information.


The Indian and Pacific Oceans surround the Maritime Continent (MC). Major modes of sea surface temperature variability in both oceans, including the Indian Ocean Dipole (IOD) and El Niño–Southern Oscillation (ENSO), can strongly affect precipitation on the MC. The prevalence of fires in the MC is closely associated with precipitation amount and terrestrial water storage in September and October. Precipitation and terrestrial water storage, which is a measurement of hydrological drought conditions, are significantly modulated by Indian Ocean Dipole (IOD) and El Niño events. We utilize long-term datasets to study the combined effects of ENSO and the IOD on MC precipitation during the past 100 years (1900–2019) and find that the reductions in MC precipitation and terrestrial water storage are more pronounced during years when El Niño and a positive phase of the IOD (pIOD) coincided. The combined negative effects are produced mainly through an enhanced reduction of upward motion over the MC. Coincident El Niño-pIOD events have occurred more frequently after 1965. However, climate models do not project a higher occurrence of coincident El Niño-pIOD events in a severely warming condition, implying that not the global warming but the natural variability might be the leading cause of this phenomenon. Farmers in the MC set fires to clear land for planting palm trees. Although they usually manage to clear lands safely, fires can get out of control in exceptionally dry years and cause catastrophe. In 2015, schools and offices in the MC faced closures and planes were unable to take off due to the toxic smog caused by severe wildfires. Studies have revealed that MC fires usually begin in the boreal summer and peak in September and October (SO)on5 Fig. . Due to 10. During its positive phase, the IOD  is characterized by positive SST anomalies in the western Indian Ocean and negative SST anomalies in the eastern Indian Ocean. Although the magnitude of SST anomalies associated with the IOD is typically smaller than those associated with El Niño events, the IOD is essential in modulating precipitation across the MC1. The pIOD events can affect the precipitation not only by inducing anomalous Walker subsidence but also by reducing water vapor transport into the MC from the Indian Ocean10. The consensus of previous studies is that both El Niño and the pIOD can reduce MC precipitation12, because their warm SST anomalies weaken the Pacific and Indian Ocean branch of the Walker circulation resulting in anomalous subsidence over the MC region. Previous studies have shown the combined role of the El Niño type and the IOD phase in affecting the fire activities over the MC regions from 1979 to 201611. In addition, in the past several decades, fire events have been increased dramatically associated with more frequent droughts. Whether such frequent droughts are related to the occurrence of both El Niño and pIOD events has not yet been studied.ENSO is the leading mode of sea surface temperature (SST) variability in the Pacific and is characterized by warm SST anomalies in the equatorial eastern-to-central Pacific during its El Niño phase. El Niño events typically develop during boreal summer and mature during boreal winter. The first leading mode of SST variability in the Indian Ocean is the basin-wide monopole, and the IOD is the second leading mode statistically13 and internally produced by the Indian Ocean–atmosphere coupling16, and the ENSO can also be forced by IOD17. Typically, an El Niño event forces a pIOD event during its developing year via the oceanic channel18 and the atmospheric bridge mechanism21. Model experiments suggested that approximately one-third of IOD events can be explained by ENSO variability while the other two-thirds are internally generated16. It has also been increasingly recognized that ENSO properties have changed in recent decades, possibly due to global warming or natural decadal variability24. Different types of ENSO, such as the Eastern Pacific and Central Pacific types27, produce different impacts on the Walker circulation28. Also, the degrees of drought caused by ENSO get more severe over the past decades29. The ENSO-IOD relationship may have changed as a result of the change in ENSO properties and/or the internal atmosphere–ocean coupling in the Indian Ocean. It is, therefore, unknown whether (1) coincident El Niño-pIOD events have occurred more or less frequently during recent decades, and (2) the combined effects of coincident El Niño-pIOD events are similar or very different from the effect of El Niño-only and pIOD-only events. In this study, we utilize long-term (more than 100 years) observational datasets and model results, mainly focusing on September and October that is the mature time of IOD event and the strongest fire seasons to address these two questions. Notice that the MC area is defined as the region from 10° S to 10° N and 90° E to 150° E in this study.It is known well that the IOD can be externally forced by ENSOFigure 5  analysis was applied to the SO precipitation for the period 1911 to 2015. The first EOF mode explains approximately one-third (34.2%) of the total variance in the precipitation  controlling the multi-decadal change of the relationship between ENSO and IOD, which will be critical for understanding the fire occurrence frequency in this region in the future. Except for the impacts of coincident El Niño-pIOD events, the recent 2019 drought is caused by an extreme pIOD-only event. The 2019 extreme pIOD-only event is associated with global warming39, implying the importance of future pIOD events.Previous studies showed that the relationship between ENSO and IOD has inter-decadal variations, and the strengths of ENSO are also highly correlated with IODion Fig. a. In facion Fig. . A serie40, which was constructed from ground observations, the 1° × 1° Hadley Centre Sea Ice and Sea Surface Temperature dataset version 1 (HadISST1)41, the 0.25° × 0.25° Global Fire Emissions Database version 4 (GFEDv4s)42 and the 1.25° × 1.25° Twentieth Century Reanalysis dataset43 from the European Centre for Medium-Range Weather Forecasts. We select the period 1900 to 2019 to examine precipitation and SST and the period 1900 to 2010 for the atmospheric circulation analysis. Another monthly gridded precipitation dataset from the Climatic Research Unit (CRU)44, and the fifth version of Extended Reconstructed Sea Surface Temperature (ERSSTv5)45 were also examined. The details of these reanalysis datasets are listed in Table 46, which was forced with atmospheric observations. The experiment period was 1900 to 2015, and simulations of the last 105 years—that is, from 1911 to 2015—were utilized. To explore whether global warming might have a substantial impact on precipitation variations in the MC in the future, we examined the results from 18 models  Full Data Monthly Analysis Version 202010 and Niño3.4 index47 to represent IOD and ENSO activities, respectively. A pIOD event was identified when the mean values of the IOD index exceed one standard deviation for the June-July–August (JJA) or September–October-November (SON) season. An ENSO event was identified when the mean value of Niño3.4 SST exceeds one standard deviation for the November–December-January season. When an El Niño event and a pIOD event occur during the same year, they are referred to as a coincident El Niño-pIOD event. Conversely, El Niño-only and pIOD-only events refer, respectively, to an El Niño event occurring without an IOD event and a pIOD event occurring without an ENSO event. Table Long-term linear trends and annual cycles were removed from the original datasets. We use the dipole mode index (DMI)Supplementary Information.


The gammacoronavirus avian infectious bronchitis virus (IBV) is a highly contagious respiratory pathogen of primary economic importance to the global poultry industry. Two IBV lineages (GI-11 and GI-16) have been widely circulating for decades in South America. GI-11 is endemic to South America, and the GI-16 is globally distributed. We obtained full-length IBV genomes from Argentine and Uruguayan farms using Illumina sequencing. Genomes of the GI-11 and GI-16 lineages from Argentina and Uruguay differ in part of the spike coding region. The remaining genome regions are similar to the Chinese and Italian strains of the GI-16 lineage that emerged in Asia or Europe in the 1970s. Our findings support that the indigenous GI-11 strains recombine extensively with the invasive GI-16 strains. During the recombination process, GI-11 acquired most of the sequences of the GI-16, retaining the original S1 sequence. GI-11 strains with recombinant genomes are circulating forms that underwent further local evolution. The current IBV scenario in South America includes the GI-16 lineage, recombinant GI-11 strains sharing high similarity with GI-16 outside S1, and Brazilian GI-11 strains with a divergent genomic background. There is also sporadic recombinant in the GI-11 and GI-16 lineages among vaccine and field strains. Our findings exemplified the ability of IBV to generate emergent lineage by using the S gene in different genomic backgrounds. This unique example of recombinational microevolution underscores the genomic plasticity of IBV in South America. Nidovirales, family Coronaviridae) are enveloped, positive-sense single-stranded RNA viruses with the largest genome (26–32 kb) of all known RNA viruses so far [Coronavirus  has been the most studied avian coronavirus since its discovery in the 1930s . It causAvian infectious bronchitis must be controlled by extensive vaccination programs using attenuated and inactivated IBV strains that provide a high degree of cross-protection . HoweverLike other coronaviruses, IBV has a relatively large RNA genome (~27 kb) with well-characterized open reading frames (ORFs) coding for proteins involved in replication and transcription of the viral genome (nsp2-16) and the structure of the virion . The genome also contains ORFs that code for accessory proteins not directly required for virus viability in cell culture but interfere with cellular processes or modulate the infection process in the natural host .IBV exhibits an extraordinary degree of serological and genetic diversity ,14 that Genotyping of IBV has become a great aid in epidemiologic studies. It is commonly achieved by sequencing the coding region of S1, the highly variable subunit of the S protein. The increasing variability detected in IBV strains has led to a continuous re-evaluation and update of the nomenclature system. The most recent classification system based on S1 identifies six main genotypes (GI-GVI) and 32 viral lineages (1–32) .IBV variants are epidemiological diverse and appear unevenly distributed in different continents; some are widespread and have a global and substantial economic impact, while others are restricted and affect specific geographical areas .Two lineages of the GI genotype 11 and 16) have been widely circulating for decades in South America  have bee,20,21,22Despite differences in the S1 sequences, two single genome sequences obtained from a representative strain of the GI-11 and GI-16 lineages from Uruguay (South America) were surprisingly similar in the rest of their genome . This fiWe obtained 16 new full-length IBV genomes from South American outbreaks in Argentine and Uruguayan farms. Our comparative study with global strains contributes to understanding the epidemiology and genomic evolution of South American lineages and their connections with other continents. Our findings also showed the role of mutation and recombination in generating IBV lineages with new genome characteristics; the impact of these mechanisms is discussed in the context of the microevolution of other coronaviruses.A total of 16 Argentine and Uruguayan strains were isolated from IBV outbreaks during 2001–15 . Some stw/v) sucrose cushion. The resuspension pellets were purified with a continuous gradient of 30 to 55% (w/v) sucrose in PBS or were filtered using 0.45 µm membrane filters.The strains were propagated in 9–11 day-old embryonated chicken eggs by allantoic route inoculation to obtain many IBV virions. Then, 40 mL of allantoic fluid was harvested 72 h post-inoculation and then concentrated through a 20%  and 200 µL of purified viral particles. Reverse transcription and double-stranded synthesis were carried out using the Maxima H Minus Double-Stranded cDNA Synthesis kit  and 13 µL of extracted RNA. Nextera™ DNA Flex Library Preparation kit  was used from 1 ng of double-strand cDNA. The libraries were purified with AMPure XP  and quantified with the Qubit dsDNA HS assay kit . The library’s quality and length were assessed with the Agilent high-sensitivity DNA kit  using a Fragment Analyzer™ System . Sequencing was performed on the MiniSeq and MiSeq platforms .RNA extraction was performed using the Quick-RNAWe assembled IBV genomes by aligning raw Illumina reads to GI-11 and G-16 Uruguayan strains  using DNASTAR’s Lasergene Genomics Suite with the default workflow . Assemblies were visually inspected and manually optimized to obtain a single contig. Genome sequences obtained were aligned with MAFFT , and annThe sequences were deposited in the GenBank database; the accession numbers are given in A full-length genome dataset was built using 15 GI-11 sequences, 9 GI-16 sequences , and 29 Sequences were aligned using the MUSCLE algorithm implemented in MEGA-X . The besIdentifying potential recombinant and parental sequences and localization of possible recombinant breakpoints were performed using the RDP4 program, which implements seven distinct algorithms to characterize recombinant sequences .https://www.rstudio.com/ (accessed on 1 January 2020)).Maximum-likelihood trees, with approximate likelihood ratio tests for internal node support, were inferred using PhyML. Tree visualization was performed with the ggTree package in RStudio  matched with Uruguayan IBV strains (accession numbers: MF421319 and MF421320). The reads were assembled, and the genome sequences were obtained for all the strains. All genome sequences have 13 ORFs (1a-1b-S-3a-3b-E-M-4b-4c-5a-5b-N-6b) and 5′and 3′UTRs.The Illumina sequencing generated about 1 × 10Phylogenetic analysis of the S1 sequences was used for genotype classification. Twelves strains belong to the GI-11 lineage, and four strains to the GI-16 lineage .Genomes obtained here were compared with sequences already available for the GI-11 and GI-16 lineages. There were six additional genomes for the GI-11 lineage, one from Uruguay and five from Brazil. For the GI-16 genotype, there was one genome for Peru, one from Uruguay, two from Italy, and one from China .All the GI-11 and GI-16 have identical ORFs. Still, there are some differences in the coding sequences caused by small insertion/deletion of three or multiples of three nucleotides or alternative codons in the 3b, 3c, and 6b ORFs of Brazilian GI-11 and Chinese GI-16 strains.Sequence differences (low nucleotide identity) are mainly located in the 5′ of the nsp3 coding region (identity range: 83.0–100%) and the 3′-terminal end of the genome, particularly the ORF S (identity range: 84.2–99.1%) and accessory ORF 6b (identity range 78.0–100%). Variability in the ORF S is higher in the S1 coding region. The cleavage site is also variable. GI-11 strains have the cleavage site RXRR, where R can be F, S, or A. All GI-16 strains have RTGR, except the Chinese strains with an RMGR site.p-value lower than 2 × 10−13. Region 1 encompassed ORF 1a and ORF 1b, region 2 covered most of ORF S, and region 3 encompassed the last part of the S (450 nt), and ORFs 3a, 3b, E, M, 4b, 4c, 5a, 5b, and N  and an intergenic breakpoint . The strSequence analyses of S1 are extremely useful for identifying IBV genotypes and lineages and are the basis of current gold-standard classification. The complete genome sequence is a complementary tool to explore genome structure and function and infer virus evolution. It also provides additional information for tracking the virus spreading, performing comprehensive recombination analysis, and identifying genetic markers associated with pathogenicity and vaccine design. The increasing relevance of genome analysis has been recently exemplified by the massive efforts made by the scientific community that obtain hundreds of thousands of SARS-CoV-2 genomes only a few months after its first detection . MoleculHere, we used Illumina deep sequencing and bioinformatic analyses to assemble millions of sequence reads into sixteen completed genomes of IBV from Argentina and Uruguay to provide new insight into the microevolution of IBV in South America.IBV is quite variable in the coding region of the N-terminus of Nsp3, the most significant multi-domain protein produced by coronaviruses, and the GI-11 and GI-16 strains are no exception. Nsp3 is released from pp1a/1b by a protease domain part of Nsp3 itself. This protein has several roles in the viral life cycle and is essential for the replication and transcription complex associated with membrane vesicles . Nsp3a eWe detected reduced nucleotide identity in ORF6b between GI-11 and GI-16 strains. The ORF6b encodes a putative accessory protein that is not always annotated in the IBV genome. Several strains have variations in this region, including the appearance of stop codons that result in proteins of different sizes. This region might not express in all the strains, but the presence of transmembrane domains suggests that this region is functional in some cases .Argentine and Uruguayan genomes of GI-11 and GI-16 lineage differ basically in the ORF S, except for the last 450 nucleotides, which are relatively similar. Much of the genome of these strains of both lineages is closely related to the Chinese and Italian strains of the GI-16 lineage. Thus, the here obtained strains of GI-11 lineage acquired most of the backbone genome of European/Asian GI-16 lineage by recombination. The GI-11 Argentine and Uruguayan strains constitute a recombinant form that emerged once  during the IBV evolution in South America. This recombinant event is supported by the phylogenetic trees derived from all IBV ORFs . RecombiThe GI-16 lineage is one of the four most distributed lineages of IBV, together with GI-1 (Massachusetts), GI-13 793B), and GI-19 (QX) . GI-16 e93B, and The recombinogenic nature of the IBV genome has significant evolutionary consequences for coronavirus. It has been associated with the expansion of viral host ranges, the emergence of new variants, increases in pathogeneses and virulence, the alteration of tropisms, the immune escape, and resistance to antivirals ,52.The genomic plasticity of the GI-11 lineage is an explicit example of recombinational microevolution in IBV. It shows the ability of IBV to generate emergent lineage by combining the S gene with different genomic backgrounds. This ability is also associated with a notorious high mutation rate evidenced by the heterogeneity observed in some genome regions. In particular, the ORF 1a targets mutational pressure that suggests adaptive evolution. Our results show that the mechanism of S1 transfer by recombination and adaptive mutation has been relevant to generating genomes of successful lineages. It has also been proved that several coronaviruses use a similar macroevolution mechanism to host-shift in several species .In addition to observing the existence of adaptive evolution by ancestral recombination within IBV, we also identified several examples of unique recombination events . Most reRecombinant strains pose challenges to nomenclature and our ability to refer to these variants consistently. Following the nomenclature system described for HIV, recombinants that share identical mosaic structures  should be considered circulating recombinant forms (CRFs) if they are found in at least three different outbreaks . If a reSarbecovirus (betacoronavirus).Microevolution of IBV resembles general evolution trends among coronaviruses of different genera; variability in nsp3 and S protein, variation in cleavage sites in S, and recombination that includes most of the S region. In this sense, IBV is a prototype of avian pathogenic coronaviruses to analyze evolution in the subfamily. The information obtained in several years of evolutionary research can be used as a reference for studies on other coronaviruses, including SARS-related viruses of the subgenus The biological significance of the recombination detected in the present study remains to be resolved. Two points should be considered further. The first is the pathogenic potential of the recombinants . The second question is whether the current IBV vaccines could efficiently protect chickens from infection by such recombinants. Our results reinforce the need to expand full-length genomic studies in South America and worldwide to estimate the overall proportion of inter-genotype recombinant in IBV epidemiology.The current IBV classification scheme based on whole-length S1 sequencing proposed by Valastro et al. (2016)  was a si