Moreover the influence of chlorogenic acid on the inhibition of lipid peroxidation process in the hemp and milk thistle seed oils was also investigated. The tested oils showed different antioxidant properties which was related to the their different chemical composition. The main phenolic compounds present in hemp seed oil were vanillic, ferulic and p-coumaric acids, (-)epicatechin, catechin, kaempferol and procyanidin B2, whereas in milk thistle seed oil—catechins, procyanidin B2, procyanidin C1, p-coumaric acid, phloridzin, quercetin, protocatechuic acid, kaempferol, and syringic acid. The methanolic extracts of hemp and milk thistle seed oils showed the highest antiradical activity, whereas the ethanolic extracts revealed the best reducing properties. The obtained antioxidant parameters for hemp seed oil were: the IC50 = 3.433 ± 0.017 v/v (DPPH test), the percent of ABTS•+ inhibition = 93.301 ± 1.099%, FRAP value = 1063.883 ± 39.225 µmol Fe2+, CUPRAC value = 420.471 ± 1.765 µmol of Trolox. Whereas the antioxidant parameters for milk thistle seed oil were: the IC50 = 5.280 ± 0.584 v/v (DPPH test), 79.59 ± 3.763% (ABTS test), 2891.08 ± 270.044 µmol Fe2+ (FRAP test), 255.48 ± 26.169 µmol of Trolox (CUPRAC assay). Chlorogenic acid effectively inhibited the lipid peroxidation process in hemp and milk thistle seed oils. In the study the cold-pressed, natural  vegetable oils: hemp and milk thistle seed oils were tested for their chemical composition and antioxidant properties. The physico-chemical parameters, content of saturated and unsaturated fatty acids were determined. Solid phase extraction and simple extraction with the use of methanol, ethanol, 80% methanol, 80% ethanol were used to obtain the extracts for the analysis of antioxidant activity and phenolic compounds in oils. The composition of phenolic compounds was studied by means of high-performance liquid chromatography (HPLC–DAD) and spectrophotometric test with the Folin-Ciocalteu reagent. The antioxidant property of extracts was established by means of the following methods: with the DPPH Phenolic compounds belong to the main group of plant antioxidants, common components of plant-based diet, which are extensively studied as a chemopreventive compounds as well as potential drugs to treat the free radical-induced diseases, including cancer, diabetes, asthma, neuro-degenerative diseases, and skin aging2. The antioxidant defense mechanism of phenolic compounds involves among others the free radical scavenging activity, protection against lipid peroxidation or chelation of toxic metals3.Nowadays, there is an increased interest in antioxidants from plant sources. Because of their natural origin they are considered as non-toxic, safe for human and environment as well as biodegradable compared to the synthetic antioxidants. Moreover the extraction of antioxidants from plants does not require the use of large amounts of chemical reagents compared to expensive (and sometimes harmful) organic synthesis. Therefore the application of natural antioxidants is more environmentally friendly and more acceptable by consumers e.g. as food antioxidants, diet supplements or functional food ingredients4. Cold-pressed oils, unlike refined oils, retain higher amount of valuable substances in their composition, including phenolic compounds, phytosterols, tocopherols, carotenoids or polyunsaturated fatty acids. Moreover, the method of obtaining oils by cold pressing is simple, does not require high energy inputs and addition of chemicals which makes it an economical and green method. The cold-pressed oils can only be purified by means of water, sedimentation, filtration and centrifugation. On the other hand, the difficulty of this process is obtaining a product of consistent quality. An important factor limiting the wide use of cold pressing is the low efficiency of pressing and the difficulty in obtaining a product with constant parameters and high quality which depends on several factors. The quality of the oils is determined by the type of raw material and its variety, the degree of seed maturity, climatic conditions of cultivation, agrotechnical treatment, transport and storage conditions, grain moisture, cleanliness and no damage of the grains. Moreover, the oxidative stability of cold-pressed oils obtained from seeds is very often lower than their refined counterparts. This can be explained by the removal of pro-oxidizing chemicals during refining  what improves the oxidative stability of refined oils. For this reason, adding selected natural antioxidants to the cold-pressed oils can improve their quality. It should be remembered that the use of cold pressing alone, without further processing—refining, allows the final product to retain many valuable compounds including antioxidants The presence of high content of bioactive compounds in the cold-pressed oils gives them a chance to be called functional food as they have health benefits in addition to their nutritional properties5.Hemp (Cannabis sativa L.) from the Cannabis family (Cannabaceae) is a versatile annual herbaceous plant that has been cultivated for many years mainly in Central Asia. Hemp seeds contain about 20–25% protein, 20–30% carbohydrates, 25–35% oil and 10–15% insoluble fiber, wide range of minerals, nutrients and bioactive compounds including antioxidants, vitamins6. Hemp products strengthen the immune system, prevent diabetes, cardiovascular disease, and fight inflammation7. The chemical composition of hemp oil, including the high content of essential fatty acids, has led to the name of hemp oil as the best-balanced vegetable oil8. Hemp oil is used, among others, to treat atopic dermatitis, psoriasis, osteoporosis, PMS , menopause, inflammation of the bladder and bowel diseasesor stomach cramps, because its diastolic effect9. In addition, hemp seed oil shows weak antibacterial activity against Bacillus cereus, Bacillus subtilis subsp. subtilis, Micrococcus luteus, Staphylococcus aureus subsp. aureus (Gram+), Citrobacter freundii, Enterococcus faecalis, Escherichia coli, Salmonella enterica (Gram-) tested by the disc diffusion method10. Currently, hemp is one of the plant raw materials of great interest due to its versatile applications in the food, cosmetic, pharmaceuticaly, energy, textile, paper industries or as a building bio-material.The cold-pressed edible oil market is constantly expanding with new oils extracted from a wide range of plants. Some of the oils that are currently popular in the market are hemp oil, milk thistle oil, evening primrose oil, pumpkin seed oil, black cumin oil, or flaxseed oil. The global cold-pressed oils market is estimated to reach a value of US$ 36.40 billion by 2026, compared to a value of US$ 24.62 billion in 2018. This is attributed to the growing consumer interest in unprocessed food from natural sources, which is rich in antioxidant compoundsSilybum marianum L.) is an annual or biennial plant that belongs to the Asteraceae family. The plant is native to the Mediterranean region of Europe. Today, thistle has spread to other countries in Europe, Asia, Australia, North America, and South America11. Thistle fruits contain 30-35% oil in their composition13. The extract of milk thistle—silymarin—is a mixture of flavonolignans which is reach in silybin A, silybin B, silydianin, silycristine, isosylibin A, and isosylibin B. Sylibin is the component with high biological activity and accounts for 50-70% of silymarin15. Thistle is recommended primarily for dyspeptic ailments and liver diseases, including toxin-induced liver damage, cirrhosis, or in an adjunctive therapy for chronic inflammation19. The milk thistle oil is rich in bioactive compounds, such as phenolic acids, tocopherols, fatty acids and phytosterols21. The extracts from seed oil effectively prevent oxidative stress and restore normal levels of cholesterol, triglycerides, low-density lipoproteins, and liver markers associated with liver pathologies22. The thistle seed oil is a natural source of vitamin E and is often recommended as a beneficial edible oil17. Therefore it is used as an ingredient of dietary supplements, medicines, and cosmetics. Importantly, the health-promoting properties of oils depend on their chemical composition , which may vary from year to year and strongly dependents on plant variety, cultivation method, climate conditions and storage method23.Thistle . Furthermore, they established the Fe2+ chelating, antiradical activity against DPPH• radicals and ABTS•+ cation radicals as well as antioxidant potential in the ORAC assay. Callaway et al.27 reported that hemp oil has higher content of PUFA acids (82%) compared to olive oil (7%)27. Grajzer et al.28 reported content of PUFA acids in pumpkin oil (50.7%), milk thistle oil (58.4%), camelina oil (66.6%), flaxseed oil (68.8%), walnut oil (70.3%) and rosehip oil (74.6%)28. Milk thistle oil is a little-known oil yet (Table 2 kg of oil) obtained by the standard thinning method,the oxidative stability index, determined by the Rancimate method(55.7 h at 80°C and 12.9 h at 100°C),the contents of fatty acids , phytosterol (2520 mg/kg) and squalene (9.35 mg/kg) determined by gas-liquid chromatography (GLC),alpha-tocopherol content (237.4 mg/kg), total phenolics (1.16 GAE mg/g oil) determined by high-performance liquid chromatography (HPLC). The milk thistle oil possessed the scavenging capacity of the DPPH• radicals (IC50 = 3.34 mg/mL) and revealed the total antioxidant capacity (2.29 GAE mmol/L)19. The main fatty acids in milk thistle oil were linoleic acid (C18: 2) (57.0–60.3%) and oleic acid (C18: 1) (15.5–22.4%). Moreover, milk thistle oil was rich in α-tocopherol, and the phenolic content was 1.59–4.73 GAE mg/g, and three phenolic acids were identified . The above results indicate that milk thistle oil can be a source of natural antioxidants and is rich in PUFA and MUFA, and that the chemical composition of the oil changes with the change of the place of cultivation19.Study on the antioxidant activity and phenolic content of unrefined hemp seed oil are limited Table . Most of•  radical), ABTS•+  cation radical) as well as FRAP (ferric ion reducing antioxidant parameter) and CUPRAC (cupric ion reducing antioxidant capacity) assays. To this aim the oils were extracted by the use of methanol and ethanol by means of simple and solid phase extraction (SPE). The best way to extract antioxidants from oils was discussed.The aim of this study was to perform the quantitative and qualitative analysis of chemical composition  and antioxidant activity of virgin, unrefined and cold-pressed oils from hemp and milk thistle seeds manufactured in Poland and provided by the oil manufacturer. Four different spectrophotometric antioxidant assays were employed: DPPH3OH), ethanol (C2H5OH), n-hexane (C6H14), Folin-Ciocalteu reagent, deionized H2O, sodium carbonate (Na2CO3), acetate buffer, phosphate buffer pH=7, iron(III) chloride (FeCl3·6H2O), iron(II) chloride (FeCl2), copper(II) chloride (CuCl2), neocuproine , ammonium acetate (C2H7NO2), ammonium thiocyanate, potassium persulfate (K2S2O8), Tween 20 (C58H114O26), TPTZ , DPPH , ABTS , hydrochloric acid 37% (HCl), acetic acid (CH3COOH), chlorogenic acid, Trolox , o-coumaric acid, m-coumaric acid, p-coumaric acid, caffeic acid, ferulic acid, isoferulic acid, sinapic acid, chlorogenic acid, quercetin, epicatechin, phloridzin, L-ascorbic acid, BHA (butylated hydroxyanisole), BHT (butylated hydroxytoluene), acetonitrile for HPLC, standards of phenolic compounds: gallic acid, protocatechuic acid, procyanidin B1, 2,5-hydroxybenzoic acid, catechin, chlorogenic acid, vanillic acid, caffeic acid, syringic acid, procyanidin B2, (-)epicatechin, procyanidin C1, p-coumaric acid, ferulic acid, rutin, procyanidin A2, quercetin-3-glucoside, quercetin, kaempferol, phloridzin were bought from Sigma-Aldrich . Chemical reagents were of analytical purity. Additionally, the purity of these compounds was confirm by FT-IR spectroscopy and melting point measurement.The cold-pressed, virgin and unrefined oils from hemp and milk thistle seeds were obtained from Olejowe Smaki company . The seeds used to produce the oils came from Polish plantations. The use of plant materials in the present study complies with the international, national, and institutional guidelines and legislation. The seeds were gently crushed and then pressed in a screw press at a temperature not exceeding 40 ° C. The seeds were crushed only once. The oils have only been cleaned by sedimentation, not exposed to high temperatures, chemicals or water. No preservatives, additives or dyes were added. Methanol  were dissolved in 10 mL of methanol, filtered through membrane filters with a pore diameter of 0.45 µm, and then analyzed by liquid chromatography.The qualitative and quantitative chromatographic analyses of phenolic compounds present in extracts of hemp and milk thistle seed oils were performed on an Agilent 1260 Infinity liquid chromatograph with a UV spectrophotometric detector, using a Zorbax Eclipse Plus C18 Analytical precolumn column . A mixture of acetonitrile and acetic acid with a concentration of 2% and a mobile phase velocity of 1 mL/min was used for the gradient elution, as described in Kalinowska et al.36.The melting point, flash-point, density, total acid number, iodine value and saponification number were established according to the methodology described in37. The total content of phenolic compounds was expressed as gallic acid equivalent, i.e. mg GAE/g oil. Equation of the standard curve in ethanol: y= 0.008x + 0.0142; R2= 0.9941 and methanol: y= 0.0055x + 0.0014; R2= 0.9963. In order to determine the standard curves: 5 mg of gallic acid was weighed and dissolved in ethanol using a 25 mL flask. In this way, a solution with a concentration of 200 mg/L was obtained. Nine successive dilutions of gallic acid were made: 100 mg/L; 75 mg/L; 50 mg/L; 25 mg/L; 15 mg/L; 10 mg/L; 7 mg/L; 5 mg/L; 2.5 mg/L. Then the following reagents were mixed: 250 µL of gallic acid solution with a defined concentration; 250 µL of Folin-Ciocalteu reagent diluted 1:1 by volume with deionized water; 500 µL 14% sodium carbonate; 4 mL of deionized water. The test tubes were mixed and incubated for 1 h in dark at room temperature. Then the absorbance of the solutions was measured at the wavelength λ= 760 nm against a blank sample, where the solvent was used instead of gallic acid solution. In order to measure the TCA in oil, 250 µL of oil extract, 250 µL of Folin-Ciocalteu reagent, 500 µL 14% sodium carbonate and4 mL of deionized water were mixed. Samples were incubated for 1 hour at room temperature, protected from light. After this time, the absorbance was measured at the wavelength λ= 760 nm against the blank using UV/VIS/NIR Agilent Carry 5000 spectrophotometer . The experiment was performed in five replications for three independent experiments.TCA was determined using the method described by Singleton and Rosi38. The FRAP working solution was prepared in a 10:1:1 volume ratio by mixing 1000 mL of acetic buffer (300 mM), 100 mL of TPTZ  and 100 mL of FeCl3∙6H2O solution (20 mM). Then the FeSO4∙7H2O standard curve was determined. The concentration of the stock solution was 1.5 mM. Serial dilutions of the iron(II) sulfate solution were made: 0.5; 0.45; 0.4; 0.35; 0.3; 0.25; 0.2; 0.15; 0.1; 0.076; 0.05 mM. Then, 0.4 mL of the solution with a given concentration of FeSO4 and 3 mL of the FRAP solution were incubate in a dark place at room temperature for 8 minutes. After this time, the absorbance of the solutions was measured at a wavelength of 595 nm against a blank containing 0.4 mL of distilled water and 3 mL of FRAP solution ). The determination was performed in triplicate. Standard curve equation was: y= 0.0019x–0.0169; R2= 0.9973. In order to establish the reducing activity of oils, the samples of the extracts were diluted as follows: 0.1 mL of the oil extract; 0.3 mL of the solvent; 3 mL of FRAP solution. The samples were incubated for 8 min in the dark and then the absorbance was measured at wavelength λ= 595 nm against the blank (0.4 mL solvent and 3 mL FRAP solution). The antioxidant activity was expressed as Fe2+ equivalents [μM].FRAP activity was determined according to Rice-Evans et al.39. 250 mL of CuCl2  was mixed with 250 mL of ammonium acetate  and neocuproine . Then, 3 mL of the CUPRAC mixture was added to 0.5 mL of oil extract and 0.6 mL of distilled water. The samples were vortexed and incubated for 60 min at room temperature (23°C). The absorbance of the samples was measured with the use of UV/VIS/NIR Agilent Carry 5000 spectrophotometer  at λ= 450 nm against a blank (solvent instead of the sample). Cupric reducing antioxidant activity was estimated using the calibration curve obtained for Trolox (in the concentration range 50–350 µM). Standard curve equation: y= 1.7302x–0.0042; R2= 0.9988. Activity was expressed as Trolox equivalents [µM].The experiment was conducted according to method de-scribed by Apak et al.• in methanol was prepared. The oil extract, 2 mL DPPH• solution and solvent (methanol or ethanol) were added to the test tube, so that the volume percent concentration of the oil extract in the sample was in the range of 0.66–33.33% [v/v]. The test tubes were mixed using Vortex and kept in the dark for 1 hour. The absorbance at 517 nm was then measured against a blank and the activity was calculated from the formula: I%= [(A517 control–A517 sample)/A517 control] × 100 [%], where A517 control—the absorbance of control sample measured at 517 nm, A517 sample—the absorbance of test sample measured at 517 nm using UV/VIS/NIR Agilent Carry 5000 spectrophotometer  against methanol as blank. In the next step, the percent inhibition curves were plotted for the individual oil extracts and the IC50 values were calculated38.Briefly, a 60 µM solution of DPPH38. First, 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and potassium persulfate (K2S2O8) were dissolved in distilled water to a final concentration of 7 mM and 2.45 mM, respectively. Then, the solutions were mixed in volume ratio of 1:1 (v/v) and left for 16 h at room temperature (23°C) to produce ABTS cation radical (ABTS•+). After that, ABTS•+ was diluted with methanol to obtain a solution with an absorbance of ~0.700 at 734 nm. Then, 1 mL of the oil extract was added to 1 mL of the ABTS•+ solution, and after 7 min the absorbance was read at 734 nm wavelength against solvent with the use UV/VIS/NIR Agilent Carry 5000 spectrophotometer . The percentage of inhibition of the ABTS•+ cation radicals was estimated according to formula: %I = [(A734 control—A734 sample)/A734 control]×100 [%], where A734 control—the absorbance of control sample measured at 734 nm, A734 sample—the absorbance of test sample measured at 734 nm.The antiradical activity of oils was measured by the use of the ABTS method according to40.It was determined according to the method described in4SCN was weighed and dissolved in 50 mL of distilled water and 0.1988 g of FeCl2 in 50 mL of 3.5% HCl. Then a milk thistle or hemp seed oil emulsion was prepared by taking 2 mL of appropriate oil and 2 mL of Tween 20 and diluting with phosphate buffer to a volume of 50 mL. The concentration of chlorogenic acid in the trials was: 4.00, 0.40 and 0.04 mM. The samples consisted of: 1.5 mL of emulsion and 1mL of chlorogenic acid solution with successive concentrations. Samples were prepared in triplicate for each concentration of chlorogenic acid, screwed, mixed and left for incubation at 40°C. After 1 hour, the first measurement was made. For this purpose, 0.1 mL of solution from the incubated samples was taken, and 4.7 mL of 75% methanol and 0.05 mL of a 30% ammonium thiocyanate solution were added to the same test tubes. After 3 minutes the 0.05 mL of a 0.02 M solution of iron(II) chloride in hydrochloric acid was added. The absorbance at 500 nm was read immediately in relation to 75% methanol using UV/VIS/NIR Agilent Carry 5000 spectrophotometer . Measurements were made through the next 4 days every 24 hours. The degree inhibition of lipid peroxidation was calculated from the formula: %I= [(A500 control—A500 sample)/A500 control]×100 [%], where A500 control—the absorbance of control sample measured at 500 nm, A500 sample—the absorbance of test sample measured at 500 nm.In order to determine the lipid peroxidation, a 30% water solution of ammonium thiocyanate and 0.02 M solution of iron(II) chloride in 3.5% hydrochloric acid were prepared. In order to prepare the appropriate concentration of the solutions, 15 g of NHAll antioxidant assays were performed in five replications for three independent experiments. The results were expressed as the means of the values obtained for the replications. Average, standard deviation calculation, and graphs were completed using Microsoft Excel 2019.In Table oil to 1.476 ± 0.001 μg/goil. The highest content of phenolic compounds (1.476 ± 0.001 μg/goil) was obtained in the 80% methanolic extract epicatechin was determined there). On the other hand, the lowest content of phenolic compounds was in the ethanolic extract (0.303 ± 0.049 μg/goil). The use of 80% ethanol and methanol as a solvent made it possible to extract the largest number of various phenolic compounds in terms of the number. Methanolic extract (simple extraction) was abundant in kaempferol, vanillic and ferulic acids. 80% methanol is good solvent for (-)epicatechin extraction from hemp seed oil. Whereas the highest amount of procyanidin B2, catechin and p-coumaric acid was obtained in 80% ethanolic extract of hemp oil , vanillic acid (2.0 ± 0.10 µg/100 goil), p-coumaric acid (2.0 ± 0.15 µg/100 goil), ferulic acid (1.0 ± 0.08 µg/100 goil) and sinapic acid (3.0 ± 0.05 µg/100 goil)24. Moreover, Smeriglio et al.39, defined the presence in methanolic extract of cold-pressed Italy hemp seed oil some of phenolic acids, flavanones, flavonols, isoflavones, flavones and flavanols. In other study, the identified compounds in hemp seed oil were vanillic acid, catechin and epicatechin41. Only p-coumaric acid and vanillic acid have been identified in Tunisian milk thistle oil, and the amount of p-coumaric acid ranged from 0.26 mg to 0.9 mg/100 goil. Meddeb et al. found three phenolic compounds in silybine, vanillic and p-coumaric acids in Tunisian milk thistle seed oil. Vanillic acid was predominant, its concentration reached 40.2 and 83 mg/100 goil (depending on the origin of the oil)17. In other studies of Tunisian milk thistle seed oil, the following compounds were identified: homovanillic acid /kgoil), vanillin (2–4.40 mg QE/kgoil), p-coumaric acid (0.70 and 1 mg QE/kgoil), quercetin-3β-glucoside (0.8 and 1 mg QE/kgoil), quercetin (1.20–2.50 mg QE/kgoil), apigenin (0.9–1.8 mg QE/kgoil). The total content of polyphenols in the milk thistle oil was 12.8 mg equivalent quercetin/kgoil12.HPLC analysis showed that the content of phenolic compounds in the extracts differs in terms of qualitative and quantitative composition depending on the type of extraction and the solvent. In both cases, the extracts obtained by the simple extraction method showed a higher content of phenolic compounds than the extracts obtained by solid phase extraction (SPE). The total content of phenolic compounds in hemp seed oil varied from 0.303 ± 0.049 μg/goil to 3.271 ± 0.070 μg/goil. The highest content of phenolic compounds (3.271 ± 0.070 μg/goil) was obtained in the 80% methanolic extract. More phenolic compounds were identified in the thistle seed oil extracts compared with the hemp seed oil extracts. The milk thistle seed oil extracts were abundant in phloridzin. In the extract obtained by simple extraction with the use of 80% methanol the content of phloridzin was on the level of 1.755 ± 0.009 μg/g of extract, which constituted ~49% of the total content of phenolic compounds in the extract. The SPE technique gave the extract with 1.362 μg phloridzin per gram of extract, which was approx. 80% of the total phenolic compounds in this extract. The milk thistle seed oil was more abundant in catechin, procyanidins B2 and C2 compared with hemp seed oil. The literature data suggests that phenolic compounds identified in hemp and milk thistle seed oils  < 80% ethanolic extract (0.011 ± 0.0004 mg GAE/goil), 80% methanolic extract (0.014 ± 0.0008 mg GAE/goil) < ethanolic extract (0.047 ± 0.0018 mg GAE/goil) < methanolic extract (0.154 ± 0.0035 mg GAE/goil). In the case of milk thistle oil: 80% methanolic extract (0.008 ± 0.002 mg GAE/goil) < 80% ethanolic extract (0.026 ± 0.004 mg GAE/goil) < methanolic extract SPE (0.039 ± 0.002 mg GAE/goil) < ethanolic extract (0.088 ± 0.004 mg GAE/goil) < methanolic extract (0.144 ± 0.006 mg GAE/goil). However, in both oils, the highest total content of phenolic compounds was determined in the methanolic and ethanolic extracts obtained by the simple extraction technique. Only in the case of ethanolic, 80% ethanolic and SPE-methanolic extracts the higher content of phenolic compounds in milk thistle seed oil was estimated by means of Folin-Ciocalteu reagent. It was not the first time where the result of the spectrophotometric assay with Folin-Ciocalteu reagent did not agree with the content of phenolic compounds established by HPLC method35. Non-phenolic compounds such as some vitamins and their derivatives , nucleotide bases (e.g. guanine), amino acids  and simple inorganic ions  react as well with the Folin-Ciocalteu reagent45. Nevertheless the assay is commonly applied for the study of the total content of phenolic compounds in food products.The total content of phenolic compounds in oils, expressed as gallic acid equivalents per g of oil, was depicted in Figure oil46. For comparison, the total phenolic content determined for other oils was 1.48 mg CAE /100g in soybean oil, 1.31 mg CAE/100g in canola oil, 1.20 mg CAE/100g in sunflower oil, 1.26 mg CAE/100g in corn oil, and 1.48 mg CAE/100g in pumpkin oil24, which is much lower total TCA compared to studied in this work milk thistle and hemp seed oils In other study, cold-pressed sunflower oil (192.8 mg GAE/100goil) and rapeseed oil (155.9 mg GAE/100goil), extra virgin olive oil (342.1 mg GAE/100goil) possessed higher TCA than studied herein plant oils47.According to the literature data the total content of polyphenols in cold-pressed hemp seed oil from Finola and Fedora cultivar ranged from 0.02 to 2.67 mg GAE/100g• and the cation radical ABTS•+ is measured spectrophotometrically. The reducing activity of oil measured in FRAP (ferric ion reducing antioxidant parameter) and CUPRAC (cupric ion reducing antioxidant capacity) test rely on SET mechanism. These tests base on the ability of the antioxidant to reduce iron Fe(III) or Cu(II) ions. It is important to use different tests to study the antioxidant capacity of compounds due to their different antioxidant mechanism which depends on the type of radical, pH value, type of solvent, time of measurement, redox potential. The result of the antioxidant assays were gathered in Table The antioxidant activity of oil extracts can be carried out by several tests that rely on different antioxidant mechanism, e.g. DPPH, ABTS, FRAP, CUPRAC tests. The DPPH and ABTS methods determine the antiradical activity based on the mixed HAT (hydrogen atom transfer) and SET (single electron transfer) mechanisms. According to the both tests, the ability of hydrophilic or lipophilic antioxidants from the oils to reduce the stable radical DPPH50= 3.43 ± 0.02 v/v methanolic extract; IC50= 3.39 ± 0.04 v/v ethanolic extract) showed higher antioxidant activity in DPPH assay than milk thistle seed oil   revealed also higher activity against ABTS•+ compared to milk thistle seed oil . Generally, the methanolic extracts from the studied oils showed higher antiradical activity (in DPPH and ABTS assays) compared to the ethanolic and the other extracts. This is in accordance with the results obtained by the use of HPLC and spectrophotometric Folin-Ciocalteu analysis. The FRAP and CUPRAC assays showed that the both oils had the iron(III) and copper(II) reducing capacity. In this case the ethanolic extracts of the oils possessed higher reducing properties than the methanolic ones. Generally, the antioxidant activity of plant extracts correlates with the phenolic compound content48. But it should be bare in minds that not only the total amount of phenolics, but also other parameters including rate of reaction with radicals, pH of reaction, type of solvent, lipophilic character of antioxidant, redox potential, stability of phenoxyl radicals affect the antioxidant capacity of plant extract or chemicals as well.Hemp seed oil (IC50= 3.29 ± 0.29 µM), quercetin (IC50= 3.90 ± 0.01 µM), and epicatechin (IC50= 4.90 ± 0.20 µM) . The o-, m-, and p-coumaric acids showed the lowest antiradical activity (IC50 > 13360.55 µM). Phloridzin, which was found to be the major phenolic compound in milk thistle seed oil extracts, showed lower DPPH• radical scavenging properties (IC50 = 837.6 ± 29.40 µM) compared to caffeic acid or quercetin  had higher antioxidant capacity compared to cinnamic acids substituted by hydroxy and methoxy (–CH3O) group (i.e. sinapic and ferulic acids). Moreover sinapic acid which aromatic ring is substituted by two methoxy and one hydroxy group occurred better antioxidant compared to ferulic acid with one methoxy and one hydroxy group in the aromatic ring. The same trend was also reported in the study of Tan and Shahidi49, Karamać et al.50, and Kikuzaki et al.51. In the work of Shimoji et al.52, sinapic acid isolated from Kurosu (unpolished rice vinegar) also showed higher antiradical activity than ferulic acid . The presence of two hydroxyl (–OH) groups in the molecular structure of caffeic acid affects its higher antioxidant activity compared to the rest of the compounds from the above series. Phenolic compounds with a large number of –OH groups in their structures, especially 3’,4’-o-dihydroxy- groups  usually show greater efficacy in protecting LDL (low-density lipoprotein) from oxidation53. Sinapic acid has one more methoxy group (in the ortho- position) than ferulic acid. This group increases the electron transfer capacity, which increases the stability of the phenoxyl radical and its antioxidant activity54. Due to the presence of the –CH3O group placed in the position ortho- to the -OH group, ferulic acid shows stronger antioxidant properties than p-coumaric acid56. The results of our DPPH assay have shown that p-coumaric acid has a greater antiradical activity that o-coumaric acid. The studies carried out by Karamać et al.50, and Strazisar et al.57 showed the following increase in the inhibition of the DPPH• radicals: m-coumaric acid < o-coumaric acid < p-coumaric acid, which confirms the obtained in our work relationship. Ortho- and para- isomers, due to the electron-donating effect of the COOH-CH=CH- group on the aromatic ring, show resonance stabilization of the phenoxyl radicals, and thus have a higher antioxidant activity than meta- isomers58. Among all tested compounds, quercetin possessed the strongest FRAP (566.23 ± 19.47 µM Fe2+) and CUPRAC  antioxidant capacity. This activity is closely related to the molecular structure of this flavonoid, in particular to the presence of -OH groups in the 5 and 7 positions of the B ring, which are responsible for the reducing properties of quercetin 59. Significant reducing activity of Fe(III) ions was also demonstrated by epicatechin (267.80 ± 8.70 µM Fe2+), sinapic acid (254.02 ± 20.74 µM Fe2+), caffeic acid (243.70 ± 4.80 µM Fe2+), and chlorogenic acid (216.09 ± 4.15 µM Fe2+). According to these results, the total antioxidant capacity determined in FRAP assay follows the order: m-coumaric acid ≤ o-coumaric acid ≤ phloridzin (40.95 ± 6.29 µM Fe2+) ≤ p-coumaric acid < BHT ≤ BHA < L-ascorbic acid ≤ isoferulic acid < ferulic acid < chlorogenic acid < caffeic acid < sinapic acid ≤ epicatechin < quercetin. L-ascorbic acid and studied isomers of coumaric acid showed the lowest antioxidant properties in the CUPRAC assay. The cuprac reducing antioxidant power of the studied substances can be arranged in the following series: L-ascorbic acid < p-coumaric acid ≤ m-coumaric acid ≤ o-coumaric acid ≤ BHT ≤ ferulic acid ≤ isoferulic acid ≤ BHA < sinapic acid < caffeic acid < chlorogenic acid < quercetin. The results of the ABTS assay demonstrated that all tested compounds showed significant scavenging activity against ABTS•+. The percentage of inhibition of ABTS•+ cation radicals ranged from 51.93 to 91.44%.The antioxidant activity of selected individual phenolic compounds (including phenolics found in hemp and milk thistle seed oils)was measured by DPPH, FRAP, CUPRAC, and ABTS tests. We chose on purpose the series of hydroxy- and methoxycinnamic acids and their derivatives, as well as natural (L-ascorbic acid) and synthetic antioxidants  applied in food industry to check whether the natural products are better antioxidants compared with the synthetic ones and to study the dependency between chemical structure of phenolics and their antioxidant potential. The studied compounds were commercially purchased chemicals . The obtained results were gathered in Table Milk thistle seed oil contained higher amount of saturated fatty acids 17–20%) compared with hemp seed oil (9–10%) . It is speculated that chlorogenic acid may play a key role in the regulation of lipid metabolism and thus help in the treatment of cardiovascular disease and obesity63. Therefore, addition of 5-CQA to the oil would not only slow down its oxidation (it would act as an antioxidant), and spoilage, but also would be a valuable pro-health food additive, making the food functional. The obtained results pointed that 5-CQA inhibited lipid oxidation and therefore the oil spoilage  on the lipid peroxidation. 5-CQA was added to hemp and milk thistle seed oils in various concentrations. The 5-CQA was chosen because of its high antioxidant potential (on the basis of the above described studies), good solubility in polar and non-polar solvent and natural origin• radicals and ABTS•+ cation radicals. The hemp seed oil was more abundant in unsaturated acids compared to milk thistle seed oil epicatechin and catechin, and contained vanillic and ferulic acids which were not detected in the extract obtained from milk thistle seed oil. Milk thistle seed oil was abundant in phloridzin, catechin and procyanidin C1, and additionally contained quercetin, protocatechuic and syringic acids—not determined in hemp seed oil. Phloridzin, epicatechin and quercetin possessed high antioxidant properties compared to hydroxy- and methoxycinnamic acids found in the studied oils. The higher content of phenolic compounds in milk thistle seed oil compared to hemp seed oil determined by the use of HPLC method not correlated with the antioxidant potential determined by the use of ABTS and DPPH assay. Hemp seed oil showed higher antioxidant potential determined by the use of these two spectrophotometric methods probably caused by the participation of unsaturated acids in quenching the DPPHl Figure .Figure 7Hemp and milk thistle seed oils were rich in phenolic antioxidants and unsaturated fatty acids which are sensitive to oxidation. The addition of chlorogenic acid to these oils inhibited the oil peroxidation process, extending its service life.


A Li3P–Li2S metastable solid solution was identified, with the phasesadopting the fluorite (Li2S) structure with P substitutingfor S and the extra Li+ ions occupying the octahedral voidsand contributing to the ionic transport. The analysis of the experimentaldata is supported by extensive quantum-chemical calculations of bothstructural stability, diffusivity, and activation barriers for Li+ transport. The new solid electrolytes show Li-ion conductivitiesin the range of established materials, while their composition guaranteesthermodynamic stability toward lithium metal anodes.All-solid-state batteries based on non-combustible solidelectrolytesare promising candidates for safe energy storage systems. In addition,they offer the opportunity to utilize metallic lithium as an anode.However, it has proven to be a challenge to design an electrolytethat combines high ionic conductivity and processability with thermodynamicstability toward lithium. Herein, we report a new highly conductingsolid solution that offers a route to overcome these challenges. TheLi–P–S ternary was first explored via a combinationof high-throughput crystal structure predictions and solid-state synthesis of the most promising compositions, specifically,phases within the Li In addition, dendrite-free platingof lithium metal remains a problem.Key goals for designing next-generationenergy storage systemsinclude improving energy density, safety, reliability, and cost. Lithium-ionbatteries (LIBs) are among the most promising storage systems dueto their high energy densities and voltages.8 One class involvesoxides such as cation-substituted Li7La3Zr2O12 (LLZO)-derived compounds, which feature highionic conductivity, kinetic stability toward lithium metal, and canbe handled in ambient atmosphere.9 However,due to their mechanical stiffness, they are prone to cracking, mechanicalinstability, and are not resilient toward lithium dendrite formation,particularly at high current densities.7A wide range of promisingSEs have emerged and attracted considerableand widespread interest.10 Their superior conductivity arises from thereplacement of oxide anions with larger, more polarizable sulfideanions, leading to decreased Li-ion jump barriers.11 Extremely high ionic conductivities similar to those measuredfor liquid electrolytes were found in materials containing thiophosphatepolyhedra as key building blocks. While initial work focused on glassesand crystalline phases within the pseudo-binary (Li2S)x–(P2S5)x1–,13 doped crystallinederivatives are among the most conductive SEs to date, including Li10GeP2S12 ,14 Lix6–PSx5–ClBrx (σ = 24 mS cm–1),15 and Li9.54Si1.74P1.44S11.7Cl0.3 (σ = 25 mS cm–1).16A second promising class of materials contains sulfide ions.Althoughmoisture and air sensitivity make their use in practical applicationschallenging, their extremely high ionic conductivity and stress-accommodationhas motivated activity world-wide to identify new materials.3– possessingeven larger polarizabilities than sulfide anions. These phases arebased on anionic TtP4 tetrahedra with Tt = Al, Si, Ge,Ga, or Sn and show conductivities of up to 3 mS cm–1 for Li9AlP4 at room temperature.23 Their large numbers of charge carriers and low density make themattractive potential SEs, albeit being sensitive to oxygen and moisture.More recently,lithium-rich ternary phosphides were introducedas SE candidates, with the phosphide anions P27 For example, thiophosphates degradeto mixtures of lithium sulfides and phosphides through reduction ofphosphorus ions from formal oxidation states of +5 to −3 inLi3P. The resulting lithium binaries are mediocre electrolytes at best and thus form a conductivity bottleneckat the SE–lithium metal interface. Furthermore, other involvedcations might eventually lead to short circuiting due to reductionto lithium intermetallics. For example, Ge4+ present inLGPS28 and phosphidogermanates can resultin the formation of electronically conductive germanides,27 which pose the risk of propagated decomposition and short circuitsvia the formation of mixed ionic-electronic conducting interphases.28Despite numerous advantages, the introduced SE materials sufferfrom a unifying drawback; they are inherently thermodynamically unstabletoward lithium metal, as demonstrated for metal/metalloid-containingoxides, sulfides, and thiophosphates.+-ion conductive SE thatis thermodynamically stable toward lithium metal, both the structuraland thermodynamic characteristics and the method used to synthesizethe material have to be considered. Many transport processes in solid-statematerials are enabled by the presence of defects, the various processesbeing generally described by vacancy, interstitial, and collectivemechanisms.29 Aside from introducing vacanciesor interstitial carrier sites by doping aliovalent lattice ions, asynthetic approach that introduces a manifold of general defects,such as ball milling, may lead to inherently high conductive electrolytesfor two reasons. First, amorphization and partial reorganization duringsubsequent annealing allows control of the desired level of crystallinity.Consequently, many sulfide SEs are synthesized via ball milling ofLi2S and sulfides such as P2S5, B2S3, and GeS2, often followed by a subsequentheat treatment to increase crystallinity. Second, low-temperatureapproaches such as ball milling can also allow the formation of metastableelectrolytes with superior ionic conductivity compared to their moreordered thermodynamically stable counterparts, as observed for thematerials discussed in this paper. Phase formation can occur eitherdirectly during the milling process or at low subsequent annealingtemperatures that allow only partial reorganization of the latticeions.In searching for a highly Li32 Moreover, DFT is oftenused jointly with crystal structure prediction (CSP) approaches, includingstochastic , evolutionary, and particle-swarm optimization methods in the in silico discovery of novel functionalmaterials.34 Even though the latter toolswere used to predict chemically-doped thiophosphide electrolytes , solid-state nuclear magnetic resonance (ssNMR) spectroscopyand relaxometry, and potentiostatic electrochemical impedance spectroscopy(PEIS). DFT calculations combined with structure search based on AIRSSand structure model configuration enumeration (CE) were first utilizedto explore the Li–P–S phase diagram and then to validateproposed model structures and study material properties of the newLi–P–S phases.Here, weshow that we can combine stability with high conductivityby introducing forced disorder into lithium (pseudo)-binaries: high-energyball milling of mixtures of Lip(O2)/p0 < 1 ppm, p(H2O)/p0 < 1 ppm] or VAC glovebox .All handling of the materialswas conducted in an argon-filledUNIlab (M. Braun) . The well-defined maxima indicate a homogenous structurewith defined diffusion pathways, in contrast to other mechanochemicallysynthesized SEs that can feature broad maxima extending over up to100 °C.75 Due to the resolved maxima, aBPP-type T1ρ relaxation expression,which is dependent on the spectral densities at 2ω1, ω0, and 2ω0 (see Supporting Information),76 can be used to extract not only the activation energy but also theArrhenius-type pre-exponential factor τc,0–1, defining τc–1 viakB denotesBoltzmann’s constant, and T is the temperature. Thecorresponding fits are shown as solid line parabolas, as shown in x = 0.50, 0.67, and 0.75, respectively (Supporting Information Table SA5). The similarity betweenthe activation energies measured for the two different timescales(T1ρ versus T1 measurements) suggests jumps between connected sites, contributingto long-range diffusion instead of just a localized motion such ashopping between a finite number of nearby sites. To estimate Li-ionmobility at finite temperatures, we calculate values for the Li-ionjump rate, τc–1, at 25 °C, leading to 2.8, 4.1, and 3.1 ±0.5 × 105 s–1 for samples with x = 0.50, 0.67, and 0.75, respectively. The highest τc–1 and thelowest activation energy are found for the x = 0.67composition. In the model used here, a slightly lower decrease of R1ρ at the low temperature flank was accountedfor by using a stretched exponential correlation function, indicatingcorrelated motion. This may be attributed to a vacancy diffusion mechanismand/or structural disorder introduced by ball milling, which can cause(apparent) correlation effects such that the data can no longer befit using a single exponential NMR correlation function.78Maxima of the temperature-dependent relaxation rateswere observedfor all samples when measuring the relaxation times in the rotatingframe, corresponding to a timescale in the order of 31 kHz [= ωR1, R2, Q2, and a2) and ionic conductivity, and the values of the parametersobtained from the fit are listed in Supporting Information Table SA3. A better fit was achieved using a constantphase element Q , which describes an imperfect capacitor;this is tentatively attributed to the inherent disorder in the bulkor porosity, leading to electrode surface heterogeneity. The totalconductivity ,79 and Li3P,which was verified under identical experimental conditions . We note that reported conductivities of lithium binariesor ternaries can range over 1–4 orders of magnitude.79 The ionic conductivity of Li3P isstill under debate, with reported values ranging from around 10–4 to 10–8 S cm–1 (see below),80 requiring further investigationon the origin of this discrepancy.Since NMR relaxometryinherently measures more local ion movements, we also used VT EISto probe the Li ion mobility further. The bulk conductivity of eachof the SE was studied using Li|SE|Li symmetric cells. Room temperatureimpedance plots of all four samples are shown in x = 0.5 are shown in Supporting Information Figure SA9 with impedance parameters for all the samples at differenttemperatures tabulated in Supporting Information Table SA3. The activation energies Ea derived from the Arrhenius plots are in the range of 0.2–0.15eV .Representative VT EIS plots for temperatures from100 °C toRT and RT to −50 °C for the sample with x = 0.67 composition was initially chosen (yielding the “Li8P2S” stoichiometry), given the computationalfeasibility  of the structural supercellgenerated with this composition. For Model 1 1), a 3 × 3 × 3 cell is needed to preserve the molar P/S= 2:1 ratio (2(P0.5S0.25□0.25)1), a2 × 2 × 2 cell can account for the Li/S = 8:1 ratio  are seen. Thus, vacancies in the anionlattice will likely yield phases that are significantly more energeticallyunfavorable, residing well above the hull, disfavoring structure Model2. The presence of additional Li ions in the octahedral voids in Model1 leads to only very minor changes in the intensities of the reflectionsin the XRD pattern, compared to the parent structure Li2S .Giventhat the original structure search had not included solid solutionsof the two end members of the tie line, new structure searches wererequired so as to compare the experiment and theory. The :1 ratio 4b1, wher:1 ratio 4b2. The Supporting Information Figure SB9), structures following Model 1 are predictedto exhibit a wide band gap (Eg ≥1.6 eV), while structures following Model 2 are predicted to be metallic.Metallic conductivity is not supported by the yellow to red colorof the synthesized samples.From the electronic density of states  occupying the tetrahedral and octahedral sites, andthe vacancies allowing Li ion hopping. This assignment is supportedby known isostructural phosphide ternaries exhibiting a ccp P3– lattice with cations occupying 8c sites completely and 4b sites partially.66Thus, based on the combined findings of X-ray andneutron diffraction,NMR experimental data, and quantum-chemical calculations, we proposean anti-fluorite-based structure model with a fully occupied mixedS2S–Li3P pseudo-binary phasediagram, containing the low-lying configurations within 250 meV/atomfrom the hull , a property desired for a battery electrolyte . Considering the knowntendency of DFT to underestimate the band gaps, we expect the synthesizedcompounds to have even larger band gaps.The electronic conductivity in the new ternaries wereagain analyzedby computing the electronic band structures and DOS. Each ternaryshows a wide, direct band gap (2S and Li3P (Supporting Information Table SB2), it can be predicted thatthe defect disorder stabilizes the ternaries by 1.2–10.5 meV/atomat 298 K. Finally, as shown for the samples with x = 0.67 , the ternary structures correspond to local minima  on thecorresponding potential energy surfaces, and they will thus be metastableover a finite temperature range.All ternary compoundsare metastable at 0 K, against the parentmaterials, Liand Li3P 2. Howeve3P in the solidsolution  provide a good match with the corresponding experimentaldata.The DFT-optimized lattice parametersshow an increase in the unitcell volume with increasing amount of Lisolution 3,resembsolution 3. The XR7Li and 31PNMR spectra to the experimental data . The calculated 31P spectra resemble theexperimental ones and, of note, predict the increased linewidths forincreasing phosphorus content, indicating the wider distribution ofchemical environments of the phosphide ions. The predicted changeof the center of gravity of the shift distribution with increasingphosphorus content is not, however, found in the experimental spectra,possibly because the calculations do not account for the mobilityof the Li sublattice, which may affect the average local environment“seen” by the 31P nuclei.To validate the predicted Li–P–S ternarystructuresfurther, we compare the computed tal data 13. The cEa) were computedusing AIMD simulations and compared the results to well-known ternariesin the Li–P–S system with high conductivity, for example, Li7P3S11 and Li3PS4. The results are compiled in Supporting Information Table SB3 and Supporting Information Figure SB14 and reveal a reasonable agreement between our Ea valuescomputed for the reference materials  and the previous computational and experimental reports.While the values converge at higher phosphorus contents, the AIMD-derivedactivation energies are higher than those found by NMR and EIS  and Li3P . The constituents withhighest Li3P contents, that is, “Li8P2S” (x = 0.67) and “Li11P3S” (x = 0.75), display the highesttotal conductivities and lowest activation energies, comparable tothose of the known superionic conductors Li7P3S11 (Ea = 160 meV) and β-Li3PS4 (Ea = 337 meV)(see Supporting Information Table SB3 fordirect comparisons). The experimentally determined Li ion conductivityof Li3P (1.81 × 10–7 S cm–1) is similar to the recently reported conductivity of 3.0 ×10–8 S cm–1 at 50 °C. However,this study also reported a much higher activation energy of 0.53 eV.62 By contrast, in an early study of Li3P conductivities of 6.6 × 10–4 S cm–1 at close to room temperature were reported, now with a much loweractivation energy of 0.18 eV that is close to the value measured inour work.80 The large discrepancies betweenthe two studies suggest that defects and/or non-stoichiometry mayenhance the transport properties within the Li3P structuretype. Consistent with this, AIMD simulations of the stoichiometricphase give an even higher activation barrier of approximately 700meV with such low Li+ conductivities that higher valuescould only be estimated above 500 K. These studies motivate furtherstudies of samples with the composition beyond x =0.75 and on the role of non-stoichiometry on the transport propertiesof Li3P.The presented solid solutions all show enhancedLi ion conductioncompared to the endmembers Li2S material) even at low temperaturescan be ascribed to a knock-on mechanism, facilitated by the interstitialLi defects, as demonstrated for similar solid ion-conducting systems.86 As determined for other lithium phosphide ternaries with ccp phosphidelattices,23 lithium hopping is expected to occur via face-sharingoctahedral and tetrahedral sites. For higher x, increasedlattice parameters lower the hopping activation barrier, and phosphideanions stabilize higher energy octahedral sites through Coulomb interactions.The enhanced conductivity in the new Li–P–Sternaries(compared to the parent LixLi3P–(1 – x)Li2S solid solution, have been reported in this work. Upon high-energyball-milling of the two binary compounds, a solid solution of theLi2S anti-fluorite structure type is formed within thecompositional range of 0.39 ≤ x ≤ 0.75,which features a disordered anion lattice with tetrahedral and octahedralvoids  filled by mobile Li ions. These materials combinehigh conductivity with inherent redox stability toward Li metal andcan thus be regarded as potential SEs for Li metal batteries.The synthesis, structural elucidation,and ionic conductivity measurements,supported by quantum-chemical calculations, of a new 3P solubility in Li2S can occur to form phases that are close to the thermodynamicground state; these phases may be accessible under electrochemicalconditions where metastable phases are often formed if they are morekinetically accessible. Furthermore, since Li2S and Li3P are known to form as degradation products at the interphasebetween Li metal and P- and S-containing SEs, these solid solutionsmay form as part of the SEI/degradation process, with implicationsfor the conductivity of the SEI.87 Theformation of these phases has particular importance when evaluatingpotential degradation products of different Li–P–S ternariesand quaternaries involving other metal ions  inthe various solid-state conductors used for LIBs. Indeed, 31P NMR spectra exhibiting similar chemical shifts and line shape wererecently measured for interphase products between the SE Li7SiPS8 and Li metal, indicating that the metastable solidsolutions reported here might intrinsically arise at thiophosphate–Liinterfaces.88 Finally, we note that Li2S–Li3P intergrowths, as identified via high-throughputCSPs, suggest potential intergrowths or grain boundaries between theLi2S and Li3P phases that should be consideredwhen analyzing the highly heterogeneous SEIs that form in these systems.The work illustrates that Li3P–Li2S solid solutions, either as part of a composite structureor when formed electrochemically. We anticipate that this class ofmaterials with a fully reduced anion lattice opens a new pathway towardstable, high-capacity solid-state batteries based on lithium metalanodes.Future work on the solid solutions will investigate the electrochemicalreactions at the interface of Li metal, decomposition at high potentials,and the overall performances of these solid solutions in SSBs. Whilefurther studies are needed to explore this, the conductivity bottleneckand contact issues between sulfide SEs and lithium metal anodes maypotentially be reduced by using the Li


The results showed that grain yield increased with increasing K under the same N level, and top-dressing K further increased the grain yield. K application increased starch content significantly at N180 and decreased significantly at N225, while the protein content increased significantly at the two N levels. The grain starch content with the K75 + 75 treatment increased by 5.8% and 9.0% compared with K150 at the two N levels, and the protein content decreased by 2.9% and 4.7%. Application of K increased the retrogradation enthalpy (ΔHret) and retrogradation percentage (%R) at N180. At N225, the ΔHret and %R of K75 and K150 decreased, while those of K75 + 75 increased. The ΔHret and %R under K75 + 75 at N180 were lower than N225. Under these experiment conditions, 75 kg ha−1 K2O at sowing date and top-dressed 75 kg ha−1 K2O at jointing stage (V6) under the conditions of appropriate N reduction could not only effectively improve the pasting and thermal properties of waxy maize flour, but also stabilized the grain yield.Reasonable fertilization of nitrogen (N) and potassium (K) had significant effects on maize growth. In this experiment, two N levels (N180 and N225 kg ha Waxy maize is a special crop, characterized by a unique structure of starch, which creates enormous possibilities for its application . Among tHgel), ΔHret and %R [To), FV and setback viscosity (SB) of rice flour, but decreased the PV and BD [Nitrogen (N) and potassium (K) are essential nutrients for crop growth and development. N participates in plant physiological processes as important components such as enzymes, hormones and amino acids . K is bet and %R ,14. N fet and %R . K fertit and %R . Additiot and %R . The suiV and BD . StudiesV and BD . ExcessiV and BD . ApproprV and BD . K enhanV and BD .Since the 20th century, breeding and industry of waxy maize has developed rapidly in China, and its planting area has exceeded 800,000 ha , which hTherefore, the objectives of this study were: (1) to investigate the interactive effects of N and K on grain component content, yield and its components of waxy maize; (2) to investigate the interactive effects of N and K on the pasting property and thermal property of waxy maize flour; and (3) to determine the appropriate application of N and K fertilizer in the production of spring-sown waxy maize in Southern China.There were significant differences in kernel number of ears, 1000-kernel weight and grain yield of waxy maize between K, year (Y), and the interaction (K × Y). N, K and Y have significant effects on grain yield, and Y has the largest effect. The interaction (N × K) and the interaction (N × K × Y) have little or no significant effect on grain yield . AccordiThe trends of 1000-kernel weight and grain yield were identical at the two N levels, and showed K75 + 75 > K150 > K75 > K0. At the same K level, 1000-kernel weight and grain yield in N180 were significantly lower than in N225. N225K75 and N180K150 significantly increased the kernel number of ears and grain yield with additional N or K fertilizer compared with N180K75. N225K150 significantly increased the 1000-kernel weight and grain yield with additional N and K fertilizer compared with N180K75. Compared with K150, the kernel number of ears, 1000-kernel weight and grain yield were further increased by K75 + 75, but the difference in kernel number of ears was not significant.There were significant differences in starch and soluble sugar contents between N, K, Y, the interaction (N × K), the interaction (K × Y) and the interaction (N × K × Y), while K, the interaction (N × K), the interaction (N × Y), the interaction (K × Y) and the interaction (N × K × Y) have significant effects on protein content . N had nAccording to Compared with N180K75, the starch and protein content of N225K75 and N180K150 significantly decreased and soluble sugar content increased. Compared with N180K75, the starch content of N225K150 significantly decreased and soluble sugar content increased, and there was no significant difference in protein content. Under the two N levels, starch content of K75 + 75 increased by 5.8% and 9.0%, and protein content decreased by 2.9% and 4.7% compared with K150.The size distribution of starch granules showed a typical bimodal pattern, and the variation of the effect of different N–K interactions on starch size distribution in 2020 was sharper than that in 2021 , which mPtemp) between fertilization treatments were less affected by N–K interactions . At the same K level, the pasting viscosity under N180 was lower than N225.Interactions of N and K had significant effects on the flour pasting properties of waxy maize . PV betwractions . The PV,Compared with N180K75, N225K75 increased the PV, TV, BD and FV of waxy maize flour and decreased the SB with additional N fertilizer; N180K150 decreased the PV, TV, BD and FV, and increased the SB with additional K fertilizer; N225K150 decreased PV, TV, BD and FV, and increased SB with additional N and K fertilizer.Hgel, and gelatinization temperature  between K treatments, no significant differences in ΔHret between N treatments, and significant differences in %R between the interaction (N × K). Overall, the ΔHgel and gelatinization temperature  in 2020 were higher than those in 2021, but the ΔHret and %R were lower. At N180 level, the ΔHgel decreased in K75 and K150 compared with K0, and the differences between treatments of To, Tp and Tc were not significant, while the ΔHret and %R were increased. At the N225 level, compared with K0, the ΔHgel, To and Tp were increased in K75, and the ΔHret and %R were decreased; in the K150 treatment, the ΔHgel, ΔHret and %R was decreased, while the To and Tp were increased.According to Hgel of N225K75 and N180K150 treatments increased, and the ΔHret and %R decreased. Compared with N180K75, ΔHgel of N225K150 increased, and ΔHret and %R significantly decreased. In comparison with N180K150, N180K75 + 75 showed significantly lower ΔHgel, and higher ΔHret and %R. In comparison with N225K150, ΔHgel, ΔHret and %R of N225K75 + 75 significantly increased.Compared with N180K75, the ΔPtemp, ΔHgel, gelatinization temperature  and ΔHret. There was a negative correlation between soluble sugar content, SB, Ptemp, ΔHgel, and gelatinization temperature. Protein content was negatively correlated with PV and BD, and positively correlated with Ptemp and gelatinization temperature. The starch volume percentage of particle size > 15 μm has a significant positive correlation with the mean particle size. The PV, TV, BD and FV have a significant positive correlation between pairwise comparisons. The gelatinization temperature had a significant positive correlation between pairwise comparisons.Correlation analysis  showed tPrevious studies have shown that reasonable N, P and K fertilization could significantly improve crop yield . In termReasonable fertilization is one of the most effective measures to improve maize quality . CompareHgel, and significant effects of year and treatment on ΔHret and %R. In most situations, combination of N and K fertilizers reduced the ΔHgel and increased the ΔHret and %R. Under the two N levels, the application of K fertilizer with K75 + 75 increased the ΔHret and %R of waxy maize flour compared with K150, indicating that K supplementation at V6 promoted the regeneration of waxy maize flour. Previous studies in wheat have reported that larger starch granules inhibited the gelatinization of starch, thereby increasing the gelatinization temperature [Starch granule size and distribution were influenced by years, growing areas and environment . In thisperature . Our res−1, 0.86 g kg−1, 66.3 mg kg−1, 6.98 mg kg−1 and 67.25 mg kg−1 in 2020, and 13.08 g kg−1, 1.01 g kg−1, 58.97 mg kg−1, 6.06 mg kg−1 and 60.49 mg kg−1 in 2021.The field experiment was conducted at Jiangxinsha farm  in Nantong City, Jiangsu Province, China, in 2020 and 2021. Suyunuo5 (SYN5) and Suyunuo11 (SYN11) were used as experimental materials. SYN5 is the control waxy maize variety of the regional test in southern China. SYN11 is widely planted in Jiangsu Province. The tested soil was sandy loam, and the soil nutrient content prior to sowing was as follows: average contents of organic matter, total N, alkali hydrolysable N, available P, and exchangeable K in the plow layer (0–20 cm) were 10.05 g kg−1 (N225). The four K treatments were 0 (K0), 75 (K75), 150 (K150) and 75 + 75 kg K2O ha−1 (K75 + 75). All treatments had a basal application of 75 kg N ha−1 and 75 kg P2O5 ha−1 at sowing date. N180 and N225 treatments were top-dressed 105 and 150 kg N ha−1 at V6. K75 and K150 treatments were applied 75 and 150 kg K2O ha−1 at sowing date. K75 + 75 treatment was applied 75 kg K2O ha−1 at sowing date and V6, respectively. Urea (46% N), superphosphate (12% P2O5) and potassium chloride (60% K2O) were used as the N, P and K fertilizer sources.A split-plot randomized complete block design (RCBD) was used, with two N fertilizer levels (main plots) and four K fertilizer treatments (sub-plots). The two N levels were 180 (N180) and 225 kg N ha2 (3.6 m × 12.0 m), and the planting density was 60,000 plant ha−1 with double row planting . The maize was sown on April 2 and harvested on August 5 in 2020, with a total growth period of 126 days. In 2021, it was sown on April 6 and harvested on August 8, with a total growth period of 125 days. The meteorological conditions of the field experiment were shown in Each plot was 43.2 mAt maturity stages (R6), 30 ears were harvested from the middle row of each treatment using a continuous sampling method to determine the yield (standard water content of 14%) and its components.Grain starch and soluble sugar content were measured by the anthrone–sulfuric acid method ; grain N3 solution for 48 h at room temperature, and the starch was isolated following the method described in previous study [Three uniform growth ears were selected for each treatment, and the stripped grains were mixed thoroughly. Then, 200 g of grains were randomly selected for the preparation of starch and flour samples. The grains (100 g) were steeped in 500 mL of 1 g/L NaHSOus study . The graThe starch granule size and distribution were determined using a laser diffraction particle size analyzer  with anhydrous ethanol as the dispersion medium, following the previously reported method of Yang et al. . The meaw/w, dry basis) was equilibrated at 50 °C for 1 min, heated to 95 °C at 12 °C/min, maintained at 95 °C for 2.5 min, cooled to 50 °C at 12 °C/min, and then maintained at 50 °C for 1 min. The paddle speed was set at 960 rpm for the first 10 s, and then decreased to 160 rpm for the rest of the analysis.According to the method of Yang et al. , the pasTo), peak of gelatinization temperature (Tp), conclusion temperature (Tc) and the enthalpy of gelatinization (ΔHgel). After conducting thermal analysis, the samples were stored at 4 °C for 7 days for retrogradation studies. The sample pans containing the starches were reheated at the rate of 10 °C /min from 20 to 100 °C after 7 days to measure retrogradation. The enthalpies of retrogradation (ΔHret) were evaluated automatically and the retrogradation percentage (%R) was calculated as ΔHret/ΔHgel × 100.A differential scanning calorimeter (DSC 200 F3 Maia) from NETZSCH (Germany) was used to determine the thermal properties of the flour. Each sample  was loaded into an aluminum pan  and distilled water was added to achieve a flour–water suspension containing 66.7% water. Samples were hermetically sealed and allowed to stand for 24 h at 48 °C before heating in the DSC. The DSC analyzer was calibrated using an empty aluminum pan as a reference. Sample pans were heated at a rate of 10 °C/min from 20 to 100 °C. Thermal transitions of flour samples were defined as onset temperature , and the obtained coefficients were tested by t-test for significance levels of 0.05% and 0.01%. Figures were generated with OriginPro .Data were analyzed for analysis of variance (ANOVA) with SPSS . The data were homogeneity-tested prior to ANOVA. The homogeneity of the outputs was satisfied for running further ANOVA. Differences in grain yield and quality, as affected by N fertilizer, K fertilizer, year and their interactions, were examined using a three-factor model of ANOVA. The three-factor variance model of ANOVA was used in −1 K2O at sowing date and top-dressed 75 kg ha−1 K2O at V6 could not only effectively improve the pasting and thermal properties of waxy maize flour, but also stabilize the grain yield under the conditions of appropriate N reduction. Therefore, we recommend optimized K application (75 kg ha−1 at both sowing date and V6) and moderately reduced N application (from 225 to 180 kg ha−1) in the production of spring-sown waxy maize in Southern China.Appropriate application of N and K fertilizers could improve the yield and its components of waxy maize, 75 kg ha


Since the beginning of the Russia-Ukrainian war, more than 50,000 people have come to Israel from Ukraine as “repatriates” (able to acquire citizenship) or “tourists” , about 30% are older adults, for whom forced migration poses serious challenges. The aim of this presentation is to describe the socio-demographic characteristics of older migrants’ fragmented families, who turn to one of the Volunteer Help Center in Northern Israel. Descriptive statistics on older adults from the database of about 900 families who received support during April-June 2022 were performed. Data revealed: 189 migrants aged 55+ (M(SD)=69.7(8.2); range 55–89); 148 (78.3%) were female; 106 (56%) had Israeli citizenship or were in the process of receiving it; 75 (40 %) were “tourists”. Among “tourists”, about half (Nf30) came to visit their adult children. “Repatriates” and “tourists” did not differ in age (F(159)=1.574, p=ns). As a rule, older adults came with family members (Nf136(72.7%)). However among “tourists” 41.9% came alone and only 17.1% among “repatriates” . Older adults were mostly accompanied by daughters (Nf80(58.8%)), usually without husbands, in 127 fragmented families (78.2%) were children under 18. Only 15% came with a spouse , and five came with mothers. This preliminary data draws attention to the unique family fragmented structure of older displaced people, as this should be considered when developing an assistance program.


Saccharomyces cerevisiae, the 20S and 23S RNA viruses are well-studied members of the narnaviridiae, which are present at low intracellular copy numbers, unless induced by stress or unfavorable growth conditions, and are not known to affect host fitness. In this study, we describe a new S. cerevisiae narnavirus that we designate as N1199. We show that N1199 is uniquely present as a double-stranded RNA at a high level relative to other known members of this family in 1 strain background, YJM1199, and is present as a single-stranded RNA at lower levels in 98 of the remaining 100-genomes strains. Furthermore, we see a strong association between the presence of high level N1199 and host phenotype defects, including greatly reduced sporulation efficiency and growth on multiple carbon sources. Finally, we describe associations between N1199 abundance and host phenotype defects, including autophagy.RNA viruses are a widespread, biologically diverse group that includes the narnaviridiae, a family of unencapsidated RNA viruses containing a single ORF that encodes an RNA-dependent RNA polymerase. In the yeast  Saccharomyces cerevisiae is a host for RNA viruses. S. cerevisiae RNA viruses are vertically transmitted, cytoplasmic genetic elements, which can be divided into 2 families—Totiviridiae  and Narnaviridiae  of N1199 in the other 100-genomes strains, demonstrated cytoplasmic inheritance, and characterized the effects of high copy number N1199 on host phenotypes. Our work demonstrates that a narnavirus, when present in high copy number, can significantly alter host fitness. Our key findings and their implications are described and discussed below.Previous surveys of the 100-genomes strains collection revealed extensive natural variation in their nuclear, mitochondrial, and 2-µm genomes .The n et al. , and Vijnd SV343 , purifieyeast extract, 2% bacto peptone) contained 2% dextrose and 2% ethanol (added after autoclaving), respectively. Similarly, SD and SE (0.67% yeast nitrogen base) contained 2% dextrose and 2% ethanol (added after autoclaving), respectively. YPD, YPE, SD, and SE plates contained 2% agar. Phenotypic analysis was performed on subsets of strains by 10-fold spot dilutions onto 100 mm diameter plates. Media containing other carbon sources at a 2% final concentration were prepared as above. For autophagy induction, cultures were grown in liquid YPD containing 0.1–250 nM rapamycin  for 2−3 days, and subsequently streaked to YPD plates to allow recovery. Single colonies from the YPD plate were subsequently analyzed for further assays as described below and in the text. For galactose induction experiments, transformants were grown on YP +2% galactose and 100 μg/mL nourseothricin for 2 days and subsequently plated to YPD. Single colonies from YPD plates were analyzed for further experiments as described in the text. The same schematic was followed for analyzing strains transformed with either the test plasmid or an empty vector.YPD and YPE (1% 2EDTA (pH 7.5). Cell pellets were then resuspended in 1 mL of 50 mM Tris-H2SO4 (pH 9.3)/1% 2-mercaptoethanol solution and incubated for 15 min at room temperature. Cells were subsequently collected by centrifugation and resuspended in 1 mL of 0.1 M NaCl/10 mM Tris–HCl (pH 7.5)/10 mM Na2EDTA/0.2% sodium dodecyl sulfate solution. 0.7 mL of phenol (pH 8.0) was added, and samples were incubated on a shaking platform for 1 h at room temperature, after which 0.7 mL of the aqueous phase was recovered by 5-min centrifugation. Nucleic acids were precipitated by the addition of 70 µL of 3 M potassium acetate and 0.7 mL of ice-cold isopropanol, followed by incubation for 5 min at room temperature and centrifugation at 14,000 rpm for 10 min. The precipitated nucleic acids were washed with 70% ethanol, dried using a SpeedVac (Eppendorf), dissolved in 70–100 µL of water, and stored at −80°.Total nucleic acids were extracted using a slight modification of the phenol-mediated method described by , in accordance with the manufacturer's protocol, using 5 µL aliquots of nucleic acid samples as a template. PCR reactions were performed with OneTaq DNA polymerase (NEB) in accordance with the manufacturer's protocol. The primers used to detect the presence and, where present, their PCR genotypes, are listed in For gel electrophoresis, 5 μL aliquots of total nucleic acid samples (to detect abundant RNA viruses) or RT-PCR-derived nucleic acid samples (to detect PCR products of sequenced RNA viruses) were mixed with 1 × gel-loading buffer and loaded on 1.3% agarose gels prestained with GreenGlo SAFE DNA dye (Denville) and/or ethidium bromide (Bio-Rad) according to the manufacturer's instructions. Electrophoresis was carried out in 1 × TAE buffer at room temperature at a constant voltage of 6 V/cm for 45–60 min. Gels were subsequently imaged under UV light using the Alpha Innotech Red gel documentation system.f, RNaseH, or ShortCut RNaseIII  at 37°C and samples were analyzed on 1.2% TAE-agarose gel stained with ethidium bromide. For subsequent analyses, treated samples were column purified using standard PCR purification kits (Qiagen), reverse transcribed with Maxima first-strand cDNA synthesis kit (Thermo), and subjected to PCR to test for the presence/absence of various genomic and viral elements, as described above. PCR samples were analyzed on 2% TBE (Tris-borate EDTA) gels at 4–6 V/cm for 75–90 min in prechilled 0.5 × TBE buffer.Nucleic acid samples were treated with DNaseI, RNaseISequences were aligned with MUSCLE (v3.8.31) configured for the highest accuracy (MUSCLE with default settings). The phylogenetic tree was reconstructed using the maximum likelihood method implemented in the PhyML program (v3.1/3.0 aLRT). The WAG substitution model was selected assuming an estimated proportion of invariant sites (of 0.002) and 4 gamma-distributed rate categories to account for rate heterogeneity across sites. The gamma shape parameter was estimated directly from the data (gamma = 1.211). Reliability for internal branch was assessed using the aLRT test (SH-Like). Graphical representation and edition of the phylogenetic tree were performed with TreeDyn (v198.3) .Full-length sequence of the N1199 dsRNA from YJM1199 was determined by various modifications to the previously described FLAC (full-length amplification of cDNA) method (The oligo-ligated dsRNA was subsequently purified using the NucleoSpin Gel and PCR Clean-up kit (Machery-Nagel) and denatured at 99° for 3 min in the presence of 1 M betaine (Sigma) and 3% DMSO. cDNA synthesis was carried out using ∼ 300 ng denatured oligo-ligated RNA with M-MuLV reverse transcriptase  for 5 min at 25° followed by 60 min at 42° and 20 min at 65°. RNA removal was performed by adding 5U RNaseH (NEB) directly to the cDNA reaction, followed by incubation at 37° for 25 min and 95° for 5 min. The primary cDNA strands were annealed without further purification by incubating the above mix at 95° for 4 min, and by a gradual lowering of temperature to 80° at the rate of 1°/min, 50° at the rate of 3°/min and finally to 4° at 0.1°/min.in silico using the SnapGene software . DNA and protein sequence homologies were determined using NCBI Blast (https://blast.ncbi.nlm.nih.gov/Blast.cgi).PCR amplification of the cDNA was carried out using Phusion DNA polymerase with PC2 primer (5′-CCGAATTCCCGGGATCC-3′) per the manufacturer's protocol, with a single additional 2 min incubation step at 72° immediately following initial denaturation. PCR products were analyzed on a 1%TAE-agarose gel. The 2.6 kb PCR amplicon from the previous step was phosphorylated with polynucleotide kinase and blunt-ligated into pAG25  lineariz2O, and subsequently with PSB  and SB . Cells were treated with 10 mg/mL Zymolyase 20T in 1 mL SB at 30°C with rotation until spheroplasts were observed. Spheroplasts were harvested at 2000×g for 5 min at 4°C, washed twice with SB, and resuspended in 500μL EBX . To lyse the plasma membrane, the suspension was treated with 0.5% Triton X-100 and incubated on ice with gentle mixing for 10 min, after which a small aliquot of the lysate was removed for gel analysis and the remainder layered over 1 mL NIB  and centrifuged at 12,000×g for 15 min at 4°C. The upper layer containing the cytoplasmic fraction was stored for gel analysis, while the pellet containing the nuclear fraction was resuspended in 500μL EBX and treated with 1% Triton X-100 to lyse the nuclear membrane. After 10 min of incubation on ice, the sample was spun down as previously to yield a supernatant containing the nucleoplasm fraction and a chromatin-containing pellet. The pellet was washed with EBX 3 times and resuspended in 50 μL Tris pH 8.0. Each fraction was phenol:chloroform extracted to remove proteins and lipids followed by nucleic acid precipitation with 3 M potassium acetate and isopropanol. All fractions were analyzed on 1.3% TAE-agarose gels as described earlier.Cell fractionation was performed according to hi grown on YPD plates were scraped off and resuspended in water, generating 7 independent cell suspensions. Appropriately diluted aliquots of each suspension were plated on selective maltose-containing plates  and control dextrose-containing plates  and incubated for 2–3 days at 30°C. N1199 loss frequencies (f) were calculated as a ratio of Mal+ cells in a colony to the number of total viable cells in a colony. Using median f value and median total number of viable cells in a colony (Nt), N1199 loss rate per cell per generation (m) was calculated by Drake's equation: m = f/ln(Nt•µ)  . 95% nonated per  and tranρ0 by DAPI staining , plated to YPD, and then tested for petite phenotype on YPEG . Petites were confirmed as staining .7 cells/mL to SD, SD-N (lacking a nitrogen source), and SD + 100 nM rapamycin media and incubated with shaking at 30°C for 48 h. All cultures contained 100 μg/mL nourseothricin throughout the experiment for plasmid maintenance. 20 μg/mL phenylmethylsulfonyl fluoride  was added to cultures to prevent vacuolar degradation and increase the intensity of the GFP signal. Since ammonium sulfate can interfere with aminoglycoside antibiotics, L-glutamate  was instead used as a nitrogen source in SD cultures /GFP-AUT7(416) (Addgene plasmid #49425) into a pAG36 backbone linearized with NsiI-EcoRI digestion create an episomal vector with a NAT marker expressing GFP-Atg8 under the control of the endogenous Atg8 promoter. Transformants were selected on YPD containing 100 μg/mL nourseothricin. The assay was performed as described in www.graphpad.com). Data were plotted as the mean and standard deviation of 3 independent replicates. Statistical significance was analyzed by 2-way ANOVA correcting for multiple comparisons using statistical hypothesis testing (Sidak test). Multiplicity-adjusted P-values were reported for each comparison.Graphical data analysis was performed using GraphPad Prism version 8 for macOS, GraphPad software .By RT-PCR with multiple primer pairs, the homothallic diploid strain YJM1199 was found to be L-Aidentity . Among tf, which is specific for ssRNA, and found that both the putative 4.6 kb L-BC band and the 2.6 kb band were unaffected. In contrast, known ssRNA species including the ribosomal RNAs, as well as the single-stranded narnaviruses 20S and 23S, were depleted by RNaseIf treatment, as confirmed by agarose gel and RT-PCR analyses, respectively , none of the 99 remaining 100-genome strains exhibited a high abundance 2.6 kb gel band. Therefore, we used RT-PCR with 5 primer pairs (see low level (N1199lo); that is, N1199lo is not visible via total nucleic acid gel but is detectable by RT-PCR while N11990 is detectable by neither total nucleic acid gel nor by RT-PCR. BLAST analysis of the original genome sequences of the 100-genome strains showed no DNA sequences with similarity to N1199; thus, widespread genomic N1199 cDNA(s) were excluded as a source of N1199 PCR products. Based on the presence/absence of amplicons from the 5 primer pairs, which is hypothesized to be due to N1199 sequence variation, there were 8 RT-PCR N1199 genotypes . Instead, we tested the inheritance of N1199lo by crossing YSV698 (isogenic with YJM682 (L-A0 L-BC+ 20S+ 23S+ N1199lo)) with YSV706 (isogenic with YJM1463 (L-A+ L-BC+ 20S0 23S0 N11990)), sporulating the resulting diploid, and analyzing spore clones from independent tetrads  results in the rapid elimination of [PSI + ]  in all YJM1199 spore clones, both by agarose gel analysis and RT-PCR , trisaccharides , pentose sugars , and glycerol. In contrast, isogenic N11990 derivatives showed markedly improved growth  were grown to stationary phase in minimal media containing 0–250 nM rapamycin, followed by outgrowth on YPD. The resulting isolates were subsequently analyzed to monitor the effects on N1199 RNA abundance. We found that even subnanomolar concentrations of rapamycin-induced N1199 loss, with all the N1199-cured cultures having phenotypes like those produced in other experiments  pathway . In contrast, under conditions of nitrogen starvation, N1199hi cells displayed poor enrichment of the GFP signal compared to N11990 isolates , with substantial vacuolar fragmentation . Similar to previous reports  and 23S (14 strains) are found less frequently in the 100-genomes strains (manuscript in preparation). There are 4 competing hypotheses for the near-universal frequency of N1199 vs the much lower frequencies of 20S and 23S. First, N1199hi loss in YJM1199 is frequently accompanied by loss of 20S and/or 23S, which implies that host pathways required for maintenance of all 3 narnaviruses overlap, at least in part; autophagy is likely 1 such pathway. Second, 20S and 23S are present in low levels in YPD-grown cultures and the same is true for N1199 in 98 of the 100-genomes strains. However, in YPD-grown parental YJM1199, N1199 copy number is high while 20S and 23S copy numbers are low, which implies different mitotic growth copy number regulation of N1199 vs 20S and 23S, in at least 1 genetic background. Finally, while we did not examine N1199 copy number in sporulation media, 20S and 23S copy numbers increase in sporulation media. For example, a previous study showed that 20S, when present in strains, accumulated to high levels during sporulation; notably, the 20S0 strain Y55 did not accumulate a narnavirus-sized species during sporulation (0 strain Y55 is isogenic with YJM627 (N1199lo 20S0 23S0)  , 1 of thHO YJM1199 N1199hi and all HO YJM1199 segregants being N11990, we were not able to introduce N1199hi into N11990 strains. Thus, we were not able to determine whether the level of N1199 in the parental YJM1199 N1199hi was due to host genome sequences or N1199 genome sequences, or a combination of both. That said, the YJM1199 N1199hi vs YJM1199 N11990 phenotypes, such as the sporulation and carbon source utilization defects of YJM1199 N1199hi, clearly show that N1199hi is deleterious to both N1199 itself and to the YJM1199 host. Finally, as exemplified by these YJM1199 N1199hi results, RNA virus-dependent phenotypes, if not known or considered, have 2 implications. First, undetected RNA viruses may confound genotype association analyses. Second, undetected loss of RNA virus(es), which may occur during growth on different carbon sources  or drug treatments (such as rapamycin), may confound phenotypic analyses.Due to sequence quality near the ends of N1199, we were not confident in our ability to construct an N1199 launching vector. In addition, due to the sporulation defect of jkac337_Supplementary_DataClick here for additional data file.