Oryza sativa L., cv. Sakha 105) grown in salt-affected soil under three irrigation intervals , as well as soil properties and nutrient uptake under field conditions during the 2019 and 2020 seasons. Our findings showed that dual application of biochar and glycine betaine (biochar + glycine betaine) reduced the soil pH, electrical conductivity, and exchangeable sodium percentage. However, it enhanced the K+ uptake which increased in the leaves of treated-rice plants. Additionally, biochar and glycine betaine supplementation enhanced the photosynthetic pigments  and physiological attributes  of osmotic-stressed rice plants. Biochar + glycine betaine altered the activity of antioxidant-related enzymes . Moreover, it improved the yield components, biological yield, and harvest index, as well as the nutrient value of rice grains of osmotic-stressed rice plants. Collectively, these findings underline the potential application of biochar and glycine betaine as a sustainable eco-friendly strategy to improve plant resilience, not only rice, but other plant species in general and other cereal crops in particular, to abiotic stress, particularly those growing in salt-affected soil.Osmotic stress is a major physiologic dysfunction that alters the water movement across the cell membrane. Soil salinity and water stress are major causal factors of osmotic stress that severely affect agricultural productivity and sustainability. Herein, we suggested and evaluated the impact of integrated biochar-based soil amendment and exogenous glycine betaine application on the growth, physiology, productivity, grain quality, and osmotic stress tolerance of rice ( Oryza sativa L.) is the main imperative food crop that feeds most people all over the world [Rice  of rainfed lowland, and around eight million hm2 of upland rice exposed to drought stress [Concerning the current global climate change, drought stress became the most imperative and serious limiting factor for rice production in rainfed ecosystems . More tht stress ,14. Ricet stress ; howeverRecovering soil quality is an important issue for improving withholds water and nutrients via soil as well as rice growth and productivity particularly under environmental stressors including salt and drought stressors . Soil am+ accumulation, reduce Na+, and improve the activity of antioxidant enzymes, therefore improving photosynthesis and reducing oxidative damage [Various management practices including exogenous application of plant growth regulators, bio-stimulative compounds, or osmoprotectants can play a pivotal role in protecting plants from salt- and drought stresses that improve tolerance in plants . Glycinee damage . Additioe damage . HoweverThere are no reports about the dual application of biochar and glycine betaine, especially under soil salinity and water stress conditions together in the field. To contribute to this goal, in this study, we are supposed to determine the dual application of biochar and glycine betaine for mitigating soil salinity and water stress by using rice as a test plant. It is expected that the research findings will be effective for formulating novel management strategies for enhancing growth and rice productivity under soil salinity and water stress under field conditions. Moreover, the findings of this study might be beneficial to grow rice drought-affected areas, as well as upland rice areas worldwide.PIR < 0.0001) in both seasons, soil amendment using biochar and/or glycine betaine application significantly decreased all studied properties at all tested irrigation intervals (PTR < 0.0001) (PIR×TR = 0.0384) compared with non-treated control  (PIR×TR = 0.0352).The effect of exogenous application of biochar and glycine betaine on the soil physicochemical properties including pH, electrical conductivity (EC), and exchangeable sodium percentage (ESP) were studied in two separate field trials . Althoug 0.0001) . Brieflyctively) A. Intere−1, respectively; −1; −1, respectively) and 2020 seasons . Similarly, in 2019, ESP was significantly reduced due to the dual application of biochar and glycine betaine together when rice plants watered every 6, 9, or 12 days  compared with control , chlorophyll b (0.95 ± 0.03 and 1.02 ± 0.03 mg g−1 FW), and total carotenoids (0.75 ± 0.02 and 0.85 ± 0.03 mg g−1 FW) during the 2019 and 2020 seasons, respectively, followed by biochar alone, then glycine betaine. Although all photosynthetic pigments were significantly reduced at 12 days irrigation intervals, the dual application of biochar and glycine betaine had the highest levels of chlorophyll a (0.78 ± 0.05 and 0.68 ± 0.03 mg g−1 FW), chlorophyll b (0.29 ± 0.03 and 0.35 ± 0.03 mg g−1 FW), and total carotenoids (0.25 ± 0.04 and 0.33 ± 0.02 mg g−1 FW) during the 2019 and 2020 seasons, respectively.Although osmotic stress reduced the levels of photosynthetic pigments when the irrigation intervals increased from 6 days to 9 or 12 days, soil amendment using biochar and exogenous glycine betaine supplementation enhanced the levels of chlorophyll a A,B, chloa  of osmotic-stressed rice plants was diminished when the irrigation intervals increased from 6 days to 9 or 12 days  during the 2019 and 2020 seasons (2 m−2 s−1) compared with control plants (16.36 ± 0.38 and 17.25 ± 0.36 μmol CO2 m−2 s−1) during 2019 and 2020, respectively.In general, during both seasons, the net photosynthetic rate (Ps) was significantly reduced when the irrigation intervals increased from 6 days to 9 or 12 days , followed by biochar alone (48.69 ± 0.66 and 49.14 ± 1.03 mol m−2 s−1), and glycine betaine alone (46.47 ± 1.34 and 47.25 ± 0.51 mol m−2 s−1), whereas the control plants had the lowest stomatal conductance (42.25 ± 0.82 and 43.36 ± 0.61 mol m−2 s−1) during 2019 and 2020, respectively . The dual application of biochar and glycine betaine together enhanced the relative water content of rice leaves at 6 (92.98 ± 0.77 and 93.25 ± 1.23%), 9 (82.85 ± 1.65 and 83.45 ± 1.28) and 12 days irrigation intervals (73.44 ± 1.07 and 74.66 ± 0.86%) during the 2019 and 2020 seasons, respectively.The relative water content of rice leaves was decreased when the irrigation intervals increased from 6 days to 9 or 12 days , soil amendment using biochar and exogenous glycine betaine supplementation reduced the activity of CAT , 1000-grain weight (29.33 ± 0.40 and 29.76 ± 0.32 g), number of grains per panicle (132.92 ± 1.98 and 135.61 ± 0.81 grains per panicle), grain yield (8.22 ± 0.12 and 8.46 ± 0.10 tonnes/hectare), straw yield (14.76 ± 0.09 and 15.01 ± 0.15 tonnes/hectare), and harvest index (35.76 ± 0.23 and 36.04 ± 0.04%) when the rice plants were irrigated every 6 days during both seasons 2019 and 2020, respectively , biological yield (grain and straw yield), and harvest index.Generally, osmotic stress negatively affected the yield and its components including the number of panicles per m2 A,B, 10002 C,D, numb2 E,F, grai2 A,B, stra2 C,D, and 2 E,F. Neveectively . On the PIR < 0.0001), soil amendment using biochar and exogenous glycine betaine supplementation enhanced the nitrogen (PTR < 0.0001). Our findings showed that rice grains had the highest N (1.57 ± 0.01 and 1.56 ± 0.02 g kg−1), P (0.76 ± 0.02 and 0.78 ± 0.01 g kg−1), and K contents (2.93 ± 0.06 and 2.94 ± 0.06 g kg−1) during both seasons 2019 and 2020, respectively, when rice plants were treated with biochar and glycine betaine together and irrigated every 6 days. On the other hand, rice grains harvested from control rice plants had the lowest N (1.28 ± 0.02 and 1.35 ± 0.01 g kg−1), P (0.51 ± 0.02 and 0.56 ± 0.01 g kg−1), and K contents (1.09 ± 0.11 and 1.05 ± 0.04 g kg−1) during both seasons 2019 and 2020, respectively, when irrigated every 12 days  and photosynthetic attributes  were positively correlated with the dual application of biochar and glycine betaine together at all tested irrigation intervals of 6, 9, and 12 days  was carried out . The PCAIn agreement with our PCA findings, the HCA and its associated heatmap revealed the differences between treatments . Briefly+ content, and APX activity, which were all higher in ‘Cluster B’ of the treatments. Cluster ‘II’ included chlorophyll a, number of panicles per m2, grain yield, number of grains per panicle, harvest index (%), chlorophyll b, total carotenoids, net photosynthetic rate (Pn), stomatal conductance (gs), relative water content, leaf K+ content, grain N content, 1000-grain weight, straw yield, grain P content, and grain K content, which were all higher in ‘Cluster A’ of the treatments. It is worth mentioning that the leaf Na+/K+ ratio was clustered separately in the middle of the dendrogram between Cluster ‘I’ and Cluster ‘II’ during 2019 , proline content, POX activity, exchangeable sodium, electrolyte leakage (EL), malondialdehyde content, CAT activity, leaf Naing 2019 A and 202ing 2019 B.Rice is among the field crops susceptible to environmental stressors like water stress and soil salinity which restraint plant growth and development owing to the decline in the water holding capacity by plant roots due to the osmotic pressure alongside ionic toxicity ,26. In t+ content has negatively affected soil structure, physicochemical properties, increased soil pH, and EC that limit root growth and plant development alongside constraint water uptake and nutrient imbalance owing to an antagonistic impact between Na+ and other elements such as Ca2+, K+, and other cations [+, Ca2+, and Mg2+ contents in soil solution while declined Na+ content alongside soil pH and EC values resulting in decreased greatly exchangeable sodium percentage (ESP) due to biochar’s porous structure, high water holding capacity, large surface area, and negative surface charge that holds more nutrients under soil salinity and water stress conditions [+, Ca2+, and Mg2+ contents in soil solution that positively affected soil physicochemical properties causing better plant growth in rice under soil salinity and water stress conditions in both years of the study [Soil salinity and water stress combined with high Na cations ,26. Applnditions . In the he study ,36.+ and Cl− as found in the present work [+ uptake and increase K+ uptake. Biochar application increased cell formation and size in the root and allowed rice roots to absorb more K+ and hold Na+ ions away under salt stress avoiding ion toxicity, reducing the transport of Na+ into the xylem isolating Na+ into the vacuole [+ ion toxicity and improved plant growth under harsh conditions compared to plants that were not treated with glycine betaine. In this context, the combined application of biochar [The harmful impacts of soil salinity and water stress disturb rice plants through two faces; the first is owing to the high osmotic stress and the second is ion toxicity as a consequence of more accumulation of toxic ions like Naent work . The app vacuole ,39. Appl vacuole . Applica biochar  and glycWater deficit causes a great increment in the enzymatic activity, i.e., CAT, APX, and POX, as well as lipid peroxidation (MDA) and electrolyte leakage (EL %) with a defense line versus the harmful impacts of water deficit and salt-affected soil in rice plants ,43. Augma, b, and carotenoids), and various physiological traits, such as the photosynthetic rate, stomatal conductance, and relative water content in rice plants due to deficient biosynthesis, as presented by Zhang et al. [a, chlorophyll b, and carotenoids as well as physiological measurements like photosynthetic rate, stomatal conductance, and relative water content under water stress and soil salinity in rice plants through nutrient content and hormone-like compounds [Combined water stress and salt-affected soil impede plant growth and development by detrimentally damaging osmolytes such as proline content, chlorophyll pigments , and the number of grains/panicles which negatively affecting grain yield, straw yield, and harvest index under water stress and soil salinity was ascribed to the inhibition in the absorption and transfer of the nutrition materials through the growth of grains and their filling periods. Furthermore, soil salinity can trigger harsh injury to the ovary, and thus may lead to a decrease in the yield [2, the number of grains/panicles, and 1000 grain weight (g) as a consequence of mitigating the harmful impact of water stress and soil salinity compared to untreated plants (control treatment) that can increase its panicle sterility under such a condition [In the present investigation, the decrement in the number of panicles/mhe yield . In termhe yield . Moreoveondition . The higondition . Howeverondition . In thisondition . All of + ions. It was shown that nutrient uptake in terms of N, P, and K contents was negatively impacted by water stress and soil salinity [Grain yield formation is the final product during plant growth and development that particularly relies on vigorous vegetative growth. Water stress and soil salinity reduced nutrient uptake from the soil as a consequence of overflowing Nasalinity . The datsalinity . Biocharsalinity , incremesalinity , cell disalinity . Glycinesalinity . The mag1), 9 (I2), and 12 (I3) days) on soil properties, physiological parameters, yield-related traits, productivity well as grain quality of rice  in salt-affected soil. The experiment was set up in a split-plot design with four replicates. The main plots were assigned to three irrigation intervals; irrigation every 6, 9, and 12 days after transplanting which is indicated as I1, I2, and I3 respectively. The subplots were allocated to four treatments namely, control (neither biochar nor glycine betaine applied and was done with distilled water); biochar as soil application (at the rate of 10 t ha−1); foliar application of glycine betaine (at the rate of 50 mM); and combined (biochar + glycine betaine). Main plots including irrigation intervals were tightly detached by trenches, 2 m width and 1 m depth. Treatment of glycine betaine had been sprayed thrice, at mid-tillering, panicle initiation, and full heading. Biochar added in this present investigation was prepared through slow pyrolysis of rice husk and corn stalk (1:1) at 350 °C under no oxygen conditions with an average residence time of 3 h [Two field trials were performed during the two summer growing years of 2019 and 2020 at the Experimental Farm of El-Karada Water Requirements Research Station, Sakha, Kafr Elsheikh (North Delta), Egypt (Latitude: 31°6′ N/ Longitude: 30°56′ E). The preceding cultivated plant was wheat during the two years of the study. The objective was to report the dual effects of foliar spraying with glycine betaine and soil amendment with biochar under three irrigation intervals (every 6 (Ie of 3 h . Biochar−1) were soaked in fresh water for 24 h and incubated for another 48 h to stimulate seedlings) and were broadcasted homogenously by hand in the nursery on 1 and 5 of May in the 2019 and 2020 seasons, respectively. Four seedlings per hill at thirty days old were transplanted in the permanent field’s experimental plots at a 20 cm × 20 cm distance between hills and rows in 67.5 m2 (13.5 m × 5.0 m) size plots. Weeds were controlled mechanically using hands and chemically using Saturn 50% (active ingredient Thiobencarb 800 g L−1) at the rate of 5 L ha−1 at 5 days post transplanting. Nitrogen was applied in the form of urea (46% N) on three equal doses during rice vegetative growth at the rate of 165 kg ha−1. The recommended phosphorous and Potassium fertilizers in the form of calcium superphosphate (15% P2O5) at a rate of 37 kg P2O5 ha−1 and potassium sulfate (48% K2O) at the rate of 50 kg K2O kg ha−1 were added during soil preparation. During the 2019 and 2020 seasons, representative soil samples were collected at the depth of 0–30 cm from the experimental site. The physical and chemical properties of the experimental soil were measured based on [Pre-germinated seeds (at the rate of 120 kg habased on  and demoe (dS m−1) was calculated in soil paste extract, whilst pH was estimated in 1:2.5 soil: sanitized water suspension, whereas pH was estimated by pH-meter . The ECe was computed by EC-meter . The content (meq L−1) of Na+, K+, Ca2+, Mg2+ ions was estimated in soil paste extract via Atomic Absorption Spectrophotometer  with a detection limit of 100 ppb [+, Ca2+, and Mg2+ were computed in meq L−1.At rice maturity from a 0–30 cm depth, representative soil samples were assembled via an auger. Soil samples were dehydrated in the open air and delivered across a 2-mm strainer for chemical traits assessment. The EC 100 ppb . Exchang 100 ppb :ESP = 1.ds, 1954 :(2)SAR=[+ and K+ contents (mg kg−1 DW) via ultra-pure water the volume of the sample was brought to 50 mL in a volumetric flask. Based on Temminghoff and Houba [+ and K+ contents were estimated using AAS (Perkin Elmer 3300) with a detection limit of 100 ppb.At the anthesis stage, representative samples of five plants were randomly collected from each experimental unit to measure Nand Houba , Na+ anda, b and carotenoid; mg g−1 FW) were measured at the heading stage based on Peng, 1992 [Photosynthetic pigments  (μmol CO2 m−2 s−1) and stomatal conductance (gs) (mol m−2 s−1) were measured at the heading stage via a portable photosynthesis measurement system  at 09:30–11:30 a.m. from flag leaves . Air relative humidity ranging between 45–55%, ambient CO2 concentration was about 370 µmol CO2 mol−1 and a leaf temperature of 30 °C, respectively during collecting the data.The net photosynthetic rate (PThe relative water content (RWC) of rice leaves was assessed according to the method of Barrs and Weatherley . Fresh rw/v) aqueous sulfosalicylic acid and sieved. To 2 mL of the filtrate was put on 2 mL of ninhydrin acid; later 2 mL of glacial acetic acid was put on the mixture, and the mixture was heated for 60 min. The absorbance readings of the toluene layer were read on a spectrophotometer at 520 nm. Proline content was computed as a standard curve. Proline content was described as µg g−1 fresh weight.Proline content was estimated based on the method of Bates et al. . Roughly−1 protein) activity was estimated by the suggested technique of Aebi, 1984 [2O2 (5.9 Mm) and 1000 µL buffer combined with the 100 µL of plant extract. The absorbance of samples was measured at 240 nm by spectrophotometer. For ascorbate peroxide  activity, the combination included 100 µL enzymes extracts, 100 µL ascorbate (7.5-mM), 100 µL H2O2 (300 mM), and 2.7 mL potassium buffer (25 mM), 2-mM EDTA having 7.0 pH. The content of APX was estimated at 290 nm wavelength by spectrophotometer. Peroxidase  activity was estimated by the technique of Vetter et al. [v/v) guaiacol and 100 mM H2O2. 100 µL was utilized for the measurement. The plant sample (0.5 g) was mixed with 5-mL potassium phosphate buffer (50 mM) with 7.0 pH under ice-cold circumstances and centrifuged at 15,000. The absorbance of the extract was measured at 470 nm for 2 min. Lipid peroxidation was assayed as Malondialdehyde (MDA) content in rice leaves according to Rao and Sresty [−1 FW.The catalase  were sampled and cut into small pieces, and placed into 20 mL of distilled water and the rate of leakage was read at 1-min intervals for 60 min via a conductivity meter . The leakage rate was measured as the slope of the line to the leaf dry weight by the method of Sullivan .The electrolyte leakage (EL%) was calculated using Equation (4):2. Panicles number was measured from every hill, each panicle was hand-threshed and the unfilled panicles were separated from filled panicles using a blower. Ten panicles were randomly collected from each experimental unit to estimate the number of grains/panicles and 1000 grain weight (g). The biological yield (both grain and straw yield t ha−1) was measured from a 6-m2 area in each experimental unit except the outer border, and the standard grain moisture content of 14% was added to yield computation, as expressed by Yoshida, 1981 [At the maturity stage, panicles of five random hills from each experimental unit were counted then converted to the number of panicles/mda, 1981 . Harvest−1). The dried samples were ground with a stainless-steel grinder and digested with HNO3 (70% v/v):H2O2 (30% v/v) solution (2:1). P content was calorimetrically estimated based on the method of Sparks et al. [At harvest, five panicles were taken randomly from the inner of the experimental unit. Thirty kernels were taken from each plot, washed with distilled water, and oven-dried for 48 h at 70 °C to estimate grain N, P, and K contents  p-value for the whole plots (reported as PIR) to compare between different irrigation intervals, (2) p-value for the strips (reported as PTR) to compare between treatments, and (3) p-value of the interaction between irrigation intervals and treatments (mentioned as PIR×TR). Moreover, Tukey’s honestly significant difference (HSD) test was used for post-hoc analysis (p < 0.05). ANOVA and Tukey’s test were carried out using JMP Data analysis software-Version 15 . Moreover, the data matrix of all individual response variables was used to perform the principal component analysis (PCA) and its associated scatter and loading plots. Finally, the standardized means of all individual response variables were used for two-way hierarchical cluster analysis (HCA). Similarities and variations between treatments are presented as a heat map.In the current study, a full factorial experiment design (strip-plot) was used as an experimental layout in all experiments. Our experimental design consists of two factors: (1) three levels of irrigation intervals  as the whole plots and (2) four treatments  as strips. All experiments were repeated twice in two different seasons with at least three biological replicates for each treatment. The analysis of variance (ANOVA) was used to test the significant differences among treatments. Based on the strip-plot ANOVA, three Our findings suggest that incorporated biochar-based soil amendment and exogenous glycine betaine foliar application can be an effective, sustainable, eco-friendly strategy to improve the resilience of rice plants growing in salt-affected soils especially in arid and semiarid regions. Dual application of biochar + glycine betaine significantly enhanced the growth, physiology, productivity, grain quality, and osmotic stress tolerance of rice plants, as well as soil properties and nutrient uptake during two successive seasons under field conditions. Our findings show that the beneficial role of biochar and glycine betaine might be due to the activation of the enzymatic antioxidant defense machinery to maintain reactive oxygen species (ROS) homeostasis within stressed plants. However, further studies are required to deeply investigate the long-term effect(s) of biochar and glycine betaine on both plant and soil ecosystems.


A high concentration of specific amino acids in the control tempe explained a strong ammonia smell, saltiness, and darker golden-brown sensory attributes. Tempe soaked in vinegar contained high concentrations of metabolites associated with a roasted aroma and cooked meat. In conclusion, most RATA panelists who were being introduced to tempe preferred that soaked in vinegar to the control that was not.Tempe is a fermented soybean food that is globally renowned for its high protein content. Methods of preparing tempe vary worldwide, and include soaking in vinegar before fermentation. This study aimed to determine the effects of soaking in vinegar by metabolome analysis, gas chromatography/mass spectrometry, and sensory attribute evaluation. Vinegar affected metabolism during tempe fermentation, which led to altered metabolite profiles in the final product. We validated the metabolite profiles of two types of tempe using triangle tests and rate-all-that-apply (RATA) tests, which revealed that the sensory attributes of a golden-brown color, ammonia smell, pleasant smell, salty flavor, and acceptance significantly differed ( Rhizopus spp. as the starter mold , in , in 8], g for 3 min, then 400 µL of supernatant was vortex-mixed with ultra-pure water (400 µL) in 1.5-mL microtubes to increase separation between the polar and non-polar phases. The mixture was centrifuged for 3 min at 4 °C, 11,740× g for 3 min. The aqueous phase (400 µL) was transferred to fresh 1.5 mL microtubes with pierced caps. Pooled quality controls (QCs) were prepared by mixing 200 µL of the aqueous phase from all samples. The solvent was evaporated using a Spin Dryer Standard  for 2 h at room temperature. The evaporated samples were lyophilized overnight (Taitec Co.). Methoxyamine hydrochloride  was added on the following day and incubated at 30 °C and 161× g for 90 min. Subsequently, 50 µL N-methyl-N-(trimethylsilyl)-trifluoroacetamide (MSTFA)  was added and the mixture was incubated at 37 °C and 161× g for 30 min before GC-MS analysis.Frozen tempe was milled into a fine powder, then 15 mg of lyophilized tempe was vortex-mixed with 1 mL of a 5:2:2 (v/v/v) ratio of methanol, ultrapure water  and chloroform  and the internal standard ribitol (100 µg/mL). The mixture was separated by centrifugation at 4 °C and 11,740× g in an incubator at 30 °C for 90 min. The samples were silylated with 50 µL of N-methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA) (GL Sciences Inc.), then incubated at 37 °C for 30 min at 161× g.Samples were derivatized before GC/MS to analyze non-volatile metabolites. Lyophilized samples were oximized with 100 µL of methoxyamine hydrochloride (20 mg/mL in pyridine)  then shaken at 161× Metabolites were profiled using a GC/MS-QP2010  equipped with a 0.25 × 30 mm, 0.25-µm Inert Cap 5 MS/NP column , and an AOC-20i/s autosampler  under the conditions applied in a previous study of coffee metabolomics . The refhttp://prime.psc.riken.jp/compms/msdial/main.html, accessed on 23 December 2021). Data patterns were searched based on differences and similarities by multivariate analysis using SIMCA-P+ v.13  for PCA without data transformation. The relative intensity of each metabolite was normalized by comparison with that of the internal standard as an explanatory variable. The data were auto-scaled to reduce the mask effect of abundant metabolites. The class for each annotated metabolite was determined using the Human Metabolome Database .The GC/MS data were converted into ANDI files (*cdf) using GCMS Solution software , then peaks were detected, the baseline was corrected, and peak retention times were aligned using MS-DIAL v. 3.70  . Peaks wWe prepared tempe as described in sub-n = 12; male, n = 13 men; Japanese, n = 17; non-Japanese n = 8) were trained on the five-basic taste test. Three tempe samples were simultaneously presented in the triangle test. Two were the same type, and one was a different type [A panel of 25 assessors  participated in these tests. During selection, the panelists were questioned about their familiarity with the sensory characteristics of tempe. Regardless of their responses, the panelists were familiarized for 30 and 60 min with the 16 sensory attributes of tempe. Tempe samples that were soaked without  and with (B) vinegar were presented along with a questionnaire with a list of 16 sensory attributes. The combinations and order of the samples were randomized (AB and BA) for each panelist, and they rated the intensity of each attribute using a 5-point scale. A panel of 27 assessors (average age, 29.5 y; female,


Nuphar) bioactive extracts have been widely used in traditional medicine owing to their multiple applications against human ailments. Phyto-active Nuphar extracts and their purified and synthetic derivatives have attracted the attention of ethnobotanists and biochemists. Here, we report that 6,6′-dihydroxythiobinupharidine (DTBN), purified from extracts of Nuphar lutea (L.) Sm. leaves, is an effective inhibitor of the kinase activity of members of the protein kinase C (PKC) family using in vitro and in silico approaches. We demonstrate that members of the conventional subfamily of PKCs, PKCα and PKCγ, were more sensitive to DTBN inhibition as compared to novel or atypical PKCs. Molecular docking analysis demonstrated the interaction of DTBN, with the kinase domain of PKCs depicting the best affinity towards conventional PKCs, in accordance with our in vitro kinase activity data. The current study reveals novel targets for DTBN activity, functioning as an inhibitor for PKCs kinase activity. Thus, this and other data indicate that DTBN modulates key cellular signal transduction pathways relevant to disease biology, including cancer.Water lily ( Nuphar lutea (L.) Sm. (Nymphaeaceae) have been widely used for treating inflammatory conditions in ethnic medicine [Nuphar products is still largely unexplored by modern research [Nuphar extracts (NUP) have indicated potential applications. We have previously published that a semi-purified leaf extract from yellow water lily, Nuphar lutea (L.) Sm. (NUP), is enriched in sesquiterpene thioalkaloids, such as nupharidine and bi-nupharidine isomers [Natural products of medicine ,2. A sysresearch . Neverth isomers . A varie isomers ,6, antiv isomers , against isomers , against isomers ,9,10, an isomers ,11,12. W isomers . Lastly, isomers . We hypo isomers . Being pλ and ζ) isoforms [The protein kinase C (PKC) family comprised a group of serine/threonine kinases which transduce a multitude of signals that control diverse cellular processes, such as proliferation, migration, invasion, differentiation, apoptosis, transcription, and translation ,17. Aberisoforms ,22,23.Numerous observations from basic studies and clinical trials have implicated PKC in the pathophysiology of diverse types of diseases, including cancer ,25. PKCsOur results show that DTBN inhibits PKC isoforms, preferentially conventional PKCs. In silico molecular docking analysis of DTNB to these enzymes confirmed our results, and provides a structural basis for the interaction of DTNB with PKCs.50), the relative amounts of p32-MBP/MBP were plotted at several DTBN concentrations: IC50 = 0.174 μM (PKCα), 0.168 μM (PKCγ), 14.23 μM (PKCε), 18.6 μM (PKCζ), and >19 μM (PKCη and PKCδ). Immunoblotting was performed with antibodies against HA-tagged different PKCs and against Myelin Basic Protein (MBP) as a loading control.To test the effect of DTBN on the activity of PKC isoforms, in vitro kinase assays were performed in a dose–response experiment, and results are presented in Using molecular docking of DTBN to individual PKCs, we demonstrate binding of DTBN to the ATP binding pocket of PKCs, with PKCα exhibiting the deepest insertion into the ATP binding pocket, followed by PKCγ. Post mm-GBSA refinement binding energy  strongly50 = 0.174 and 0.168 μM, 50 = 14.23 μM, 50 >19 μM each, 50 = 18.6 μM, The inclusion of DTBN inhibited the ability of PKCs to phosphorylate their substrate MBP, myelin basic protein, in a dose-dependent manner . The inhNuphar semipurified extracts (NUP) and purified DTBN. Our studies provided evidence for the phytoactive ability of NUP in modulating various cell signaling cascades. For instance, it enhanced ERK activation, which resulted in a decreased activity of TNFα-induced NF-κB. DTBN also was shown to covalently inhibit human type II topoisomerase activity, an observation that highlighted the possibility of DTBN behaving as a potent inhibitor of human enzymes by targeting their active sites [As mentioned in the introduction, we previously reported the anti-inflammatory, anticancer, and antibacterial properties of ve sites . Here, wve sites ,28,29,30ve sites . Thus, P2) terminal lobe, which is made up of β-sheets and contains the ATP binding domain, which is rich in glycine and has a consensus sequence of GXGXXG and an invariant lysine. The carboxylic (–COOH) end domain mostly comprises α-helical structures and contains an activation loop. Multiple sequence alignment and ATP binding site of all PKC isoforms  are highly conserved (GKGSFG), except for PKCζ  is stabilized by four hydrogen bond interactions [DTBN binds deep into the ATP binding cleft of PKCα, surrounded by β-sheets on its sides , with biractions . On the ractions . These s···π and π···S interactions at the binding site  of octahydro-1H-quinolizine ring in DTBN and carboxylic –OH of Asp493 residue. This weakens the ligand–enzyme interaction and contributes to a reduced binding score  group of DTBN and the amino group (–NH) of Asp645 present in the loop next to the binding pocket (···S interactions (50: 9 nM) is stabilized by three hydrogen bonds [Similarly, a smaller binding pocket of PKCη to accommodate DTBN leads to a steric clash between the octahydro-1g pocket . A smallg pocket . At the ractions . In compen bonds , whereasThe low binding score of PKCη in comparison to PKCε is attributed to the presence of a single-turn α-helix near the PKCε binding pocket, which results in several weak interactions with DTBN, thus providing additional stabilization for PKCε as compared to PKCη.Alike PKCε and PKCη the binding pocket of PKCδ and PKCζ are also smaller to accommodate DTBN in the ATP binding pocket without steric clash , leadingIn Nuphar lutea extracts and purchased from Sigma /Merck , cat. SMB00609. DTBN was dissolved in DMSO.6,6′-dihydroxythiobinupharidine (DTBN) was purified from 2, and 0.1 mM EGTA). The immunoprecipitates were then equally divided to Eppendorf safe-lock tubes, followed by the addition of indicated concentrations of DTBN (μg/mL) and subsequent incubation in a kinase reaction mixture , also including 100 μM cold ATP and 1.5 mM CaCl2 , and incubated for 30 min at 32 °C with gentle shaking. Reactions were terminated by the addition of 5× sample buffer and boiling for 5 min, followed by SDS-PAGE (10% acrylamide gels) under reducing conditions. Samples were then transferred to nitrocellulose membranes (Sigma-Aldrich) that were developed by immunoblotting and autoradiography. After the detection of the phosphorylated substrate (32P-MBP) by autoradiography, the membranes were probed with anti-HA and anti-MBP mAbs , (loading controls). 32P-MBP and MBP protein bands were quantified using Image Lab software (5.2.1).PKC kinase assays were performed, as described , using mhttps://swissmodel.expasy.org/) (accessed on 20 December 2020) [Molecular docking studies were performed using Schrödinger Maestro Suite 2020-3 . DTBN was docked to the ATP binding site of PKC isoforms. X-ray crystallographic structures of PKCα (PDB ID: 3IW4) and PKCη (PDB ID: 3TXO) are available in RCSB PDB. The structures of other PKC isoforms, δ, γ, ε, and ζ were generated using a homology modelling approach using a Swiss model online server (er 2020) . The homer 2020) ,37.mean scores of the generated models were above −2.0. Qmean score provides the “degree of nativeness” of the model [mean score near ‘0′ represents a good agreement between the predicted model structure and experimental structures of similar size. It also helps in the estimation of the quality of the generated models. Structure assessment details are given in Since the crystal structures of PKC isoform δ, γ, ε, and ζ were not reported earlier, the homology modelling approach was adopted to develop their structures. The templates chosen for model generation are given in he model . A Qmean3. Further, mm-GBSA refinements were carried out on the docked poses of each isoform, with a flexible residue distance of 5.0 Å.All the 3D X-ray crystallographic and homology modelled structures were optimized before docking using Schrödinger in-built protein preparation wizard module. Structural inconsistencies, such as missing hydrogen or incorrect bond orders, were rectified during this process. PKCα and PKCη both had co-crystallized ligand, which was chosen as the centre of the receptor grid for docking. However, in the case of homology-based models, active sites were predicted using SiteMap. Active sites with binding scores < 0.9 were rejected. The internal grid size of x × y × z was fixed as 15 × 15 × 15 ÅA sitemap module was used to identify the potential binding pockets. Sitemap score is based on the potential hydrogen bonding, hydrophobicity, and pocket volume. A site score greater than 0.9 is considered as a cut-off to distinguish between the potential drug binding site and non-drug binding sites . The sitTaken together, the present in vitro results reveal that DTBN inhibits the kinase activity of PKCs. Classical PKCs, PKCα and PKCγ, were significantly the main targets for DTBN inhibition. Molecular docking analysis of the interaction of DTBN with PKCs active sites further corroborated our results. These data point to an affinity-based effect of DTBN on PKC isozymes. The key factor to be noted here is the drug’s varying behavior to mediate stabilizing interactions at the active site of individual PKCs. In silico analysis revealed a higher binding score of DTBN upon docking with PKCα and PKCγ. This score was reduced in the cases of PKCε and PKCη, and was further reduced in the cases of PKCδ and PKCζ. The selectivity of DTBN towards PKCs could guide us in targeting pathological conditions impaired in classical PKC signaling, including cancers.


Many neurodegenerative diseases are associated with the self-assemblyof peptides and proteins into fibrillar aggregates. Soluble misfoldedoligomers formed during the aggregation process, or released by maturefibrils, play a relevant role in neurodegenerative processes throughtheir interactions with neuronal membranes. However, the determinantsof the cytotoxicity of these oligomers are still unclear. Here weused liposomes and toxic and nontoxic oligomers formed by the sameprotein to measure quantitatively the affinity of the two oligomericspecies for lipid membranes. To this aim, we quantified the perturbationto the lipid membranes caused by the two oligomers by using the fluorescencequenching of two probes embedded in the polar and apolar regions ofthe lipid membranes and a well-defined protein–oligomer bindingassay using fluorescently labeled oligomers to determine the Stern–Volmerand dissociation constants, respectively. With both approaches, wefound that the toxic oligomers have a membrane affinity 20–25times higher than that of nontoxic oligomers. Circular dichroism,intrinsic fluorescence, and FRET indicated that neither oligomer typechanges its structure upon membrane interaction. Using liposomes enrichedwith trodusquemine, a potential small molecule drug known to penetratelipid membranes and make them refractory to toxic oligomers, we foundthat the membrane affinity of the oligomers was remarkably lower.At protective concentrations of the small molecule, the binding ofthe oligomers to the lipid membranes was fully prevented. Furthermore,the affinity of the toxic oligomers for the lipid membranes was foundto increase and slightly decrease with GM1 ganglioside and cholesterolcontent, respectively, indicating that physicochemical propertiesof lipid membranes modulate their affinity for misfolded oligomericspecies. This phenomenon seems to begeneric, as it has been found for oligomers of the amyloid βpeptide,13 α-synuclein,14 islet-amyloidpolypeptide,15 calcitonin,17 as well as model oligomers.19Many neurodegenerative diseases, including Alzheimer’s disease(AD), Parkinson’s disease (PD), Creutzfeld-Jacob disease (CJD),Huntington disease (HD), frontotemporal dementia (FTD), familial amyloidpolyneuropathy (FAP), and many others are associated with the self-assemblyof specific peptides or proteins into misfolded fibrillar aggregatesknown as amyloid fibrils.23 One of such pairs is that obtained from the 91-residue N-terminaldomain of [NiFe]-hydrogenase maturation factor HypF (HypF-N) from E. coli.19 Oligomer formation of HypF-Ncan be readily and reproducibly directed into two morphologicallysimilar forms, previously named type A (toxic) and type B (nontoxic)oligomers , by altering the solution conditions.Although this protein domain has no known link to human disease, ithas proved to be a valuable model system as its toxic OAs have beenshown to have effects indistinguishable from those of Aβ42 oligomers associated with AD at the biochemical, electrophysiologicaland animal model levels.29The study of the structural elements of misfolded protein oligomersresponsible for neuronal dysfunction and of the mechanism throughwhich they cause neurotoxicity has benefited from the isolation ofpairs of oligomers of the same protein having a toxic and benign effect,respectively.30 This interaction induces a series of downstream events associatedwith cytotoxicity, including an influx of calcium ions (Ca2+) from the cell medium to the cytoplasm, generation of intracellularreactive oxygen species (ROS), lipid peroxidation, perforation ofcell membranes with exit of intracellularly trapped large molecules,caspase-3 activation, and mitochondrial damage.19 OAs also induce loss of cholinergic neurons when microinjected intorat brains with an associated impairment of spatial memory in rats,colocalization with synapses in primary neurons, and inhibition oflong-term potentiation (LTP) in rat hippocampal slices.26 By contrast, OBs were found to bind to lipid membranes on the surface,without infiltrating their lipid bilayer, and were not found to haveany of the deleterious biological effects observed for OAs.26OAs manifest a strong ability to bind and penetrate the lipid bilayerof cell membranes.31 By contrast, other experiments with supportedlipid bilayers (SLBs) devoid of proteins treated with OAs and OBsand monitored with atomic force microscopy (AFM) revealed that numerousOAs were bound strongly with both the gel and liquid-disordered phasesof SLBs, whereas few OBs were found to have this ability.30 It was therefore concluded that although bothspecies bind to cell membranes, only OAs bind to, penetrate, and crossthe lipid bilayer of the membranes, thus manifesting their toxic behavior.31 In particular, the GM1 concentration dependence of the OA toxicityin cell cultures and of OA binding to SLBs showed that the oligomersbinding to the gel-phase (Lβ or So) ofthe lipid bilayer are responsible for the OA toxicity.30A recent interactome approach to detect all the membrane proteinsof microglia N13 cells interacting with OAs and OBs showed that OBshad a higher affinity for membrane proteins relative to OAs.33 In spite of these structural similarities, characterization of thesespecies at a molecular level revealed important differences.32 By labeling the oligomers with pyrene at various sequence positions,it was shown that nontoxic OBs are stabilized by intermolecular interactionsbetween the three major hydrophobic regions of the sequence, suchthat a lower fraction of the hydrophobic residues are solvent-exposedon the oligomer surface relative to the toxic OA species.18 Such interactions are weaker in the toxic oligomersso that a larger fraction of hydrophobic residues are solvent exposed.18 The binding of the fluorescent reporter 8-anilinonaphthalene-1-sulfonicacid (ANS) was also found to be higher in OAs, confirming a higherexposure of hydrophobic clusters.18 Solution-stateand solid-state nuclear magnetic resonance (NMR) spectroscopy andsite-directed fluorescence resonance energy transfer (FRET) experiments,involving residues labeled with a donor and acceptor at various positions,showed that toxic OAs have a greater compactness and structural rigidity,so that structural constraints are generated that cause a number ofthe hydrophobic residues to interact less strongly with each other,with a fraction of them becoming exposed to the solvent.32 Accordingly, FRET efficiency values were, onaverage, higher in toxic OAs than nontoxic OB species, except whendonor and acceptor labeling involved hydrophobic residues, when theopposite situation was observed.32In terms of their morphology and structure, both oligomer typeswere found to be highly stable, spheroidal, or discoidal under atomicforce microscopy (AFM) with a diameter of 2–6 nm, to possessa similar β-sheet core structure, and to display weak thioflavinT (Th-T) binding.19 However, giventhe complexity of cellular systems, methods for quantitative measurementsof oligomer-membrane binding, for example, in terms of associationor dissociation constants, are not well established. In addition,it is not clear whether the oligomers change their structures uponinteracting with cell membranes. To address these issues, we haveused liposomes in the form of large unilamellar vesicles (LUVs) witha variable and biologically compatible lipid composition and OAs/OBsof HypF-N. Our results indicate that OAs are characterized by a higherand measurable affinity than OBs for the LUV bilayer and that thenative protein does not bind to the lipid membranes. We also foundthat neither oligomer type changes its structure upon interactionwith the LUVs and that OAs do not feature a preferential binding toany of the lipids contained in LUVs. We then reveal quantitativelythe effects of trodusquemine, a promising and previously studied smallmolecule that binds to the membrane, on the OA–membrane affinityand on the mechanism of displacement of these toxic oligomers fromthe bilayer and, again in a quantitative manner, how the lipid compositionof LUVs can influence the affinity of toxic oligomers for the membranes.The binding of OAs to the bilayer of cell membranes representsan important event in the mechanism through which these oligomericspecies manifest their toxicity to neuroblastoma cells and primaryneurons.33 LUVs were prepared using DOPC and SM in a molar ratio of 2:1 (mol/mol),1% (mol) CHOL and 1% (mol) GM1, as previously reported.34 LUVs were prepared at various mass concentrations(mg/mL); at a total lipid concentration of 1 mg/mL, for example, molarconcentrations were 836 μM DOPC, 418 μM SM, 13 μMCHOL and 13 μM GM1.Toxic OAs and nontoxic OBs were preformedfrom purified HypF-N at a total concentration of 0.5 mg/mL, correspondingto 48 μM , as previously reported.β or So) and to the liquid-disorderedphase (Lα or Ld) of the SLBs with 1% GM1,whereas only few OBs were found to be bound to them , in agreement with previous results obtained with5% GM1 as the only difference in LUV composition relative to our LUVpreparations.30 Furthermore, the differencein the thickness between the Lβ and Lα domains (ΔZ) is altered by the presence ofOAs, but not OBs, clearly indicating the presence of structural changesof the overall bilayer (Table S1), againin agreement with the result obtained with 5% GM1.30We first checked whether OAs and OBsbound to LUVs. To this purpose, we formed separately supported lipidbilayers (SLBs) with the same lipid composition as LUVs, treated themwith 12 μM OAs or 12 μM OBs , andthen imaged them with AFM. The images show that OAs bind to the gel-phasedomains  with TMA-DPH-and DPH-labeled LUVs  causeda marked and concentration-dependent reduction of the fluorescenceemission of both fluorescent probes, with a more consistent quenchingof TMA-DPH , with KSV values of 3.7± 0.7 and 2.2 ± 0.4 mM–1, respectively,which reflected a lower binding to the membrane and the absence oflipid membrane alteration ,with KSV values of 1.2 ± 1.1 mM–1 and 1.6 ± 0.4 mM–1, respectively,reflecting the absence of binding to the membrane , indicating that it consists of a LUV-induced fluorescence drift,most probably arising from light scattering as the LUV concentrationincreases. The 15 min incubation of OAs with increasing concentrations of unlabeled LUVs  caused a marked decrease of OA fluorescence from0 to ca. 0.4 mg/mL LUVs , followed by the same drift at higher LUV concentrations(KD) value of 0.09 ± 0.04 mg/mL, correspondingto 0.12 ± 0.05 mM of total lipids, indicating binding of OAsto the LUV bilayer. The fluorescence of OBs also decreased significantlywith LUV concentration, to an extent lower relative to that of OAs,but larger relative to native HypF-N or GSH  with increasingconcentrations of unlabeled LUVs  caused a weak decrease of protein fluorescence thatwas found to correlate linearly with LUV concentration 1C. A simtrations6, we obtKSV values of fluorescence quenching of membrane-embeddedTMA-DPH and DPH caused the oligomers  and the oligomer-membrane KD values . Although KSV–1 has been shown to correspondto KD,37 thesevalues are not immediately comparable in our system because they weremeasured in different conditions, that is at constant LUV concentration(0.3 mg/mL) upon varying OA concentration and at constant OA concentration(20 μM), upon varying that of LUVs, respectively. The molaritiesof KSV–1 and KD also refer to protein and total lipids, respectively,and are not, therefore, comparable. In both cases, however, the bindingaffinity of the toxic OAs for the membrane appears to be ca. 20–25times higher than nontoxic OBs. Albeit with much lower affinity, nontoxicOBs also bind to the LUV membrane, unlike the native protein.Hence, under the conditions used here, we have quantified the bindingaffinity of toxic OAs and nontoxic OBs of a sample protein for thebilayer of lipid vesicles (LUVs) by measuring the 38 a lipid density of a membrane bilayer estimated from LUVs of 425± 3 ng/cm2,34 and a celldensity value of neuroblastoma SH-SY5Y cells commonly used to testthe toxicity of OA/OB species of ca. 7.5(±0.7)104 cells/cm2,29 andthen extrapolating this value to the three-dimensional space, onecan determine a lipid concentration of 0.06 ± 0.01 mg/mL in SH-SY5Ycell cultures where OA/OB species are tested. The KD value of 0.09 ± 0.04 mg/mL lipids measured herefor OAs, and referred to total lipid concentration, implies that asignificant fraction of OAs (40 ± 15%) are bound to the lipidmembranes of cells, as soon as the equilibrium between membrane-boundand membrane-unbound OAs is established and before they enter intothe cells. By contrast, the KD value of∼2.5 mg/mL lipids measured for OBs, implies that a very minorfraction of OB species interact with the cell membrane (∼2%).Can we relate the data obtained here with LUVs to cell culturesand brain tissues? By using a mean diameter of 7.5 ± 0.5 μmknown for human neuroblastoma SH-SY5Y cells,One of the questions that is often raisedwhen studying the structure–toxicity relationship of misfoldedprotein oligomers is whether the structural characteristics determinedfor the oligomers in aqueous suspension are maintained or changedupon the interaction with biological membranes. Difficulties to addressthis issue arise from interferences by cellular or membrane proteinsthat make it very difficult to monitor the structural characteristicsof the oligomers with conventional spectroscopic probes. Here we circumventedthis problem using protein-free LUVs and three optical probes to whichLUVs are spectroscopically silent, making it possible to monitor thesecondary and tertiary structure of the oligomers before and aftertheir binding to the membrane.We first acquired far-UV CD andintrinsic fluorescence spectra of OAs, OBs and native HypF-N incubatedwith increasing concentrations of LUVs. The far-UV CD spectra of OAs,OBs, and native HypF-N  were notfound to be significantly different in the absence or presence ofthe various LUV concentrations (0–1.5 mg/mL), indicating thattheir secondary structure was maintained upon interaction with LUVs2A–C.TheE values determined by analyzing the resulting fluorescencespectra acquired following 10 min-incubation with unlabeled LUVs (0–0.7mg/mL) were not found to significantly change when varying LUV concentration,either for OAs or for OBs  OAs labeled with 1,5-IAEDANSas a donor probe (OA-D) and 0.3 mg/mL LUVs containing one of the fourlipids labeled with BODIPY-FL as an acceptor probe (Lipid-A). Theseexperiments were carried out separately by using each of four lipidscontained in the LUVs labeled with A. The FRET r 15 min 4A and wer 15 min 4B.39 involves a change of the bilayer physical properties, without adirect preferential interaction of the lipid with the oligomers. Indeed,it is clear that GM1 increases the overall negative net charge ofthe membrane,34 increases the thicknessof the membrane, particularly of the Lβ phase,40 and decreases lateral diffusion,41 allknown to contribute to a facilitated oligomer insertion.This analysis indicates that OAs bind to LUVs but do not have apreferential interaction with any of the four lipids. Therefore, therole played by GM1 in the oligomer–membrane interaction, observedhere and previously,Figure S4), which has been reported to displace toxic oligomersfrom lipid membranes,43 could induce a variation of the affinity of OAs for the membrane,as measured with TMA-DPH and DPH fluorescence quenching and OA fluorescencechange upon LUV binding. To this aim, we prepared TMA-DPH and DPH-labeledLUVs  containing 5 μMtrodusquemine, and we incubated them with increasing concentrationsof unlabeled OAs . Previousexperiments have shown that trodusquemine has a high affinity forLUVs of this type and partitions completely in the bilayer at thisconcentration.34 The presence of trodusqueminein the bilayer caused a significant reduction of the TMA-DPH fluorescencequenching, with an almost complete absence of quenching at low concentrationsof OAs up to ca. 10 μM (S2C).We then investigated whether trodusquemine S4, which27 and populates the membrane to a lower extent. However,under these excess OA concentrations and in the presence of 5 μMtrodusquemine, the KSV constant remainslower than that observed in the absence of the small molecule. Thisphenomenon indicates that at the toxic OA concentrations and protectivetrodusquemine concentrations the molecule is largely effective asa protective factor but partly loses its protective action in thepresence of excess oligomers.Interestingly, the absence of TMA-DPH and DPH fluorescence quenchingat the concentrations of OAs that normally cause dysfunction and toxicityto cell cultures  and at theconcentration of trodusquemine that causes protection (5 μM)indicates that this small molecule provides protection by preventingOA-LUV binding. By contrast, at higher OA concentrations, the oligomer-displacingeffect of the molecule is overcome, most probably because in the excessof OAs trodusquemine partitions to OAs more markedlyKD, from a value of 0.09± 0.04 mg/mL in its absence (corresponding to 0.12 ± 0.05mM lipids) to a value of 0.86 ± 0.65 mg/mL in its presence (correspondingto 1.10 ± 0.83 mM lipids), therefore reducing the binding affinityof OAs to LUVs by 1 order of magnitude (KSV and KD with and without trodusquemine are reportedin We then incubated BODIPY FL-OAs (20 μM) with increasing concentrationsof unlabeled LUVs containing the same molar fraction of trodusquemine, and we repeatedthe analysis described above in the absence of the small molecule,but this time with the molecule 5C. Troduagnitude 5C. Using46 particularly GM1 and CHOL.49 In order to better mimic the physiological contentof GM1 and CHOL in neuronal plasma membranes, and in light of thefact that the content of these two lipids plays a crucial role inthe interaction with LUVs and toxicity of misfolded protein oligomers,50 we decided to explore whether the variation of these two lipidsin LUVs could affect the affinity of OAs for LUVs , whichwas then found to increase with GM1 content , confirming the crucial role of this lipid in the membrane-oligomerinteraction  containing 100 μg/mL of ampicillin (Sigma-Aldrich).The cells were then diluted 1:20 in 4 l of fresh medium and grownat 25 °C under shaking until ∼0.6 optical density at 600nm (OD600), monitored with a Jasco V-630 UV–visspectrophotometer . Protein expression was induced overnightat 25 °C under shaking by the addition of 1 mM isopropyl β-d-thiogalactopyranoside . The bacterial cells were then harvested by centrifugation for15 min at 7000g at 4 °C; the pellet was resuspendedin ∼30 mL of lysis buffer  and stored at −20 °C overnight.The cell suspension was defrosted at 37 °C in a Thermo HaakeC25P water bath  and then incubated for 1 h with1 mg/mL lysozyme (Sigma-Aldrich) in ice under shaking followed byfive cycles of sonication at 50 kHz for 30 s alternated to 30 s inice. The cell lysate was then centrifuged for 45 min at 38 700g at 4 °C and the supernatant containing the proteinwas filtered using filters with a cutoff of 0.45 μM. The filteredsupernatant was applied to an affinity chromatography column packedwith HIS-Select Nickel Affinity Gel (Sigma-Aldrich), previously equilibratedwith the lysis buffer at 4 °C. The column was then washed withthe washing buffer , equilibrated with the cutting buffer , incubated with 50 units of human thrombin (Sigma-Aldrich)dissolved in 5 mL of cutting buffer for 1 h at 37 °C, and thenincubated overnight at 4 °C under slight shaking. The pure wild-typeand mutated HypF-N were then eluted using 50 mM phosphate buffer,50 mM NaCl, 10 mM imidazole, pH 8.0. Wild-type and mutant HypF-N werethen buffer-exchanged and concentrated in 5 mM acetate buffer, 2 mMdithiothreitol (DTT), pH 5.5, and in 20 mM phosphate buffer, 2 mMtris(2-carboxyethyl)phosphine hydrochloride (TCEP), pH 7.0, respectively,using an ultrafiltration cell with a 3000 Da molecular weight cutoff(MWCO) cellulose membrane  at 4 °C.The final solution containing the pure protein was centrifuged for10 min at 16 100g to eliminate any aggregatesand/or impurities, and its concentration was measured with a JascoV-630 UV–vis spectrophotometer using ε280 =12490 M–1cm–1. Samples were checkedfor their purity with SDS-PAGE and DLS, as described below, and thenstored at −80 °C until use.Cultures of Samples of the variousHypF-N purification steps were denatured with a 4× sample buffer and then incubated at 98 °C for 2min. The resulting samples and a molecular weight marker  wereloaded in a precast gel with a gradient of 4–20% of acrylamide and then run for about 90min at 20 mA, using the Bio-Rad Laboratories electrophoresis kit . The running chambers were filled with a running buffer . The gel was then stained withCoomassie Blue dye  for 30 min at 37 °C with slow agitation and then washedin a destaining solution  for at least1 h at room temperature to remove the excess of dye. The purifiedprotein featured a single band at 10 kDa, in agreement with its expectedmolecular weight of 10 464 Da.51The monomeric state of purifiedHypF-N was assessed acquiring its size distribution on a ZetasizerNano S DLS device from Malvern Panalytical , thermostated at 25 °C with a Peltier temperature controllerand using a 10 mm reduced-volume plastic cell. The refractive indexand viscosity were 1.33 and 0.89 cP, respectively. The measurementwas acquired with the cell position 4.65, attenuator index 8, at 25°C. The DLS distribution in volume mode featured a single populationwith a hydrodynamic diameter of 5.4 ± 1.0 nm, which is compatiblewith a monomeric folded HypF-N, as determined with X-ray crystallography.N-(2-aminoethyl)maleimide, previouslydissolved at high concentration in dimethyl sulfoxide (DMSO) for 2h at 25 °C in the dark on a mechanical shaker. The labeled samplewas dialyzed (membrane MWCO of 3500 Da) in the dark against 1.5 lof 20 mM phosphate buffer, pH 7.0, overnight and then centrifugedto remove any precipitate. The concentration of the dye in the BODIPYFL-labeled HypF-N mutant was determined spectrophotometrically, usingε505 = 76 000 M–1 cm–1, whereas the concentration of the protein was determinedusing ε280 = 12490 M–1cm–1. The labeling degree of the sample was then estimated by determiningthe ratio between the measured dye and protein molar concentrations.The C7S/C65Amutant of HypF-N (containing only Cys40) was diluted to a final concentrationof 150 μM in 20 mM phosphate buffer, pH 7.0, and incubated with2.25 mM BODIPY FL 32 The C18 variant was dilutedto a final concentration of 180 μM in 100 mM potassium phosphatebuffer, pH 7.0, with 2.7 mM 1,5-IAEDANS (15-fold molar excess of dye)and 3 M guanidine hydrochloride (GdnHCl), whereas the C34 variantwas diluted to the same final concentration of 180 μM in 100mM potassium phosphate buffer, pH 7.0, with 1.8 mM 6-IAF (10-foldmolar excess of dye) and 3 M GdnHCl. Both dyes were previously dissolvedin dimethylformamide (DMF) at high concentration. The two labelingmixtures were left in the dark under shaking for 2 h at 30 °Cand then overnight at 4 °C. They were then dialyzed in the dark(membrane MWCO of 3000 Da) against: (i) 0.25 L of 100 mM potassiumphosphate buffer, pH 7.0, with 1.5 M GdnHCl for 4 h, (ii) 0.25 L of100 mM potassium phosphate buffer, pH 7.0 for 4 h, (iii) 0.5 L of50 mM potassium phosphate buffer, pH 7.0, overnight, and (iv) 1.0L of 20 mM or 5 mM potassium phosphate buffer , at pH 7.0, for 6 h. The samples were then centrifugedto remove any precipitate. The concentrations of the dye in the 1,5-IAEDANS-C18variant and in the 6-IAF-C34 variant were determined spectrophotometrically,using ε336 = 5700 and ε491 = 8200M–1 cm–1, respectively. The proteinconcentration was determined spectrophotometrically using ε280 = 12 490 M–1 cm–1 after subtraction of the absorbance contribution of the 1,5-IAEDANS/6-IAFprobe at the same wavelength of 280 nm. The labeling degree was estimatedas the ratio between the two measured dye and protein molar concentrations.The C7S/C40S/C65A/Q18C (named C18) and C7S/C40S/C65A/N34C (namedC34) mutants of HypF-N  were labeled with 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonicacid  and 6-iodoacetamidofluorescein (6-IAF) dyes , respectively, as previouslyreported.18OAs and OBs of wild-typeHypF-N were obtained at a protein concentration of 0.5 mg/mL, correspondingto 48 μM , by incubating the protein for4 h at 25 °C in (i) 50 mM acetate buffer, 12% (v/v) trifluoroethanol(TFE), 2 mM DTT, pH 5.5 and (ii) 20 mM trifluoroacetic acid (TFA),330 mM NaCl, pH 1.7, respectively, as previously described.BODIPY FL-labeled OAs and OBs used forthe binding experiments were obtained under the same conditions, butusing 4.4 μM of BODIPY FL-labeled C7S/C65A HypF-N and 43.6 μMof unlabeled C7S/C65A HypF-N, in order to obtain a 1:10 molar ratiofor labeled/unlabeled protein.OAs and OBs for intraoligomer FRET were formed under the same conditionsby 24 μM C18 variant labeled with the donor dye 1,5-IAEDANS(18D) and 24 μM C34 variant labeled with the acceptor dye 6-IAF(34A), at a molar ratio 18D:34A of 1:1. These oligomers were named18D_34A. Oligomers 18D_10 and 10_34A  were also produced in a 1:1 molar ratio betweenlabeled and unlabeled variant.336 = 5700 M–1 cm–1, whereas the concentration of the protein was determined using ε280 = 12 490 M–1 cm–1 after subtraction of the absorbance contribution of 1,5-IAEDANSat 280 nm. The labeling degree was estimated as the ratio betweenthe measured dye and protein molar concentrations.OAs and OBs for LUV-oligomer FRET were initially formed under thesame conditions using C7S/C65A HypF-N. They were then centrifugedat 16,100 g for 15 min at 20 °C. The supernatants were removed,the pellets were gently dried with nitrogen flow and then resuspendedin 20 mM phosphate buffer, 2 mM TCEP, pH 7.0, to a final HypF-N concentration of 160 μM. They were then incubated witha 12.5-fold molar excess of 1,5-IAEDANS, previously dissolved at highconcentration in DMF for 2 h at 25 °C in the dark on a mechanicalshaker. The labeled samples were dialyzed (membrane MWCO of 3500 Da)in the dark against 1.5 l of 20 mM phosphate buffer, pH 7.0, overnightand then centrifuged to remove any precipitate. The concentrationof the dye in the samples was determined spectrophotometrically, usingand ε34 These lipid speciesare known to be present in the neuronal membranes.54 The non-natural percentages of the various lipidswere chosen to favor well-separated Lβ domains andLα regions.55 In particular,the CHOL percentage of 1% was already adopted in other works58 and it is necessary to obtainmodel membranes with well distinct Lα and Lβ phases and relatively extended Lβ domains. It wasdemonstrated that the Lβ domains significantly decreasedtheir size by increasing the CHOL concentration, becoming indistinguishablefor microscopic analyses.55 The ordereddomains of LUVs, enriched in GM1 and CHOL, partially mimic the complexfeatures of lipid rafts in neurons.59 Moreover,the progressive increase of CHOL and GM1 content in our experiments,aim at better mimicking the real composition of neuronal membranes.LUVs were prepared using DOPC andSM in a molar ratio of 2:1 (mol/mol), 1% (mol) CHOL and 1% (mol) GM1,as previously reported.sn-glycero-3-phosphocoline  and sphingomyelin  ina molar ratio of 2:1 (mol/mol), 1% (mol) cholesterol and 1% (mol) monosialotetrahexosylganglioside 1 ; (ii) DOPC and SM in a molar ratio of 2:1 (mol/mol), 1% (mol)CHOL; (iii) DOPC and SM in a molar ratio of 2:1 (mol/mol), 1% (mol)CHOL and 5% (mol) GM1; (iv) DOPC and SM in a molar ratio of 2:1 (mol/mol)and 1% (mol) GM1; (v) DOPC and SM in a molar ratio of 2:1 (mol/mol),5% (mol) CHOL and 1% (mol) GM1; (vi) DOPC and SM in a molar ratioof 2:1 (mol/mol), 10% (mol) CHOL and 1% (mol) GM1. LUVs containingtrodusquemine were obtained by adding the aminosterol during the hydrationphase to obtain final trodusquemine and total lipid concentrationsof 5 μM and 0.3 mg/mL, respectively.LUVs were obtained by dissolving the desired lipid mixture in chloroform/methanol(2:1) and by removing the organic solvent by evaporation in vacuo for 3 h. The mixtures werehydrated with distilled water to form multilamellar vesicles (MLVs)to a total lipid concentration of 2 mg/mL for quenching experiments,3.5 mg/mL for binding experiments, 1 mg/mL for LUV-oligomer FRET experiments,and 3 mg/mL for circular dichroism (CD), tryptophan fluorescence andintraoligomer FRET experiments (mother solutions). MLVs were leftto swell for 1 h at 60 °C and then extruded 17 times througha polycarbonate membrane with 100 nm pores using a mini-extruder (AvantiPolar Lipids) at the same temperature, to form LUVs. After coolingto room temperature, LUVs were stored at 4 °C for a maximum of1 week. The lipid mixtures were: (i) 1,2-dioleoyl-2 freshlycleaved mica substrate with 10 μL of a 10 mM CaCl2 solution. The samples were kept for 15 min at room temperature andthen incubated for 15 min at 60 °C in a close chamber with 100%relative humidity to form a uniform SLB. Subsequently, the sampleswere cooled at room temperature and gently rinsed three times withMilli-Q water. A Nanowizard III  mountedon an Axio Observer D1  inverted optical microscopewas used to acquire the AFM images. V-shaped DNP silicon nitride cantilevers, with a typical tip curvature radius of 20–60nm, nominal spring constant 0.24 N/m, and a resonance frequency inair ranging from 40 kHz to 75 kHz were used. The measurements werecarried out in water using the intermittent contact mode in the constant-amplitudemode, working with an oscillating frequency of 10–20 kHz. Theamplitude set point was kept above 70% of free oscillation amplitudein all cases. OAs and OBs were administered under the AFM head ata final concentration of 12 μM and left standing to interactwith the SLBs for 30 min. AFM images (512 × 512 image data points)were processed using the JPK Data Processing software . The difference in thickness (ΔZ)between gel (Lβ) and fluid (Lα)lipid domains was determined by considering image height distributions.The distributions were fitted to the sum of two Gaussian functions,and the ΔZ value was determined as the differencebetween the peaks of the two Gaussian functions. This procedure wasrepeated for at least 10 different images for each experiment.Theinteraction was tested on SLBs with three different lipid mixtures:(i) DOPC/SM 2:1 (mol/mol), 1% (mol) CHOL; (ii) DOPC/SM 2:1 (mol/mol),1% (mol) CHOL, 1% (mol) GM1; (iii) DOPC/SM 2:1 (mol/mol), 1% (mol)CHOL, 5% (mol) GM1. Aliquots (40 μL) of LUV suspensions (0.1mg/mL) were deposited onto a 1.0 × 1.0 cm18 Intrinsictryptophan fluorescence spectra were then acquired at 25 °C from300 to 450 nm (excitation at 280 nm) using a 3 mm × 3 mm blackwall quartz cell on a PerkinElmer LS 55 spectrofluorimeter  equipped with a thermostated cell-holder attached to a HaakeF8 water-bath , or on an Agilent Cary Eclipsespectrofluorimeter  equippedwith a thermostated cell holder attached to a Agilent PCB 1500 waterPeltier system.OAs, OBs, andnative HypF-N were diluted in 20 mM phosphate buffer, pH 7.0, in thepresence of different concentrations of LUVs  to a final HypF-N concentration of 1.9 μM (OAs and OBs) and 20 μM(native HypF-N) for 15 min at 25 °C. DLS was used to assess theintegrity of LUVs upon change of solution conditions from distilledwater (in which they were prepared) to 20 mM phosphate buffer, pH7.0. The structural integrity of OAs and OBs upon change of solutionconditions was assessed previously.2 dmol–1, θ is the ellipticity in mdeg, optical path is in cm, concentrationis in g/l, and molecular weight is in g/mol.OAs, OBs and native HypF-Nwere diluted in 20 mM phosphate buffer, pH 7.0, in the presence ofdifferent concentrations of LUVs , to a final HypF-N concentration  of 20 μM for 10 min at 25 °C. The far-UV CDspectra were collected over the 190–260 nm wavelength rangeat 25 °C using a using a 1 mm path length cell on a Jasco J-810spectropolarimeter  equipped with a thermostated cellholder attached to a Thermo Haake C25P water bath .All spectra were truncated at HT > 700 V, blank-subtracted and normalizedto mean residue ellipticity usingE) valuesbetween 18D and 34A in OAs and OBs were calculated asAA and AD represent the absorbancevalues at 336 nm of acceptor (AA = 0.05) and donor (AD = 0.07),respectively, obtained in the presence of a concentration of dye of120 μM; FDA and FA represent the acceptor fluorescenceemission at 1 mM (excitation 336 nm) obtained in the presence andin the absence of donor, respectively, determined from the area between490 and 600 nm below the corresponding curves.32OAs and OBs  formed at a total monomer concentration of 48 μM werediluted in 20 mM and 5 mM potassium phosphate buffer , pH 7.0, to obtain a final HypF-N concentration of 20 μM, and in the presence of differentconcentrations of LUVs  prepared asdescribed above. Oligomers and LUVs were incubated for 10 min at 25°C. The samples were diluted to a final HypF-N concentrationof 2 μM immediately before fluorescence acquisition. Fluorescenceemission spectra were recorded on a PerkinElmer LS55 spectrofluorimeter equipped with a thermostated cell-holder attachedto a Haake F8 water-bath . The measurements wereperformed using a 2 mm × 10 mm quartz cell at 25 °C withexcitation at 336 nm. The FRET efficiency , BODIPY-FL cholesterol , BODIPY-FL-sphingomyelinor BODIPY-FL-DOPC  used as acceptors, with a molar fraction of each labeled lipidof 1% relative to total lipids in all cases. OAs from the C7S/C65Amutant labeled on their surface with 1,5-IAEDANS were prepared ata total protein concentration of 160 μM, as described above,and used as donor (OA-D). Fluorescence spectra of 0.3 mg/mL nonlabeledLUVs incubated with 20 μM OA-D (OA-D spectra), 0.3 mg/mL LUVscontaining lipid-A incubated with 20 μM nonlabeled OAs (Lipid-Aspectra), and 0.3 mg/mL LUVs containing lipid-A incubated with 20μM OA-D (OA-D+Lipid-A spectra) were acquired after 15 min ofincubation on a PerkinElmer LS 55 spectrofluorimeter equipped witha thermostated cell-holder attached to a Haake F8 water bath. Thespectra were acquired from 350 to 600 nm using a 3 × 3 mm blackwall quartz cell at 25 °C, with excitation at 336 nm. The FRET F0 and F are the integrated fluorescence intensityareas at 400–500 nm in the absence and presence of the quencher, respectively; [Q] is the concentrationof the quencher and KSV is the Stern–Volmerconstant. The plot of quenching of TMA-DPH and DPH in 0.3 mg/mL LUVscontaining 5 μM trodusquemine was analyzed from 12.5 μMOAs with an equation derived from the Stern–Volmer equationq isthe intercept and all the other parameters have the same meaning asin 1,6-Diphenyl-1,3,5-hexatriene and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatrienep-toluenesulfonate  were dissolvedin chloroform/methanol (2:1) and added to the lipid mixture to obtaina probe:lipid molar ratio of 1:300. LUVs were then prepared at 2 mg/mLas described above, diluted with distilled water to 0.3 mg/mL andincubated with increasing concentrations of OAs, OBs, and native protein(0 to 32.5 μM) at 25 °C for 15 min in the dark. The fluorescencespectra of the resulting samples were acquired at 25 °C from380 to 550 nm (excitation 355 nm) using a 3 mm × 3 mm black wallsquartz cell on an Agilent Cary Eclipse spectrofluorimeter  equipped with a thermostated cellholder attached to an Agilent PCB 1500 water Peltier system. The quenchingof TMA-DPH and DPH was then analyzed with the Stern–VolmerequationF isthe observed fluorescence at 512 nm, [OA] is the molar concentrationof OAs , fU and fB are the fluorescence emission of the unboundand bound OAs at unitary concentration of OAs, respectively, m is the dependence of F on [lipid] afterbinding (drift), [lipid] is the molar concentration of total lipidsin LUVs, and KD is the dissociation constant.OAs, OBs and native HypF-N formed by BODIPY FL-labeled and unlabeledHypF-N C7S/C65A mutant (molar ratio 1:10) were diluted in 20 mM phosphatebuffer, pH 7.0, to obtain a final HypF-N mutant concentration of 20μM , and were incubated for 15 min at 25°C, with increasing concentrations (from 0 to 2.0 mg/mL) of LUVsprepared as described above. In experiments involving trodusquemine-containingLUVs, the concentration of the small molecule was variable but thetrodusquemine/lipid molar ratio was maintained and corresponded to5 μM in 0.3 mg/mL LUVs. The fluorescence spectra were acquiredat 25 °C from 490 to 560 nm (excitation 480 nm) using a 3 mm× 3 mm black walls quartz cell on the Agilent Cary Eclipse spectrofluorimeterdescribed above. The fluorescence emission at 512 nm was then plottedversus LUV concentration and analyzed with


Cladonia cariosa and Himantormia lugubris: protolichesterinic acid (1), fumarprotocetraric acid (2), and lichesterinic acid (3). The maleimide cysteine labeling assay showed that compounds 1, 2, and 3 inhibit at 50 µM, but compounds 2 and 3 are statistically significant. Based on its inhibition capacity, we decided to test compound 2 further. Thus, our results suggest that compound 2 remodel soluble oligomers and diminish β sheet content, as demonstrated through ThT experiments. Hence, we added externally treated oligomers with compound 2 to demonstrate that they are harmless in cell culture. First, the morphology of cells in the presence of aggregates does not suffer evident changes compared to the control. Additionally, the externally added aggregates do not provoke a substantial LDH release compared to the control, indicating that treated oligomers do not provoke membrane damage in cell culture compared with aggregates alone. Thus, in the present work, we demonstrated that Michael’s acceptors found in lichens could serve as a scaffold to explore different mechanisms of action to turn tau aggregates into harmless species.Neurodegenerative disorders, including Tauopathies that involve tau protein, base their pathological mechanism on forming proteinaceous aggregates, which has a deleterious effect on cells triggering an inflammatory response. Moreover, tau inhibitors can exert their mechanism of action through noncovalent and covalent interactions. Thus, Michael’s addition appears as a feasible type of interaction involving an α, β unsaturated carbonyl moiety to avoid pathological confirmation and further cytotoxicity. Moreover, we isolated three compounds from Antarctic lichens  These two motifs are within the microtubule-binding domain (4R) of tau and are prone to form a cross β structure [Alzheimer’s disease (AD) is the most prevalent form of dementia , involviInterestingly, the first stage of oligomer formation can find a mixture of a random coil and β sheet structure. However, as long the tau polymerization occurs, mature oligomers are formed whose content is a majority of β sheet content . These o2 can prevent cell membrane damage and further LDH leaking.Additionally, tau is related to FTD linked to chromosome 17, where the neuronal loss occurs in the absence of Aβ . Thus, c2 and 3. As a control, we used dimethyl sulfoxide (DMSO) as an oxidizing agent capable of interacting with cysteine, as shown in The lichens compound isolated , tested 2 showed fewer illuminated elements, suggesting that compound 2 can interfere with cysteine interactions that promote further assembly of soluble oligomers  selectively illuminates the fluorophore linked covalently to cysteine. The control aggregation image  were placed in 200 mL of chloroform . After filtration, the organic solutions were concentrated under reduced pressure to obtain a chloroformic extract (2 g). All chloroformic extract was dissolved in a minimum volume of boiling dichloromethane. The solution was allowed to cool to crystallize the fumarprotocetraric acid 2 (100 mg). The di-chloromethane solution was then concentrated (1.5 g) and submitted to column chromatography (CC) on silica gel 60  and eluted with CHCl3/MeOH mixtures (200 mL each) of increasing polarity (9:1 up to 1:9) and MeOH. The eluates were monitored and pooled according to TLC analysis and then concentrated under reduced pressure to produce the isolation of usnic acid (250 mg) and protolichesterinic acid 1 (15 mg), as described [escribed .Himantormia lugubris (20 g) was dried, powdered, and extracted with dichloromethane . The organic solutions were filtered and concentrated under reduced pressure, yielding 1.5 g of extract. The DCM extract was dissolved in methanol and subjected to Sephadex LH-20 using mobile phase MeOH. Some 70 fractions (15 mL) were monitored by TLC and combined to give two fractions, A-B. Fraction A (1.0 g) was chromatographed on silica gel  using n-hexane/EtOAc mixtures (0 up to 100%) to yield 120 mg of usnic acid and 20 mg of lichesterinic acid 3, as described [2, 63–200 µm) using DCM/MeOH mixtures (0 up to 100%) and giving barbatolic acid (400 mg).escribed . Fractio2HPO4, and 500 mM NaCl. The purity of the protein was verified on a Coomassie brilliant blue-stained SDS-polyacrylamide gel. The protein was concentrated and stored at −80 °C until use. The concentration of purified 4R was determined using the extinction coefficient at 280 nm (1520 M−1 cm−1).Tau fragment 4R htau244-372) was amplified by using the plasmid for htau40 as a template. The PCR amplified sequence was subcloned into a pET-28a vector (Novagen) to produce a His-tagged protein. The recombinant fragment 4R was expressed in Escherichia coli strain BL21 (DE3), as described [4-372 was2. After 48 h of incubation, 100 μL of a 25 μM of ThT was added to the solution and incubated for 1 h at room temperature before fluorescence reading. Then, fluorescence was measured in a Biotek H1 multimode reader  with an excitation wavelength at 440 nm and emission wavelength at 485 nm in a 96-well plate, as described. Each experiment was done at least in triplicate, and background fluorescence was subtracted as needed.The ThT fluorescence was done as described . BrieflyAll atomic force images were obtained in a Nanoscope IIIa, in a fluid cell using the tapping mode through a SiN cantilever, whose stiffness constant ranges from 0.12 to 0.15 N/m. The tapping mode provides the phase information, making it possible to distinguish between the sample elements and establishing their presence or absence. In addition, AFM images in the tapping mode allow for determining the membrane surface’s topographic details, verifying its thickness using a cross-section obtained from the topographic image.Protein samples were reduced with TCEP (tris(2-carboxyethyl) phosphine) by using a 10 M excess (TCEP 400 µM:4R 40 µM). Then, the mix was incubated for 2 h at 37 °C and eluted by using an Amicon Ultra centrifugal filter unit at 3500 rpm for 5 min at 4 °C . After that, the protein was incubated with the fluorophore Alexa 488 Maleimide molar ratio (2:1) for 2 h at 30 °C. The excess of the fluorophore was eluted as described before. The induction of the aggregation of the protein labeled was done as described ,25. BrieSamples were analyzed in a microscope Olympus IX81 linked to a color camera PL-B778F . Briefly, samples were diluted at 1:10 in ultrapure water. Then, 5 µL were placed over the Menzel-Glaser coverslips (1.5 × 24 × 60 mm) over Ig TIRF (TIRF LAB). The laser, 465 nm, was turned on linked to a side-end excitation light launcher (TIRF LAB) to excite the fluorophore attached to the 4R . Then, we selected the proper filter (FITC/EGFP/Fluo 3/DiO Acridine Orange(+DNA)). The samples were irradiated, acquiring image scanning of the surface of the coverslips by using the software Pixelink Capture OEMa (exposure 250 ms and gain 8 db).2, using MMM Group® Stove, Standard CO2 Cell 190, in DMEM D1152 plus F-12 medium supplemented with 10% fetal bovine serum (FBS) , 100 μg/mL ampicillin, and 100 μg/mL streptomycin, in T25 cell culture flasks. The cells were cultured and differentiated in Petri dishes, 35/10 mm over coverslips, then FBS was added at 2.5% plus retinoic acid of 10 μM for five days, replacing the medium culture every 48 h for neurite growth. Treated oligomers and oligomers were added on day four for 24 h. After that, cells were fixed for immunofluorescence and the supernatant recovered for LDH measurement. Cells previously incubated with either tau oligomers treated or oligomers alone were transferred to glass coverslips. The cells were then washed twice with 1X PBS buffer , fixed for 30 min with 4% paraformaldehyde at room temperature, washed three times with PBS, and permeabilized for 10 min with Triton X100 0.5%  diluted in PBS. Finally, coverslips were washed three times with PBS. After that, the cells were incubated with 1% BSA  for 30 min at room temperature. Subsequently, samples were incubated at 37 °C for one hour with the supernatant of α tubulin monoclonal TU-01 [N2a neuroblastoma cells were cultured at 37 °C in a humidified atmosphere at 5% COal TU-01 ,27, dilual TU-01 . The enzData analysis was performed using GraphPad Prism 6.0. Data are presented as mean ± SD and analyzed using one-way ANOVA. The analysis of TIRFM images was performed by using MATLAB and FIJI software. The experiments were done in triplicate.


Leishmania and spread by sandflies. The standard therapy for this ailment is the first-line medication of pentavalent antimonial and the second drug line of pentamidine amphotericin B. All are practiced over the years and exhibit adverse toxicity effects. Herbal product-derived medicine is a promising potential source for treating parasitic diseases. Xanthatin, a xanthanolide sesquiterpene lactone, is isolated from Xanthium strumarium L. treats several ailments in many countries. In the present study, we investigated the leishmanicidal activity of the xanthatin by using a metabolomics-based analysis in J774 macrophages and amastigotes phases in Leishmania major. Xanthatin was isolated and identified by NMR spectroscopy. Macrophage toxicity of xanthatin performed by MTT assay. Macrophages infected by the L. major’s promastigote stationary phase, the infection rate (IR), and multiplication index (MI) were calculated. Axenic amastigotes were treated with xanthatin. Cell quenching and metabolite extraction were performed, and the metabolome profile was analyzed with NMR spectroscopy. Outliers were classified by using multivariate statistical analysis software, and relevant metabolites and pathways were worked out. The xanthatin IC50 rate defined 0.75 µg/mL base on macrophages viability and also in-vitro activity of xanthatin on amastigotes showed the best leishmanicidal activity in IR and MI values of 53% and 62.5%, respectively. Xanthatin altered amino sugars and nucleotide sugars metabolism, starch and sucrose metabolism, cyanoamino acid, and galactose metabolism. Our finding revealed that the main target of xanthatin is carbon metabolism, which is an essential step for amastigotes virulence. Cutaneous leishmaniasis is caused by protozoa of the genus  Leishmania and transmitted by sandflies to various mammal hosts, including humans. CL has an annual incidence of 0.6 to 1 million cases, with a mortality rate of 60000 per year , meglumine antimoniate (glucantime), and the second drug line, pentamidine amphotericin B, which used for over the years, and they show adverse reactions and toxicity (Cutaneous leishmaniasis (CL) is considered a protozoan zoonotic disease induced by protozoa of the genus toxicity , 3.Xanthium strumarium L., an annual herb that belongs to the family Asteraceae genus Xanthium  . Preparation of plant crude extractsX. strumarium (4 kg) were macerated with an Ethanol-water solution (80%) at room temperature for 72 h. The resulting extract was mixed with active charcoal, centrifuged to isolate plant pigments. This mixture was filtrated and concentrated under reduced pressure in rotavapor at 45 °C to obtain a dark brown syrup (450 g) (The air-dried leaves of  (450 g) . Isolation and characterization of xanthatin et al.  was cultured in RPMI 1640 medium (Atocel), containing 10% FBS (Gibco), Penicillin100 U/mL - Streptomycin 100 µg/mL (Sigma) as antibiotics at 37 °C with 5% COParasite cultureL. major (strain MRHO/IR/75/ER) from the lymph node of infected BALB/c mice was grown in M199 medium  supplemented with 10% FBS, Penicillin100 U/mL - Streptomycin 100 µg/mL, two mM L-glutamine, 0.1 mM adenosine, 0.5 μg⁄mL hemins, and 40 mM HEPES at 23-26 ºC for 5-6 days. The stationary phase of promastigotes was centrifuged at 3000 rpm for 10 min at 4 °C and used to infect J774 macrophages , sterilized by 0.22 μm micro-filters as stock solution and kept at 4 °C until use.Macrophage toxicity assay 3cell/well) were seeded in every well of 96-well culture plate (in triplicates) and incubated in RPMI 1640 supplemented with 10% FBS 37 ºC with 5% CO2 to attach. After 24 h, all wells’ medium was replenished with different xanthatin fractions (1050 to 0.007 µg/mL). The wells with no xanthatin (only cells and medium) and the wells without cells and xanthatin were considered negative control and blank sequentially. Also, Amphotericin B was used as positive control. The plate was incubated at 37 °C for 48 h. After adding 20 µL thiazolyl blue tetrazolium bromide (MTT) reagent (5 mg/mL in PBS) into each well, the plate was kept at 37 °C until the purple formazan crystals were visible under a microscope after 4 h. All medium was then discarded, and added 100 µL of DMSO into each well, including controls, shacked gently to dissolve formazan crystals and observe purple. Every well’s absorbance was recorded at 570 nm against 630 nm as a reference using a plate reading spectrophotometer. The formula Below determined the value of 50% inhibitory concentrations (IC50) and Percentage macrophage viability (Equation 1) (J774 macrophages (8 × 10ation 1) :Macrophage infection4 cells of J774 macrophages seeded in 24-well culture plates equipped with 10 mm coverslips. The coverslips containing cells were incubated 24 h to adhere in the M199 medium at 37 °C with 5% CO2. Then, non-adherent cells were washed with phosphate-buffered saline (PBS) and infected with a stationary phase of L. major (10:1 parasite/macrophage) at 37 °C 5% CO2 for 4 h. Non-internalized promastigotes were washed with PBS (4 times), and the plate was incubated again in M199 medium supplemented with 2% FBS for 24 h  once. The three wells, amastigotes without any xanthatin, considered negative controls, and amphotericin B, was used as a positive control to compare the efficacy of xanthatin. All M199 medium and xanthatin concentrations were replenished every 24 h for three days. Then, wells were washed with PBS, fixed with methanol, and stained with 10% Giemsa. The infection rate (IR Equation 2) and multiplication index  in each stained coverslip were determined microscopically and calculated. Using two Formulas Below , 22:Treatment of amastigotes with xanthatin7 J774 macrophages (in each 175 cm cell culture flask) infected with 4 × 108 stationary promastigotes of L.major . All samples were incubated in an M199 medium containing 2% FBS at 37 °C with 5% CO2 for 24 h. Based on the xanthatin IC50 value, every experimental flask was treated with 1.575 µg/mL of xanthatin. The medium was replenished daily for three days in every flask. After five days, cells in each flask were washed once in PBS 1X (pH 7.4) gently, trypsinized, and transferred to 50 mL falcon tubes  was washed using ice-cold PBS (three times) to remove any impurities and residual media. The quenching process was done by tubes immersing into liquid nitrogen until freezing and thawing in an ice bath (0 °C)  was added, followed by 5 min the sonication in an ice bath to disrupt the amastigotes membrane. All samples were kept in an ice bath for one hour to allow the precipitation of potassium perchlorate. After centrifugation at 12000 rpm at 4 °C for 10 min, the supernatant’s pH adjusted to 7.2, the samples were lyophilized and stored at −80 °C until future analysis  . The celanalysis . 1HNMR Spectroscopy2O buffer  containing 1 mM trimethylsilyl propionate (TSP) as a chemical shift reference (δ = 0 ppm) and two mM imidazole as pH indicator (δ = 5.50 to 8.80 ppm). Finally, samples were centrifuged at 18000 rpm for 15 min at 4 °C, and 500 µL supernatants were transferred into an NMR probe. Lyophilized sample (n = 10) dissolved in 600 µL phosphate D1HNMR spectra were recorded using a 10-µs pulse, 0.1 s mixing time, 3.0 s relaxation delay, 6009.6 Hz spectral width, and 3000 transients with standard 1D NOESY pulse sequence to suppress the water peak  code in MATLAB (v.7.8.0.347) to convert spectra into a suitable format for multivariant analysis. Spectra binned into 0.005 ppm chemical shift, and water peak at 4.7 ppm eliminated. Data normalization and Pareto scaling were performed before data classification. A supervised Partial Least Square- Discriminate Analysis (PLS-DA) classification technique was used to identify notable outliers between experimental groups. Human Metabolome Database (HMDB) and LeishCyc databases were utilized to discover the corresponding metabolites to spectral outliers. Metabolic pathways worked out by using online MetaboAnalyst Ver. 3.0  a metabolomics analysis software and SPSS to determine the p values.Isolation and identification of xanthatinet al. (Adequate quantities of xanthatin (15 mg) were collected by column chromatography, followed by TLC techniques . Crystalet al.  Cytotoxicity effect of Xanthatin on Macrophages J77450 defined as 0.75 µg/mL. Nevertheless, xanthatin with our stock concentration (1050 µg/mL) showed the highest cytotoxic activity (100%).Cell viability results of different xanthatin concentrations on J774 macrophages are described in The results for the activity of xanthatin and amphotericin B on amastigotes are depicted in 1H- NMR spectroscopy analysisp-value of pathways value, less than 0.05 was taken for discussion in this article.The NMR spectroscopy analysis revealed a spectral peak variation in two groups of experiments; changes are more confined to regions 3, 6, and 8 ppm of spectra. PLS-DA score plot revealed that both groups had accurately classified , and thei.e., amphotericin B, pentamidine, Miltefosine, and paromomycin, are known as efficient drug medications leishmaniasis therapy . Therefore, the main drug target of pentavalent antimonies is based on the inhibition of  pathway , 31. The pathway , 32. It  pathway , and par pathway , 27. . Based on our results, as the strength of anti leishmanicidal activity of drugs maters, xanthatin may show equal potency compared to amphotericin B, and the affected pathways are confined to amino sugar and nucleotide sugar metabolism, starch and sucrose metabolism, cyanoamino acid metabolism, Galactose metabolism, and pentose phosphate pathway only.Amphotericin B binds to membrane sterols and phospholipids and kills the parasite by altering membrane permeability, cellular potassium, magnesium, glucose, and water disbalance . ResearcLeishmania’s energy demand is provided by carbon transformation in glycolysis, Krebs cycle, gluconeogenesis, and pentose phosphate pathway, which is called central carbon metabolism. It is believed that the living and prime proliferation of Leishmania amastigotes in mammalian hosts depend on phagolysosome metabolism.Generally, Leishmania genomes (35). Various types of sugars in phagolysosomes have emerged from the turnover of extracellular matrix compositions such as host glycoproteins, glycosoaminoglycans, and proteoglycans (34). Also, several genes related to hexosamines metabolism are reported in et al. showed that scavenged hexosamines are vital sugars in leishmania phagolysosomes. Their catabolism is necessary to maintain critical biochemical pathways and prevent hexosamine toxicity (36). Naderer et al. postulated that some phosphorylated hexosamines produce surface glycoconjugates like GPI and mannogens in glycosomal glycolysis and pentose phosphate pathways (30). So, these pathways play essential roles in the pathogenesis of amastigotes.Malcolm J. p-value ≤ 0.05) within our findings, some hexosamines metabolites like; D-glucosamine 6-phosphate, N-Acetyl-alpha-D-glucosamine in amino sugar, and nucleotide sugar metabolism were the most changed metabolites. Based on statistical significance (L. major amastigotes is utilized as sources of carbon and energy (36). Also, enough a concentration of amino sugars like glucosamine and N-acetyl glucosamine is needed to maintain the lowest hexosamines in the phagolysosome. They are transported to the glycosome and, after phosphorylation, convert to fructose-6-phosphate by some enzymes such as glucosamine 6-phosphate deaminase (GND).Amino sugars pathway in Leishmania parasite could be targeted by GND, which plays an essential role in the growth and development of leishmania infection in susceptible animals (36).Former results demonstrated that in a situation in which hexosamines are considered the primary source of carbohydrate, glycosomes of the  Fructose-6-phosphate is a crucial metabolite in glycoconjugate biosynthesis, and it is finally exported to the cytoplasm to produce compositions like mannogens and N-glycan. Our results showed that treated amastigotes by xanthatin exhibited some variations in hexose metabolites such as alpha-D-glucose, glucose 1-phosphate, beta-D-fructose, fructose 6-phosphate, and mannose 6-phosphate. Our results are then approved by earlier studies and suggest that any variation in the first metabolites of the amine sugar pathway or GND results in subsequent metabolite alterations of parasites. Moreover, our findings revealed that some metabolites, including; beta-D-fructose, alpha-D-glucose, and glucose 1-phosphate, have changed in starch and sucrose and Galactose metabolism pathways. Thus, it seems that the central role of xanthatin in amastigotes metabolism might have been performed by these critical metabolites, although further investigation should conduct to confirm this statement.L.major amastigotes. Amastigotes de novo pathway synthesizes non-essential amino acids like aspartate, alanine, and glutamate. On the contrary, with promastigotes, fatty acids have an essential role in de novo biosynthesis in phagolysosomes (34). Intracellular amastigotes are dependent on fatty acid β-oxidation and Krebs cycle for energy production. It is also stated that acetyl-CoA result from fatty acid β-oxidation is expensed in the Krebs cycle to produced intermediates for biosynthesis of non-essential amino acids, and they are used for biosynthesis of amino sugars, nucleotides, and thiol in amastigotes (36-38). The viability and virulence of amastigotes in phagolysosomes effect via disruption of key enzymes in the Krebs cycle for non-essential amino acid biosynthesis (29), because of the inhibition of some genes and proteins involved in β-oxidation and mitochondrion respiratory chain . Our metabolomics-based study also revealed that the amastigote concentration of aspartate and asparagine amino acids in cyanoamino acid metabolism has changed as two targets for xanthatin. Some studies reported that the rate of amino acid oxidation elevates in Leishmania species. Several studies focused on the pentose phosphate pathway role and its enzymes in some trypanosomes and Leishmania species . Mehlotra et al. declared that there are not enough antioxidant enzymes available in Leishmania promastigotes, so they are sensitive to oxidative stress (44), and Deschacht focused on the relationship between oxidative stress mechanism and Leishmania infection (45). Moreover, Mukherjee et al. postulated that amphotericin B could affect macrophages’ antioxidant system . Therefore, some researchers concluded that the pentose phosphate pathway could be considered as drug target for the treatment of leishmaniasis . Our research revealed that Beta-D-glucose 6-phosphate, 6-D-phosphogluconate, and D-ribose 5-phosphate in the pentose phosphate pathway varied significantly between the two groups of study. Hence, our finding is commonly in line with prior reports. Glucose 6-phosphate and 6-phosphogluconate in the pentose phosphate pathway are catalyzed by oxidative branch enzymes, including glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase to produced Ribose 5-phosphates and NADPH. Ribose 5-phosphates needed for DNA and RNA, another nucleotide biosynthesis (41), and NADPH plays a crucial role in antioxidant defenses against XanthiumStrumarium extracts on leishmaniasis. This present pilot study was conducted for the first time to survey the antileishmanial activity of xanthatin and its impact on macrophages and amastigotes’ metabolism. Our finding suggests that the main target of xanthatin in amastigotes is carbon metabolism, which is an essential step for amastigotes virulence. Variations in the concentration of some essential metabolites in amino sugars and nucleotide sugars metabolism, starch and sucrose metabolism, cyanoamino acid, and galactose metabolism pathways might be considered valid drug targets. Nevertheless, additional investigations need to elucidate the biochemical and molecular mechanisms of action of xanthatin.Currently, there is a strong trend in herbal medicine for the treatment of leishmaniasis worldwide. To date, several research studies have focused on the therapeutic effects of The authors have no conflict of interest to declare.