Musa acuminata (MA) is a popular fruit peels in the world. Non-food parts of the plant have been investigated for their antioxidant and anti-ulcerative colitis activity. Metabolomic approaches were found to be informative as a screening tool. It discovered different metabolites depending on statistical analysis. The antioxidant activity content was measured by colorimetric method. Seventy six investigated metabolites were observed. The identities of some of these markers were confirmed based on their MS2 fragmentation and NMR spectroscopy. These include: cinnamic acid and its dimer 2-hydroxy-4-(4-methoxyphenyl)-1H-phenalen-1-one beside; gallic acid and flavonoids; quercetin, quercetin-3-O-β-d-glucoside, luteolin-7-O-β-d-glucopyranoside. GC/MS analysis of MA peels essential oil led to identification of 37 compounds. The leaves, pseudostem and fruit peels extracts were tested for their safety and their anti-ulcerative colitis efficacy in rats. Rats were classified into: normal, positive, prednisolone reference group, MA extracts pretreated groups (250–500 mg/kg) for 2 weeks followed by induction of ulcerative colitis by per-rectal infusion of 8% acetic acid. Macroscopic and microscopic examinations were done. Inflammatory markers  were measured in sera. The butanol extracts showed good antioxidant and anti-inflammatory activities as they ameliorated macroscopic and microscopic signs of ulcerative colitis and lowered the inflammatory markers compared to untreated group. MA wastes can be a potential source of bioactive metabolites for industrial use and future employment as promising anti-ulcerative colitis food supplements. Inflammation occurs as a defensive mechanism against spread of traumatic insult of irritating agent which leads to occurrence of inflammatory activity and subsequent threat to affected organ. Inflammatory diseases can affect the patient’s life quality adversely as it may lead to progression to more serious conditions that hinder normal activity as neuritis, osteoarthritis and digestive system inflammation that may progress to ulceration, which necessitates great care and continuous treatment with specific regimens3. One of the well-established characteristic of UC is chronic, colonic mucosal inflammation that is usually relapsing and is manifested by episodic attacks of severe abdominal pain that persist for a period of time and is associated with catarrhal or bloody diarrhea4.The most serious digestive system inflammatory diseases are those of bowel diseases including ulcerative colitis which affect a considerable range of population around the world. The rate of occurrence of ulcerative colitis (UC) is very high and is continuously increasing yearly. It affects patients at early adolescence and continues to progress throughout life5, which may end up by total or partial colectomy6. That’s why there is need for introduction of supplementary herbal medicines in the regimen of treatment of UC provided that they don’t have evidenced side effects1. Musa spp. (bananas) is a good sources of carbohydrates, proteins, other vitamins and minerals. They contain different amino acids like threonine, tryptamine, tryptophan, as well as flavonoids, dopamine, beta-carotene and sterols7. Studying the biological activities of banana different parts was carried out in various studies; where stem were studied as antidiabetic supplements which improved the level of insulin and reduced blood glucose as well as glycosylated haemoglobin through modulating carbohydrate metabolizing enzymes activity9, also they have glycaemic effects oweing to their high content of sodium and potassium, fruit peels were studied as healing and anti-ulcerative agents, moreover methanolic as well as aqueous extracts of Musa paradisiaca (banana) were reported to produce wound healing in rats10, peel were studied for their immunomodulatory effects11. Additional evidence of the healing activity of Musa paradisiaca is that when ulcer was induced in experimental animals using non- steroidal anti-inflammatory drugs or steroid or histamine, oral administration of banana pulp powder showed marked antiulcerogenic effect12.Unfortunately as a whole current medication aren’t always to the expectations and have adverse effects on patient’s health, as treatment of mild or moderate UC is achieved by 5-aminosalicylates or sulfasalazine, while moderate or severe UC are treated by high-dose of oral or intravenous corticosteroid. However primary remission may not be accessed for all patients and consequently corticosteroid dependence or resistance may occur2 fragmentation. These include quercetin-O-rhamnoside-O hexoside, kaempferol-3-O-rutinoside, quercetin-O-hexoside, isorhamnetin-O-rutinoside and hexadecanoic acid.Phytochemical profile studying using a metabolomic approach of Leaves of bananas in previous studied using UPLC–QToF–MS technology showed that thirty-one compounds were identities of some of these markers based on their MS7.Though accessions by soxhlet gave better yield (20.0–60.0%) than by sonication (18.4–23.0%),neither motherland nor methods of extraction had any significant role in the separation processThis research was carried out to investigate the chemical composition, and phytochemicals of some banana by-products; leaves, stem and fruit peels and study their potential protective effects against colonic inflammatory insult induced by acetic acid in rats, to mimic signs and symptoms of the serious devastating inflammatory bowel disease “ulcerative colitis” aiming at introducing a new functional anti-inflammatory food supplement.Guideline ethics for plant usage in the phytochemical study The present study complies with local and national guidelines as permission was obtained for collection of plant material.Guideline ethics for experimental animal handling in the in vivo pharmacological study The study was done in accordance with the guide for care and use of laboratory animals Experiments were performed according to the National Regulations of Animal Welfare and the Institutional Animal Ethical Committee (IAEC) in Egypt, and is reported in accordance with Animal Research: Reporting of in vivo Experiments (ARRIVE) guidelines.Ethical approval was obtained from National Research Centre ethics committee under number 16/138.ABTS, DPPH, Trolox and FCR were purchased from Sigma Aldrich (GmbH). All chemicals and solvents used in this study were supplied by Fisher Scientific UK  with high analytical grade.Musa acuminata waste parts  were collected from a private farm at Anshase, Sharqia Governorate, Egypt and identified by Dr. Mohamed El Gebally, Former researcher of plant taxonomy, National Research Center and Senior of of plant taxonomy at Orman Botanical garden, Giza, Egypt. Herbarium specimens no S7570, S7571 and S5772, respectively were deposited at the herbarium of the National Research Centre, Dokki, Cairo, Egypt. The powdered MA wastes (0.5 kg) individually were exhaustively extracted three times with 5L of methanol at room temperature, then filtered and concentrated under reduced pressure at 45 °C using rotary evaporator to yield crude extracts 75.5 g (stem), 81.7 g (leaves) and 87.2 g (fruit peels). The three crude residues were suspended in 1L water, left overnight and then it was successively partitioned with 0.7 L of petroleum ether, chloroform three times, followed by 0.7 L ethyl acetate then 0.7 L n-butanol three times repeatedly.13. From 50% of alcohol gives five compound flavonoids and phenolics acids and one compound from 70% alcohol that identified by spectral tools data.Chromatographic procedure for isolation and identification of some phenolics compounds from three kg of powdered leaves were extracted with EtOH (80%) by soaking at room temperature. The combined alcoholic extracts were concentrated under reduced pressure at 45 °C, which yield 365 g of residue. The crude residue was suspended into water then overnight partitioned with petroleum ether, methylene chloride, ethyl acetate and n-butanol. The n-butanol fraction was chosen for further purification. The n-butanol fraction (g) was loaded on a column (3.5 cm × 150 cm) of Polyamide, and the column was stepwise eluted with water, 30, 50, 70, and 100% methanol at a flow rate of 50 mL/min to yield five sub fractions. 50% and 70% methanol sub-fraction was further purified using a sephadex LH-20 column chromatography and eluted with 50% methanol, to yield 6 compound. The jar of different elution solvent systems were used . After air-drying, the spots were visualized under UV light14. The resulted essential oil was separately dehydrated with anhydrous sodium sulphate and kept in deep freezer at − 20 °C until GC/MS analysis. The analysis was done in triplicate and the mean values of the oil content (%) were recorded. The components of essential oil in MA were identified by GC/MS analysis instrument stands at the Department of Medicinal and Aromatic Plants Research, National Research Center with the following specifications. Instrument: a TRACE GC Ultra Gas Chromatographs , coupled with a THERMO mass spectrometer detector (ISQ Single Quadrupole Mass Spectrometer). The GC/MS system was equipped with a TG-WAX MS column . Analyses were carried out using helium as a carrier gas at a flow rate of 1.0 mL min−1 and a split ratio of 1:10 using the following temperature program: 60 °C for 1 min; rising at 4.0 °C min−1 to 240 °C, and held for one minute. The injector and detector were held at 210 °C. Diluted samples  of one μL of the mixtures were always injected. Mass spectra were obtained by (EI) at 70 eV, using a spectral range of m/z 40–450. Most of the compounds were identified using the analytical method: mass spectra 15.The volatile oil of waste fresh fruit peels MA was extracted by water distillation method as Solvent-Assisted Flavour Evaporation (SAFE). The homogenate was continuously steam-distilled by diethyl ether (25 mL) extracted in 3 h in a Likens-Nickerson apparatus(a) Quantitative of phenolic compounds from Butanol fractions of three parts of MA were measured using authentic fifteen standards. The HP 1100-HPLC system  with an auto-sampler (G1329B), quaternary pump and a diode array detector.The measurements were integrated by chemstation chromatographic software interfaced to a personal computer. The analytical column was ZORBAX Eclipse XDB C18 column . Operative conditions were: mobile phase A, 2% acetic acid; mobile phase B, acetonitrile; flow rate, 0.85 mL/min; fixed wavelength, 280 and 360 nm; injected quantity, 10 µL; elution program (%), A:B as followed: 0 min 90/10; 10 min 50/50; 15 min 20/80; 20 min 90/10; 25 min 90/10. Identification of phenolic compounds was performed by comparison with the retention times of standard substances.16. The second instrument; UPLC  coupled to Q-Exactive hybrid MS/MS quadrupole—Orbitrap mass spectrometer . The system of separation Chromatography was carried out using solvent (A) water acidified with 0.1% formic acid and (B) acetonitrile (solvent B). The flow of mobile phase 0.4 mL/min was adapt to the following sequences: 0–15 min 95:5, 15–22 min 50:50, 5 min for maintained the conditions 0.2:98, then system returned to the starting conditions and was re-equilibrated for 3 min. with column C18 . Q-Exactive MS was operated upon following settings: the HESI ion source voltage (− 3 kV or 3 kV). The sheath gas (N2) flow 48 L/min, auxiliary gas flow 13 L/min, ion source capillary temperature 250 °C, auxiliary gas heater temperature 380 °C. The CID MS/MS experiments were performed using collision energy of 15 eV. Data recording and processing were performed using the Xcalibur 4.0 software with accuracy error threshold at 5 ppm and Imported data from raw MS data to export (abf) format were packaged in MS-DIAL 4.61 an enhanced standardized untargeted lipidomics and metabolomics by using (MSP) format libraries databases17.(b) Instrument stands at the department of Metabolomics Groups, Institute of Plant Genetics of the Polish Academy of Sciences, Poznan, Poland, with the following specifications: first instrument; ion-trap Esquire 3000 mass spectrometer equipped with ESI was operated in negative ion mode with scan range was 15–3000 m/z and scan resolution was 13,000 m/z/s18. ABTS+ dissolved in water to a 7 mM concentration. ABTS stock solution with 2.45 mM potassium persulfate  and allowing the mixture to stand in the dark at room temperature for 24 h before use Oxidation of the ABTS was performed according to Dinkova-Kostova et al.19. The inhibition of the DPPH and ABTS radical were calculated using the following formula: % Inhibition = [(A control − A sample)/A control] × 100. Where; A is the absorbance at 517 nm in DPPH and 734 nm in ABTS by using UV spectrophotometer (Agilent Technologies Carry 100 UV–Vis) was used for absorption measurements.DPPH and ABTS free radical-scavenging activity method was adopted to measure the in vitro antioxidant activity of MA ethanolic, butanolic, ethylacetate, chloroform and petroleum ether extract; at different concentration  of the three parts extract; ascorbic acid and trolox was used as a positive control. The radical scavenging model for antioxidant activity, using 1,1-diphenyl-2-picrylhydrazyl , was performed according to Shimada et al.Animals The present study used male Wistar albino rats, of body weights (bwt) (150–175 g) obtained from the animal house colony of the National research centre, Dokki, Giza, Egypt. The rats were kept in standard metal cages in an air conditioned room at 22 ± 3˚C, 55 ± 5% humidity and provided with standard laboratory diet and water ad libitum. The present study complies with local and national guidelines, as it was done in accordance with the guide for care and use of laboratory animals and obtained ethics committee approval certificate from National Research Centre ethics committee numbered 16/138. Experiments were performed according to the National Regulations of Animal Welfare and the Institutional Animal Ethical Committee (IAEC) in Egypt,and is reported in accordance with Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines.Drugs and chemicals (a) Prednisolone  used as reference anti-inflammatory drug and was given orally by gastric tube. (b) Acetic acid  injected per rectum (Pr) for induction of colonic inflammation and ulceration; a model mimicking ulcerative colitis. (c) Diethyl ether , for animal anaesthesia during blood sampling from retro-orbital plexus of veins and for euthanasia under anaesthesia. (d) Formaldehyde , for fixation of postmortem tissues dissected for histopathologic examination.Diagnostic kits (a) Kits for determination of Liver function tests  and Kidney function tests (urea and creatinine) in serum were purchased from Biodiagnostic company, Dokki, Giza, Egypt. (b) Serum highly sensitive C-Reactive Protein (CRP) was measured according to the manufacturer kit using rat hs-CRP ELISA kit from Wuhan Fine Biotech. Ltd. Co., China. (c) Serum Interleukin-6 was measured according to the manufacturer kit using rat IL-6 ELISA kit Cat. no. ER0042 from Wuhan Fine Biotech. Ltd. Co., China. (d) Serum Anti-Neutrophil Cytoplasmic Antibodies (ANCA)was measured according to the manufacturer kit using rat ANCA ELISA kit Cat. no. SL1417Ra from SUNLONG Biotech. Ltd. Co., China.In vivo biological studies were conducted to investigate some pharmacological activities of anti-inflammatory and colonic anti-ulcerative activities of MA fruit peels, leaves and pseudo-stem extracts, after ensuring their safety.Acute and subchronic toxicity studies Determination of safety of the tested herbal extracts was done by performing acute and subchronic toxicity studies.Experimental design acute toxicity The butanol extracts of MA leaves, pseudo-stem and fruit peels were dissolved in distilled water then given orally (Po) in a dose of 5000 mg/kg to three groups of rats each consisted of five rats. A fourth group acted as negative control group and received the same volume of distilled water. The percentage of mortality was recorded 24 h later. No mortality occurred after 24 h. Close monitoring of animals’ change in body weight, bowel habits, hair colour or behaviour, was noticed during the next 2 weeks20.Subchronic toxicity According to the results of acute toxicity study the selected doses for chronic toxicity study were 250 and 500 mg/kg.Fifty six male rats were classified equally into seven groups: Negative control group given one ml of distilled water orally (Po). Treated groups received different three parts MA leaves, pseudo-stem, fruit peels extract in two doses 250,500 mg/kg, All extracts were given orally for 14 days.Detection of the effect of treatment on body weight All rats in all groups were weighed before starting the experiment, after acute toxicity study and after subchronic toxicity study. Precaution was taken that the amount of daily chaw was fixed and equal for all groups.Biochemical parameters Two days after ulcer induction, The animals in all groups were kept fasting for 12 h, on the fifteenth day of the subchronic study, blood was obtained from all groups of rats after being lightly anaesthetized with ether by puncturing retro-orbital plexus, the blood was allowed to flow into a clean dry centrifuge tube and left to stand 30 min before centrifugation for 15 min at 2500 rpm with RCF = 1048 gf, to avoid haemolysis21. The clear supernatant serum was separated and collected for determination of serum levels of liver function tests  according to Reitman and Frankel22; blood urea and serum creatinine were measured by the methods described by Patton and Crouch23 and Young24, respectively.Efficacy study: Experimental design Seventy two male Wister albino rats were divided equally into 9 groups as follows: Negative control group given one ml of distilled water orally (Po). Positive control group for which colonic inflammation and ulceration were induced without previous treatment. MA pretreated groups received leaves extract , pseudo-stem extract  and fruit peels extract . The six MA pretreated groups were given the extracts orally for two successive weeks, which is the same duration of pretreatment with the standard drug prednisolone. Prednisolone pretreated group in a dose of 5 mg /kg given orally for two successive weeks26. The last dose of each treatment was administered 2 h before ulcer induction. The selected efficacy experimental dose used in the present study depended on the results of the acute toxicity and subchronic toxicity studies which had proven the safety of tested extracts.Method of induction of colonic ulcer All rats were fasted overnight with access to water only, before being anesthetized with ether inhalation. A polyethylene catheter (2 mm diameter) was inserted 8 cm into the lumen of the colon via the rectum (Pr). For all treated and positive control groups, an acetic acid solution  was slowly infused into the colon through the catheter. The acid solution was then aspirated and 2 mL of phosphate buffer solution (pH = 7) was infused into the rectum of each rat27. Negative control rats received an equi-volume saline solution devoid of acetic acid.Biochemical parameters Two days after ulcer induction, blood sampling and centrifugation was done21. The clear supernatant serum was separated and collected for determination of serum levels of liver function tests  according to Reitman and Frankel22; blood urea and serum creatinine were measured by the methods described by Patton and Crouch23 and Young24, respectively. systemic inflammatory marker C-reactive protein (CRP) and determination of interleukin beta six (ILβ6) and ANCA following manufacturer’s instructions according to the methods described by Sibiya et al.28; Sen et al.29 and Hauschild et al.30 respectively.Macroscopic examination All animals were sacrificed with ether and laparotomy was performed. Colonic segments  were excised, opened along the mesenteric border, washed with saline, and scored macroscopically. Gross mucosal lesions were recognized as hemorrhage or erosions with damage to the mucosal surface. The number and severity of mucosal lesions were noted and lesions were scaled as follows: Almost normal mucosa = 0, Petechial lesions = 1, one or two lesions or lesions less than 1 mm = 2, severe lesions or lesions between 1 and 2 mm = 3, very severe lesions or lesions between 2 and 4 mm = 4, Mucosa full of lesions or lesions more than 4 mm = 5. Mean ulcer score for each animal was expressed as ulcer index (U.I) and the percentage of inhibition against ulceration was determined using the expressions in the following equation32:UI = Ulcer index, UN = Ulcer number, US = Ulcer severity, UP = Percentage of ulcerated colons.Histological assessment of liver and kidney tissue for MA extracts acute and sub-chronic toxicity studies and Colon mucosa for efficacy of the MA extracts in anti-ulcerative colitis study Different sections from the liver, kidneys and colon were cut and fixed in 10% formalin. The tissues were then dehydrated in ethanol and embedded in paraffin bocks. The liver and kidney tissues were cut into sections of 4-μm thickness, while the colon tissues were cut into 5 µm thick sections. All tissues were stained with hematoxylin and eosin (H&E), and conventional histopathological examination was carried out under light microscopy by a pathologist who was blinded to the therapeutic strategy. Images were acquired with a Leica ICC50 HD digital camera attached to a Leica motorized light microscope system33.34. The grading system is scaled from 0 to 5 and the details of this grading system are illustrated in Table 134.For assessment of colonic tissue damage, the tissue sections were examined blindly and the lesions were semiquantitively evaluated in ten random low power fields, as described by Amir Rashidian et al.Immunohistochemistry Immunohistochemical procedures for the demonstration of myeloperoxidase immune reactivity, a marker of neutrophil infiltrstion, in the colon were performed according to the method of Hassan et al.33. Briefly, the paraffin-embedded colon sections were deparafinized and rehydrated in ethanol. The sections were then incubated with rabbit monoclonal anti-myeloperoxidase antibody . The sections were stained with diaminobenzidine (DAB) for the demonstration of the immune reaction. Finally, counterstaining with hematoxylin was carried out. MPO immunohistochemical staining was semi quantitively assessed in the colonic mucosa and submucosa in ten random high microscopic power fields, according to the % of positively stained cells. A grading system scaled from 0 to 4 was used; in which 0 = no immune staining; 1 = positive staining in ˂ 25% of cells in HPF; 2 = positive staining in 25–50% of cells in HPF; 3 = positive staining in 51–70% of cells in HPF; and 4 = positive staining in ˃ 70% of cells in HPF33. The assessment of immunohistochemical analysis was performed using image J 1.8.0 (https://imagej.nih.gov/ij/download.html).Statistical analysis The data were expressed as means ± SE for each group. Results were analyzed using one-way analysis of variance, followed by the Tukey–Kramer test for multiple comparisons; P value of less than 0.05 was considered significant in all types of statistical tests. Graph Pad Software  (version 6) was used to carry out the statistical tests.Musa Wastes are a potential source of bioactive metabolites that’s why they were subjected to phytochemical and pharmacological studies for evaluating their anti-ulcerative colitis effects. They provided a considerable promising supplement against ulcerative colitis Fig. .The crude leaf extract of MA  was applied to polyamide column chromatography and eluted with water methanol mixtures in the order of decreasing polarity.All collected fraction was investigated individually by TLC chromatography.β-d-glucoside, luteolin-7-O-β-d-glucopyranoside and Gallic acid, cinnamic acid. On the other hand, the 70% alcohol subfraction was applied to Sephadex LH-20 column chromatography and eluted with 80% ethanol/water to give one pure compound as 2-hydroxy-4-(4-methoxyphenyl)-1H-phenalen-1-one : δ ppm = 7.63, the remaining phenyl ring resonate at δppm 7.61–7.57  and 7.42–7.38 , 6.51  and 13C NMR : δ ppm :171.65 (C=O), 145.05 (C-β), 136.16 (C-1), 131.05 (C-4), 129.93 (C2 & C-6), 129.00 (C-3 & C-5), 120.99 (C-α) in  δ ppm: 6.97 (S). 13C NMR: (δ ppm) 165.67  144.10 (C3 & C5), 138.99 (C4), 118.82 (C1) 108.86 (C2 & C6) : δ 7.74 , 7.62 , 6.88 , 6.39 , 6.18 ” and 13C NMR: δ (ppm) 93.2 (C-8), 98.0 (C-6), 102.9 (C-10), 115.0 (C-2′), 115.4 (C-5′), 119.8 (C-6′), 121.8 (C-1′), 135.5 (C-3), 144.9 (C3′), 146.7 (C-2), 147.5 (C-4′), 156.0 (C-9), 160.6 (C-5), 163.8 (C-7), 176.7 (C-4). Compounds 1,2 and 3 were eluted with mobile phase benzene:methanol:acetic acid, 45:8:4 , 7.62 , 6.87 , 6.39 , 6.19 , 5.43 : 156.39 (C-2), 133.31 (C-3), 178.18 (C-4), 161.12 (C-5), 98.66 (C-6), 163.23 (C-7), 94.13 (C-8), 156.14 (C-9), 104.75 (C-10), 120.88 (C-1′), 117.04 (C-2′), 145.25 (C-3′), 148.78 (C-4′), 114.28 (C-5′), 122.01 (C-6′), 101.09 (C-1″), 74.16 (C-2″), 76.73 (C-3″), 70.89 (C-4″), 77.65 (C-5″), 61.18 (C-6″)  revealed signals at δ ppm 7.45 , 7.42 , 6.9 , 6.8 , 6.7 , 6.4  and signal appeared as doublet at δ ppm 5.08  assignable for the anomeric proton of the sugar moiety and 13C-NMR spectrum (DMSO-d6). 13C NMR  δ (ppm): 181.92 (C-4), 164.99 (C-7), 163.14 (C-2), 161.51 (C-5), 157.97 (C-9), 149.95 (C-4′), 147.12 (C-3′), 118.88 (C-6′), 122.12 (C-1′), 116.59 (C-5′), 113.86 (C-2′), 103.89 (C-10), 103.19 (C-3), 99.82 (C-1′′), 99.58 (C-6), 94.49 (C-8), 77.14 (C-5′′), 76.71 (C-3′′), 74.02 (C-2′′), 70.11 (C-4′′), 61.17 (C-6′′) -1H-phenalen-1-one, it soluble in chloroform and gave light blue under UV lamb, 254 nm. with RF: 0.82 eluted by mobile phase methanol: chloroform, 9.5:0.5.1H NMR , δppm: 3927.05 , 7.34, 7.42 ,7.58 , 7,81, 7.95 , 8.27 , 8.78 ; 13CNMR(CDCI3 ppm: 55.60 (OCH3), 112.89 (C-3), 114.15 (C-3′ and C-5′), 125.04 (C-9b), 126.63(C-3a), 127.77(C-8), 129.84 (C-9a), 130.14 (C-5), 131.38 (C-6), 131.65(C-6a), 131.72(C-9). 132.00 (C-2′ and C-6′), 132.39 (C-1′), 136.64 (C-7), 143.99 (C-4), 149.49 (C-2), 159.75(C-4′), 179.98 (C-l)  and n-hexadecanoic acid 22.05%) followed by myristicine 9.31% and isovaleric acid amounted to 8.06% of the oil. Occasionally, our results are relatively similar to that identified from banana reported by Facundo et al.2.05% fol in Fig. .Figure 2∆ less than 5 ppm, mass spectra and retention times) with those of the standard compounds available in PubChem, ChEBI, Metlin, KNApSAck, HPLC, NMR and literature data. Different types of phenolic compounds of MA extracts were recorded in  and literature data  Fig. .43.Results of the present study revealed that the highest concentrations of ICIn the present acute toxicity study MA leaves, pseudo-stem and fruit peels extracts given to three groups rats in a single dose of 5000 mg/kg; all were given once; exhibited no mortalities during the first twenty four hours after administration. The percentage of body weight change of the group that received pseudo-stem extract showed significant decrease while the group that received fruit peels extract showed significant increase compared to negative control group Table . HoweverMoreover histopathologic examination of both liver and kidneys revealed normal hepatic parenchyma and normal hepatocytes Fig. a, and noAccordingly the selected doses for testing the sub-chronic toxicity of all extracts were 250 and 500 mg/kg given orally for fourteen successive days, which is the same duration of the efficacy study. Observation of rats for any marked change in body weights Table , or grosAssessment of both liver and kidney functions in the subchronic toxicity study by measuring ALT, AST, Urea and Creatinine levels in sera of treated rats Table , in the In the present study, results of both acute and subchronic toxicity studies denoted the safety of MA leaves, pseudostem and fruit peels to be used in the efficacy study as protective agents against inflammatory model of rat distal part of colon mimicking ulcerative colitis in humans.In the present study the efficacy of MA leaves, pseudo-stem and fruit peels extracts was evaluated as potential protective supplements against colonic inflammatory disease in a rat model mimicking ulcerative colitis in human patients.Ulcerative colitis was induced by per rectal injection of 2 ml 8% acetic acid. Treatment with MA extracts was given orally to rats in doses of 250 and 500 mg/kg of each extract for 14 days prior to induction of ulcerative colitis. The doses were selected according to the results of toxicity studies formerly done in the present work.The weights of all treated rats involved in the study were within normal and didn’t show any significant difference from the negative control group, also the % change of weights at the end of experiment compared to those before starting was minimal. The non significant change in body weights denotes that the extracts don’t alter normal bowel habits and don’t affect the appetite of rats as food consumption was constant throughout the experiment Table .It was noticed that untreated positive control rats suffered severe diarrhoea within the twenty four hours period following acetic acid per rectal infusion for induction of UC. This finding varied in intensity from mild to absent in all other groups, which denotes that acetic acid led to severe irritation.Evaluation of the effects of pretreatments was performed by macroscopic examination of dissected colons by naked eyes Table , and by . Microscopic examination of mucosa of colons of rats in this group was normal and the lamina propria was normal with few eosinophils and normal crypts that were lined by mucin-secreting cells Fig. a,b, and Fig. a,b were On the other hand colons dissected from untreated rats that received only acetic acid per rectum were severely ulcerated to the degree of perforation with grossly detected haemorrhagic areas in 100% of rats, which was also confirmed by histopathologic examination as severe deleterious histopathological lesions were demonstrated in the colon of Positive Control (C+ ve) group, with increased pathologic lesion scoring. These histopathological lesions were characterized by diffuse ulcerative colitis with diffuse necrosis and desquamation of mucosal epithelium and complete necrosis as well as fragmentation of the crypts which are intensely infiltrated by neutrophils in addition to severe congestion of mucosal blood vessels Fig. c in addiOn the other hand macroscopic examination of group treated with prednisolone which was used as a standard drug revealed significant reduction in ulcer index as 62.5% of rats were affected and showed significant increase in percentage of ability of protection against UC 41.81%) compared to untreated group,the results were consistent with histopathology, which revealed pronounced improvement with significant decrease of pathologic lesion scoring, which revealed small multifocal ulcerative lesions with focal necrosis and desquamation of mucosal epithelium, focal mononuclear inflammatory cell infiltration , by gross examination as the ulcer index was significantly reduced and the percent of protection of the high dose extract was 28.31% which was significantly higher than both low leave extract and positive control group as ulceration was detected only in 75% of pretreated rats, yet it was significantly less than prednisolone group. Consistently histopathologic examination revealed large focal erosive lesion and few proprial hemorrhage Fig. i,j. But 00 mg/kg,The group pretreated with pseudostem 250 mg/kg showed better macroscopic examination profile as the number and severity of ulcers was less as they were detected in only 75% of pretreated rats which consequently significantly reduced the ulcer index compared to leaves pretreated group by low and dose and also to positive control group, but its protective effect was significantly less than prednisolone and almost the same as leaves pretreated group with high dose as the % of protection was 28.52% in pseudostem low dose group. The histopathologic examination of pseudostem low dose revealed pronounced attenuation of the pathological lesions with decreases pathologic lesion scoring,small focal necrosis of mucosal epithelium associated with mild proprial edema and few leukocytic cell infiltration were demonstrated Fig. k,l. MildRegarding the gross examination of the high dose of pseudostem 500 mg/kg) and low dose of fruit peels extract (250 mg/kg), ulcers were detected in only 62.5%, which is the same percentage of affected rats in the prednisolone (standard),also the number and severity of ulcers were approximately close to each other leading to non significant differences in ulcer indices and consequently % of protection of both pseudostem extract high dose 40.34%) and fruit peels extract low dose (41.13%)on one side, and prednisolone (41.81%) which is the standard treatment on the other side, however their protective effects were significantly higher than those of leaves low and high doses as well as pseudostem low dose, and of course the positive control group. On the other hand they showed significant lower protective effects than fruit peels extract high dose (500 mg/kg), whose protective effect was the highest of all pretreatments when compared to positive group and each pretreatment with pronounced significant % of protection of 53.33% and least number of ulcers, severity and affection of only 50% of rats, consequently exhibiting the least ulcer index among all other groups. The histopathologic photomicrographic findings were consistent with the macroscopic examination of these groups, where normal colonic mucosa in most examined sections was frequently demonstrated in high dose stem-treated groups stem group. Regenerative activity of the mucosal epithelium and minimal leukocytic cell infiltration as well as scant proprial hemorrhage were demonstrated in high dose stem-treated groups  pass directly to the large intestine (colon)48. where fermentation occurs by colonic microbiota, leading to elaboration of the positive effect of phenolics on colon’s health by reducing its pH as anti-inflammatory effects49 which was emphasized in our study and consequently suppression of cancer cells.The anti-inflammatory effects of phenolics present in MA extract when they were given orally in our study are due to serial enzymatic reactions that take place in the digestive system. After being absorbed in the small intestine they conjugate with glucuronic acid and sulfonate, then 5–10% pass to the plasmaIn the present study an animal model mimicking ulcerative colitis was induced by using per rectal acetic acid infusion, it produced severe inflammation which could be prohibited by pretreatment with natural plant extracts rich in Flavonoids and phenolic acids due to their protective effects on the colon as 90–95% of phenolic acids are metabolized in the colon by microorganisms that lead to their anti-inflammatory activity.This was clear in our study as characterization of secondary metabolites in MA revealed high contents of these compounds.It is noteworthy mentioning that the MA fruit peels had the best effect regarding the ability to protect against development of severe ulcerative colitis, which introduces a promising natural supplement that can be used in future clinical studies for further evaluation of its effect as a protective pretreatment in vulnerable patients that are susceptible to have ulcerative colitis.Supplementary Information.


PRF imparted the highest cell viability at 48 h (p < 0.001), followed by MTA, Theracal PT, and Tetric N-Bond. Similarly, PRF had the highest potential to enhance cell proliferation and differentiation (p < 0.001), followed by Theracal PT, MTA, and the bonding agent at the end of 24 h and 72 h, respectively. Finally, PRF sustained the viability of human primary dental pulp stem cells more effectively than Theracal PT and MTA; however, the application of a Tetric N-Bond as a pulp capping agent was ineffective.The present research study assessed the cell viability and cytotoxic effect of mineral tri-oxide aggregate (MTA), Tetric N-Bond Universal bonding agent, Theracal PT (pulpotomy treatment), and platelet-rich fibrin (PRF) as pulp capping agents on human dental pulp stem cells (hDPSCs). The cells were isolated from the pulp tissue of an extracted healthy permanent third molar. After four passages in Dulbecco’s Modified Eagle’s Medium, the primary cells were employed for the investigation. The test materials and untreated cells (negative control) were subjected to an Methylthiazol-diphenyl-tetrazolium (MTT) cytotoxicity assay and assessed at 24-, 48-, and 72-h intervals. The Wilcoxon matched-paired  Direct pulp capping (DCP) is regularly performed following deep caries treatment to stimulate reparative dentin, a physiological barrier that acts as a “biological seal” to protect and preserve the underneath pulp tissue’s vitality . Direct Mineral tri-oxide aggregate (MTA) is a bioactive and biocompatible self-setting hydrophilic calcium silicate cement which promotes the proliferation/differentiation of human dental pulp cells and showed calcified tissue-conductive activity, allowing for rapid dentine bridge construction and new hard tissue synthesis ,9. DentiTheraCal PT (pulpotomy treatment) is a newly introduced biocompatible, dual-cured, resin-modified calcium silicate cement that acts as a barrier to the dental pulpal complex, preserving and promoting tooth viability. When TheraCal PT is directly applied to the pulp chamber, the setting of this dual-cured cement enables the immediate placement of restorative materials. After light curing the material for 10 s, clinicians can immediately place the desired adhesive with any bonding technique , base, and/or restoration . PlateleCytotoxicity screening assays are used to detect whether materials or their extracts promote cell death. A material’s effect on cell survival seems to be a determinant of biocompatibility. Cell culture determines in vitro cytotoxicity testing, which is a crucial component of bio-mechanical development . A threeMTA, Tetric N-Bond Universal bonding agent, Theracal PT, and PRF have effects on human dental pulp tissues, either alone or in combination with other materials, and with differing success rates. These materials had not been compared for cell viability before this investigation. The purpose of this in vitro research study was to assess the functional differentiation potential of MTA, Tetric N-Bond Universal bonding agent, Theracal PT, and PRF placed in direct contact with human dental pulp stem cells (hDPSC) at 24 h, 48 h and 72 h. The null hypothesis was that MTA, Tetric N-Bond Universal bonding agent, Theracal PT, and PRF are cytotoxic to the human dental pulp stem cells (hDPSCs).The study was conducted at the Institute of Dental Sciences in Bhubaneswar, Odisha, in collaboration with the Maratha Mandal Dental college and Research Centre in Belgaum, Karnataka. The IMS and SUM Hospital Siksha ‘O’ Anusandhan Ethics Committees approved the study and Institutional Ethical Clearance Ref. No.DRI/IMS.SH/SOA/2021/145.The extraction of hDPSCs from the third molar was carried out according to “Goorha and Reiter’s” protocols . Four heThe obtained pulp stem was transported in Dulbecco’s Modified Eagle’s Medium (DMEM)  for fibroblast culture. Isolation and harvesting of the hDPSC were performed as discussed in Bulbule et al. and Goorha and Reiter ,22.2 to obtain single-cell suspensions. Every three days, the culture media was replaced until cell confluence was attained. When the outgrown cells reached confluence, they were subcultured in growth media containing DMEM supplemented with 10% foetal bovine serum (FSB), 2 mmol L-1 of L-glutamine (a source of energy for rapidly dividing cells), and 1% penicillin, streptomycin, and amphotericin (PSA)  antibiotic solution to prevent bacterial contamination of the cell culture and incubated at 37 °C in a humidified atmosphere of 5% CO2 and 15% O2. Subculturing the outgrown cells at a 1:4 ratio generated hDPSCs. The cells were divided into five groups after the fourth passage based on test materials and a control group treatment.Using a sterile blade, pulp tissues were minced into small pieces in a petri dish and placed in a 37 °C washing medium  to remove all debris. The pulp was placed in a centrifuge tube along with the washing media and spun at 800 rpm for 5 min at room temperature to produce a tissue pellet. The pellet was washed for 1 h at 37 °C in 4 mL of culture medium with 3 mg/mL of collagenase and 4 mg/mL of dispase II , until the tissue underwent enzymatic dissociation. Cell pellets were obtained by enzymatically digesting the cell cultures in four steps and resuspending them in 5–10 mL of phosphate buffered saline. To create single-cell suspensions, these pellets were resuspended in 5 mL of DMEM and grown in 24-well microtiter plates for 7 days at 37 °C in 5% CO2 for 20 s) with a light-emitting diode curing lamp . The sample discs were immersed in respective Dulbecco’s Modified Eagle’s Medium and grouped as Group I-MTA, Group II-Tetric N-Bond Universal bonding agent, and Group III-Theracal PT. All three groups were kept for 24 h at 37 °C and 5% CO2 in a humid atmosphere.In this investigation, hDPSCs were identified in the fourth linear passage and subsequently tested for viability. The negative control cells were the subcultured DMEM stem cells. Next, 5 mm × 2 mm diameter and height disc of test materials  are prepared using sterilized cylindrical rubber moulds  and stored in an incubator   to ensure complete setting. As per the manufacturer’s instructions, one scoop of MTA powder and a drop of distilled water were mixed for 30 s to obtain a homogeneous consistency. The mixed MTA is poured into the rubber moulds and allowed to set completely. Tetric N-Bond Universal (drops placed in the rubber moulds) and premixed Theracal PT were injected into different rubber moulds and photocured (1200 mW/cmPreparation of PRF (Group IV): 10 mL of healthy participants’ venous blood was collected and split into 5 mL sterile vacuum blood tubes without anticoagulants. Blood samples were centrifuged at 3000 rpm for 15 min. PRF was made before introducing test materials into cultured pulp stem cells. The control group (Group V) is a subcultured (DMEM) hDPSC, and no test materials were added.5 cells/well of the stem cell culture. For the PRF group, the PRF was cut into 1 × 1 mm pieces using a scalpel and applied directly in triplicate. The number of viable cells after 24-, 48-, and 72-h incubation at 37 °C in a humidified environment of 5% CO2 and 95% air was evaluated using the MTT  assay [MTA, Tetric N-Bond Universal, and Theracal PT, each weighing one thousand milligrams, were dissolved in one thousand microliters of dimethyl sulfoxide to produce 100 percent solutions. Using Eppendorf tubes, 100 mL of the test agents and the control were administered in triplicate to 96-well microtiter plates containing 2 × 102 were used to maintain the stem cell lines (hDPSC) in 96-well microtiter plates at 37 °C for 48–72 h.MTT solution preparation (stock solution): At pH 7.4, 5 mg of MTT powder was dissolved in 1 mL of Dulbeco’s phosphate buffered saline (PBS). MTT solution was filtered via a 0.2 micron filter and stored in a light-resistant, sterile container. Minimum Essential Media (MEM) with 10% heat-inactivated foetal calf serum (FCS) and 5% of a mixture of Gentamicin (10 μg), Penicillin (100 units/mL), and Streptomycin (100 g/mL) in the presence of 5% of CO2, a stock solution of MTT was added to each well  for an additional 4 h. The supernatant was aspirated carefully, and 100 µL of DMSO was added to dissolve the “Formazan blue” crystals that had precipitated, and the optical density (OD) at 570 nm was measured using a spectrophotometer. The values are the mean of five readings. The concentration at which treated cells’ OD decreased by 50% compared to the untreated control. For accuracy, the OD was measured in triplicate at 540 and 720 nm, and the mean was used. The live cell percentage is calculated as follows: Surviving cells (%) = Mean OD of test compound × 100/mean OD at control [In this investigation, 10, 20, 25, 30, and 50 µL of the stock solution (10 mg/mL produced in dimethyl sulfoxide (DMSO)) were applied to wells containing 100 µL of medium, and the final concentrations were 10, 20, 25, 30, and 50 µg/mL. After 48 h of incubation at 37 °C in a humidified atmosphere containing 5% CO control .2, with 98% humidity for 4 h. At the end of the incubation time, the MTT-containing media was withdrawn and 100 μL of dimethyl sulfoxide was added to each well. The purple MTT formazan was dissolved utilizing a microplate shaker. Dental pulp stem cell viability was expressed as the colour intensity of experimental wells relative to control. The same method was used three times to confirm the readings. On a microplate reader, absorbance was measured at 492 nm with background subtraction at 620 nm . Evaluations at 48 and 72 h were conducted using the same method.To evaluate cell viability in all groups after 24 h, 48 h, and 72 h, 20 μL of 5 mg/mL MTT was added to fifteen wells  on a 96-well microtiter plate and incubated at 37 °C, 5% COt-test (p 0.05) was used to assess intra-group mean differences. The cytotoxicity was measured in percentage as shown in Descriptive and analytical statistics were computed in SPSS Version 24.0 . The Shapiro–Wilk test verified the normality of the data. The one-way ANOVA test compared group mean differences, and post-hoc analysis used Tukey’s HSD (Honest Significant Difference). A paired sample p < 0.001) differences in mean OD values among the groups. The group IV had the highest mean OD values of 0.575 ± 0.003, followed by group III (0.526 ± 0.002), group I (0.5464 ± 0.003), group V (0.435 ± 0.004), and group II (0.391 ± 0.007) (p < 0.001) between pairwise comparisons shown in One-way ANOVA showed statistically significant (± 0.007) . The ratThe group IV had the highest mean OD values of 0.627 ± 0.006, followed by group I (0.576 ± 0.004), group III (0.527 ± 0.001), group V (0.455 ± 0.003), and group II (0.423 ± 0.004) .The group IV had highest mean OD values of 0.575 ± 0.003, followed by group III (0.564 ± 0.002), group I (0.456 ± 0.004), group V (0.399 ± 0.001), and group II (0.382 ± 0.002) .p = 0.00 1) at 48 h (0.575 ± 0.004) than 24 h (0.464 ± 0.003), and the 72 h results (0.456 ± 0.004) were significantly lower (p 0.001) than the 48 h (0.575 0.004). In group II: results at 48 h (0.423 ± 0.004) were significantly higher (p = 0.038) than at 24 h (0.391 ± 0.007) and, at 72 h (0.382 ± 0.002), they were significantly lower (p = 0.001) than 48 h. Group III: There was an insignificant difference (p = 0.580) at 24 and 48 h; however, 24 h (0.526 ± 0.002) was significantly higher (p = 0.001) than 72 h (0.564 ± 0.002). Group IV: 48 h (0.627 ± 0.006) was significantly higher (p = 0.012) than at 24 (0.575 ± 0.003) and 72 h (0.575 ± 0.003). In group V: at 72 h (0.399 ± 0.001) there was a significant decrease than at 24 (0.435 ± 0.004) and 48 h (0.455 ± 0.003), as shown in Groups were compared at 24 versus 48 h, 48 versus 72 h, and 24 versus 72 h. Group I was significantly higher /regenerative endodontic therapies recommend treating pulp–dentine wounds with calcium hydroxide to preserve damaged tissue, induce reparative dentine production, and sustain pulp. Advances in material science have led to improved pulp capping materials for deciduous and permanent teeth . DPC heaMTT was used to examine the effects of bioinductive compounds on hDPSC viability and proliferation. Mitochondrial succinate dehydrogenase decreased the amount of yellow 3--2,5-diphenyl tetrazolium bromide. MTT transforms into an insoluble, dark purple formazan in the mitochondria ,31. DuriThe MTT assay measures the metabolic activity of stem cells in culture conditions over a set period of time. As the absorbance value rises over time, it might be read as a higher level of cellular activity and viability, and thus as an indirect measure of cell proliferation. Intra-group comparisons of MTT assays were performed for individual groups at 24 h, 48 h, and 72 h to obtain the best results. The results suggested that, in all time periods, the cell proliferation rate was the significant. Moreover, the 72-h MTT assay results for Theracal PT were substantially higher than the 24- and 48 h results. This suggested that cell growth was constant across time. After 24 and 72 h, Group IV (PRF) had the highest mean OD value, followed by group III , group I (MTA), group V, and group II (Tertric N-Bond). In a recent work by Rodrguez-Lozano et al. (2021) , MTA demIn the current study, group II  showed the least viability of cells . Cell deGenerally, dentin bonding hybridization and adhesive diffusion into dentin tubules may protect the dentin from bacterial leakage, reducing recurrent pulpal inflammation ,14. A seGroup IV showed maximum viable cells, as platelet concentrates (PRF) contain growth factors that promote and modify cellular functions, as a pulp capping agent plays a significant role in hard and soft tissue healing, exhibiting chemotactic and mitogenic properties that stimulate and change cellular processes . They haThe biocompatibility of PRF on hDPCs has been confirmed in the present in vitro study. PRF was as effective as the widely accepted MTA when in direct contact with the cells, and were even superior to MTA in promoting cell proliferation . Chen etThe study’s limitation is that the cell viability and cytotoxicity of pulp capping agents (PCA) were investigated in an in vitro controlled environment with no inflammation in the cells being treated. Further clinical studies can be conducted to determine the efficacy of inflamed pulp caps.To summarize, PRF demonstrated greater cell viability than MTA at all intervals. Theracal PT bonding agent preserved the hDPSC better than the Tetric N-Bond Universal bonding agent. Cytotoxicity (cell death) was only observed in the Tetric N-Bond Universal bonding agent group. More research is needed to determine the effect of the dentin bonding agent on the viability of pulp tissue cells.


Neuropathic pain reduces GABA and glycine receptor (GlyR)-mediated activity in spinal and supraspinal regions associated with pain processing. Interleukin-1β (IL-1β) alters Central Amygdala (CeA) excitability by reducing glycinergic inhibition in a mechanism that involves the auxiliary β-subunit of GlyR (βGlyR), which is highly expressed in this region. However, GlyR activity and its modulation by IL-1β in supraspinal brain regions under neuropathic pain have not been studied. We performed chronic constriction injury (CCI) of the sciatic nerve in male Sprague Dawley rats, a procedure that induces hind paw plantar hyperalgesia and neuropathic pain. Ten days later, the rats were euthanized, and their brains were sliced. Glycinergic spontaneous inhibitory currents (sIPSCs) were recorded in the CeA slices. The sIPSCs from CeA neurons of CCI animals show a bimodal amplitude distribution, different from the normal distribution in Sham animals, with small and large amplitudes of similar decay constants. The perfusion of IL-1β (10 ng/mL) in these slices reduced the amplitudes within the first five minutes, with a pronounced effect on the largest amplitudes. Our data support a possible role for CeA GlyRs in pain processing and in the neuroimmune modulation of pain perception. Neuropathic pain is a condition that involves a complex combination of physiological symptoms, including the sustained perception of pain ,2. The cDuring the establishment of neuropathic pain, the CeA shows increased excitability associated with reduced inhibitory control ,6. CurreA, glycine is less abundant in the brain and presents an uneven distribution but has similar inhibitory properties [A receptors and GlyR regulate neuronal excitability in regions that are critical for central nociceptive processing, including the spinal cord [Glycine is the primary inhibitory neurotransmitter in the ventral and dorsal horn of the spinal cord and brainstem . Compareoperties . Both GAnal cord , thalamunal cord , prefronnal cord , nucleusnal cord , hippocanal cord , periaqunal cord , and amynal cord . Both renal cord .GlyRs are located at the postsynaptic membrane, anchored to gephyrin. This scaffold protein can modulate the balance towards more or less inhibitory strength, by regulating the number of available GlyRs at the membrane ,19. The We recently reported that spontaneous glycinergic currents in CeA slices are modulated by IL-1β . The desp ˂ 0.001)  of the sciatic nerve is a model of neuropathic pain in which the sciatic nerve is loosely ligated to induce constriction, inducing neuropathic pain and hyperalgesia, which can be measured as allodynia (pain response to tactile stimulation) in response to hind paw plantar mechanical stimulation ,24. Afte˂ 0.001) . These dt-test, ** p > 0.01, p = 0.105, p = 0.265; Ten days post-CCI surgery, the animals were euthanized, their brains extracted, cut into slices, and then whole-cell voltage-clamp recordings were performed in CeA neurons . We isolTo understand the physiological relevance of IL-1β modulation in neuropathic pain, we recorded glycinergic sIPSCs in CeA neurons from brain slices of CCI animals and perfused them with IL-1β 10 ng/mL . We plot(5min): 14.2 ± 0.3 pA vs. +IL-1β(15min): 18.7 ± 0.4 pA; one-way ANOVA followed by Bonferroni post-hoc test *** p < 0.001, (5min): 0.19 ± 0.03 Hz vs. +IL-1β(15min): 0.17 ± 0.01 Hz; p = 0.64, (5min): 0.45 ± 0.05 Hz vs. +IL-1β(15min): 0.53 ± 0.05 ms; p = 0.07, Notably, the perfusion with IL-1β almost completely eliminated the bimodal distribution within the first 5 min; the distribution became unimodal, with the lower amplitudes mainly represented. This new distribution lasted over time, with peaks at 11 pA and 14 pA after 5 min or 15 min of IL-1β application, respectively A,B. The In the present work, we studied the spontaneous glycinergic currents in CeA slices from animals under neuropathic pain using the CCI neuropathic pain model. Our results show that CCI induces a bimodal distribution of spontaneous glycinergic current amplitudes, utterly different from the obtained from Sham animals. In normal conditions, the spontaneous glycinergic currents have a normal distribution. However, in peripheral pain, the currents are distributed in two populations, one lower amplitude peak at 9 pA, which likely represents unitary currents, as shown elsewhere , and a lOur data show that neuropathic pain can alter ionic conductances allocated in supraspinal brain centers. In particular, the inhibitory conductance generated by GlyR in CeA appears to be persistently modulated. The increase in the average current amplitude found after CCI could be explained by the number of events of high amplitudes. The increased GlyR expression or increased channel permeability are not discarded, but given the amplitude distribution, the explanation of the higher amplitudes by clustering seems the most plausible. CCI does not affect other kinetic parameters like the frequency that depends on the released vesicles from presynaptic sites or the decay time that affects the time the channel is open.In consequence, our results may point to the idea that neuropathic pain could induce aggregation into clusters of GlyRs in CeA interneurons. Clustering of GlyRs depends on interactions with the scaffolding protein gephyrin, modulated by different GlyRα and β subunits ,26,27. F2+ entry, and the increase of gephyrin-binding of GluRs at the membrane [Neuropathic pain induced by the CCI surgery, like other inflammatory processes, causes the increment of systemic inflammatory factors, such as proinflammatory cytokines , most of which can reach the central nervous system . The cytmembrane .In a previous study, we reported that in CeA neurons, IL-1β induces a short-lasting increment in glycinergic currents five minutes after the addition of IL-1β. However, fifteen minutes later, the whole range of amplitudes diminished, suggesting that IL-1β disaggregates the GlyR clusters and induces endocytosis . Here, iThe external perfusion of IL-1β onto the CeA slices reduces all the current amplitudes, including large-amplitude events. If the CCI had produced ten days of a permanent inflammatory response mediated by the release of IL-1β at the CeA, we would have expected no large amplitude currents still present. Hence, it is unlikely that the bimodal response seen after CCI is, at least directly, produced by only the continuous local release of IL-1β. We speculate that a new equilibrium is established during those ten days that could include other proinflammatory cytokines and GABA conductances. Inhibitory conductances are still needed to keep the balance with excitation, therefore is a matter of determining how GABA currents change while glycinergic currents decrease by the action of IL-1β. More studies are required to test this hypothesis.The central amygdala is one of the nuclei of the amygdaloid complex, associated with the emotional processing and perception of pain ,5,8,27. Surprisingly, CeA contains unusually high expression levels of the βGlyR subunit, the modulatory auxiliary subunit of GlyR, compared to other brain regions . There iIn the present work, we evidenced the state of GlyR currents in CeA under a neuropathic pain condition. Although the perfusion of IL-1β can explain the acute effect on GlyR currents, a persistent condition such as neuropathic pain would better resemble chronic inflammation’s neurological status. The pain sensation caused by nerve injury lasts long and causes detrimental effects on quality of life and emotional aspects ,25,27. TMale Sprague-Dawley (SD) rats (250–350 g) were obtained from the Animal Facility of the University of Chile. Every effort was made to minimize animal suffering. Animals were bred and housed in controlled laboratory conditions, received a standard rat chow diet and water ad libitum, and were housed on a 12-h light/dark cycle at a constant room temperature of 23 °C. Animal care and experimental protocols for this study were approved by the Institutional Animal Use Committee of University of Chile (N°17027-MED-UCH) and of Universidad Andrés Bello (N°11-2016).The sciatic chronic constriction nerve injury in Sprague Dawley rat is a validated model for the study of neuropathic pain . BrieflyRats were placed into individual chambers with wire mesh floors and transparent covers. After 15 min of habituation, their mechanical threshold was manually evaluated using the von Frey filaments. A perpendicular force was applied to the mid-plantar zone of each paw using filaments that reached a pre-determined pressure (32–512 mN) before bending, ordered from thinner to thicker until a withdrawal response was elicited. The mechanical withdrawal threshold was determined using the Up-Down method with three up-down cycles per measure .2PO4, 25 NaHCO3, 10 dextrose, 3.5 MgSO4, 0.5 CaCl2, 3 sodium pyruvate, 0.5 sodium L-ascorbate and 3 Myo-inositol . Coronal slices were cut (300 µm thick) with a vibratome. After 1 h at 36 °C, the slices were changed to ‘recording solution’, composed of (in mM): 126 NaCl, 3.5 KCl, 1.25 NaH2PO4, 25 NaHCO3, 10 dextrose, 1 MgSO4, 2 CaCl2, 3 sodium pyruvate, 0.5 sodium L-ascorbate and 3 Myo-inositol  at room temperature (22 °C). Slices were recorded in a submerged-style chamber solution with ACSF at 30–32 °C under an upright infrared-differential interference contrast (IR-DIC) fluorescence microscope  equipped with a 40X water objective. We performed voltage-clamp whole-cell recordings in the soma of visually identified neurons allocated in the central amygdala. We used a borosilicate glass electrode  pulled on a P-97 Flaming/Brown Micropipette Puller . The glass pipette, ranging from 3.5 to 4.2 MΩ, was filled with intracellular solution containing the following (in mM): 130 Cs-gluconate, 3.5 CsCl, 4 ATP-Mg, 0.3 GTP-Na, 10 Na-phosphocreatine, 1 EGTA, 10 HEPES, and 0.4% Biocytin . After seal formation and successful transition to whole-cell configuration, access resistance was usually between 4–15 MΩ and series resistance was continuously monitored and compensated between 75–80%. We isolated glycine-mediated currents in the presence of synaptic blockers CNQX (10 μM), APV (50 μM), and bicuculline (10 μM) to block AMPAR, NMDAR, and GABAA receptors, respectively. We recorded the spontaneous inhibitory postsynaptic currents (sIPSCs) obtained as outward currents at a holding potential of +20 mV. The remaining sIPSCs obtained after further application of bicuculline (10 µM) were considered glycine-based sIPSC. At the end of the recording, we applied strychnine to block glycinergic currents. We used PClamp detection events to choose single events of about 3 to 10 pA, showing greater than peak-to-peak noise to analyze the area and decay constant. Spontaneous synaptic events were detected over 100 s of continuous recording two times after the application of IL-1β (10 ng/mL). The signals for both voltage-clamps were acquired using a MultiClamp 700B amplifier , low-pass filtered at 10 kHz, digitally sampled at 30 kHz, and recorded through a Digidata-1440A interface  and PClamp 10.3 software. The analysis was performed from the recordings of 9 cells from 5 Sham rats and 10 cells from 5 CCI rats. In most cases, 2 cells per animal (one cell per slice) were recorded. Slices from each animal were considered replicates and averaged together. As the CeA is a small brain region, only 2 slices per hemisphere were obtained per animal.For electrophysiological recordings, male rats that underwent CCI or control (Sham) surgery were used 10 days after the surgery, and the treatment that each animal underwent was not known to the person who performed the recordings. Every day, one animal was anesthetized with isoflurane and then decapitated. The brain was quickly removed, and placed in a beaker with cold artificial cerebrospinal artificial solution (ACSF)-modified with sucrose, composed of (in mM): 85 NaCl, 75 sucrose, 3 KCl, 1.25 NaHt-test when two groups were analyzed and a one-way analysis of variance (ANOVA) when more than two groups were compared, followed by the Bonferroni’s post hoc test. The threshold for statistical significance was p < 0.05, with a 95% confidence interval (CI). Statistical analyses were performed using Prism software .The results were expressed as the mean ± standard error (SEM). We used a In the model of neuropathic pain induced by CCI, the spontaneous glycinergic currents in the CeA are distributed in two populations of small and large current amplitudes. The exposure to IL-1β reduces both populations’ amplitudes but with a more substantial effect on the population with larger current amplitudes.


However, most reported MoS2 phototransistors are fabricated using the mechanical exfoliation method to obtain micro-scale MoS2 flakes, which is laboratory- feasible but not practical for the future industrial fabrication of large-scale pixel arrays. Recently, wafer-scale MoS2 growth has been rapidly developed, but few results of uniform large-scale photoelectric devices were reported. Here, we designed a 12 × 12 pixels pixel array image sensor fabricated on a 2 cm × 2 cm monolayer MoS2 film grown by chemical vapor deposition (CVD). The photogating effect induced by the formation of trap states ensures a high photoresponsivity of 364 AW−1, which is considerably superior to traditional CMOS sensors (≈0.1 AW−1). Experimental results also show highly uniform photoelectric properties in this array. Finally, the concatenated image obtained by laser lighting stencil and photolithography mask demonstrates the promising potential of 2D MoS2 for future optoelectrical applications.Two-dimensional molybdenum disulfide (MoS TD transitio up to 9 [10,11, to 109) ,13. Ther devices ,15,16 an devices . Most proliation ,19. Sucholiation ,20.2 devices has become mainstream since various wafer-scale bottom-up growth methods have been developed, such as CVD, atomic layer deposition (ALD), and metal-organic CVD (MOCVD). For example, Peng et al. presented CVD-grown MoS2 phototransistors with a high photoresponsivity of 6650 AW−1 and detectivity of 1.23 × 1012 [16 cm Hz1/2 W−1 [2 phototransistors [2 photodetectors. Chen et al. presented a bilayer MoS2/graphene heterostructure array with the photoresponsivity of 32 mAW−1 [2/n-MoS2 phototransistors with a photoresponsivity of 1.7 × 104 AW−1 [2 image sensors. Park et al. reported a 4 × 4 multilayer MoS2 phototransistors array grown using a post-sulfurization process, which gives rise to a high uniformity but at the cost of a relatively low photoresponsivity of 3.7 AW−1 [2 film with a maximum photoresponsivity of 119.16 AW−1 [2 film. Compared with previous work, all 144 individual pixels exhibit the desired optoelectrical properties  with a high uniformity. Moreover, the MoS2 image sensor, placed under photolithography masks, was exposed to lights of visible-band wavelengths from a laser emitter. By illumination with a different wavelength and different stencils, three sets of photocurrent data were collected and converted to a visualization image, respectively. Thus, this work introduces a new platform for optoelectronic application of wafer-scale 2D-TMDs such as ultra-thin image sensors, transparent image sensors, artificial intelligence photo sensors, and selective light-detecting imagers.In recent years, the large-scale fabrication of MoS3 × 1012 . Chu et z1/2 W−1 . Guo et 32 mAW−1 . Jeong e104 AW−1 . However3.7 AW−1 . Hong et.16 AW−1 . In thes2 film, as shown in 2) substrate without any transfer processes [2 film. The difference between two dominant peaks, i.e., the in-plane (E12g) vibration mode at ~384.3 cm−1 and the out-of-plane (A1g) vibration mode at ~404.7 cm−1 was around 20 cm−1, which was consistent with previous work [2 film. 2 with silicon substrate [2 film was measured by AFM (Bruker Dimension Edge). The average height difference along the red line, as shown in 2 film [2 and the grain boundaries could be clearly recognized from SHG images. Such capability of direct visualization of the grain information in MoS2 is attributed to the suppressed SHG signal at the grain boundaries. The difference in crystal orientations resulted in the destructive interference and annihilation of the nonlinear waves [2 phototransistor device fabrication.Our pixel array image sensor was formed using a 2 cm × 2 cm CVD-grown monolayer MoSrocesses . Compareous work . Moreoveubstrate . Furthern 2 film . Since tar waves ,30. Abov2 film is demonstrated in 2 phototransistors with the same geometric size. A more detailed single device structure was illustrated in 2 channel with W/L of 30 μm/20 μm and Au electrodes for source and drain contacts. 2 pixelarray image sensor. As described before, the monolayer MoS2 film was grown on a SiO2/Si substrate, Au (35 nm), as the source/drain (S/D) electrodes were deposited using an electron-beam evaporator and photolithography via a lift-off technique, and then the channel isolation was realized by the CF4 reactive ion etching using a photoresist mask, and a MoS2 channel was etched to the designated dimensions. Finally, Aluminum oxide (40 nm) was deposited as the protective insulator via ALD. The details of the device fabrication are presented in the The mature back-gate phototransistor structure was adopted here, and the detailed process is presented in 2 is directly grown on a SiO2/Si substrate without additional processing , which 2 phototransistors. The typical transfer characteristics (ID-VBG) of a single MoS2 phototransistor was plotted in Ion/Ioff) of ≈106 and the threshold voltage (VTH) of −9.2 V at a drain voltage (VDS) of 1 V indicated the strong gate modulation of the designed MoS2 device. 2 phototransistor in the pixel array image sensor. The drain current (ID) was saturated at a high drain bias (>20 V) because of pinch-off at the drain region. Due to a satisfied contact formation between the monolayer MoS2 film and the S/D electrodes (Au), the output characteristics, ID-VDS, in the insets of ID at a low drain bias [μeff could also be extracted from the linear region of the transfer curve using the following equation gm is the transconductance, Lc and Wc are the length and width of the channel, respectively, Cox is the capacitance of the gate insulator, and VDS is the drain voltage [Then, we tested the electrical properties of as-fabricated MoSain bias . Field-e voltage .μeff), threshold voltage (VTH), and on/off current ratio (Ion/Ioff) measured from all 12 × 12 phototransistors, as summarized in μeff of 6.07 cm2 V−1 s−1, a VTH of −10.2 V, and an Ion/Ioff of 4.87 × 106. According the equation D could be replaced by μeff, VTH and Ion/Ioff. The standard deviations (STD) of carrier mobility μeff is 23%. The STD of threshold voltage VTH and current ratio Ion/Ioff are 2.8% and 25.2%, respectively. Compared with previous works [To further demonstrate the uniformity of our image sensor in terms of electrical properties, histogram and corresponding fitted Gaussian distribution were also calculated for mobility  ranging from 14.7 to 285.9 μW/cm2. The photocurrent curve of IPH = Iillumination − Idark. The photocurrent of the monolayer MoS2 phototransistor gradually increased with increasing Pin according to the photocurrent value under light with different power densities. Moreover, the photoinduced transfer characteristics and photocurrent curve under light with changeable excitation wavelength are presented in 2 is more sensitive to visible wavelength band (420–680 nm) compared with near-infrared band (700–1200 nm). RPH as a function of incident power density and excitation wavelength, which were important figures of merit for phototransistors. The RPH was extracted from the transfer characteristics in RPH = IPH/Pin (unit: AW−1), where IPH and Pin are the photocurrent and incident power density, respectively. The main mechanism for the high RPH of the monolayer MoS2 phototransistor is the photogating (PG) effect by the formation of trap states near the valance band due to the structural imperfection and defects of MoS2 [In addition to the electrical characteristics, the key opto-electric characteristics of a single MoS of MoS2 ,35,36,372 array. IPH and response time are all positively correlated to incident power, which is also attributed to the photogating effect [IPH under different VDS values. The photoresponsivity increases at the expense of a longer response time. The switching characteristics under different back-gate voltages (VBG) are displayed in VBG was over-high (>10 V) or over-low (<−10 V), the recovery time would be longer for photo-excited carriers to detrap from the subgap state [VBG would improve greatly the response speed of our image sensor array. As shown in VBG was injected simultaneously with a short-time (≈1 s) pulse voltage from 8 V to 15 V to suppress carriers as soon as possible. As a result, the falling time decreased by 90% compared with 2 image sensor [Finally, g effect . Figure ap state , wherease sensor ,39.2 phototransistors was confirmed in the mapping images that presented the current level of each phototransistor under illumination with Pin of 285.9 μW/cm2 (VDS of 1 V. The detectivity (D*) is obtained by the equation of A is the channel area, q is unit electric charge, ID is dark current, and RPH is photoresponsivity. The average photoresponsivity was 364.00 AW−1 with a standard deviation of 99 AW−1. The detectivity was 2.16 × 1010 cm Hz1/2 W−1 with a standard deviation of 3.23 × 109 cm Hz1/2 W−1. The photocurrent maps under illumination with different wavelengths  are demonstrated in To demonstrate the overall uniformity of our image sensor, a statistical distribution of the photoelectrical properties, i.e., responsivity and detectivity of 12 × 12 MoS9 μW/cm2 e,f, resp2 image sensor array. These character masks were sequentially placed on the image sensor array during light projection . The 2D photocurrent data, collected under light stencil, was firstly normalized from float type to then concatenated horizontally to form the final 36 × 12 image . Due to the uniform photo-related properties of all 12 × 12 phototransistors, the photosensitivity mapping result could display three characters clearly. Moreover, three sets of masks (each was 12 × 12 pixels) with different characters  were prepared and patterned using a laser cutting system, as shown in 2 film was fabricated to present the potential of the next generation photodetector. The integrated pixel number is significantly increased without sacrificing photodetector performance. The fabricated MoS2 devices showed high uniformity in electrical properties, including carrier mobility (≈6.07 cm2 V−1 s−1), Ion/Ioff (≈4.875 × 106), and threshold voltage VTH (≈−10.19 V). The measured photoresponsivity RPH (≈364 AW−1) and detectivity (≈2.13 × 1010) were superior to traditional CMOS image sensors [2. These results provide a blueprint for the future development of wafer-scale 2D TMD optoelectrical application and suggest further application scenarios requiring a high dynamic range, such as with artificial retinas.In this paper, a 12 × 12 phototransistor pixel array image sensor based on a wafer-scale monolayer MoS sensors ,41, whic


Glycine max) xylem sap via o-chlorophenol extraction and conducted an in-depth peptidomic analysis using a mass spectrometry (MS) and bioinformatics approach. We successfully identified 14 post-translationally modified peptide groups belonging to the peptide families CEP , CLE (CLAVATA3/embryo surrounding region-related), PSY (plant peptides containing tyrosine sulfation), and XAP (xylem sap-associated peptides). Quantitative PCR (qPCR) analysis showed unique tissue expression patterns among the peptide-encoding genes. Further qPCR analysis of some of the peptide-encoding genes showed differential stress-response profiles toward various abiotic stress factors. Targeted MS-based quantification of the nitrogen deficiency-responsive peptides, GmXAP6a and GmCEP-XSP1, demonstrated upregulation of peptide translocation in xylem sap under nitrogen-deficiency stress. Quantitative proteomic analysis of GmCEP-XSP1 overexpression in hairy soybean roots revealed that GmCEP-XSP1 significantly impacts stress response-related proteins. This study provides new insights that root-to-shoot peptide signaling plays important roles in regulating plant stress-response mechanisms.Increasing evidence has revealed that plant secretory peptides are involved in the long-distance signaling pathways that help to regulate plant development and signal stress responses. In this study, we purified small peptides from soybean ( Terrestrial plants require constant adaptation to the changing environment. To maintain quick and systematic responses to environmental changes, plants utilize a variety of signaling molecules that are involved in specific signaling pathways to coordinate the optimum shoot–root balance and development strategies. The vascular tissues of plants play a crucial role as long-distance-traveling conduits for long-distance signaling throughout the plant body ,2. DecadLotus japonicus, on rhizobia infection, the CLE-root signal (CLE-RS) genes, LtCLE-RS1 and LtCLE-RS2, are upregulated in the roots to initiate a long-distance negative feedback loop for the autoregulation of nodulation . Le. LeArabiTwo peptides with a length of 12 aa and that matched three precursor genes were identified to be CLE members. In this group, GmXAP4 and GmCLE-XSP1 are CLE peptides that differ by only 1 aa at position 2 . Both peTo discover the potential root-to-shoot signaling candidates, we next examined the tissue expression patterns of polypeptide genes via RT-qPCR. From soybean plants in the V1 growth stage, the stage in which the first trifoliate is fully unfolded, we collected the leaves, second internode (between the first trifoliate and unifoliate), hypocotyl, and root tissues for RNA extraction and cDNA synthesis. We compared the relative expression levels between tissue types. Diverse tissue expression patterns were observed among the peptide precursor genes in the CEP and PSY groups, but not in the CLE group, which was predominantly expressed in the root tissue . To idenGmCEP-XSP1, GmXAP6a, and GmXAP6b were found to be significantly upregulated during nitrogen deficiency. In contrast, GmXAP1, GmXAP2, GmXAP4, GmCLE-XSP1a, and GmCLE-XSP1b were significantly downregulated during nitrogen deficiency. The expression of GmXAP3a, GmXAP3b, GmPSY-XSP1, and GmPSY-XSP3 was significantly upregulated during PEG-induced drought stress. GmXAP3a, GmCLE-XSP1a, and GmCLE-XSP1b were significantly upregulated under iron deficiency. Meanwhile, GmXAP1, GmCEP-XSP1, GmCEP-XSP2, GmXAP6a, and GmXAP6b were significantly downregulated under iron deficiency. Our RT-qPCR results indicated diverse stress response patterns among the xylem sap peptide genes at the transcript level, suggesting the broad involvement of xylem sap peptide genes in abiotic stress responses.Long-distance signaling plays a crucial role in the systemic stress adaptations of plants by transmitting stress signals from sensing tissues to distant organs ,7. To unGmCEP-XSP1 and GmXAP6a were increased during the nitrogen deficiency test , CEP-XSP1-D2 , and GmXAP6-D2 (Hyp11) A. We treAgrobacterium-mediated soybean hairy root transformant overexpressing GmCEP-XSP1 for a gain-of-function analysis. We quantified the differences in the root proteomes in control and GmCEP-XSP1ox soybean hairy root tissue using label-free MS-based quantification , including 303 upregulated and 125 downregulated proteins. To gain an overall understanding of the proteomic changes, we analyzed the gene ontology (GO) annotations for the differentially expressed protein (DEP) genes via GO term enrichment analysis. The results indicated that the top-four (ranked by -log FDR) enriched biological process GO terms were “response to stimulus”, “response to stress”, “secondary metabolic process”, and “response to external stimulus” , soybean (Glycine max), rice (Oryza sativa), and legumes (Medicago truncatula) were frequently studied in early xylem sap proteomic research, but many of the frontier studies relied on gel-based approaches that limited the analysis of small and low-abundance peptides . Wereatment C. Recentficiency ,47. Thisficiency ,47. Takeem (SAM) . The stuPlants continuously conduct long-distance signaling via their vascular structure to regulate the whole-plant physiological state, to adapt to the changing environment and balance organ development. With technological improvement and cumulative effort by the research community, various xylem sap signaling peptides have been discovered, revealing the vast diversity of the signaling peptides involved in the stress response ,8,10,12.Glycine max) were germinated in vermiculite under greenhouse conditions. One week after germination, the seedlings were transferred to a hydroponic culture in half-strength Hoagland solution, which was changed twice a week. Soybean plants were cultured to the V3 growth stage, in which the third trifoliates were fully unfolded. At the V3 growth stage, the xylem sap samples were collected via the root pressure method described by Djordjevic et al., with some modifications [To collect xylem sap exudate, the seeds of soybean cultivar C08  was mixed with the equilibrated xylem sap by vortexing for 1 min. After centrifugation at 18,000× g for 5 min, the phenol phase was collected, and the extraction step was repeated twice. Immediately, the extracted phenol phase was supplemented with one-tenth the volume of 1% Ficoll as a co-precipitant, and was then precipitated with nine volumes of acetone overnight in a refrigerator set at −20 °C. The pellet was recovered by centrifugation at 18,000× g for 30 min at 4 ℃, followed by two ice-cold acetone washes with centrifugation at 18,000× g for 15 min. The obtained protein pellet was dried in vacuo and resuspended in water. Lastly, the sample solution was passed through an Amicon Ultra 10K MWCO filter  to collect the small peptide fraction as filtrate.Xylem sap peptides were purified and enriched using the ications . BrieflyTo discover xylem sap peptides by LC-MS/MS, the enriched peptide fraction was fractionated using a high-pH reversed-phase fractionation kit . Briefly, the xylem sap peptide sample was first equilibrated to 0.1% trifluoroacetic acid (TFA) and then loaded on reversed-phase resin on spin columns. The bound peptides were sequentially eluted with a high-pH buffer (0.1% triethylamine) with increasing concentrations of acetonitrile (ACN): 7.5%, 12.5%, 17.5%, and 50%, divided into four fractions by centrifugation. The collected fractions were then vacuum-dried and resuspended in 0.1% formic acid (FA) before being loaded onto an LC column.The peptide samples were separated and analyzed with an Ultimate 3000 Nano-LC system  coupled with an Orbitrap Fusion Lumos Tribrid Mass Spectrometer . Fractionated peptide samples were first quantified using the quantitative fluorometric peptide assay . Next, 0.5 µg of each peptide fraction was dissolved in 0.1% FA and injected into the Thermo C18 PepMap 100 trap reversed-phase column . Separation was achieved at 300 nL/min when using buffer A (0.1% FA) and buffer B (0.1%FA in ACN) as a mobile phase for gradient elution with a Thermo Acclaim PepMap RSLC C18 column  at 50 °C. Peptide elution employed a 90 min long-flow gradient method that started with an equilibration step of 0–5 min, followed by 0–30% buffer B for 5–70 min, a washing step of 80% buffer B for 70–80 min, 80–0% buffer B for 80–80.1 min, and 0% buffer B for 80.1–90 min. The duty cycle was set for MS1 scanning at a 60,000 resolution in the range of 375–1500 m/z followed by a 3-s-long data-dependent MS2 scan at a 15,000 resolution using higher-energy collisional dissociation (HCD) fragmentation. Dynamic exclusion of precursor ions was set to 60 s with mass tolerance of 10 ppm.The generated MS raw data were analyzed using Proteome Discoverer (version 2.4). The MS files were searched against a soybean database with the enzyme set as “no-enzyme”. Methionine oxidation, proline oxidation, tyrosine sulfation, and proline triarabinosylation were set as variable modifications. Precursor and fragment ion mass tolerances were set to 10 ppm and 0.02 Da, respectively. The minimum peptide length was set to 5, and the maximum length was set to 30. Peptide validation was performed under “percolator” based on q-values with FDR <0.05.Glycine max genome. qPCR was performed using SYBRTM Select Master Mix  and conducted on a CFX96 System . The PCR condition was set as follows: a UDG activation step at 50 ℃ for 2 min and then a denaturing step at 95 ℃ for 2 min, followed by 45 cycles of 95 ℃ for 15 s, 57 ℃ for 15 s, and 72 ℃ for 1 min. For the tissue expression study, the soybean ELF1b gene was used as a reference gene for normalization. For abiotic stress response analysis, the soybean Bic-C2 gene was used as a reference gene for normalization [For tissue expression pattern analysis, the soybean seeds were germinated in wet vermiculite under greenhouse conditions. One week after germination, the seedlings were transferred to a hydroponic culture in half-strength Hoagland solution. When the soybean plants reached the V1 growth stage, in which the first trifoliates were fully unfolded, total RNA was collected from the leaves, second internode (between the first trifoliate and unifoliate), hypocotyl stem, and root tissues using an RNeasy Plant Mini Kit , and cDNA was synthesized using the QuantiTect Reverse Transcription Kit  following the manufacturer’s protocols. The primers were generated with NCBI online Primer-BLAST against the lization .3, 1 mM MgSO4, 0.5 mM KH2PO4, 2.5 mM CaCl2, 30 µM Fe-EDTA, 46.25 µM H3BO4, 9.1 µM MnCl2, 0.765 µM ZnSO4, 0.32 µM CuSO4, and 0.5 µM H2MoO4. At the V1 growth stage, in which the first trifoliates were fully unfolded, all of the plants were transferred to control or treatment nutrient solutions for a 48-h-long treatment. The control plants were cultured in nutrient solution without modification. For the nitrogen-, phosphate-, and iron-deficiency conditions, the treated plants were cultured in a nutrient solution without KNO3, KH2PO4, or Fe-EDTA, respectively. For the high-nitrogen supply treatment, the KNO3 concentration was increased by 10-fold, to 25 mM, in the nutrient solution for the treated plants. For the salinity treatment, the nutrient solutions for the treated plants were supplemented with 0.6% NaCl. For the PEG-induced drought treatment, the nutrient solution was supplemented with 10% PEG 6000 to induce water-deficit stress. After 48 h of treatment, the root tissues of the plants were harvested, frozen with liquid nitrogen, and stored at −80 °C until RNA extraction for cDNA synthesis.For stress-response analysis, soybean cultivar C08 seeds were germinated in vermiculite under greenhouse conditions. One week after germination, the seedlings were transferred to a hydroponic culture in a nutrient solution comprising 2.5 mM KNOFor the PRM assay, 7 days after germination, the soybean plants were transferred to a hydroponic culture supplied with the nutrient solution described above, and the solution was changed twice a week. At the V3 growth stage, when the third trifoliates were fully unfolded, the soybean plants were then treated with either normal nutrient solution or a nitrogen-depleted solution for 48 h. Xylem sap was then collected from the plants by the root pressure method described above, and the collected sap samples were frozen with liquid nitrogen and stored at −80 °C until downstream analysis.The synthetic peptides with hydroxylated proline used in the experiment were ordered from GL Biochem Ltd.  and had a purity >95%. Four days after germination, soybean seedlings were transferred to half-strength Hoagland’s solution either with or without synthetic peptides (1 µM) for a 7-day-long treatment under greenhouse conditions with natural light. Treated plant roots were measured to determine the root growth conditions.MS analysis was performed with an Ultimate 3000 nano-LC system connected to a Orbitrap Fusion Lumos Tribrid mass spectrometer, as stated before. Peptide elution followed a 60 min-long flow gradient with an equilibration step for 0–8 min, 0–20% buffer B for 8–50 min, 20–80% buffer B for 50–51 min, 80% buffer B for 51–55 min, 80–0% for 55–55.5 min, and 0% buffer B for 55.5–60 min. Synthetic peptides of GmCEP-XSP1/GmXAP6a-D1, GmCEP-XSP1-D2, and GmXAP6a-D2 carrying different degrees of proline hydroxylation were first analyzed under the DDA mode to obtain the MS/MS spectra. The raw data were searched against the database using Proteome Discoverer. The peptide-matching result files were then imported into Skyline software (version 21) for ion chromatogram extraction and used to generate a peptide library [o-chlorophenol/acetone method, as previously mentioned. Purified and enriched xylem sap peptides were first quantified using the quantitative fluorometric peptide assay . Then, equal amounts of the peptide samples (0.75 µg) from the untreated and nitrogen-starved soybean xylem sap were injected into a column in each run. Xylem sap peptide samples were analyzed by a mass spectrometer under the target MS mode following the assigned precursor isolation list. The precursor ions of the targets were monitored in an unscheduled PRM setting. The duty cycle was first set as an MS1 scan at 120,000 resolutions in the 400–1500 m/z range and with a maximum injection time of 246 ms. The MS1 scan was followed by a targeted MS2 scan targeting a pre-assigned precursor isolation list. The MS2 scan was performed with the following parameters: isolation window of 1.4 m/z; Orbitrap resolution of 60,000; mass range set by “define first mass” to 100 Da; maximum injection time of 118 ms; loop control setting of “All”. The raw MS files that were obtained were first searched against the database in Proteome Discoverer for peak identification. The peptide-matching result files (.msf) and raw MS data were then processed by the Skyline software to generate extracted ion chromatograms and to perform peak integration using the synthetic peptide library. The MS2 peak areas were generated by the three most intense fragment ions (transitions), and the coefficient of variation (CV) values for each individual fragment ion were calculated within the technical replicates (n = 3) of each sample.Small peptide samples were extracted from the xylem sap collected from the low-nitrogen treatment experiment using the Agrobacterium rhizogenes K599, and empty vector pGWB2 was used as a control. For hairy root induction, 4 days after soybean seed germination, the seedlings were infected with a transformed K599 culture via needle injection into the hypocotyl region. The original roots of the infected plants were removed 2 weeks after infection, leaving only the induced hairy roots. The plants were then transferred to a hydroponic culture with half-strength Hoagland’s solution for another 2 weeks for hairy root development. Afterward, root tissues were harvested and frozen with liquid nitrogen before protein extraction.Target genes were first cloned and inserted into entry vector pENTR/D-TOPO  according to manufacturer’s protocols, and then transformed into destination vector pGWB2 using the Gateway LR Clonase Enzyme mix . The resulting pGWB2 gene constructs were transformed into g for 15 min at 4 °C. The supernatant was discarded, and the tissue pellets were washed twice with chilled acetone, with each wash followed by centrifugation at 18,000× g for 15 min at 4 °C. The tissue pellets were briefly dried in vacuo. Then, the dried pellets were incubated in SDS extraction buffer , 1 mM EDTA, 100 mM DTT, and protease inhibitors  for 15 min at room temperature, and the mixture was centrifuged at 18,000× g for 10 min at 4 °C to collect the supernatant in new tubes. The extraction step was repeated once, and the SDS buffer extracts were combined. The SDS extract was mixed with an equal volume of phenol (pH 8.0) by vortexing for 5 min. The phenol phase was collected by centrifugation at 18,000× g for 10 min at 4 °C. The collected phenol phase was then cleaned up with wash buffer  by vortexing for 5 min, followed by 10 min of centrifugation to collect the phenol phase. Finally, the phenol phase containing the proteins was precipitated by nine volumes of 0.1 M ammonium acetate in methanol for 30 min at −20 °C, then was subsequently centrifuged at 18,000× g for 15 min at 4 °C to obtain pellets. The pellets were washed again with 0.1 M ammonium acetate in methanol, and then twice with 80% acetone. Lastly, the protein pellet was air-dried andresuspended in 8 M urea and 10 mM ammonium bicarbonate (AB) for the downstream workflow.Hairy root tissues from soybean transformants were collected 4 weeks post-infection and ground into fine powder in liquid nitrogen. Total protein extraction was performed via the SDS/phenol extraction method . In brieg for 10 min to remove salt and small molecules. The proteins retained on the filter were then reduced and alkylated with DTT and IAA in urea buffer, respectively, and the reagents were removed by centrifugation. Buffer exchange was performed with 10 mM AB by carrying out successive centrifugation three times. Finally, the protein samples retained on the filter were digested overnight with 1 µg sequencing-grade modified trypsin  at 37 °C, and the yielded peptide solution was collected from the filter by reverse spinning, before being dried in vacuo. Finally, the peptide samples were resuspended in 300 µL of 0.1% TFA and were further purified by using Peptide Desalting Spin Columns . The elutes were dried and then resuspended in 0.1% FA for LC-MS.Tryptic digestion of proteins was performed by the filter-aided sample preparation (FASP) method . In brieThe LC-MS system for label-free proteomic analysis was set up under the same conditions as stated above. Tryptic peptides were first quantified using the quantitative fluorometric peptide assay . Peptide samples of 0.75 μg resuspended in 0.1% FA were injected for each technical run. Peptide elution employed a 180 min-long gradient comprising an equilibration step for 0–5 min, 0–35% buffer B for 5–160 min, 35–80% buffer B for 160–165.5 min, a washing step of 80% buffer B for 165.5–175 min, 80–0% for 175–176 min, and 80–0% buffer B for 175–180 min. The duty cycle was set with an MS1 scan at a 120,000 resolution in the range of 300–1800 m/z, followed by a 3-s data-dependent MS2 scan at 30,000 resolution using HCD for fragmentation. The dynamic exclusion of precursor ions was set to 30 s with 10 ppm mass tolerance.p-values <0.05 was considered to determine DEPs.Label-free quantitative proteomic data analysis was performed with Proteome Discoverer, and the MS/MS spectra were searched against the soybean database with the enzyme set as trypsin, with one missed cleavage allowed. Carbamidomethyl cysteine was set as a fixed modification, and oxidized methionine and proline were set as variable modifications. Precursor and fragment ion mass tolerances were set to 10 ppm and 0.02 Da, respectively. The minimum peptide length was set to 6, and the maximum length was set to 30. Peptide validation was performed under “percolator” based on q-values with FDR <0.05. Identified proteins were filtered for quantification as follows: unique peptides greater than or equal to 1; “Confidence” achieved at least “Peak Found” in all samples and “High” in at least two samples. The statistical significance of the differentially expressed proteins was analyzed using the Benjamini–Hochberg method, where the protein abundance ratio between control and treatment groups with adjusted https://www.soybase.org, accessed on 11 March 2022) tool for GO term enrichment [The GO annotations of the proteins from the label-free quantification analysis were obtained through the SoyBase (richment . We perfrichment . The enr