The sustainable production of polymers and materialsderived fromrenewable feedstocks such as biomass is vital to addressing the currentclimate and environmental challenges. In particular, finding a replacementfor current widely used curable resins containing undesired componentswith both health and environmental issues, such as bisphenol-A andstyrene, is of great interest and vital for a sustainable society.In this work, we disclose the preparation and fabrication of an all-biobasedcurable resin. The devised resin consists of a polyester componentbased on fumaric acid, itaconic acid, 2,5-furandicarboxylic acid,1,4-butanediol, and reactive diluents acting as both solvents andviscosity enhancers. Importantly, the complete process was performedsolvent-free, thus promoting its industrial applications. The curedbiobased resin demonstrates very good thermal properties (stable upto 415 °C), the ability to resist deformation based on the highYoung’s modulus of ∼775 MPa, and chemical resistancebased on the swelling index and gel content. We envision the disclosedbiobased resin having tailorable properties suitable for industrialapplications. Here, plant biomass has the potential to be one of the mostrenewable feedstock.28 It consists of thevital components cellulose, hemicellulose, and lignin, which can providevery useful monomers for further processing.29 Various physical,30 chemical,32 and biological processes34 have been developedto extract useful biobased monomers from lignocellulosic biomass.37The current environmentaland greenhouse gas emissions challengespromote the advancement of renewable technologies and products.38 low cost, potential biodegradability,39 and tunable properties.40 There have been several reports demonstrating the development ofentire biobased resins; for instance, Brännström etal. used itaconic acid (IA), succinic acid (SA), and 1,4-butanediol(BD) to prepare curable polyesters. The obtained biobased polyesterdemonstrated a low melting point (∼45 °C) and a very lowglass transition temperature (Tg = −31to −38 °C).41 Nevertheless,the cured resin demonstrated increased Tg ∼ 12 to ≤100 °C. Dai and coauthors prepared abiobased polyester from the monomers IA, 2,5-furandicarboxylic acid, SA, and 1,3-propanediol (PD), and the final resin wasobtained by adding the reactive diluent guaiacol methacrylate.42 The cured resin demonstrated thermal stabilityup to 330 °C, Tg of 73.5–141.7°C, and a flexural strength of 41.9–116.8 MPa. Moreover,a biobased resin combining curable polyester made from IA and PD withreactive diluents such as dialkyl itaconates such as dimethyl itaconatehas also been reported.43 The cured resinshowed a Tg of 65–118 °C,a storage modulus of 0.37–1.4 GPa, and a stress-at-break between21 and 54 MPa.The family of polyesters has several advantages in providingmacromolecularcomponents in thermoset resins, such as facile preparation,45 and create alternative technological solutionsto tackle vide supra challenges, we envisioned designing an all-biobasedsolvent- and styrene-free curable resin through a careful rationaldesign. We hypothesize that a novel combination of IA, fumaric acid(FA), and 2,5-FDCA, with BD as the linker, will provide the curability,rigidity,46 and thermomechanical properties47 needed to design an all-biobased resin withsimilar or improved properties to a commercially available equivalent.To get a quick understanding of the differences in mechanical propertiesbetween our devised cured resin and commercially available resin,we performed initial tensile tests according to the ASTM D638 standard.The rational design, as depicted in 48 and have shown a significant increase in usagein the scientific community  containing styrene (<50wt %).59The use of solvents in resins to dissolve the variouscomponentswithin the resin generally does not add any properties to the finalresin and results in waste;enhancer 1.25,57,52p-toluenesulfonic acid and mixedat 160 °C for 7 h (1H NMR) analysis,where the various components could be identified .35 Moreover, the NMR spectrum also showedsmall traces of additional peaks at 6.75 and 2.28 ppm that correspondto the mesaconic moiety , which is the isomerization product of itaconicacid formed at high temperatures.60 TheFourier transform infrared spectrometry (FTIR) analysis further corroboratedthe successful synthesis and the peaks corresponding to the carbonylgroup (C=O) at 1714 cm–1 and a double bond(C=C) at 1637 and 813 cm–1 (Mw) of the polyester was ∼4700g/mol with a dispersity (D̵ = Mw/Mn) of 2.09, as determinedthrough size exclusion chromatography (SEC)  2c.61 For instance, having a too high concentrationof the accelerator can lead to deactivation of the reactive MEKP.In our case, using 0.25 wt % of the accelerator and 5.0 wt % of theMEKP generated the highest degree of cross-linking (DC) of 65% (64The fabrication and curing of the biobased resinare presentedin ) of 65% 3b. Next,Tonset) and temperature with the highest degradation rate (Tmax) were higher for the biobased resin (Tonset = 137.0–181.4 °C and Tmax = 413.5–415.2 °C) (Tonset = 129.8–132.1°C and Tmax = 393.2–392.8°C) (Tonset (181.4 °C) and Tmax (415.2 °C) values (Tg was higher for the VERsamples (VER: Tg = 101.9 and 104.2 °Cand biobased resin: Tg = 74.1–99.6°C). Despite that, the VER resin demonstrated a higher Tg, and the biobased resin demonstrated betteroverall thermal stability and was more stable at higher temperatures.The results are also in accordance with the SI and GC analyses. Afurther increase in the DC of the biobased resin would most likelylead to a higher Tg.65The thermal properties of the devised cured resinswere evaluatedthrough thermogravimetric analysis (TGA) and differential scanningcalorimetry (DSC) analysis 2. The stP < 0.0001) higher tensile stressat break (37.58 ± 4.72 MPa) compared to the cured biobased resin(14.08 ± 7.11 MPa) (P <0.0336) higher Young’s modulus obtained (biobased resin = 774.99± 199.85 MPa and VER = 610.92 ± 67.66 MPa) , and no significant difference was observedin the Young’s modulus .The mechanical properties were evaluated by tensiletesting 5a.The c.11 MPa) 5b–d. How.66 MPa) 5e. This .66 MPa) 2.66,67 MOverall, key performance properties put forth thesynthesized FA/IA/2,5-FDCA/BDpolyester as a feasible, curable, all-biobased resin competitive withthe assessed commonly used fossil-based counterpart.Tg. This could be accomplished,for instance, by further increasing the MEKP catalyst concentrationduring the curing, evaluating various temperatures for the curing,etc. Moreover, increasing the concentration of the monomer 2,5-FDCAwithin the polyester would most likely promote the overall mechanicalperformance of the biobased resin. Furthermore, increasing the concentrationof the reactive viscosity enhancer relative to the reactive diluentmight also impact the overall performance of the resin, which is thereforeimportant to further investigate. Additionally, dynamic mechanicalanalysis (DMA), which is more sensitive, should be performed to geta more profound understanding of the mechanical performance.The findingsin the presented work provide a promising and interestingfuture for continuing the design of an all-biobased tunable and curableresin for various industrial applications. For instance, future researchendeavors within the presented topic would focus on further increasingthe DC of the biobased resin to improve its properties, such as mechanicalproperties and 3Tg (upto 104.2 °C); nevertheless, the biobased cured resin demonstratedhigher overall thermal stability with Tmax up to 415.2 °C, starting degradation temperature <181.4°C, and Tg up to 99.6 °C (VER: Tmax up to 392.8 °C and Tonset up to 132.1 °C). The tensile stress of thecured VER resin was significantly higher (37.6 MPa) than that of thecured biobased resin (∼15.1 MPa); nevertheless, the biobasedresin showed to be significantly stiffer with a Young’s modulusof ∼775 MPa (VER ∼ 611 MPa). Furthermore, the SI andGC for the cured biobased resin further corroborated its better thermalstability and chemical resistance. The entire process was conductedwithout the use of solvents, which is essential for improving theprocess’s climate footprint and advantageous for scaling upand industrial implementation. Instead, a biobased reactive diluentwas shown to be a viable substitute. In conclusion, the presentedrational design provided an all-biobased curable resin in the absenceof any solvent, free from styrene, and with a tailorable viscositysuitable for industrial application.An all-biobased solvent-and styrene-free curable resin is fabricated,comprising a polyester moiety and reactive diluents. The polyesteris synthesized through a green acid-catalyzed solvent-free condensationreaction with rationally selected biobased monomers. The final curableresin is fabricated by combining the polyester with a reactive diluentand a reactive viscosity enhancer. These combinations would allowfor further tuning of the viscosity of the final resin for a suitableapplication. The thermal and mechanical performance of the fabricatedbiobased resin was compared to the commercial epoxy bisphenol A vinylester urethane resin (VER) containing styrene (<50 wt %). The VER-basedcured resin demonstrated higher 44.1p-toluenesulfonicacid monohydrate , 4-methoxyphenol , sodium hydroxide ,potassium permanganate ,cobalt(II) 2-ethylhexanoate solution ,and 2-butanone peroxide [methyl ethyl ketone peroxide (MEKP)]. 5-(Hydroxymethyl)furan2-carbaldehyde (98%) was purchased from AmBeed .X500  and X600 were used as received from the supplier (Sigma-Aldrich). VER was gracefullyreceived as a gift from AOC Nederland B.V.The following chemicalswere purchased from Sigma-Aldrich: methacrylic anhydride (≥94.0%),1,4-butanediol , itaconic acid ,fumaric acid , 4.268 A round-bottomedflask was charged with NaOH  and dissolved by theaddition of H2O (30 mL). Subsequently, 5-(hydroxymethyl)furan 2-carbaldehyde  and then KMnO4  were added. The mixture was stirred for 30 min at roomtemperature, and then, the solid KMnO4 was filtered off.Afterward, HCl (concentrated) was added to the solution dropwise tokeep the pH at 1.0 or less. The precipitated product was filteredoff, washed with deionized water, and dried at 60 °C for 24 h,providing the 2,5-FDCA product  as a light brownpowder.The 2,5-FDCA was prepared following the method in a previous reportwith minor modifications.4.341 The biobased polyester was synthesized by mixing the monomers IA, FA , 2,5-FDCA, and BD ;the catalyst p-toluenesulfonic acid ; and the radical inhibitor 4-methoxyphenol  in a round-bottomed flask. Subsequently,the mixture is heated to 160 °C under stirring and purging withN2-gas during the entire reaction time. After 7 h, thereaction is cooled to room temperature and subsequently diluted withdichloromethane . Next, the solutionis poured into a beaker containing cold methanol, and the productimmediately precipitates. The precipitate is filtered off, washedwith methanol, and dried under vacuum, providing the product (13.3g) as an off-white powder.The unsaturated polyester was synthesized following the method ina previous report with minor modifications.4.42) atmosphere for 5 h for curing tocomplete.The biobased resin was preparedby mixing the polyester (2.5 g) in the reactive diluent X500  and the reactive viscosity enhancer X600 under sonication until fully dissolved and homogeneous. Subsequently,cobalt(II)-ethylhexanoate  was added and mixed.Next, the MEKP  was added to the mixture andimmediately mixed until homogeneous. Afterward, the mixture was keptunder a nitrogen (N4.5–1 and with 4cm–1 resolutions. Data were recorded and processedusing the software PerkinElmer Spectrum (2015). The degree of conversion(DC) corresponds to the percentage of C=C bonds converted tosingle bonds (C–C) during curing to form the polymer resin.The curing of the biobased resin was calculated by following the decreaseof the peak centered at 1637 or 813 cm–1 correspondingto the C=C stretching vibration. To quantify the DC, the peakat 1714 cm–1 representing the C=O stretchingvibration was taken as internal standard since it is not affectedby the curing process. For the VER resin, the peak at 1720 cm–1 corresponding to the C=O or the aromatic C=Cbond at 1601 cm–1 was taken as internal standardssince these are not affected by the curing process and following thedecrease of the peak centered at 1630 cm–1. Thecalculations were performed following FTIR analysis was carried out on a PerkinElmer Spectrum 100 FT-IRSpectrometer equipped with a single reflection [attenuated total reflection(ATR)] accessory unit (Golden Gate) from Graseby Specac LTD  and a TGS detector using the Golden Gate setup. The spectrawere collected based on 16 scans averaged in the transmittance modeat regions between 4000 and 600 cm4.62-atmosphere (rate 50 mL/min). The thermal behaviorof the samples was investigated by using two repeated heating–coolingcycles. The temperature program was as follows: the temperature wasfirst ramped from 25 to 200 °C and kept at 200 °C for 2min, followed by a cooling cycle from 200 to −30 °C. Afteran isotherm at −30 °C for 2 min, a second heating cyclewas performed from −30 to 200 °C. The glass transitiontemperature (Tg) was determined from thesecond heating curve. Data analysis was performed on Mettler STAReevaluation software.DSC analysis was performed using a Mettler-Toledo DSC1 STARe system.Samples having masses of ≈6 mg were inserted in 100 μLaluminum pans with pierced lids. The applied heating rate was 10 °C/minin a N4.72-atmospherewith a purge rate of 50 mL/min at the temperature range 30–600°C. The samples were kept isothermally at 120 °C for 10min to remove solvent residues and then cooled to 30 °C, followedby heating at 600 °C, and the starting degradation temperature(Tonset), temperature with the highestdegradation rate (Tmax), final degradationtemperature , and residual amountat 600 °C were determined. Data analysis was performed on MettlerSTARe evaluation software.TGA wasperformed with a Mettler-Toledo TGA/SDTA 851e instrument. Sampleshaving masses of ≈8 mg were used, and the experiment was performedat a heating rate of 10 °C/min under an N4.81H NMR analysis was performedat 298 K on a 400 MHz Bruker Avance III HD spectrometer using deuteratedchloroform (CDCl3) or deuterium oxide (D2O)as the solvent. Spectra were based on 128 scans and reported in partsper million relative to the solvent residual peak at 7.26 ppm forCDCl3 or at 4.79 ppm for D2O. MestReNova 9.0software was used for data analysis.4.9SECanalysis to determine the molecular weight was performed on a MalvernGPCMAX instrument equipped with an autosampler, a PLgel 5 μmguard column (7.5 mm × 50 mm), and two PLgel 5 μm MIXED-D(300 mm × 7.5 mm) columns. The polymer sample (4–5 mg/mL)was dissolved in chloroform containing 2% v/v toluene, which was alsoused as an eluent. The flow rate was 0.5 mL/min, and the temperaturewas kept at 35 °C. Narrow disperse polystyrene standards withmolecular weights in the range of 1200–400,000 g/mol were usedfor calibration. OmniSEC version 5.10 software was used for data analysis.4.10Mechanical testingwas performed on an Instron 5944 Universal Testing Machine with asingle column, a 2 kN load cell, and a crosshead speed of 10 and 2mm/min. The tensile test was performed according to the standard ASTMD638 using dog-bone-shaped specimens with nominal dimensions of 60× 10 × 1 mm. All of the samples were conditioned at 23 °Cand 50% relative humidity for 2 days before testing. The results ofthe tensile tests were based on the average of 10 (for the biobasedresin) and 7 (for VER) individual measurements. The tensile stress-at-breakwas obtained at the failure point, and Young’s modulus wasdetermined from the slope in the linear region of the stress–straincurve. Bluehill software was used for the test control and data acquisition.4.1162 Three separate samples from each group (∼150mg) were immersed in THF for 24 h separately. Subsequently, the massof each sample was determined after swelling in THF. The SI was calculatedfollowing m1 is the mass of the material after swelling,and m2 is the initial mass of the samplesThe SI and GC were measuredfollowing the methods in a previous report with minor modifications.m3 is the mass of the samples after oven drying, and m2 is the initial mass of the samples.Next, the samples were dried in anoven at 70 °C for 24 h. The GC was calculated following 4.12–1 with 10 points/decade).The viscosity was determined at an initial shear rate of 0.5 s1.The viscositywas determined by rheological analysis with a TA Instrument modelDHR-2. About 0.300 mL of the samples were used for the tests. Theviscosity was recorded using a parallel plate of plate steel (25 mmdiameter) with a gap of 100 μm and by performing a shear ratesweep at 0.1–1000 s4.13t-test andcompared the means of the two groups. The software GraphPad Prism6 was used to calculate the statistics. Values are means ± standarddeviation (SD), where p < 0.05 indicates significantdifferences and ns = no significance.The experimentswere analyzed using the Student’s


Herein, we applied the Illumina MiSeq pyrosequencing platform to amplify the V3–V4 hypervariable regions of the 16 S rRNA gene of the gut microbiota (GM) and a gas chromatograph–mass spectrometer to detect the metabolites after supplementation with pumpkin oligosaccharides (POSs) to determine the metabolic markers and mechanisms in rats with type 2 diabetes (T2D). The POSs alleviated glucolipid metabolism by decreasing the serum low-density lipoprotein (LDL), total cholesterol (TC), and glucose levels. These responses were supported by a shift in the gut microbiota, especially in the butyric-acid-producing communities. Meanwhile, elevated total short-chain fatty acid (SCFA), isovaleric acid, and butyric acid levels were observed after supplementation with POSs. Additionally, this work demonstrated that supplementation with POSs could reduce TNF-α and IL-6 secretion via the FFA2-Akt/PI3K pathway in the pancreas. These results suggested that POSs alleviated T2D by changing the SCFA-producing gut microbiota and SCFA receptor pathways. The polysaccharides extracted from pumpkin  have attracted increasing attention in recent years because of their antidiabetic, antihyperlipidemic, and oxidation-resistance effects, their functional values, and their variety of nutrients ,2. HowevAs a metabolic disorder, T2DM is associated with metabolic disturbances with regard to proteins, fats, and carbohydrates, and it induces low-grade systemic inflammation and peripheral insulin resistance in response to a high-fat diet (HFD) ,6,7. AddDietary supplements or diabetes drugs alleviate T2DM associated with SCFA-producing gut micro-organisms. However, the effects of POSs on the GM in T2DM remain unclear, and no direct evidence regarding the relationship between SCFAs, the gut microbiota, and POSs is available. Thus, to compare the changes in the GM, gut metabolic profile, and SCFAs induced by POSs in HFD/STZ-induced T2DM rats, we used next-generation sequencing (NGS) via the Illumina MiSeq pyrosequencing platform. This systematic analysis might provide a novel understanding of the interactions between micro-organisms and the host, the key biomarkers, and the underlying mechanisms induced by POSs in metabolic diseases. As described in our previous study , we prepn = 50). All the animals were kept in a controlled SPF environment at 22 ± 1 °C, at 42–47% humidity, and under a 12/12 h light/dark cycle (lights on 06:00–18:00). Additionally, the animals had free access to food and water during this study. After one week of acclimatization, the rats received a high-fat diet (HFD) composed of 66.5% pulverized standard rat pellet, 1% sodium cholate, 2.5% cholesterol, 20% sucrose, and 10% lard for four weeks. After the rats had fasted overnight, 30 mg/kg STZ (Sigma) was given to the HFD-fed rats via intravenous (i.v.) injection. A vehicle control buffer was given to the normal control rats. Two weeks after the STZ injection, we examined the fasting blood glucose (FBG) concentration, and the rats with a FBG > 11.1 mmol/L (n = 30) were selected for further experiments. The animal experiments were carried out according to the guidelines of China’s Institutional Animal Care and Use Committee, and all the procedures performed were consistent with the European Community guidelines (2010/63/EU) for the care and use of experimental animals. In order to eliminate the effect of sex on feed intake and the results of glucolipid metabolism, we conducted the animal experiments using adult male Wistar rats : rats fed with HFD during the experiment; (2) an HFD + POS group (n = 10): HFD-fed rats treated intragastrically with 500 mg/kg pumpkin oligosaccharides once daily according to a toxicology test; and (3) an HFD + metformin (MET) group (n = 10): HFD-fed rats treated intragastrically with 200 mg/kg metformin once daily [Next, the T2D rats were divided into three groups: (1) a nontreated group . Then, rapid freezing of the pancreatic tissue and fecal samples was carried out using liquid nitrogen. The blood glucose (GLU), HDL-C, triglyceride (TG), total cholesterol (TC), and LDL-C contents of the serum were examined by the Health Science Center of Peking University as previously described [We collected blood samples via ocular sampling once the rats were anesthetized. Afterward, they were beheaded and fresh fecal samples (rectum content) and pancreatic tissue were collected at the end of the experiment. As previously described , to collescribed . The sho2O, 2 μL of DNA template, 1 μL of reverse primer (10 µM), 1 μL of forward primer (10 µM), 2μL of dNTP (2.5 mM), 5 μL of 5 × GC buffer, and 5 μL of 5 × reaction buffer. The PCR procedure was as follows: 98 °C, 2 min for initial denaturation; 98 °C, 15 s for denaturation; 55 °C, 30 s for annealing; 72 °C, 30 s for extension, repeated for 25 cycles; and 72 °C, 5 min for the final extension. After purification of the PCR product using gel electrophoresis followed by AxyPrep DNA Gel Extraction , a TruSeq Nano DNA LT Library Prep Kit  was used to construct the DNA library. After testing the results using an Agilent High Sensitivity DNA Kit , Illumina MiSeq was used to sequence the samples.Based on a previous description , a TIANa2O and mixed at 4 °C for 90 s. After the addition of heptadecanoic acid , 2-chloro-lphenylalanine , and −20 °C pre-cooled methanol (1000 μL), the samples were vortexed for 30 s. Then, the samples were treated for 10 min with ultrasound at RT and cooled on ice for 30 min. Next, centrifugation was carried out for 10 min at 12,000 rpm and 4 °C. Then, the supernatant (1.2 mL) was blow-dried using vacuum concentration. Then, methoxyamine pyridine solution  was added, and the reaction was carried out for 120 min at 37 °C. Next, the mixture was reacted for 90 min at 37 °C with 60 μL of BSTFA reagent containing 1% TMCS. Next, after the 10 min centrifugation at 12,000 rpm and 4 °C, the supernatant was transferred and stored for analysis.GC–MS was used to detect the profiles of the metabolites and SCFAs. Briefly, 100 mg fecal samples were placed in 5 mL centrifuge tubes with 500 μL of ddHFor the separation of the derivatives, an HP-5MS capillary GC column  was applied. For the injection of the samples (1 µL), an autosampler was used in split mode and a 20:1 split ratio. The injection, interface, and ion source temperatures were 280, 150, and 230 °C, respectively. The temperature was increased as follows: initial temperature: 60 °C for 2 min; 10 °C/min until 300 °C; and 300 °C for 5 min. To determine the results of the mass spectrometry, the full-scan method was used from 35 to 750 (m/z).g was conducted to collect the supernatant (800 μL). Then, after a 2 min oscillation of the supernatant with ethyl acetate (800 μL), the samples were centrifuged again . After mixing 600 μL of the organic phase with 500 μM 4-methylpentanoic acid, each sample was collected into a gas-phase flask to analyze the SCFA contents.We mixed standard concentrations of caproic acid, isovaleric acid, valeric acid, isobutyric acid, butyric acid, propionic acid, and acetic acid with ethyl acetate to 200, 100, 50, 20, 10, 5, 1, and 0.1 μg/mL, respectively. After resuspension of the colon contents (100 mg) with 0.5% phosphoric acid (1 mL) in 2 mL sterile tubes, 10 min centrifugation at 17,949× An Agilent 7890A/5975C system equipped with a DB-WAX MS silica capillary column (30 m × 0.25 mm × 0.25 μm) was used for the GC–MS analysis. After 1 min of maintenance of the oven temperature at 90 °C, the temperature was raised to 120 °C (10 °C/min). The oven temperature was then increased to 150 °C (5 °C/min) and then to 250 °C (25 °C/min). The injection port temperature was set at 250 °C. During each run, 1 μL of the prepared sample was injected using helium as the carrier gas at a 1.0 mL/min flow rate with a split ratio of 10:1.2, and 500 ng of total RNA. For the PCR, an SYBR green master mix  was employed in ABI7500 [The gene expression was assayed using real-time quantitative PCR (RT-qPCR) . Briefly ABI7500 . GAPDH e ABI7500 .p < 0.05. QIIME  was used to analyze the Illumina MiSeq sequences. Based on the linear discriminant analysis (LDA), which was conducted using the Wilcoxon rank sum and Kruskal–Wallis tests (p < 0.05), we employed mothur software   and identified the key communities for separating the different treatment groups . Spearman analysis (p < 0.05) was carried out to evaluate the connections of the SCFAs to key GM communities. One-way variance analysis in SAS (version 9.4) was carried out to analyze the effects of POSs on the SCFAs, gene expression, and biochemical parameters. Data are listed as the square means ± SD. Means with different letters are significantly different, p = 0.03) with POS supplementation for four weeks compared to the NC group (p = 0.03). We also detected a remarkable reduction in the TG concentration (p = 0.01) when POSs and MET were administrated intragastrically for four weeks. The changes in the lipid metabolism parameters were similar to those in a study in which T2DM rats were treated with a hypoglycemic diguanide drug, presenting decreased serum TG, TC, and LDL-c levels [p = 0.06) in the serum LDL-c content was detected under POS supplementation, no differences were detected in the MET group. The serum HDL-C did not differ (p = 0.69) between the control, POS, and metformin groups. There is no direct evidence regarding the effects of POSs on T2D in terms of lipid metabolism and glucose attenuation. However, as a hypoglycemic diguanide drug for oral usage, MET is significantly connected to glycemic control [To investigate the hypoglycemic and hypolipidemic effects of POSs, STZ-induced T2D rats were continued on an HFD. The concentration of serum GLU significantly decreased  were identified, belonging to Bacteroidetes, Proteobacteria, and Firmicutes (p < 0.05) (p < 0.05), and the POS group showed the selective enrichment of Prevotella, Paraprevotellaceae, Prevotellaceae, Phascolarctobacterium, rc4_4, Deltaproteobacteria, and Bilophila   D. The LDctively) . Salgaçoctively) . This reNext, we compared and overlayed the total ion chromatography (TIC) spectra of the quality control samples. There was an overlap between the retention time and response intensity of the quality control samples, which suggested that this model had low variation, good stability, and robustness, and a low instrumental error A. The ovp < 0.05 and VIP > 1 were defined as significantly differential, while 0.05 < p < 0.1 and VIP > 1 were considered differential [We identified the key metabolites in the different treatments based on the VIP (variable importance in the projection) value. For the biomarkers differentially expressed in the varied treatments, metabolites with erential . Our stuerential . Similarerential . p < 0.01) after POS treatment in the HFD T2D rats, consistent with the metabolite detection results. We also observed a significant increase (p < 0.01) in the acetic acid concentration under POS supplementation compared to the control group. Valeric acid, isobutyric acid, and propionic acid also increased. An evident elevation in the total SFCAs (p = 0.03) was detected after POS treatment in the T2DM rats. The results of the gut metabolic analysis indicate that SCFAs, especially butyric and isovaleric acids, are the key biomarkers of the antidiabetic effect of pumpkin oligosaccharides in T2DM rats.To investigate whether SCFAs were key mediators of POSs within the GM in the improvement of T2DM, the SCFAs in the colon contents were analyzed. The concentrations of caproic acid, isovaleric acid, valeric acid, isobutyric acid, propionic acid, butyric acid, and acetic acid are shown in p < 0.05) correlation with butyric acid . These key species presented the same trend for the acetic and isovaleric acids. Meanwhile, Streptococcaceae, Xanthomonadaceae, and Xanthomonadales presented negative correlations (p < 0.05) with isovaleric acid, butyric acid, and acetic acid. The enrichments of Prevotellaceae and Paraprevotellaceae were correlated with propionic acid with coefficients of 0.49 and 0.55. NGS and metabolomics profiling were used to evaluate the effects of POSs on the gut microflora and metabolites in T2DM. We found that butyric acid was strongly correlated with the key communities under POS treatment, consistent with similar experiments on the cross-feeding between Akkermansia muciniphila and butyrate-producing bacteria promoting butyrate production [Many studies have shown that microbes are significantly connected to the metabolism of SCFAs ,39. The oduction ,42. Addioduction . Elevateoduction . The sigp < 0.01). In the present study, the pancreatic tissues from POS-treated rats showed dramatically reduced TNF-α and IL-6 expression at the transcriptional level (p < 0.01). This result is supported by another study using chitosan oligosaccharides, indicating decreased TNF-α and resistin levels, as well as increased adiponectin, during treatment in T2DM patients [SCFAs play their roles by binding to FFA2 and FFA3, which are considered G-protein-coupled receptors and act as free fatty acid receptors . There ipatients . TNF-α apatients . Simultapatients . Based oOur present work demonstrated the beneficial effects of POSs in an STZ HFD-induced T2DM rat model. The administration of POSs decreased the serum GLU, TC, and TG levels; decreased the pancreatic IL-6 and TNF-α gene expression; and increased the gut SCFAs and the pancreatic mRNA levels of Akt and PI3K. Simultaneously, we detected the key species through microbiome analysis using 16s rRNA sequencing and the key metabolites using GC–MS after POS treatment in T2DM rats. The Spearman correlation analysis revealed that POSs fermented by enteric micro-organisms induced the generation of SCFAs. SCFAs circulated to peripheral tissues play an important role by binding to the FFA2 receptor, which might be the mechanism underlying the improvements in inflammation in the pancreas. The changes in Akt/PI3K supported this notion. The selective enrichment of the SCFA-producing communities and the FFA2-Akt/PI3K pathway indicated that POS treatment has a potential therapeutic effect on T2DM by improving glucolipid metabolism. Further research could focus on the deep macromolecular mechanisms underlying the effects of macromolecular polysaccharides and oligosaccharides on biomarkers from the gut microbiota.


Additionally, dithianon at 0.01 and 0.1 µgmL−1 downregulated the expressions of glutamatergic (slc17a6b), GABAergic (gad1b), and dopaminergic (th) neuronal markers. Contrarily, dithianon upregulated the expression of the oligodendrocyte marker (olig2) at concentrations of 0.001 and 0.01 µgmL−1, concurrently suppressing the gene expression of the glucose transporter slc2a1a/glut1. Particularly, dithianon-induced increase in reactive oxygen species (ROS) production was reduced by both N-acetylcysteine (NAC) and betaine; however, only NAC prevented dithianon-induced mortality of zebrafish embryos. Moreover, NAC specifically prevented dithianon-induced alterations in glutamatergic and dopaminergic neurons while leaving GABAergic neurons unaffected, demonstrating that the major neurotransmission systems in the central nervous system differentially respond to the protective effects. Our findings contribute to a better understanding of the neurotoxic potential of dithianon and to developing preventive strategies.Despite the widespread agricultural use of dithianon as an antifungal agent, its neurotoxic implications for humans and wildlife have not been comprehensively explored. Using zebrafish embryonic development as our model, we found that dithianon treatment induced behavioral alterations in zebrafish larvae that appeared normal. Detailed quantitative analyses showed that dithianon at ≥0.0001 µgmL The negative effects of chemical contaminants on the ecosystem and animal life have become a global concern. Despite the contribution of xenobiotics, such as pesticides and pharmaceutical compounds, to the growth of agricultural industries and the control of infectious diseases, they are harmful to the environment and endanger the health of humans and aquatic non-target organisms via surface or underground water contamination . IdentifSaccharomyces cerevisiae) dithiin),evisiae) , exert delegans) , and redN-acetylcysteine (NAC) is an antioxidant, anti-inflammatory, and tumor-inhibiting agent [N,N,N-trimethyl glycine) is an antioxidant that serves as an osmolyte to maintain cell volume, reverse stress-induced aberrant cellular methylation [ROS act as cell signaling molecules in both normal and pathological biological processes. Mitochondria are among the most sophisticated responsive systems in the body. They are the principal generators of ROS, and failure in their function can induce oxidative stress and damage to other cellular compartments and molecular machinery, resulting in the progression of neurological diseases ,11,12. Nng agent ,14 that ng agent . Betainehylation , reduce hylation ,18,19.Neurons communicate with each other through neurotransmitters. The three most important neurotransmitters in the CNS are glutamate, gamma-aminobutyric acid (GABA), and dopamine. Glutamate and GABA are the major excitatory and inhibitory neurotransmitters regulating neuronal excitability in the brain, and dopamine plays a role in the brain’s reward system and coordination of body movement. The intricate interplay among these neurotransmitters is crucial for the precise development and proper functioning of the brain. Disruption of neuromodulation has been linked to various neurological conditions, including depression, schizophrenia, Parkinson’s disease, and other neurodegenerative disorders ,21. In aDanio rerio) is a useful vertebrate model in studies on hazard identification, particularly in toxicological research of environmental chemicals [Zebrafish  at 28.5 °C. The embryos were staged as hours post-fertilization (hpf) and days post-fertilization (dpf) according to Kimmel staging [Wild-type AB zebrafish were obtained from the Zebrafish International Resource Center . Adult zebrafish were raised in balanced saltwater at 27.5 °C under a 14/10 h light/dark cycle. Zebrafish eggs were obtained from 3–12-month-old adult fish by natural spawning and reared in E3 medium  was dissolved in dimethyl sulfoxide (DMSO)  and diluted with E3 medium. Zebrafish embryos in the 50% epiboly stage (5 hpf) were treated with dithianon at 0, 0.0001, 0.001, 0.01, 0.1, 0.2, and 0.5 µgmL−1 dithianon. Treatments were administered in 5 mL of 0.5% DMSO/E3 medium in 35 mm Petri dishes, with 25 embryos per dish. Each experiment was repeated at least thrice.We tested the ability of NAC  and betaine  to alleviate dithianon-induced toxicity in zebrafish embryos using previously described methods ,31. BrieMorphological abnormalities in zebrafish embryos were monitored using bright-field imaging. Briefly, the embryos were anesthetized with 0.02% tricaine (Sigma-Aldrich Corp.) in E3 medium, mounted with 3% methylcellulose (Sigma-Aldrich Corp.), and bright-field images of the embryos were captured using a SZX16 fluorescent stereoscope  equipped with an AxioCam camera  and Zeiss Zen 3.4 (Blue edition) software (Carl Zeiss) .−1 dithianon in the 50% epiboly stage was monitored at 5 dpf with or without co-treatment of NAC or betaine. All experiments were repeated at least three times.The mortality of the embryos treated with dithianon from the 50% epiboly stage was examined until 4 dpf using the Olympus stereoscope. Survival rate of the 25 embryos treated with 0.1 µgmLBehavioral tests of zebrafish larvae treated with dithianon were performed in the 6 dpf stage . Before b-actin (actb1). The transcripts were quantified using the 2−ΔΔCt method. The qPCR primers are listed in the Twenty-five embryos from each exposure group were collected in a 1.5 mL tube at the 28- or 72 hpf stage, and then 500 µL of TRI Reagent™ solution was added . Thereafter, 100 µL of chloroform was added to separate the organic and inorganic phases, followed by 250 µL of isopropanol to precipitate the RNA. The total RNA was extracted using the Qiagen RNeasy Mini Kit , and RNA concentration was measured using a Nanodrop Lite Spectrophotometer (Thermo Fisher Scientific). cDNA was synthesized using Superscript III Reverse Transcriptase  according to the manufacturer’s instructions. Quantitative PCR (qPCR) was performed on the QuantStudio 1 Real-Time PCR system (Thermo Fisher Scientific) using SYBR Green with low ROX ; b-actin was used as the control. Each sample was run in triplicate. Gene expression was normalized to the expression level of −1 2′,7′-dichlorofluorescein diacetate  in E3 medium with 0.1% DMSO, and incubated in the dark at 23 °C for 30 min. The embryos were anesthetized with 0.02% tricaine, mounted with 3% methylcellulose on microscope slides, and imaged using a SZX16 fluorescence microscope (Olympus). Fluorescence intensity in the region of interest of each sample was measured using ImageJ 1.53 software .The control and treated embryos were dechorionated at 50 hpf, placed in a small Petri dish (eight embryos per group) with 5 mL of 1 µgmLp < 0.05, ** p < 0.01, and *** p < 0.001.All statistical analyses were performed, and graphs were created using GraphPad Prism . The data are presented as quantitative values, which are expressed as mean ± standard deviation. To ensure statistical robustness, each experiment had a minimum of three independent tests. Differences between control and treatment groups were assessed after checking for homogeneity of variances with Levene’s test using a one-way analysis of variance (ANOVA) followed by Bonferroni post hoc multiple comparisons. All statistical analyses were carried out using SPSS version 24. To determine statistical significance, the levels of significance were set as follows: * −1 dithianon was examined from 5 hpf to 4 dpf  at 25 hpf, and small eyes (43% ± 5%) and shrunken swim bladders (60% ± 5%) at 4 dpf , an upstream regulator in antioxidant genes (gpx1a (glutathione peroxidase 1a) and sod1 (superoxide dismutase 1) at 0.0001 µgmL−1 and that of sod2  at 0.001 µgmL−1. Moreover, the expressions of gpx1a, sod1, and sod2 were downregulated in embryos exposed to dithianon at 0.1 µgmL−1 (We investigated the molecular basis of the dithianon-induced oxidative stress response in zebrafish using qRT-PCR. Dithianon exposure caused a dose-dependent decrease in the mRNA expression of nt genes A. Dithia1 µgmL−1 A. These −1 dithianon upregulated the expressions of twnk (mtDNA helicase twinkle mtDNA helicase) and polg (mtDNA polymerase (DNA directed) gamma), which are essential for mtDNA replication. Furthermore, the expression of tk2 (deoxyribonucleoside kinase thymidine kinase 2) for mtDNA synthesis considerably increased under 0.01 µgmL−1 dithianon treatment. Exposure to 0.001 µgmL−1 dithianon significantly upregulated the expression of tfam  (p < 0.01), which is essential for mtDNA transcription. On the contrary, the expressions of both polg and tk2 were significantly downregulated following 0.1 µgmL−1 dithianon treatment   content and metabolism B. Exposuctively) B.ndufs4 (NADH–ubiquinone oxidoreductase iron–sulfur protein 4 subunit) of complex I, sdha (succinate dehydrogenase A) of complex II, uqcrc2b (ubiquinol–cytochrome c reductase core protein II) of complex III at 0.001 and 0.01 µgmL−1, and that of cox5ab (cytochrome c oxidase subunit 5ab) of complex IV at 0.001 µgmL−1 compared with those in the untreated controls. On the contrary, the expressions of uqcrc2b, cox5ab, and atp5fa1  of complex V were significantly downregulated under 0.1 µgmL−1 treatment  compared with those in the untreated controls  and mfn2 (fusion marker mitofusin 2) were slightly upregulated in embryos exposed to 0.0001 µgmL−1 dithianon but dose-dependently downregulated in embryos exposed to 0.01 and 0.1 µgmL−1 , pro-apoptotic gene baxa (BCL2 associated X apoptosis regulator a), tp53 (tumor protein p53), pro-apoptotic gene casp9 (apoptosis-related cysteine peptidase 9), and activator of mitochondrial apoptotic signaling apaf1 (apoptotic peptidase activating factor 1) compared with those in the untreated controls. Furthermore, the expression of casp3a (apoptosis-related cysteine peptidase a) was slightly upregulated under 0.001 µgmL−1 dithianon treatment, and those of both casp3a and casp8 (apoptosis-related cysteine peptidase) were significantly downregulated under 0.1 µgmL−1 treatment (p < 0.001), compared with those in the untreated controls (ripk3 (receptor-interacting serine–threonine kinase 3) was significantly downregulated under 0.01 and 0.1 µgmL−1 dithianon treatments , and that of ripk1l (receptor (TNFRSF)-interacting serine–threonine kinase 1, like) was also significantly downregulated under 0.1 µgmL−1 treatment (p < 0.01), compared with those in the untreated controls (sqstm1 (sequestosome 1) and stress-induced molecular chaperone hsp70l  significantly increased in embryos exposed to 0.1 µgmL−1 dithianon (p < 0.001) compared with those in the untreated controls (We examined the effect of dithianon on cell death A. The excontrols A. The excontrols B. In parcontrols C. These −1 dithianon significantly reduced the level of slc2a1a (solute carrier family 2 member 1a)/glut1 (p < 0.05), which is expressed in the cranial vasculature, endothelial cells, otic vesicles, and liver, compared with that in the untreated controls. In contrast, the level of slc2a2 (solute carrier family 2 member 2)/glut2, a glucose sensor that is expressed in the brain, muscle, and liver, was unaffected in embryos treated with 0.01 µgmL−1 dithianon and significantly decreased with 0.1 µgmL−1 (p < 0.001) compared with that in the untreated controls , a glutamatergic excitatory neuronal marker, gad1b (glutamate decarboxylase 1b), a GABAergic inhibitory neuronal marker, and th (tyrosine hydroxylase), a dopaminergic neuronal marker, were downregulated in a dose-dependent manner (olig2 (oligodendrocyte lineage transcription factor 2), a marker of oligodendrocyte progenitor, was considerably upregulated in the embryos exposed to 0.001 and 0.01 µgmL−1 dithianon, whereas expressions of olig1 (oligodendrocyte transcription factor 1), a marker of oligodendrocyte progenitor, and mbpa (myelin basic protein a), a myelination marker, were not markedly altered compared with those in the untreated controls (olig1 and mbpa were significantly downregulated in embryos exposed to 0.1 µgmL−1 dithianon (p < 0.05) compared with those in the untreated controls. These results suggest that dithianon reduces the populations of glutamatergic excitatory, GABAergic inhibitory, and dopaminergic neurons but increases the oligodendrocyte progenitors in the developing central nervous system at 0.01 µgmL−1.Deficiencies in  release ,35. Thert manner B. Interecontrols C. The ex−1 dithianon (−1 dithianon (p < 0.01)  A. Howeveithianon B and the slc2a1a C. These slc17a6b in glutamatergic excitatory neurons and th in dopaminergic neurons in the central nervous system of larvae treated with 0.1 µgmL−1 dithianon was only significantly restored with NAC (p < 0.05); however, both antioxidants failed to restore the expression of gad1b in GABAergic inhibitory neurons (olig1 (p < 0.05) and olig2 (p < 0.05) but not that of mbpa in the larvae treated with 0.1 µgmL−1 dithianon  is required for the formation of the blood–brain barrier [slc2a2 (glut2) is important for glucose sensing in neural circuits of the central nervous system [Furthermore, dithianon exposure concentration-dependently interrupted mitochondrial homeostasis. A previous study showed that dithianon at 100–1000 µgmLin worms . Althoug barrier , and slcs system . Our resbcl-2a to baxa, a predictor of apoptotic susceptibility, indicating dithianon-induced apoptotic pressure without cell death. In contrast, the expression of most apoptotic and necroptotic markers was downregulated by dithianon at high concentrations. The expression of the molecular chaperone hsp70l and autophagy adaptor sqstm1 was considerably upregulated, which could have protected zebrafish against dithianon-induced cellular toxicity [Moreover, in this study, the expression of apoptotic markers was upregulated by dithianon at low concentrations; however, there was no change in the ratio of toxicity .C. elegans worms at very high concentrations [olig2 oligodendrocyte progenitors in the larvae treated with 0.001 and 0.01 µgmL−1 dithianon, suggesting that the glial cells that support neuronal populations may compensate for dithianon-induced neuronal loss. Nonetheless, dithianon-induced developmental alterations in neurotransmission systems may disrupt neural connections and communication, possibly resulting in neurological and psychiatric disorders.In this study, we demonstrated dithianon-induced toxicity in the three major neurotransmission systems of the central nervous system, whereas it was recently shown that dithianon caused only dopaminergic neuronal toxicity in trations . In cont−1 dithianon treatment. Dithianon-induced decrease in glutamatergic and dopaminergic neuronal populations and olig1 and olig2 glial cell populations was also significantly restored with NAC but not with betaine. However, the populations of GABAergic neurons and mbpa myelinated glial cells were not restored with NAC or betaine under similar conditions. These findings indicate that the neuronal populations of different neurotransmission systems respond differentially to dithianon-induced oxidative stress and neurotoxicity and that neurotoxicity is also differentially alleviated by NAC and betaine. Dithianon is known to react with thiol groups in peptides, including glutathione, which modulates the physiological levels of ROS and the cellular oxidative stress response [Both antioxidants NAC and betaine reduced dithianon-driven ROS production in the brain; however, early embryonic mortality was reduced only with NAC, suggesting that ROS generation is not the direct cause of embryonic death under 0.1 µgmLresponse . AccordiDevelopmental neurotoxicity from dithianon is poorly understood, particularly in vertebrates. To this end, dithianon was recently shown to induce dopaminergic neurotoxicity in worms, and NAC alleviated this effect . In the Our findings distinguished the differential toxicity of dithianon at different concentrations and the potency of the protectants NAC and betaine in embryonic development using zebrafish. In particular, behavioral and molecular assessments demonstrated that dithianon exposure altered the major neurotransmission systems in zebrafish larvae. Moreover, the altered GABAergic neurons were less responsive to NAC than glutamatergic and dopaminergic neurons, indicating the differential potency of protectants against the toxicity of environmental chemicals. Developing neuronal populations interact with each other to generate neuronal circuits with proper connections and functions in the central nervous system. Therefore, it is essential to develop a strategy for the integrative assessment of chemical toxicity and protection at the organismal level.


Udi et al. present a new method for quantitative subcellular fractionation of a wide range of mammalian cells while maintaining nuclear integrity. This method provides a fast and reliable means to perform mass spectrometry and DeDuvian enrichment analyses for the different cellular compartments. Subcellular fractionation in combination with mass spectrometry–based proteomics is a powerful tool to study localization of key proteins in health and disease. Here we offered a reliable and rapid method for mammalian cell fractionation, tuned for such proteomic analyses. This method proves readily applicable to different cell lines in which all the cellular contents are accounted for, while maintaining nuclear and nuclear envelope integrity. We demonstrated the method’s utility by quantifying the effects of a nuclear export inhibitor on nucleoplasmic and cytoplasmic proteomes. The eukaryotic nucleus is surrounded by the nuclear envelope (NE) and contains most of the cell’s genetic material in the form of chromosomes . It reprThe transport of molecules in and out of the nucleus is mediated by nuclear pore complexes (NPCs) embedded within the NE. A single NPC is composed of multiple copies of ∼30 different proteins, termed nucleoporins Nups; , and is Given the importance of proper protein localization and its effect on pathological states, methods to fractionate mammalian cell lines for further biochemical studies into nuclear and cytoplasmic fractions are potentially of great utility. Ideally, such methods should be rapid, straightforward, reproducible, and be easily adaptable to multiple mammalian cell types, requiring only modest amounts of starting material. The method should ideally produce a manageable number of final fractions without loss-inducing wash steps. Each fraction should represent a single subcellular compartment  or sets of subcellular compartments  and be recovered in high yield concentration and purity. For nuclear studies, the fractions should include morphologically intact nuclei and NEs, both to ensure retention of nuclear and NE proteomes and to allow ultrastructural analyses. Many published methods and commercially available kits lack some or all of these desirable characteristics, and thus are of particular but not general utility . For exaSaccharomyces cerevisiae and Trypanosoma brucei cells, using the stabilizing agent polyvinylpyrrolidone (PVP) in our lysis buffer , although, due to the difference in characteristics between mammalian cells, S. cerevisiae and T. brucei a significant redesign of these protocols for the subcellular fractionation of mammalian cells was needed , HeLa , HOS (human osteosarcoma cells), HT1080 (human fibrosarcoma cells), and N2A (mouse neuroblastoma cells). The protocol was slightly adjusted for each cell line based on the three parameters mentioned above. Each fraction was analyzed by SDS-PAGE and Coomassie blue staining . NotablyOne of the major challenges in cellular fractionation is maintaining the integrity of the nuclei, avoiding leakage of native nucleoplasmic assemblies . In ordeWe next used a one-step, high-yield approach to release the NEs by combining DNase, RNase, and Heparin  to minimTo further assess the enrichment of each fraction, we performed quantitative label-free MS analyses of the cytoplasmic, membrane, NE, and nucleoplasmic fractions of HEK293T cells, providing a measure of the degree to which each identified protein partitions between these four fractions. 2-tagged reporter bearing both an NLS and a Crm1-recognized NES. After even a relatively brief (1 h) incubation with 20 nM LMB, all the GFP signal that was originally in the cytoplasm was localized in the nucleus , for three reasons. First, because it mediates the bulk of nuclear protein export (proteins carrying nuclear export sequences [NESs]), we might expect inhibition of Crm1-mediated export to generate a significant nucleocytoplasmic proteome alteration. Second, this alteration—provided that its duration is relatively brief—should be largely due to nucleocytoplasmic redistribution rather than general pleiotropic changes in total protein levels due to loss of cell viability, and so detectable only through a reliable nucleocytoplasmic fractionation protocol . Third,  nucleus , and so  nucleus . The res nucleus . Fig. 5  nucleus . We also nucleus . The abu nucleus . A signi nucleus . As for  nucleus . In mamm nucleus . Interes nucleus , was fou nucleus . Another nucleus . Other n nucleus , which w nucleus . In the  nucleus , is incr nucleus . The MYC nucleus . MYC pro nucleus . MYC pro nucleus . The red nucleus ; hence, We have developed a rapid, reproducible, and efficient method to fractionate cultured mammalian cells. This protocol does not require any unusual equipment or reagents. In addition, this method is applicable to a range of cell lines. Importantly, the protocol does not require large amount of cells; cells from a confluent 100-mm tissue culture plate are sufficient for a successful fractionation. Finally, this protocol produces only four samples, a sample for each fraction of the cell, and all the cell content is accounted for since there are no wash/discard steps. These two factors allow easy downstream processing for traditional biochemical analyses as well as for in-depth proteomic analyses. Moreover, since this method does not use extreme conditions, the resulting fractions are compatible for further downstream applications where the subcomplex interactions within each compartment are maintained. Since we have demonstrated that the fractions obtained are highly enriched, this provides a reliable means of monitoring proteins of interest in the different cellular compartments.There are many fractionation methods and commercial kits available; however, they are usually either lengthy, expensive, or require special equipment. The limitations of the commercially available kits have been described above: in particular, limited and incomplete enrichment of the nuclear fraction , proteinAs mentioned above, mislocalization of proteins within the cell contributes to the pathogenesis of many human diseases . The avaOverall, we found a strong interplay between import and export through the NPC and how mislocalization of transport factors plays a significant role in the process. Thus, while one might expect simply to see accumulation of a large set of proteins in the nucleus as a result of Crm1 inhibition, instead we show here that (i) Crm1 export inhibition exerts a subtle yet significant redistribution of a relatively small set of proteins, as was also previously reported in frog oocytes  and (ii)Finally, although each cellular compartment has its own characteristic set of marker proteins, numerous proteins display a complex distribution throughout the different fractions, likely not just simply due to inefficient fractionation but, based on the faithful fractionation of other such markers, reflective of intracellular communication between the different cellular compartments and distributed functionalities of those proteins in the different compartments.Tissue culture cell lines (HEK 293T (CVCL_0063), HeLa (CVCL_0030), HOS (CVCL_0312), and HT1080 (CVCL_0317)) were maintained in growth media, Dulbecco’s modified Eagle’s medium  with penicillin–streptomycin , and 10% (v/v) fetal bovine serum . N2A cells were maintained in growth media, Dulbecco’s modified Eagle’s medium: Nutrient Mixture F-12  with penicillin–streptomycin , and 10% (v/v) fetal bovine serum . Transfections into cells for transient and stable transgene expression were performed with the Lipofectamine LTX with Plus Reagent  according to the manufacturer’s protocol. Once transfected, stable cells were selected using Geneticin  in growth media.The pHR39-CMV-GFP-LacZ plasmid backbone with or without the nucleoplasmin NLS sequence was a generous gift from Melissa Kane (University of Pittsburgh). We then replaced the EGFP sequence in these plasmids with an mCherry sequence from the pRS426-GPD-mCherry-4xMS2 plasmid using Gibson cloning procedure, as per manufacturer specifications .The pcDNA3-EGFP plasmid was a kind gift from Sigi Benjamin-Hong . This plasmid was used to prepare new plasmid with NES/NLS/NLS-NES sequence according to To generate pcDNA3-EGFP-NES, an insert DNA fragment was prepared using oligonucleotides encoding LALKLAGLDI from human PKIα NES. Two oligonucleotides, 5′Phos-AATTTAGCCTTGAAATTAGCAGGTCTTGATATCG-3′ and 5′Phos-GATCCGATATCAAGACCTGCTAATTTCAAGGCTA-3′, were annealed and ligated into EcoRI- and BamHI-digested pcDNA3-EGFP.To generate pcDNA3-EGFP-NLS, an insert DNA fragment was prepared using oligonucleotides encoding PKKKRKV from SV40 large T antigen. Two oligonucleotides, 5′Phos- GATCTCCAAAAAAGAAGAGAAAGGTACA-3′ and 5′Phos- AGCTTGTACCTTTCTCTTCTTTTTTGG-3′, were annealed and ligated into HindIII and BglII digested pcDNA3-EGFP.To generate pcDNA3-EGFP-NLS-NES, an insert DNA fragment was prepared using oligonucleotides encoding PKKKRKV from SV40 large T antigen. Two oligonucleotides, 5′Phos- GATCTCCAAAAAAGAAGAGAAAGGTACA-3′ and 5′Phos- AGCTTGTACCTTTCTCTTCTTTTTTGG-3′, were annealed and ligated into HindIII and BglII digested pcDNA3-EGFP-NES.g for 40 min at 4°C in a SW55 rotor (Beckman ultracentrifuge). The interface and the sucrose underlay were collected as the membrane fraction and the pellet constituted the nuclear fraction. To further extract the NE fraction, the pelleted nuclei were resuspended in DNase buffer . Nuclei were vortexed briefly and incubated at room temperature for 15–30 min. The digestion progress was monitored by phase contrast microscopy, where the NE was visualized as “C”-shaped structures. The suspension was underlaid with 100 µl of 30% sucrose in 20 mM HEPES, pH 8, 0.1 mM MgCl2 and 1:100 Solution P, and the NE was pelleted at 18,000 g for 10 min at 4°C in Optima Ultracentrifuge. The supernatant was then collected and considered to be the nucleoplasm fraction, while the pellet contained the NE fraction. For downstream SDS-Page analyses and Western blot samples were methanol precipitated: 900 μl methanol was added to 100 μl of the sample. Samples were vortexed and incubated in −20°C overnight. Next, samples were pelleted at 20,000 g for 20 min at 4°C, resuspended by 2 s sonication in 500 μl methanol, incubated at −20°C for 20 min, and pelleted again. The resulting pellet was resuspended in 50 μl buffer A and sonicated for 5 s.Each fractionation procedure was carried out using cells from a 90 to 95% confluent 10 cm plate. Cells were washed with PBS and harvested with 1 ml trypsin (Trypsin-EDTA (0.25%) at 37°C for 1 min. Cells were pelleted at 10 g for 10 min at 4°C and resuspended in 1-ml pre-chilled PBS and pelleted again at 50 g for 2 min at 4°C. The supernatant was carefully removed, and cells were gently resuspended in lysis buffer , vortexed for 5 s, and left on ice for 5 min to swell. Cells were next lysed with needle strokes using a 1-ml insulin syringe with a 28 gauge needle. Lysis was monitored by phase contrast microscopy for completion and dispersal of cytoplasmic material away from nuclei. Lysed cells were underlaid with 200 μl 20% sucrose in 8% PVP buffer and pelleted at 3,500 g for 10 min 4°C. The supernatant, including the sucrose phase, was collected (cytoplasmic fraction). The pellet was resuspended with 1 ml 6% PVP supplemented with 1:100 solution P and 1 mM DTT and processed with a handheld polytron (PT 1200 E) twice for 10 s at mid power setting. In the absence of a polytron, it is possible to resuspend the nuclei pellet with repeated pipetting up and down of a 1.0 ml Pipetteman tip. While not as efficient as the Polytron, this method is suitable for many purposes. The dispersed nuclei was underlaid with 1 ml of 2.01 M Sucrose in 8% PVP and 1 mM DTT and centrifuged at 50,000 Solution P: 20 mg/ml phenylmethylsulfonyl fluoride, 0.4 mg/ml Pepstatin A in ethanol.2. pH adjusted to 6.53. with concentrated H3PO4.8% PVP buffer: 8% PVP-40, 20 mM K-Phosphate, 7.5 μM MgClLysis buffer: 6% PVP, 0.015% Digitonin, 0.015% Triton X-100, 4 μM Cytochalasin B, 1:100 Solution P , 1 mM DTT (0.045% detergent is required to lyse HeLa and HT1080 cells).2, 10 μM CaCl2, 1 mM DTT, 100 μg/ml Heparin, 0.1 μg/ml RNase, 1:1,000 DNase : Resuspended at 5 mg/ml in buffer containing 50% glycerol, 10 mM Tris–HCl (pH 7.4), 50 mM NaCl, 1 mM DTT, and 2 mM MgCl2), 1:100 Solution P.DNase buffer: 10% sucrose in 20 mM HEPES, pH 8 and 0.1 mM MgClBuffer A: 0.5 M TRIS base, 5% SDS.A handheld Polytron is highly recommended for this protocol as it is a low-cost laboratory instrument; however, in the absence of a polytron nuclei, can be resuspended with repeated pipetting.Hela cells were fractionated using #78833, Thermo Fisher Scientific; #AR0106, Boster; and #ab109719, Abcam as per manufacturer’s instructions.Proteins were measured using a capillary-based electrophoresis instrument . Protein amounts (0.5 mg/ml) were preoptimized for and were denatured using manufacturer-supplied reagents and loaded into multi-well plates. Protein separation and detection were performed via capillary electrophoresis, antibody binding, and HRP-conjugated visualization following the manufacturer’s instructions. Antibody optimization was completed for all proteins for which Wes analysis was performed to determine ideal dilution conditions. Antibodies used are listed in HEK293T cells were transiently transfected in 24-well plate on poly-L-lysine-coated cover slips to express mCherry-LacZ fusion proteins with or without NLS . Cells wHEK293T cells were fractionated according to protocol above. Nuclear permeability was measured using 1 μM Tetramethylrhodamine Dextran . Fluorescent images were obtained with the Revolve R4, Model: RVL2-K2 with Olympus 60× Plan Fluorite Oil IRIS Phase 3 objective with an NA of 1.25 at room temperature. Images were acquired with Echo Pro version 6.4.1. and processed with Photoshop.Methanol precipitated fractions were reduced and alkylated (25 mM iodoacetamide in the dark) protein samples were run ∼5 mm into a 10% bis-Tris SDS-polyacrylamide gel, and gels were Coomassie-blue stained.Proteins in gel plugs were digested and peptides were extracted as described in t test function in Microsoft Excel was used to calculate the P values, with parameters for a two-tailed distribution and two-sample unequal variance (https://doi.org/10.5281/zenodo.7630027.SpectroMine (Biognosys AG) software was used for label-free quantitation (LFQ). The protein LFQ outputs from SpectroMine were further analyzed using Microsoft Excel. To compare LFQs across samples from a cell fractionation, including biological replicates and technical replicates, normalization was applied so that the sum of the normalized LFQs for 6–8 abundant proteins from each LCMS run was the same. After normalization, the relative standard deviations of LFQs across samples for these 6–8 proteins were within 20%. Data distribution was assumed to be normal but this was not formally tested. Student variance . The heavariance , as distNuclei and NE fractions were fixed in 2% glutaraldehyde (VWR 100503-966) in 0.1 M sodium cacodylate buffer (pH 7.2) for >1 h at room temperature and then overnight at 4ºC, postfixed in 1% osmium tetroxide 1%, en bloc stained in 1% uranyl acetate in 0.05 M sodium maleate buffer (pH 5.2), and processed for Epon embedding.Ultrathin sections (60–65 nm) were counterstained with uranyl acetate and lead citrate and imaged on a Tecnai 12 electron microscope , equipped with an AMT BioSprint29 digital camera.2-NLS-NES were treated with 20 nM Leptomycin B  or vehicle (70/30 methanol/water) for 1 h at 37°C and the reporter fluorescence was examined with the Revolve R4, Model: RVL2-K2 with Olympus 40× Plan Fluorite Phase Ph2 NA: 0.75 at room temperature.Cellular fractionation: HEK293T cells stably expressing GFP2-NLS-NES were seeded at low density on poly-L-lysine-coated cover slips in 24-well plates. At the desired confluence, the cells were treated with 20 nM LMB or vehicle for 1 h at 37°C. Cells were washed with PBS, fixed in 4% PFA at room temperature for 15 min, washed with PBS, and permeabilized using 0.1% Triton X-100 in PBS for 15 min followed by three PBS washes. Next, cells were blocked with blocking buffer  at room temperature for 1 h and subsequently incubated with primary antibodies LTV1  and FOXK1  diluted in blocking buffer at room temperature for 1 h. After PBS wash, cells were incubated with secondary antibody conjugated to Cy3  for 1 h at room temperature. After incubation, cells were washed with PBS and nuclei were stained with  in PBS at room temperature for 10 min. Coverslips were mounted with ProLong Gold Antifade. Fluorescent images were obtained with the Revolve R4, Model: RVL2-K2 with Olympus 60× Plan Fluorite Oil IRIS Phase 3 objective with an NA of 1.25 at room temperature. Images were acquired with Echo Pro version 6.4.1. and processed with Photoshop.Immunofluorescence: HEK293T cells stably expressing GFPCell nuclei (stained with DAPI) were segmented using the Cellpose software , model: Table S1lists the proteins and their abundance in Fig. 4.Click here for additional data file.Table S2lists the proteins and their abundance in Fig. S4 A.Click here for additional data file.Table S3lists the proteins and their abundance in Fig. S4 B.Click here for additional data file.SourceData F2is the source file for Fig. 2.Click here for additional data file.SourceData F3is the source file for Fig. 3.Click here for additional data file.


In summary, EVs from patients with cGVHD can serve as a biomarker for the cGVHD state. Placental EVs may be used to regulate dermal inflammation and fibrosis, warranting further investigation of their therapeutic potential.Chronic graft-versus-host disease (cGVHD) presents with dermal inflammation and fibrosis. We investigated the characteristics of extracellular vesicles (EVs) obtained from cGVHD patients, and their potential effects on human dermal fibroblast (NHDF) cells. The anti-inflammatory and anti-fibrotic effects of placental EVs were also explored given their known anti-inflammatory properties. Fourteen cGVHD patients’ EVs contained higher levels of fibrosis-related proteins, TGFβ and α-smooth muscle actin (αSMA), compared to EVs from thirteen healthy subjects. The exposure of NHDF cells to the patients’ EVs increased the NHDF cells’ TGFβ and αSMA expressions. Placental EVs derived from placental-expanded cells (PLX) (Pluri Inc.) and human villous trophoblast (HVT) cells expressing the mesenchymal markers CD29, CD73, and CD105, penetrated into both the epidermal keratinocytes (HACATs) and NHDF cells. Stimulation of the HACAT cells with cytokine TNFα/INFγ (0.01–0.1 ng/µL) reduced cell proliferation, while the addition of placental EVs attenuated this effect, increasing and normalizing cell proliferation. The treatment of NHDF cells with a combination of TGFβ and placental HVT EVs reduced the stimulatory effects of TGFβ on αSMA production by over 40% ( An allogeneic hematopoietic cell transplant (HCT) is a vital therapeutic option for a variety of hematologic disorders. Graft-versus-host disease (GVHD) remains the most frequent and serious complication following allogeneic HCT. Chronic GVHD (cGVHD) is a multi-organ, immune-mediated, life-threatening disorder that occurs in 30–70% of patients undergoing HCT . cGVHD iMesenchymal stem cells (MSCs) are multipotent, non-hematopoietic stem cells that can be isolated from multiple tissues and be applied for tissue repair and immunomodulatory responses . ImmunosExtracellular vesicles (EVs) are membrane vesicles found in circulating blood and in other biological fluids under normal physiologic conditions, and their levels increase in a variety of diseases . Both smThis is a two-part study. The first part investigates the characteristics of cGVHD patients’ EVs compared to those of heathy controls. The second part analyzes EVs derived from placental cells and characterizes their therapeutic properties. Specifically, the current study explored the therapeutic potential of EVs originating from two distinct sources of placental cells: (1) human term placental-expanded (PLX) mesenchymal-like adherent stromal cells and (2) early pregnancy stage of human villus trophoblast (HVT) cells that line the placental villi and are in direct content with maternal circulation. We assume that placental EVs express the immunosuppressive antigen characteristic of their parental cell surfaces, and may possibly reduce the inflammation and fibrosis related to cGVHD disease.cGVHD severity was determined according to the NIH Consensus Criteria . EVs werNanoparticle tracking analysis (NTA) displayed similar concentrations and sizes of EVs in platelet-poor plasma (PPP) obtained from cGVHD patients and healthy controls (HCs) a,b. The p = 0.027, p = 0.065, p = 0.0011, p= 0.0036), but not with the αSMA levels.The EVs derived from patients with cGVHD contained significantly higher levels of fibrosis-related proteins compared to the EVs obtained from the HC . The TGFp = 0.0068, Exposure of dermal fibroblasts to EVs obtained from patients with cGVHD, for 48 h, induced statistically significant higher levels of TGF-β (2.76 ± 1.99) compared to that observed following exposure to HC-derived EVs  compared to HC EVs (4.78 ± 3.67), while addition of EVs from the other three patients induced a reduction in SMAD7 production compared to the HC EVs  and HC EVs (1.56 ± 1.38). However, exposure of NHDF cells to two of the patients’ EVs induced >10 times higher expression of αSMA compared to the other five patient EV samples  or compared to HC EVs (1.56 ± 1.38) g.The characteristics of placental EVs derived from two sources of placental cells (HVT and PLX) were assessed via transmission electron microscopy (TEM). The images from both placental EV samples representing EVs of varied sizes are shown in p < 0.05, The expression of the MSC markers was measured via flow cytometry using specific fluorescent antibodies. The PLX and HVT placental cells and their related EVs expressed the CD73, CD105, CD29, and PDL1 markers, while the EVs demonstrated non-significantly lower levels of CD73 and CD105 expression a,b compaThe ability of placental EVs to bind and penetrate dermal cells was assessed through fluorescence microscopy. The images demonstrated that green placental EVs originating from calcein-AM-labeled placental cells bound to dermal cell membranes. The cell membranes were pre-labeled with DID (red), and the co-localization of the green EVs with red membranes is depicted in yellow and the penetration of the green EVs into the cell nucleus pre-labeled with Hoechst (blue) is depicted in pink staining in p = 0.008), 34% (p = 0.008), and 45% (p = 0.0139), respectively, compared to non-treated cells , as evidenced by the Annexin/PI assay (p = 0.0112), 43% (p = 0.0179), and 48% (p = 0.0179), respectively, compared to untreated controls, as measured by RT-PCR (p = 0.0117), and a trend of increment in SMAD7 levels  cells were exposed to increasing doses of two specific cytokines, TNFα and IFNγ, which are known to be associated with the inflammatory state in cGVHD. As measured by an XTT assay, exposure to increasing doses of TNFα and IFNγ  for 24 h reduced HaCaT cell proliferation to 70% (ed cells a. ExposuPI assay . Exposury RT-PCR c. Exposu7 levels .p = 0.0159). The addition of TNFα and INFγ (0.1 ng/µL) to HaCaT cells reduced their proliferation from 100% to 34 ± 30%, and the combination of cytokines with PLX EVs increased cell proliferation to 87 ± 19% (p = 0.0571). The addition of HVT EVs was less effective, with an increasing trend of proliferation having been documented solely for the combination of HVT EVs with 0.1 ng/µL TNFα and INFγ, compared to this dose of cytokines alone. However, placental EVs of both types did not alter the anti-proliferative effects induced by the high dose of TNFα and INFγ (1 ng/µL) (The addition of PLX EVs to cells stimulated with increasing doses of cytokines (TNFα and INFγ 0.01 ng/µL and 0.1 ng/µL) attenuated the cytokines’ anti-proliferative effects, resulting in an increased proliferation of HaCaT cells. Specifically, while TNFα and INFγ (0.01 ng/µL) reduced proliferation from 100% to 70 ± 17%, the combination of cytokines with PLX EVs increased cell proliferation to 102 ± 10% (1 ng/µL) a.p = 0.0571) and HVT EVs induced a trend toward an increase. In combination with the median cytokine dose (0.1 ng/µL) with PLX EVs or with HVT EVs, BCL2/BAX expression was increased by 32% (p = 0.0571) and by 73% (p = 0.0571), respectively, compared to cells exposed to cytokines alone. Only HVT EVs affected the high dose of cytokines (1 ng/µL) and increased the BCL2/BAX expression by 38% (p = 0.0571)  was non-significantly reduced (>30%) when PLX EVs or HVT EVs were added to the high cytokine dose (TNFα and INFγ 1 ng/µL) b. Howeve 0.0571) c. p = 0.0286) (We have found that patient EVs contained both significantly higher levels of TGF-β compared to HC EVs and increased TGF-β expression in NHDF cells . We next 0.0286) b.The current study aimed to characterize the EVs of cGVHD patients and validate whether those EVs could serve as potential biomarkers for a cGVHD state. In addition, we aimed to explore the therapeutic potential of placental EVs in dermal inflammatory and fibrotic conditions. The study findings showed that cGVHD patients’ EVs contained cargo with pro-fibrotic characteristics that affected dermal cell function, and that placental EVs can attenuate this cytokine-induced dermal cell inflammation and fibrosis. We also studied the effects of cGVHD patient EVs on dermal cell culture models that could predict the response or lack of response to treatment of cGVHD in an in vitro setting for, what we believe to be, the first time . cGVHD is the most common and severe complication in patients surviving more than 100 days after allogeneic HCT. cGVHD involves various immune cells and molecules that affect multiple organs . The expTGF-β is a crucial cytokine involved in the activation of fibroblasts into myofibroblast phenotypes that express αSMA, as well as in the production of collagen. In the setting of cGVHD, TGF-β is secreted from mononuclear cells, and it has been recognized as a valuable prognostic marker of cGVHD . The TGFIn the current study, while 71% of the cGVHD patients were treated with glucocorticoids, known as an inhibitor of TGF-β1 and -β2 production , the EVsTo assess the involvement of EVs in cGVHD pathology, we studied the effects of patients’ EVs compared to EVs of HC, as well as the effects of TNFα, INFγ, and TGF-β that are known to be involved in the inflammatory and fibrotic stages of cGVHD. The findings of the current study revealed that increasing doses of the cytokines TNFα and INFγ significantly reduced cell proliferation, increased cell apoptosis, and reduced anti-apoptosis-related genes (BCL2/BAX ratio) in stimulated HaCaT cells. Stimulating fibroblast cells with either cGVHD patient EVs or TGF-β increased the fibrotic protein pathway. We consider that a combination of therapeutic EVs originating from placental cells may attenuate these effects.Mesenchymal stem cells (MSCs) are multipotent, non-hematopoietic stem cells that can be isolated from multiple different tissues, such as the umbilical cord, bone marrow, adipose tissue, and placenta . Human MIn the current study, we found that placental EVs from PLX and trophoblast cells counteracted the inhibitory effects of cytokines (TNFα and INFγ 0.01 ng/µL and 0.1 ng/µL) on cell proliferation in dermal keratinocyte cells. Cell apoptosis increased and anti-apoptotic capabilities decreased at high cytokine doses, while this effect was attenuated when either PLX EVs or HVT EVs were added. Additionally, exposure of dermal keratinocyte cells to high-dose cytokines (TNFα and INFγ 1 ng/µL) increased cell apoptosis and reduced cell anti-apoptotic capabilities, as indicated by the decreased ratio of BCL2 (anti-apoptotic) to BAX (pro-apoptotic) genes compared to non-treated cells. However, when combined with PLX EVs or HVT EVs, these effects were attenuated and the moderate dose reduction effects of cytokines on the anti-apoptotic gene expression and the ratio of BCL2 to BAX were overcome.This study has some limitations. Its findings are based upon small groups of patients, and samples were obtained from patients with the severe form of cGVHD and not from mild or moderate disease forms, precluding our ability to claim that EVs represented varying disease severity. Due to the limited volume of each sample, samples were examined on most, but not all the assays. Additional studies in larger cohorts with several stages of disease severity are warranted., Tel Aviv, Israel. All patients participated in phase 1/2 study of administration PLX-PAD for patients with steroid-refractory cGVHD. The study was approved by the committee . In addition, thirteen healthy individuals, sex- and age-matched, age ≥ 18 years old, served as a control group registered on clinicaltrials.gov  [The study was conducted between the years 2019 and 2023 at the BMT Unit, Tel Aviv (Sourasky) Medical Centerry 2021) . All patg, 24 °C) within one hour of collection and frozen in aliquots at −80 °C, as we have done in previous studies [g, 1 h, 4 °C, max acceleration, zero declaration. Patient blood samples were obtained through an intravenous 20–22-gauge cannula. Blood was collected in sodium citrate (1:10) and in EDTA. EVs were isolated as previously described ,44, acco studies . Single  studies . For EV g, 4 °C). The samples were adsorbed on carbon-coated grids and stained with 2% aqueous uranyl acetate. Samples were examined using a JEM 1400 plus transmission electron microscope .For transmission electron microscopy (TEM) imaging, the EV pellets were washed with PBS and re-pelleted . The software version build 3.1.54., Camera Type sCMOS was used . Software settings for analysis were kept constant for all measurements. Capture settings were as follows: camera level = 7, temperature = 25 °C, viscosity = 0.86 cP, and syringe pump speed = 20. Five 30 s videos were recorded per sample in light scatter. Samples were diluted (1:200) with phosphate-buffered saline (PBS) filtered through a 0.1 um membrane. The filtered PBS served as control for each measurement.EV protein content and exosome markers were analyzed through a Western blot. Thirty microliters of EV pellets obtained from similar PPP volumes (500 µL) or 50 µg of placental EV pellets isolated from cell culture media were combined with x2 lysis buffer , supplemented with 1% proteinase inhibitor and 1% phosphatase inhibitors (Sigma) containing β-mercaptoethanol  . The samTo evaluate placental EV membrane antigens, EVs were assessed by flow cytometry  using Size Mega mix beads  to set the EV size gate. Fluorescent antibodies  were use2. Passages 6–11 were used for EV isolation. Forty-eight hours before EV isolation, the medium was replaced with a serum-depleted medium, low-glucose DMEM. The reagents for cell biology were obtained from BI Biological Industries, Israel, unless otherwise declared.Primary cells of the second trimester were purchased from Scien-Cell . HVTs were cultured in a complete medium with DMEM/F-12 (HAM) 1:1, 10% heat-inactivated fetal bovine serum (FBS), 1% Pen-strep solution , supplemented with insulin-like growth factor 1 , and fibroblast growth factors . The cells were grown at 37 °C, 5% COPLX) cells were manufactured by Pluri Inc.  from term placentas, as described previously . Thawed g for 5 min and the cell pellets were discarded. The supernatant was then centrifuged for 15 min at 1500× g and the cell debris was subsequently discarded. The supernatant was then submitted to ultracentrifugation (UC)  at 20,000× g for 1 h at 4 °C, max acceleration, zero declaration. The supernatant was discarded, and the EV pellets were divided to aliquots of 50 µg and frozen at −80 °C.For placental EV production, cell media were changed to a medium without serum and EVs were isolated after 24 and 48 h of starvation by several centrifugation steps. First, the cell medium was centrifuged at 400× The expression of mesenchymal markers on placental cells and their related EVs was validated by fluorescent antibodies  and anal2 at 37 °C in a complete medium, including DMEM supplemented with 10% heat-inactivated FBS, 1% Pen-strep solution . HaCaT cells are the recommended model for investigating anti-inflammatory interventions/therapies for skin diseases . Cells wFor all experiments with HaCaT, the cells were seeded at a density of 5000–20,000 cells/well in a 96-well plate. After 24 h, the cell medium was changed to a medium depleted serum. TNFα and INFγ , PLX EVs (50 ug/well), HVT EVs (50 µg/well), and a combination of cytokine and placental EVs were added to the cells and compared with non-treated cells. Cell proliferation (after 24 h of stimulation) and cell apoptosis (after 48 h of stimulation) were assessed. The proliferation rate was measured by XTT, a colorimetric assay , according to the manufacturing protocol. Apoptotic rates were validated by an annexin/PI assay  using fluorescent microscopy and flow cytometry analysis. Levels of gene expression were studied after 6 h of cell exposure to EVs, with or without stimulators. Cells were washed with PBS and the total RNA was extracted with a TRI-Reagent kit . RNA concentration and purity were determined by UV absorption at 260 and 280 nm (NanoDrop). cDNA was constructed with a Maxima First Strand cDNA Synthesis Kit for RT-qPCR (Cat. K1641 Thermo Fisher Scientific). mRNA expressions of BCL2 and BAX were validated by a Taqman gene expression assay and evaluated with a quantitative StepOnePlus real-time PCR system (Applied Biosystems), and compared to the human housekeeping GAPDH gene. All reagents were obtained from Applied Biosystems . Results were expressed as a relative quantification. 2 at 37 °C. For all experiments, NHDF cells were seeded at a density of 20,000 cells/well in a 96-well plate. After 24 h, the cell medium was changed to a medium depleted serum. TGF-β  was added in duplicate, with or without PLX EVs (50 µg/well) or HVT EVs (50 µg/well), and compared with non-treated cells or to cells that were treated with placental EVs only for 48 h. Patient EV vs. HC EV pellets obtained from 120 uL PPP, added in duplicate for 24 h. At the end of each stimulation, the cells were washed and proteins were extracted by buffer lysis x2 (Ray Biotech) for Western blot analysis. Protein concentrations were measured by a BCA Protein Assay Kit . WB analysis was performed as described above with EV samples (4.2.2), using specific antibodies for fibrosis-related proteins  and 2 mM glutamine. The cells were maintained in a humidified atmosphere of 95% air and 5% COproteins . n = 7000 cells) for 24 h, then cells were washed and labeled with Hoechst  and with Vybrant DiD Cell-Labeling Solution , according to the manufacturer’s instructions, ending with a cell wash. Green PLX EVs and green HVT EVs were added to HaCaT cells on the cover glass for 12 h. The interaction between placenta EVs and HaCaT cells was documented with fluorescent confocal microscopy . PLX or HVT cells were washed and exposed to calcein AM  in a serum-free medium at 37 °C for 2 h, ending with a wash. A new, serum-free medium was added to the cells. After 24 h, EVs were isolated from the cells as previously described. HaCaT cells were seeded on a cover glass (n = 7000) were seeded on a 96-well plate for 24 h in complete HaCaT or NHDF medium, and then a new medium free of serum, with 0.34 × 1010 particles/mL of green placental EVs, was added to the cells for overnight incubation. The supernatant from PLX or HVT EVs isolation with the green exosome phase was added to the cells as a negative control. Interaction of green placental EVs with non-labeled HaCaT or NHDF cells were analyzed by flow cytometry (FACS CANTO BD).HaCaT or NHDF cells  and 95% confidence intervals. Fisher’s exact test was used to determine whether or not there was a significant association between two categorical variables. Effect size analysis was performed using Cohen’s d method to characterize the sizes of the differences between the groups. Small, moderate, and large effects were defined as 0.20, 0.40, and 0.80, respectively [n = the number of samples that were validated in each subgroup in each figure.Statistical analysis was performed with the GraphPad Prism 5 software . Results were assessed by a one-way ANOVA, non-parametric Kruskal–Wallis test, and Dunn’s multiple comparison test. The non-parametric Mann–Whitney U test and Student’s ectively ,48. n = The EV profile of cGVHD patients differs from that of EVs obtained from healthy individuals, and may serve as a biomarker for disease status. Placental EVs may regulate dermal inflammation and fibrosis. Further research is needed to explore the therapeutic properties of placental EVs.


NOL12 5′-3′ exoribonucleases, conserved among eukaryotes, play important roles in pre-rRNA processing, ribosome assembly and export. The most well-described yeast counterpart, Rrp17, is required for maturation of 5.8 and 25S rRNAs, whereas human hNOL12 is crucial for the separation of the large (LSU) and small (SSU) ribosome subunit rRNA precursors.nol12 mutant to stress factors, including heat and pathogen Pseudomonas syringae, enhances the observed molecular phenotypes, linking pre-rRNA processing to stress response pathways. The aberrant rRNA processing, dependent on AtNOL12, could impact ribosome function, as suggested by improved mutant resistance to ribosome-targeting antibiotics.In this study we demonstrate that plant AtNOL12 is also involved in rRNA biogenesis, specifically in the processing of the LSU rRNA precursor, 27S pre-rRNA. Importantly, the absence of AtNOL12 alters the expression of many ribosomal protein and ribosome biogenesis genes. These changes could potentially exacerbate rRNA biogenesis defects, or, conversely, they might stem from the disturbed ribosome assembly caused by delayed pre-rRNA processing. Moreover, exposure of the Despite extensive studies, the pre-rRNA processing pathway in plants remains insufficiently characterized. Our investigation reveals the involvement of AtNOL12 in the maturation of rRNA precursors, correlating this process to stress response in Arabidopsis. These findings contribute to a more comprehensive understanding of plant ribosome biogenesis.The online version contains supplementary material available at 10.1186/s12870-023-04561-9. In eukaryotic cells, the processing of the long, polycistronic ribosomal RNA precursor (pre-rRNA) involves a series of events that begin co-transcriptionally and occur simultaneously with ribosome assembly to ensure timely and accurate formation of the ribosomes , 2. The The processing of pre-rRNA takes place mainly in the nucleolus, and nucleoplasm, and the final steps of maturation are carried out after the export of the pre-ribosomes to the cytoplasm , 3. A laThe known plant ribosome biogenesis factors (RBFs) include enzymes that directly cleave or trim pre-rRNAs, namely endo- and exoribonucleases, and many other factors that facilitate the processing , 6. In AS and the long 5.8SL, which differ at their 5′ ends   as per manufacturer’s instructions, and then ethanol precipitated. RNA was denatured at 80°C and then hybridized to 1 pmol of labeled primer by slowly cooling the reaction mixture to 50°C. Reverse transcription was performed using SuperScript IV Reverse Transcriptase (Invitrogen) for 15 min at 50°C in 20-µl reactions containing 1 mM dNTPs, 10 units of RiboLock (Thermo Fisher Scientific) and 1x SuperScript IV buffer (Invitrogen), and stopped by 10 min. incubation at 80°C. Products of the reaction were denatured at 95°C for 5 min and separated on 6% acrylamide/7 M urea sequencing gels.Bacterial infection assays were performed with virulent Pseudomonas syringae pv. tomato strain DC3000 (Pst). Bacteria for inoculation were grown overnight in LB medium with rifampicin at 28°C, centrifuged at 3500 g for 7 min, washed, and resuspended in 10 mM MgCl2 with density adjusted to 106 cfu ml− 1 (OD600nm = 0.003). 6-week-old plants were inoculated by spraying with the Pst suspension in 0.05% Silwet L-77 or with 10 mM MgCl2/0.05% Silwett L-77 (control plants) and then covered with plastic lids overnight. The material was harvested from at least 10 plants for each time point, frozen in liquid nitrogen, and used for RNA extraction. Bacterial growth was quantified by assessing the number of dividing bacterial cells 24 and 72 h after infection (hpi). Leaves were harvested and surface-sterilized . Samples (four-leaf discs) were taken using a cork-borer (4 mm) from 2 leaves, ground in sterile Mili-Q water, diluted and plated on a selection medium. Plates were incubated at 28°C and colonies were counted after 48 h.PAMP assays: sterilized seeds were grown on MS plates as described above. Five days after stratification seedlings were transferred to a 24-well plate with liquid MS . MS was exchanged for a fresh medium after 8 days (500 µl per well) and the next day 500 µl of flg22  or elf18  solution was added to each well to a final concentration of 100 nM. Seedlings were harvested at the indicated time points and frozen in liquid nitrogen. Samples collected from each experiment were subjected to RNA isolation and northern blot analysis.Stress tests: In the case of high-temperature stress, seeds were surface-sterilized and grown on MS medium supplemented with 1% (w/v) sucrose and 0.3% phytagel under long-day conditions, then after 2 weeks plates were transferred to a 37 or 42°C growth chamber. Samples were collected at the indicated time, blotted dry, and frozen in liquid nitrogen. For salt treatment, seeds were grown on an MS medium containing 100 mM NaCl, 1% (w/v) sucrose, and 0.3% phytagel (Sigma-Aldrich). After 14 days, the seedlings were harvested and frozen in liquid nitrogen. RNA was then isolated and analysed by northern blotting.Antibiotic tests: the seeds were initially germinated on MS medium supplemented with 1% (w/v) sucrose and 0.3% phytagel, without antibiotics, under long-day conditions, for 5 days, and then transferred to the antibiotic-containing plates. Root length and dry weight measurements were performed after 14 days of growth in antibiotic-containing media. Dry weight measurements of plants grown on CHX media were performed after drying seedlings in 70°C overnight. The experiment was conducted in three biological replicates, 27 plants were analyzed for each replicate.Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2