Whey protein isolate (WPI)-derived bioactive peptide fractions  were for the first time used as emulsifiers in nanoemulsions. The formation and storage stability of WPI bioactive peptide-stabilized nanoemulsions depended on the peptide size, enzyme type, peptide concentration, and storage temperature. The highly bioactive <10 kDa fractions were either poorly surface-active or weak stabilizers in nanoemulsions. The moderately bioactive >10 kDa fractions formed stable nanoemulsions (diameter = 174–196 nm); however, their performance was dependent on the peptide concentration (1–4%) and enzyme type. Overall, nanoemulsions exhibited better storage stability (less droplet growth and creaming) when stored at lower (4 °C) than at higher (25 °C) temperatures. This study has shown that by optimizing peptide size using ultrafiltration, enzyme type and emulsification conditions (emulsifier concentration and storage conditions), stable nanoemulsions can be produced using WPI-derived bioactive peptides, demonstrating the dual-functionality of WPI peptides. Whey proteins and their hydrolysates are important emulsifiers in food emulsions ,2,3,4 anThe utilization of bioactive peptides in foods requires the design of suitable food matrices, capable of protecting and delivering bioactive peptides in foods, while minimizing interactions with other food components . MoreoveAlternatively, bioactive peptides may be applied as emulsifiers in food or pharmaceutical nanoemulsions. Protein hydrolysates are effective emulsifiers in conventional food emulsions ,17,18, aThe current work investigated the emulsifying properties of WPI bioactive peptidic fractions in nanoemulsions. Using WPI-derived bioactive peptides as emulsifiers in nanoemulsions may increase the incorporation of bioactive peptides in food systems and lead to products with modified texture, taste, and sensory attributes. The study had two objectives; first, to investigate the emulsifying ability of WPI-derived bioactive peptide fractions as emulsifiers in nanoemulsions, demonstrating their dual functionality in food systems. Secondly, to study the effects of peptide concentration, storage conditions, and peptide fractionation on the stability of WPI bioactive peptide-stabilized nanoemulsions.w/v basis unless otherwise stated.Whey protein isolate (WPI) was purchased from MyoPure Pty Ltd., Petersham, Australia, and canola oil was obtained from a local supermarket. α-Chymotrypsin, pepsin, Tween 40, 6-hydroxy-2,5,7,8-tetramethylchroman-2-acid, and 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) were purchased from Sigma-Aldrich . All other chemicals were of reagent grade. Concentrations are calculated on a w/v) was suspended in 10 mM phosphate buffer, pH 7.00. The suspension was adjusted to pH 2.60 for pepsin or 7.80 for chymotrypsin hydrolysis. Enzymes were then added at an enzyme: substrate ratio of 1:40 and the suspensions were incubated at 37 °C for 3 h with constant stirring. After the hydrolysis, the enzymes were inactivated by heating the hydrolysates at90 °C for 15 min. The crude hydrolysates were subsequently fractionated by ultrafiltration (UF). Ultrafiltration was carried out in a Model 8200 Amicon Stirred Ultrafiltration cell, fitted with 1, 3, 5, and 10 kDa molecular weight (MW) cut-off cellulose membranes , under nitrogen gas. Ultrafiltration was performed sequentially and four peptide fractions  were collected, concentrated by rotary evaporation, and freeze-dried. For this discussion, the >10 kDa fractions are labelled as UC–10F and UP–10F for the chymotrypsin and pepsin fractions, respectively. The designations, 1–3, 3–5, 5–10 kDa, are unchanged for the other fractions.Hydrolysis was carried out as previously described . Briefly50), and surface hydrophobicity (So), using the anionic fluorescence probe, 8-anilino-naphthalene-1-sulfonate (ANS). Details of these assays are described in Adjonu et al. [The fractions were assessed for their free radical scavenging antioxidant (µmol TE/mg peptide) and ACE-inhibition activities  and distribution of (nano)emulsion droplets were measured by dynamic light scattering using a Zetasizer Nano ZS . Measurements were performed at 25 °C with duplicate determinations for each sample. Before measurement, the (nano)emulsions were diluted in phosphate buffer  at a dilution ratio of 1:100.The ζ-potential of the (nano)emulsions was measured using a universal ‘Dip cell’  on the Zetasizer Nano ZS. (Nano)emulsions were diluted (1:50) in the phosphate buffer and then equilibrated at 25 °C for 60 sec before measurement. The ζ-potential was also measured for the 1%, 2%, and 4% aqueous peptide solutions. Duplicate determinations were performed on each sample.The stability of the (nano)emulsions was determined by monitoring changes in droplet size, size distribution, ζ-potential, and cream development over 7 d of storage at 4 °C and 25 °C . Creamin® Centurion XVI, StatPoint Technologies, Inc., Warrenton, VA, USA) and mean separation was performed using Fisher’s least significant difference (LSD) test (p < 0.05).All experiments were performed in duplicate, and results were reported as the mean ± standard deviation. Analysis of variance was performed on the bioactivity and surface hydrophobicity data (Statgraphicsp < 0.05). No other mean comparisons were significantly different. Fractions UC–10F and UP–10F had lower antioxidant activity than the <10 kDa fractions; however, the activities were about 63 to 86% of the average activities of the <10 kDa fractions. Except for the fraction UP–10F, the antioxidant activities were comparable to those reported for unfractionated WPI hydrolysates [All WPI peptide fractions showed antioxidant activity, influenced by the peptide size and the enzyme used . The 1–3olysates .50 values for ACE-inhibition of peptides are also presented in p < 0.05). Fraction UC–10F exhibited greater ACE-inhibition than the fraction UP–10F (p < 0.05). Like the antioxidant activity, the smaller to medium size <10 kDa fractions possessed greater inhibitory potencies than larger size >10 kDa fractions, which is consistent with previous studies [50 values for the peptide fractions were comparable to those reported by da Costa et al. [Whey protein peptides can inhibit the activity of ACE . To comp studies ,21. Morea et al. . Overallp < 0.05)  . The <10p < 0.05)  . DiffereEmulsions containing 2% peptide emulsifiers were produced with the 1–3, 3–5, and 5–10 kDa WPI fractions. None of the pepsin WPI fractions formed nanoemulsions; diameter = 1233–4122 nm , 0 h. ThThe magnitude of the ζ-potentials was greater for the chymotrypsin fraction nanoemulsions than the pepsin fraction emulsions . RegardlAs shown in Emulsion formation and stability are two separate processes, governed by different properties of the peptide emulsifier. Emulsion formation is governed by adsorption behavior , whereas stability depends on the size and proportion of larger peptides present . LikewisFractions UC–10F and UP–10F formed stable nanoemulsions because they contained a more diverse range of larger and surface-active peptides , capableVarying the concentration (1 to 4%) of fractions UC–10F and UP–10F had different effects on the (nano)emulsions. Droplet sizes were larger at the 2 and 4% UP–10F concentrations than at 1% but remained relatively constant over the range of concentrations for the UC–10F nanoemulsions (The UP–10F (nano)emulsions displayed a pH dependency at the 2 and 4% peptide concentrations. Fraction UP–10F failed to form a nanoemulsion at the 2 and 4% concentrations due to the proximity of the peptide solution pH (4.98–5.88) to the isoelectric point (pI) of WPI proteins (pH 4.00–5.50). Conformational changes around the pI facilitate peptide–peptide hydrophobic interactions , diminisConversely, the UC–10F nanoemulsion droplets were smallest at 2% but comparable at the 1 and 4% peptide concentrations  and −35.9 mV (25 °C)), whereas the converse holds for the UC–10F nanoemulsion  and −20.7 mV (25 °C)). Better stability for the UC–10F nanoemulsion at 4 °C than at 25 °C was due to: (i) more enhanced charge stabilization of the droplets at 4 °C than at 25 °C and (ii) increased viscosity in the nanoemulsions stored at 4 °C, due to stronger solvent–peptide interactions ,37. MoreIrrespective of the peptide fraction, enhanced stability occurred at lower peptide concentrations and storage temperature . With inCreaming in emulsions causes visible rings in beverage emulsions and leads to products with an uneven consistency, texture, and sensory characteristics . The 1% In a previous study, unfractionated WPI hydrolysate obtained by pepsin hydrolysis acted poorly as an emulsifier in a nanoemulsion . In the The fractions UC–10F and UP–10F nanoemulsions were more stable against droplet size growth than the unfractionated WPI hydrolysate nanoemulsions, especially at lower peptide concentrations (<2%), but comparable stabilities existed at the higher peptide concentration (4%) . Thus, UIn conclusion, WPI-derived bioactive peptide fractions were successfully applied as emulsifiers in food-grade nanoemulsions with varying outcomes. Nanoemulsion formation and stability depended on the peptide size range, concentration, storage temperature and time, and the hydrolytic enzyme type. Peptide size determined the short (<12 h) and long (>24 h) term stability of nanoemulsions by influencing the peptides’ interfacial viscous effect and charge-stabilizing properties. The highly bioactive <10 kDa peptide fractions acted poorly as stabilizers in nanoemulsions, but the moderately bioactive >10 kDa fractions exhibited enhanced stability in nanoemulsions. Ultrafiltration enriched larger and more surface-active peptides in fractions UC–10F and UP–10F, possessing better functionality in nanoemulsions than unfractionated WPI hydrolysates. Thus, utilizing WPI and other food proteins’ bioactive peptides as emulsifiers in nanoemulsions provides an alternative to incorporating bioactive peptides as functional and nutraceutical ingredients in food systems. Thus, WPI peptides serve a dual function as bioactive compounds and emulsifiers in nanoemulsion. Further investigation is ongoing to enrich specific WPI peptide fractions with excellent bioactive and interfacial activities, capable of forming and stabilizing nanoemulsion droplets.


However, changes in soil N availability and NO15N natural abundance (δ15N) of leaves, soils, and different-sized aggregates at the three stages of secondary succession were analyzed. The δ15N compositions of soil organic nitrogen (SON) in aggregates and soil to plant 15N enrichment factor (EF = δ15Nleaf −δ15Nsoil), combined with soil inorganic N contents and δ15N compositions were used to indicate the alterations of soil N availability and NO3–loss potential following secondary succession.In the Karst Critical Zone Observatory (KCZO) of Southwest China, croplands, shrub-grass lands, and secondary forest lands were selected to represent the three stages of secondary succession after agricultural land abandonment by using a space-for-time substitution approach. The contents and δ15N values of SON also significantly decreased, mainly affected by plant δ15N composition and N mineralization. SON content in macro-aggregates and soil NH4+ content significantly increased while δ15N values of NH4+ decreased, implying increases in SON stabilization and improved soil N availability following secondary succession. Leaf δ15N values, the EF values, and the (NO3–-N)/(NH4+-N) ratio gradually decreased, indicating reduced NO3– loss following secondary succession.Leaf N content and SON content significantly increased following secondary succession, indicating N accumulation in the soil and plant. The 3– leaching loss reduces following secondary succession after agricultural land abandonment in the KCZO.Soil N availability improves and NO Input al N pool .4+ and NO3−) from chemical N fertilizer (2 fixing (15N-abundance of synthetic fertilizer (δ15N: 0.3 ± 0.2‰) and atmospheric N2 (δ15N: 0‰) can significantly affect the δ15N composition of crops (15N-depletion in NH4+ and nitrification produces 15N-depletion in NO3− (15N-depleted and is easily absorbed or lost compared to soil organic nitrogen (SON)  . Denitrimosphere . Ammoniaual NH4+ . As the 15N natural abundance can provide a clearer understanding of soil N dynamics under land-use change including secondary succession after agricultural land abandonment  . However, the δ15N values of the plants and soils are higher and the EF value is closer to zero with the long-term loss of the 15N-depleted NO3− in N-saturated ecosystems  . In clososystems . The appatchment . However3− loss reduces after agricultural land abandonment in the KCZO. The 15N natural abundance in soil aggregates and the EF values are important research methods in this study. The study objectives were to: (1) identify the changes of δ15N compositions of SON in different-sized aggregate and the EF values at different stages following secondary succession and (2) estimate soil N availability and soil NO3− loss potential following secondary succession after agricultural land abandonment in the KCZO.In the Karst Critical Zone Observatory (KCZO) of Southwest China, many croplands have been abandoned and naturally recovered with the promulgation of the Grain for Green Project (GGP) program in the 1990s . Changes2. The Chenqi catchment is one of the key research areas of the KCZO in Guizhou province, Southwest China. The climate in the catchment is mainly sub-tropical monsoonal, with a mean annual temperature (MAT) of 15.1 °C and mean annual precipitation (MAP) of 1,315 mm , with an area of 1.54 km1,315 mm . Seasona1,315 mm . This ca1,315 mm . The alt1,315 mm . The soi1,315 mm , and cla1,315 mm . Quatern1,315 mm .Zea mays), potato (Solanum tuberosum), oilseed rape (Brassica napus), and peanut (Arachis hypogaea) are planted in rotation from spring to autumn, while the fields lie fallow in winter  after removing obvious gravel and fresh coarse roots. A part of the samples was crushed by hand to make all particles pass through a 10 mesh-steel sifter (2 mm), which was stored as the sample of bulk soil (<2 mm). The remaining soil samples were not crushed and were used for soil aggregate separation by using the improved wet-sieving method . Macro-a3−, NH4+, and dissolved organic N  were soaked in a 2 mol/L KCl solution for 24 h to remove NOrganic N . The trei.e., the measured SON content is multiplied by the ratio of the sample mass after and before treatment  in the Surficial Environment and Hydrological Geochemistry Laboratory, China University of Geosciences . Standard soil substances (OAS B2152) were repeatedly measured to monitor reproducibility. The relative standard deviations were less than 3%. Actual SON content could be calibrated because of sample mass reduction after removing dissolved inorganic and organic N, reatment .15N/14N) of leaf, SON in bulk soils and aggregates were determined by an isotope mass spectrometer  in the Central Laboratory for Physical and Chemical Analysis, Institute of Geographic Sciences and Natural Resources Research, Chinese Academy of Sciences. The measurements are expressed in standard δ notation (‰) to indicate the differences between the 15N/14N ratio of the samples and accepted standard materials (atmospheric N2), where  was monitored and repeatedly measured to evaluate the precision of the measurements (<0.1‰).Reference material  software program and the figures were drawn using SigmaPlot 12.5  software program.One-way ANOVA with the least significant difference (LSD) test was conducted to identify the significant differences in SON contents of aggregates, Foliar N contents in the croplands were higher than those in the shrub-grass lands and much higher than those in the secondary forest lands . Foliar The SON contents of bulk soils and macro-aggregates at the 0–20 cm depth in the croplands were slightly lower than those in the shrub-grass lands, and significantly lower than those in the secondary forest lands . While tδ15N values in the croplands (mean 3.37‰) were significantly higher than those in the shrub-grass lands (mean −2.43‰), and much higher than those in the secondary forest lands (mean −7.06‰)  were significantly higher than those in the shrub-grass lands (mean −8.48‰), and much higher than those in the secondary forest lands (mean –10.40‰)  . Similar–10.40‰) . Foliar δ15N values of SON in bulk soils at the 0 −10 cm depth under the croplands (mean 6.56‰) were significantly higher than those under the shrub-grass lands (mean 4.99‰) and secondary forest lands (mean 5.16‰), while the δ15N values in the soil of the 10–30 cm depth were not significantly different among the three land-use types   under the three land-use types was 6.56‰, 4.99‰, and 5.16‰, respectively  N following secondary succession, indicating an intensive N mineralization process. In summary, increases in N input from plants, biological N fixation, and soil N mineralization rate determine soil N availability during secondary succession.Soil NHccession , which sligible) . However biomass . Thus, iectively , while tectively . The δ15e plants , and is lization . Soil N ted NH4+ . SON in δ15N values and soil to plant 15N EF values significantly decreased following secondary succession /(NH4+-N) ratio gradually decreased following secondary succession in the study area  at all stages of secondary succession must be balanced  from soils (Soil NObalanced . Nitrifited NO3− . The δ15ccession , indicatndonment . Soil NOoil NO3−  graduallom soils . In the om soils , increasδ15N values during secondary succession after agricultural land abandonment under GGP as shown in 3− loss following secondary succession. Our study highlights the positive implications of the GGP program for karst ecosystem restoration. As the GGP program in China progresses, this conceptual model can provide basic scientific guidance for policymakers in the karst region, and even in the non-karst region. However, considering the differences in climate, soil type, and vegetation restoration approach in different regions, the key factor that affected soil N transformation processes and N loss may differ (In the present study, the diagram exhibits the changes in plant and soil N content and y differ . For fut3− loss potential following secondary succession after agricultural land abandonment were estimated in the KCZO, Southwest China. During secondary succession, soil N input from plant and biological N fixation promote SON accumulation in the surface soils. Increases in soil SON stock and N mineralization rate determine soil N availability. Decreases in foliar δ15N values and soil to plant 15N EF values indicate reduced soil N losses. Improved SON stability by increasing the C/N ratio of litter and aggregate protection and increased soil NO3− uptake by plants and microbes are beneficial for reducing soil NO3− loss potential. This study highlights the positive implications of the GGP program for karst ecosystem restoration. In future works, the evaluation of soil N availability and the mechanism of soil NO3− loss potential following secondary succession after agricultural land abandonment should be studied in other environments, particularly non-karst.Under the GGP program, the changes in soil N availability and soil NO10.7717/peerj.14790/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.14790/supp-2Supplemental Information 2Click here for additional data file.


The abnormal expression of Transient Receptor Potential cation channel subfamily V member 4 (TRPV4) is closely related to the progression of multiple tumors. In addition, TRPV4 is increasingly being considered a potential target for cancer therapy, especially in tumor metastasis prevention. However, the biological correlation between TRPV4 and tumor metastasis, as well as the specific role of TRPV4 in malignant melanoma metastasis, is poorly understood. In this study, we aimed to examine the role of TRPV4 in melanoma metastasis through experiments and clinical data analysis, and the underlying anticancer mechanism of Baicalin, a natural compound, and its inhibitory effect on TRPV4 with in vivo and in vitro experiments. Our findings suggested that TRPV4 promotes metastasis in melanoma by regulating cell motility via rearranging the cytoskeletal, and Baicalin can inhibit cancer metastasis, whose mechanisms reverse the recruitment of activated cofilin to leading-edge protrusion and the increasing phosphorylation level of cortactin, which is provoked by TRPV4 activation. Metastasis is the leading cause of cancer-related death and the treatment strategies have not substantially progressed in the last few years . MetastaIon channels are crucial for normal cellular processes including cell proliferation, migration, and apoptosis ,9. The aMetastasis is initiated and maintained by signaling pathways that mediate cytoskeletal dynamics in the tumor cells . The dynScutellaria baicalensis Georgi, a widely used traditional herbal medicine with various pharmacological effects including antitumor functions .A total of 5 × 10g for 20 min at 4 °C. The supernatant was collected; the protein concentration was determined by the bicinchoninic acid (BCA)  method and the proteins were separated by SDS gel electrophoresis for western blot analysis.A solution of the Baicalin (200 μM) in Phosphate-Buffered Saline (PBS)/dimethyl sulfoxide (DMSO) or PBS/DMSO as the vehicle, was added to the cell extract at a final concentration of 1% PBS/DMSO. Then, the cells of the two groups were incubated for 40 min at 37 °C. After incubation, the two groups were divided into four parts of 100 μL each in 0.2 mL PCR tubes. Thus, one sample contained the compound and the other contained the vehicle. Pairs of one control and one experimental aliquot were heated at 44 °C, 46 °C, 48 °C, 50 °C, 52 °C, 54 °C, 56 °C, 58 °C, 60 °C, or 62 °C for 3 min, followed by a 3 min incubation at room temperature. Subsequently, the extract was centrifuged at 12,000× Cells were collected and lysed in the lysis buffer containing protease and phosphatase inhibitors. Protein concentrations were determined to ensure an equal amount of protein lysate per sample using the bicinchoninic acid protein assay kit . Cell lysates were incubated with Baicalin for 15–30 min using a thermomixer at room temperature. Each cellular lysate sample was proteolyzed at 4 °C for 30 min with different pronase concentrations  . Subsequently, the reaction was suspended in EDTA and incubated on ice for 10 min. The samples were then loaded on SDS-PAGE and the gel was stained with Coomassie Brilliant Blue. Ultimately, the specific gel bands that were seen in treatment samples were compared with controls, cut out, and further processed for western blotting analysis.Cells were seeded in 12-well plates and cultured in a medium supplemented with 10% FBS for 24 h. After fixing with 4% paraformaldehyde for 30 min, the cells were blocked with 1.5% bovine serum albumin (BSA) for 1 h. Then, cells were incubated with primary antibodies against TRPV4 , phospo-cortactin , and cofilin  at 4 °C overnight followed by incubation with FITC-labeled goat anti-rabbit IgG for 1 h at room temperature. DAPI  was used to stain the cell nucleus. All the images were captured with the fluorescence microscope .5 cells/plate). After the cells reached complete confluence, they were starved overnight and scratched with a sterile 200 μL pipette tip. They were washed using PBS buffer, subsequently treated with reagents, and cultured for 24 h. They were imaged before and after 24 h of culture.Cell migration was evaluated by wounding healing assays. Briefly, A375 cells were plated in 6-well plates . The tissue sections (5 mm) were stained with hematoxylin and eosin (H&E). The slides were imaged using the Fluorescence Inversion Microscope System .5 A375 cells/dish were added with 5 μL dye and incubated at 37 °C for 15 min. After staining, the cells were centrifuged, cleaned twice with PBS, and re-suspended in DMEM complete medium and inoculated in special dishes. After 4 h, the cells adhered to the wall and the supernatant was aspirated. Basal medium: serum: matrix glue = 2:2:1 on 300 μL clear liquid. Baicalin with the final concentration of 8 μM, 16 μM, 32 μM, and DMSO solution was added into the culture supernatant. After culturing for 24 h, confocal microscopes were used to observe and photograph.In brief, 4 × 10® , Rabbit mAb , Phospho-Cortactin (Tyr 421), Anti-Bax antibody , Anti-Bcl-2 antibody , Anti-Caspase-3 antibody , Anti-Cdc42 antibody , Anti-RhoA antibody , Anti-Rac1/2/3 antibody , Anti-m-TOR antibody .A375 and A375M cells were lysed with RIPA buffer , and total proteins of approximately 25 ng were separated by SDS-PAGE and then transferred to polyvinylidene fluoride membranes . After labeling with antibody and Goat Anti-Rabbit IgG (H þ L) HRP antibodies, the membranes were scanned using a BIORAD imaging system . Anti-TRPV4 antibody, Akt (pan) (11E7) , Rabbit mAb , Phospho-Akt (Ser473) (D9E) XPp < 0.05, ** p < 0.01, *** p < 0.001.All statistical analyses were carried out with the GraphPad Prism software version . Values were expressed as the mean ± SD of at least three independent experiments. One-way analysis of variance (ANOVA) or t-tests were used to compare in groups. The results were considered statistically significant at * In the present study, we found that Baicalin inhibited calcium influx induced by TRPV4 activating and inhibiting melanoma cell migration and invasion. Additionally, we found that Baicalin inhibits melanoma metastasis by modulating TRPV4-Src-cofilin axis signaling. Collectively, targeting TRPV4 is now a promising area of cancer therapy, and Baicalin is a potential therapeutic drug targeting TRPV4 for the prevention and therapy of melanoma metastasis.


Anemarrhenaasphodeloides Bunge, has been reported to affect various diseases, such as cancer, Alzheimer’s disease, and allergies. However, the underlying molecular mechanisms and impacts are largely unknown. In the present study, we hypothesized that TA3 induces apoptosis through the inhibition of c-Myc expression via CNOT2 or MID1IP1 in HCT116. An MTT assay and colony formation assay were used to measure cell viability and proliferation. The protein expression of apoptotic markers and oncogenes was measured using immunoblotting and immunofluorescence assays. The interaction between MID1IP1 and c-Myc was confirmed by performing an immunoprecipitation assay. TA3 markedly inhibited colon cancer cell proliferation. Consistently, TA3 regulated the apoptotic proteins pro-PARP and caspase 3. TA3 inhibited the half-life of c-Myc and suppressed its expression in response to serum stimulation. In addition, TA3 enhanced the apoptotic effects of doxorubicin and 5-FU in colon cancer cells. Altogether, our results reveal a mechanism by which TA3 induces apoptosis through inhibiting c-Myc expression via CNOT2 or MID1IP1 in HCT116, which may help in the development of new therapies for colon cancer based on TA3 in the future.Timosaponin A3 (TA3), extracted from the rhizome of  Colorectal cancer is the third most common malignancy worldwide, and diet control, weight management, and exercise can help to reduce the risk for develop colorectal cancer. However, over the past 25 years, morbidity and mortality have increased in adults aged under 50 years worldwide, and approximately 50,000 colorectal cancer patients pass away every year in the United States . AlthougTo overcome this issue, many studies have been conducted on relatively low-toxicity, natural product-derived compounds to explore candidate materials to reduce the negative effects while increasing the treatment efficiency of chemotherapies such as 5-FU and DOX. In addition, treatments that inhibit their function by targeting specific molecules such as c-Myc, CCR4-NOT2 (CNOT2), and midline1 interacting protein 1 (MID1IP1), which have recently been identified as oncogenes, have been widely proposed as novel treatments for various cancers, including colorectal cancer ,7,8. Witc-Myc, a prototype member of the Myc family, is a transcription factor that is thoroughly controlled in normal cells, while it is overexpressed in human cancer and plays an essential role in cell metabolism, growth, proliferation, and apoptosis ,11,12. ICNOT2 is a subunit of the CCR4-NOT complex that controls mRNA metabolism and the stability of eukaryotic cells and has been reported to play a role in autophagy, apoptosis, proliferation, and angiogenesis in various cancer cells, including those of the colon, lung, and breast ,15,16,17Anemarrhena asphodeloides Bunge is a herbal medicine used for a long time to treat high fever and diseases such as diabetes [Anemarrhena asphodeloides, through cell cycle arrest and apoptosis, has been reported in colon, pancreatic, gastric, and liver cancers [Anemarrhena asphodeloides Bunge, with effectiveness against various diseases, such as cancer, diabetic osteoporosis, Alzheimer’s disease, allergies, and viral infection [diabetes . In addi cancers ,22,23,24nfection ,26. HoweOur results indicate that TA3 suppresses the expression of c-Myc via downregulation of CNOT2 and MID1IP1 in colorectal cancer cell lines, thereby inducing apoptotic markers. In addition, it was shown that the co-administration of 5-FU or DOX with TA3 has a combined effect that further enhances apoptosis.p53−/−, HT-29, and DLD-1 cells  group  group B.p53+/+ cells treated with TA3 or DMSO by serum stimulation  and HCT116p53−/− cells were kindly provided by Dr. Wonchae Choe . Each cell line was cultured in RPMI-1640 containing 1% antibiotics and 10% fetal bovine serum at 5% CO2 and maintained at 37 °C.HCT116p53−/− cells were distributed in 96-well plates (1 × 104 cells/well), and, the next day, they were treated with TA3 at a concentration range of 0–25 µM for 24 h.The cytotoxicity assay was performed as described in a previous study . HCT116pp53−/− cells treated with TA3 at a concentration of 0, 6.25, and 12.5 μM were distributed in 6-well plates (1 × 103 cells/well) and cultured for a week to form colonies at 5% CO2 and 37 °C. After this, the cells were washed with PBS and fixed for 10 min using Diff-Quik fixation, and the colonies were stained using Diff-Quik solution 2 for 20 min.Colony formation assays were performed using the Diff-Quik kit , as previously described . Brieflyp53−/− cells (1 × 105 cells/well), the DeadEnd™ Fluorometric TUNEL system kit  was used. TUNEL-stained cells were visualized using the EVOSR Cell Imaging System .To confirm whether TA3 treatment causes apoptosis in HCT1165 cells/well) were lysed using a lysis buffer . Cell lysates were separated by SDS-PAGE (8–12%) and transferred to nitrocellulose membranes. Subsequently, the immunoblotting process was implemented according to a previous study [The drug-treated cells (2 × 10us study . The dilp53+/+ cells (1 × 105 cells/well) treated with or without TA3 were fixed with 4% paraformaldehyde for 20 min and at room temperature for 10 min, permeabilized with 0.1% Triton X-100, and blocked with 3% BSA for 1 h. Cells were then labeled with a specific antibody against c-Myc (1:100) at 4 °C overnight. Subsequently, the cells were incubated with Alexa Fluor 488 goat anti-rabbit IgG antibody (1:500)  at 4 °C for 2 h. The experiment was performed according to a previous study [HCT116us study .p53+/+ cells were distributed in a 6-well plate (7 × 104 cells/well) and cultured overnight. The next day, transfection was performed with control, MID1IP1, and CNOT2 siRNA using INTERFERin®  transfection reagent, according to the manufacturer’s protocol.Control, MID1IP1, and CNOT2 siRNAs were purchased from Bioneer , and RNA interference was performed as described previously . Brieflyp53+/+ cells (2 × 105 cells/well) were exposed to TA3 for 24 h and then treated with the protein synthesis inhibitor cycloheximide at a concentration of 50 µg/mL for 0, 30, 60, and 90 min. Western blotting was performed to confirm the expression levels of c-Myc and β-actin.The cycloheximide chase assay was described in a previous study . HCT116pp53+/+ cells in 6-well plates (2 × 105 cells/well), the cells were starved in serum-free medium for 24 h. Subsequently, TA3 diluted in a medium containing 20% FBS was incubated at a concentration of 12.5 µM for 0, 6, 12, and 24 h and then the cells were harvested using a cell scraper.After seeding HCT11630. Briefly, TA3-treated or untreated HCT116p53+/+ cells (2 × 105 cells/well) were lysed in lysis buffer. Cell lysates were then incubated overnight with MID1IP1 antibody. The next day, Protein A or G beads  were added and incubated on a rotator at 4 °C for 5 h, and then washed three times with lysis buffer and boiled. Finally, the bound protein was subjected to Western blot analysis.Immunoprecipitation was performed as previously describedAll experiments were performed with at least three individual repetitions. The significance of each comparison was analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s test using GraphPad Prism software , and comparisons between the two groups were analyzed by an unpaired t-test. All experimental data are expressed as the mean ± standard deviation.Our new findings provide further insights into whether the inhibition of c-Myc expression by TA3 might involve cross-talk with the ribosomal proteins or respond to yet-unidentified signals. In addition, the relationship between TA3 and c-Myc can be better understood by creating a biological model system. In conclusion, our findings could be helpful for the discovery of anti-colon cancer drugs in the future.


In the current study, lipid-polymer hybrid nanoparticles (LPHNPs) fabricated with lipoid-90H and chitosan, sunitinib malate (SM), an anticancer drug was loaded using lecithin as a stabilizer by employing emulsion solvent evaporation technique. Four formulations (SLPN1–SLPN4) were developed by varying the concentration of chitosan polymer. Based on particle characterization, SLPN4 was optimized with size (439 ± 5.8 nm), PDI (0.269), ZP (+34 ± 5.3 mV), and EE (83.03 ± 4.9%). Further, the optimized formulation was characterized by FTIR, DSC, XRD, SEM, and in vitro release studies. In-vitro release of the drug from SPN4 was found to be 84.11 ± 2.54% as compared with pure drug SM 24.13 ± 2.67%; in 48 h, release kinetics followed the Korsmeyer–Peppas model with Fickian release mechanism. The SLPN4 exhibited a potent cytotoxicity against MCF-7 breast cancer, as evident by caspase 3, 9, and p53 activities. According to the findings, SM-loaded LPHNPs might be a promising therapy option for breast cancer. Breast cancer is considered one of the leading types of cancer, surpassing lung cancer, as per worldwide cancer incidence in 2020 . Breast Sunitinib malate (SM) is a multiple tyrosine kinases inhibitor, used effectively in the cancer of the stomach, bowel, and esophagus, generally called gastrointestinal stromal tumors (GST), an abnormal proliferation of cells in gastrointestinal tract tissues ,11. It iMoreover, breast cancer treatment failure could be surfaced due to poor drug solubility, low-bioavailability, permeability, cell uptake, drug resistance, and systemic toxicity. The cost of therapy and adverse drug effects could be lowered by adopting and aligning with new technologies. Nanotechnologies ensure it reduces adverse systemic toxic effects; thereby, the cost of therapy will also be reduced ,21,22. NSunitininb malate (SM) was purchased from “Mesochem Technology” Beijing, China. Chitosan and polyvinyl alcohol (PVA) were procured from Sigma Aldrich, St. Louis, MO, USA. Lipoid 90H was a generous gift from Lyon, France. Human breast cancer cell line (MCF-7 cells) with estrogen, progesterone, and glucocorticoid receptors was procured from the American Type Culture Collection (ATCC). All other chemicals used were of analytical grade, and Milli-Q water was used wherever needed.w/v acetic acid (Lipid–polymer hybrid nanoparticles (LPH-NPs) were prepared by emulsification solvent evaporation technique . Brieflytic acid . Furthertic acid .Freshly prepared SM-loaded LPHNPs were diluted 200 times with milli-Q water, sonicated for 5 min, and transferred into plastic cuvette, then analyzed for particle size and PDI using Malvern Zetasizer . Three measurements were carried out for 11 runs with 10 sec durations each run at 25 °C temperature. The same procedure was followed for ZP measurements as particle size except a glass electrode sample holder was used instead of a plastic cuvette . Each samax 250 nm [Percent entrapment efficiency (%EE) was measured indirectly . Freshlyx 250 nm  . Samples 5 mg  (SLPN4) were packed in a hemispherical aluminium pan, separately. A pan filled with the sample was kept in a heating chamber against the blank. The temperature was raised from 50 °C to 350 °C at a rate of 20 °C/min; additionally, nitrogen gas was supplied at flow rate of 20 mL/min  . Endothe−1 . Peaks at the fingerprint region were interpreted, and additional or absent peaks were then studied for possible functional group interactions between drug and excipients. Spectrums were then collaged and presented for compatibility study [FTIR spectra of SM, lipoid 90H, chitosan, and optimized LPHNPs (SLNP4) were taken using the KBr technique. The samples were mixed with crystalline KBr and the mixture was then compressed into transparent pellets using a hand-held compression machine. Thin transparent sample film enclosed in the die was fixed into the sample holder and scanned in the range of 400–4000 cmty study .Sample (pure SM and SLPN4) were characterized by XRD to identify the nature of solid-state. X-ray diffractometer  was used with the following set parameters: Cu-kα radiation at 40 kV/40 mA, scan rate of 0.500°/min in the 0–60 (2θ) range, at a fixed monocromator (U4), attached with scintillation detector .Comparative in vitro release studies of pure SM and optimized LPHNPs (SLPN4) were carried out using a dialysis bag (cut off mol wt. 12 kD) as per our previously reported method . BrieflyThe morphology and size of images of optimized LPHNPs (SLPN4) were viewed by Scanning Electron Microscopic (SEM) . In a thin film coater under vacuum, the sample was homogeneously dispersed and coated with gold-metal . The pre-treated sample was then bombarded with an electron beam, resulting in the creation of secondary electrons known as auger electrons. Only the electrons scattered at ≥90 degrees were picked from this interaction between the electron beam and the specimen’s atoms, and surface topography was obtained at 15 kV acceleration voltage and 7.58 K × magnification.® . The cells were grown at 37 °C in 50 cm2 tissue culture flasks in a 5% CO2 humidified incubator. The cells were seeded into 96-well cell culture plates in DMEM.The Human breast cancer cell line (MCF7) was procured from the American Type Cutler Collection . The cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) with phenol red supplement with 10% Fetal Bovine Serum (FBS), with Penicillin (100 units/mL), Streptomycin (100 μg/mL), and Amphotericin B (250 ng/mL), Gibco50 values were calculated using Log (inhibitor) versus normalized response on variable slope by GraphPad Prism V-5.1 .To determine the dose dependent cell viability of MCF-7 cells, they were incubated with SM and SLPN4 ranging from 0.78 to 100 µg/mL  for 48h. The data presented demonstrate relative cell viability after the treatment, since MTT assay determines the viable cells through activity of mitochondria. This is primarily targeted through mitochondria-mediated apoptosis. Thus, this approach was adopted to determine the activity of SM and SLPN4. The IC50 value of SM (10.79 µg/mL) equivalent to SLPN4 (8.24 µg/mL) was taken as the dose of treatment and morphological features were manifested by phase-contrast microscopy. Morphological features such as membrane blebbing, cell shrinkage, and necrosis were determined.The cytotoxic effect of pure STB and SLPN4 was also determined by visualizing the morphological changes in MCF7 cells . The IC52. The SM, SLPN4-treated, and untreated control cells were then allowed to equilibrate at room temperature in 96-well plates. Each well of plate  containing 100 µL of culture media received 100 µL of caspase-3 and 9 reagents. The plate was covered and the contents were stirred at 500 rpm for 30 s. After 30 min of incubation at room temperature, the optical density was measured at 405 nm using an an ELISA reader.Caspase-3, caspase-9, and p53 assay ELISA kits were used to measure caspase activity . The MCFTo analyze the change in formulation over storage or shelf life, a stability study was conducted for optimized LPHNPs (SLPN4). The formulation was sealed in a glass vial and stored for three months at 25 ± 0.5 °C/65 ± 5% RH and 40 ± 2 °C/75 ± 5% RH in a stability chamber, and physical appearance, particle size, PDI, ZP and entrapment were examined in samples obtained at 0, 1, 2, and 3 months ,37.p < 0.01) was considered significant.The experimental data were analyzed statistically using one way ANOVA followed by Tukey’s multiple comparison test using SPSS 16 software  (SM-loaded LPHNPs (SLPN1-SLPN4) were prepared by the single emulsification method. The developed LPHNPs were characterized for their particle size, PDI, and ZP and measured in the range of 218–439 nm, 0.269–0.504, and +18 to +34 mV, respectively . AccordiThe %EE of SM-loaded LPHNPs (SLPN1–SLPN4) were measured in the range of 45.71 ± 3.3–83.03 ± 4.9% . The higDSC spectra of pure SM, Lipoid 90H, chitosan, and optimized LPHNPs (SLPN4) are presented in −1. The FTIR spectra of pure SM showed many intense peaks at 3324 cm−1 for the acidic O-H, 2981 cm−1 for the acidic –CH=CH- (aryl) str, 2884 cm−1 for C-H  str, 1635 cm−1 for the –NHCO str, and 1073 cm−1 bands correspond to the (C–F stretching) [−1 (-OH str), 2873 cm−1 (CH2 str) [−1 and 2861 cm−1 for –C-H- str present in long fatty acid chain, 1722 cm−1 for –C=O str, and 972 cm−1 for P=O str [−1, 2981 cm−1, and 2884 cm−1 were found to have disappeared. The FTIR peaks of SLPN4 represents no significant modifications in the functional peaks of the SM in the lipid–polymeric NPs matrix, thereby retaining drug’s physicochemical properties and efficient chemical stability for the encapsulated SM in the fabricated nanocarriers. As there is no evidence of drug–polymers interaction, the selected lipid, polymer, and drug have the compatibility and suitability for the SM-loaded HNPs.etching) . FTIR spCH2 str) . The FTI P=O str . In the Pure drug sunitinib (SM) showed intense X-ray diffractions at 13.3°, 19.5°, 22.4°, and 25.6° at 2θ, which represents the crystalline state of the drug . HoweverIn vitro drug release studies were conducted at pH 6.8, i.e., the pH of cancer cells . A rapid2 value in the prediction of the release mechanism. The kinetics analysis of regression coefficient of all the four models used indicated R2 values for zero order (0.612), first order (0.9359), Higuchi model (0.8494), and Korsmeyer–Peppas (0.9406) with diffusion coefficient n (0.271). The optimized LPHNPs (SLPN4) followed the Fickian diffusion (n < 0.5) and mechanism of release from the Korsmeyer–Peppas kinetic model [Release kinetics were assessed for optimized LPHNPs (SLPN4). The goodness of fit models was selected by evaluating Ric model .SEM images of optimized LPHNPs (SLPN4) are shown in the 50 values for pure drug SM and SLP4 were found as 10.79 and 8.24 µg/mL for MCF-7 cells, respectively. The formulation SLP4 showed a significant reduction in cell viability  in comparison with pure drug SM , respectively, against MCF-7 cells. Based on the results of MTT assay, it was observed that SLP4 exhibited potential anticancer activity against breast cancer cell lines, probably due to the enhancement of the release of SM from the SLP4 formulation. SM-loaded LPHNPs (SLPN4) could be used as a potent carrier for the treatment of breast cancer.The MTT assay showed concentration-dependent reduction in cell viability for SM and optimized SM-loaded LPHNPs (SLPN4) against MCF-7 cell lines . The IC5The morphological changes on MCF-7 cell lines after treatment of control, pure SM, and SLPN4 are presented in Apoptosis is a type of programmed cell death that involves the disassembly of intracellular components while avoiding injury and inflammation to nearby cells ,53. The p < 0.01) in particle size and PDI were noted when stored at 25 ± 0.5 °C/65 ± 5% RH and 40 ± 2 °C/75 ± 5% RH, which indicated that high temperature can lead to aggregation of the nanoparticles.Following ICH guidelines, the stability of optimized LPHNPs (SLPN4) was assessed for three months in terms of particle size, PDI, ZP, and entrapment efficiency after storage. No research has been reported on SM as a breast cancer treatment using lipid–polymer nanoparticles. In vitro release and cell line investigations showed that the developed optimized SM-loaded LPHNPs (SLPN4) significantly enhanced the release and accessibility of SM at the breast cancer cells. Importantly, the potential cytotoxicity of SLPN4 on the MCF-7 breast cancer cell line was determined using the MTT test for cytotoxicity, ELISA activity of caspase-3, -9, and p53 in comparison to free SM or control. Thus, the developed formulation could provide an attractive nanoplatform for the treatment of breast cancer and may be the focus for the future chemotherapeutic investigations.


Postnatal heart maturation is the basis of normal cardiac function and provides critical insights into heart repair and regenerative medicine. While static snapshots of the maturing heart have provided much insight into its molecular signatures, few key events during postnatal cardiomyocyte maturation have been uncovered. Here, we report that cardiomyocytes (CMs) experience epigenetic and transcriptional decline of cardiac gene expression immediately after birth, leading to a transition state of CMs at postnatal day 7 (P7) that was essential for CM subtype specification during heart maturation. Large-scale single-cell analysis and genetic lineage tracing confirm the presence of transition state CMs at P7 bridging immature state and mature states. Silencing of key transcription factor JUN in P1-hearts significantly repressed CM transition, resulting in perturbed CM subtype proportions and reduced cardiac function in mature hearts. In addition, transplantation of P7-CMs into infarcted hearts exhibited cardiac repair potential superior to P1-CMs. Collectively, our data uncover CM state transition as a key event in postnatal heart maturation, which not only provides insights into molecular foundations of heart maturation, but also opens an avenue for manipulation of cardiomyocyte fate in disease and regenerative medicine.The online version contains supplementary material available at 10.1007/s13238-022-00908-4. Functional maturation with concomitant, near complete loss of proliferative potential, are hallmarks of mature cardiomyocytes. Understanding the mechanisms underlying heart maturation is not only essential to filling our knowledge gaps of cardiac biology, but also paves the way for artificial manipulation of cardiomyocyte fate in cardiac disease intervention and regeneration.in vivo maturation.At the cellular level, cardiac maturation is generally considered a process where cardiomyocytes undergo a series of maturational changes, including the transition from hyperplastic to hypertrophic growth, cell cycle arrest, sarcomeric protein isoform switching, and a shift from glycolysis to oxidative phosphorylation. Early gene expression studies directly compared the fetal or neonatal heart to their adult counterpart as a means to elucidate mechanisms of cardiac maturation , have greatly facilitated our understanding of postnatal heart development  encompassed nearly all well-known cardiac-specific structural genes and transcription factors, including Tnnt2, Myh6, and Nkx2-5  by fluorescence-activated cell sorting (FACS). In line with previous results, the expression of representative cardiac genes  was reduced in P7 cardiomyocyte nuclei compared to their P1 counterparts  on freshly isolated cardiomyocytes (CMs) from mouse hearts at postnatal days 1 (P1), 7 (P7), 21 (P21) and 56 (P56). Quality control of isolated cardiomyocytes was performed to ensure the viability and purity of isolated cells . Clustering of differentially expressed genes (DEGs) revealed 4 major expression patterns (Clusters 1–4)  almost diminished at P7, consistent with our observations at the transcription level  exhibited a marked increase at P7, suggesting a transcriptionally unsettled state in CMs  on cells isolated from hearts at P1, P4, P7 and P14 after birth Figs. A and S4Ahigh-CMs was 6.67% at P1, reached a peak level of 24.00% at P7, and sharply declined to 0.18% and 0.07% at days P14 and P21, respectively  staining against ACTN2 (CM marker) and JUN (one of top-ranked markers) at various developmental stages. Strikingly, the percentage JUNNext, we aligned CMs of different postnatal days along pseudotime using Monocle to characterize their developmental status. P7-CMs and P14-CMs were situated at two distinct ends of the trajectory, indicating large transcriptional differences between them Fig. H. SimilaMyh6-Cre/Esr1;R26-tdTomato transgenic mice  by gene silencing using lentivirus, which effectively infects proliferating neonatal cardiomyocytes, to determine their importance in CM transition state in vivo , in neonatal mouse hearts (P1), respectively, and evaluated their impact on heart development and cardiac function at various time points  or all of these 3 AP-1 components (shAll) resulted in nearly 20.12% or 18.08% decline in cardiac ejection fraction at P14, and 21.28% or 19.23% at P28, respectively . We further partitioned CMs into 8 subtypes which closed resembled 7 CM subtypes in hearts of P28 wild-type mouse (P28_CM1 to P28_CM7)  than in those injected with P1 cells (0.30%), indicating better engraftment of P7-CMs -ligated, infarcted mouse hearts Fig. A. IntrigMyh6-RFP+ cells, compared to their P1 counterparts, displayed molecular features closer to P28-CMs  .Myh6 labeling were achieved by crossing Myh6-cre/Esr1 mice and R26-tdTomato. Four micrograms of tamoxifen were orally delivered to maternal mice on postnatal days (P) 0–4 after delivery of offsprings. For cell transplantation experiments, RFP+ cells (mainly MYH6+ cells) from Myh6-Cre/Esr1;R26-tdTomato mice were sorted by a flow cytometer .Mice with –25 g, 8–10 weeks) underwent myocardial infarction surgery through permanent ligation of the left anterior descending artery (LAD). Immediately after ligation, 2 × 105 of freshly isolated unlabeled cardiomyocytes or RFP+ (sorted by flow cytometry) cells, or 20 μL PBS vehicle control, were injected directly into the border zones surrounding infarct regions. Two weeks post injection, ejection fraction was measured by echocardiography. On the same day, mice were sacrificed, and the RFP+ cells were sorted by a flow cytometer  for scRNA-seq and hearts were collected for further analysis.Wild-type male C57BL/6 mice  for mouse heart injection. Ten microliters of concentrated shNT, shJun, shTcf4, shKlf2, or shAll (Jun + Tcf4 + Klf2) lentivirus were injected into left anterior ventricles at 3 points in P1 mouse hearts, respectively. The time point of injection was chosen to allow sufficient time of viral integration, and to be sufficiently early so as to block the gradual state transition from P1 to P7. Expression of Jun, Tcf4 and Klf2 was assessed 28 days post injection. Expression of KLF2 in CMs was examined to validate inhibition of the transition state. Heart function was evaluated 28 days post injection. Afterwards, the cells from the shNT and shJun P28 mouse hearts were isolated using the Langendorff method for scRNA-seq.For gene knockdown experiments, JUN, TCF4 and KLF2 shRNA oligos were purchased from the Beijing Genomics Institute with target sequencing. The shRNAs were cloned into pLKO.1 vector according to the protocol, and knockdown efficiency of lentivirus was tested in P1 CMs g, 2 min at room temperature) three times to separate cardiomyocytes from non-cardiomyocytes. Cardiomyocytes were collected for further experiments. Aside from C57BL/6 mice, this method was also applied to KM mice, NIH mice, Sprague Dawley rats and Wistar rats.For isolation of cells from neonatal mouse ventricles, P1 and P7 C57BL/6 mice were sacrificed, and hearts were transferred to PBS-containing heparin sodium (1000 units in 50 mL PBS) for washing. Ventricles were chopped into small pieces, and digested with the Pierce™ Primary Cardiomyocyte Isolation Kit  according to the manufacturer’s instructions with minor modifications. Specifically, P7 tissues were cut into relatively larger tissue chunks compared to P1, and a shorter digestion time of 20–25 min was applied. To obtain relatively pure cardiomyocytes and non-cardiomyocytes, we used the differential velocity adherence method to culture isolated cells for 1 h, and non-adherent cells were collected as cardiomyocytes. For isolation of cells from P21 and P56 mouse hearts, mice were injected with heparin sodium 100 μL (1000 units in 50 mL PBS) 20 min before sacrifice, and hearts were transferred to Tyrode’s solution for washing, after which they underwent digestion using the Langendorff perfusion technique. In brief, hearts were perfused with Tyrode’s solution for 5 min using the Langendorff apparatus, followed by digestion with the heart dissociation enzyme stock solution (0.7 mg/mL Collagenase type II and 0.7 mg/mL Bovine Serum Albumin Tyrode’s Solution) for 30 min. Tissues from ventricles were collected, chopped into pieces, and gently dissociated into single cells. Digestion was stopped by Tyrode’s solution containing 10% FBS. Finally, cell suspension was centrifuged , H3K4me1 , H3K27ac , H3K4me3  and H3K27me3  were performed as described previously . One μg of RNA was used for the generation of sequencing libraries using the TruSeq RNA Library Prep Kit V2  according to manufacturer’s instructions. All libraries were sequenced on the NextSeq500 sequencer  using the 35 nt paired-end sequencing protocol.Two biological replicates were performed.Immunostaining was performed according to standard protocols for paraffin embedded samples and frozen sections, respectively. For paraffin sections, hearts were collected in PBS on ice in 4% PFA at room temperature for 12–24 h depending on tissue size, followed by tissue dehydration and embedding. Embedded hearts samples were sectioned to a thickness of 4 μm. Prior to staining, sections were dewaxed, and antigen was retrieved with an EDTA buffer or citric acid solution using a pressure cooker. For frozen sections, fresh heart tissues were embedded in OCT Compound  and then stored at −80 °C until sectioning to a thickness of 10 μm. Frozen sections were washed in water several times to remove OCT, then fixed in 4% PFA at room temperature for 10 min, and stored at −80 °C until use for immunostaining. For immunostaining, tissue sections were blocked with PBSST (0.3% Triton X-100 in goat serum working solution) for 90 min at room temperature, followed by incubation with the primary antibody overnight at 4 °C. The next day, tissue sections were washed 3 times with PBS, incubated with Alexa-Fluor-conjugated secondary antibodies for 1.5 h at room temperature, washed 3 times with PBS, and mounted with mounting medium containing the nuclear stain DAPI. The following antibodies were used (ratios indicate antibody dilution): tdTomato-RFP , TNNT2 , ACTN2 , PCM1 , CDH5 , KLF2 , DCN , CD68 , NPPB , S100A1 , JUN  and ATP1A2 , GAPDH . Alexa Flour488 Rabbit IgG, Alex Fluor and 594-AffiniPure Goat Anti-Mouse IgG, and Alex Fluor 647-AffiniPure Goat Anti-Rat IgG secondary antibodies  were used for secondary antibody incubation. Fluorescent images were captured using a laser scanning confocal microscopy .Quantification of immunohistochemical staining was performed using the image analysis software ImagePro Plus. Specifically, the integrated optical density (IOD) is obtained summing all optical density values of each brown dot within an image. Mean density is calculated by dividing IOD by the area of the distribution area of the target of interest.Hearts were collected and fixed in 4% paraformaldehyde in PBS overnight at 4 °C and then processed for either paraffin or cryo embedding. Haematoxylin and eosin and Masson’s trichrome staining were performed according to standard procedures on paraffin sections using Automatic Dyeing Machine (Thermo Scientific Gemini AS).®R37610) by adding 2 drops (~80 µL) of each dye per mL of cells, mixed carefully, and incubated at 37 °C for 20 min. Then, the reaction was stopped by adding the same volume of pre-warmed 1× PBS, and cells were pelleted by centrifugation at 100 ×g for 3 min at room temperature. To achieve maximum single cell collection, the cell suspension was diluted to approximately 2,200 cells/100 µL with Second Diluent 2× (WaferGen 430-000239-0001), RNase Inhibitor (Invitrogen AM2694), and pre-warmed 1× PBS . After dilution, cells were dispensed into the ICELL8 Chip (WaferGen430-000245-0001) by the MultiSample NanoDispenser (MSND), and the chip was imaged by an imaging system. “DAPI” (to ensure single cell selection) and “Texas Red” (to exclude dead cells) were applied to select single live cells for subsequent analysis. After single cells selection, the reverse transcription (RT) reaction mix was prepared in a 220 µL total volume containing 5 mol/L Betaine Solution (Sigma B0300-1VL) 56 µL, 25 mmol/L dNTP mix (Clontech639132) 24 µL, 1 mol/L MgCl2 (Thermo Fisher ScientificAM9530G) 3.2 µL, 100 mmol/L DTT (Sigma43816-10ML) 8.8 µL, SMARTScribeTM5× First-Strand Buffer (Clontech639537) 61.9 µL, SeqAmpTM2× PCR Buffer (Clontech638504) 33.3 µL, 100 µmol/L RT E5 Oligo (WaferGen430-000240-0001) 4.0 µL, Amp Primer (WaferGen430-000241-0001), 100% Triton X-100 (Sigma T8787) 1.6 µL for cDNA synthesis. Reverse transcription was performed on a Chip Thermal Cycler (BIO-RAD 1861096 ), with a lid temperature of 72 °C and the following reaction program: 53.5 °C 5 s, 48.5 °C 180 s, 4.0 °C 360 s, 46.5 °C 5 s, 40.5 °C 5400 s, 51.7 °C 5 s, 48.5 °C 120 s, 38.5 °C 5 s, 39.8 °C 120 s, GOTO step 6 and 1 cycle, 73.0 °C 5 s, 69.5 °C 900 s, 98.0 °C 5 s, 96.0 °C 60 s, 100.0 °C 10 s, 99.5 °C 10 s, 60.0 °C 5 s, 63.5 °C 30 s, 68.0 °C 3 s, 67.6 °C 180 s, GOTO step 15 and 23 cycles, 72.0 °C 600 s, 4.0 °C hold. After reverse transcription, the cDNA was collected and purified using the DNA Clean & Concentrator kit (Zymo Research D4004) and Agencourt AMPure XP magnetic beads  according to the manufacturer’s instructions. The quality of the cDNA product was analyzed by Bioanalyzer (Agilent 2100) and quantified with the Qubit® dsDNA HS Assay Kit (Invitrogen Q32851). When necessary, the cDNA product was diluted to about 0.5 ng/µL for library generation. The libraries were generated with the Nextera XT Library Preparation Kit  following the manufacturer's instructions, and were purified using Agencourt AMPure XP magnetic beads . The resulting libraries were sequenced on the NextSeq500 sequencer .Single-cell RNA-Sequencing was performed using SMARTer ICELL8 Single-Cell System (Takara Bio) as previously described. In brief, single cell suspension was filtered with a 100-μm nylon cell strainer  and counted. Afterwards, cells were stained with Hoechst 33342 and Propidium Iodide  . For Myh6-Cre/Esr1;R26-tdTomato samples (P4 to P28) , a total of 13,317 cells were isolated from 9 mice, 11,168 of which passed the filtering criteria. For shNT and shJun samples, a total of 7,482 cells were collected, 7,049 of which passed the filtering criteria. To assess the effect of sequencing depth on the number of genes detected, and to learn the sequencing saturation level, we isolated different cell types from P1 samples, and calculated the number of genes with a TPM-like value > 0 in each cell. Then, a scatter plot was generated and a smooth trend line was added. As anticipated, the number of detected genes was saturated at our sequencing depth for the main cell types in P1 (data not shown).For C57BL/6 samples (P1 to P14), a total of 5,010 cells were isolated from 20 mice with an average raw read depth over 0.1 mol/L reads per cell, of which 4,231 cells passed our filtering criteria as described above , to describe the CM cell clusters in a finer level, cells were clustered for each time point separately. First, the most variable genes among cells were selected by their average expression level and dispersion: average log TPM-like value was restricted to be between 0.01 and 3, and the dispersion was set to be greater than 1. Then, the selected genes were used for cell clustering. The FindClusters function of Seurat based on shared nearest neighbor (SNN-cliq) clustering method was utilized with default parameters (k = 30). Main cell types, including CM, EC, FB and MP, were identified by the expression of known markers. Simultaneously, sub-clusters of CMs were recognized for each time point. CM subclusters and non-CMs were visualized by UMAP. Then we collected CM subclusters from all time points of genetic lineage labeling samples, identified reporter-positive CMs, and combined them with P1-CMs to learn the developmental trajectory of CMs. For shNT and shJun samples, to avoid potential batch effect among individuals, and to retain true differences between shNT and shJun samples, we applied an iterative clustering strategy following these steps: 1) for each individual, cells were clustered in each individual as described above. The first 10 PCA components were used to do SNN clustering with a resolution value of 1. After clustering, the data were visualized by UMAP. Main cell types, including CM, EC, FB, MP, T and pericyte (PC), were determined based on the expression of known markers. 2) We collected each type of cells from all four individuals, examined the expression of Jun in shNT and shJun groups in each cell type, and performed a second round of dimensionality reduction and clustering for CM cells in isolation.Clustering was conducted using the Seurat package (v2.3.4) . Ninety-seven genes were detected in our data of C57BL/6 samples and 96 in genetic lineage labeling samples. Using the housekeeping genes, we generated a RLE  plot for each animal sample. The distribution of RLE values was very similar between samples and the median RLE value was zero in most samples. PCA analysis on these genes showed that all samples were mixed, and no obvious distinct pattern was observed. Moreover, any cluster in UMAP analysis comprising cells from a single animal sample was considered an outlier cluster, and therefore was removed from downstream analysis. From the UMAP result, all clusters contained cells from multiple animal samples. Hence, the difference among cell clusters represented true biological difference rather than systematic technical issues.As described by Butler et. al. , the FinMonocle2 (v2.6.0) was used to study pseudotime trajectories of cells  Risso, . Then DeTo further learn cell-to-cell heterogeneity in gene expression, we used the R package kohonen (v3.0.6) to construct SOM (self-organizing maps) for exhibiting the similarity relationships of genes in each transcriptome in a two-dimensional heat map, in which spatial neighborhood reflects expression pattern similarity  to quantitatively predict a tipping point as a critical transition state based on decrease of correlation between cells and concomitant increase of correlation between genes at each time point, as illustrated by Mojtahedi et al |〉 is the absolute value of all Pearson’s correlation coefficients of all pairs of genes, and 〈R〉 is the absolute value of all Pearson’s correlation coefficients of all pairs of cells.where 〈|P value of < 0.05. Then the expression of each pair of genes, which was composed of one P1 TF and one P28 TF, was plotted for each time point. TF value was defined as the product of expression of P1 TF and P28 TF in each pair of genes. Then the TF values of each pair of genes at each time point were plotted to compare the activities of TFs during postnatal development.To describe the dynamic expression pattern of TFs (transcription factors) from P1 to P28, we compared gene expression in P1CM and P28CM, and selected representative TFs of P1CM or P28CM with Pairwise discreteness test was used as described by Su et al. to test the discrete or continuous levels between Myh6 lineage tracing cell P1 to P28 and injected P1 and P7 cells in the MI model  in each condition using CellphoneDB   and RNA Polymerase II for each time point was determined via chromatin immunoprecipitation followed by high-throughput sequencing. Reads were mapped to genomes of mm10 by subread and mapped reads from biological replicates were merged for peak calling by MACS2 (v2.1.1) . Then, the input value was subtracted from the ChIP value as background signal. Since expression data were computed at the gene level, the value of each chromatin mark was defined as the mean value observed across all transcripts of a gene. Chromatin marks at TSSs in different gene categories were calculated as a median value of genes in each category (Table S2).P value < 0.05 were taken as significantly differentially expressed genes, which were then clustered according to their expression patterns through the sampling series by hclust and cutree functions in R  Below is the link to the electronic supplementary material.


Malbranchea pulchella (MpXyn10) in various chemical supports and evaluated its potential to produce xylooligosaccharides (XOS) from hydrothermal liquor of eucalyptus wood chips. Values >90% of immobilization yields were achieved from amino-activated supports for 120 min. The highest recovery values were found on Purolite (142%) and MANAE-MpXyn10 (137%) derivatives, which maintained more than 90% residual activity for 24 h at 70 °C, while the free-MpXyn10 maintained only 11%. In addition, active MpXyn10 derivatives were stable in the range of pH 4.0–6.0 and the presence of the furfural and HMF compounds. MpXyn10 derivatives were tested to produce XOS from xylan of various sources. Maximum values were observed for birchwood xylan at 8.6 mg mL−1 and wheat arabinoxylan at 8.9 mg mL−1, using Purolite-MpXyn10. Its derivative was also successfully applied in the hydrolysis of soluble xylan present in hydrothermal liquor, with 0.9 mg mL−1 of XOS after 3 h at 50 °C. This derivative maintained more than 80% XOS yield after six cycles of the assay. The results obtained provide a basis for the application of immobilized MpXyn10 to produce XOS with high purity and other high-value-added products in the lignocellulosic biorefinery field.Xylooligosaccharides (XOS) are widely used in the food industry as prebiotic components. XOS with high purity are required for practical prebiotic function and other biological benefits, such as antioxidant and inflammatory properties. In this work, we immobilized the recombinant endo-1,4-β-xylanase of  In recent years, the biomass biorefinery concept has been extended beyond the technological application of bioethanol production. Innovative and efficient technologies for lignin, cellulose, and hemicellulose fractionation allow the implementation of integrated processes for the co-production of bioenergy and higher value-added bioproducts, such as cello- and xylooligosaccharides and lignin derivatives ,2,3,4. Tp-coumaroyl side-chain groups [p-coumaric acid esterases (EC 3.1.1.-) [Xylan is the most common hemicellulosic polysaccharide in the plant cell wall and the second most abundant polysaccharide in nature . It is cn groups ,7. The c3.1.1.-) ,7.Among the enzymes of the xylanolytic system, the interest in xylanases has grown remarkably due to their application in the hydrolysis of lignocellulosic materials (LCM), releasing XOS (sugar oligomers composed of xylose units linked with β-(1→4) bonds) ,8,9. XOSRecent works have demonstrated the use of free and immobilized xylanases in the hydrolysis of xylan, mainly from agricultural crop residues ,19,20,21Malbranchea pulchella [Aspergillus nidulans and immobilized in different supports activated with different functional groups. The activity, thermostability, and operational stability of the MpXyn10 immobilized were studied, and the analysis was applied in the XOS production from xylan of diverse sources. Finally, as a proof of concept in the LCM biorefinery, the potential of MpXyn10 in producing XOS from hydrothermal liquor of eucalyptus wood chips  was evaluated.Here, the thermostable MpXyn10 xylanase from ulchella  was overAspergillus nidulans A773 was cultivated in a minimal medium supplemented with 5% corn syrup for 60 h at 37 °C. The xylanase-rich crude extract was filtered and concentrated, and as expected, high levels of xylanase activity (>200 U mL−1) were obtained. Afterward, MpXyn10 was purified from concentrated crude extract using two chromatography steps, as described in Methods. SDS–PAGE analysis  at 1370 cm−1, and of glycosidic linkage (C-O-C) at 1059 cm−1, even as bands corresponding to epoxy rings around 1252, 930, and 890 cm−1 were observed [−1 was attributed to the elongation mode (=C-H) of the aldehyde (glyoxyl) group. For agarose activated with amino groups, a new band was formed at 3077 cm−1 attributed to amine stretching (N-H) [−1 confirms the presence of the imine (C=N) in the support arising from the bond between glutaraldehyde and amino groups of ethylenediamine (EDA)  stretching at 1717 cm−1, alkene (C=C) stretching at 1640 cm−1, alkene methylene group (C-H) bending at 1455 cm−1, alkene methyl group (C-H) bending at 1388 cm−1, amine (C-N) stretching at 1255 cm−1, aliphatic ether (C-O) stretching at 1147 cm−1, and alkene (C=C) bending at 960 cm−1 [MpXyn10 was immobilized on commercial Purolite and agarose-activated supports. These approaches were chosen to evaluate the best support/protocol to preserve the enzymatic activity and improve operational features of the immobilized MpXyn10, such as pH and temperature stability. Initially, hydroxyl groups on agarose surface were activated with epichlorohydrin, forming epoxy/diols groups, and then activated with several functional groups and characterized using FTIR spectroscopy. As shown in observed ,29. The ng (N-H) . For GLUne (EDA) A [32,33,960 cm−1 .2) present in the surface of the support [Aspergillus oryzae and β-xylosidase from Bacillus subtilis [ support ,40. Its  support . Similarsubtilis ,42.The activity recovery was low for glyoxyl- and GLUT-agarose derivatives . It suggests that the immobilization of MpXyn10 from covalent attachment (one- and multipoint) via aldehyde groups, even at mild conditions (pH 7.0 and low ionic strength), promoted a negative effect on the enzyme activity upon immobilization on these supports . The negConsidering the retention of activity , the MANThermal stability assays were performed at 65 and 70 °C. Thermal inactivation profiles of the free and MpXyn10 derivatives after incubation for 24 h are shown in We also assessed the effect of pH on the stability of the MpXyn10 derivatives with incubation in buffers ranging from pH 4.0 to pH 6.0 C, conditbeechwood as a substrate (−1) for free and immobilized MpXyn10 preparations assayed  and wheat arabinoxylan (8.9 mg mL−1) after 180 min of the assay  were used, and the chemical composition of raw material was performed  as the highest product obtained  present in EWC hydrothermal liquor was used to obtain XOS with lower xylose DP (2–4), which are preferentially used as prebiotic components in functional foods. Initially, free and immobilized MpXyn10 preparations were applied in the hydrolysis of EWC hydrothermal liquor, and the profile of the enzymatic hydrolysis products was analyzed by TLC and quantified by HPLC. As observed in obtained B. These beechwood and EWC hydrothermal liquor. The quantification of produced XOS was performed by HPLC, and XOS yield was determined for each cycle. As shown in As one of the main disadvantages of enzyme processes to produce XOS is the cost of enzymes, therefore, the use of immobilized xylanases to increase catalyst efficiency is attractive. Generally, this strategy allows the development of more stable biocatalysts that can be recovered and reused several times. Thus, in order to evaluate the operational stability of the Purolite-MpXyn10, this derivative was used in several cycles of hydrolysis of xylan from beechwood xylan (~90%), birchwood xylan (~90%), oat spelts xylan (~75%), xylose (>95%), arabinose (>95%), glucose (>95%), furfural (99%), and 5-(Hydroxymethyl) furfural, HMF (99%) were purchased from Sigma-Aldrich . Wheat Arabinoxylan (~95%), xylobiose (>95%), xylotriose (>95%), xylotetraose (>95%), xylopentaose (>95%), and xylohexaose (>95%) were acquired from Megazyme . Silica gel plates DC-Alufolien Kieselgel 60 were purchased from Fluka . Bradford protein assay and Precision Plus protein TM standards were purchased from Bio-Rad Laboratories . Hiprep Q FF and Superdex 75 10/300 GL columns were acquired from GE Healthcare . Chromatography HPLC-column Rezex™ ROA-Organic Acid H+ was purchased from Phenomenex . Amino C2 methacrylate beads (Purolite) support was kindly provided by Purolite S.A . All other reagents used for the assays were of analytical grade.Agarose 6 BCL was purchased from GE Healthcare Bio-Sciences AB . Epichlorohydrin, iminodiacetic acid, ethylenediamine, polyethyleneimine, 3,5-dinitrosalicilic acid (DNS), orcinol, Eucalyptus grandis wood chips were kindly donated by the International Paper Company . Before use, the raw material was dried at 50 °C and milled in a 25-mesh sieve knife mill (SL-32–SOLAB).Aspergillus nidulans A773 recombinant strain carrying the construction pEXPYR/Mpxyn10 was used to produce the endo-1,4-β-xylanase from Malbranchea pulchella (Mpxyn10) [Mpxyn10) . The str®/CelliGen® 310 . Briefly, 20 mL of a spore suspension (1 × 108 spores mL−1) of A. nidulans A773 was inoculated in 500 mL of minimal medium, pH 6.5 [−1 and 300 rpm. After 60 h of cultivation, the crude extract was filtered in a Büchner funnel with Whatman n° 1 filter paper and concentrated using the QuixStand Benchtop Systems tangential filtration concentrator with a 10 kDa Hollow Fiber Cartridge . The concentrated crude extract was loaded on an anion exchange column (Hiprep Q FF 16/10) equilibrated with 50 mmol L−1 phosphate buffer, pH 7.5, integrated with an ÄKTA Purifier 900  chromatography system. Protein elution was monitored at 280 nm, with a linear gradient from 0 to 1 mol L−1 of NaCl. The protein samples were analyzed using SDS-PAGE and selected, pooled, and concentrated using Vivaspin ultra-filtration devices with a 10 kDa membrane . The recovered piece was loaded in a size exclusion column (Superdex 75 10/300 GL), equilibrated with phosphate buffer 50 mmol L−1, pH 7.0, plus NaCl 150 mmol L−1. Protein elution was monitored at 280 nm, and the fractions of interest were selected as described above.Production of MpXyn10 was carried out in a 5 L stirred tank bioreactor BioFlo, pH 6.5 , supplemm/v) and distained with a methanol/acetic acid/water solution (5/1/4 v/v/v).Electrophoresis SDS-PAGE (15%) was performed according to Laemmli , using a−1, pH 5.0), and 100 µL of 1% xylan beechwood (w/v). The assay was incubated in a Thermomixer comfort  for 10 min at 50 °C and 1300 rpm. After incubation, 200 µL of DNS were added to the assay mixture and boiled at 98 °C for 5 min. Afterward, 1 mL of distilled water was added to the mix, and 100 μL samples were collected and analyzed for reducing sugar at 540 nm using microplate readers . A standard curve of D-xylose was used to estimate the reducing sugar concentrations equivalents. One unit of enzyme activity (U) was defined as the amount of enzyme that catalyzes the release of 1 μmoL of reducing sugar per minute under the assay conditions.Xylanase activity was determined with the reducing-sugars method using 3,5 dinitrosalicylic acid (DNS) . The ass4, and 40 mL of acetone. The mixture was kept in suspension by stirring with paddles in an ice bath, and 27.5 mL of epichlorohydrin was added dropwise. Afterward, the suspension was gently stirred for 16 h at 25 °C. Finally, epoxy-activated agarose was washed with distilled water, filtered using a glass filter, and stored at 4 °C.Epoxy-agarose support was prepared according to ,57. Firs−1 H2SO4 and gently stirred for 2 h at 25 °C. Then, the support was washed with distilled water, filtered, and resuspended in 200 mL of 100 mmol L−1 NaIO4 for 2 h at 25 °C. Afterward, the glyoxyl-agarose support was washed with the excess distilled water, filtered, and stored at 4 °C.Glyoxyl-agarose support was prepared as previously described ,45. Firs−1 ethylenediamine (EDA) at pH 10.5. The suspension was gently stirred for 2 h at 25 °C. Afterward, NaBH4 (10 mg mL−1) was added to the mixture, which was kept under gentle stirring for 2 h at 25 °C. The support was then washed with 1 L of 100 mmol L−1 sodium acetate, pH 4.0, and 1 L of 100 mmol L−1 sodium borate, pH 9.0. Finally, MANAE-agarose support was washed with the excess distilled water, filtered using a glass filter, and stored [Monoaminoethyl-N-aminoethyl (MANAE)-agarose support was prepared according to Fernández-Lafuente et al. . Brieflyd stored ,62 at 4 v/v) and added in 9 mL of 200 mmol L−1 of phosphate buffer, pH 7.0. The mixture was kept under gentle stirring for 18 h at 25 °C. Afterward, GLUT-agarose support was washed with the excess distilled water, filtered using a glass filter, and stored at 4 °C.Agarose-activated with glutaraldehyde groups (GLUT-agarose) was prepared as previously described by ,63. MANA−1 sodium bicarbonate buffer, pH 11, containing 10% (w/v) of polyethyleneimine (PEI). The mixture was kept gently stirred for 3 h at 25 °C. Afterward, 1 g of NaBH4 was added, and the mixture was incubated for a further 2 h at 25 °C. PEI-activated agarose was washed with distilled water, filtered using a glass filter, and stored at 4 °C.PEI-agarose support was prepared according to . Glyoxyl−1 with a resolution of ±1 cm−1 and 120 scans. FTIR spectra were not used as a quantitative relationship but as a qualitative reference.The chemical groups on the untreated and treated support were identified by FTIR using a Perkin Elmer model Frontier spectrophotometer equipped with a diamond ATR module . Measurements were taken in the range of 200 to 4000 cm−1, pH 7.0. For Glyoxyl-agarose, sodium bicarbonate buffer 100 mmol L−1, pH 10.5, was used, and after the incubation, it was reduced by adding NaBH4 (1 mg mL−1) for 30 min. The time course of immobilization was evaluated by determining the residual activity in aliquots of the supernatant and suspension removed over time. The parameters of the immobilization were defined as the immobilization yield (IY), calculated according to Equation (1):−1) of the supernatant before immobilization and Utf is the activity (U) remaining in the supernatant at the end of the immobilization procedure.Immobilization assays were carried out using a standard protocol for all supports. Briefly, 1 g of support was suspended in 10 mL of a solution containing an enzyme loading of 25 U per g of support (~0.2–0.3 mg of protein loading). The immobilization was performed under mild stirring (80 rpm) at 25 °C. For MANAE-agarose, PEI-agarose, and Purolite support, the enzyme was added to a phosphate buffer at 10 mmol Lo is the observed activity of the immobilized derivative (U g−1 of support) and UT is the theoretical activity of the immobilized preparation (U g−1 of support), calculated based on the difference between the initial activity and the removed activity from the supernatant during the immobilization procedure.The activity recovery (AR) was calculated according to Equation (2):−1 citrate buffer, pH 5.0, at 65 and 70 °C. For pH stability assays, free and immobilized MpXyn10 was incubated in a 50 mmol L−1 citrate buffer in the pH range of pH 4.0–6.0 for 24 h at 25 °C. Aliquots were withdrawn periodically to quantify residual enzymatic activity as described in 2.4. The residual activities for both experiments were calculated concerning controls treated identically but without incubation.Thermal stabilities of free and immobilized MpXyn10 were assessed with incubation in 50 mmol L−1) and HMF (0.63 g L−1) in the reaction medium corresponding to 5 mmol L−1. The relative activities for free and immobilized MpXyn10 were calculated concerning controls treated identically but without adding the compounds.The effect of furfural and 5-hydroxymethyl-furfural compounds on MpXyn10 activity was determined by adding furfural . The assays consisted of 0.5% (w/v) xylan in 50 mmol L−1 citrate buffer, pH 5.0, and 0.1 U of xylanase activity in the reaction medium for both preparations. The assays were incubated in a Thermomixer comfort  at 50 °C, 1300 rpm. Aliquots were removed over time (0 to 180 min) and boiled at 98 °C for 5 min. The reducing sugars were quantified with the DNS method, and the XOS produced were analyzed with TLC and HPLC. Controls were carried out to evaluate the xylan-derived products in the reaction medium under the same assay conditions.Free- and immobilized MpXyn10 were applied in the hydrolysis of xylans from diverse sources (xylan from −1); fX2 (0.936) and fX3 (0.956) are stoichiometric factors [The XOS yield, % (xylan conversion to xylobiose (X2) and xylotriose (X3) was calculated according to Equation (3): factors ,53,65.Eucalyptus grandis wood chips (EWC) was carried out according to Silva et al. [w/v) of solid loading. The reactor was immersed in an oil bath  at 160 °C for 60 min (severity factor = 3.54). These conditions were used to obtain a better recovery of the xylan-rich liquor after the pretreatment avoiding the formation of unwanted products, such as xylose, furfural, and HMF [The LHW pretreatment of a et al. . Briefly and HMF ,67. Aftev/v) sulfuric acid, at 121 °C, for 20 min [2SO4 in ultrapure water, and the flow rate was 0.7 mL/min.The chemical composition of in natura and pretreated EWC was determined according to the US National Renewable Energy Laboratory (NREL) ,69. The r 20 min . HPLC an−1 citrate buffer (50% v/v), pH 5.0. The assay was carried out as described in item 3.10. For the operational stability of the immobilized MpXyn10 derivatives, EWC liquor was used as the substrate to produce the low DP XOS. Immobilized preparations (2 U) were added in a 2-mL vial containing the reaction medium (1 mL) at 50 °C, 150 rpm. After each reaction cycle, the immobilized preparations were recovered with filtration using a glass filter and washed thrice with 50 mmol L−1 citrate buffer, pH 5.0, and then reused on a new reaction cycle. XOS yield was determined for each cycle.After LHW pretreatment, enzymatic hydrolysis of xylan present in EWC liquor was carried out by employing free or immobilized MpXyn10. Initially, the reaction medium consisted of 1.5 mL containing EWC liquor in 50 mmol Lv/v) [g for 10 min. Then, ten microliters of each supernatant were applied to TLC plates. After two runs with the mobile phase, the plates were pulverized with a solution composed of orcinol 0.04% (w/v) dissolved in ethanol/sulfuric acid  and heated at 150 °C until the appearance of dark spots. Xylose (X1), xylobiose (X2), xylotriose (X3), xylotetraose (X4), xylopentose (X5), xylohexose (X6) were used as TLC standards.Thin-layer chromatography (TLC) analyses were performed on silica gel plates (10 × 10 cm)  at room temperature using a mobile phase composed of ethyl acetate, acetic acid, formic acid, and water in a ratio of 9:3:1:4 (v/v) ,71. Befo+ column Rezex™ ROA-Organic (8%) (300 × 7.8 mm diameter), at 80 °C. Samples were filtered in a 0.22-μm filter and injected using 0.005 mmol L−1 H2SO4 as the mobile phase at a flow rate of 0.5 mL min−1 and a run time of 25 min. Standards were dissolved in the mobile phase at different concentrations and analyzed under the same conditions of samples. XOS were quantified using average peak areas compared with a mixture of standards, xylobiose (X2) and xylotriose (X3), and expressed in mg mL−1.XOS and sugar profiles of hydrothermal liquor were determined using a High-Performance Liquid Chromatography system  equipped with a YL9170 refractive index detector and acid HIn the current work, we successfully immobilized and applied the recombinant MpXyn10 xylanase to produce XOS from different xylan sources. The immobilization process does not alter the biochemical properties of MpXyn10 and the hydrolysis patterns. Besides, it allows the obtention of biocatalysts with high activity and thermal- and operational stability, critical features for application in bioprocesses. Our results also show that the liquor produced from hydrothermal pretreatment of the eucalyptus wood chips residues could be used as a source to produce XOS. The liquor, rich in soluble xylan, was used as a substrate in the efficient production of XOS by Purolite-MpXyn10 in various hydrolysis cycles, which shows the potential of the immobilized recombinant xylanase MpXyn10 in integrated bioprocess in a biorefinery for complete conversion of lignocellulosic materials. The results obtained in this work provide a basis for applying MpXyn10 in the hydrolysis of xylan from EWC hydrothermal liquor but also open the possibility of its application (together with cellulases) in pretreated solids, even for other LCM.


Osteoarthritis (OA) is a common chronic degenerative disease characterized by the loss of articular cartilage, which causes loss of joint function and reduce quality of life. Resolvin D1 (RvD1) has shown interesting anti-inflammatory effects; however, the mechanism of action of RvD1 in OA remains unclear. The aim of this study was to investigate the potential mechanism of RvD1 in OA by bioinformatics and partial in vitro mechanisms. Here, 106 shared differentially expressed genes (DEGs) were identified based on the GSE82107, GSE55235, GSE55457 dataset; 700 DEGs were identified based on GSE169077. Enrichment analyses of these genes were then successively conducted. RvD1-targeted genes and KEGG pathways are identified by STITCH. 27 shared KEGG pathways were identified among RvD1-targeted pathways and OA. Furthermore, cell apoptosis assay, western blotting, real-time fluorescent quantitative PCR (qRT-PCR), enzyme linked immunosorbent assay (ELISA) were used to confirm the expression levels of the key genes of shared Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways between RvD1-targeted and OA in IL-1β treated rat knee chondrocytes. The results showed that RvD1-targeted pathways and the expression of nuclear p65, p53, and p-JNK were inhibited in the RvD1 group compared with the IL-1β group. Thus, the findings indicate that RvD1 may inhibit the development of OA through NF/kB, p53, MAPK/JNK, PI3K-AKT signaling pathways, and act as a treatment for OA.  Osteoarthritis (OA) is a chronic, degenerative joint disease, which is mainly characterized by degeneration and destruction of articular chondrocytes, as well as bone, subchondral bone, and synovial membrane. This leads to joint deformity and dysfunction, seriously affecting the quality of life of middle-aged and elderly people ,2. StudiResolvins are lipid derivatives generated from omega-3 polyunsaturated fatty acids, more specifically, after sequential enzymatic transformations of eicosapentaenoic acid and docosahexaenoic acid producing a series of E resolvins (RvE) and D resolvins . ResolviThe Gene Expression Omnibus (GEO) database stores gene profiles containing microarray and sequence-based functional genomics ,13. To fwww.ncbi.nlm.nih.gov/geo/) was searched to download OA-related gene expression and data for synovial membrane using GSE82107, GSE55235, GSE55457 datasets, thirty OA patients and twenty-seven normal controls were downloaded using GSE169077 dataset. The R software  and limma package [GEO database ( package  were use package .http://stitch.embl.de/), which is a web‐based tool, that can identify and predict interactions between proteins, chemicals, and their related genes [The RvD1-targeted genes were searched using STITCH (ed genes . By impohttp://david.abcc.ncifcrf.gov/) was used to analyze the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) using R ‘ggplot2’ packages with statistically significant results (p < 0.05) [The Database for Annotation Visualization and Integrated Discovery (DAVID 6.8) ( < 0.05) .http://stitch.embl.de/) search function [https://cytoscape.org/index.html) was used to reconstruct and visualize the network [The protein-protein and protein-chemical interactions for RvD1 were determined using the STITCH  [Gene ontology analysis was used to retrieve human OA KEGG pathways. Venn diagram   was used2 in fresh medium until they coalesced and were ready for this experiment. All experiments were conducted using third passage cells. To identify chondrocytes by cell morphology, toluidine blue staining was used for glycosaminoglycans  of the cultured cells.To further verify the effects of RvD1 on OA, rat knee joint chondrocytes were isolated and cultured. 15 male Sprague Dawley (SD) rats (7–10 days old) were purchased from the Experimental Animal Centre of Jinan, Shandong province, China. All animals were housed in the animal room of the Sports Medicine Institute of Shandong First Medical University. This study was approved by the Research Ethics Committee of Shandong First Medical University  . First, cartilage tissue was obtained under aseptic conditions from rats and dissolved in Dulbecco’s modified eagle Medium: Nutrient Mixture F-12 (DMEM/F12) containing 0.25% collagenase II  at 37°C for 2 h. Chondrocytes were extracted and cultured in DMEM/F12 supplemented with 10% fetal bovine serum , 100 units/mL penicillin and 100 µg/mL streptomycin . The cells were cultured at 37°C and 5% COThe chondrocytes were treated with DMEM/F12 or 100 nM RvD1 , 10 nM NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC)  or the 10 nM JNK inhibitor SP600125  or 10 μM p53 inhibitor PFTα  before specific tests. In addition to the control group, chondrocytes were seeded in culture plates and activated with DMEM/F12 containing 10 ng/mL IL-1β  for 24 h to stimulate OA [IL-8) forward, 5′-TCCCGAATTGGAAAGGGAAATA-3′; reverse, CTGTTGGCCCAATTACTAACAG; tumor necrosis factor-α (TNF-α), forward, 5′-GCGCCCAGCCTTCCTTAC-3′, reverse, 5′-GCCCCGGCCT TCCAAATAAATAC-3′; glyceraldehyde-3-phosphate dehydrogenase (GAPDH), forward, 5′-GAAGGTCGGTGTGAACGGATTTG-3′, reverse, 5′-CATGTAGACCAT‐3′.Total RNA was isolated from the cells using a simply P Total RNA Extraction Kit  according to the manufacturer’s instruction, Total RNA (1 μg) was reverse transcribed to cDNA with starscript II First-stand cDNA synthesis Mix with gDNA Remover  as detailed in the manufacturer’s instructions. The amplification products were then detected using StepOne-Plus system . The primer sequences of genes were as follows: Interleukin-8  and IL-8 , following the manufacturer’s instructions.The cytoplasmic and nuclear proteins were extracted using the nucleoprotein and cytoplasmic protein extraction kit . The nuclear protein was used to detect the expression of p65, p53, Bax, p-JNK and JNK, and the total protein was extracted using highly efficient RIPA tissue/cell rapid lysate  to test the expression of p65, p53, Bax, p-JNK and JNK and actin. Protein samples (30 µg) were loaded on a 12% sodium dodecyl sulfate-polyacrylamide gel for electrophoresis and then transferred to a polyvinylidene fluoride membrane. Subsequently, the membrane was blocked with blocking buffer for 1 h, then incubated with the following primary antibodies: p65 , p53 , Bax , p-JNK , JNK , actin , and proliferating cell nuclear antigen  at 4°C overnight. It was then incubated with secondary antibodies for 1 h.To measure chondrocyte apoptosis, the Annexin V-FITC/propidium iodide (PI) apoptosis detection kit  was used for flow cytometry. The steps are outlined as follow: after treatment, chondrocytes were collected and immediately washed twice with ice-cold PBS, resuspended by 100 µL 1× Binding Buffer, and incubated with 5 µL Annexin V‐FITC and 5 µL PI for 10 min in dark at 26°C. Next, 400 µL 1× Binding Buffer was added. Finally, the total cell suspension was analyzed using flow cytometry. The results were calculated as the percentage of apoptotic cells (Annexin V+ PI−).All experiments were repeated at least thrice. Data were represented as means ± standard deviation; the SPSS 22.0 statistical software  was used to determine significant differences between the groups. Multiple sample means were compared using analysis of variance, and pair comparisons among multiple groups was performed using the Student–Newman–Keuls test. Statistical significance was set at p < 0.05.To further explore the potential effect of Resolvin D1 in OA, bioinformatics analysis was used to identify the RvD1 targeted genes and signaling pathways that highly related to OA. Differentially expressed genes and enrichment analysis, the shared KEGG pathways of RvD1-targeted genes and OA analysis were performed. The biological effects of RvD1 and selected hub genes were verified by cell apoptosis assay, western blotting, qRT-PCR, ELISA. The aim of this study is to help us better understand the therapeutic effect of RvD1 in OA.The expression of genes was determined by processing the data from GSE82107, GSE55235, GSE55457 and GSE169077 were analyzed. The results were significant at the level of p < 0. 05 and |log2 fold change (FC)| >0.5. The level of differentially expressed genes (DEGs) is depicted by volcano plot, 829 were upregulated and 2269 were down-regulated in GSE82107 ), 1655 whttp://stitch.embl.de/) was used to input the Resolvin D1 and the search was conducted for Homo sapiens. STITCH is a database that integrates various chemicals. This process highlighted nine relevant genes in the first shell. The genes or chemicals were then expanded to the second and third levels. Sixteen target genes and twelve chemicals were thus identified. RvD1 targeted genes including: IL8, TRPA1, PTGS2, MYOF, FER1L6, GPR32, FPR2, DYSF, OTOF, RELA, CXR2, CXR1, TP53, NFKB1, AITC, DARC. The interactions of protein-protein and protein-chemical were constructed using Cytoscape  [Venn diagram was used to identified the shared KEGG pathways. A total of 37 related KEGG pathways of RvD1-targeted genes were identified; 135 human OA‐related KEGG pathways were retrieved from synovial tissue, 297 human OA‐related KEGG pathways were retrieved from cartilage by using the DAVID database. There were 27 shared KEGG pathways of RvD1-targeted genes and OA related genes as depicted with Venn diagrams . The topay.html)  was usedTo identify the cultured chondrocytes, toluidine blue staining and immunofluorescence staining for type-II collagen were performed. In the optical microscopy results of the cultured chondrocytes, most chondrocytes were observed to be spherical ). In addTo verify the anti-apoptotic effect of RvD1 in OA, cell apoptosis was assessed using flow cytometry after the chondrocytes were treated with DMEM/F12 ), IL-1β To evaluate the anti-inflammatory effect of RvD1 in OA according to the RvD1-targeted genes and signaling pathways, cells were incubated with DMEM/F12, IL‐1β, RvD1+ IL‐1β, or PDTC+IL‐1β; qRT-PCR was used to detect the expression of related genes by extracting total RNA ). In addTP53, NFKB1, RELA are the key RvD1-targeted genes, NF-κB signaling pathway is one of top signaling pathway. To verify these results, the level of nuclear p65, total p65 were detected using western blotting. After treatment of chondrocytes with IL-1β, the expression level of nuclear p65 was higher than that in the control group . The expressions of p53  and Bax To further identify the mechanism of action of RvD1, the expression of JNK and p-JNK was analyzed using western blotting. As shown in OA is a degenerative bone and joint disease that usually occurs in middle-aged and elderly individuals ,26. StudIn this study, synovial tissue and cartilage samples from patients with OA were analyzed using GSE82107, GSE55235, GSE55457, and GSE169077. RvD1-targeted genes and KEGG signaling pathways were identified by STITCH. In comparision with previous studies, we analyzed the differential gene expression of synovial tissue and cartilage in OA as well as potential shared signaling pathways among synovial tissue, cartilage in OA, and RvD1-targeted genes. Additional experiments were conducted to verify these results which could help to understand the pathological mechanism of OA and provide data supporting the use of RvD1 in the treatment of OA.TP53, NFKB1, RELA (IL-8 (https://www.genome.jp/kegg/pathway.html) was used to analyze these top KEGG pathways. We found that they include nuclear factor-κB (NF-κ B), p53, MAPK, PI3K-Akt signaling pathways and others. Therefore, we supposed that RvD1 exerts biological effects mainly through NF-κB, p53, MAPK, PI3K-Akt signaling pathways. These pathways are involved inflammation, apoptosis, glucose transport, and cell signal transduction. The results validated using qRT-PCR and ELISA showed that the mRNA levels and expressions of TNF-α and IL-8  and RELA (p65) are the numbers of the Rel/NFkB family, and the p65 (RelA) plays a key role in the classical NF-κB signaling pathway , NF-κB rp53 and NF-κB are major important transcription factors that control cell death and survival . p53 is MAPK is a serine-threonine protein kinase family that includes p-JNK/JNK, p-p38/p38, and p-ERK/ERK. MAPK regulates cell differentiation, proliferation, inflammation, apoptosis, and innate immunity . PreviouThe PI3K-Akt signaling pathway is important for the cell cycle and is an intracellular signaling pathway, that also plays a role in cell survival, inflammation, apoptosis, and metabolism . When PIHere, bioinformatics analysis was used to identify the shared hub genes and signaling pathways between RvD1-targeted network and OA, and the results were validated using qRT-PCR, ELISA, and western blotting; These results provide evidence for possible treatment of RvD1 in OA and help further understand the mechanism of OA. This study is limited by the fact that the hub genes and signaling pathways were verified using IL-1β-induced chondrocytes, and these results need to be validated in human OA chondrocytes.TP53, NFKB1, and RELA as the hub genes. Our results show that RvD1 may suppress the development of OA via the NF‐κB, p53, MAPK/JNK, and PI3K-Akt signaling pathways. These findings indicate that RvD1 may serve as a therapeutic agent for OA.In this study, the shared genes and signaling pathways between targeted RvD1 and OA were identified with Click here for additional data file.