The glass transition temperature (Tg) of room-temperature curing epoxy adhesives is limited by the temperature used during curing. It is already known that the excess of epoxy groups can undergo a homopolymerization reaction initiated by tertiary amines at elevated temperatures, resulting in an increase in Tg. However, there is no evidence of this reaction occurring at room temperature. In the present work, the influence of formulation stoichiometry on Tg and mechanical properties was investigated. Dynamomechanical, rheological and mechanical properties of epoxy adhesives were determined by DSC, DMA, rheometer and tensile and shear strength testing. It has been probed that an excess of epoxy resin combined with a complex curing system composed of a primary amine, a polymercaptan and a tertiary amine leads to an increase in Tg up to 70 °C due to the homopolymerization reaction that takes place at room temperature. However, as the excess of epoxy resin is increased, gel time becomes slower. Regarding mechanical properties, it has been proven that an excess of epoxy resin provides a tighter and tougher material but maintains flexibility of the stoichiometric formulation, which is meant to enhance the resistance to impact-type forces, thermal shock and thermal cycling. Epoxy resin is a type of reactive prepolymer and polymer containing epoxide groups. This material has many industrial applications for a variety of purposes ,3,4,5,6.Since epoxy adhesive systems are mainly used for structural purposes, one of the properties to be considered is the glass transition temperature (Tg). Tg is the temperature at which the transition between the glassy and rubbery state of amorphous solids occurs. Above this value, the polymer behaves like a rubbery material, and below this value, the polymer chains have relatively low mobility, and the material becomes hard and rigid. Thus, Tg it is considered an extremely useful yardstick for the reliability of this type of structural adhesive.There are several factors contributing to Tg of epoxy adhesives: the chemical structure of epoxy resin, the type of hardener, the degree of cure and the curing conditions (time and temperature (Tcure)) ,17,18,19This work led to a long-established rule of thumb that Tg cannot exceed Tcure by more than 25 °C. That is the reason why commercial room-temperature curing epoxy adhesives always have a Tg limited to 40–50 °C. On the other hand, high-temperature curing epoxy adhesives can reach values up to 160 °C . The higRegarding this matter, Fernandez-Francos et al. studied a new dual-cure system based on epoxy/thiol formulations catalyzed by tertiary amines . The stu®.In order to study a possible approach to increase the Tg of room-temperature curing epoxy adhesive formulations without the use of an external heat source, a new formulation has been proposed. This new formulation consists of a on a nonstoichiometric epoxy adhesive using a complex curing based on a commercial adhesive developed by Dr. Sails® PY 302-2, Huntsman, Pamplona, Spain) with an equivalent molecular weight of 170 g/eq was used as the base epoxy resin. It is a highly crystallization-resistant, low-viscosity liquid epoxy resin with superior chemical resistance. It presents a viscosity between 6500–8000 mPa-s−1 and a density of 1.17 g/cm3. The basic chemical structure of epoxy resin used in this investigation is shown in ® 90, Hunstman, Pamplona, Spain polymercaptan with tertiary amine was also supplied by Huntsman. It presents a viscosity between 10,000–160,000 mPa.s−1, a H+ active equivalent of 200 g/Eq and a gel time against Araldite GY 302-2 of 4–5 min. Ethylenediamine was purchased from Aldrich. All chemicals were used as received.Diglycidyl ether of bisphenol-A (DGEBA)  with a flow rate of 50 mL/min. Dynamic experiments to determine the curing enthalpies of the prepared mixtures were carried out at a heating rate of 10 °C/min between 30 °C and 300 °C.Calorimetric studies were carried out using Mettler Toledo DSC equipment, Barcelona, Spain. Samples were analyzed using perforated aluminum capsules under an inert atmosphere (NCuring time characterization was made with a AR2000 rheometer (TA Instrument), Cerdanyola, Spain using a 20 mm steel plate. The gap was set at 300 microns. An oscillatory procedure was applied using a constant strain of 5% at 1 Hz. Analyses were carried out isothermally at 25 °C.Gel time was determined as the crossing between the G’ and G’’ curves; at this point, the system stores the same or a very similar amount of energy to that dissipated.A TA instrument dynamic mechanical analyzer (DMA) Q800 apparatus  was used in this study, along with a 3-point bending clamp. Tested specimens were rectangular-shaped, 60 mm × 15 mm × 2 mm. The linear viscoelastic region (LVR) of the material was determined by an amplitude sweep test, from 10 to 60 μm at a constant temperature of 25 °C. Tg was determined using a temperature ramp test from 30 to 100 °C with a heating rate of 5 °C/min, an amplitude of 30 μm and a frequency of 1 Hz. The tan δ peak was used as a viscoelastic parameter to calculate the Tg of each formulation.Tensile tests were conducted following ASTM D638 standard protocol  and usinUNE-EN 22643 test standard protocol  was foll−1 and 700 cm−1. The first one, at 911 cm−1, is attributed to the C-O deformation of the oxirane group. The second band is located at approximately 3057 cm−1 and is attributed to the C-H tension of the methylene group in the epoxy ring, although it is also related to asymmetrical and symmetrical C-H stretch of aromatic ring. The presence of these two peaks at the spectre states that the epoxy resin has not reacted yet. However, once the epoxy resin starts curing, the oxirane ring concentration decreases, and this was observed in the spectra as the decrease in the two characteristic absorption bands until all oxirane rings are depleted, as can be seen in Before studying the curing mechanism, the curing behavior of epoxy was analyzed by FTIR. The FTIR spectra are presented as Kinetic analysis of the reactions that take part in the studied systems were analyzed by DSC. The aim of the present study is to develop a complex curing system able to generate enough heat to activate the homopolymerization of the excess of epoxy resin.®PY302-2 epoxy resin with ethylenediamine on one hand and Araldite®PY 302-2 epoxy resin with Aradur®90 on the other hand were studied in order to determine characteristic exotherm peaks of epoxy-amine and epoxy-thiol crosslinking reactions. As shown in Stoichiometric mixtures of AralditeOnce the curing profiles of the reactions involved in the curing of epoxy systems were described and identified, different formulations  were studied in order to determine differences in between stoichiometric and nonstoichiometric formulation reactions .The results obtained showed the different reactions occurring for each tested formulation. When the ratio between the crosslinker’s active groups and epoxy groups is stoichiometric (D01), two separated reaction peaks are detected. The first one corresponds to the reaction between the epoxy and thiol crosslinkers, and the second to the reaction with the amine crosslinkers. As the epoxy ratio in the formulation increases, the appearance of a third peak is detected at around 130 °C, overlapped with the amine curing peak. This phenomenon indicates that a third reaction is taking place during the curing, and such a reaction can only be due to the homopolymerization of the excess of DGEBA , thiol-epoxy and amine-epoxy condensation takes place, while the excess of epoxy homopolymerizes in the second stage. The curing rate of the second reaction that takes place at room temperature is much lower than thiol/amine-epoxy condensation reaction curing rate. This difference should be explained by the different reaction mechanism undergone by the two reactions, since on one hand, epoxy-amines and epoxy-thiols suffer a step growth polymerization mechanism that processes rapidly at the start. On the other hand, the excess of epoxy resin undergoes a chain polymerization mechanism catalyzed by the tertiary amine that requires high initiator content in order to be activated rapidly ,37,38. TA ratio between the epoxy resin and crosslinker was optimized to generate enough exotherm to activate the homopolymerization (D03 formulation), which increases the Tg of the product without requiring any post-treatment. This formulation gave rise to an epoxy adhesive formulation with a Tg of 70 °C, maximizing the sustainable operating temperature of the material, and breaking the stablished thumb rule of Tg ≈ Tcure + 25 °C. Regarding mechanical properties, the product developed is tighter and tougher but maintains flexibility of the stoichiometric formulation (D01), which is meant to enhance the resistance to impact-type forces, thermal shock and thermal cycling.


Cardiovascular diseases are the leading cause of morbidity and mortality worldwide. Numerous cardiac pathologies result in myocardial damage or dysfunction, inevitably leading to heart failure. A fundamental roadblock in heart failure treatment is the inability of the adult human heart to repair itself after injury, which results from permanent cardiomyocyte cell cycle arrest shortly after birth. As such, mechanism(s) that regulate cardiomyocyte cell cycle progression have been the focus of intense research over the past two decades.,2. Canonical WNT signaling has low activity in the adult heart. However, this signaling pathway has also been shown to mediate cardiac pathology, such as cardiomyocyte hypertrophy and death.WNT/β-Catenin pathway is an evolutionarily conserved signaling pathway with essential roles in cell proliferation and differentiation. Canonical WNT signaling, mediated by the binding of WNT ligands to its membrane receptors, Frizzled and LRP5/6, and subsequent activation of downstream β-Catenin-dependent transcription has a pivotal role in the heart. During embryogenesis, WNT/β-Catenin signaling regulation is essential for the proper specification and proliferation of cardiac progenitor populations in the first and second heart fieldset al. provide a deep analysis of the short-term transcriptional changes taking place upon WNT/β-Catenin signaling modulation in the adult mouse heart. By using cardiomyocyte-specific inducible gain (GOF) and loss (LOF) of function models of the canonical WNT pathway, the authors found that WNT/β-Catenin signaling governs the expression of cell cycle and oxidative phosphorylation-related genes in adult cardiomyocytes. Enhanced β-Catenin transcriptional activity in the GOF model resulted in increased expression of cell cycle re-entry and cytokinesis genes, while a decrease in the transcriptional activity of β-Catenin in the LOF model led to a modest increase of genes related to oxidative phosphorylation. Interestingly, none of the genetic models caused functional alterations in the heart, in terms of fibrosis, lipogenesis, cardiac function, cell size, or cardiomyocyte number.In this issue, Olcum Moreover, GOF animals did not show an increase in mitotic cardiomyocytes by means of pH3 immunostaining , a mitosis marker considered a gold standard for assessment of cardiomyocyte cell cycle entry, indicating that increased canonical WNT signaling may not be sufficient to induce cardiomyocyte proliferation under basal conditions. Additional studies, including S-phase and cytokinesis makers, would help clarify the effect of GOF on cardiomyocyte cell cycle. In addition, it would be important for future studies to determine the effect of GOF on cardiomyocyte cell cycle following ischemic injury.Interestingly, the LOF model showed a modest increase in transcripts related to mitochondrial oxidative phosphorylation. These results, even if modest, would be interesting to pursue. For example, some functional experiments in the LOF model, such as mitochondrial activity, morphology, and substrate utilization, would be important to delineate the role of canonical WNT signaling in the mitochondrial biology of adult cardiomyocytes. Conceptually, a shift towards an oxidative phenotype is generally considered a facet of cardiomyocyte maturation. If true, although the picture is not complete at this stage, it would suggest that WNT signaling might regulate a cardiomyocyte maturation program involving metabolism and cell cycle regulation.,7. However, inhibition of the same pathway in epicardial cells and cardiac fibroblasts led to worsening of cardiac function and wound healing following myocardial infarction. This highlights the importance of cell type-specific studies in regard to canonical WNT signaling in the adult heart. In this regard, Olcum et al. performed all the analyses using cell-specific genetic models, providing a cardiomyocyte-specific signature of transcriptional changes upon β-Catenin GOF and LOF in adult cardiac myocytes.Previous studies in pathological cardiac conditions showed contradictory roles of WNT/β-Catenin signaling depending on the cell type studied. For example, inhibition of the canonical WNT pathway upon cardiac injury improves heart function and reduces fibrosis and aging. Given the increased β-Catenin levels achieved in the GOF model in the current study, despite the fact that there does not seem to be any overt electrical or contractile consequences, it would be interesting to determine whether β-Catenin levels are also increased at the level of intercalated disks and whether some of the transcriptional changes or long-term effects are dependent on it.Previous studies have also demonstrated that β-Catenin is a component of the intercalated disk, and that it accumulates in that location during cardiac hypertrophy. In that regard, it is possible that some of the de-regulated pathways in the GOF and LOF models are due to alterations in the non-canonical branch of the pathway. Perhaps additional studies using double mutants of both β-Catenin and non-canonical WNT pathway members would help dissect the role of each signaling cascade in the adult cardiomyocytes.WNT signaling is known to have a non-canonical pathway that acts independently of β-Catenin. However, there is increasing evidence that both WNT canonical and non-canonical WNT signaling interact and coordinately regulate several cellular processeset al. conducted a series of elegant studies that suggest a degree of regulation of cell cycle genes and cell cycle progression pathways by canonical WNT signaling in adult myocytes. As mentioned above, this does not appear to be sufficient for bona fide induction of cardiomyocyte mitosis in the adult heart under basal conditions; however, additional long-term and injury-related studies may be needed to further delineate the role of WNT signaling in myocardial regeneration following injury. From a mechanistic standpoint, it would be important to determine whether WNT signaling acts as a nodal point for the regulation of a cardiomyocyte maturation program involving metabolism and cell cycle.In conclusion, Olcum


Then, LMT-28 was administered by gavage at 0.23 mg/kg once every 5 days for 2 weeks to type 2 diabetic rats with peri-implantitis induced by LPS injection and silk ligature. The expression of IL-6 and RANKL was evaluated by immunohistochemistry, and the bone resorption around implants was evaluated by microcomputed tomography. The results showed that LMT-28 downregulated the expression of RANKL through the JAK2/STAT3 signaling pathway in osteoblasts stimulated by LPS and AGEs, reduced bone resorption around implants with peri-implantitis, decreased the expression of IL-6 and RANKL, and decreased osteoclast activity in type 2 diabetic rats. This study confirmed the ability of LMT-28 to reduce LPS-induced bone resorption around implants in diabetic rats.In this study, we examined the effect of the GP130-targeting molecule, LMT-28, on lipopolysaccharide- (LPS-) induced bone resorption around implants in diabetic models using in vitro and rat animal experiments. First, LMT-28 was added to osteoblasts stimulated by LPS and advanced glycation end products (AGEs), and nuclear factor- Peri-implantitis is an irreversible inflammatory change of hard and soft tissues around implants, which is caused by an imbalance of microorganisms in the peri-implant microenvironment . The mosPorphyromonas gingivalis) in dental plaque, is shown to facilitate PBL due to a significant inflammatory response and persistent bone resorption activity [κB ligand (RANKL), which induces osteoclast precursors to become mature osteoclasts [Based on existing research, the mechanism of bone loss in peri-implantitis has been explored from multiple angles. The host immune inflammatory response is considered to be the basic mechanism of peri-implant destruction in peri-implantitis . This inactivity . In addiactivity . IL-6, aactivity , 9. IL-6eoclasts .According to previous studies, LMT-28, a derivative of oxazolidinone, has been shown to significantly inhibit IL-6 activity through direct binding to GP130 and is nontoxic with oral availability . LMT-28 To evaluate the effects of LMT-28 on bone resorption in peri-implantitis, an appropriate animal model is needed that can simulate human diseases, diabetes mellitus, dentition defects, and peri-implantitis. In this study, we chose Sprague-Dawley (SD) rats, which are an ideal model for implant placement and induction of peri-implantitis –14. BecaPorphyromonas gingivalis (BNCC337441) obtained from BeNa Chuanglian Biotechnology Research Institute  was grown at 37°C in brain heart infusion (BHI) broth medium in an anaerobic incubator. A bacterial LPS extraction kit  was used to extract LPS from 50 to 200 mg of P. gingivalis, and the LPS concentration was measured by a total polysaccharide content measurement kit .μm. The volume concentration of DMSO in the cell culture medium was 0.1%. To examine its in vivo effects, LMT-28 was dissolved in 10% DMSO and 90% corn oil  at 2.5 mg/mL.LMT-28  was provided by MedChemExpress Inc. . For in vitro experiments, LMT-28 was dissolved in dimethyl sulfoxide  to obtain a final concentration of 0.1–1,000 2) at 37°C.Mouse embryonic osteoblasts  were cultured in low-glucose Dulbecco's modified Eagle medium  supplemented with 10% fetal bovine serum  containing 1% streptomycin/penicillin  and incubated in a humidified incubator  of AGEs . After 2 h, the expression of RANKL was measured by RT-qPCR to determine the optimal concentration of AGEs and LPS. Then, osteoblasts were stimulated by the optimal concentration of LPS and AGEs for different periods . Western blot (WB) and RT-qPCR were used to measure the expression of RANKL to determine the time at which RANKL expression was upregulated by LPS and AGEs and peaked. In addition, various concentrations of LMT-28  were added to the osteoblasts, and the cells were collected 2 h after stimulation. The expression of RANKL was measured by RT-qPCR to obtain the optimal stimulus concentration of LMT-28. The optimal concentration of LMT-28 was added to osteoblasts, and cells were harvested at different times . The expression of RANKL was measured by RT-qPCR and WB for determining the optimal stimulation time of LMT-28.Various concentrations of LPS  were added to the osteoblasts, and the cells were harvested 2 h later. Real-time fluorescence quantitative PCR (RT-qPCR) was used to detect the expression of RANKL mRNA expression levels were evaluated using RT-qPCR; and RANKL protein, JAK2 phosphoprotein, and STAT3 phosphoprotein expression levels were evaluated using WB. In addition, the optimal concentrations of LPS, AGEs, baricitinib, and LMT-28 were added to the cultured osteoblasts. After the optimal stimulus time, the osteoblasts were harvested; the RANKL mRNA expression levels were evaluated using RT-qPCR; and RANKL protein, GP130 phosphoprotein, JAK2 phosphoprotein, and STAT3 phosphoprotein expression levels were determined using WB.The optimal stimulus concentration of dexamethasone  and baricitinib  was evaluated by CCK8. Then, dexamethasone and LMT-28 were added to the cultured osteoblasts at their respective optimal concentrations. After the optimal stimulus time, the osteoblasts were harvested; the n = 30, 241–282 g) were obtained from the Laboratory Animal Center of Southwest Medical University. The rats were fed in a ventilated room with a high-fat and high-sugar diet and water ad libitum. All surgical procedures were preapproved by the Animal Ethics Committee of Southwest Medical University (SWMU20210414).Eight-week-old male SD rats  (30 mg/kg) to induce diabetes mellitus type 2 (T2DM). The body weight was recorded every week, and the plasma glucose concentration was measured at 3, 7, and 14 days postoperatively. Rats with three plasma glucose concentration measurements >16.7 mmol/L accompanied by weight loss were considered T2DM rats. The procedure and group assignment for the in vivo experiment are shown in Subsequently, the maxillary right first molar was extracted under general anesthesia with isoflurane . After 4P. gingivalis LPS. A 10 μL P. gingivalis LPS (0.1 mg/mL) suspension was injected three times consecutively at 10 min intervals for each treatment  were seeded into 96-well plates in 100 μL of plain medium, treated with dexamethasone  or baricitinib , and incubated for 1, 3, 5, or 7 d. Subsequently, 10 μL of CCK8 solution (Solarbio) was added to each well followed by incubation at 37°C in a 5% CO2 incubator for 1 h, and the absorbance was immediately measured at 450 nm.To measure cell proliferation, osteoblasts  expression levels were quantified and used to normalize gene expression levels. The 2−∆∆CT method was used to analyze relative gene expression. Each experiment was replicated at least three times. RT-qPCR was performed with the following primers: RANKL (5′-CTGATGAAAGGAGGGAGCACG-3′ for sense and 5′-CTGATGAAAGGAGGGAGCACG-3′ for antisense) and GAPDH (5′-CCACTGGCGTCTTCACCAC-3′ for sense and 5′-CCTGCTTCACCACCTTCTTG-3′ for antisense).Total RNA was extracted from mouse osteoblasts using a nonchloroform RNA extraction kit  following the manufacturer's protocol. An aliquot of total RNA was reverse transcribed using a ReverTra Ace qPCR RT kit  and amplified using SYBR Green real-time PCR master mix (TOYOBO). 6 cells/well. After stimulation was complete, cells were treated with RIPA lysis buffer (Bioteke) with 1 mm phenylmethylsulfonyl fluoride (PMSF) (Solarbio), 1% phosphatase inhibitors , and 1% proteinase inhibitor (Roche) for 30 min on ice. Cell lysates were resolved using 8–15% sodium dodecyl-sulfate polyacrylamide gel electrophoresis followed by transfer to polyvinylidene fluoride (PVDF) membranes. Membranes were then blocked with skim milk (Solarbio) at room temperature for 1 h and then incubated with the appropriate primary antibodies against RANKL, GAPDH, p-GP130, p-STAT3, STAT3, p-JAK2, or JAK2  on an orbital shaker at 4°C overnight. Then, membranes were washed  with tris-buffered saline with 0.1% Tween and incubated for 1 h with horseradish peroxidase-conjugated secondary antibodies diluted 1: 6,000. After washing , membranes were incubated with an enhanced chemiluminescence reagent (Affinity), and the chemiluminescent signals were visualized using an iBright CL1000 imaging system . The semiquantitative analysis was performed using ImageJ v1.8.0 .Osteoblast cells were seeded into six-well plates at a density of 1 × 10μm; tube voltage, 80 kVp; tube current, 500 μA; and total rotation, 360°. After reconstructing the region of interest in three dimensions, the BAH was evaluated in sagittal sections of the implants as the distance from the implant platform to the first bone of implant contact. In addition, Inveon Research Workplace was used to calculate the BAV, which was defined as bone loss within 1 mm around the implant.Micro-CT  was performed to examine the bone absorption height (BAH) and bone absorption volume (BAV) using the following specifications: spot size, 50 The harvested maxillary alveolar bones were fixed in 4% paraformaldehyde and then decalcified in 10% ethylene diamine tetraacetic acid (EDTA) (Solarbio). The EDTA solution was replaced every 5 days for 5 weeks. Samples were embedded in paraffin after unscrewing the implants. The central part of the specimens was sectioned into 7 mm thick slices in the mesiodistal direction. Slides were stained using tartrate-resistant acid phosphatase (TRAP) staining according to the manufacturer's directions (BestBio). The slides were incubated with the primary antibodies overnight at 4°C after incubating in a goat serum-blocking solution for 20 min. Subsequently, the slides were washed with phosphate-buffered saline (PBS) three times for 5 min each time before staining with the secondary antibodies for 1 hr. The slides were washed with PBS three times for 5 min each time again. After incubating in DAB chromogenic solution for a few minutes, slides were counterstained with hematoxylin for 3 min. The investigator was blinded to the immunopositive outcome when photographing the slides using a camera system , and semiquantitative analysis was performed using ImageJ v1.8.0 (NIH).p < 0.05.Statistical analyses were performed using SPSS statistical software . Data were presented as means ± standard deviation (SD). Differences between groups were analyzed using one-way ANOVA, and least significant difference tests were used for post hoc tests. Statistical differences were considered significant at RANKL in osteoblasts increased significantly (p < 0.05), but when the LPS concentration was higher than 100 ng/mL, RANKL expression tended to be constant, so the optimal concentration of LPS to stimulate RANKL expression in osteoblasts was set to 100 ng/mL (RANKL in osteoblasts increased significantly with an increase in the AGE concentration (p < 0.05), but when the concentration of AGEs was higher than 100 pg/mL, the expression of RANKL tended to be constant. Therefore, the optimal concentration of AGEs to stimulate the expression of RANKL in osteoblasts was chosen as 100 pg/mL (RANKL in osteoblasts decreased significantly with an increase in the LMT-28 concentration (p < 0.05), but when the concentration of LMT-28 was higher than 100 nm, the expression of RANKL tended to be constant. Therefore, the optimal concentration of LMT-28 to attenuate the expression of RANKL in osteoblasts was 100 nm . However, 1,000 nmol/L, 10,000 nmol/L, and 100,000 nmol/L dexamethasone at 3, 5, and 7 days, respectively, significantly reduced the proliferation activity of osteoblasts (p < 0.05), while 100 nmol/L dexamethasone had no effect , while 10 nmol/L baricitinib had no effect on the proliferation of osteoblasts , and the BAH in the LMT-28 group was lower than that in the peri-implantitis+LMT-28 group  and peri-implantitis+LMT-28 group (1.69 ± 0.47 mm3) was significantly higher than that of the control group (0.46 ± 0.19 mm3) (p < 0.05), while the BAV of the peri-implantitis+LMT-28 group was significantly lower than that of the peri-implantitis group , while there was no significant difference in IL-6 expression between the peri-implantitis+LMT-28 group and the control group , and the expression of RANKL in the peri-implantitis+LMT-28 group was significantly lower than that in the peri-implantitis group  and interleukin-1β (IL-1β) in mice with arthritis [α titer in mouse serum induced by IL-6 was reduced significantly by LMT-28 [β and TNF-α [β and TNF-α in lung tissues from mice with sepsis were markedly decreased by LMT-28 [β, IL-6, and TNF-α are the most commonly studied functional polymorphisms for peri-implantitis and peri-implant bone loss [LMT-28 is one type of IL-6 inhibitor and acts by binding directly to GP130 and selectively inhibiting IL-6-induced phosphorylation of GP130, which is a common receptor and signal transducer for the IL-6 family of cytokines. To date, it has been reported that LMT-28 was used to treat many diseases with positive results –29. Severthritis , 26. In y LMT-28 . In the nd TNF-α . Furtherone loss , 33, 34.In the present study, IL-6-mediated inhibition of RANKL-induced osteoclast formation was achieved by LMT-28, which interfered with IL-6/IL-6R/GP130 trans-signaling. Moreover, the combination of IL-6/IL-6R and GP130 in osteoblasts, which subsequently activated the JAK signaling pathway and preferentially induced tyrosine phosphorylation of STAT3, also correlated with RANKL levels. The JAK2/STAT3 signal transduction pathway is one of the main pathways through which IL-6 exerts its biological roles and plays a critical part in inflammatory bone disease . To studκB in osteoclast, which may indicate the potential mechanism of LMT-28 on bone resorption [One limitation of the current study is that, although the SD rat model is an ideal animal model for T2DM and peri-implantitis, it does not completely recapitulate the characteristics of T2DM and peri-implantitis in humans and shows different immune responses. Furthermore, LMT-28 inhibits the secretion of RANKL in osteoblasts through the IL-6/GP130 signaling pathway, but the effect of LMT-28 on osteoclast differentiation and maturation remains to be demonstrated. Studies reported that inflammatory bone resorption was related to RANKL- and LPS-mediated NF-sorption . RANKL iOur study highlighted the potential of LMT-28 to reduce LPS-induced bone resorption around implants in a diabetic rat model with peri-implantitis. LMT-28 also significantly inhibited the mRNA and protein expression of RANKL in osteoblasts under costimulation by LPS and AGEs. These findings provide a rationale for the critical role of IL-6 in LPS-induced bone resorption and in vitro and in vivo evidence of LMT-28 therapeutic effects on peri-implantitis in diabetics.


This study aimed to develop functional fruit wines using whole fruit, pulp, and peels from green bananas. The boiled banana homogenates were mixed with cane sugar before wine fermentation. Quality parameters, phenolic compounds, flavor components, and antioxidative properties of the green banana peel wine (GBPW), green banana pulp wine (GBMW), and whole banana wine (GBW) were determined. High-performance liquid chromatography was used to determine the phytochemical compounds in three wines, and the flavor components were further analyzed using headspace solid-phase microextraction combined with gas chromatography–mass spectrometry. The flavor components and in vitro antioxidant activities were, respectively, determined using the relative odor activity value and the orthogonal projections on latent structure discrimination analysis (OPLS-DA). In vitro antioxidative capacities for these wines were evaluated using antioxidant chemical assays and cell culture methods. The total phenolic and total tannin content of the GBPW, GBMW, and GBW showed reducing trends with increasing fermentation days, whereas the total flavonoid content of the wine samples exhibited downward trends. The antioxidant capacities of the three wine samples were higher than those of the raw fruit samples, except for the metal chelation rate (%). Additionally, the main flavor component in the wine samples was 3-methyl-1-butanol. Its percentages in the GBPW, GBMW, and GBW were 72.02%, 54.04%, and 76.49%, respectively. The OPLS-DA results indicated that the three wines presented significantly different antioxidant activities. The cell-culture-based antioxidant analysis showed that these wine samples had protective effects against the oxidative stress of the 3T3-L1 preadipocytes induced by hydrogen peroxide. This study provided a theoretical basis for defining the antioxidant characteristics of banana wines and expanding novel channels for using banana peels to develop nutraceuticals. Bananas are one of the most cultivated and consumed fruits in the world. They are also one of the crucial fruit varieties grown in South China, with an average annual output of 11,130.02 million tons from 2016 to 2020, accounting for 27.23% of the total fruit output. The skin or peel accounts for about 40% of the total fruit weight of a ripe banana. Banana processing often generates many by-products, such as peels, which are rarely eaten directly owing to their bitter taste. Banana peels are by-products or waste, which poses serious environmental hazards [Bananas have been developed into different functional foods to date. Although banana peels are not commonly used as food, they are typically used as a carbon-based adsorbent for wastewater treatment , fuel 33, and anStudies on green bananas can be divided into fundamental research and product development, of which the product characteristic determination includes physicochemical and digestive properties ,9. The aA large volume of by-products is produced annually during banana production and processing because no special recycling treatment is available. Therefore, fully utilizing these by-products to improve the economic benefits of the banana industry is necessary and urgent. Applying green banana peels in wine making can reduce waste production and increase resource utilization. Furthermore, determining the flavor components and antioxidative capabilities of green banana wines is also helpful for functional product development and recycling banana by-products or alternative uses.Musa acuminate L.) of the ‘Guijiao 9’ variety were harvested from the banana germplasm resource nursery of Jinghong city, Xishuangbanna, Yunnan, in April 2021. Green bananas were obtained during the immature stage, packed, and transported to a wine fermentation laboratory. One kilogram of each whole fruit, pulp, and peel was prepared, pulverized, and homogenized with 3.5 L of distilled water. Then, 500 mL of each sample was separately collected from the 3.5 L mixture as a fresh fruit sample. The leftover 3.0 L was boiled for 10 min to denature all the enzymes and destroy the microbes. The hot banana samples were mixed with sugar (136 g/L), homogenized, and cooled to 40 °C before adding 0.5 g/L pectinase and 0.3 g/L cellulase. Then, the mixture was hydrolyzed for 2 h and added with 0.02% dried wine yeast K1-V1116 (Saccharomyces cerevisiae). The wine fermentation was conducted at room temperature, and test samples were collected at 0, 3, 6, 9, 15, and 30 days of fermentation. The sterilization was performed by heating the fermentation medium to 65 °C and maintaining it for 15 min.The unripe green bananas , pH value, total soluble solids (TSS) content, and alcohol content, were determined according to the methods described by Li et al. . The totThe phenolic compounds in the banana wine samples were determined using ultrahigh pressure liquid chromatography (UHPLC) coupled with a mass spectrometer (MS) on an Acquity UPLC HSS T3 column . The mobile phase A was 0.1% aqueous formic acid, and the mobile phase B was acetonitrile . The floThe flavor components in the banana wine samples were analyzed using a headspace solid-phase microextraction combined with gas chromatography–mass spectrometry  . The GC The method for relative odor activity value (ROAV) was used to quantify the flavor components in the banana wine samples. The odor activity value (OAV) was calculated according to Equation (1):max, with the highest contribution rate, was marked as 100. The ROAV values of the other remaining aroma components were calculated according to Equation (2):The ROAViC is the relative content of the aroma components, iT is the sensory threshold of the aroma components, and SC tan and ST tan were the relative content and sensory threshold of the aroma components contributing the most, respectively.The sensory threshold of the flavor components was determined by Shi et al. . AccordiThe antioxidant activities of the banana wine samples were determined using the DPPH radical scavenging rate, hydroxyl radical scavenging ability, superoxide anion radical scavenging ability, and metal chelation rate. The DPPH radical scavenging rate was measured using the DPPH free radical scavenging assay , and theThe antioxidant capacities and inhibition abilities of the banana wine samples were determined using the oxidative stress model of 3T3-L1 preadipocytes induced by 30% hydrogen peroxide. A cell-culture-based oxidative stress model was developed and established by Kim et al. , and thep-value less than 0.05. The antioxidant activities of the three green banana wine samples, determined using four in vitro antioxidant activity assays, were analyzed with OPLS-DA.All the data are presented as the means of three replicates. The statistically significant differences in the antioxidant activities between the samples were determined using the analysis of variance (ANOVA) coupled with a posthoc LSD test. SPSS version 24  was used to analyze the mean differences at a The TSS, alcohol content, pH value, and TAC are important quality indexes of fermented fruit wines. These quality parameters are closely related to the taste and flavor of wines. These quality parameter values for the GBPW, GBMW, and GBW samples are shown in After the third day of fermentation, the increment of the alcohol content and the reduction in the TSS became slower, probably because the yeast multiplied rapidly at the early fermentation stage, and the metabolism rate increased. It digested soluble solids in a fermentation solution to provide sufficient nutrients and convert them into alcohol, organic acids, and other flavor substances. At a later fermentation period, soluble solids in the wine samples were lower. The low amount of soluble carbohydrates could not provide enough energy for the growth and survival of the yeast. Therefore, yeast entered the aging period and finally lyzed. The yeast produced increasing amounts of alcohol and organic acids during fermentation. Additionally, the GBMW showed the most notable decreasing TSS level. The TSS in the GBPW was lower than the other wine samples, and the alcohol content in the GBPW increased the least.The changes in the pH value and TAC of the fruit wine samples are depicted in At the early fermentation stage, the TAC drastically increased because the yeast rapidly multiplied, and a high metabolic rate produced a higher organic acid content. It caused the pH value of the sample to rapidly decrease to a pH of 4. Due to the influences of a low pH value, substrate concentration, and other related factors, yeast metabolism slowed down at the later stage of fermentation. The production rate of the organic acids also decreased; it was easier for the acids to undergo an esterification reaction with alcohols, and the degradation of organic acids occurred and produced ethanol. The reduced concentration of the organic acids led to a lesser increase in the pH value. During wine fermentation, the GBMW had the most notable increase in the TAC, but the pH values of the three fruit wines showed no remarkable differences.p < 0.01). The increased TPC during the early stage of fermentation could be due to the digestion of carbohydrates, thus releasing free phenolic compounds.The TPC, TTC, and TFC in the GBPW, GBMW, and GBW during fermentation are shown in The reduction in the TPC of the GBMW and GBW could be due to the increased concentration of the soluble polysaccharides in the extracts. Similarly, the TPC in the GBPW was reduced by about 5 mg/g. This could have been due to the same reason. The further digestion and utilization of banana starch released more free polyphenolic compounds, except for the GBW. The GBW showed a slight decrease in the TPC from day 15 to day 20 of fermentation. It showed that the minor degradation of polyphenols occurred during fermentation at a temperature of 40 °C. The reduced TPC might also be attributed to the binding and adsorption of secondary metabolites produced by microbial fermentation with polyphenolic compounds or the precipitation and oxidation of polyphenols during fermentation .p < 0.01). The TTC in the GBPW increased on the first three days of fermentation due to the rapid digestion of complex tannin in the banana peel. The reduced TTC could be due to the polymerization reaction of tannins. This result showed that the TTC in the GBPW was significantly higher than in the GBMW and GBW. It could be related to a higher TTC in the green banana peel. Additionally, changes in the TPC and TTC of the fruit wine samples had similar trends.The mass spectrometric parameters of 18 phenolic standards are shown in Apigenin, coumarin, and luteolin are flavonoids that were not detectable in these wine samples. Kaempferol was the only flavonoid detected in all the banana wine samples, where the GBPW exhibited the highest concentration. Additionally, the initial screening of the phenolic compounds was performed using the UHPLC method coupled with a UV-DAD detector. The results showed that gallic acid was not detected in the banana wine samples. The differences in the types and phenolic compound concentrations of the banana wine samples might be related to plant origin, microbial species, and several fermentation parameters .The relative contents of the flavor components in the GBPW, GBMW, and GBW are shown in The ROAV values of the flavor components in the three wine samples are shown in The flavor component is an essential index of fruit wine quality. The sensory quality of a fruit wine is attributed to the volatile and aromatic compounds in the wine. These flavor components indirectly affect the commodity value of the fruit wine. The flavor substances in a fruit wine mainly consist of alcohols, esters, acids, and alkenes, which are related to fruit aroma. Among them, alcohols and acids have relatively high flavor thresholds and contribute little to the flavor of fruit wine. On the other hand, esters have relatively low flavor thresholds and thus contribute more to the aroma of fruit wine.p < 0.01). The green banana peel (GBP) had the significantly lowest DPPH radical scavenging activity (p < 0.05), whereas the GBMW had the highest activity. We also found no significant differences between the DPPH radical scavenging activities of green banana pulp (GBM) and whole green banana (GB) (p > 0.05). Moreover, no statistical differences were observed between the scavenging activities of Vc and green banana wine samples (p > 0.05).The DPPH radical scavenging activity, hydroxyl radical scavenging ability, superoxide anion radical scavenging ability, and metal chelation rate of the GBPW, GBMW, and GBW are shown in p < 0.01). The scavenging ability of the GBPW was also statistically the highest among the banana fruit parts and wine samples (p < 0.01). Moreover, hydroxyl radical scavenging ability of Vc was not as high as the green banana wines. Moreover, significant differences were observed between the scavenging abilities of the different green banana wine samples.The hydroxyl radical scavenging abilities of the banana wine samples are depicted in p < 0.01). The GBPW had the significantly highest scavenging ability (p < 0.05). We found no significant differences between the scavenging abilities of the different fruit parts (p > 0.05). On the contrary, the scavenging ability of Vc was significantly the highest among all the samples (p < 0.05).Based on the data depicted in p < 0.05), indicating that the banana pulp had a lower metal chelation rate than the peel. This finding revealed that the fermentation conditions and types of raw materials altered the metal chelation rate of the fermented wine. The metal chelation rate of the green banana fermented wine was significantly lower than that of its raw material (p < 0.01), whereas the metal chelation rates of the green banana skin, pulp, and fruit samples were significantly different from each other (p < 0.01).The metal chelation rates of the fruit samples were remarkably lower after the wine fermentation D. The meOPLS-DA data are depicted in The antioxidant capacities of the green banana wine samples were determined by an inhibition assay of hydrogen-peroxide-induced oxidative stress on 3T3-L1 preadipocytes. The antioxidant capacities of these wine samples are shown in 50 value of the GBPW was significantly the lowest, followed by the GBW and GBMW. The highest IC50 value of the GBMW indicated that it had the least inhibitory effect on the growth of 3T3-L1 preadipocytes. The results showed that the wine produced from the green banana peel had the highest protective effect against cellular oxidative stress compared to the fruit pulp-based wines. Additionally, the in vivo antioxidant capacity of the GBPW was the highest at sample concentrations between 25 and 100 mg/mL. In this study, 3T3-L1 preadipocytes could be specifically induced to differentiate into mature adipocytes [The protective effects of the three wine samples against hydrogen-peroxide-induced oxidation to 3T3-L1 preadipocytes are shown in ipocytes . TherefoGreen bananas are rich in polyphenolic and other compounds with high antioxidant properties. Green banana waste, such as banana peels, has been used as a raw material to produce functional materials. Banana peels are prebiotics and biosubstrates . They coThe quality of green banana wines was influenced by the other chemical properties in addition to their bioactive antioxidants. The GBPW had a remarkably low alcohol content compared with the other two wine samples. The results showed that wine fermentation using a banana peel had a low alcohol content in the wine sample. The possible reason could be that it had a carbohydrate content lower than the banana pulp. A previous study supported our findings that the acidity and TSS of the GBMW reduced gradually during the 30 days of fermentation . The TSS50 value than the GBMW. The production of fruit wine using bananas with the peel attached maintained a high antioxidant level in addition to improving the wine quality. Therefore, the study on developing banana wines by applying different parts of green bananas warrants the wine quality. This study may also provide a theoretical basis for expanding the channel of banana industrialization, increasing the economic value of bananas, and reducing the waste of resources. The reason for using green bananas was that green banana pulp had higher phenolic content than ripe yellow bananas [Different parts of bananas, such as the peel, pulp, and whole fruit, contain different percentages of phenolic compounds and flavor components. This study compared the fruit wines produced from three parts of bananas because banana pulp wine has a better flavor, but its antioxidant activity could be lower than banana peel wine. The use of the whole banana in wine production somehow improved its aroma and antioxidant activity. The banana peel wine (GBPW) had higher antioxidants than its pulp wine, but the whole banana fermented wine (GBW) had a lower IC bananas .The aroma of the GBPW was unique because the main flavor component of the wine produced from banana peels was 3-methyl-1-butanol. The literature reported that isoamyl butyrate was the main volatile in banana peels, but the level of its isoamyl alcohol, such as 3-methyl-1-butanol, was low . The higWe identified 22 flavor substances in the GBMW. Most of these substances were fatty acid esters. This study was preliminary work on developing green banana wines using different parts of green bananas. A few other physicochemical parameters and analyses of the wine samples have not been included in this study. Although we have not performed a sensory evaluation for these wine samples, we noted that each green banana wine sample had a unique color and appearance. The GBPW had a yellowish brown hue, the GBMW had a milky yellowish appearance, and the GBW was a pale golden-yellow-colored liquid. Among the three wine samples, the GBPW had the most pleasant odor. It had a fresh and fruity smell and taste due to its higher percentages of 2,4-di-tert-butylphenol and 3-methyl-1-butanol. Only the banana-pulp-based fermented wines had a pungent smell due to the acetic acid in these wines.The production of the GBMW is more costly because the banana pulp has a higher commercial value than its peel. The antioxidant quality of the GBMW was also lower than the GBPW, although the GBMW had a better aroma. Since this study did not cover sensory evaluation and some other in vitro bioassays for the wine samples such as the GBPW, they should be included in future studies. The GBPW is one of the potent functional alcoholic beverages with high antioxidants for promoting a healthy drinking lifestyle. The wine sample can be used as an alcoholic tonic drink to prevent metabolic diseases among the population with unhealthy lifestyles because salicylic acid is its key compound.The chemical compositions and bioactive phenolics of the GBPW, GBMW, and GBW had remarkable changes during the first few days of fermentation. The changes in these values were minor during the following days until the end of fermentation. During the fermentation process, the TSS content of the GBPW, GBMW, and GBW showed reducing levels with increasing fermentation time, whereas the alcohol and TAC had increasing rates but with some exceptions. The alcohol and TAC of the GBW had a downward trend. The TPC and TTC of the GBMW and GBW were decreased during the fermentation. However, the changes were not notable after day 10 of fermentation. The GBPW had the highest TPC, TTC, and TFC compared to the GBMW and GBW. This shows that adding a banana peel to the sugar fermentation improved the quality of the fermented wine, such as by further reducing its alcohol and TSS content and increasing its total phenolic components a few times higher than the banana-pulp-based wine.These wine samples had higher antioxidant activities than the different banana parts, except the wine samples had a lower metal chelation rate than the fruit samples. The OPLS-DA revealed that the antioxidant activities of the green banana wine samples were notably different, and the antioxidant activity assay that discriminated based on the antioxidant activities of these wine samples was the superoxide anion radical scavenging assay. The three fermented wine samples had a protective effect against oxidative stress induced by hydrogen peroxide in 3T3-L1 preadipocytes. The GBPW had a better inhibitory effect than the other two wine samples at the same concentrations. Based on these findings, adding different banana parts with sugar for wine fermentation improved the wine quality. The addition of the whole banana in wine fermentation is recommended because the full utilization of the banana peel reduces waste production, the banana peel is rich in antioxidants, and banana pulp has a more favorable aroma. Wine is good for preventing cardiovascular diseases among heavy drinkers. Future studies can consider performing a large-scale sensory evaluation of these green banana wines and their physicochemical characteristics.


Optimizing the pigment volume concentration of zinc phosphate pigments can protect Al alloy 6101 from alkaline media. Additionally, zinc phosphate pigments form a shielding film on the substrate and facilitate stopping the penetration of aggressive corrosion ions. The efficiency of eco-friendly zinc phosphate pigments is almost 98% during the corrosion analysis. A comparative study of physical aging of neat epoxy and Zinc Phosphate (ZP) pigment-modified epoxy coatings on Al alloy 6101 was conducted in Xi’an, China, for one year in all four seasons, where in summer for 3 months, results degraded more due to high UV radiation and humidity; it is found that peeling force of ZP pigments modified epoxy coatings is 50% higher of than that of the neat epoxy coatings though both peel-off adhesion strength and scratch test visibility decreased in both coatings; The electrochemical resistance of ZP pigments modified epoxy coatings is about 30% higher of than that of neat epoxy coatings, the corrosion rate of ZP pigments modified epoxy coatings is about 70% lower of than that of neat epoxy coatings, moreover the gloss retention is 20% higher in the modified epoxy; Optical surface observation of the coatings showed that the ZP modified epoxy coating could effectively restrict the crack and shrinkage in coatings after aging experimentation in the natural environment.  Epoxy resins are thermosetting polymers frequently used in electrical appliances, paints, varnishes, and adhesives . Epoxy iUsually, mechanical and chemical barrier properties of the epoxy coatings degraded due to the water adsorption , the chaAluminum is the 13th element in the periodic table and the 3rd most chemical element after oxygen and silicon . It is wThe function of coatings protection is to prevent the substrate from corrosion, scratch, peel-off, etc., which mainly depends upon the chemical and mechanical properties of the coating and adhesive performance with the substrate . A signiPhysical aging is known as structural relaxation, a kinetic phenomenon in which organic polymers relax when not in equilibrium because they are present at temperatures lower or higher than their glass transition temperature (Tg) . AdditioIn the last 30 years, many publications dedicated to the physical aging of polymeric materials, contrary to organic coatings, even of their huge significance, have not gained the same attention . There aIn general, many factors affect the durability and stability of the coatings, which include radiation, sunlight, pollutants, dust, oxygen, humidity, temperature, and wind speed. In most cases, the influential factors simultaneously affect coatings’ properties and accelerate mutually, such as natural exposure . The potThis study elaborated on the efficiency of a neat epoxy coating modified with ZP pigments on Al alloy 6101, which was not previously explored. Moreover, physical aging under natural weather, including heavy/light rain  during the whole year with a time of three months, including four seasons, while at high temperatures and humidity for both coatings, is being investigated by different experimental techniques in which the peel off test, scratch test, and electrochemical test, water contact angle test and gloss measurements are performed. Moreover, surface characterization is done by the scanning electron microscope and EDS analysis to confirm the distribution and elemental presence of the Zinc phosphate pigments. FTIR analysis was used to verify that no structural changes were observed after the epoxy modification with ZP pigments. The results showed that the protective efficiency of the modified coating with ZP pigments was comparatively higher than that of the neat epoxy.As to the comparative study of the aging effects of protective properties of neat epoxy and ZP-modified epoxy coatings, both coatings on Al alloys are prepared by dipping methods. Al alloy 6101 used in this study is with compositions of Si: 0.3%–0.7%, Fe ≤ 0.50%, Cu ≤ 0.10%, Mn ≤ 0.03%, Mg: 0.35%–0.8% and Cr ≤ 0.03%.The epoxy resin was brought from  and a solvent mixture of butyl acetate, ethylene glycol, and toluene with a mass ratio of 0.5: 0.5: 3. All these chemicals are with the purity of 99% from Wuxi Yasheng Chemical co., Ltd. The optimized ratio of epoxy to the solvent mixture is 1:4.5.3PO4)2.4H2O , and their technical information is listed in 4)2.4H2O . Figure 1. Immersing the Al alloy 6101 substrates into the solution (30 mL) for a half-hour;2. Draw the substrate at a speed of 0.05 mm/s from the immersed solution to prepare a wet-coated sample;3. The wet sample is dried at room temperature (about 24°C and 55% moisture) first; its weight change is recorded to evaluate the liquid evaporation rate of the epoxy coating every 2 min simultaneously; The typical evaporation process of the coated sample with the ratio of epoxy to a solvent mixture of 1:4.5 is shown in .4. On the 3rd day, the samples were further cured in an oven at 80°C for 12 h till no weight changes. The real dry film thicknesses were 40 ± 10 μm by the thickness gauge of the coatingBoth neat epoxy resin and modified epoxy coating are prepared by dipping process according to the following procedure.As coatings were ready, the coated specimens were placed tightly in a fixed place under a natural outdoor environment for every 3 months of each season  totally for 1 year. Data collection (temperature and humidity) of weather was recorded daily by the software weather version 11.01.524 of Xian city Beilin district. There is immense impact of different seasons on the coatings due to the varitions of the temperature and humidity.A property test was conducted gradually with aging to characterize mechanical and chemical properties degradation of coatings due to aging. As aging went a month, experiments analysis of peel-off, scratch, water contact angle, gloss measurements and electrochemical test were performed with three specimens. Simultaneously surface analysis was done by the optical microscope (VHX-5000) to observe the appearance of the coating. Perkin Elmer-Spectrum One conducted the FT-IR analysis on KBr disks in the region of 400–4,000 cm−1. The assessments for these tests are as follows.Peeling—off test is a method to quantify the adhesion of a coating to a substrate, which reflects the binding force required to remove the adhesive film from the substrate , Figure The following formula is used to compute interfacial energy.G: interfacial energy, N/m.P: critical force of film peeling to detach the film, N.D: width of film at peeling point.Θ: peeling angle.Gα: plastic bending dissipation force, N/m.β: participating strain force, N/m.GGα = 0, hence, film thickness on the aluminum alloy surface is very thin so that it can be estimated as Gβ = 0, so Eq. In the peeling-off test, there is no change in the length and width, so −cosθ/dSo, the The Scratch test is a simple method to assess the anti-wear property. To conduct the scratch test, weighted sandpaper glides on the surface of a coated substrate at a certain speed to a certain distance, and the critical weight corresponding to a visible abrasive mark appearing was then detected for a unit width of sample Water drop contact angle measurements were done by a goniometer device to determine the liquid surface contact with each other. A drop of distilled water is deposited on a substrate surface with the help of a dropper at room temperature, and contact angles are measured by a micro charged-coupled device (CCD) camera for respective images.2 Pt (platinum) as a counter electrode.PARSTAT 2273 (Princeton Applied Research Inc.) performs EIS  and potentiostatic polarization. A three-electrode system was used to reduce and compensate for the potential changes caused by large currents passing through the working  electrode, saturated calomel electrode as a reference, and 0.1 cmThe surface of the coated Al alloy 6101 specimens was also analyzed by the SEM and EDS (Uanta FEG 250) characterization to confirm the complete mixing, dispersion, elemental, and phase identification of the pigments in the epoxy coatings. Moreover, the specimens’ optical images were also taken after the three-month aging period, so the final surface of the neat and ZP modified epoxy coatings were analyzed.The perception of the gloss can relate to a product (substate) finish, texture and how the sample is illuminated and viewed. Surface with high reflectance and perceived as a gloss, shiny or lustrous, while the less reflective surface is perceived as a semigloss or matt . NeverthRs(t) represents the specular resistance divided by the highly polished reflectance, black glass standard RSTD and multiplied by 100. The calibration of the gloss meter was done by the very highly polished black surface having a refractive index of 1.611 for the D sodium line assigning the value of 100 with all geometries. However, the initial surface brightness of the substrate was measured after sample preparation and the (%) change in gloss measurements before and after the aging test is known as gloss retention. Gloss retention was calculated by the following formula .Gloss r-1 absorption bands demonstrate vibration stretching of H-O adsorption peaks , corrosion potential (Ecorr), Tafel slopes , and annual corrosion rate (mm/a) by extrapolating cathodic and anodic polarization curves to Ecorr. In both cases, the decreasing trend of the corrosion potential of the coated surface of Al-6101 is shifting towards negative values (corr of ZP pigment-modified samples in the presence of an alkaline solution with pH 11. Similarly, the corrosion protection efficiency of ZP-modified samples is very high, 0.070 mm/a while 0.198 mm/a for neat epoxy, which is much lower after the summer aging experiments. The corrosion rate of ZP pigments modified epoxy coatings is about 70% lower than that of neat epoxy coating (Polarization curves obtained for Al-6101 in the alkaline media with pH 11 deviations are shown in e values . In addi coating .5 to 1.69 × 104, and 7.76 × 105 to 1.63 × 104 in summer months aging. The most common and typically useful technique to test the corrosion events occurs on the surface of the working electrode, where pH becomes highly basic at cathodic and acidic at anodic sites. EIS measurements were conducted to evaluate the corrosion protection performance of the neat epoxy coatings with the modified ZP pigments. This analysis is performed in an alkaline solution of NaOH with pH 11 for all specimens. Gloss measurements conclude that surface degradation and deprivation due to environmental factors progressively decrease. The shine or gloss of the coated samples after the coating and after the physical aging was very different; gloss was just like removed from the samples, and surfaces seemed to be more rough, uneven and dull. A comparison can be seen in 1. Peeling force of ZP pigments-modified epoxy coatings is 50% higher than that of the neat epoxy coatings though both peel-off adhesion strength and scratch test visibility decreased in both coatings2. Totally, the electric resistance of ZP pigments-modified epoxy coatings is about 30% higher than that of neat epoxy coatings, and the corrosion rate of ZP pigments-modified epoxy coatings is about 70% lower than that of neat epoxy coatings.3. Optical surface observation of the coatings showed that the ZP-modified epoxy coating could effectively restrict the crack and shrinkage in coatings after aging.4. Gloss measurements and surface analysis also proved that the neat epoxy coating lost 50% of its gloss, and the surface became rougher after aging compared to the ZP-modified epoxy coating.5. Maximum degradation and surface deterioration occur in the summer compared to other seasons; the main reason could be the Influence of the maximum UV radiation, temperature and high humidity in the atmosphere.Through the comparative study of neat epoxy and ZP pigment-modified epoxy coatings on Al alloy 6101 under natural weathering conditions after aging in Xi’an, China, for 12 months, the following conclusion is drawn.


Computational analysis predicts subtype specificity of 11-2 F among PRMTs. Structural analysis of ligands in the binding pockets is performed to compare poses of 11-2 F and its redesigned analogs and identifies three new analogs predicted to have significantly better potency. One of them, after synthesis, is ~4 fold more efficient in inhibiting PRMT5 catalysis than 11-2 F, with strong MTA-synergy. These data suggest the feasibility of employing near-atomic resolution cryo-EM structures and computational analysis of ligand poses for small molecule therapeutics.Projected potential of 2.5–4.0 Å cryo-EM structures for structure-based drug design is not well realized yet. Here we show that a 3.1 Å structure of PRMT5 is suitable for selecting computed poses of a chemical inhibitor and its analogs for enhanced potency. PRMT5, an oncogenic target for various cancer types, has many inhibitors manifesting little cooperativity with MTA, a co-factor analog accumulated in MTAP−/− cells. To achieve MTA-synergic inhibition, a pharmacophore from virtual screen leads to a specific inhibitor (11-2 F). Cryo-EM structures of 11-2 F / MTA-bound human PRMT5/MEP50 complex and its  Cryo-EM map-guided drug design and computational comparison of ligand poses yield a PRMT5 inhibitor or derivatives that are MTA-synergic and show enhanced potency. Many of these structures represent biomacromolecules complexed with small molecule agonists, antagonists, or modulators9. Although single particle cryo-EM often lags behind crystallography in resolution because a majority of cryo-EM structures are still of 3.5 Å resolution or worse, it is still expected to usher a new era in structure-based drug design (SBDD), which has been successful with high-resolution crystal structures and fragment-based design. Although atomic resolutions (~1.2 Å) for high-symmetry complexes were achieved recently11, most near-atomic resolution cryo-EM structures of low- or no-symmetry have been resolved at 2.5–4.5 Å. These developments promised a high potential of cryo-EM SBDD, which may be realized for many important biological or pharmacological targets; but such potential is still not well materialized, in part because most cryo-EM structures (>85%) are not resolved to 1.0–2.5 Å, where X-ray SBDD has been quite successful2. With advancements in computational analysis of ligand configurations (poses)13, virtual screening, docking and energy minimization of ligands could be utilized to enhance cryo-EM SBDD. We started to test this possibility in 2015, using human protein arginine methyltransferase 5 (PRMT5) as a target.The so-called “resolution revolution” in single particle cryo-EM has produced hundreds of structures of 2.5–4.5 Å resolutions in ten years16, because it regulates gene transcription17, cell signaling18, RNA splicing19, DNA repair20, chromatin remodeling21 and cell cycle22. It is overexpressed in various human cancers28, and its inhibition causes cell death30, especially in cancer cells lacking 5′-methylthioadenosine phosphorylase (MTAP). ~15% of all human cancers lack MTAP (MTAP-) and accumulate by 5–20 folds a metabolic derivative—5′-methylthioadenosine (MTA)—of S-adenosyl-l-methionine (SAM), the cofactor for PRMT5 catalytic activity. High MTA sensitizes MTAP−/− cells to partial PRMT5 inhibition that spares MTAP+ cells33. PRMT5 is therefore a compelling anti-cancer target35.PRMT5 is epigenetically important36 and non-histone proteins, such as p53 and NF-κB38. It consumes SAM to form an ω-NG-monomethyl arginine (MMA), releasing an S-adenosyl-l-homocysteine (SAH) as a byproduct. A second SAM molecule is used to generate ω-NG,N’G-symmetric dimethylarginine (SDMA) as the final product41. PRMT5 activity requires a crucial partner, called methylosome-associated protein 50 (MEP50)42, which dramatically augments PRMT5 catalysis by increasing substrate recognition and presentation44. X-ray structures of PRMT5 from C. elegans, X. laevis and H. sapiens45 revealed a conserved triosephosphate isomerase (TIM) barrel at the N-terminal half, a Rossmann-fold domain for cofactor binding and a β-sandwich domain between the two for substrate binding and dimerization. MEP50, a 7-WD40 β-propeller protein, binds PRMT5 via contacts with the TIM barrel, making a hetero-octameric PRMT5:MEP50 complex one of an apparent D2 symmetry39. The octamer methylates a broad spectrum of substrates, including COPR546, SWI/SNF47, pICln48, Riok149, Menin50, etc., and making targeted PRMT5 inhibition a potential cancer therapy.A type II PRMT, PRMT5, catalyzes symmetric arginine dimethylation of histone proteins 51, which are SAM analogs with ribose and adenine moieties strongly favored by the Rossmann-fold domain. The second class is substrate-site competitive inhibitors with high potency and selectivity52. The third class is allosteric modulators indirectly altering the canonical binding sites53. The fourth class includes inhibitors that suppress binding of substrate adapter proteins (SAPs) or decrease PRMT5:MEP50 stability55. Many inhibitors have been discovered and designed56. Two molecules, JNJ-64619178 of the first class and GSK-3326595 of the second class, entered clinic trials for multiple cancer types in 201851. They appear less potent in preclinical studies than recently-identified class 2 molecules, MRTX1719 and its analogs (like MRTX9768), from high-throughput fragment screen and X-ray SBDD58. Moreover, a nucleoside-based inhibitor targeting a conserved cysteine residue (C449) inside the SAM-binding site59 lacks specificity among SAM-binding enzymes. On the other hand, most class 2 inhibitors rely on SAM or its analogs for PRMT5 inhibition60, but are less potent in the presence of MTA61. For MTAP−/− cancer cells, class 2 inhibitors working in synergy with MTA are desired to suppress or kill them more specifically while sparing MTAP+ cells.Currently, all PRMT5 inhibitors fall into four classes based on their modes of action. The first class is cofactor-site competitive inhibitorsapo C. elegans PRMT5:MEP50 complex differs significantly from that of the liganded human complex45, whereas the apo human complex has been resistant to crystallization. The first structure of the human complex with a SAM analog A9145C and a histone H4 substrate peptide (PDB: 4GQB) was a milestone and triggered competition in X-ray SBDD58. However, all published crystal structures of human PRMT5 were obtained in complex with one or more ligands37 and reveal little on their low synergy with MTA.Available structural data are insufficient for understanding interactions between a Class 2 inhibitor and MTA. A crystal structure (PDB: 3UA4) of 58, we asked whether combination of near-atomic resolution cryo-EM structures with computational analysis of compound poses constitutes a different, but much less expensive, approach for selecting the most probable pose and using it to design higher-potency inhibitors. The first cryo-EM structure of PRMT5:MEP50 complexed with a cofactor analog (dehydrosinefungin) was solved at a 3.7 Å resolution62, which would be insufficient for our purpose. We reasoned that the high-quality phase information in cryo-EM structures of ~3.0 Å resolutions may provide sufficient constraints for large ligands and allow proper docking and selection among possible poses optimized by computational analysis9. The top candidate(s) will be accurate enough for structure-based redesign and reselection, ultimately leading to more potent inhibitors. In this paper, we tested this strategy on a PRMT5 inhibitor initially discovered by virtual screen63. We determined its stably-bound pose, unraveled the chemical basis for its synergy with MTA, designed and reselected derivatized compounds, and synthesized one of the designed compounds to confirm its higher potency in enzyme inhibition. Our results suggest that after more tests this non-crystallographic strategy probably has broader applications to cryo-EM-based molecular therapeutics.Search for MTA-synergic PMRT5 inhibitors that work more potently in MTAP−/− cancer cells asks for a different strategy. Although chemical screen against PRMT5/MTA complexes may provide a practical, yet costly, approach (as Mirati Therapeutics did)29. The resulted pharmacophore was modified manually based on the crystal structure of the catalytic pocket to increase its non-covalent interactions with the residues lining the binding pocket and was synthesized in the Li lab . Surface plasma resonance (SPR) studies detected that 11-2F binds to PRMT5:MEP50 with an apparent KD ~13.6 μM without MTA, but its affinity increased drastically to ~82 nM in the presence of 25 μM MTA   Fig. . How to 64. An X-ray structure (PDB: 6CKC) was fitted into the cryo-EM map by real-space refinement and manual adjustment  on ice for 30 min before being applied to glow-discharged QuantiFoil grids. Movies collected in a Titan Krios were processed as outlined in the supplementary information  MTA-binding induces a change in the binding pocket, probably due to induced fit and the ordering of the flexible loop. 2) The substrate-binding pocket in the apo state has a larger volume so that a substrate or an inhibitor (11-2 F) experiences significant freedom in testing different poses before becoming securely bound with the flexible loop making part of the pocket  to the ring. (3) The cyclo-alkylated indole ring of 11-2F is relatively flexible and may be stabilized by introducing H-bonds or electrostatic interactions with the binding pocket, especially with the residues on the flexible loop. We used these principles to guide the design of dozens of 11-2F derivatives and utilized computational analysis to predict their most stable poses before ranking them and selecting the most stable ones. Three 11-2F analogs predicted to have higher potency introduce more interactions with the binding pockets  more potently than 11-2 F does  It is important to retain the quinoline ring backbone to maintain the positive cooperativity with MTA. (2) The pi-stacking interactions of Trp579 and Phe327 with the quinoline ring may be enhanced to keep the inhibitor properly oriented, which could be achieved by introducing small groups  were ranked high, and their top poses presented in Fig. 71. Our results showed that a 3.1 Å cryo-EM map of the MTA/11-2F-bound PRMT5/MEP50 complex contains densities in the two ligand-binding pockets with clear shapes that enable the selection of top poses delivered by computational analysis through molecular docking and energy minimization. In doing so, a key step is probably the separation of the densities of two ligands from that of the protein so that the refinement of the protein model does not eclipse the optimization of the ligand models. At ~3.1 Å resolutions, two-fused rings (10-atoms) of MTA are sizeable enough for accurate modeling. The arrangement of the binding pocket residues and the accurate positioning of the adenine ring in MTA or the quinoline ring of 11-2F are important for computational analysis to gain sufficient sensitivity in differentiating the docking energy on a relative scale and allow the top cluster of compound poses to approximate true (most stable) solutions with good accuracy. For 11-2F, the density corresponding to the cyclo-alkylated indole rings is another determinant for its accurate positioning. Different software packages for molecular docking generate similar top poses, although their relative energetic levels vary, probably because of the same stereo chemistry parameters used for proteins and ligands. Expectedly, the results will probably be even better with cryo-EM maps of 2.0–3.0 Å resolutions, whereby individual atoms of certain multi-member rings in ligands and some of ordered water molecules at the binding pockets will become recognizable.We showed a strategy to redesign and improve potency of an inhibitor for an oncogenic target, PRMT5, by using a cryo-EM structure to select computed compound poses. Near-atomic resolution structures by single particle cryo-EM, which are often resolved to 2.5–4.5 Å, were proposed to be useful for SBDD by multiple investigators recentlyThe strategies we tested above appear to work well for the redesign of 11-2F based on accurate modeling of the protein-ligand interactions and the computational analysis to select three different ways out of virtual modifications to decrease binding energy. The energy minimization defines the energetically favored pose with the 11-2F quinoline ring being next to the tail of MTA. One of three hits from virtual modification, 11-9F, showed significantly better potency in enzyme inhibition assay and strong MTA-synergy, close to the predicted enhancement based on relative binding energy Table . Because51. Given that the MTA and 11-2F binding sites are next to each other, a chimera harboring the key groups of the two may be prepared for the same purpose.Understanding the catalytic mechanism of PRMT5 may also be helpful in development of potent inhibitors. So far, all well-characterized PRMT5 inhibitors in published studies require the nucleoside component of the cofactor SAM, indicating that they are either SAM analogs or SAM-dependent molecules. Our work depicted that the cofactor binding induces a conformational change of the loop region (residues 292–329) by forming a short helix α1 (residues 320–329), which stabilizes the nucleoside via Tyr324, followed by the formation of a “lid” (residues 314–319) and a substrate-binding tunnel (residues 295–313), where Phe327 sandwiches the aromatic ring of the substrate against Trp579. These provide a direct physical connection to achieve positive cooperativity between MTA in the cofactor-binding site and the inhibitor at the substrate-binding site Fig. . MTA hasOur work showcased the feasibility of the proposed strategy in using cryo-EM structures of ~3.0 Å resolutions and computational analysis of compound poses for SBDD. It led to the development of a different class of substrate-competitive inhibitors that preferentially bind the PRMT5:MEP50 complex with MTA and may be used to pharmacologically exploit the PRMT5-related vulnerability in MTAP−/− cancer cells. We expect that the same or similar strategy can be tested for other cryo-EM maps of near-atomic resolutions, where the energetic calculations without ordered water molecules in the binding pockets will allow selection of the top poses by extensively sampling ligand configurations to distinguish the most stable ones from the rest. With more successful applications, this strategy will likely expand our capacity in developing new molecular therapeutics.39. Briefly, full-length human PRMT5  and human MEP50  were co-expressed in Sf9 cells using a Bac-to-Bac expression system (Invitrogen). The cells were harvested and lysed. After removal of cell debris, supernatants were collected for FLAG-affinity chromatography  and fractionated by size exclusion chromatography in a 10/300 Superose 6 column (GE Life Sciences). The purified PRMT5:MEP50 complex was characterized by SDS-PAGE and sedimentation velocity analytical ultracentrifugation (SV-AUC) using a ProteomeLab™XL-I system  BSA and 0.01% (vol/vol) Tween-20) with final DMSO concentration at 5% (vol/vol). The enzymatic inhibition assay was performed in a solid white low-volume 384-well plate  with a total reaction volume of 16 µl and in the presence of 100 nM PRMT5:MEP50 enzymes, 10 µM SAM , 2 µM histone H4 peptide (1–21) as the substrate , and 11-2F of indicated concentrations. Reactions without enzyme were conducted as negative control and reactions without 11-2F were included as positive control in every experiment. Methyltransferase reaction was started by adding 4 µl of SAM/H4 substrate mixture to each well that contains 8 µl enzyme and 4 µl 11-2F pre-mixed and incubated for 10 min. The reaction was performed at room temperature for 60 min followed by the addition of 4 µl 5x MTase-Glo Reagent to produce SAH and concomitantly convert it to ADP. After shaking for 2 min and incubation at room temperature for 30 min, 20 µl room temperature MTase-Glo Detection Solution was added and mixed well before incubation for another 30 min and luminescence recording. Luminescence was measured in a Synergy Neo2 HTS multimode microplate reader (BioTek). Each data point represents the average of three replicates; the error bars represent the standard deviations. Data are analyzed in GraphPad Prism 8. For inhibitor studies, IC50 was determined by nonlinear regression (curve fitting) using the equation for the sigmoidal dose response (variable slope).Enzymatic inhibition activity of 11-2F (or other compounds) was determined by MTase-Glo™ methyltransferase assay . 11-2 F was serially diluted by 5-fold from 500 µM to 6.4 nM in DMSO then added into reaction buffer  at 25 °C. PRMT5:MEP50 proteins were directly immobilized onto a 500,000 Da carboxymethyl dextran sensor chip at pH 5.5 using a standard amine-coupling approach. The small molecule analyte (11-2 F as an example) was injected at a flow rate of 30 μL/min of different concentrations in the running buffer . The association and dissociation times were set to be 1 min. In the competitive binding experiment, 25 μM MTA was added into the running buffer to saturate the cofactor-binding pocket of immobilized PRMT5: MEP50 protein before the injection of the analyte. The dissociation time for 11-2 F in MTA-containing running buffer was elongated to be 3 min to achieve complete dissociation. Sensorogram data were processed using the TraceDrawer software package to calculate the equilibrium dissociation constant apo form except that the protein was incubated with the inhibitor for 30 min at a 1: 5 molar ratio of protein: inhibitor with a final protein concentration of 0.05 mg/ml right before grid preparation.The frozen protein sample was thawed and diluted by 1:30 (vol/vol) with a buffer containing 10 mM HEPES at pH 7.5, 150 mM NaCl and 1.0% (vol/vol) DMSO to a final concentration of 0.4 mg/ml. Aliquots of 4.0 μL of the diluted proteins were applied to plasma-cleaned , 300 mesh holey carbon-coated gold grids . After sample loading, the grids were incubated for 30 s in a stable environment of 10 °C and 100% humidity inside a plunge-freezing device  before being sandwich-blotted for 4.0 s with a blot force of 8.0. Grids were plunge-frozen in liquid nitrogen-bathed liquid ethane and were transferred into grid-boxes and stored in liquid nitrogen before cryo-EM imaging. Grids were first screened and checked in a CM120 microscope before good grids were selected for data collection in a Titan Krios. CryoEM grids for the compound-bound PRMT5:MEP50 (PRMT5: MEP50 / 11-2 F/MTA) were prepared in a similar way as that for the −/Å2 per movie.A total of 1535 micrograph movies were recorded inside a Titan Krios operated at 300 kV utilizing a Gatan K2 Direct Electron Detector (Gatan Inc.) in the super-resolution electron-counting mode at a nominal magnification of 105,000 ×. The calibrated pixel size under such conditions was 0.66 Å, and the defocus range was set in −0.75 to −3.0 μm. Each movie was collected in 12 s with an exposure time of 0.3 s per frame. The total electron dose was approximately 40 e−/Å2 per movie.A total of 4153 micrograph movies were recorded inside a Titan Krios operated at 300 kV utilizing a Gatan K3 Direct Electron Detector (Gatan Inc.) in the super-resolution electron-counting mode. The calibrated pixel size under such conditions was 0.56 Å, and the defocus range was set in −0.75 to −3.0 μm. Each movie was collected in 12 s with an exposure time of 0.3 s per frame. The total electron dose was ~40 e72, resulting in a pixel size of 1.32 Å. Parameters for the contrast transfer function (CTF) of each movie were estimated by CTFFind473. Relion3.074 was utilized for particle picking, extraction, 2D/3D classification, and refinement. LoG-based particle picking mode was utilized to select an initial set of particles, which were classified and averaged as templates for subsequent template-based auto-picking. The auto-picked 597,002 particles were subjected to three rounds of 2D classification to remove junky or damaged particles, yielding a sub-set of 151,554 particles for 3D classification. An initial de novo 3D model was generated in Relion3.0. Two rounds of 3D classification were performed to identify distinct conformational states or sub-stoichiometric assemblies of the PRMT5:MEP50 complexes. Unbinned particles from good 3D classes exhibiting 1:1 stoichiometry and high-resolution structural features were re-extracted. A cleaned set of 113,466 particles were used to calculate a 3.9 Å reconstruction based on the gold-standard FSC (Fourier shell correlation)75 with the 0.143 criterion with D2 symmetry imposed. The particle stack was then subjected to 2 iterations of CTF refinement and beam tilt correction, resulting in a 3.8 Å resolution structure. The resolution was improved to 3.6 Å by applying a soft mask around the entire protein complex during 3D auto-refinement. Bayesian polishing and another iteration of CTF refinement further improved the resolution to 3.4 Å. The refined particles were extracted and further refined in the cisTEM76 which slightly improved the resolution to 3.17 Å. The final map was sharpened within a soft mask with an automatically calculated B-factor of −79.7 Å2. The local resolution variation of the apo form was assessed using ResMap78 and colored in Chimera79.The general workflow and some intermediate results are showed in Supplementary Fig. 72, resulting in a pixel size of 1.11 Å. CTF parameters for each movie were estimated by CTFFind473 and 1918 micrographs with good CTF fitting were selected. CisTEM74 was utilized for particle picking, extraction, 2D/3D classification, and refinement. The auto-picked 866,240 particles were subjected to two rounds of 2D classification to remove junky or bad particles, yielding a sub-set of 461,119 particles. Three rounds of 3D classification were performed to identify distinct conformational states or remove sub-stoichiometric assemblies of the PRMT5: MEP50 complexes using the apo map as a starting reference. A cleaned dataset of 213,221 particles were auto-refined in cisTEM, yielding a structure of a nominal 3.14 Å resolution based on the gold-standard FSC75 using a threshold of 0.143 with D2 symmetry imposed. The final map was sharpened within a soft mask with an automatically calculated B-factor of −90.0 Å2. The local resolution variation of the final map was assessed using ResMap78.The general workflow and some intermediate results are showed in Supplementary Fig. 80 with secondary structure and geometry restraints and manually adjusted in COOT81. A molecular dynamics (MD)-based optimization was performed using ISOLDE82 with the technical assistance by Dr. Tristan Croll (CCPEM)83. Overfitting and overinterpretation of the model were monitored by refining the model against one of the two independent half-maps and testing the refined model against the other map. The final structure was assessed in MolProbity and optimized to minimize clashes84. Parameters for cryo-EM data collection and modeling statistics are summarized in Table apo and inhibitor-bound PRMT5 complexes.The crystal structure of an inhibitor-bound (LLY-283) form of PRMT5:MEP50 dimer (PDB: 6CKC) was used as the initial model and docked into the density map using Chimera. The model was subjected to real-space refinement using PHENIX69. The X-ray crystal structure of human PRMT5 in complex with a substrate and a co-factor , structures of different PRMTs  and the 3D structures of MTA, SAM and SAH were retrieved from the RCSB Protein Data Bank (www.rcsb.org)85. All input files were prepared using the AutoDockTools (ADT) 1.5.4 package. To carry out the docking simulations, a 50 Å ×  50 Å × 50 Å grid box with a lattice spacing of 0.375 Å was defined with its center at the 11-2 F position that was modeled and refined in cryo-EM map. The grid box enclosed fully the catalytic center of PRMT5. The AutoGrid program was used to construct the grid maps for energy minimization and scoring. The three-dimensional locations and orientations of the various inhibitor configurations were analyzed from 25 million configurations with randomly sampled seed parameters using a Lamarckian genetic algorithm (LGA)87. After energy minimization, the best poses from 2000 individual runs were generated, which represent 2000 typical configurations of the inhibitor and were grouped into clusters by a threshold in root mean square deviations (RMSD) of 2.0 Å. These clusters were ranked by relative binding energy. The poses of the lowest energy  or the cluster of the largest sizes  were chosen for further analyses in the PyMOL molecular graphics system (https://pymol.org/2/). Usually at least 3–5 poses were examined. More details on the three separate steps are provided in the following.Molecular docking was performed mainly by using AutoDock 4.2.687. No water molecules were included. Top poses from 2000 runs were clustered into discrete groups using a Root Mean Square Distance (RMSD) threshold. Usually for each ligand we obtained 8–10 clusters and used the pose with the lowest energy from each cluster as its representative one. The top pose has the lowest docking energy (ΔΔG) among a large number (billions or more) of different configurations.The initial model of a ligand was generated in Chimera and went through energy minimization. The model is then docked to overlap with the ligand density in the cryo-EM map, which served as a starting point. The refined protein model based on the cryo-EM map (without the ligands) was used as a rigid model for docking of different inhibitors in AutoDock 489. The top poses for the same ligand from three different software packages against the same protein structural model were very similar, if not the same, suggesting that the large number of starting positions sampled by this protocol were able to reflect almost all possible configurations of the ligand within a small range of errors.Similarly, the same scheme was implemented in AutoDock Vina and an online server, SwissDock67. The refinement may slightly improve the model for better match with the density. For MTA, the refinement introduced small changes of the ligand model. For 11-2 F, the refinement led to a 180° flipping of the quinoline ring around a C-C bond connected to its 6th position.The refinement of the top pose of a ligand was performed by simplified all-atomistic molecular dynamics (MD) calculations implemented in ISOLDE6 cells/dish and allowed to adhere overnight. The cells were then treated with indicated doses of the test compounds (11-9F) in complete growth medium for 96 h. The cells were harvested and lysed in cold RIPA lysis buffer (Pierce #89900) containing protease inhibitor cocktail and phosphatase inhibitor cocktail (Pierce #78441). The protein concentrations were determined using the BCA Protein Assay kit (Pierce #23227). After being boiled in loading buffer at 95 °C for 10 min, equivalent amounts of proteins were resolved in a 10% SDS-PAGE gel, and then were transferred to a PVDF membrane (Bio-Rad #1620174) at 110 V for 1 h. The membrane was treated in a blocking buffer (Pierce #37530) and incubated with corresponding primary antibodies: anti-SDMA , rabbit anti-ADME-R , and anti-β-actin  in the blocking buffer at 4 °C overnight. After three washes with TBST buffer (Pierce #28352), the blots were incubated with goat anti-rabbit secondary antibody  in the blocking buffer for 1 h at RT. After three washes with the TBST buffer, the membrane was analyzed using ECL Western blotting detection reagents (Pierce #34096) and imaged in a ChemiDoc MP imaging system (Bio-Rad). Digitization of the blot bands was done in ImageJ -7′-methyl]--3-carboxamide, and 11-9 F: N-[-7′-methyl]--3-carboxamide.The two compounds were synthesized, purified and verified in Dr. Li’s lab. After HPLC purification the compound purity was more than 95%. The molecular mass and structural details were verified by mass spectrometry and NMR analysist test to compare experimental data from separate experiments done in parallel or in repeated fashions. Data from three or more repeated experiments were included to enhance reproducibility. For structural modeling and comparison, the statistical analysis was performed in different software packages.We used two-tailed Student’s Further information on research design is available in the Peer Review FileSupplementary InformationReporting SummaryAuthor List Change