Artemisia annua L., has been used in malaria treatment for a long time. Recently, many studies have indicated that DHA also exhibits potent anti-rheumatoid arthritis (RA) activity. In this study, collagen-induced arthritis (CIA) in DBA/1J mice and inflammatory model in THP-1 cells were established to evaluate the modulatory effects of DHA on joint destruction and to explore the underlying mechanisms. Our results showed that DHA decreased the serum levels of IL-1β and IL-6, alleviated paw oedema, and reduced bone destruction in DBA/1J mice with CIA. Further exploration with the inflammatory model in THP-1 cells indicated that DHA reduced the protein expression of hypoxia‐inducible factor (HIF)‐1α and the phosphorylation in Janus kinase (JAK) 3 and signal transducer and activator of transcription (STAT) 3 protein, which resulted in a decrease in NOD-like receptor protein (NLRP) 3 expression and interleukin (IL)-1β release. Consequentially, the inflammatory activation in THP-1 cells was inhibited. Therefore, we concluded that DHA efficiently alleviated the inflammation and arthritic symptoms in CIA mice and downregulated inflammation in part by inhibiting NLRP3 expression via the HIF‐1α and JAK3/STAT3 signaling pathway. Thus, DHA may be considered as a potential therapeutic agent in RA treatment.Dihydroarteannuin (DHA), the primary element of artemisinin extracted from the traditional Chinese herb  Rheumatoid arthritis (RA) is an autoimmune inflammatory disease characterized by chronic synovitis and progressive cartilage and bone destruction and ultimately leads to joint damage or even irreversible disability. Relief of synovial inflammation has long been the key to RA treatment, as persistent synovial inflammation is the dominant pathological manifestation of RA . Disease. In 2006, data from Tu Youyou’s group showed that DHA inhibited the secretion of TNF-α by blocking the nuclear factor (NF)-κB signaling pathway, which may be beneficial for the treatment of systemic lupus erythematosus related nephritis (via the Nrf2-p62-Keap1 feedback loop (Dihydroarteannuin (DHA) is an active metabolite of artemisinin and its derivatives, which has been demonstrated to be an effective antimalarial drug with low toxicity . Numerouephritis . Besidesack loop  and inhiack loop . Anotherack loop . STAT3 iack loop . TherefoPrevious studies showed that hypoxia‐inducible factor (HIF)‐1α, the most direct regulatory factor of the adaptive response to alterations in oxygen tension , took pavia the HIF‐1α and JAK3/STAT3 signaling pathway. In the present study, the role of DHA in modulating the inflammation and consequently protecting against bone erosion in CIA mice was examined.On the basis of the above information, we assumed that DHA reduced inflammatory response in RA by inhibiting NLRP3 expression DHA (Cat. No. S2290) was purchased from Selleck Chemicals. Lipopolysaccharide  and phorbol myristate acetate  were purchased from Sigma. Complete Freund’s adjuvant , incomplete Freund’s adjuvant , and bovine type II collagen (Cat. No. 20021) were purchased from Chondrex. Roswell Park Memorial Institute (RPMI) 1640 medium and fetal bovine serum (FBS) were obtained from Gibco. Cell Counting Kit 8  was purchased from Med Chem Express. RIPA buffer was purchased from Solarbio Life Sciences (Cat. No. R0020). BCA detection kit was purchased from Thermo Fisher Scientific (Cat. No. 23225). The primary antibodies against STAT3 (Cat. No. 9139), Phospho-STAT3 (Cat. No. 9145), JAK3 (Cat. No. 8863), Phospho-JAK3 (Cat. No. 5031), and GAPDH (Cat. No. 2858) were purchased from Cell Signaling Technology. The primary antibodies against NLRP3 (Cat. No. PA5-79740) were purchased from Invitrogen. The primary antibodies against HIF‐1α (Cat. No. sc-13515) were purchased from Santa Cruze. Goat Anti-Rabbit IgG (H + L) HRP (Cat. No. 98164) and Horse Anti-Mouse IgG (H + L) HRP (Cat. No. 91196) were purchased from Cell Signaling Technology.Eight-week-old male DBA/1J mice  were used for this experiment. All mice were raised in standard cages under a controlled light/dark cycle of 12 h at 22 ± 2°C and a humidity of 45 ± 5%. All the experimental studies were strictly in accordance with Guangzhou University of Chinese Medicine Animal Ethics Committee guidelines for the rational use of animals.After a week of acclimatization, all mice were randomly divided into two groups: control group (n = 6) and arthritis-induced group (n = 18). Bovine type II collagen was fully emulsified with an equal volume of CFA. 0.1 ml of the emulsion was injected intradermally at approximately 1.5 cm distal to the base of the tail. A booster injection of 0.1 ml emulsion of Bovine type II collagen and IFA was administered intradermally into the back on day 21. Mice in the control group were injected with normal saline at the same location and frequency as the arthritis-induced group.From day 21 after the second immunization, mice in the arthritis-induced group were randomly divided into three groups (n = 6 per group): model group, DHA group, and MTX group. 20 mg/kg of DHA was orally given for mice in the DHA group every day. 2 mg/kg of MTX was orally given for mice in the MTX group every 3 days as the positive control. DHA and MTX were dissolved in corn oil. Mice in the control and model groups were orally given an equal volume of corn oil in parallel.From day 21 after the second immunization, clinical arthritis scores were determined every week using a scoring system of 0–4 for each limb: 0, normal; 1, definite redness and swelling of the ankle or one digit; 2, two joints involved; 3, more than two joints involved; and 4, severe arthritis of the entire paw and all digits. Meanwhile, swelling of hind paws was measured using digital calipers, and body weight was recorded during the experiment.At the end of the experiment, the right hind limb of each mouse was collected and scanned using the Skyscan 1,176 Micro-CT Imaging System . The scanning was carried out using the following settings: voltage, 80 kV; source current, 88μA; pixel size 4 μm. Two- and three-dimensional images were generated using Data-viewer and CTvol softwares , respectively. The bone mineral density (BMD) and bone volume/tissue volume (BV/TV) were evaluated using CT Analyzer program .5 cells/ml in a humidified chamber with 5% CO2 at 37°C. In all experiments, THP-1 cells were cultured in 6-well plates treated with 100 ng/ml PMA for 24 h to transform them into adherent macrophages. Then, THP-1–derived macrophages were pretreated with different concentrations of DHA for 2 h, followed by stimulation of 100 ng/ml LPS for 24 h to establish the in vitro inflammation model.THP-1 cells, a pro-monocytic cell line, were obtained from American Type Culture Collection (ATCC) and cultivated in RPMI-1640 media supplemented with 10% FBS. THP-1 cells were cultured at a density of 5×102 at 37°C for 1–4 h. The absorbance at 490 nm was measured using a microplate reader for the detection of cell viability.THP-1–derived macrophages were incubated with different concentrations of DHA for 24 h. Then, CCK-8 solution was added, and the cells were incubated in a humidified chamber with 5% COAt the end of the experiment, blood samples of mice were collected. Serums were harvested by spinning of whole blood at 3,000 rpm for 15 min at 4°C. Serum levels of IL-1β  and IL-6  were determined using ELISA kits. After DHA and LPS treatment for 24 h, the supernatant was collected from cell cultured medium and its IL-1β level was measured using ELISA kit .After being treated with DHA and LPS, cells were harvested and lysed by RIPA buffer for the total protein collection. After the protein concentration was evaluated using a BCA detection kit, the protein was mixed with loading buffer, and denaturation was carried out by heating under 97°C for 7 min. The target protein was separated by SDS-PAGE, transferred to a PVDF membrane, sealed with 5% skimmed milk at room temperature for 2 h, and incubated with one of the following primary antibodies at 4°C overnight: rabbit anti-STAT3, rabbit anti-Phospho-STAT3, rabbit anti-JAK3, rabbit anti-Phospho-JAK3, rabbit anti-NLRP3, mouse anti-HIF-1α, and rabbit anti-GAPDH. Then, the membranes were incubated with the corresponding secondary antibodies (goat anti-rabbit IgG or horse anti-mouse IgG) for 2 h following visualization through the Enhanced Chemiluminescence (ECL) method. Last, the images were recorded in a gel documentation system .p < 0.05 indicates statistical significance.Statistical data were shown as means ± SD. SPSS software (Version 18.0) was used to analyze data, while GraphPad Prism software (version 7.0) was used to draw graphs. One-way ANOVA with Newman-Keuls multiple comparisons test was applied for normal distribution and homogeneous variance. Dunnett’ T3 test was used for non-normal distribution. Each mouse was weighed weekly to assess whether the treatment of DHA (20 mg/kg·d) or MTX (2 mg/kg·3 d) influenced weight gain in CIA mice. As illustrated in p < 0.05). Mice in the DHA group showed significant reduction in paw oedema between day 28 and day 49 compared with mice in the model group (p < 0.05). Mice in the MTX group also showed significant reduction in paw oedema between day 35 and day 49 compared with mice in the model group (p < 0.05). Additionally, a significant reduction in arthritis index scores also occurred in CIA mice with DHA or MTX treatment from day 35 and day 49, when compared to the model group (p < 0.05). There was no significant difference between the DHA and MTX groups when it came to paw oedema or arthritis index scores.After the secondary immunization (day 21), paw oedema appeared dramatically in mice with CIA . Hind pavs. 0.48 ± 0.06 mm2, p < 0.001; 24.93 ± 1.05 vs. 10.95 ± 2.65%, p < 0.001; ankle: 0.69 ± 0.07 vs. 0.55 ± 0.06 mm2,, p < 0.05; 33.27 ± 4.73 vs. 18.06 ± 3.47%, p < 0.001). DHA or MTX treatment ameliorated the bone loss in CIA mice, as the BMD  and BV/TV  in these two groups were significantly higher than those in the model group. No meaningful difference between the DHA and MTX groups was observed in BMD or BV/TV.For measuring of bone mineral density (BMD) and bone volume/tissue volume , the articular structure of hind knees and ankles were scanned by micro-CT . As show 0.04 mm , 0.65 ± vs. 88.64 ± 29.14 pg/ml, p < 0.05; IL-6: 3.29 ± 0.66 vs. 1.32 ± 0.11 pg/ml, p < 0.05). Both the DHA and MTX groups had a much lower level of IL-1β and IL-6 than the model group . No significant difference between the DHA and MTX groups was observed in circulating levels of IL-1β or IL-6.Serum was harvested at the end of the experiment and the circulating levels of IL-1β and IL-6 were detected by ELISA. As shown in The cytotoxicity of DHA at concentrations of 0.1–2.5 μM in THP-1–derived macrophages were detected by CCK-8 assay . The resp < 0.001), whereas DHA was able to partially attenuate the secretion of IL-1β at the doses of 0.2 and 0.4 μM. The maturation of IL-1β is mainly mediated by NLRP3 inflammasome. Subsequent Western blotting with anti-NLRP3 demonstrated that LPS increased NLRP3 levels in these cells. However, DHA at the doses of 0.2 and 0.4 μM decreased the NLRP3 levels in macrophages under LPS triggering  on LPS-mediated IL-1β release were evaluated by ELISA . After Liggering .The protein levels of HIF‐1α, JAK3, p-JAK3, STAT3, and p-STAT3 were detected by Western blotting . The expArtemisia annua L.) has been used as a traditional Chinese medicine for more than 2,000 years. Artemisinin, an extract of Qinhao, was discovered by Tu Youyou and has been known around the world for its potent treatment of malaria  facilitated histone H3 and H4 acetylation on Nlrp3 promoter, which promoted the expression of NLRP3 . STAT3 ivia its receptor G protein-coupled receptor-91 (via the HIF‐1α and JAK3/STAT3 signaling pathway.HIF-1α is a direct regulatory factor of the adaptive response to alterations in oxygen tension . With reeptor-91 . In thiset al. (However, interaction between STAT3 and HIF-1α is still obscure. Some studies have indicated that HIF-1α is a downstream regulatory signal of STAT3. As showed by Wang et al. , the STAIn this study, DHA alleviated the inflammation and arthritic symptoms in CIA mice. Consistently, DHA impeded inflammatory response in THP-1 cells derived macrophages by decreasing the NLRP3 expression and IL-1β release. Additional research found that the HIF‐1α and JAK3/STAT3 signaling pathway involved in NLRP3 expression was also inhibited by DHA. Our data provided evidence supporting the potential of DHA in RA treatment and shed some light on the underlying mechanisms: DHA inhibited the NLRP3 expression through the HIF‐1α and JAK3/STAT3 signaling pathway in macrophages, and as a consequence, the following inflammatory response was prevented.


Human mesenchymal stem cell (hMSC) and extracellular vesicle (EV) therapy is a promising treatment for discogenic low back pain (LBP). Although promising, major obstacles remain to be overcome. Cellular senescence reduces self-renewal and multipotent potentials, and the senescence-associated secretory phenotype creates an inflammatory environment negatively affecting tissue homeostasis. Reducing senescence could therefore improve regenerative approaches. Ortho-Vanillin (o-Vanillin) has senolytic activity and anti-inflammatory properties and could be a valuable supplement to MSC and EV therapy. Here, we used direct co-culture experiments to evaluate proteoglycan synthesis, inflammatory mediators, and senescent cells in the presence or absence of o-Vanillin. EV release and transfer between hMSCs and intervertebral disc cells (DCs) was examined, and the effect on hMSC differentiation and DC phenotype was evaluated in the presence and absence of o-Vanillin. This study demonstrates that o-Vanillin affects cell communication, enhances hMSC differentiation and improves DC phenotype. Co-cultures of DCs and hMSCs resulted in increased proteoglycan synthesis, a decreased number of senescent cells and decreased release of the cytokines IL6 and 8. Effects that were further enhanced by o-Vanillin. o-Vanillin profoundly increased EV release and/or uptake by hMSCs and DCs. DC markers were significantly upregulated in both cell types in response to conditioned media of o-Vanillin treated donor cells. Collectively, this study demonstrates that o-Vanillin affects hMSC and DC crosstalk and suggests that combining hMSCs and senolytic compounds may improve the outcome of cell supplementation and EV therapy for LBP. Low back pain (LBP) is the leading cause of disability worldwide with a lifetime prevalence exceeding 84% ,2. LBP-rIntervertebral disc (IVD) degeneration is a major etiology of LBP . DegenerAlthough current hMSC therapy shows promising results in some preclinical and clinical studies , severalv/v) fetal bovine serum (FBS) , 100 µg/mL PrimocinTM, 2 mM GlutaMAXTM , and incubated at 37 °C with 5% CO2.Degenerate disc tissue from consenting patients undergoing spinal surgery for LBP was collected according to the procedures approved by McGill University ethical review board (IRB#s A04-M53-08B and Tissue Biobank 2019–4896). Patient demographics are presented in v/v) FBS, 2 mM GlutaMAXTM, and 0.25 mg/mL Gentamicin ) at 37 °C and 5% CO2. hMSCs were cultured in the same DMEM supplemented with recombinant human basic fibroblast growth factor 2  at 5 ng/mL during the expansion phase [Monolayer culture: Human-bone-marrow-derived MSCs were purchased from RoosterBio, Inc.,  (Cat. #MSC-001). One million cells (hMSCs or DCs) were seeded in T75 cell culture flasks  and cultured in DMEM media  supplemented with 10%  at 37 °C and 5% CO2. Pellets were generated and left to mature for 2 days, following which they were treated for 4 days with 100 μM o-Vanillin  or vehicle 0.01% (v/v) DMSO  and then cultured for an additional 21 days with a media change in every 4 days. Media from the 21-day period were collected and stored at −20 °C until analyzed. The o-Vanillin concentration resulting in the highest senolytic effect has been determined in our previous work [Pellet culture: Three hundred thousand cells were pelleted with different cell proportions at the following combination ratios: 100% hMSCs, 100% DCs and DCs:hMSCs 1:1, 2:1, and 3:1. Pellets were cultured in 1 mL DMEM media  , fixed with 4% paraformaldehyde  and prepared for cryopreservation according to our previous work . Pelletsw/v) Safranin-O  for 5 min at room temperature and rinsed with running tap water for 10 min. Dehydration was performed with 75% and 95% ethanol and samples were mounted with PermountTM Mounting Medium  and imaged using a Leica DMRB microscope  with a pre-fixed Olympus DP70 digital camera  under visible light.Safranin-O staining: Pellet sections were exposed to 50 °C for 30 min followed by a PBS rehydration at room temperature. Samples were then immersed in 0.1%  Triton X-100, Sigma-Aldrich, Oakville, ON, Canada). Pellet sections were blocked with hydrogen peroxide, rinsed with 1X CINTec Wash Buffer , then blocked with PBS-T-Tween 20 (1% (v/v)) with 1% (w/v) bovine serum albumin  and 1% (v/v) goat serum. Samples were exposed to p16INK4a antibody solution (50 mmol/L)  at 4 °C overnight. Detection of p16INK4a immunopositivity was performed using HRP/DAB Detection IHC Kit . Meyer’s hematoxylin  was used as a nuclear counterstain. Samples were dehydrated with 75% and 95% ethanol and then mounted, imaged, and analyzed as described for Safranin-O staining.p16Sulfated glycosaminoglycans (GAGs) released in the CM of hMSC and DC pellets was quantified using DMMB assays as previously described . CM of hProtein concentrations of inflammatory cytokines IL-6 and IL-8 present in the collected culture media were measured using ELISA kits  according to the manufacturer’s instructions. Post-treatment CM of hMSC or DC pellets were pooled. Absorbance was measured using a TECAN Infinite M200 PRO plate reader with i-control 1.9 Magellan software . Protein levels of untreated and o-Vanillin-treated samples were compared.TM reagent  and extracellular vesicle (EV) uptake was examined under a LSM780 confocal microscope and ZEN software . The florescent intensity was measured using Fiji Image J software [hMSCs and DCs were released with trypsin, collected, resuspended and the plasma membrane was labeled with a Vybrant™ DiI cell-labeling solution  according to the manufacturer’s instructions. One million labeled or unlabeled cells were seeded on 24 mm × 50 mm sterile cover glass  which were placed in a 10 cm-cell culture dish . Cells were cultured in DMEM media with or without o-Vanillin and successful labeling was confirmed . Unlabelsoftware . One milv/v) DMSO) or 100 µM o-Vanillin for 4 days. The cell culture media were changed to fresh DMEM without o-Vanillin on days 5 and 8. CM of days 5 and 8 were collected, and debris was removed by centrifugation. CM samples and standard cell culture media (set as control) were diluted with filtered PBS at 1:80 and prepared for nano-flow cytometry. Sample acquisition was performed using a CytoFLEX flow cytometer (Model No. A00-1-1102) and a CytExpert software  according to the manufacturer’s guidelines. Briefly, the acquisition was set to 2 min with a 10 µL/min flow rate, < 2% abort rate, and < 10,000 event/second speed to ensure a good resolution. ApogeeMix calibration beads  were used as size references. The total event window was set to 110–1300 nm. The area of interest was gated using cell culture media that had not been in contact with cells. The data were analyzed using the CytExpert software and particles from the DMEM media were subtracted.One million of DiI-labeled hMSCs and DCs were exposed to vehicle (0.01% (v/v) DMSO or 100 µM o-Vanillin for 4 days, were then cultured in DMEM without o-Vanillin for 21 days, with media change every 4 days. Media from the provider group was transferred to the recipient group: from hMSC to DC pellets and from DC to hMSC pellets  and RNA was extracted according to the manufacturer’s instructions as previously described [® Veriti® 96-Well Fast Thermal Cycler . qPCR was performed in PROGENE® 96-Well Half-Skirt ABI Fast PCR Plates  with PROGENE® Adhesive Routine PCR Sealing Film  using an Applied Biosystems® StepOnePlus™ Real-Time PCR System  with PowerUp™ SYBR™ Green Master Mix . Primers of hMSC and NP markers and a housekeeping gene are presented in beta-ACTIN) and presented in fold-changes calculated with the 2−ΔΔCt method [Pellet samples were lysed in TRIzolescribed ,27. Extrt method .t-test (paired or unpaired) and one-way ANOVA were used for data analysis. All assessments were conducted with three or more independent experiments, indicated by “n” in the figure legends. Each independent experiment was conducted with cells from one donor and three or more technical replicates from the same donor. The statistically significant difference was set at p-value < 0.05. Data are presented as mean ± SEM. The graphical abstract and schematic illustrations .Data were analyzed using GraphPad Prism version 9.3.1(350) . A D’Agostino-Pearson omnibus K2 normality test was conducted for data of all experiments. All the data are not inconsistent with a Gaussian distribution. Two-tailed Student’s trations  were creTo evaluate the optimal ratio of DCs:hMSCs co-cultures, GAG production was measured in the presence or absence of o-Vanillin. We evaluated three ratios, 1:1, 2:1 and 3:1 of DCs:hMSCs. An optimal concentration of o-Vanillin for DCs has previously been determined; here we verified that this concentration has no cytotoxicity on hMSCs . Safrani5 DCs. The GAG concentration of untreated 1:1 co-cultures (4.52 ± 0.83 ng/µL) was 55.17% higher than that of untreated 100% DCs (2.88 ± 0.50 ng/µL) and significantly higher than any other untreated groups (p = 0.0070)  (p = 0.0006) than that of untreated 1:1 co-cultures and significantly greater than any other o-Vanillin-treated groups (p = 0.0001)  (p = 0.0386), 100% DC (p = 0.0202), and 2:1 (p = 0.0426) cultures. There was no significant difference in total DNA between the groups following 21 days of pellet culture  B. The ac0 ng/µL) B. In add culture .Taken together, these data indicate that the co-culture combination of 1:1 (DCs:hMSCs) resulted in the most robust GAG production, which was significantly improved by o-Vanillin treatment. Therefore, the subsequent experiments were performed with the co-culture ratio 1:1.INK4a staining (INK4a positive senescent cells (p = 0.0121) reduction in senescent cells in untreated co-cultures (7.28% ± 1.43) compared to the mean of each cell type alone. When treated with o-Vanillin, the number of senescent cells further decreased to 3.74% ± 0.96 (p = 0.0064) in 100% hMSCs and to 6.79% ± 0.46 (p = 0.0121) in 100% DCs and to 6.54% ± 1.30 (p = 0.0126) in co-cultures. Together the results demonstrate that co-culture of hMSCs and DCs (1:1) result in a lower total number of senescent cells and that o-Vanillin treatment reduced both senescent hMSCs and DCs.Senescent cells accumulate in degenerating disc tissues and aging stem cells. The number of senescent cells in pellet cultures was evaluated using p16staining A. The unnt cells B. There p = 0.0060), 71.78% (p < 0.0001), and 81.91% (p < 0.0001), respectively, in o-Vanillin-treated 100% hMSCs (from 5422 ± 99 to 631 ± 101 pg/mL), 100% DCs (from 978 ± 78 to 276 ± 88 pg/mL), and 1:1 co-cultures (from 1421 ± 22 to 257 ± 61 pg/mL) (p = 0.0001), 80.54% (p < 0.0001), and 85.55% (p < 0.0001), respectively, in o-Vanillin-treated 100% hMSC (from 4247 ± 401 to 1436 ± 170 pg/mL), 100% DC (from 3526 ± 112 to 686 ± 145 pg/mL), and 1:1 co-cultures  (The release of inflammatory cytokines IL-6 and IL-8 was measured in the CM by ELISA. IL-6 concentrations decreased by 88.36% (1 pg/mL) C. While 0 pg/mL) D.To evaluate how cell phenotype is affected by co-culture, hMSCs were labeled with a red-lipophilic-fluorescent-membrane-dye DiI. Labeled hMSCs were cultured with unlabeled DCs (1:1 DCs:hMSCs). The goal was to separate red from unlabeled cells after co-culture. However, all cells were red after a 21-day culture period (p = 0.0447) and the 501–1000 nm  groups (p = 0.4803) and the 1001–1500 nm  groups (p = 0.0170) than those generated by DCs   groups D. No sig) groups D. These ) groups D. The qu,609/µL) E.p = 0.0126) (p = 0.0236) (p = 0.0016) (p = 0.0242)  B in DCs  0.0236) B when ex 0.0236)  in cells 0.0016) D in hMSC 0.0242) D when ex 0.0242) B was hig 0.0242) D.p = 0.0250) and from DCs by 32.96%   E.CD73 showed no change , whereas expression of CD105  significantly increased in hMSCs exposed to DC CM  compared to the control pellets, but was significantly increased by 0.64 ± 0.23-fold (p = 0.0146) compared to hMSCs exposed to DC CM. The expression of CD105  significantly increased compared to the control pellets, while it was not significantly changed  compared to hMSCs exposed to DC CM  , SOX9, and ACAN increased significantly by 1.23 ± 0.39-fold (p = 0.0103), 30.26 ± 12.24-fold (p = 0.0005), 1.76 ± 0.59-fold (p = 0.0127), 2.54 ± 1.27-fold (p = 0.0327), and 170.8 ± 65.3-fold (p < 0.0001), respectively, in hMSCs exposed to DC CM, while the change of HIF-1α  expression was not significantly affected , 238.34 ± 111.39-fold (p = 0.0011), 3.99 ± 0.75-fold (p = 0.0001), 5.45 ± 2.11-fold (p = 0.0004), 2.36 ± 1.54-fold (p = 0.0275), and 551.15 ± 155.80-fold (p < 0.0001), respectively, in hMSCs compared to the control pellets , TEK , SOX9 , HIF-1α , and ACAN  was significantly increased by o-Vanillin treatment, while no statistically significant difference was found in PAX1  in hMSCs exposed to CM of o-Vanillin-treated DCs compared to those of hMSCs exposed to DC CM , 1.51 ± 0.38-fold (p = 0.0006), and 3.08 ± 0.64-fold (p < 0.0001), respectively, while the expression of PAX1 , TEK , and ACAN  was not significantly changed , 0.88 ± 0.30-fold (p = 0.0118), 2.35 ± 0.49-fold (p = 0.0004), and 4.19 ± 0.61-fold (p < 0.0001), respectively, while TEK  and ACAN  showed no changes in DCs compared to the control pellets , TEK , SOX9 , and HIF-1α  was significantly increased by o-Vanillin treatment, while no statistically significant difference was found in PAX1  and ACAN  in DCs exposed to CM of o-Vanillin-treated hMSCs compared to those of DCs exposed to hMSC CM  in adults [3 might be a candidate density range of hMSCs for adult patients. Estimating the volume of NP and iAF compartments in a human IVD is 4 cm × 2.5 cm × 1 cm, the candidate hMSC dose may range from 22 × 106 to 27 × 106 cells. The doses of implanted hMSCs are highly variable in published studies. For example, Orozco et al. and Elabd et al. implanted 10 ± 5 × 106 and 15.1–51.6 × 106 (with autologous platelet lysate) autologous MSCs, respectively, in patients in 18–65 years of age with chronic LBP [6 allogenic MSCs in patients in 18–75 years of age with chronic LBP [This study investigated the potential of o-Vanillin in improving hMSC-based therapy for IVD repair. The results confirmed previous studies showing that hMSCs enhance GAG production and deposition in co-cultures with DCs ,36,37. Pn adults . Given sonic LBP ,40. In aonic LBP . Our finCo-culture of DCs with hMSCs reduced the number of senescent cells after 21 days in the pellets. This could be explained by an increased secretion of trophic factors from the hMSCs that restored and rejuvenated the disc cell phenotype. o-Vanillin is known to remove senescent IVD cells, and the data demonstrated that it also has senolytic effects on senescent hMSCs. o-Vanillin has senolytic effects as well as anti-inflammatory properties. Suppression of the inflammatory microenvironment in degenerating IVDs may further improve GAG deposition and disc cell phenotypes.Cell transplantation carries some risks such as competition for the low nutrient and oxygen supply in the center of a human IVD. Degenerating IVDs possess progenitor cells; therefore, if the microenvironment could be improved, it would allow them to regenerate, thereby avoiding the need for cell transplantation. To investigate the potential for a cell-free system EV production and transfer between hMSCs and DCs was evaluated. Here, qualitative and quantitative data show that o-Vanillin promoted EV formation and/or uptake by both hMSCs and DCs.COL2A1, ACAN, VCAN, ELN, and SOX9, in NP and hMSC co-cultures with and without direct cell–cell contact. Whereas ACAN, SOX9, HIF-1α, FOXF1, PAX1, and TEK in DC and hMSC pellets with CM transfer were evaluated in the current study.The results demonstrated that hMSCs differentiated toward disc-like cells when exposed to DC CM in the absence of a discogenic differentiation media  or direcex vivo in a controlled environment, may overcome some of these shortcomings as the cell-free therapy provides therapeutic molecules with less immunogenicity [Clinical studies have demonstrated that hMSC implantation attenuates discogenic LBP ,40,41; hgenicity . Other agenicity ,53. Howein vivo.Treating EV donor cells with o-Vanillin, a senolytic and anti-inflammatory natural compound, improved hMSC differentiation and reduced hMSC senescence. We have previously demonstrated that o-Vanillin can remove senescent IVD cells and improve tissue heath in intact human IVDs . The finThe findings presented here suggest that o-Vanillin improves trophic effects of hMSCs and promotes hMSC differentiation toward a disc-like cell phenotype. This was evident in direct and indirect 3D pellet cultures of hMSCs and DCs from degenerate IVDs. The data indicate that the senolytic and anti-inflammatory properties of o-Vanillin have the potential to improve stem cell-based therapies. Enriched and purified EVs or secretome generated ex vivo in a controlled environment, may overcome some of the shortcomings of cell-based therapies. The advantages of lower immunogenicity, extended shelf life and storage time, simpler and more feasible transportation, and lower tumorigenesis risk make the cell-free therapy a promising treatment in IVD repair.


Liquid chromatography-mass spectrometry (LC-MS) is the method of choice for the untargeted profiling of biological samples. A multiplatform LC-MS-based approach is needed to screen polar metabolites and lipids comprehensively. Different mobile phase modifiers were tested to improve the electrospray ionization process during metabolomic and lipidomic profiling. For polar metabolites, hydrophilic interaction LC using a mobile phase with 10 mM ammonium formate/0.125% formic acid provided the best performance for amino acids, biogenic amines, sugars, nucleotides, acylcarnitines, and sugar phosphate, while reversed-phase LC (RPLC) with 0.1% formic acid outperformed for organic acids. For lipids, RPLC using a mobile phase with 10 mM ammonium formate or 10 mM ammonium formate with 0.1% formic acid permitted the high signal intensity of various lipid classes ionized in ESI(+) and robust retention times. For ESI(−), the mobile phase with 10 mM ammonium acetate with 0.1% acetic acid represented a reasonable compromise regarding the signal intensity of the detected lipids and the stability of retention times compared to 10 mM ammonium acetate alone or 0.02% acetic acid. Collectively, we show that untargeted methods should be evaluated not only on the total number of features but also based on common metabolites detected by a specific platform along with the long-term stability of retention times. N-oxide amino]-dodecanoic acid, CUDA), shaken (30 s), centrifuged , and used for LC-MS analysis.For the metabolomic profiling, 70 µL of the bottom aqueous phase was collected, evaporated, and resuspended in 70 µL of an acetonitrile/water (4:1) mixture with internal standards , shaken (30 s), centrifuged , and analyzed using the HILIC metabolomics platform. Another 70 µL aliquot of the bottom aqueous phase was mixed with 210 µL of an isopropanol/acetonitrile (1:1) mixture, shaken (30 s), centrifuged , and the supernatant was evaporated. The dry extracts were resuspended in 5% methanol/0.2% formic acid with internal standards , shaken (30 s), centrifuged , and analyzed using the RPLC metabolomics platform.In total, 20 mg of tissue samples were homogenized (1.5 min) with 275 μL of methanol using a grinder, followed by adding 1 mL of MTBE with an internal standard and shaking (30 s). Then, 275 μL of 10% methanol containing internal standards was added, and the tubes were shaken (1 min) and centrifuged .For the lipidomic profiling, 100 µL of the upper organic phase was collected, evaporated, and resuspended using 300 µL of methanol with an internal standard (CUDA), shaken (30 s), centrifuged , and used for LC-MS analysis.A volume of 275 µL of methanol and 275 µL of 10% methanol containing internal standards was put directly on the cells (in a 6-well plate) and then scraped off and transferred to an Eppendorf tube with a milling ball. After homogenization (1 min), 1 mL of MTBE with the internal standard was added, and the tubes were shaken (1 min) and centrifuged .For the metabolomic profiling, the procedure was the same as for the biofluids.The LC-MS systems consisted of a Vanquish UHPLC system  coupled to a Q Exactive Plus mass spectrometer .The polar metabolites were separated on an ACQUITY UPLC BEH Amide column  coupled to an ACQUITY UPLC BEH Amide VanGuard pre-column  . The column was maintained at 45 °C at a flow rate of 0.4 mL/min. The mobile phase consisted of (A) water with the following mobile phase modifiers; (i) 0.1% formic acid; (ii) 10 mM ammonium formate and 0.125% formic acid; (iii) 10 mM ammonium formate; (iv) 10 mM ammonium acetate; (v) 10 mM ammonium acetate and 0.01% ammonium hydroxide (pH 9.3); (vi) 10 mM ammonium bicarbonate (pH 8.8); (vii) 0.001% ammonium hydroxide (pH 10.0); and (B) acetonitrile/water (95:5) with the same mobile phase modifiers. In the case of 10 mM ammonium bicarbonate, acetonitrile/water (90:10) was used due to solubility issues. Separation was conducted under the following gradient: 0 min 100% (B); 0–1 min 100% (B); 1–3.9 min from 100% to 70% (B); 3.9–5.1 min from 70% to 30% (B); 5.1–6.4 min from 30% to 100% (B); 6.4–7.5 min 100% (B) +1 min preinjection steps. An injection volume of 1 μL was used. The sample temperature was maintained at 4 °C.m/z 60–900; MS1 resolving power, 35,000 FWHM (m/z 200); the number of data-dependent scans per cycle, 3; MS/MS resolving power, 17,500 FWHM (m/z 200). A spray voltage of 3.6 kV and −2.5 kV for ESI(+) and ESI(−) were used. Normalized collision energies of 20, 30, and 40% were used.The ESI source and MS parameters were: sheath gas pressure, 50 arbitrary units; aux gas flow, 13 arbitrary units; sweep gas flow, 3 arbitrary units; capillary temperature, 260 °C; aux gas heater temperature, 425 °C. For the untargeted metabolomic profiling, the mass spectrometer was operated under the following conditions: MS1 mass range, The polar metabolites were also separated on an ACQUITY UPLC HSS T3 column  coupled to an ACQUITY UPLC HSS T3 VanGuard pre-column  . The column was maintained at 45 °C using a ramped flow rate. The mobile phase consisted of (A) water with 0.2% formic acid, (B) methanol with 0.1% formic acid, or acetonitrile with 0.1% formic acid. Separation was conducted under the following gradient: 0 min 1% (B) 0.3 mL/min; 0–0.5 min 1% (B) 0.3 mL/min; 0.5–2 min from 1% to 60% (B) 0.3 mL/min; 2–2.3 min from 60% to 95% (B) from 0.3 mL/min to 0.5 mL/min; 2.3–3.0 min 95% (B) 0.5 mL/min; 3.0–3.1 min from 95% to 1% (B) 0.5 mL/min; 3.1–4 min 1% (B) 0.5 mL/min; 4–4.1 min 1% (B) from 0.5 mL/min to 0.3 mL/min; 4.1–4.5 min 1% (B) 0.3 mL/min + 1 min preinjection steps. An injection volume of 5 μL was used. The sample temperature was maintained at 4 °C.m/z 60–900; MS1 resolving power, 35,000 FWHM (m/z 200); the number of data-dependent scans per cycle, 3; MS/MS resolving power, 17,500 FWHM (m/z 200). A spray voltage of −2.5 kV for ESI(−) was used. Normalized collision energies of 20, 30, and 40% were used.The ESI source and MS parameters were: sheath gas pressure, 50 arbitrary units; aux gas flow, 13 arbitrary units; sweep gas flow, 3 arbitrary units; capillary temperature, 260 °C; aux gas heater temperature, 425 °C. For the untargeted metabolomic profiling, the mass spectrometer was operated under the following conditions: MS1 mass range, The lipids were separated on an ACQUITY UPLC BEH C18 column  coupled to an ACQUITY UPLC BEH C18 VanGuard pre-column  . The column was maintained at 65 °C at a flow rate of 0.6 mL/min. For the LC-MS analysis, the mobile phase consisted of (A) 60:40 acetonitrile/water with six different mobile phase modifiers: (i) 10 mM ammonium formate; (ii) 10 mM ammonium formate and 0.1% formic acid; (iii) 10 mM ammonium acetate and 0.1% formic acid; (iv) 10 mM ammonium acetate; (v) 10 mM ammonium acetate and 0.1% acetic acid; and (vi) 0.02% acetic acid (B) 90:10:0.1 isopropanol/acetonitrile/water with the same type of mobile phase modifiers. Separation was conducted under the following gradient for LC-ESI(+)-MS: 0 min 15% (B); 0–1 min from 15% to 30% (B); 1–1.3 min from 30% to 48% (B); 1.3–5.5 min from 48% to 82% (B); 5.5–5.8 min from 82% to 99% (B); 5.8–6 min 99% (B); 6–6.1 min from 99% to 15% (B); 6.1–7 min 15% (B) +1 min preinjection steps. For the LC-ESI(−)-MS, the following gradient was used: 0 min 15% (B); 0–1 min from 15% to 30% (B); 1–1.3 min from 30% to 48% (B); 1.3–4.8 min from 48% to 76% (B); 4.8–4.9 min from 76% to 99% (B); 4.9–5.3 min 99% (B); 5.3–5.4 min from 99% to 15% (B); 5.4–6.3 min 15% (B) +1 min preinjection steps. An injection volume of 1–5 μL was used based on the extract type. The sample temperature was maintained at 4 °C.m/z 200–1700; MS1 resolving power, 35,000 FWHM (m/z 200); the number of data-dependent scans per cycle, 3; MS/MS resolving power, 17,500 FWHM (m/z 200). For ESI(+), a spray voltage of 3.6 kV and a normalized collision energy of 20% was used; for ESI(−), a spray voltage of −3.0 kV and normalized collision energies of 10, 20, and 30% were set up.The source and MS parameters were: sheath gas pressure, 60 arbitrary units; aux gas flow, 25 arbitrary units; sweep gas flow, 2 arbitrary units; capillary temperature, 300 °C; aux gas heater temperature, 370 °C. For the untargeted lipidomic profiling, the mass spectrometer was operated under the following conditions: MS1 mass range, m/z library and MS/MS libraries available from various sources .The LC-MS instrumental files from the metabolomic and lipidomic profiling were processed through MS-DIAL v. 4.92 software . The metThe total number of molecular features was obtained by processing the repeated injections of biological samples and method blanks. For the untargeted metabolomics using HILIC-ESI(+), a series of 10 injections of serum extracts was conducted. For the untargeted lipidomics using RPLC-ESI(+) and RPLC-ESI(−), six injections of serum extracts, three injections of liver extracts, and three injections of tea extracts were performed. First, only the molecular features detected in all replicates for a particular platform and matrix were kept. Then, features passing the criterion “sample maximal signal intensity/blank average signal intensity” >10-fold change (available in MS-DIAL v. 4.92 software) were kept as reliable features for the evaluation of the mobile phase modifiers.The scoring for the untargeted metabolomics (the HILIC platform) was performed using the (i) retention time , (ii) peak height intensity , and (iii) peak width . In addition, for leucine/isoleucine, the ability to resolve these isomers was also considered . The total score for each metabolite was then calculated as a sum of all the scoring values .m/z but should also take into account the common metabolites detected and annotated for particular platforms. In addition, the long-term stability of the retention times should be assessed.We evaluated commonly used mobile phase modifiers in analyzing polar metabolites and simple and complex lipids during fast LC-MS. Our findings support that during untargeted metabolomics and lipidomics, the method should not be evaluated just purely based on the total number of features characterized by their retention times and For untargeted metabolomics with an ACQUITY UPLC BEH Amide column, using a mobile phase with 10 mM ammonium formate with 0.125% formic acid provided the best performance for separating amino acids, biogenic amines, sugars, nucleotides, acylcarnitines, and sugar phosphate. However, none of the mobile phase modifiers evaluated performed well for organic acids in ESI(−). For the fast separation and detection of this metabolite class, an ACQUITY UPLC HSS T3 column performed well.For untargeted lipidomics with an ACQUITY UPLC BEH C18 column, using a mobile phase with 10 mM ammonium formate or 10 mM ammonium formate with 0.1% formic acid permitted a high signal intensity of various lipid classes ionized and detected in ESI(+) along with robust retention times during long runs. For ESI(−), the mobile phase with 10 mM ammonium acetate with 0.1% acetic acid represented a reasonable compromise regarding the signal intensity of the detected lipids and the stability of the retention times compared to 10 mM ammonium acetate alone or 0.02% acetic acid.Overall, the analysis time between 5.5 and 8.5 min was achieved using optimized LC gradients, permitting fast untargeted LC-MS-based metabolomics and lipidomics methods.


Cyclin-dependent kinase 5 (Cdk5) is a crucial regulator of neuronal signal transduction. Cdk5 activity is implicated in various neuropsychiatric and neurodegenerative conditions such as stress, anxiety, depression, addiction, Alzheimer’s disease, and Parkinson’s disease. While constitutive Cdk5 knockout is perinatally lethal, conditional knockout mice display resilience to stress-induction, enhanced cognition, neuroprotection from stroke and head trauma, and ameliorated neurodegeneration. Thus, Cdk5 represents a prime target for treatment in a spectrum of neurological and neuropsychiatric conditions. While intracranial infusions or treatment of acutely dissected brain tissue with compounds that inhibit Cdk5 have allowed the study of kinase function and corroborated conditional knockout findings, potent brain-penetrant systemically deliverable Cdk5 inhibitors are extremely limited, and no Cdk5 inhibitor has been approved to treat any neuropsychiatric or degenerative diseases to date. Here, we screened aminopyrazole-based analogs as potential Cdk5 inhibitors and identified a novel analog, 25–106, as a uniquely brain-penetrant anti-Cdk5 drug. We characterize the pharmacokinetic and dynamic responses of 25–106 in mice and functionally validate the effects of Cdk5 inhibition on open field and tail-suspension behaviors. Altogether, 25–106 represents a promising preclinical Cdk5 inhibitor that can be systemically administered with significant potential as a neurological/neuropsychiatric therapeutic. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed kinase predominantly expressed in mature neurons . Unlike 2+ homeostasis. Increased intracellular Ca2+ can activate the protease calpain, which then cleaves Cdk5’s physiological coactivator p35 into a truncated aberrant coactivator p25 and a p10 fragment , Parkinson’s disease (PD), ischemia, and brain injury . This isfragment . The p25fragment . Furtherfragment . These cfragment . For thefragment . Elevatefragment . Deletiofragment . Thus, CTherapeutic infusion of preclinical inhibitors of Cdk5 into the brain has been successfully shown to ameliorate neurological disease phenotypes in mice. However, none of these compounds has proven effective in clinical trials . A majorin vivo and displays high potency for inhibition in vitro. Furthermore, this inhibitor modulates behavioral phenotypes linked to Cdk5 through previous characterizations of conditional knockout mice. These studies further advance our understanding of Cdk5’s role in neuropsychiatric behavior, bring forth a new pharmacological tool to assess Cdk5 activity, and suggest a promising new preclinical drug.Here, by screening a small panel of aminopyrazole analogs, we identify analog, 25–106, that can be delivered systemically, penetrate the blood–brain barrier, and pharmacologically inhibit Cdk5/p35 activity in the brain. These data demonstrate that 25–106 inhibits Cdk5 activity in vivo, ex vivo, and behavioral studies. This age range was selected to be consistent with previous studies of conditional Cdk5 knockout mice , coupling constant in hertz (Hz), and integration. The purity of the final compounds was determined by analytical HPLC and was found to be ≥95% pure. HPLC was performed on a Waters Alliance 2,690 system equipped with a Waters 2,996 photodiode array detector and an auto-sampler under the following conditions: column, Phenomenex Luna-2 RP-C18 ; solvent A, H2O containing 0.1% formic acid (FA); solvent B, CH3CN containing 0.1% FA; gradient, 60% B to 100% B over 8 min followed by 100% B over 6 min; injection volume, 25 μL; flow rate, 1 ml/min retention times and purity data for each target compound are provided in the Experimental Section. The purified compound was further confirmed by MS analysis using a Quattro Micro triple–quadrupole mass spectrometer using an electron spray ionization (ESI) technique and a TOF mass analyzer.All reagents were purchased from commercial sources and were used without further purification. Flash chromatography was carried out on silica gel (200–400 mesh). Thin-layer chromatography (TLC) was run on pre-coated ANALTECH uniplate and observed under UV light at 254 nM and with a basic potassium permanganate dip. Column chromatography was performed with silica gel . 1H NMR (400 MHz) and 13C NMR (100 MHz) spectra were recorded in chloroform-d or DMSO-Synthesis of 2- acetyl chloride . To a so2Cl2, washed with brine, and dried with MgSO4. The crude product was purified using silica gel column chromatography .Synthesis of tert-butyl 3-amino-5-cyclobutyl-1H-pyrazole-1-carboxylate (2) . 3-amino2. The reaction mixture was cooled to 0°C and freshly prepared 2-acetyl chloride  dichloromethane was added dropwise at 0°C. Following addition, the reaction temperature was allowed to rise to room temperature and, the mixture was stirred for an additional 3 h. The crude mixture was washed with brine, extracted with dichloromethane, dried with MgSO4, and concentrated. The crude mixture was purified using silica gel column chromatography ; 1H NMR  δ 10.31 , 6.87 , 6.57 , 3.91 , 3.87 , 3.72 , 3.55 , 2.41–2.14 , 2.12–1.85 , 1.72–1.56 .Synthesis of tert-butyl 5-cyclobutyl-3-[2-acetamido]-1H-pyrazole-1-carboxylate (3) . Compounin vacuo, and the resulting solid was recrystallized to yield a colorless solid . Purity was assessed by HPLC (>98%); 1H NMR  δ 12.04 , 10.41 , 6.64 , 6.31 , 3.76 , 3.63 , 3.51 , 3.48–3.37 , 2.35–2.18 , 2.18–2.02 , 1.95 , 1.89–1.73 ; 13C NMR  δ 168.1, 153.1, 147.8, 147.5, 136.7, 132.2, 107.0, 93.8, 60.4, 56.3, 43.3, 31.6, 29.4, 18.6; MS calculated for C18H23N3O4 m/z 345.16, found mass: 346.14.Synthesis of N-(5-cyclobutyl-1H-pyrazol-3-yl)-2-acetamide (25–106). To a stirred solution of tert-butyl 5-cyclobutyl-3-[2-acetamido]-1H-pyrazole-1-carboxylate  in dichloromethane (5 ml) at 0°C, trifluoroacetic acid (1 ml) was added dropwise, and the reaction mixture was stirred at room temperature for 3 h. After completion, the reaction mixture was concentrated 2) linear regression analysis of the 25–106/IS peak area ratio vs. the 25–106 concentration from 1.0–80.0 ng/ml (ng/g for tissue) for the low curve and 80.0–10,000 ng/ml (ng/g for tissue) for the high curve. The concentrations of incurred and quality control samples were calculated with the same regression analysis, and the results were reported in ng/mL (plasma) or ng/g (tissue) of 25–106.A quantitative determination of 25–106 in mouse plasma and tissue homogenate was accomplished by the use of protein precipitation and high-performance liquid chromatography with tandem mass spectrometry detection (LC-MS/MS). 5- amiloride (HMA) was used as the internal standard (IS). 25–106 and IS were typically extracted from 50 µL of mouse plasma or tissue homogenate using protein precipitation with methanol. The extracts were analyzed by reverse-phase chromatography using an ACE Excel 3 C18-PFP column under isocratic conditions at a flow rate of 500 μL/min. The column temperature was maintained at 40°C. The mobile phase A consisted of 0.1% formic acid in water, and the mobile phase B was acetonitrile. A triple–quadrupole mass spectrometer  equipped with TurboV IonSpray operating in positive-ion mode was used. Column effluents were analyzed by multiple reaction monitoring (MRM). The precursor/product transitions are 346.1 to 137.1 m/z for 25–106 and 312.37 to 129.5 m/z for IS. The calibration curve was fit using weighted  crystal structure was retrieved from the Protein Data Bank and analyzed using Maestro version 12.3.013 (Schrödinger Inc.). The protein preparation was performed using Protein Preparation Wizard in which missing hydrogen atoms, side chains, and loops were added, followed by grid generation, ligand preparation, and docking. The protein structures were minimized using the OPLS3e force field to optimize hydrogen bonding networks and converging heavy atoms to an RMSD of 0.3 Å. The prepared protein structure was subjected to SiteMap for binding site analysis as implemented in Schrödinger Suite 2020–1. At least 15 site points per reported site were required. A more restricted definition of hydrophobicity together with a standard grid was used. Site maps at 4 Å or more from the nearest site points were cropped. The structures of 25–106 and roscovitine were subjected to Lig Prep to generate conformers and possible protonation at pH of 7 ± 2, which serves as an input for the docking process. The receptor grid was generated using the receptor grid generation tool in Maestro (Schrödinger Inc.). GLIDE XP was used to perform all the dockings with the van der Waals radii of nonpolar atoms for each of the ligands being scaled by a factor of 0.8 and the partial charge cutoff of 0.15. The docked poses from GLIDE XP were analyzed using Maestro version 12.3.013 (Schrödinger Inc.).3, 1.25 mM KH2PO4, 1.5 mM MgSO4, 10 mM d-glucose, and 1.5 mM CaCl2). The brains were subsequently sectioned into normal Kreb’s solution at 350 µ thickness. The brain slices were transferred to a 30°C solution of normal Kreb’s containing 10 µM deaminase for a 1-h recovery period. The sections were then incubated in normal Kreb’s solution with and without the indicated compounds across the dose and time ranges indicated. The sections were rapidly snap-frozen in dry ice to terminate treatment regimes.Brain slice pharmacology was performed as described . BrieflyImmunoblotting was performed as previously described . Mouse bIn vitro phosphorylation reactions were conducted as dose-response kinase inhibition assays with three highly homologous CDKs, namely, Cdk2/CyclinE (1.5 nM), CDK5/p25 (0.35 nM), and Cdk9/CyclinT1 (9 nM). Briefly, the abovementioned kinases were incubated with ATP (30 μM), the corresponding substrate (20 μM), and an increasing concentration of 25–106 (0–5 μM) for 30 min. The kinase activity of the abovementioned reactions was graphed against the concentration of 25–106 to determine the IC50 and Ki values for each kinase.The OFT was performed as described . With thThe tail suspension test was conducted as previously described . The micThe physicochemical properties of five FDA-approved CNS drugs   Ghose e, were esvia immunoblotting the phosphorylation of established Cdk5 sites, phospho-Thr75 DARPP-32 and phospho-Ser1116 NR2B , and brain striatal lysates from various time points were subjected to quantitative immunoblot analysis for phosho-Ser549 Synapsin I levels. Three compounds, 20–107, 20–132, and 20–143, had no effect on Cdk5 activity compared to that of vehicle alone (data not shown). In contrast, 25–106 significantly reduced phospho-Ser549 synapsin I levels in the striatum 2 h after I.V. injection (Each of the four compounds tested in brain slices was further screened for their ability to penetrate the blood–brain barrier (BBB) and inhibit Cdk5-dependent activity njection . Moreovein vitro selectivity of 25–106, we conducted a dose–response kinase inhibition assay with three highly homologous Cdks, Cdk2/CyclinE, Cdk5/p25, and Cdk9/CyclinT1  detection method to assess the distribution, concentration, and elimination of 25–106 throughout several organ systems. For these studies, 5- amiloride (HMA) was used as an internal standard. Mass spectra product transitions were observed as 25–106 displayed a prominent peak of the parent compound at 346.6 m/z, with the product transition for 25–106 displaying a larger peak at 137.9 m/z , elimination rate constant (Ke), half-life (T1/2), last measurable concentration (Clast), 24-h area-under-curve (AUC24h), distribution volume (V), and clearance (CL), were calculated  (R2 = 0.9454)  . The CL  0.9454) . Of noteinjected .max results of 101.13, 118.25, 376.88, and 668.75 ng/g for 10, 50, 100, and 200 mg/kg injections, respectively (1/2 of 25–106 in the brain remained relatively constant (excluding the lowest dose of 10 mg/kg), with an average half-life of 6.23, 5.03, and 4.39 h for 50, 100, and 200 mg/kg injections, respectively , reaching Cectively ; Table 3ectively ; Table 3max, Clast, and AUC could be determined for these samples  activity and protein kinase A (PKA) signaling (Previous studies have demonstrated striatal Cdk5 knockout or inhibition results in reduced anxiety-like behaviors in mice ignaling . Impaireignaling . Here, wignaling . Therefoignaling . Since tignaling . In addiignaling . Cdk5 acignaling .As perhaps the first robust systemic inhibitor, 25–106 represents an exciting and expandable and translatable pharmacological tool to study the function of Cdk5 activity in wild-type animals. Achieving systemic applicability may be considered a step forward toward the testing of Cdk5 inhibitors to treat neuropsychiatric and neurodegenerative diseases. This provides a promising landscape for future studies to assess the effects of brain-permeable Cdk5 inhibitors to combat stress, anxiety, depression, addiction, cancer, and neurodegeneration.


Phosphate-solubilizing bacteria (PSB) can enhance plant growth and phosphorus (P) solubilization, it also has been reported to reduce the negative effects of overused agricultural fertilizer in farmland and protect the soil environment. However, the mechanism behind this interaction has not been fully elucidated.Pseudomonas moraviensis, Bacillus safensis, and Falsibacillus pallidus which can both solubilize P efficiently and produce indole-3-acetic acid (IAA) from sandy fluvo-aquic soils. The yield of wheat (Triticum aestivum) under PSB inoculation significantly increased up to 14.42% (P < 0.05) compared with the control treatment in phosphate fertilizer-used farmland. Besides promoting wheat growth, we found the labile P fraction in soil was significantly increased by over 122.04% (P < 0.05) under PSB inoculation compared with it in soils without, in parallel, the stable P fraction was significantly reduced by over 46.89% (P < 0.05). Furthermore, PSB inoculation increased the soil microbial biomass and activity, indicating that PSB screened out in this work performed a remarkable ability to colonize the soils in the wheat field.In this study, we screened out PSB from sandy fluvo-aquic soil improve wheat growth and crop productivity by increasing the labile P fraction and IAA content in the greenhouse and wheat field. Our work provides an environment and economy-friendly bacterial resource that potentially promotes sustainable agricultural development in the long term.The online version contains supplementary material available at 10.1186/s12866-022-02715-7. Phosphorus (P) is one of the essential elements in plant growth and nutrient cycling in soil systems . AlthougTo obtain sufficient P for grain production, P fertilizer is applied to most farmlands, even though its efficiency of P uptake by plants appears low as 10–25% mostly due to P fixation or loss from soils –9. PhospPhosphate-solubilizing bacteria (PSB) play an essential role in P cycling and promoting plant growth by increasing its P uptake in rhizosphere soils. Most PSB produces indole-3-acetic acid (IAA) which enables plant cells to grow, RNA/protein synthesis thus increasing plant growth . MoreoveOur study aims to reveal the role of PSB in solubilizing P, enhancing P fertilizer efficiency, and promoting plant growth. In this study, we isolated PSB that can both solubilize P and produce IAA from sandy fluvo-aquic soil. We evaluated the effects of PSB on plant growth, and soil P cycling in pot experiments. Furthermore, we tested the ability of PSB on enhancing P fertilizer efficiency in fields. The present work provides a full-picture description of studying the P solubilization ability of PSB and fills the gap in understanding the mechanisms of interaction between microbes and plants in natural soil systems.3 (PO4)2 solubilization and IAA production 2 in large quantities, the AP concentration in the supernatant of strains X2, X3, and X21 were 401.25, 476.60, 421.15 mg L−1, respectively. The IAA concentration with strains X21, X2, and X3 were 45.65, 40.42, and 34.78 μg mL−1, respectively.Based on the Caon Fig. S, three sPseudomonas moraviensis CCM7280 (AY970952)    with X2, X3, and X21 inoculation, and the total MLP and SP content were significantly reduced by 8.95%–14.80% and 45.35%–46.89% (P < 0.05), respectively , while the content of NaOH-Pi, dilute HCl-P, concentrated HCl-Po, concentrated HCl-Pi, and concentrated H2SO4-P were significantly reduced by 8.09%–15.91%, 9.57%–17.51%, 43.23%–54.74%, 37.53%–45.54%, and 47.68%–64.66% (P < 0.05), respectively . The proportion of the MLP fractions declined from 55.14% to 46.94%–50.25% and the SP fractions declined from 29.53% to 15.69%–16.12%, respectively , the total LP content increased by 122.04%-142.17% (P < 0.05), among which inoculation with X3 performed best in enhancing the root growth. Inoculation with X21 significantly increased the root diameter by 5.06% (P < 0.05) , respectively , indicating that bone meal has no obvious effect on soil microbial biomass. In the wheat seedling growth stage, the MBC and MBN contents with X2 and X3 inoculation were significantly higher than those of the two controls (FP and BM) (P < 0.05). In the wheat stem elongation stage and ripening stage, the application of three PSB strains could significantly improve the soil microbial biomass (P < 0.05). Furthermore, during the whole growth period of wheat, inoculation with X2, X3 and X21 significantly improved the soil base respiration (SBR) compared with controls (P < 0.05). The microbial metabolic quotient (qCO2) was significantly influenced by the inoculation with PSB at the stem elongation stage and ripening stage although not at the seedling growth stage  and Microbial biomass nitrogen (MBN) contents showed differently along with each growth stage of wheat Fig. SA, B. TheP < 0.05). In the wheat stem elongation stage and ripening stage, the application of three PSB significantly increased AP content (P < 0.05) compared with FP and BM . For grain number per spike, there was no significant difference among the treatments (P > 0.05). Compared with FP treatment, the yield increase rates of inoculation with X2, X3, and X21 were 11.18%, 14.42%, and 13.21%, respectively 2 was selected as the phosphate resource. The concentration of phosphate solubilized by P. moraviensis, B. safensis, and F. pallidus reached the highest as 401.25, 476.60, and 421.15 mg L−1, respectively (P source 5000 mg L−1 Ca3(PO4)2), while the solubilized phosphate reached 8.02%, 9.53%, and 8.42% respectively. The large quantities of phosphate solubilized by P. moraviensis, B. safensis, and F. pallidus make them powerful tools to release fixed P in soil systems, which is of significant value to promote grain yield [in soils –27. P. mr plants , 29. At in yield .P. moraviensis, B. safensis, and F. pallidus in pot experiment and found that the LP fraction increased while SP fraction declined, which is important for plant root promotion [Different P fractions drive P cycling and uptake of plants in most soils, labile and some moderately labile P are easy for plants to absorb, which are key to plant root development, and further makes better growth of above-ground parts of plants , 31, 32.romotion , 33. In romotion , 35.Bacillus firmus MAJ PSB12 collected from the rhizosphere soil of the castor-oil plant showed high efficiency in solubilizing insoluble calcium triphosphate by producing a variety of organic acids, resulting in a decrease in the pH of the medium. Moreover, it could adapt to pH 5.4 and also be functional [The diversity and function of PSB have been found highly associated with environmental abiotic conditions, such as pH, temperature, stoichiometric allocation, and artificial interaction –38. Basenctional . Our resnctional .P. moraviensis, B. safensis, and F. pallidus improved the soil microbial biomass, which potentially suggests that the PSB we investigated could survive better in wheat field and perform good cooperation with indigenous microorganisms. The soil microbial biomass has been regarded as a sensitive indicator of environmental changes [The field experiment showed that  changes , particu changes , which i changes . It has  changes . We founP. moraviensis, B. safensis, and F. pallidus showed a higher adaptation and colonization in the field under long-term fertilizer application than other bacteria isolated from rhizosphere soils [Besides increasing the content of LP fraction, PSB in this study can produce IAA and jointly promote the yield of crops by increasing above-ground biomass, spike number, and 1000-grain weights of wheat. Therefore, re soils .P. moraviensis, B. safensis and F. pallidus isolated in our study showed superior ability to promote plant growth and high adaptation to different soil environmental conditions. We optimized the P solubilizing abiotic conditions and explored the P solubilizing process of PSB in soils. Pot experiments show that PSB can solubilize the SP and MLP fraction in the soil and PSB in the field experiments increased the utilization rate of P fertilizer. The PSB inoculation promote the wheat growth promotion and increased the crop productivity. Our work preliminary fills the gap in understanding the mechanisms of interaction between microbes and plants in the natural soil systems  . The samples were collected at 0–20 cm depth, in which the rocks, stubble, and other debris were removed. After that, soils were passed through a 2 mm sieve and divided into two parts, and placed into separate sterile bags. One group was kept for PSB screening and the other was air-dried for basic physical and chemical property measurements.−1 of organic matter, 30.02 mg kg−1 of available nitrogen, the total P was 2.95 g kg−1 including 31.2 mg kg−1 of AP, the total potassium was 19.60 g kg−1 including 50.39 mg kg−1 of available potassium, soil pH (H2O) was 7.37.The physicochemical properties of soil samples showed 8.89 g kg3(PO4)2 5 g, (NH4)2SO4 0.5 g, MgSO4· 7H2O 0.3 g, MnSO4 0.03 g, FeSO4· 7H2O 0.03 g, distilled water 1000 mL, pH 7.0–7.2, autoclave (30 min). The solid medium: 15–20 g agar was amended to the liquid medium.Lysogeny broth (LB) and Pikovskaya (PKO) mediums were used to screen PSB and test the phosphate-solubilizing capacity . LB mediA single colony of bacteria from the soil was isolated by the dilution method on the plate . BacteriThe selected strains were cultured on LB liquid medium at 30 ℃ in a rotary shaker (180 rpm) for 24 h (v/v) and the activated strain was then transferred into a 250 ml flask containing 100 mL sterilized PKO liquid medium. The samples were put in an incubation shaker (180 rpm) at 30 ℃ for 96 h (3 replications per strain). The 10 mL of each culture was centrifuged for 5 min at 12,000 rpm, 4 ℃, and the supernatant was collected to measure the amount of P solubilized by each strain. The concentration of AP was determined by the molybdenum antimony colorimetric assay method . Strains−1) LB liquid medium at 30 ℃ shaker (180 rpm) for 96 h. The 2 mL of bacterial suspension was centrifuged at 12,000 rpm for 10 min and obtained the supernatant. The 100 μg mL−1 IAA standard solution was serially diluted to 0, 10, 20, 30, 50, and 75 μg mL−1. An equal volume of Salkowski colorimetric solution (50 mL 35% HClO4 + 1 mL 0.5 M FeCl3) was added. After that, the suspensions were kept in the dark for 30 min, and the absorbance values at 530 nm wavelength were measured by spectrophotometry . The content of IAA per unit volume of fermentation broth was calculated according to the standard curve.IAA production of PSB was measured according to the method developed by Gordon and Weber . The strThe selected strains were inoculated on LB plates at 30 ℃ for 24 h. The appearance of strain colonies including size, shape, color, gloss, consistency, and transparency was observed under a microscope  and identified by the Gram staining method .2O. The 30%, 50%, 70%, 80%, and 90% ethanol aqueous solutions were used for gradient dehydration (15 min each). The samples were dropped onto 5 × 5 mm tinfoil paper and air-dried naturally [The purified strains were inoculated on LB medium and cultured on a shaker at 160 rpm and 30 ℃ to the logarithmic phase. The 5 mL of sample was centrifuged at 4000 rpm for 5 min. The supernatant was discarded and the precipitate was washed three times with 0.1 M Phosphate buffer, fixed with 2.5% glutaraldehyde for 3 h, washed twice with 0.1 M phosphate buffer, 10 min each time, and then washed twice with ddHThe physiological and biochemical characteristics of each strain were analyzed by the aerobic test, contact enzyme test, M.R, V-P test, starch hydrolyzed gelatin liquefaction, nitrate reduction and citrate utilization test .16S rDNA was used as a barcode gene for classifying the different PSB strains. The total genomic DNA was extracted by the method of SDS-CTAB . PCR ampDifferent carbon sources , nitrogen sources , culturing time , temperatures , pH  and liquid volumes  were set to evaluate the effect of different PSB on P solubilizing.7 CFU mL−1, then inoculated into the soil at 106 CFU g−1 dry soil.The sandy fluvo-aquic soil was collected from 0–20 cm depth of field soil and filtered by a sieve (2 mm). A total of 8.4 kg of soil were collected in this study and 12 repetitive pots were conducted with 700 g of soil each. The strains prepared for the pot experiment were cultured in LB medium for 48 h at 30 ℃ and shaken at 150 rpm, then the samples were centrifuged . The pellets were washed twice with sterilized water and resuspended in sterilized water to a density of 10The treatments were marked as X2, X3, X21 which refers to inoculation with each strain, and the CK were added with sterilized water that was equivalent to each bacteria suspension (3 replicates each). The moisture was adjusted to approximately 60% of the field capacity  .2O2 for 20 min. They were rinsed 3–5 times with sterile distilled water and dried on a sterile plate. The seeds were germinated for 2 days at 25 ℃. For each treatment, three wheat seedlings were planted in each pot one day after the bacteria were inoculated. The day of planting was marked as the first day. The pots were then placed in a greenhouse under a day/night regimen of 10/14 h at 22/19 ℃, the photon flux density was 150 mmol m−2 s−1. The pots were watered every day to maintain the soil moisture at 24.8% (60% of the field capacity).Seeds of wheat were surface-sterilized with 20% H2O-P (includes resin P), 0.5 M NaHCO3-Po, 0.5 M NaHCO3-Pi, 0.1 M NaOH-Po, 0.1 M NaOH-Pi, 1 M dilute HCl-P, 12 M concentrated HCl-Po, 12 M concentrated HCl-Pi, 65% concentrated H2SO4-P) were measured according to the Hedley’s method [2O-P + NaHCO3-Po + NaHCO3-Pi, MLP = Ʃ NaOH-Po + NaOH-Pi + dilute HCl-P, SP = Ʃ concentrated HCl-Po + concentrated HCl-Pi + concentrated H2SO4-P.After 30 days of planting, the content of various P components in soils , and the root-related parameters  were evaluated with Win-rhizo software . The plant height (from the root neck to the top leaf tip of the seedling) and dry weight , and plant total N, P and K were determined according to the method of Cresser and Parsons .11 CFU g−1.The field experiment was conducted at Wheat–Maize Rotation Nutritional Fertilization Scientific Observation Station in the North China Region of the Ministry of Agriculture  . The field experiments test site was a semi-arid, semi-humid, warm temperate monsoon climate zone, the average annual rainfall was 676 mm. The present study was mainly conducted in July–September . The soil type was sandy fluvo-aquic and wheat and corn rotation perennial farming was applied. The selected strains were made into inoculants with the bone meal as a carrier, which contains viable bacteria of 102). FP treatment used 600 kg hm−2 compound fertilizer (N: P: K = 15: 15: 15) as base fertilizer, ditching and topdressing 120 kg hm−2 of urea at the stem elongation stage of wheat, other treatments were treated with 40 kg hm−2 bone meal or a microbial agent made from the same amount of bone meal  based on base fertilizer. The tested wheat variety was Aikang 58, the sowing rate was 150 kg hm−2, and other management measures during the growth period were the same as those in local high-yield wheat fields.A total of 5 treatments were set in the field experiment: (1) Farmer conventional fertilization control (FP), (2) Bone meal application control (BM), (3) Bacteria agent X2 (X2), (4) Bacteria agent X3 (X3), (5) Bacteria agent X21 (X21). The experiment was set for 3 repetitions and arranged in random blocks (area of 6 × 3 m2 = SBR/MBC) was calculated with the formula of Anderson and Domsch [−1 NaHCO3 and measured by the ammonium molybdate method [2 to measure the actual yield.The 0–20 cm soil samples were collected between wheat rows at the seedling growth stage, stem elongation stage, and ripening stage, respectively, to determine soil microbes and nutrient indexes. The MBC and MBN were determined by using a chloroform fumigation-direct extraction method , 58. SBRd Domsch . Soil APe method . Soil IAe method . At the P < 0.05). Pearson correlation analyses were performed to determine the correlation among soil P fraction components, wheat root architecture and growth indices in the pot experiments, among soil microbial indices, soil AP and IAA contents, wheat yield indices in the field experiment. All of the statistical analyses were performed in SPSS 16.0 . All the data were log-transformed before PCA  analysis. ClustVis was employed to create a PCA plot and heatmaps [The significance of differences among treatments was analyzed by one-way ANOVA with a least significant difference (LSD) test . Different lowercase letters indicate significance at the 5% level in the phosphate solubilizing ability, different uppercase letters indicate significance at the 5% level in the IAA producing ability. Fig. S2. (A) Phylogenetic trees of maximally similar species representing strains X2 (The bar represents 2 nucleotide substitutes per 1000 nucleotidesin16S rDNA sequences), (B) X3 (The bar represents 2 nucleotide substitutes per 1000 nucleotidesin16S rDNA sequences) and (C) X21 (The bar represents 5 nucleotide substitutes per 1000 nucleotidesin16S rDNA sequences). Fig.S3. Phosphate solubilizing ability of different strains  under different carbon source (A), nitrogen source (B), incubation time (C), temperatures (D), pH values (E) and liquid volume (F). Data are shown as Mean ± SD. Fig. S4. Correlation analysis of each soil phosphorus fraction components correlated with the wheat root architecture and growth indices. Red means positive correlation, blue means negative correlation. * indicates significant correlation at 0.05 level, ** indicates significant correlation at 0.01 level. A-I: the compounds correlated with the H2O-P, NaHCO3-Po, NaHCO3-Pi, NaOH-Po, NaOH-Pi, HCl-P, Conc.HCl-Po, Conc.HCl-Pi, Conc.H2SO4-P. Fig. S5. Effects of different treatments  on soil microbial biomass carbon (A), soil microbial biomass nitrogen (B), soil respiration (C) and microbial metabolic quotient (D) at different wheat growth period in the field experiment. Different lowercase letters indicate significance among different treatments at the same stage at the 5% level. The same below. Fig. S6. Principal component analysis (PCA) showing (A) pair plot, (B) score plot, (C) loading plot of different attributes of the treatments  on soil MBC, MBN, respiration, qCO2, AP, IAA content and wheat yield indices at different wheat growth period in the field experiment. Pair plot represents different explained variance ratio. Score plot represents separation of treatments. Loading plot shows the loading indices to PC1 and PC2. PC1, first principal component; PC2, second principal component. The abbreviations are as follows: MBC: microbial biomass carbon; MBN: Microbial biomass nitrogen; qCO2: Microbial metabolic quotient; AP: Soil available phosphorous; IAA: Indole-3-acetic acid; S: Seedling growth stage; E: Stem elongation stage; R: Ripening stage.


Mechanistically, ATRA directly binds in a deep hydrophobic pocket of the receptor binding domain (RBD) located on the top of the SARS-CoV-2 spike protein (S) trimer. The bound ATRA mediates strong interactions between the “down” RBDs and locks most of the S trimers in an RBD “all-down” and ACE2-inaccessible inhibitory conformation. In summary, our results reveal the pharmacological biotargets and structural mechanism of ATRA and other retinoids in SARS-CoV-2 infection and suggest that ATRA and its derivatives could be potential hit compounds against a broad spectrum of coronaviruses.Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent of the global pandemic and life-threatening coronavirus disease 2019 (COVID-19). Although vaccines and therapeutic antibodies are available, their efficacy is continuously undermined by rapidly emerging SARS-CoV-2 variants. Here, we found that all- Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the etiological agent of coronavirus disease 2019 (COVID-19), has caused a severe pandemic and public health threat worldwide 13. Increa5–trans retinaldehyde , which is further metabolized to all-trans retinoic acid (ATRA) by retinaldehyde dehydrogenases (trans-4-OH retinoic acid (AT-4-OH RA) and all-trans-4-OXO retinoic acid (AT-4-OXO RA) through cytochrome P450 family members  of the S trimer adapt “up” and “down” two distinct conformations, of which only the “up” RBDs bind the human host receptor protein hACE2 –12. Vita–13–ogenases . ATRA ca members .10.1128/mbio.01485-22.8TABLE S1Table S1, DOCX file, 0.04 MB.Primers for qRT-PCR. Download Copyright © 2022 Tong et al.2022Tong et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 50) of ATRA determined with Vero cells was approximately 4.07 μM , ATRA had the highest viricidal activity in these retinoids (cis retinoic acid (13-cis RA) (cis retinoic acid (9-cis RA)   and 9-ci-cis RA) . AltogetWe next assessed the antiviral effect of ATRA with a human colon carcinoma cell line (Caco-2)  and humaTo assess the infection stage inhibited by ATRA, we treated Caco-2 cells with ATRA 1 h before (pretreatment), simultaneously with (cotreatment), or 1 h after (postinoculation) SARS-CoV-2 inoculation . After tWe next exploited a SARS-CoV-2 S protein-mediated cell-cell fusion assay to assess the role of ATRA during SARS-CoV-2 cellular entry. The 293T cells that express the SARS-CoV-2 S protein were used as effector cells; 293T/hACE2 cells expressing human ACE2 were used as target cells. The effector cells aggregated around a target cell to form a typical syncytium  and F. HDK) of 3.44 × 10−6 M  and enzyme-linked immunosorbent assay (ELISA). ATRA directly interacted with the trimeric spike protein, with an equilibrium dissociation constant (× 10−6 M . Notably× 10−6 M  and coim× 10−6 M . To expl× 10−6 M  with 40 × 10−6 M  with preectively ) as init10.1128/mbio.01485-22.1FIG S1t test. Experiments were performed independently with at least three biological replicates with comparable results. Download FIG S1, TIF file, 1.4 MB.ATRA directly impaired the interaction between human ACE2 protein with the SARS-CoV-2 spike protein but not with RBD. (A) ATRA impaired the interaction between human ACE2 and spike proteins. The interaction between the SARS-CoV-2 spike and human ACE2 was evaluated by coimmunoprecipitation assay. (B) The interaction between the SARS-CoV-2 RBD and human ACE2 was not blocked by incubation with ATRA. The interaction between the SARS-CoV-2 RBD and human ACE2 was evaluated by an ELISA. About 0.1 μg/well of SARS-CoV-2 RBD and an hACE2-precoated microplate were used in the assay. The human ACE2 antibody served as a positive control. The data are presented as the mean ± SEM. The data were analyzed using an unpaired Copyright © 2022 Tong et al.2022Tong et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.01485-22.2FIG S2FIG S2, TIF file, 1.0 MB.Electron microscopy analysis of the ATRA- and DMSO-treated spike protein. The cryo-EM structural analysis pipeline is shown. Download Copyright © 2022 Tong et al.2022Tong et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.01485-22.3FIG S3FIG S3, TIF file, 1.4 MB.Cryo-EM structure validation. (A) FSC curves (top) and directional FSC curves (bottom) for the ATRA-treated spike (left panel) and the DMSO-treated spike (right panel) reconstructions with C3 symmetry imposed; (B) cryo-EM density map of the ATRA-spike (left panel) and DMSO-spike (right panel) colored according to local resolution; (C) densities and corresponding models from portions of the N-terminal domain (NTD) (blue), RBD (green), and S2 domain (pink). Residues are shown as sticks, with oxygen atoms colored red and nitrogen atoms blue. The cryo-EM density map is shown as gray mesh. The upper panel shows the ATRA-spike map/model, and the lower panel shows the DMSO-spike map/model. Download Copyright © 2022 Tong et al.2022Tong et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.01485-22.9TABLE S2Table S2, DOCX file, 0.1 MB.Cryo-EM data collection, refinement, and validation statistics. Download Copyright © 2022 Tong et al.2022Tong et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Additional elongated densities that did not belong to the polypeptide chain could be found near the contact interfaces among the “down” RBDs on top of the trimeric spike protein, which had one distal end embedded in a hydrophobic pocket in one RBD and the other distal end exposed outside the pocket and in contact with a neighboring RBD . These d10.1128/mbio.01485-22.4FIG S4FIG S4, TIF file, 1.4 MB.Binding affinity of retinol derivatives with the SARS-CoV-2 spike protein. The interaction between the SARS-CoV-2 spike and AT-4-OH retinoic acid (A), AT-4-OXO retinoic acid (B), AT-retinol (C), and AT-retinaldehyde (D) was determined by the SPR assay. The concentrations of chemicals that flowed over the chip surface ranged from 1 to 32 μM. The binding kinetics and affinity were analyzed by using the Biacore Insight Evaluation software . Download Copyright © 2022 Tong et al.2022Tong et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mbio.01485-22.5FIG S5FIG S5, TIF file, 1.6 MB.Structural comparison of ATRA- and DMSO-treated spike structures.  Superposition of S protomers of the ATRA-treated spike (in purple) and the DMSO-treated spike (in dim gray) shows consistency of the S2 domain but discrepancy in the RBD domain.  The S2 domain and RBD are illustrated as axes for comparison. (B) Orientation comparison of the RBD domains in the ATRA- and DMSO-treated spike structure; (C) measurements of the distances between centroids of RBDs. The relative rotation angle between the RBDs is indicated. Download Copyright © 2022 Tong et al.2022Tong et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the DK = 3.44 × 10−6 M), the carboxyl tail of the bound ATRA was essential for establishing strong interactions with neighboring RBDs. The RBDs of native spike proteins were supposed to be in a dynamic transformation between the “up” and “down” conformations. Once binding to the pocket, ATRA functioned like a bolt to lock the spike protein in a compact closed state and was unable to interact with ACE2.Retinol and AT-retinaldehyde had the same head and stem structure as ATRA, but differ only in the tail, of which retinol and AT-retinaldehyde had a hydroxyl group and a carbonyl group, respectively. Given that retinol and AT-retinaldehyde have much weaker binding affinity  to spikeTo further validate the structural model, we aimed to generate the spike proteins with single mutation at Arg408 to Ala (R408A) and Gln409 to Ala (Q409A), as well as the R408A Q409A double mutation. The mutants were expressed in HEK293F cells and purified with StrepTactin resins by following the same routine as for the wild-type S. Nonetheless, only the R408A mutant was expressed and purified successfully, while no expression was detected for either the Q409A or R408A Q409A mutant, suggesting that Q409 may play an essential role in folding and assembly of the spike . The com10.1128/mbio.01485-22.6FIG S6t test. Binding affinity of ATRA with R408A mutant protein and the RBD monomer is shown. The interaction between the ATRA and R408A mutant protein (C) and RBD monomer (D) was determined by the SPR assay. The concentrations of ATRA that flowed over the chip surface ranged from 1 to 32 μM. The binding kinetics and affinity were analyzed by using the Biacore Insight Evaluation software . Download FIG S6, TIF file, 1.8 MB.ATRA did not impair the interaction between mutant spike R408A and human ACE2. (A) SDS-PAGE analysis of the elution from StrepTactin resins. R408A, Q409A, and R408A Q409A (double mutation [DM]) mutants were expressed in HEK293F cells and captured by StrepTactin resins. The proteins captured by StrepTactin resins were then eluted with 2.5 mM desthiobiotin. No expression was detected for both Q409A and DM proteins. Only the R408A protein was detected by SDS-PAGE. (B) The interaction between the R408A mutant protein and human ACE2 protein was evaluated by an ELISA. Approximately 0.1 μg/well of R408A mutant protein and a microplate precoated with hACE2 were used in the assay. The human ACE2 antibody served as a positive control. The data are presented as the mean ± SEM. The data were analyzed using an unpaired Copyright © 2022 Tong et al.2022Tong et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Interestingly, linoleic acid (LA) was found to bind the same pocket of the spike proteins of SARS-CoV-2 and bat and pangolin coronaviruses in high-resolution cryo-EM structures –35. The –10.1128/mbio.01485-22.7FIG S7GAPDH. The data are presented as the mean ± SEM. The data were analyzed using an unpaired t test. Experiments were performed independently with at least three biological replicates with comparable results. Download FIG S7, TIF file, 1.2 MB.ATRA inhibited the infection of other coronaviruses. (A) ATRA presented general inhibitory activity against the entry process of SARS-CoV in 293T/hACE2 cells (green line) and MERS-CoV in Huh7 cells (blue line). The effectiveness of spike-mediated viral entry was determined by a luciferase assay. The gray dotted line represents the value of the 50% inhibition rate. The data are presented as the mean ± SEM. (B) Incubation with ATRA did not inhibit the infection of the IAV H1N1 WSN strain in A549 cells. The amount of viral RNA was normalized to human Copyright © 2022 Tong et al.2022Tong et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Retinoids, a group of compounds including vitamin A and its active metabolite ATRA, regulate serial physiological activity in multiple organ systems, such as cell growth, differentiation, and apoptosis –38. Inde–39–43–Human airway biopsy specimens were collected with written informed consent from pulmonary bulla patients with approval of the local ethics committee at Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, and No. 7 Affiliated Hospital of Zhongshan University.2. The 293T cells expressing human ACE2 (293T/hACE2) were a gift from Qiang Ding from Tsinghua University and were cultured under the same conditions. SARS-CoV-2 (GISAID accession no. EPI_ISL_413879) was propagated in Vero cells and titrated by a standard plaque-forming assay followed by storage of aliquots at −80°C until further use in the experiments. Influenza A virus (IAV) H1N1 A/WSN/33 strain was passaged in MDCK cells cultured in DMEM with 0.3% FBS and 0.5 mg/mL TPCK -treated trypsin . Plasmids expressing the SARS-CoV-2 wild-type spike protein (pcDNA3.1-SARS-2-S) were kindly provided by Lu Lu from Fudan University. Plasmids expressing the spike protein of SARS-CoV-2 variants pcDNA3.1-B.1.1.7-S, pcDNA3.1-P.1(501Y. V3)-S, pcDNA3.1-B.1.351(20HY)-S, pcDNA3.1-B.1.351-V1-S, pcDNA3.1-B.1.351-V2-S, pcDNA3.1-B.1.351-V3-S, pcDNA3.1-B.1.617.2-S, pcDNA3.1-B.1.1.529-S, and pNL4-3.luc.RE (the luciferase reporter-expressing HIV-1 backbone) was maintained in our laboratory. Plasmids expressing the spike protein of SARS-CoV and MERS-CoV were kindly provided by Linqin Zhang from Tsinghua University.Vero, Caco-2, 293T, and MDCK cells were purchased from ATCC , cultured in Dulbecco’s modified Eagle’s medium (DMEM)  supplemented with 10% heat-inactivated fetal bovine serum (FBS) , and 1% antibiotic-antimycotic  at 37°C and 5% COCompounds were dissolved in dimethyl sulfoxide (DMSO) and subsequently diluted with cell culture medium for cell incubation. Vero cells were preincubated with serial concentrations of each compound at 37°C for 1 h and then infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.01 for 1 h. After three washes with PBS, cells were cultured with compound-containing medium for an additional 48 h. Vero cells were harvested, and the viral burden was detected by qRT-PCR.GAPDH gene (GenBank accession no. NC_000012.12).Total RNA was isolated from infected cells using a Multisource RNA miniprep kit  and reverse transcribed to cDNA using an iScript cDNA synthesis kit . The viral burden was quantified with qRT-PCR using iTaq Universal SYBR green supermix  on the Bio-Rad CFX-96 Touch real-time detection system. Primer sequences are shown in For the analysis of the antiviral activity of compounds, cells were preincubated with compounds, infected, and incubated for 48 h prior to immunofluorescence staining. For the cell-cell fusion assay, 293T/hACE2 cells were incubated with 293T cells transfected with the SARS-CoV-2 spike protein in the presence or absence of ATRA. The cocultured cells were incubated for 24 h, and the state of cell fusion was observed by microscopy and immunofluorescence staining.Cells were washed three times in PBS and fixed with 4% paraformaldehyde (PFA) for 2 h at room temperature. Then, the cells were permeabilized with 0.1% Triton X-100 for 30 min, washed with PBS, blocked with 2% bovine serum albumin (BSA) for 1 h, and stained with anti-SARS-CoV-2 nucleocapsid , anti-SARS-CoV-2 spike , and anti-hACE2  antibodies for 2 h at room temperature, according to different experimental settings. The cells were washed with PBS and then stained with secondary antibodies for 1 h at room temperature. The nuclei were stained with DAPI  . Images were examined by a Zeiss LSM 880 Meta confocal microscope.4 cells per well and incubated for 12 h before the experiment. Compounds were diluted with cell culture medium at concentrations ranging from 0.78 to 50 μM and preincubated with cells at 37°C for 1 h. Cells were infected with SARS-CoV-2 at an MOI of 0.01 for 1 h, washed three times with PBS, and cultured with compound-containing medium for 48 h. The viral titer in the supernatant was determined by a plaque-forming assay. The concentration of each compound required to inhibit viral infection by 50% (IC50) was calculated by comparing the values with the DMSO-treated cells using the dose-response inhibition model in GraphPad Prism 8.0 .Vero cells were seeded in a 48-well plate at a density of 2 × 1050) was calculated by comparing the values with DMSO-treated cells using a sigmoidal nonlinear regression function in GraphPad Prism 8.0 .Cell viability was estimated by the Cell Counting Kit-8 (CCK-8) . Briefly, 10,000 Vero cells were seeded in a 96-well plate, incubated at 37°C overnight, and then administered gradient concentrations of compounds at 37°C for 48 h. The CCK-8 solution was added, followed by an additional 1-h incubation at 37°C. Absorbance was measured at 450 nm. The concentration of each compound necessary to reduce cell viability by 50% , anti-SARS-CoV-2 nucleocapsid , anti-hACE2 , and antiactin .Caco-2 cells were seeded in a 6-well plate at a density of 5 × 10Air-liquid interface (ALI) human epithelial cultures were kindly provided by Liang Li from the Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences. ALI human epithelial cultures were used to evaluate antiviral activity at day 28. Briefly, ALI cultures were preincubated with ATRA at 37°C for 30 min, infected with 5,000 PFU of SARS-CoV-2, and incubated at 37°C for 1 h. The inoculum was removed, the cultures were washed three times with PBS, and ATRA was added to the Transwell for further incubation. At 24 h and 48 h postinfection, 150 μL of PBS was added to the top surface of ALI cultures, and the cells were then harvested for viral genome copy determination. The epithelia were harvested for viral burden quantification at 48 h postinfection.2O2 in methanol for 20 min. After undergoing blocking for 30 min at room temperature, the sections were incubated with the primary antibody for 2 h at 37°C, followed by detection using a horseradish peroxidase (HRP)-conjugated secondary antibody and TSA-dendron-based fluorophores. Subsequently, the sections were incubated in retrieval/elution buffer at 95°C for 10 s to eliminate the primary and secondary antibodies. The following antibodies were used in this study: anti-β-IV-tubulin , anti-MUC5AC , anti-p63 , antipodoplanin , and anti-SPC . After all the antibodies were detected sequentially, the epithelial sections were stained with DAPI  and cleared by the clearing-enhanced three-dimensional (3D) method. Images were captured by a Zeiss LSM 880 Meta confocal microscope.ALI cultures were fixed in 4% PFA, and paraffin sections (3 μm thick) were prepared routinely. Sections were treated with xylene and a graded series of alcohol solutions and incubated in citrate buffer (pH 6.0) and microwaved at 95°C for 20 min for antigen retrieval. Sections were stained with hematoxylin and eosin for histological analysis. The NEON 7-color Allround Discovery kit for formalin-fixed, paraffin-embedded (FFPE) sections  was used for multiplex fluorescence labeling. Briefly, sections were quenched for endogenous peroxidases in 3% HTo estimate the impact of ATRA on the SARS-CoV-2 replication cycle, Caco-2 cells were infected at an MOI of 0.01 for 1 h at 37°C. ATRA was added 1 h before (pretreatment), simultaneously with (cotreatment), or 1 h after (posttreatment) SARS-CoV-2 inoculation. For the pretreatment assay, cells were first incubated with ATRA at 37°C for 1 h, followed by three washes with PBS, and then infected with SARS-CoV-2 for 1 h. For the cotreatment assay, cells were simultaneously incubated with SARS-CoV-2 and ATRA for 1 h at 37°C. Then, the mixture was removed, and the cells were washed three times with PBS before fresh medium was added. For the posttreatment assay, cells were infected with SARS-CoV-2 for 1 h, washed three times with PBS, and then incubated with ATRA-containing medium for 1 h. At 48 h postinfection, cells were harvested for viral burden detection by qRT-PCR.To determine the specific stage of the viral entry process affected by ATRA, a time-of-addition assay was performed. Briefly, for the attachment assay, Caco-2 cells were precooled on ice and then incubated with a mixture of ATRA and SARS-CoV-2 for 1 h at 4°C, followed by three washes with precooled PBS and incubation at 37°C for 48 h for viral burden detection. For the internalization assay, precooled Caco-2 cells were infected with SARS-CoV-2 at 4°C for 1 h, washed three times with PBS, and then incubated with ATRA at 37°C for 1 h. The cells were then washed and cultured in fresh medium for 48 h for viral burden detection.g for 10 min, and frozen at −80°C until further use in the experiments. Pseudotyped viruses were incubated with serial dilutions of ATRA for 30 min at 37°C, and then 40,000 Caco-2 or HEK293T/hACE2 cells were added to each well. Plates were incubated at 37°C for 48 h, washed with PBS, and lysed with 1× passive lysis buffer . Luciferase activity was analyzed using a luciferase assay system .Pseudotyped viruses were generated by the cotransfection of pNL4-3.luc.RE backbone plasmid and pcDNA3.1-SARS-2-S, pcDNA3.1-B.1.1.7-S, pcDNA3.1-P.1(501Y.V3)-S, pcDNA3.1-B.1.351(20HY)-S, pcDNA3.1-B.1.351-V1-S, pcDNA3.1-B.1.351-V2-S, pcDNA3.1-B.1.351-V3-S, pcDNA3.1-B.1.617.2-S, pcDNA3.1-B.1.1.529-S, pcDNA3.1-SARS-S, or pcDNA3.1-MERS-S plasmid to HEK293T cells. The medium was changed 12 h after transfection. At 48 h after transfection, the supernatant was harvested, centrifuged at 3,000 × For expression of the prefusion S ectodomain, a gene encoding residues 1 to 1208 of SARS-CoV-2 (GenBank accession no. MN908947) spike with proline substitutions at residues 986 and 987, a “GSAS” substitution at the furin cleavage site (residues 682 to 685), a C-terminal trimerization motif, an HRV3C protease cleavage site, and a Twin-Strep-tag were synthesized and cloned into the mammalian expression vector pCMV. For mutant plasmid construction, R408, Q409, and R408 Q409 were substituted with alanine. The constructed plasmid was confirmed by sequencing and used in transient transfection of HEK293F cells with polyethylenimine. At 48 h posttransfection, protein was purified from filtered and buffer-exchanged cell culture medium using StrepTactin resin (IBA). The eluted protein was further purified by size exclusion chromatography (SEC) with a Superose 6 10/300 column  in 20 mM HEPES (150 mM NaCl [pH 7.5]). Peak fractions from SEC were analyzed by SDS-PAGE, flash frozen with liquid nitrogen, and stored at −80°C until further use.2HPO4, 18 mM KH2PO4, 27 mM KCl, 1.37 M NaCl, 5% DMSO, 0.05% Tween 20 [pH 7.4]). Chemicals were dissolved and diluted with buffer A. Concentrations of chemicals ranging from 1 μM to 32 μM flowed over the chip surface. The binding kinetics and affinity were analyzed using Biacore Insight Evaluation software .The purified SARS-CoV-2 spike protein was captured on a CM5 sensor chip  with approximately 8,000 response units in the test flow channels, and a blank channel was employed as a negative control. The SPR experiments were performed using a Biacore 8K system at 25°C with a flow rate of 30 μL/min in buffer A , which contained an hACE2-precoated microplate. Briefly, 0.1 μg/well of SARS-CoV-2 spike protein with a streptavidin tag or the horseradish peroxidase (HRP)-conjugated recombinant SARS-CoV-2 RBD fragment was incubated with ATRA at 37°C for 30 min. DMSO was used as a negative control, and the hACE2 antibody was used as a positive control. The mixture was then added to the precoated microplate at 37°C for 15 min, and the unbound protein was removed by three washes with the wash buffer provided with the kit. For the spike binding ELISA, HRP-labeled streptavidin  was added at a dilution of 1:4,000, and the mixture was incubated at 37°C for 45 min. After four washing steps, TMB  solution was added to the microplate, and the mixture was incubated at room temperature. Then, the stop solution was added, and the absorbance was detected at 450 nm.g for 2 min and washed with 500 μL immunoprecipitation (IP) lysis buffer  6 times. The pellet was resuspended in 30 μL protein loading buffer and heated at 100°C for 10 min. Samples were then subjected to Western blot analysis.Purified human ACE2 protein with a Flag tag and purified spike protein with Twin-Strep-tag were used in the coimmunoprecipitation assay. Briefly, 2 μg of spike protein was preincubated with or without ATRA at 37°C for 30 min. The mixture was then added to 2 μg human ACE2 protein at 4°C for 2 h. About 5 μL anti-Flag antibody  was added, and the mixture was incubated for 2 h at 4°C. The mixture was incubated with 20 μL prewashed protein A/G agarose  at 4°C for 5 h with gentle mixing. The immunoprecipitated complexes were collected by centrifugation at 3,000 × ATRA, dissolved in DMSO at a concentration of 100 mM, was added to the purified SARS-CoV-2 spike protein (approximately 0.5 mg/mL), mixed thoroughly to a final concentration of 40 μM, and incubated at room temperature for 30 min. An identical volume of DMSO was added to another copy of the spike protein. The protein was then cross-linked with 0.015% glutaraldehyde on ice for 20 min. Tris stock buffer (2 M [pH 7.5]) was added to stop the cross-linking reaction. Four microliters of the spike-DMSO or spike-ATRA sample was applied onto a freshly glow-discharged Quantifoil R1.2/1.3 carbon grid. The grids were blotted for 10 s at 100% humidity and 8°C before flash freezing in liquid ethane with Vitrobot MarkIV 20 (Thermo Fisher Scientific). Data were acquired on a Titan Krios electron microscope (FEI Company) equipped with a field emission gun operated at 300 kV and equipped with a Gatan K3 Summit direct detector. Data were collected in superresolution mode at a nominal magnification of 29,000× with a virtual pixel size of 0.97 Å. A total of 4,049 micrographs of spike-ATRA and 2,089 micrographs of spike-DMSO were collected. The defocus range was set between −0.5 and −1.5 μm. A full description of the cryo-EM data collection parameters can be found in EMD-11203, EMD-11205 and EMD-11206, respectively [https://3dfsc.salk.edu) (The beam-induced motion of each micrograph was corrected with MotionCor2 . The defectively ) were usalk.edu) . The loc6VXX [UCSF Chimera  and Coot6VXX ) into tht test. Statistical analyses were carried out using GraphPad Prism 8.0. P values of <0.05 were considered significant.Descriptive statistics are provided in the figure legends. Analyses of independent data were performed by using the unpaired EMD-33600, and the accompanying atomic coordinates have been deposited in the Protein Data Bank (PDB) under accession no. 7Y42.All data are available in the main text or the supplemental material. The electron microscopy (EM) map has been deposited in the Electron Microscopy Data Bank (EMDB) under accession no.