Leishmania donovani DD8, Trypanosoma brucei brucei and Trypanosoma cruzi) using phenotypic assays. The aim of this study was to identify hit compounds, with a focus on improved efficacy, selectivity and potential to target several kinetoplastid parasites. The IC50 values of the natural products were obtained for L. donovani DD8, T. b. brucei and T. cruzi in addition to cytotoxicity against the mammalian cell lines, HEK-293, 3T3 and THP-1 cell lines were determined to ascertain parasite selectivity. Thirty-one compounds were identified with IC50 values of ≤10 µM against the kinetoplastid parasites tested. Lissoclinotoxin E (1) was the only compound identified with activity across all three investigated parasites, exhibiting IC50 values <5 µM. In this study, natural products with the potential to be new chemical starting points for drug discovery efforts for kinetoplastid diseases were identified.Kinetoplastid parasites cause vector-borne parasitic diseases including leishmaniasis, human African trypanosomiasis (HAT) and Chagas disease. These Neglected Tropical Diseases (NTDs) impact on some of the world’s lowest socioeconomic communities. Current treatments for these diseases cause severe toxicity and have limited efficacy, highlighting the need to identify new treatments. In this study, the Davis open access natural product-based library was screened against kinetoplastids ( Leishmania and Trypanosoma. Parasites belonging to the genus Leishmania are the causative agents of leishmaniasis, while Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense are the causative agents of human African trypanosomiasis (HAT). The other parasite in the genus Trypanosoma is Trypanosoma cruzi, which is responsible for Chagas disease. Around 20 million individuals are infected with kinetoplastid pathogens worldwide leading to 95,000 deaths per year  which were both initially purified from a didemnid ascidian thiazine-5,10-dione 1,1-dioxide (9) and psammaplysin F (14) have novel activity against T. cruzi parasites.On the basis of the information currently available, it seems fair to suggest that thiaplakortone A (3) is a thiazine-derived alkaloid, initially isolated from the marine sponge Plakortis lita indole-5,9-dione 4,4-dioxide  showed activity against both intracellular and extracellular forms of L. donovani DD8. Compound 12 and 13 displayed IC50 values of 11.09 ± 0.311 and 5.65 ± 0.26 µM against L. donovani DD8 intracellular amastigotes. Both of these compounds showed no significant activity against either T. b. brucei or T. cruzi. Compound 13 has shown high selectivity for L. donovani DD8 intracellular amastigotes in relation to HEK-293 cells. Compound 13 was previously reported to have moderate activity against both L. donovani (MHOM/SD/62/1S-CL2D) axenic amastigotes and T. b. brucei variant 221 with IC50 values of 16.75 ± 4.35 and 21.11 ± 3.68 µM, respectively [13 also caused a 96 ± 2% reduction in infection of L. mexicana intracellular amastigotes at 41.89 µM [L. donovani DD8 when compared to other strains [13 for Leishmania species; however, differences in assays may also play a role. Compound 13 is a promising hit as it fulfils the TCP criteria set by DNDi for anti-leishmanial drug discovery. It will be interesting to further explore this compound by synthesising analogues and assessing structure activity relationships.Compounds ectively . Compoun41.89 µM . Previou41.89 µM . An exam strains . This ma8), emetine dihydrochloride (10), and chelerythrine chloride (15) which belong to this sub-group were shown to have activity against L. donovani DD8, T. b. brucei and T. cruzi serving as internal controls. The 50% inhibitory concentration observed for mefloquine HCl (8) was 0.62 ± 0.06 and 3.96 ± 0.58 µM for T. b. brucei and T. cruzi, respectively, with moderate selectivity for T. b. brucei. Mefloquine has previously been shown to have activity against T. b. brucei in vitro and in vivo [50 of 6.1 µM against T. cruzi, with a SI of 2 to 3T3 host cells [T. cruzi activity, although higher doses would be needed to confirm the selectivity. We have previously identified this as active against T. cruzi from the Pathogen Box collection of compounds . In this study, an IC50 value could also not be determined against host cells.A small percentage (5%) of the library is comprised of commercial or synthetic compounds inspired by natural products. Mefloquine HCl ( in vivo  and has st cells . Herein 10) was found to have activity against both T. b. brucei and T. cruzi with an IC50 of 0.05 ± 0.01 and 0.09 ± 0.01 µM, respectively. This compound has shown no selectivity for HEK-293 cells in relation to T. b. brucei and T. cruzi but has shown moderate selectivity for T. cruzi in relation to 3T3 cells, indicating increased cytotoxicity against replicating cells. This compound was previously used as an antiprotozoal drug for the treatment of amoebic liver abscess and invasive intestinal amoebiasis [T. b. brucei [50 value was 7.9 µM, however it also displayed poor selectivity (3.8) toward MRC5 cells. No data is currently reported in the literature for activity of this compound against T. cruzi.Emetine dihydrochloride  exhibited an IC50 of 0.23 ± 0.04 µM against T. b. brucei with good selectivity of >18 fold for HEK-293 cells. Chelerythrine was previously discovered following testing as an oncology drug candidate [50 of 1.3 µM against T. b. brucei [Chelerythrine chloride  (THP-1 TIB-202™) were maintained in RPMI (1X) Roswell Park Memorial Institute Medium 1640 GlutaMAX™  and 10% (v/v) heat inactivated fetal bovine serum (HIFBS)  at 37 °C in a humidified atmosphere with 95% air and 5% CO2. The cells were passaged every 48 hours to maintain cell density between 2.5 × 105 and 8 × 105 cells/mL.THP-1 (human monocytic leukemia) cells obtained from American Type Culture Collection (ATCC® CCL92) were maintained in RPMI  with no phenol red, supplemented with 10% HIFBS (growth medium).3T3 host cells  were maintained in DMEM  supplemented with 10% (v/v) HIFBS at 37 °C in a humidified atmosphere of 95% air and 5% CO2. The cells were passaged as they reach 80% confluence.HEK (human embryonic kidney) 293 cells obtained from the ATCCL. donovani (MHOM/IN/80 DD8) promastigotes obtained from ATCC®, USA (ATCC® 50212™) were cultivated at 27 °C in Medium 199 (1X) (Gibco®-Life Technologies) supplemented with 100 µM adenosine (CAS Number 58-61-7) , 23 mM folic acid (CAS Number 59-30-3) , 2 mM L-glutamine 200 mM (Gibco®-Life Technologies) and 1% (v/v) BME vitamin mix 100X (Sigma-Aldrich) and 10% (v/v) HIFBS. The pH of the media was adjusted to 6.8 with 1 M hydrochloric acid (HCl) before being filter sterilised with a cellulose acetate membrane filter, pore size 0.22 µM .T. b. brucei bloodstream form strain 427 (BS427), a surrogate model for human infective species commonly used for the determination of compound activity, was kindly supplied by Dr. Achim Schnaufer,  whilst at the Seattle Biomedical Research Institute  and was cultivated in HMI-9 media at 37 °C in a humidified atmosphere with 95% air and 5% CO2, according to previosuly published protocols [rotocols ,88.T. cruzi Tulahuen epimastigotes were generously provided by Professor Frederick Buckner . The parasites were maintained in liver infusion tryptose (LIT), with preparation as described by Sykes and Avery [6 cells/mL respectively in to 10 mLof media in 25 cm2 flasks. Cells were grown at 27 °C. Parasites were differentiated into infective trypomastigotes in liquid medium before sub-culturing in 3T3 host cells, as described by Sykes and Avery [nd Avery . Cells wnd Avery .The Davis open access natural product-based library currently consists of 472 distinct compounds, the majority (53%) of which are natural products that have been obtained from Australian natural sources, such as endophytic fungi , plants www.compoundsaustralia.com), was dispensed into microtitre plates as 5 mM DMSO solutions. Library compounds were either isolated in quantities ranging from 0.2 mg to >50 mg or purchased from commercial suppliers. The natural product isolation procedures or semi-synthetic studies for the majority of compounds in this unique library have been previously published [The Davis open access natural product-based library housed within Compounds Australia  at final assay concentrations of 20 µM and 16.7 µM for the L. donovani intracellular amastigote and promastigote viability assays, respectively. Primary screening against T. b. brucei was undertaken at 5 µM and for T. cruzi at 10 µM final concentrations. Actives were defined as compounds which exhibited >70% activity at 20 µM and 16.7 µM for L. donovani DD8 intracellular amastigote and promastigote assays, respectively. Compounds exhibiting >50% activity at 5 µM for T. b. brucei and showing ≥50% activity at a concentration of 10 µM for T. cruzi intracellular amastigote were considered active. These compounds were then retested in a 14 point format to determine the IC50 value against the parasite, and against HEK-293, THP-1 and 3T3 cells to determine the selectivity to the parasite. To determine IC50 values, 14 point dose response curves with top concentrations of 20 µM and 16.7 µM were used for the L. donovani DD8 intracellular amastigote and promastigote viability assays, respectively. A top dose of 20 µM was used in the T. b. brucei assay and 18.3 µM for T.cruzi assay, in 14 point dose repsonse curves as described.For the primary screen, compounds were prepared as a stock concentration of 5 mM in 100% DMSO and tested in duplicate points for two independent experiments  HIFBS) and incubated at 27 °C. After 96 h the parasites reached the mid-log phase and were seeded in black, clear bottom 384-well plates  at a concentration of 5 × 105 parasites/mL in a volume of 55 µL using a Bravo automated liquid handling platform . Compound stocks were diluted 1:25 in M199 media without HIFBS using a dilution plate . The compounds were dispensed in 5 µL using a Bravo and parasites incubated for an additional 68 h at 27 °C. Resazurin  at 0.142 mM final assay concentration was then dispensed into plates at a volume of 10 µL/well using a Multidrop™ 384 Reagent Dispenser  and incubated at 27 °C. The plates were read after 4 h on an EnVision™ Multilabel plate reader  using fluorometry with an excitation of 530 nm and emission 590 nm. For the L. donovani DD8 promastigote viability assay, 1 µM of amphotericin B was used as positive control and 0.4% DMSO as negative control.This assay was established to assess the activity of the compound library on the extracellular form of the parasites. An initial cell density of 1 × 10T. b. brucei bloodstream form was evaluated using a resazurin-based assay, previously described in the literature [The efficacy of compounds against terature .T. cruzi was undertaken using a phenotypic, high-throughput image-based assay as previously described [The efficacy of compounds against escribed .Cytotoxicity testing was undertaken as previously described .1 displayed in vitro activity against, L. donovani DD8, T. b. brucei and T. cruzi with good selectivity against T. b. brucei. Further exploration of the activity of analogues of this compound is warranted to improve anti-parasitic selectivity. Target identification for the most promising compounds will support the future development of pan-active treatments against kinetoplastids. Further compounds from this collection have potential as new chemical starting points for drug discovery efforts against one or more of the parasites tested.The present study identifies several compounds with novel anti-kinetoplastid activities from phenotypic screening of 472 distinct compounds from the Davis open access natural product-based library. Compound


Western blotting and dual-luciferase activity assays showed that ectopic NANOG expression significantly upregulated PBX1 expression and increased PBX1 promoter activity. In contrast, PBX1 knockdown by RNA interference in hair follicle- (HF-) derived MSCs that were ectopically expressing NANOG resulted in the significant downregulation of p-AKT and the upregulation of p16 and p21. Moreover, blocking AKT with the PI3K/AKT inhibitor LY294002 or knocking down AKT via RNA interference significantly decreased PBX1 expression, while increasing p16 and p21 expression and the number of SA-β-gal-positive cells. In conclusion, our findings show that NANOG delays HF-MSC senescence by upregulating PBX1 and activating AKT signaling and that a feedback loop likely exists between PBX1 and AKT signaling.Stem cells derived from elderly donors or harvested by repeated subculture exhibit a marked decrease in proliferative capacity and multipotency, which not only compromises their therapeutic potential but also raises safety concerns for regenerative medicine. NANOG—a well-known core transcription factor—plays an important role in maintaining the self-renewal and pluripotency of stem cells. Unfortunately, the mechanism that NANOG delays mesenchymal stem cell (MSC) senescence is not well-known until now. In our study, we showed that both ectopic NANOG expression and PBX1 overexpression (i) significantly upregulated phosphorylated AKT (p-AKT) and PARP1; (ii) promoted cell proliferation, cell cycle progression, and osteogenesis; (iii) reduced the number of senescence-associated- Mesenchymal stem cells (MSCs)—which are characterized by self-renewal, multipotency, nontumorigenic properties, immune-privilege, and rich sources —are consDnmt1 promoter [KIP1. These experimental data suggest that the transcription factor NANOG plays a vital role in maintaining self-renewal and in pluripotent or multipotent differentiation in stem cells, while suppressing spontaneous senescence during stem cell culture.NANOG, a homeodomain transcription factor, is expressed in the inner cell mass of embryonic stem cells (ESCs) during early embryonic development and the developing germlines of mammals. In conjunction with Oct4 and Sox2, NANOG plays an important role in maintaining stem cell pluripotency and self-renewal and in reprogramming human somatic cells into pluripotent stem cells . In concpromoter . Studiespromoter –12. Furtpromoter  showed tMyogenin promoter [Runx2 and Osterix, while decreasing the proliferation of osteoblasts and the formation of bone nodules [NANOG and PBX1. By directly binding the proximal promoter of NANOG, PBX1 upregulates the expression of endogenous NANOG protein in human ESCs, which may activate downstream cell signaling cascades [PBX1 transcription in both human ESCs and NIH3T3 cells [PBX1, a homeodomain transcription factor from the three-amino acid loop extension family, is involved in various developmental processes such as organogenesis and hematopoiesis , 15. In promoter  and regu nodules . Moreove nodules . Studies nodules  found ancascades . MoreoveT3 cells , 23. HowThe phosphatidylinositol 3 kinase (PI3K)/protein kinase B  pathway is considered to be a key pathway in the regulation of cell metabolism-associated processes including proliferation, DNA repair, and senescence , 25. OurHowever, the cellular senescence that results from organismal aging or cell culture exerts considerable effects on the quantity and quality of MSCs, which not only compromises the therapeutic potential of the MSCs but also raises safety concerns for stem cell-based regenerative medicine. As such, it is necessary to develop novel strategies for maintaining MSCs in a highly proliferative but undifferentiated state. To this end, we isolated MSCs from hair follicles and transduced them with lentiviral vectors to express NANOG or PBX1. Our research shows that the ectopic expression of NANOG promotes cell proliferation and delays HF-MSC senescence by enhancing PBX1 expression and activating downstream AKT signaling. Moreover, we uncovered a previously unknown positive feedback loop between PBX1 and AKT in these cells.The protocols for all experiments in the present study were approved by the Institutional Review Board of the Jilin University School of Public Health.2. The culture medium was changed every 3 days. After spindle-shaped cells migrated out from the hair follicles and reached 80% confluency, cells were digested with 0.25% trypsin and subsequently subcultured . Immunofluorescence assays and flow cytometry [The isolation and culture of HF-MSCs were performed according to previously published methods , 29, 30.ytometry  were theNANOG coding region (Accession No. NM_024865) was first cloned into the pLVX-IRES-ZsGreen1 lentivirus vector .The human PBX1 forward (5′-GGAGGGTTTCTCTCCCAACG-3′) and reverse (5′-GGGAGTCTAGGACAGAGGCA-3′) primers were synthesized by Sangon Biotech , and PBX1 cDNA was obtained by PCR amplification  according to the manufacturer's protocols. After enzymatic digestion and ligation, the human PBX1 coding region (Accession No. NM_002585) was cloned into the pLVX-IRES-ZsGreen1 lentiviral vector .6 293 T cells  were seeded in a 100 mm cell culture dish  and cultured at 37°C with 5% CO2 in High Glucose DMEM  containing 10% FBS  overnight. The following day, 10 μg of lentivirus vector encoding NANOG or PBX1, 7.5 μg of psPAX2-gag/pol/tat/rev, and 2.5 μg of pMD2.G-VSVG were mixed with 22 μl of EndoFectin™ Max Transfection Reagent  in 1 ml of Opti-MEM  and added onto 293 T cells one drop at a time. After 6 h, the medium was replaced with fresh High Glucose DMEM containing 10% FBS and cultured for another 36 h. At the end of the cultivation period, the supernatant was harvested, filtered through a 0.45 μm polystyrene filter , and added to HF-MSCs supplemented with 10% FBS, 2 ng/ml bFGF  and 10 μg/ml polybrene . Subsequently, 72 h after transduction, the HF-MSCs were observed using an inverted fluorescence microscope  and Western blotting was performed to assess the expression of NANOG or PBX1.Next, 6 × 10μM dexamethasone , 0.5 mM isobutyl-methylxanthine , 10 μM insulin , and 200 μM indomethacin . After the cultivation period, cells were stained with Oil Red O  to evaluate the formation of lipid droplets and were subsequently imaged using an inverted fluorescence microscope equipped with a digital camera . To quantify adipogenesis, 200 μl of isopropanol  was added to each of the 24 wells to dissolve the Oil Red O at room temperature. After 20 min, OD values were measured at a maximum absorption wavelength of 512 nm using a microplate reader , and the concentration of Oil Red O was calculated based on a standard curve that had been created by measuring the optical density of Oil Red O using serial dilutions [For adipogenic differentiation assays, HF-MSCs ectopically expressing NANOG or overexpressing PBX1, and empty vector (control) were cultured in 24-well plates (in triplicate) for 2 weeks in adipogenic differentiation medium consisting of High Glucose Dulbecco's Modified Eagle Medium  with 10% FBS , 1 ilutions .β-glycerophosphate for 4 weeks at 37°C with 5% CO2. At the end of the cultivation period, cells were stained with Alizarin Red S  to assess the formation of mineralized nodules, and cells were imaged using an inverted fluorescence microscope equipped with a digital camera . To quantify osteogenesis, 200 μl of 10% cetylpyridinium chloride  was added to each of the 24 wells to dissolve the calcium salt–Alizarin Red S complex at 37°C for 30 min. The OD values of this complex were measured at a maximum absorption wavelength of 558 nm using a microplate reader, and the concentration of calcium salt–Alizarin Red S complexes was calculated based on a standard curve created by measuring the optical density of a CaCl2-Alizarin Red S gradient in 10% cetylpyridinium chloride [For osteogenic differentiation assays, HF-MSCs ectopically expressing NANOG or overexpressing PBX1, and empty vector (control) were cultured in 24-well plates (in triplicate) in osteogenic differentiation medium consisting of High Glucose DMEM  with 10% FBS , 0.1 mM dexamethasone , 50 mM ascorbate-2-phosphate , and 10 nM chloride .4 cells per well in a 24-well plate with culture medium containing DMEM/F12 and 10% FBS without bFGF and cultured for 5 days. Cells were washed with PBS, digested with 0.25% trypsin, and counted using a hemocytometer  at each time point.To assess cell proliferation, HF-MSCs were seeded at 2 × 106 HF-MSCs transduced with PBX1 (PBX1 group), NANOG (NANOG group), or empty vector (vector group) were harvested and fixed in 70% ice-cold ethanol at −20°C overnight. The following day, HF-MSCs were washed three times with PBS and incubated in 500 μl of propidium iodide with RNase  for 15 min at room temperature in the dark, subsequently subjected to flow cytometry  and analyzed using CellQuest Pro software . The cell proliferation index (PI) was calculated as follows: PI = (S + G2/M)/(G0/G1 + S + G2/M) × 100% [For cell cycle assays, 1 × 10) × 100% .β-gal activity assays using a cellular senescence staining kit  according to the manufacturer's instructions. After HF-MSCs reached 80% confluency in a 24-well plate, cells were fixed with the fixative from the kit for 15 min at room temperature and washed using PBS. Next, cells were cultured in Staining Solution Mix overnight at 37°C without CO2. The following day, cells were washed twice with PBS, and SA-β-gal positive cells were observed and calculated based on three randomly selected bright fields (containing at least 200 cells per field).The detection of cellular senescence was performed by SA-5 HF-MSCs were seeded in a 10 mm cell culture dish with DMEM/F12 medium supplemented with 10% FBS and 2 ng/ml bFGF. When cell confluency reached 80%, cells were digested and washed twice with cold PBS. Then, cells were lysed in 250 μl RIPA  supplemented with 1% protease inhibitor cocktail  and 1% phosphatase inhibitor cocktail  on ice for 30 min, and subsequently centrifuged at 15,000 g for 20 min at 4°C. Next, the concentration of the supernatant was examined using an Enhanced BCA Protein Assay Kit , and 15 μg of protein from each sample was loaded in each well and separated by 10% SDS polyacrylamide gel electrophoresis. Proteins were then transferred from the gels to polyvinylidene difluoride membranes , and membranes were incubated in 5% nonfat milk powder  with tris-buffered saline containing Tween 20 (TBST) at room temperature for 1 h. Next, blots were incubated with primary antibodies in Primary Antibody Dilution Buffer  according to the stated antibody dilution ratio. The primary and secondary antibodies used were as follows: AKT (pan) (C67E7) rabbit mAb , phospho-AKT (Ser473) rabbit mAb , NANOG rabbit mAb , PBX1 rabbit mAb , p16 rabbit mAb , p21Waf1/Cip1 (12D1) rabbit mAb , p53 mouse mAb , PARP1 rabbit mAb , GAPDH mouse mAb , HRP-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) , and HRP-conjugated AffiniPure Goat Anti-Mouse IgG (H+L) . Images were obtained using a chemiluminescence imaging analysis system , and band intensity was analyzed with Tanon Gis analytical software .For Western blotting, 6 × 10PBX1 promoter pGL3-Basic vector (0.9 μg/well) and pRL-TK vector . The empty pGL3-Basic vector (0.9 μg/well) and pRL-TK vector (0.1 μg/well) were used as the control conditions. Transfection reagent (1.1 μl) was added to each well . After 6 h, the medium was replaced with fresh DMEM/F12 and 10% FBS. The cells were lysed and the supernatant was then harvested, and relative light units (RLUs) were measured after 36 h using the Cytation 3 Cell Imaging Multi-Mode Reader  according to the specifications of the Dual Luciferase Reporter Gene Assay Kit .NANOG ectopic expression plasmid- and vector-expressing HF-MSCs were cultured in a 24-well plate and transfected with plasmids that included the PBX1 shRNA (AGCTGTCACTGCTACCAATGT) or AKT shRNA (ATCGCTTCTTTGCCGGTAT) to inhibit the expression of PBX1 or AKT, respectively . In addition, the appropriate shRNA control was also used. Transfection and verification were performed as previously mentioned in the section of lentiviral plasmid construction and transduction.The inhibitor LY294002  was used to block the PI3K/AKT signaling pathway in order to inhibit the phosphorylation of AKT. The optimal treatment concentration and time for LY294002 were determined by Western blotting (data not shown). HF-MSCs that were ectopically expressing NANOG were treated with t-test, whereas differences between multiple groups were compared by one-way analysis of variance (one-way ANOVA). P < 0.05 was considered to be statistically significant. All results were repeated independently a minimum of three times, except where indicated.Data were statistically analyzed using SPSS version 21.0 . The data are presented as the mean ± standard deviation (SD). Differences between two groups were compared using the independent samples' HF-MSCs migrated from human hair follicles 10-14 days after seeding in a 24-well plate and displayed a fibroblast-like morphology Figures . ImmunofEctopic NANOG expression or PBX1 overexpression via lentivirus transduction was visualized by immunofluorescence imaging and measured by Western blotting Figures . In addiP < 0.01; Figures Cell growth assays showed that the ectopic expression of NANOG or the overexpression of PBX1 significantly increased the quantity of HF-MSCs by 1.4- and 1.2-fold, respectively, in comparison to cells in the vector group (control) on day 8 . Cell cyP < 0.05 and P < 0.01, respectively) and also increased calcium nodal formation in HF-MSCs by 2.18 ± 0.05- and 1.78 ± 0.18-fold (P < 0.001 and P < 0.01), respectively  or 30.33 ± 1.73% at passage 6 and 80.33 ± 2.31% at passage 14  in comparison to 44.67 ± 2.02% at passage 6 and 98.33 ± 0.67% at passage 14 in the vector group  and at passage 10 by 0.25 ± 0.07-fold (P < 0.001) in comparison to that of passage 4. In contrast, the expression of p16, p53, and p21 was significantly increased with passage in HF-MSCs by 1.34 ± 0.01-fold at passage 7 (P < 0.001) and 1.49 ± 0.09-fold at passage 10 (P < 0.001) for p16, 1.54 ± 0.03-fold at passage 7 (P < 0.001) and 2.07 ± 0.04-fold at passage 10 (P < 0.001) for p53, and 1.34 ± 0.35-fold at passage 7 (P = 0.10) and 2.08 ± 0.54-fold at passage 10 (P < 0.01) for p21, in comparison to the expression at passage 4  and 1.75 ± 0.14-fold (P < 0.01), respectively, and the expression of p-AKT/AKT by 3.14 ± 0.65-fold (P < 0.05) and 3.35 ± 0.46-fold (P < 0.01), respectively, in comparison to the empty vector. However, NANOG and PBX1 decreased p16 expression by 0.38 ± 0.18-fold (P < 0.01) and 0.49 ± 0.26-fold (P < 0.05), respectively, p21 expression by 0.57 ± 0.24-fold (P < 0.05) and 0.58 ± 0.24-fold (P < 0.05), respectively, and p53 expression by 0.60 ± 0.16-fold (P < 0.05) and 0.58 ± 0.25-fold (P < 0.05), respectively  but increased the expression of p16, p53, and p21 to 162.91 ± 19.72% (P < 0.05), 123.43 ± 6.25% (P < 0.05), and 160.99 ± 9.29% (P < 0.01) of control levels, respectively. Furthermore, blocking p-AKT significantly increased SA-β-gal-positive cell numbers from 43.33 ± 2.40% to 82.67 ± 1.20% , 59.16 ± 1.86% (P < 0.001), and 64.36 ± 4.47% (P < 0.001) of control levels, respectively, but increased the expression of p16 and p21 to 149.17 ± 14.76% (P < 0.05) and 143.58 ± 7.84% (P < 0.01) of control levels, respectively. As expected, total AKT knockdown led to a significant increase in SA-β-gal-positive cells from 47.67 ± 1.45% to 87.33 ± 1.45% , suggesting that NANOG may increase PBX1 expression  and increased the expression of p16 and p21 to 138.30 ± 13.43% (P < 0.05) and 201.21 ± 26.56% (P < 0.01) of control levels, respectively (Figures PBX1 knockdown significantly increased the quantity of SA-β-gal-positive cells from 64.33 ± 2.33% to 83.67 ± 2.73% (P < 0.01; To further elucidate the relationship between NANOG and PBX1, HF-MSCs that were ectopically expressing NANOG were transduced with  Figures . ConsistThe hair follicle is an appendage of human skin and contains abundant MSCs. HF-MSCs play crucial roles in the generation of hair follicles and hair cycle progression. HF-MSCs can be used as cell sources for the engineering of vascular tissue and angiogenesis, neural regeneration, and hair follicle reconstruction, and the delivery and controlled release of insulin, resulting in tremendous therapeutic potential in clinical settings –35.As a homeodomain transcription factor, NANOG is crucial for maintaining the self-renewal and pluripotency of ESCs. Our results demonstrated that there was no endogenous expression of NANOG in HF-MSCs in agreement with Shahini et al.'s study . MoreoveINK4a—a gene related to antiaging—acts as a cyclin-dependent kinase inhibitor and is crucial for cell-cycle progression and cellular senescence [KIP1 and p21CIP/WAF are involved in regulating the G1 to S transition, we did not find that NANOG represses cellular senescence by downregulating p27KIP1 in HF-MSCs. It has been suggested that this process is likely cell-type specific, which could result in the induction of various patterns of p27KIP1 expression. In addition, it has been shown that p21 is downstream of p53 during the regulation of the G1 phase of cell cycle [Han et al.  suggestenescence . Althougll cycle . Furtherll cycle —and attell cycle  and thatll cycle .γ after the embryonic stage [In this study, we found that ectopic NANOG expression or PBX1 overexpression could significantly enhance osteogenesis and decrease adipogenesis, in agreement with previously published studies that show that PBX1 represses adipocyte differentiation by directly regulating PPARic stage , and thaic stage . Humans ic stage , 43, andic stage . It has ic stage . These dPBX1 was further confirmed by dual luciferase assays, as NANOG overexpression significantly increased PBX1 promoter activity, indicating that it might be upstream of PBX1. We performed RNA interference assays to confirm these findings and observed that, as expected, PBX1 knockdown in HF-MSCs that were ectopically expressing NANOG significantly reduced the expression of p-AKT, increased the expression of p16 and p21, and promoted cellular senescence. These results suggest that NANOG attenuates cellular senescence via the PBX1-mediated regulation of AKT signaling and that PBX1 plays a vital role in attenuating HF-MSC senescence. Indeed, further investigation confirmed that the expression of PBX1 was remarkably decreased over time when subculturing HF-MSCs. A study by Li and Wang [PBX1 promoter is a target of NANOG in NIH3T3 and human ESCs based on a genome-scale location analysis. Moreover, both KLF4 and PBX1 have been shown to collaboratively bind the NANOG promoter to subsequently regulate gene expression in human ESCs [Our results indicate that NANOG and PBX1 are key transcription factors involved in the antiaging process. Moreover, we found that PBX1 expression in HF-MSCs was remarkably decreased with cell subculture, suggesting that it plays a role in maintaining these cells in a proliferative and multipotent state. Most notably, we found that the ectopic expression of NANOG significantly upregulates PBX1 and results in a decrease in the expression of p16 and p21. The upregulation of and Wang  showed tand Wang  and Piesand Wang  found thWAF1 but p53 [WAF1 [Surprisingly, we found LY294002 increased p53 as well as p16 and p21; AKT knockdown increased only p16 and p21 but p53. These results are consistent with the report of Kim et al.'s group, showing that AKT directly phosphorylates p21 but p53 . This is53 [WAF1  and LY2953 [WAF1 . The res53 [WAF1 . These rAlthough stem cell therapy holds promise for regenerative medicine, stem cells that are derived from aged donors or harvested by repeated subculture tend to lose their capacity for proliferation and multipotent differentiation, which not only compromises their therapeutic efficacy but also raises safety concerns for stem cell therapy. This necessitates the development of strategies for maintaining stem cells in a multipotent and proliferative state. Collectively, our study confirms the presence of a NANOG/PBX1/AKT pathway and a feedback interaction loop between PBX1 and AKT in HF-MSCs. The NANOG/PBX1/AKT pathway plays an important role in attenuating HF-MSC senescence, while the feedback interaction loop between PBX1 and AKT is mutually beneficial for maintaining HF-MSCs in a highly proliferative state with differentiation potential. We believe that our study makes an important contribution to antiaging research and regeneration medicine because there are few previous studies on the role and molecular mechanisms of NANOG in delaying senescence of HF-MSCs.This study is the first to report the existence of a NANOG/PBX1/AKT pathway and a feedback interaction loop between PBX1 and AKT in HF-MSCs. The NANOG/PBX1/AKT pathway plays an important role in attenuating HF-MSC senescence, while the feedback loop between PBX1 and AKT is mutually beneficial for maintaining HF-MSCs in a highly proliferative state with the multipotential capacity.


AbstractLactifluussubgenusLactifluus were discovered during a three-year monitoring of the ectomycorrhizal fungi in a tropical oak forest from central Veracruz, Mexico. Systematic sampling of basidiomes allowed recording of the morphological variation of fruit-bodies in different growth stages along with their fructification season. Both new species were distinguished, based on macro- and micromorphological features and on molecular data. A phylogenetic analysis of a concatenated nuc rDNA ITS, D1 and D2 domains of nuc 28S rDNA (LSU) and the 6–7 region of the second largest subunit of the RNA polymerase II (rpb2) sequence dataset of species of Lactifluus is provided. In the phylogeny inferred, one of the new species is sister to L.dissitus Van de Putte, K. Das & Verbeken and the other belongs to the group of species of L.piperatus (L.) Kuntze, sister to an unidentified species from U.S.A. The studied taxa grow under Quercusoleoides in the study site. The species are presented and illustrated here.Two new species of  Quercus) diversity. It hosts around 174 species, over 60% of which are endemic (ECM) fungi, such as Lactifluus species, are undoubtedly a key to the growth and survival of Quercus seedlings and trees in such patches of native tropical forest under drought conditions and adverse edge effects, through greater water and nutrient absorption in degraded soils. Despite their importance, ECM fungi in such a Mexican ecosystem have received scarce taxonomic attention, excepting reports, from the area or surroundings, of a few Boletales, Lactarius s.l., Cantharellus  and recent publications and is widespread in a variety of ecosystems worldwide but with a clear predominance in the tropics, especially in tropical Africa, Asia and the Neotropical region  have been recorded, most of them from montane (above 1200 m elevation) subtropical and temperate forests, in comparison with the higher proportion of surveys focused on the subtropical and temperate diversity in this country. In western Mexico, at elevations between 2200– 2550 m, the ECM community of Quercus spp. (including Q.laurina and Q.crassifolia), studied by Lactarius but none to Lactifluus. In our weekly monitoring of two tropical Quercus forests in eastern Mexico, we have noticed the presence of milkcaps, including Lactifluus species. One of our interests is to continue documenting their taxonomic identity and, in parallel with research, such as Quercus species. In this paper, we describe two new species found in these forests, recognised with morphological information and a multilocus phylogeny.In Mexico, around 19 species of Quercus forest from Central Veracruz (eastern Mexico). The site is privately owned, at Alto Lucero Co. (450–500 m elevation). Sampling of the two Lactifluus species studied was developed in monodominant stands of Q.oleoides, surrounded by a coffee trees plantation or land used for livestock.Random visits were conducted during June-October of 2015–2017 to a remnant of the tropical KOH) and Melzer´s solution. Methods to determine basidiospore ranges are those used by X̄ representing the range of X (where X is the average of basidiospores length and width in each collection) and Q̄ refers to the range of Q (where Q is the average of the ratio of basidiospore length/basidiospore width in each collection). The methods used to produce scanning electron microscope  images of their basidiospores are those used by XAL) .Macro-morphological features and colours were recorded from fresh samples in different growth stages. Alpha-numeric colour codes in descriptions follow rpb2) were amplified with primers bRPB2 6f/fRPB2 7CR (rpb2 markers were (i) initial denaturation at 95 °C for 5 min; (ii) 35 cycles of 30 sec at 95 °C, 30 sec at 55 °C and 40 sec at 72 °C (for LSU this condition was for 60 sec); and (iii) a 5 min final elongation at 72 °C. Amplified PCR products were sequenced using a Genetic Analyzer 3730XL (Applied Biosystems). Once sequences were assembled and edited, they were deposited at GenBank , with final length of 2,423 bp, in PhyDE v.0.9971 (BIC), the Akaike Information Criterion (AIC) and corrected AIC to select the best-fit model. This later was used to generate a phylogenetic tree with the Maximum Likelihood (ML) method, with a Nearest Neighbour Interchange (NNI) heuristic, with TNe+I+G evolutionary model and Ascertainment Bias Correction (ASC). We also generated a consensus tree, calculating the Robinson-Foulds distance between the ML tree and the consensus tree, the branches being tested by means of Ultrafast Approach Bootstrap (UFBoot), SH-like approximate Likelihood Ratio Test (SH-aLRT), Approximate Bayes test (aBayes) and Bootstrap Standard (BS). A phylogenetic tree was generated also by Bayesian Inference (BI), using MrBayes v. 3.2.6 , BI (≥ 0.90), UFBoot (≥ 95%), aBayes (≥ 0.90) and SH-aLRT (≥ 80%). The ML tree with the two former values for the nodes is presented here  to L.dissitus from India and the other one, L.lorenae, clusters in a clade with L.piperatus (L.) Kuntze from Europe and related species from North America and Asia, sister (with strong support) to an undescribed species from U.S.A.Based on morphological features and supported with the grouping displayed in the phylogenetic tree, we recognised two groups of the Mexican samples studied representing two distinct new species of Lactifluus . Ectomycorrhizal, under Quercusoleoides.MEXICO, Veracruz State, Alto Lucero Co., 12 km SW Palma Sola (road Veracruz-Nautla) 25 June 2015, Montoya 5190 , MK211194 (LSU), MK258872 (rpb2).In honour of Dr. Lorena E. Sánchez Higueredo because of her interest in the conservation of tropical oak forest relicts in Veracruz, Mexico.Pileus 25–114 mm diam., convex when young, expanded to broadly infundibuliform, undulate, depressed at centre when old, smooth to irregular when old, dull whitish with yellow tinges (3A2–3A5), staining orange-brown (5C6–C7) when bruised; margin decurved when young, with edge faintly decurved to straight when old, continuous to irregular. Lamellae adnate to subdecurrent, crowded to very close, 0.5–1.8 mm broad, edge entire, bifurcate at different levels, yellowish (3–4A2), staining orange-brown when handled, with lamelullae of different sizes, approximately 1 lamelullae per two lamellae. Stipe 20–90 × 11–35 mm, eccentric, cylindrical, attenuated or broadened towards the base, robust but at times flattened; surface smooth to irregular, faintly velvety under lens, more evident towards the base, whitish to cream-white, with yellow stains (5Y8/6), staining orange-brown when handled. Context cream colour, changing to brownish-orange when exposed, compact. Odour somewhat like chlorine; taste acrid. Latex whitish, milky, at times somewhat serous, staining white paper yellow (5Y 8/2), brownish after some minutes; taste burning acrid. KOH staining the pileus and stipe yellow to pale reddish.Basidiospores (6–)6.5–8(–10) × (5–)5.5–6.5(–9) µm; X̄ = 7.0–7.3(–9.2) × 5.5–6.0(–7.6) µm; Q̄ = 1.20–1.27, broadly ellipsoid, thin-walled; ornamentation 0.2–0.4 µm high (measured under SEM), an incomplete reticulum, composed of thick and thin bands and some isolated warts, others ornamented almost with isolated warts and some unconnected bands, plage inamyloid; under SEM the relief of the bands of the basidiospores ornamentation appear with an irregular inflated shape and the plage area with reminiscences of ornamentation. Basidia 30–45 × 8–11 µm, clavate, some subcylindrical, with refractive contents, thin-walled, with 2, 4 or at times 3 sterigmata. Pleuromacrocystidia 40–53 × 7–9 µm, clavate, some cylindrical and faintly broadened towards the middle area, thin-walled, with refractive needle-like and granular contents. Cheilomacrocystidia 34–54 × 7–9 µm, cylindrical, some clavate at base, thin-walled, with refractive contents. Pseudocystidia absent. Pileipellis a hyphoepithelium; suprapellis layer of 30–60 µm thick, gelatinized, composed of periclinally orientated hyphae, in some areas the hyphae are loosely intermixed or at times projected in mounds of up to 85 µm thick, the gelatinized matrix dissolved in KOH after some minutes; hyphae 2–4 µm broad, cylindrical, septate, wall up to 0.5 µm thick, sinuous; subpellis of 50–130 µm thick, composed of subisodiametric cells, 12–35 × 10–38 µm diam., yellowish in KOH, wall up to 1.0 µm thick; dermatocystidia 37–128 × 6–8 µm, 3.6–4.8 µm diam. at base, clavate, with refractive needle-like and granular contents, wall up to 0.5 µm thick, scarce, arising from subisodiametric cells of the subpellis layer. Context hyphae 5–7 µm broad, cylindrical, thin-walled, some with walls 0.5 µm thick, with faint refractive contents, sphaerocytes 12–26 µm diam., pale yellowish, wall 0.5(–1) µm thick, frequent, laticiferous hyphae 4–7 µm diam., infrequent. Hymenophoral trama composed of hyphae which are 4–6 µm diam., septate, wall 0.5 µm thick, with sphaerocytes of 10–25 µm diam., pale yellowish, wall 0.5 µm thick, laticiferous hyphae 4–6 µm diam., infrequent. Clamp connections absent.Quercusoleoides, infrequent.Gregarious, under XAL).MEXICO, Veracruz, Alto Lucero Co., 12 km SW Palma Sola (road Veracruz-Nautla) 25 June 2015, Corona 1127, Montoya 5191; October 11, 2016, Caro 103 . Ectomycorrhizal, under Quercusoleoides.MEXICO, Veracruz State, Veracruz, Alto Lucero Co., 12 km SW Palma Sola (road Veracruz-Nautla) 11 July 2016, Montoya 5276 , MK211190 (LSU), MK258871 (rpb2).referring to Mexico.Pileus 33–125 mm diam., convex, plano convex to depressed at centre, subvelvety, smooth or at times faintly rugose at centre, at remaining disc surface smooth, vinaceous-brown or vinaceous  when young, then ferruginous-brown, cinnamon-brown, frequently pale vinaceous (7C4–6), dull vinaceous (7D6) or pinkish-wine over a yellowish base, other reddish-brown to vinaceous , at times with orange-brown  areas; margin decurved, straight in age, at times undulated, rugose to strongly venous-rugose. Lamellae 2–9 mm broad, close to very close, adnate to subdecurrent, arcuate, with entire edge, some furcate at different levels, at times sinuous especially towards the stipe attachment, pale yellowish to yellowish , straw-yellow, yellow-orange  with brown to cinnamon-brown tinges, with faint vinaceous stains or brown colour  when handled; lamelullae of different sizes, 1–4 per lamellae. Stipe 35–115 × 9–27 mm, cylindrical, faintly broadened towards the base, subtomentose, dry, solid, in general concolorous but paler than pileus surface, at apex pale pinkish-orange (5YR 8/3–4), pinkish-brown, pale orange-brown or pinkish-red , continuing in pale orange (6A2–3), brown-orange with pinkish-grey tinges  and pinkish-brown  colours, becoming darker towards the base (7C4–6) , with some dark brown areas; base whitish and with whitish mycelium. Context compact, whitish to yellowish, staining brown-vinaceous. Odour faintly disagreeable, fishy; taste mild to somewhat bitter. Latex whitish to cream colour (2.5Y 8/3–6), milky, abundant, secreting from the whole basidiome, staining the lamellae and white paper pale brown; taste mild. KOH darkens the pileus surface.Basidiospores 8–10(–11) × 7–9(–10) µm, X̄= 8.7–9.2 × 7.5–9.0 µm, Q̄ = 1.1–1.2, subglobose to broadly ellipsoid, thin-walled; ornamentation up to 0.2–1.2 µm high (measured under SEM), a rather complete reticulum with irregular ridges, at times with thin connecting lines, rarely with some isolated ridges; plage in most spores inamyloid, rarely faintly amyloid; under SEM, the basidiospores wall appears rugose and with some isolated verrucae, with a complete reticulum composed of continuous regular or irregular ridges, some parts of the reticulum having rounded or irregular nodulose elevations, these later seen in the light microscope as verrucae, plage area smooth or with ornamentation reminiscences. Basidia 38–47 × 8–13 µm, clavate to faintly cylindrical, with 3–4 sterigma (at times with 2), thin-walled, with refractive contents. Pleurolamprocystidia 47–63 × 5–8 µm, lanceolate, at times mucronate, with wall 1.0–2.0 (–3.0) µm thick . Cheilolamprocystidia 40–55 × 5–8 µm, lanceolate, some subcylindrical, at times mucronate, with wall up to 1.0 µm thick, without dense contents, hyaline. Pseudocystidia absent. Pileipellis a lampropalisade, elements of the suprapellis 45–63 × 3–6 µm, most cylindrical, others clavate, ventricose or even ovoid 10–12 × 5–6 µm, without dense contents, some septate, hyaline, compact, at times, the elements arranged in mounds, wall up to 0.5 µm thick; subpellis 42–70 µm thick, composed of cells 9–30 × 7–20 µm, inflated, some subisodiametric, others irregular in form, wall 0.5–1.0 µm thick, not gelatinzied, pale yellowish in KOH. Context hyphae in an irregular arrangement, 5.0–8.0 µm diam., cylindrical, septate, wall up to 0.5 µm thick, laticiferous hyphae 4–7 µm diam., with refractive contents, yellowish in KOH; sphaerocytes 14–20 × 16–22 µm, yellowish, wall 1–1.5 µm thick scarce. Hymenophoral trama with hyphae 4–8 µm diam., cylindrical, septate, wall up to 0.5 µm thick, with scarce refractive contents, intermixed with laticiferous hyphae 4–8 µm diam., with refractive contents, yellowish in KOH; sphaerocytes 19–27 µm diam., hyaline, with a faint yellowish tinge. Clamp connections absent.Quercusoleoides.Solitary or gregarious, under XAL).MEXICO, Veracruz, Alto Lucero Co., 12 km SW Palma Sola (road Veracruz-Nautla) 25 June 2015, Montoya 5189, 5192; 3 July 2015, Montoya 5193; 5 July 2016, Montoya 5266; 4 October 2016, Montoya 5294, 5295; 29 June 2017, Caro 109, Montoya 5329, 5330, 5331; 4 July 2017, Corona 1370, 1371; 10 July 2017 Montoya 5340; 12 September 2017, Montoya 5398; 16 September 2017, Montoya 5411, 5412; 19 September 2017, Caro125, 126; 25 September 2017 Corona 1423, 1424 . The strong support of the clades, especially of L.mexicanus and L.lorenae allow us to complement morphological results and, on this basis, we decided to describe them. Both species are members of subgenus Lactifluus, the first one falling in section Piperati and the second in section Lactifluus, according to the classification proposed by The results inferred in the multilocus phylogeny Fig. , stronglLactifluuslorenae is a white milkcap, with basidiomes showing macromorphological similarities with L.piperatus, as narrowly cicumscribed by L.piperatus in the strict sense, significant differences between the two taxa are detected. Basidiomes of the Mexican species show a uniform tendency to develop an orange-brown colouration on the surfaces when handled and in the context when exposed. The latex can be somewhat serous, staining white paper yellow and becoming brownish after some minutes. When comparing micromorphological features between L.lorenae and L.piperatus (according to the later authors), in the former, the basidiospores are more globose (Q̄ = 1.20–1.27 vs. Q̄ = 1.28–1.40) and pleurocystidia are distinctly shorter (40–53 × 7–9 µm vs. 50–70(–90) × 8–11 μm). Another difference between the taxa is the pileipellis structure, which in the Mexican species presents a thicker hyphoid suprapellis (30–60 µm thick vs. 10–30 μm thick) and with abundant dermatocystidia in the suprapellis in L.piperatus, while scarce in the subpellis in the Mexican taxon. Organoleptic differences may be noted between both taxa too, because in L.lorenae, the odour is somewhat like chlorine, while in L.piperatus, it is slightly acidic, distinctly honey- or apple-like when drying. In the inferred phylogeny . Lactifluusmexicanus, is recovered as sister species of L.dissitus from India, this latter differs by having more distant gills arrangement and clearly larger cystidia   Kuntze. According to the original description of L.corrugis  × 7.0–9.0 (–10) µm vs. 9–12 × (8.5–)9–11(–12) µm], with the basidiospore ornamentation up to 1.5 µm high vs. (0.2–) 0.4–0.7 (–0.8) µm high in L.corrugis. The cystidia and pileipellis terminal elements are shorter in the Mexican species (pleurocystidia: 4– 63 × 5–8 µm vs. (48–) 60–125 (–204) × 6–10 (16) µm; cheilocystidia: 40–55 × 5.0–8.0 µm vs. (25–) 35–78 × (2) 4–8 µm; pileipellis terminal elements 45–63 × 3–6 µm vs. 45–80 (–128) × 2.5–6 µm]. The pleurocystidia in L.corrugis, according to  5–8 µm] . Lactiflcorrugis , for whiQuercus forest, during 2015–2017, we conclude that basidiomes of the studied species are produced in June-October, with those of Lactifluusmexicanus being more abundant. Although close to other edible species (L.mexicanus in the area.From the weekly sampling in tropical  species , we haveQuercus and Pinus species in Mexico, they represent important ECM hosts, related with the milkcaps in the country. Quercusoleoides, with a wide distribution from Mexico to Costa Rica, especially represents a key ECM host for this group of fungi in its range. In Costa Rica, however, at an elevation around 215 m, associated with Q.oleoides, ECM species belonging to different genera, three of which were determined as Lactarius but no Lactifluus was recorded. Considering that the two Lactifluus species, here studied, were found in a monodominant area of Q.oleoides, we consider them as putative mycobionts of this tree species. However, this will need to be confirmed at root tip level with molecular evidence, as in other milkcaps, such as Lactariustrichodermoides Montoya, Bandala & M. Herrera and L.subplinthogalus Coker (Q.sapotifolia and Q.glaucescens, respectively, in the relicts of the tropical oak forests from central Veracruz, Mexico.Considering the high diversity of us Coker . The two


H-tetraphen-3-one) is a new peltogynoid quinone methide isolated from Palo Morado (Peltogyne mexicana Martínez) heartwood by column chromatography. Its chemical structure was elucidated by IR, NMR , 2D NMR experiments , ESI-MS, and UV-Vis spectroscopic analysis. According to HPLC quantification, this compound is the main pigment and accounts for 1.21% of Palo Morado heartwood material. The antioxidant activity of peltomexicanin and dried methanolic extract (DEx) of purple heartwood was evaluated using the radical of 2,2’-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) assay, and the corresponding values expressed as Trolox equivalents (µmol TE/mg sample) were 4.25 and 4.57, respectively.Peltomexicanin (7,10-dihydroxy-6,12-dioxa-5 Peltogyne mexicana Martínez, commonly known as Palo Morado, is one of the twenty-three species belonging to Caesalpiniaceae family, and it is an endemic tree from Guerrero State, Mexico. The principal characteristic of its heartwood is a particular purple color; native people exploit this attribute and the wood’s hardness to elaborate wooden crafts, furniture and souvenirs + ion at m/z 283.0589, (m/z expected 283.0601) in agreement with the molecular formula C16H11O5. A purple amorphous powder was obtained, with UV-Vis (MeOH) λogynoids . We obseJ = 1, 10 Hz) and H-11  in ring A. The three protons for ring B were observed as broad singlets at 7.27 ppm (H-4), 7.16 ppm (H-2) and 6.66 ppm (H-1), respectively. Methylene protons for C-5 were observed as a broad singlet at 5.06 ppm. Correspondent protonated carbons were attributed based on results from an HSQC experiment , H-9  has a three bond correlation with (C-9), proton H-9 (δ 6.25 ppm) in the same ring correlated with C-11 (δ 107.52 ppm). H-8 was correlated with the carbon located at 152 ppm (C-11a) which is the fused enol ether group of the system. The second hydroxyl group was found in ring C and shifted the corresponding ipso carbon C-7 to 146.06 ppm due its enol character.Fusion between rings ABCD was evidenced by long range 13C attached proton test (APT) analysis, methylene protons correlated with alpha carbon C-4 (δ 112.58) in ring B. In ring D we observed long range correlations 1H-13C between the methylene protons (δ 5.06 ppm) with the carbon C-7 (W) and C-12b belonging to the quinone methide structure conformed by ring B and ring D (1H-13C correlation was observed from protons H-2 and H-4 shifted to 7–16 and 7.27 ppm respectively with the carbonyl group of quinone moiety located at 179.36 ppm in 13C-NMR.Methylene carbon C-5 (δ 66.64) was evidenced by d ring D . Two bonH-tetraphen-3-one, due the observed IR, MS and 1H- and 13C-NMR data. We named the pigment as “peltomexicanin”. The proposed structure suggests that peltomexicanin represents a rare peltogynoid quinone methide compound. Thus, the structure was established as 7,10-dihydroxy-6,12-dioxa-5P. mexicana has one less hydroxy group than peltogynin or mopanin.Results obtained for pigment analysis by UV-Vis, IR, ESI-MS, and 1D/2D nuclear magnetic resonance techniques confirm that we have isolated a flavonoid not previously reported, which in its enolized form, it has a similar structure to that of peltogynin and mopanin. The molecular weights reported for those compounds are 298.05 g/mol, whereas the molecular weight determined for peltomexicanin was 282.05 g/mol, indicating that the pigment from P. mexicana.The HPLC chromatogram showed a well-defined peak at 530 nm for the purified pigment with a retention time of 11.08 min. The peltomexicanin concentration calculated in the semipolar fraction was 11.85%. This represents 9.12% of the dried extract and 1.21% of the heartwood of The antioxidant activity of peltomexicanin expressed as Trolox equivalent antioxidant capacity (TEAC) was calculated as 1.20 ± 0.04 mM, which is lower compared to that reported for other flavonoids such as quercetin (TEAC = 3.68 ± 0.12) and myricetin (3.07 ± 0.23 mM) . This maThe specific radical scavenging estimated for peltomexicanin was 4.28 ± 0.10 µmol TE/mg; this value was slightly lower than 4.57 ± 0.10 µmol TE/mg obtained with the dried extract (DEx). This may be attributable to the presence of other compounds in the extract that could increase the antiradical activity alone or synergistically.P. mexicana Martinez and identified it as a quinone methide peltogynoid. This compound, named peltomexicanin, showed an important antioxidant activity. This feature lays the groundwork for additional studies of its biological activity.In conclusion, we have isolated a new purple pigment from m/z 50 to 3000 were obtained. MS data were processed using Data analysis 1.0 . NMR experiments (1H at 500 MHz) and 13C at 125 MHz) were performed with Varian 500 equipment  using methanol-4d as a solvent and TMS as internal reference.UV-Vis spectra were obtained using a DR 5000 spectrophotometer  in a range from 230 to 700 nm. The IR spectrum was recorded on a Jasco FT-IR instrument equipped with ATR . The exact molecular mass was obtained by (ESI) analysis on a Bruker micrOTOF-Q II instrument . Samples were dissolved in methanol and were injected directly to the spectrometer. The capillary potential was −4.5 kV, the dry gas temperature 200 °C and the drying gas flow 4 L/min. Total ion chromatograms from P. mexicana Martinez  was collected in Guerrero State (17°09′32′′ N latitude and 99°30′51′′ W longitude). Plant was identified by Post-graduate College of Chapingo, Mexico and a voucher specimen  was deposited at the Herbarium CHAP.P. mexicana was crushed into a fine powder and then sieved through US standard testing sieve #45 to #100. Particles retained in mesh #100 (355 µm < particle size > 150 µm), were extracted in the dark with MeOH (1% HCl) in a solvent: solid ratio of 95:5 (v/w) at 20 ± 2 °C for 24 h in a 250 mL flask with magnetic stirring. The solution was then filtered and concentrated on a rotatory evaporator  at 40 °C. This yielded an amorphous purple powder, which was named “dried extract” (DEx). Prior to purification, DEx was added to a partition liquid-liquid in a system of water-ethyl acetate (EtOAc) in order to remove compounds that were either highly polar or water- and EtOAc-insoluble. One gram of DEx was dissolved in 100 mL of EtOAc and 30 mL of distilled water to neutral pH was added. This solution was mixed vigorously and then organic phase was recovered; the operation was repeated by adding an additional two volumes of water. The organic fraction (corresponding to the semipolar fraction of DEx) was concentrated with a rotatory evaporator at 40 °C. This fraction represented 76.93% ± 1.73% of DEx.The heartwood of 2O was used in proportions of 0:10, 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1 and 10:0. The pigment fraction of interest was concentrated and dried with a rotary evaporator at 40 °C.Subsequently, 600 mg of the semipolar fraction were dissolved in 2.0 mL of MeOH and loaded onto a column packed with Sephadex LH-20 . As eluent, a gradient of MeOH:HQuantification of peltomexicanin pigment in the heartwood was performed by HPLC analysis, using the pure pigment as standard of reference. One milligram of peltomexicanin was diluted in 1 mL of MeOH at 1, 0.8, 0.6, 0.4, and 0.2 mg/mL, and filtered with 0.2-µm pore nylon acrodisc. Sample (20 µL) was injected onto the HPLC-DAD instrument  equipped with a quaternary pump and analyzed by a ZORBAX Eclipse Plus C18 column (Agilent Technologies) 4.6 i.d. × 150 mm, 5 µm; with a gradient elution of distilled water (phase A) and acetonitrile (phase B) at a flow of 1 mL/min, starting at 100% of phase A for 5 min, reaching 50% of phase B in 12 min, and 100% of phase B in 4 min, maintaining for 3 min gradient, returning to phase A 100% in 3 min and then held for a further 3 min. The oven temperature was set at 25 °C. Chromatographic analysis was monitored at 280 nm and 530 nm in DAD channels. A calibration curve was constructed using the area data obtained for each pigment dilution. In parallel, a sample of the semipolar fraction of DEx at 5 mg/mL was analyzed by HPLC in the same conditions as above, and the corresponding area for peltomexicanin was recorded. Using the external standard method, the peltomexicanin concentration in the extract and in the heartwood was calculated.et al. [+ radical was produced by reacting 50 mL of 7 mM ABTS (A1888 Sigma) stock solution with 880 µL of 140 mM potassium persulfate (379824 Sigma) and allowing the mixture to stand in a dark room at room temperature for 16 h before use. The ABTS+ solution was diluted with methanol to obtain an absorbance of 0.70 ± 0.04 at 734 nm. Then, 1.5 mL of ABTS+ radical and 30 µL of sample or standard were mixed for 6 min and final absorbance was measured at 734 nm on a Hach DR5000UV-Vis spectrophotometer. Calibration was performed with a Trolox (238813 Sigma) stock solution as described above and the results were presented as TEAC.The antioxidant activity of the DEx and the pure peltomexicanin were determined by measuring their capacity to scavenge the pre-formed radical of 2,2’-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) according to the method described by Rufino et al. . ABTS+ r


A very high potency of DOPE-TLR7a conjugate (EC50 around 9 nM) was observed either when prepared by direct dilution from DMSO or when formulated into 400–700 nm large multilamella liposomes containing dimethyldioctadecylammonium bromide salt (DDA) and DOPE. When prepared by dissolution in DMSO followed by dilution in aqueous culture medium, 93 ± 5 nm nanoparticles were formed. Without dilution from solution in DMSO, no nanoparticles were observed and no immunostimulatory activity could be detected without this formulation step. SAXS analysis of the conjugate after DMSO dissolution/water dilution revealed a lamellar order with a layer spacing of 68.7 Å, which correlates with arrangement in groups of 3 bilayers. The addition of another immunostimulatory glycolipid, trehalose‑6,6‑dibehenate (TDB), to DOPE:DDA liposomes gave no further increase in immunostimulatory activity beyond that provided by incorporating DOPE-TLR7a. Given the importance of nanoparticle or liposomal formulation for activity, we conclude that the major mechanism for increased potency when TLR7 agonists are conjugated to macromolecules is through alteration of physical form.Conjugation of small molecule agonists of Toll-like receptor 7 (TLR7) to proteins, lipids, or polymers is known to modulate potency, and the physical form or formulation of these conjugates is likely to have a major effect on their immunostimulatory activity. Here, we studied the effect of formulation on potency of a 1,2‑di‑(9Z‑octadecenoyl)‑  Howeverith alum  and the ith alum . The incith alum . The phyith alum .in vivo adjuvant activity of TLR2 stimulatory lipopeptides was found to be strongly dependent on physical form, with particulate components primarily responsible for vaccine activity in vivo  subtypes . ExtrAvidin alkaline phosphatase conjugate, Sigmafast pNPP, PBS tablets, sodium azide and EDTA (0.5 M) were from Sigma Aldrich . The IL-12p40 capture antibody C 15.6 and biotinylated IL-12p40/70 detection antibody C17.8 were from eBioscience . Nunc® Maxisorb 96 well ELISA plates were from Thermo Fisher scientific . Reagents for chemical synthesis were purchased from Sigma Aldrich  or Fluorochem  and used without further purification.2.27, 8, 9, 9 (Supplementary information).TLR7 agonist with carboxyl site on the para-benzyl group for conjugation 7,  and DOPEH‑purin‑9(8H)‑yl]methyl}benzoic Acid ‑8‑oxo‑7 Acid 7, 1H NMR (DMSO‑d6) δ 12.94 OH), 10.03 N), 7.89 , 7.39 , 6.51 , 4.94 , 4.25 , 3.57 , 3.26 . 13C NMR (DMSO‑d6) δ167.00, 159.83, 152.17, 149.08, 147.75, 141.98, 129.83, 129.55, 127.39, 98.34, 70.15, 65.26, 58.01, 42.12. HRMS calculated for C16H18O5N5+ (MH+) 360.1302 found 360.1302.ν, cm−1): 3351, 3165, 2956 (Broad), 1703, 1630, 1603IR (R)‑3‑(((2‑(4‑((6‑amino‑2‑(2‑methoxyethoxy)‑8‑oxo‑7,8‑dihydro‑9H‑purin‑9‑yl)methyl)benzamido)ethoxy)(hydroxy)phosphoryl)oxy)propane‑1,2‑diyl dioleate (8): δ 10.35 N), 9.97 NHCH2CH2), 9.37 , 7.86 , 7.34 , 6.60 , 5.31 , 5.09 , 4.89 , 4.29 NHCH2CH2OPO3) 4.25 , 4.09 NHCH2CH2OPO3), 3.84 , 3.81  3.57 , 3.26 , 3.17, , 2.24 CH2CH2), 1.95 , 1.48 CH2CH2), 1.23 , 0.85 .13C NMR  δ 173.13, 172.88, 160.43, 152.74, 149.62, 148.45, 140.74, 134.04, 130.22, 128.01, 127.9, 99.00, 70.82, 62.93, 58.66, 46.04, 42.69, 40.59, 34.15, 33.97, 31.88, 29.70, 29.69, 29.44, 29.30, 29.23, 29.19, 29.12, 29.10, 29.03, 28.99, 19.16, 14.53.57H94N6O12P+ (MH+) 1085.6662 found 1085.6677HRMS calculated for C−1): 3438, 3352, 3193, 2927, 2856, 1742, 1708, 1674, 1638, 1617.IR : LAL Chromogenic Endpoint Assay Endotoxin quantification kit and was performed according to manufacturer's instruction, unless otherwise stated all compounds tested were below the limit of detection of 0.08 EU/mL.2.42.4.12O to form a stock solution at 150 μM. Samples were then diluted in ether H2O or complete cell culture media  from the stock solution. Solutions of DOPE-TLR7 agonist conjugates were tested at concentrations relevant for in vitro stimulation of RAW 264.7 macrophages (1–1000 nM). Unconjugated TLR7a or DOPE-TLR7a conjugates stored as either dry powder or in complete media as nanoparticle formulations showed no difference in activity when stored at −20 °C over a period of 6 months. Although not formally evaluated, we have no evidence to suggest any instability of these conjugates upon storage.Weighed amounts of DOPE-TLR7 agonist conjugates  were pre-solubilized with DMSO (50 μL) before diluting with H2.4.22 to form a thin lipid film on the bottom of a round-bottomed flask. The lipid film was hydrated in 10 mM Tris-buffer at pH 7.4 by heating for 20 min at 60 °C. The phase transition temperature (Tm) of DDA is 47 °C. This general method to formulate MLVs was used to produce a variety of liposomal formulations as listed in Multilamellar vesicles (MLV) were synthesised by using the original lipid-film hydration method . MLV werin vitro are described in more detail as follows. To produce MLVs with 20 w%/v% of TLR7a-DOPE conjugate: 125 μL of DDA (10 mg/mL) was transferred to a round bottom flask with the addition of 25 μL of DOPE-TLR7a conjugate (10 mg/mL). Following the above procedure, the lipid cake was then resuspended in 1 mL of Tris-buffer to give a final following concentration of compounds: DDA = 1.25 mg/mL or 1.98 mM, DOPE-TLR7a = 0.25 mg/mL or 230 μM, TDB = 0.25 mg/mL or 250 μM. MLV were then diluted into complete culture medium to the desired concentration for in vitro stimulation assays. When both TLR7a-DOPE conjugate and TDB were present in MLVs, the concentration of DOPE-TLR7a was used to determine dosing during in vitro stimulations assays.To clarify the final concentrations of immuno-modulatory components in each preparation and the calculations involved to determine the concentration of compounds used 2.5Samples were prepared in 1 mL volume using milliQ ultrapure water for NTA analysis, filtered through a 0.45 μm syringe filter immediately prior to use. NTA was performed on a LM10 system with LM14 top plate and 533 nm laser . Flow rate was determined by software and set to 30 AU. Videos were recorded 5 time for each sample, 60 s each, samples were repeated 3 times at a controlled temperature of 25 °C. A 200 nm particle standard was used as a calibration run diluted 1:1000 in miliQ water. For samples analysed in complete media, detection threshold was increased to 6 to reduce autofluorescece noise. All analysis was performed using NTA software v3.1 .Liposomes were characterized using DLS measured used a Zetasizer Nano-ZS . Samples were diluted into ultrapure water to appropriate concentrations in low volume plastic cuvettes (Fisher Scientific). A dispersant refractive index of 1.33 and material refractive index of 1.45 was used for all samples. Individual measurements were carried out for 10 s per run, with 12 runs per reading, repeated in triplicate. This was repeated for three separate samples at 25 °C. ξ-Potential values were also measured; using DTS-1061 folded capillary tube cuvettes . Samples were diluted 1:100 in ultrapure water to the same concentration used in sizing experiments. Samples were measured using 20 sub-runs per reading, repeating 3 times for each sample. Each sample was measured three times and the results were processed using the Smoluchowski model (Fκa = 1.50).2.62.6.1An aliquot of DOPE-TLR7a conjugate was first dissolved in DMSO then further diluted in TRIS buffered saline  to give the corresponding concentrations used for SAXS experiments. 1 wt% DOPE-TLR7a in 0.02 vol DMSO/vol TBS was diluted with TBS to give (for example) 0.25 wt% DOPE-TLR7a in 0.005 vol DMSO/vol TBS.2.6.2via an automated sample exchanger into a quartz capillary  in the X-ray beam. The quartz capillary was enclosed in a vacuum chamber, in order to avoid parasitic scattering. After the sample was injected in the capillary and reached the X-ray beam, the flow was stopped during the SAXS data acquisition. BM29 operated with an X-ray wavelength λ = 1.03 Å (12 keV). The images were captured using a PILATUS 1M detector, while data processing was performed using dedicated beamline software ISPYB (BM29).Synchrotron SAXS experiments on solutions were performed using a BioSAXS robots at beamline BM29 . Solutions were loaded into the 96 well plate of an EMBL BioSAXS robot, and then injected 2.72 in T-75 flasks until approximately 80% confluency. Cells were then detached using a solution of EDTA in PBS  per T-75. Cell suspensions were then centrifuged at 200 ×g for 5 min. The supernatant was removed then the pellet re-suspended in complete media (5 mL). Cells were then re-suspended in fresh culture media at 2 × 105/mL.Raw 264.7 macrophages were subcultured according to ATCC methods: Complete media contained RPMI 1640 Glutamax® . Cells were cultured at 37 °C 5% CO6 cells/mL freshly passaged cells were added in 100 μL to give a final volume of 200 μL. Plates were incubated for 24 h before the analysis of supernatants for pro-inflammatory cytokines. IL-12p40 concentrations in supernatants of RAW 264.7 stimulation assays were determined by sandwich ELISA. Briefly, Nunc Maxisorb 96 well ELISA plates were coated overnight with IL-12p40 capture antibody (C15.6) at 2 μg/mL in carbonate buffer , followed by 2 h with 200 μL per well block buffer . 100 μL per well biotinylated detection antibody (C17.8) at 1 μg/mL in block buffer was added. Stimulated RAW 264.7 supernatants were diluted 1:7.5 in block buffer, and compared to 1:2 dilution of recombinant IL-12p40 standards with a top concentration of 10 ng/mL in block buffer, with 150 μL sample per well. ExtrAvidin alkaline phosphatase conjugate was prepared 3:10,000 dilution in PBS.Stimulation assays were performed in tissue culture grade 96 well plates. Test compounds were diluted serially 1:2  in complete media  then 2 × 1033.1in vitro stimulation  to determine the physical form of this preparation, and found nanoparticles with a mean diameter of 93 ± 1 nm when diluted into ultrapure water. Complete cell culture medium showed background particulate matter as expected given the high protein content and heterogenous composition of heat inactivated foetal bovine serum. A background fluorescence was also observed from phenol red in the culture medium, which increased noise and reduced sensitivity with control 100 nm latex particles. The increased viscosity of complete media over H2O was taken into account by software correction viscosity values at 25 °C for water = 0.89 cP and complete media = 0.96 cP. However, when diluted from DMSO into culture medium an increase in size of the DOPE-TLR7 agonist conjugate was observed comparing to ultrapure water. The increase from 93 ± 1 nm in water compared to 155 ± 5 nm in culture medium could be attributed to agglomeration of particles, or to incorporation of media components into micelles/nanoparticles. The reduction of particle frequency in DOPE-TLR7a conjugates - not mirrored by the 100 nm latex standard - also supported the possibility of agglomeration (The poor water solubility of the lipid conjugated TLR7 agonist prevents direct addition of this compound to meration .Table 4P3.3d = 2π /qo = 68.7 and 33.9 Å (qo = position of the maxima in the scattering curve) and at a positional ratio 1:2. These peaks suggest that DOPE-TLR7a is arranged in a lamellar order with a layer spacing of 68.7 Å. The SAXS data for 1 wt% DOPE-TLR7a was modelled according to the form factor for a bilayer structure, comprising three Gaussian functions to represent the electron density variation across the two headgroups and the dense lipid core , and without functional group at the para-benzyl position (9), suggesting the alteration of activity is due to lipid conjugation not simply variation in groups on this para-benzyl position (9 to confirm for the first time the solid state structure of this important immunostimulatory compound (Supplementary information).Conjugation of TLR7a to the phospholipid DOPE has previously been shown to promote strong pro-inflammatory response chniques B and likiposomes A. Separa potency A. Similaposition . We werein vitro (Either using DMSO to dissolve the DOPE-TLR7a conjugate prior to dispersion by dilution in aqueous solvents, or using well-established lipid cake hydration method of forming MLVs showed a clear and large increase in the number of particles formed and also a strong pro-inflammatory response in vitro . Completin vitro . Changinin vitro . The incin vitro , suggestin vitro. Previous reports suggested that the immunostimulatory activity of TDB does not synergise with TLR 3 and 9 agonists,(in vitro system. Adding TDB to MLV containing 20% DOPE-TLR7a showed no increased pro-inflammatory activity (TDB is not only an immunostimulatory glycolipid, but has also been reported to have a stabilising effect on cationic MLVs constructed from DDA and DOPE . Using Dagonists, and we sactivity . Furtheractivity  showed n5in vitro, which has not been reported previously. Our data shows that the formation of TLR7 agonist containing nanoparticles distinctly modulates pro-inflammatory activity. This can easily be demonstrated by the aqueous dispersion of lipid conjugates from DMSO solution resulting in nanoparticle formation and high potency. Similarly, the formation of DOPE-TLR7a particles in the form of MLV liposomes significantly increased in vitro pro-inflammatory potency over the unconjugated TLR7a. SAXS data provided insight into the physical form of the conjugate after DMSO dissolution and aqueous dispersion, identifying a lamellar structure with repeat distance of 77 ± 25 Å, and with the modelled parameters listed in via dissolution into DMSO prior to aqueous dilution are both equally potent inducers of IL-12p40. The most significant change in pro-inflammatory activity following lipid conjugation could be attributed to the formation of sub-micron particles. Overall, this study highlights the importance of physical form and formulation on activity of immunostimulatory compounds which trigger PRR.This study links the physical form of DOPE-TLR7a conjugates in aqueous dispersion to their biological activity No competing financial interests have been declared.


H2/Ar plasma engraving could significantly increase electrochemically active O22−/O− concentration and tune the electronic structure of Co ions efficiently, and consequently tailor the intrinsic electrocatalytic ability for OER. Of note, P-Bi0.1BSCF, with unique crystalline core/amorphous shell structure, exhibits an enhanced intrinsic OER activity and higher stability than the noble metal IrO2 catalyst, which outperforms most of the reported perovskite catalysts. The present work provides new insights into exploring efficient catalysts for OER, and it suggests that, in addition to the extensively applied for surface treatment of various catalysts such as carbons and metal oxides, the plasma engraved perovskite materials also exhibits great potential as precious metal-free catalysts.The development of highly active and cost-effective catalysts based on noble metal free oxygen electro-catalysis is critical to energy storage and conversion devices. Herein, we highlight a plasma-treated Bi Given that OER is in general impeded by its intrinsically sluggish kinetics due to a multistep four electron process, and consequently requires a considerable overpotential relative to its thermodynamic potential of 1.23 V vs. reversible hydrogen electrode (RHE). To address this, precious metal-based materials, e.g. RuO2 and IrO2, are currently employed as the efficient state-of-art OER catalysts1. However, high cost, low abundance as well as poor durability during long term operation badly hinder their widespread application.The pursuit of highly active and cost-effective catalysts is of prime significance for sustainable energy conversion and storage in order to develop renewable energy production. Implementing these emerging clean energy solutions, such as water splitting, direct solar and metal air batteries 5, carbon (carbon based hybrid)7, metal oxides/carbon composite catalysts10, chalcogenides12 and phosphates14. Among these candidates, perovskite oxides have drawn much attention as competitive electrocatalysts due to their flexible states of transition metals, fascinating oxygen ion mobility and exchange kinetics, and unique tailorable properties17. For example, Suntivich et al.19 reported that Ba0.5Sr0.5Co0.8Fe0.2O3 (BSCF) perovskite provided a higher intrinsic OER activity in alkaline solution comparable to commercial IrO2 electrocatalyst, A site of Ba/Sr displays the fastest oxygen transport kinetics, while the Co-rich compositions on the B site offers faster oxygen exchange kinetic. The rational design of B-site transition metal ratio could tune the eg orbital filling , and thus benefited OER process18.Over these years, a tremendous number of alternatives based on non-precious metals materials have been intensively explored, including oxides (hydroxides)18 limit their large-scale practical application due to low gravimetric mass activity. The poor stability caused by surface amorphization of BSCF particles under OER conditions is another tough issue for industrial application20. It is known that the surface area could be increased through a nanostructure strategy or surface modification, while the stability of perovskites could be improved by intrinsic methods resort such as chemical substitution or electronic configuration modification. Jung et al.19 introduced a nano-sized Lax(Ba0.5Sr0.5)1−xCo0.8Fe0.2O3 with superior activity and stability, where the electronic states was managed by chemical substitution of A-site cations with lanthanum and particle growth was controlled by sintered temperature. Ln(Ba0.5Sr0.5)0.5Co0.8Fe0.2O3 21 and Pr0.5(Ba0.5Sr0.5)0.5Co0.8Fe0.2O322 have also been developed as highly active catalysts base on A-site chemical substitution strategy.Although BSCF is known to have superior intrinsic activity, large particle sizes rendered from traditional bottom-up method0.1(Ba0.5Sr0.5)0.9Co0.8Fe0.2O3−δ, denoted as Bi0.1BSCF) to modify the electronic structure of perovskites. As known, bismuth oxide is usually used as an oxygen-ion conducting electrolyte in solid oxide fuel cells. Bi could also be introduced to A site of BSCF perovskite and tailor oxygen electrocatalysis ability23. Furthermore, surface modification is another effective strategy to improve the activity and durability of electrocatalysts. It is reported that the cold plasma process is an efficient approach for surface modification and functionalization, which can generate roughed surface, surface vacancies, defects and other active functional groups28. Therefore, for the first time, we propose the pollution-free and facile plasma modification on perovskite oxides to tailor the near-surface structure. The optimized 5% H2/Ar plasma-treated Bi0.1BSCF catalyst, which denoted as P-Bi0.1BSCF, demonstrates a higher intrinsic activity and durability towards OER, relative to the well-known BSCF perovskite as well as commercial IrO2 catalyst. An electrochemically active amorphous layer created by plasma engraving is achieved, and its effect is furthermore elaborated for OER.In this work, we highlight a feasibility of Bi introduced into A site of BSCF lattice (Bi0.5Sr0.5Co0.8Fe0.2O3 and Bi0.1(Ba0.5Sr0.5)0.9Co0.8Fe0.2O3−δ  perovskite oxides were synthesized by using a EDTA-citrate sol-gel process17. Briefly, the starting materials of Ba(NO3)2, Sr(NO3)2·4H2O, Bi(NO3)3·6H2O, Fe(NO3)3·9H2O, and Co(NO3)2·6H2O  were mixed in deionized water in accordance with their stoichiometric amounts, employing EDTA and citric acid as chelating agents. With the introduction of aqueous ammonium hydroxide solution  into above solution, the pH value was tuned at ~7. Such solution was stirred and then heated at 100 °C to yield a gel, then calcination at 250 °C overnight to form a solid precursor. The solid precursor of BSCF was sintered in air at 1000 °C, whereas Bi0.1BSCF was calcined in air at 850 °C for 4 h to obtain the powders. The commercial IrO2  catalysts was studied for comparison.Ba0.1BSCF catalyst was subsequently conducted via a plasma cleaner  with a gas flow of air, Ar or 5% H2/Ar for the plasma ignition . Different irradiation time  with powers of ~130 W and pressure of 90 Pa was applied during plasma process. The optimized 5% H2/Ar plasma-treated Bi0.1BSCF for 3 min was used for detailed studies, which denoted as P-Bi0.1BSCF.The freshly-prepared Bi2 as the adsorptive medium. Approximately 2.0 g samples were weighed and degassed at 250 °C for 4 h before nitrogen physisorption at the temperature of liquid nitrogen (77 K). The ability of perovskites to adsorb OH− after exposure to water (100% humidity) at room temperature for 2 h was estimated from the Fourier transform infrared spectra . Oxygen temperature programmed desorption (O2-TPD) measurement was surveyed to analysis the oxygen desorption properties.Phase structures of the as-prepared catalyst powders were determined by XRD on Bruker . Program FullProf was employed for the diffraction refinement. SEM images were performed on a SU-8010 SEM, whereas high resolution TEM images equipped with EDS were conducted on a Tecnai G2 F20 U-TWIN TEM. XPS measurements of the catalysts were carried out on a Kratos Axis Ultra DLD instrument. The obtained XPS spectra were calibrated by referencing C 1 s to 284.6 eV, and simulated using the XPSPEAK41 software. We analyzed the specific surface areas by Brunauer Emmet Teller (BET) system with N0.1BSCF, P-Bi0.1BSCF and commercial IrO2  catalysts were, respectively, ground in mortar to disperse well. The mixture, including 40 mg perovskite catalyst, 8 mg Ketjen black , 5 mL ethanol and 0.25 mL 5 wt.% Nafion solution were ultrasonicating to obtain a homogeneous ink. A 2 μL catalyst ink was then deposited onto the polished glassy carbon electrode surface with a uniform loading of ~0.2832 mg cmdisk−2 . Electrodes containing IrO2 were also prepared with a similar loading of 0.2587 mg cmdisk−2. The OER electrochemical characteristics were conducted in N2-saturated 0.1 M KOH electrolyte with a standard three electrode cell configuration (Pine Research Instrumentation). 1 M Hg/Hg2Cl2 electrode and platinum sheet were applied as the reference and the counter electrode, respectively. The potentials in this work were iR corrected, where the value of R is the ohmic electrolyte resistance via high frequency AC impedance. Cyclic voltammetry (CV) curves for OER were recorded on the rotating disk electrode from 1.3 to 2.0 V vs. RHE at 1600 rpm, with the scan rate of 10 mV s−1.The as-prepared BSCF, Bi0.5Sr0.5Co0.8Fe0.2O3 and Bi0.1(Ba0.5Sr0.5)0.9Co0.8Fe0.2O3−δ . Upon Rietveld refinements, the fitted lattice parameters of BSCF and Bi0.1BSCF are summarized in Table 0.1BSCF present a cubic perovskite structure with the space group of Pm-3m. Schematic representation of A-site doping perovskite structure of Bi0.1BSCF is described in Figure 0.1BSCF in this study agrees well with the reported literature23. The typical diffraction peak (110) of Bi0.1BSCF moves to a higher angle compared to pristine BSCF, indicating that the introduction of Bi into BSCF lattice causes lattice contraction. This might be attributed to the substitution of the larger Sr2+ (0.158 nm) and Ba2+ (0.175 nm) ions by smaller Bi3+ (0.124 nm). Additionally, upon XRD analysis  as estimated via BET method BSCF interplanar spacing, respectively. In the case of Bi0.1BSCF, the lattice diffraction fringes in Fig. 0.1BSCF particles are highly crystalline with little amorphous region. After exposure in 5% H2/Ar-plasma for 3 min, the perovskite structure of P-Bi0.1BSCF is still observed in Fig. 0.1BSCF, the results reveal that no crystalline changes after plasma treatment. In addition, STEM and the corresponding EDS mapping analysis suggests that the elements of P-Bi0.1BSCF are uniformly distributed without surface segregation phenomenon after plasma engraving process. Moreover, it is apparent that P-Bi0.1BSCF displays a unique core-shell structure, a disordered amorphous shell with a thickness of approximately 8–10 nm is generated after plasma modification. Similar phenomenon is reported by Li and his co-workers that the TiO2 nanosheets shows a crystalline TiO2 core/amorphous TiO2−δ shell structure via NH3-plasma surface modification with oxygen deficient on shell region29.To further investigate the crystalline structural change of BSCF after Bi-doping and plasma treatment, HR-TEM are performed on BSCF, Bi0.1BSCF and P-Bi0.1BSCF catalysts under the OER potential window. Similar measurements are performed on the IrO2 and KB for comparison. The catalytic activity contribution of KB is subtracted according to the composition of electrode. The performance of commercial IrO2 catalyst is comparable to that reported elsewhere31. The CV of Bi0.1BSCF shows a comparably lower onset potential (1.49V) and a higher current density than the IrO2 and BSCF catalyst, indicating a better OER catalytic activity of Bi0.1BSCF. After plasma engraving, P-Bi0.1BSCF catalyst manifests an apparent enhancement in OER activity with a significantly improved current density. The optimized processing parameters for plasma engraving is 5% H2/Ar working atmosphere only for 3 min  at a current density of 10 mA cmdisk−2 of 370 mV, which is superior to that of Bi0.1BSCF (411 mV), BSCF (525 mV), and IrO2 (464 mV). Notably, the overpotential of P-Bi0.1BSCF favorably outperforms the reported perovskite-based OER catalysts39, e.g. BaCo0.7Fe0.2Sn0.1O3−δ (410 mV)36, SrNb0.1Co0.7Fe0.2O3−δ (SNCF) (500 mV)39 summarized in Table Figure min Fig. , ESI. Mo−1 for P-Bi0.1BSCF, Bi0.1BSCF, BSCF, and IrO2, respectively. Furthermore, the mass activity (MA) and specific activity (SA) are illustrated to evaluate the intrinsic activity, as shown in Fig. 0.1BSCF displays 8.9 times higher MA and 2.7 times higher SA relative to BSCF, respectively. The results confirm the positive contribution from the Bi-dopant and plasma engraving to the intrinsic activity. In addition,Corresponding Tafel plots of the investigated samples are compared in Fig. 0.1BSCF, Bi0.1BSCF, BSCF, and commercial IrO2 catalysts for 600 cycles, as shown in Fig. 2 show a 25% and 27% reduction of its 2nd activity over 600 cycles, respectively, and the poor stability of BSCF results from the leaching of A site cations to the alkaline medium20. In contrast to BSCF, only 10% and 12% reduction is observed in the Bi0.1BSCF and P-Bi0.1BSCF catalysts under the same condition, respectively. The TEM image of post-OER P-Bi0.1BSCF in Fig. 0.1BSCF is maintained and the thickness of amorphous shell did not change much after continuous CV tests compared to as-synthesized P-Bi0.1BSCF. In addition, the XPS spectra before and after long-term OER testing shows that the oxidation state of Bi3+ does not change41 , followed by the surface oxidative oxygen O22−/O− (530.5 eV), hydroxyl groups OH− or adsorbed O2 (531.5 eV), and adsorbed molecular H2O of the oxide surface (532.9 eV)42. The relative content of the lattice and surface oxygen species derived is listed in Table 22−/O− increases from 35.68% with BCSF to 42.17% with Bi0.1BSCF. Moreover, the O22−/O− concentration is further increased to ~49.23% by plasma engraving, indicating the existence of abundant active oxygen species on the amorphous region of P-Bi0.1BSCF oxide surface. Importantly, the electrochemically active O22−/O− are proved to be beneficial for catalyzing OER44. Also, the highly oxidative oxygen species on the surface are reported to be closely relevant to the oxygen vacancies45, and surface oxygen vacancies play a key role for perovskite oxides in catalyzing OER46. It reveals that the plasma engraving could create more electrochemical active sites on the amorphous shell. The similar phenomenon of surface amorphization and simultaneous increased activity for efficiently electro-catalyzing OER is also found in other catalysts50.To understand the source responsible for the excellent OER activity of P-Biaks Fig. . The fir0.1BSCF shift to lower binding energies, suggesting that the surface valence of Co slightly decrease owing to high valence Bi-substitution. After 5% H2/Ar plasma engraved, the two main peaks of P-Bi0.1BSCF shift to higher binding energies obviously, which indicates the presence of surface cobalt in a higher oxidation state. It is reported that the high valence of Co could also play a positive role in the improvement of OER activity51. No distinctive variation was observed in the Fe state of catalyst surface . It’s worth noting that, after 5% H2/Ar plasma engraved, the desorption temperature of P-Bi0.1BSCF catalyst reduce remarkably to nearly 198 °C, this indicates an excellent oxygen desorption capability35 and reflects a higher oxygen surface exchange ability for P-Bi0.1BSCF perovskite. The result is consistent with the EIS analysis that P-Bi0.1BSCF has better ionic and charge transfer abilities. Besides O2 desorption capability, the P-Bi0.1BSCF catalyst also shows good OH− adsorption. As observed by FTIR spectroscopy (Fig. −1 appeared, which corresponds to H-bonded OH− stretching vibration52. Obviously, Bi0.1BSCF exhibited a stronger OH− absorption ability than initial BSCF, and the OH− absorption ability is further improved via plasma treatment. According to the adsorbate evolution mechanism53, OH− absorption on the active sites of perovskites is the prerequisite for OER, and thereby the large OH− absorption can continually offer raw materials for the following OER process36. Due to the complex interplay between oxygen deficiency and surface-active redox centre of perovskite oxides, detailed mechanistic insights into the OER electrocatalysis of P-Bi0.1BSCF materials in alkaline media are currently limited. Nevertheless, with the superior activity, stability and cost-effectiveness, Bi0.1BSCF promises a novel precious-metal-free catalyst for the alkaline OER, and plasma engraving proved to be a facile and effective surface modification method to further improve the activity.The redox property of BSCF, Biopy Fig. , a broad2/Ar plasma engraving only for 3 min, an amorphous layer with abundant electrochemical active oxygen species is generated on Bi0.1BSCF perovskite surface. Moreover, the increased oxygen surface exchange ability and OH- absorption ability of P-Bi0.1BSCF are achieved. Owing to the unique properties via Bi-doping as well as plasma engraving, P-Bi0.1BSCF exhibits a fast kinetics process with a small Tafel slope of 68 mV dec−1 and high stability for OER, that is superior to most of the state-of-the-art perovskite-based electrocatalysts. The foregoing results open a new avenue for engineering perovskite oxides via plasma method for highly efficient OER, which is promising for a variety of electrochemical energy storage applications.In summary, a plasma engraving strategy is, for the first time, proposed for the surface modification of perovskite materials towards electrocatalyzing OER. After 5% HSupplementary Information