Multiple sclerosis (MS) is a chronic CNS autoimmune disease characterized by inflammation, demyelination, and neuronal degeneration, where myelin-specific CD4 T cells play critical roles in the formation of acute MS lesions and disease progression. The suppression of IL-7Rα expression and the upregulation of inhibitory receptors  are essential parts of the cell-intrinsic immunosuppressive program regulating T effector functions to prevent autoimmunity. However, little is known on the factors regulating IL-7Rα/PD-1 balance in myelin-specific CD4 T effector/memory cells during the development of CNS autoimmunity.We analyzed the roles of the transcription factor T-bet in regulating the expression of IL-7Rα and inhibitory receptors in myelin-specific CD4 T cells. Furthermore, we compared the effects of different inflammatory cytokines that are crucial for Th1 and Th17 development in regulating the IL-7Rα/PD-1 balance.We discovered that T-bet suppresses the expression of inhibitory receptors (PD-1 and LAG-3) and promotes IL-7Rα expression in myelin-specific CD4 T cells in vitro and in vivo. As a result, T-bet skews IL-7Rα/PD-1 balance towards IL-7Rα and promotes enhanced effector function. Furthermore, IL-12 enhances IL-7Rα expression in a T-bet independent manner in myelin-specific Th1 cells. Meanwhile, IL-6, the cytokine inducing highly encephalitogenic Th17 differentiation, suppresses PD-1 while upregulating IL-7Rα, skewing IL-7Rα/PD-1 balance towards IL-7Rα, and promoting enhanced effector function. Moreover, blocking IL-7 signaling in myelin-specific CD4 T cells by αIL-7Rα significantly delays experimental autoimmune encephalomyelitis (EAE) onset and reduces disease severity.T-bet is a major transcription factor regulating IL-7Rα/PD-1 balance in myelin-specific CD4 T cells during EAE development, and there is a positive correlation between several major determinants promoting T cell encephalitogenicity  and an IL-7Rα/PD-1 balance skewed towards IL-7Rα. Furthermore, IL-7 signaling inhibits PD-1 expression in myelin-specific CD4 T cells and blocking IL-7 signaling suppresses T cell encephalitogenicity. Therefore, interference with inhibitory pathways and IL-7Rα expression may suppress the encephalitogenic potential of myelin-specific CD4 T cells and have therapeutic benefits for MS patients.The online version of this article (doi:10.1186/s12974-016-0768-3) contains supplementary material, which is available to authorized users. Multiple sclerosis (MS) is the leading cause of neurologic disability in the US in young adults after trauma; thus, most patients suffer from the effects of MS for most of their adult life. Experimental autoimmune encephalomyelitis (EAE) is a T cell mediated autoimmune disease of the central nervous system (CNS), which has served as an animal model for MS for several decades. The formation of acute inflammatory MS lesions is mediated by myelin-specific, autoreactive T cells . PreviouThe inhibitory receptors are important immune checkpoints that negatively regulate immune responses to prevent tissue damage and autoimmunity. The roles of inhibitory receptors in the regulation of T cell effector function have been well-established in T cell exhaustion, which was identified during chronic viral infection and observed in tumor microenvironment. The axis of PD-1 and its ligand is a central regulator of T cell exhaustion, although multiple inhibitory receptors, including Lag-3, CTLA-4, Tim3, CD244/2B4, CD160, TIGIT, are involved , 10. Blo−/− mice were purchased from the Jackson Laboratory and bred in a specific pathogen-free animal facility at the Ohio State University (OSU) Wexner Medical Center. B10.PL mice transgenic for the MBP Ac1-11-specific TCR chains Vα2.3 or Vβ8.2 [B6/WT and B6/T-betor Vβ8.2  were als6 cells/well with irradiated B10.PL splenocytes (6 × 106 cells/well). Cells were activated with MBP Ac1-11 (2 μg/ml) and different combination of cytokines or neutralizing antibodies for cytokines to differentiate effector T helper cells. Cytokines and antibody concentrations were as follows: 0.5 ng/ml IL-12, 25 ng/ml IL-6, 1 ng/ml TGFβ1, 2 μg/ml anti-IFNγ, 1 μg/ml anti-IL-12, 2 μg/ml anti-IL-4, and 0.35 μg/ml anti-TGFβ [Splenocytes were prepared from naive 5–10-week-old Vα2.3/Vβ8.2 TCR transgenic mice and cultured in 24-well plates at 2 × 10+/−, or B6/T-bet−/− mice were s.c. injected over four sites in the flank with 200 μg MOG 35-55 (CSBio Company Inc.) in an emulsion with CFA (Difco); 200 ng pertussis toxin (List) per mouse in PBS was injected i.p. at the time of immunization and 48 h later.The 8–10-week-old B6/WT, B6/T-bet6 cells/well with irradiated B10.PL splenocytes (6 × 106 cells/well). After 72 h, the cells were washed with PBS and 8 × 106 cells/mouse were injected i.p. into naive B10.PL mice.Splenocytes were isolated from naïve 5–10-week-old Vα2.3/Vβ8.2 TCR transgenic mice and activated with 2 μg/ml of MBP Ac1-11 with or without rmIL-7 (10 ng/ml) or αIL-7Rα (0.5 μg/ml) in 24-well plates at 2 × 10The mice were evaluated daily for clinical signs of EAE. Mice were scored on scale of 0 to 6: 0, no clinical disease; 1, limp/flaccid tail; 2, moderate hind limb weakness; 3, severe hind limb weakness; 4, complete hind limb paralysis; 5, quadriplegia or premoribund state; and 6, death.3 (pH 8.2) at 2 μg/ml while purified anti-mouse IFNγ primary antibody was diluted in 0.1 M NaHCO3 (pH 9.5) at 2 ug/ml. Immunolon II plates (Dynatech Laboratories) were coated with 50 μl of primary antibodies per well and incubated overnight at 4 °C. The plates were washed twice with PBS/0.05% Tween 20. The plates were blocked with 200 μl of 1% BSA in PBS per well for 2 h. The plates were washed twice with PBS/0.05% Tween 20, and 100 μl of supernatants were added in duplicate. The plates were incubated over-night at 4 °C and washed four times with PBS/0.05% Tween 20. Biotinylated rat anti-mouse secondary antibody (BD Biosciences) were diluted in PBS/1% BSA, 100 μl of 1 μg/ml biotinylated antibody was added to each well, and plates were incubated at room temperature for 1 h. The plates were washed six times with PBS/0.05%Tween 20, and 100 μl avidin-peroxidase was added at 2.5 μg/ml and incubated for 30 min. The plates were washed eight times with PBS/0.05% Tween 20, and 100 μl ABTS substrate containing 0.03% H2O2 (for IL-17) or TMB substrate (for IFNγ) was added to each well. The plate was monitored for 10–20 min for color development and read at A 405. A standard curve was generated from cytokine standard, and the cytokine concentration in the samples was calculated.ELISA was performed to detect the expression of IL-17 and IFNγ in supernatant. Purified anti-mouse IL-17 primary antibody (BD Biosciences) was diluted in 0.1 M NaHCOFlow cytometric analysis was performed to evaluate the expression of surface markers and T-bet in CD4 T cells, as previously described . BrieflyP < 0.05, as determined by Mann–Whitney U test. The Mann–Whitney U test is non-parametric, and therefore accounts for the fact that EAE scores are ordinal and not interval-scaled. ELISA and quantitated flow data comparisons were performed using two-tailed unpaired student’s t tests. Differences with P < 0.05 were considered significant.GraphPad software  was utilized for statistical analysis. A statistically significant difference in EAE clinical scores was considered to be +/+ (TCR-WT) or T-bet−/− (TCR-T-bet−/−) were activated with MBP Ac1-11 for 72 h, followed by resting for 4 days, and reactivation with MBP Ac1-11 for 2 days. At the end of primary stimulation, PD-1 and LAG-3 expressing myelin-specific effector CD4 T cells were significantly higher in T-bet−/− group compared to WT group  , may exert their function through regulating IL-7Rα/PD-1 balance in myelin-specific CD4 T cells during EAE development.T-bet is the key transcription factor regulating the differentiation of Th1 cells. T-bet deficient mice were originally shown to be resistant to EAE induction by active immunization , but latAfter the identification of Th17 cells as another encephalitogenic CD4 T helper population in addition to Th1 cells in EAE, the search for the potential therapeutic targets that convey the encephalitogenicity to myelin-specific CD4 T cells becomes even more complicated. Although IFNγ producing Th1 and IL-17 producing Th17 cells are both encephalitogenic, they have distinct cytokine profile, which raises the question whether encephalitogenic CD4 T cells exert their function mainly through the production of signature cytokines. Both IFNγ and IL-17 deficient mice are still susceptible to EAE induction . On a reTargeting PD-1/PD-L1 pathway for therapeutic purposes has been explored in cancer and chronic viral infection . ContrarIn the context of autoimmunity, our data demonstrate that several major determinants of T cell encephalitogenicity, including T-bet, IL-12, and IL-6, skew IL-7Rα/PD-1 balance towards IL-7Rα, favoring an encephalitogenic phenotype of myelin-specific CD4 T cells with enhanced effector function. Our data show that IL-7 signaling inhibits PD-1 expression in myelin-specific CD4 T cells and blockade of IL-7R signaling on myelin-specific CD4 T cells significantly decreased the encephalitogenic potential of those cells. Therefore, skewing IL-7Rα/PD-1 balance towards PD-1 by either stimulating PD-1/PD-L1 pathway or suppressing IL-7Rα signaling may have therapeutic potential for the treatment of autoimmune diseases, including MS.In this study, we characterized the factors regulating IL-7Rα/PD-1 balance in myelin-specific CD4 T effector/memory cells during EAE development. We have shown that T-bet is a major transcription factor regulating IL-7Rα/PD-1 balance in myelin-specific CD4 T cells, and there is a positive correlation between several major determinants promoting T cell encephalitogenicity  and an IL-7Rα/PD-1 balance skewed towards IL-7Rα, suggesting that those major determinants critical to T cell encephalitogenicity may exert their function through regulation of IL-7Rα/PD-1 balance. Additionally, IL-7 signaling inhibits PD-1 expression in myelin-specific CD4 T cells and blocking IL-7 signaling suppresses T cell encephalitogenicity. Therefore, interference with inhibitory pathways and IL-7Rα expression may suppress the encephalitogenic potential of myelin-specific CD4 T cells and have therapeutic benefits for MS patients.


Extracellular vesicles  are approximately 30- to 200-nm-long vesicles that have received increased attention due to their role in cell-to-cell communication. Although EVs are highly anticipated to be a next-generation intracellular delivery tool because of their pharmaceutical advantages, including non-immunogenicity, their cellular uptake efficacy is low because of the repulsion of EVs and negatively charged cell membranes and size limitations in endocytosis. Here, we demonstrate a methodology for achieving enhanced cellular EV uptake using arginine-rich cell-penetrating peptides (CPPs) to induce active macropinocytosis. The induction of macropinocytosis via a simple modification to the exosomal membrane using stearylated octaarginine, which is a representative CPP, significantly enhanced the cellular EV uptake efficacy. Consequently, effective EV-based intracellular delivery of an artificially encapsulated ribosome-inactivating protein, saporin, in EVs was attained. Extracellular vesicles  are cellular vesicles (30–200 nm in diameter) with a lipid bilayer that are constitutively secreted from most cells. EVs are found in abundance in bodily fluids, such as blood, saliva, urine, and breast milk121243679in vivo treatment due to the dispersal of EVs in bodily fluid. Thus, the artificial induction of macropinocytosis stimulated by the functionalized EV itself is strongly considered very useful for the EV-based intracellular delivery of therapeutic molecules.Endocytosis has been shown to be a major route for cellular EV uptake11121316Given the importance of the artificial induction of macropinocytosis for the development of EV-based intracellular delivery systems, in this study, we demonstrate that modification of arginine-rich cell-penetrating peptides (CPPs) on EV membranes results in the effective induction of macropinocytosis and cellular EV uptake . Arginin1820212223In our system, EV membranes were modified with r8, which is a representative arginine-rich CPP189Next, the cellular uptake efficacy of the stearyl-r8-modified EV was evaluated using confocal microscopy and flow cytometry. Marked induction of macropinocytosis, which facilitated the cellular uptake of EVs into cells, was attained by surface modification with stearyl-r8 peptide, as was also observed for liposomes modified with octaarginine2728To examine the cellular uptake mechanisms of the stearyl-r8-modified EV, we tested the induction of macropinocytosis by the treatment of the EV on cells. First, we tested cellular EV uptake under low temperature (4 °C), which is an experimental condition for the prevention of endocytosis2016Next, we examined the cellular uptake of cargo molecules artificially encapsulated in EVs. First, we prepared FITC-dextran -encapsulated EVs via electroporationIn addition, we encapsulated the ribosome-inactivating protein saporin31In this study, we successfully developed an EV-based intracellular delivery system via the effective induction of macropinocytosis by the modification of the arginine-rich cell-penetrating peptide on EV membranes. The macropinocytosis route can take up large molecules from outside cells (>1 μm)All peptides were chemically synthesized via 9-fluorenylmethyloxycarbonyl (Fmoc) solid-phase peptide synthesis on a Rink amide resin with a coupling system using 1-hydroxybenzotriazole (HOBt)/2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) /N,N-diisopropylethylamine (DIEA) as previously described233(CH2)16-CO-NH-D(Arg)8-amide): MALDI-TOFMS: 1533.6 [calculated for (M + H)+: 1534.0]. Retention time in HPLC: 21.2 min (column: Cosmosil 5C18-AR-II (4.6 × 150 mm); gradient: 5–95% B in A  over 30 min; flow: 1 ml/min; detection: 220 nm). Yield from the starting resin: 28%.Stearyl-r8 (CH2-D(Arg)8-amide): MALDI-TOFMS: 1267.1 [calculated for (M + H)+: 1267.5]. Retention time in HPLC: 13.5 min (column: Cosmosil 5C18-AR-II (10 × 250 mm); gradient: 5–80% B in A  over 30 min; flow: 1 ml/min; detection: 220 nm). Yield from the starting resin: 10.2%.r8  at 20 °C for 3 h, and the product was purified via HPLC purification. Fluorescent labeling with purified peptides was performed by treatment with 1.5 equivalents of Alexa Fluor 488 (Alexa488) C5 maleimide sodium salt  in a dimethyl formamide/methanol mixture (1:1) for 1.5 h at room temperature followed by HPLC purification, as previously reported3(CH2)16-CO-NH-D(Arg)8-Gly-Cys-amide): MALDI-TOFMS: 1693.4 [calculated for (M + H)+: 1693.3]. Retention time in HPLC: 19.2 min (column: Cosmosil 5C18-AR-II (4.6 × 150 mm); gradient: 5–95% B in A  over 30 min; flow: 1 ml/min; detection: 220 nm). Yield from the starting resin: 0.4%.Stearyl-r8-GC (CH2-D(Arg)8-Gly-Cys-amide): MALDI-TOFMS: 1426.8 [calculated for (M + H)+: 1426.9]. Retention time in HPLC: 8.4 min (column: Cosmosil 5C18-AR-II (4.6 × 150 mm); gradient: 5–95% B in A  over 30 min; flow: 1 ml/min; detection: 220 nm). Yield from the starting resin: 20.8%.r8-GC (NH3(CH2)16-CO-NH-D(Arg)8-Gly-Cys(Alexa488)-amide): MALDI-TOFMS: 2391.5 [calculated for (M + H)+: 2391.0]. Retention time in HPLC: 19.8 min (column: Cosmosil 5C18-AR-II (4.6 × 150 mm); gradient: 5–95% B in A  over 30 min; flow: 1 ml/min; detection: 220 nm). Yield from the starting purified stearyl-r8-GC peptide: 0.3%.Stearyl-r8-GC(Alexa488) (CH2-D(Arg)8-Gly-Cys(Alexa488)-amide): MALDI-TOFMS: 2125.5 [calculated for (M + H)+: 2124.5]. Retention time in HPLC: 10.5 min (column: Cosmosil 5C18-AR-II (4.6 × 150 mm); gradient: 5–95% B in A  over 30 min; flow: 1 ml/min; detection: 220 nm). Yield from the starting purified r8-GC peptide: 13.5%.r8-GC(Alexa488)  cells were purchased from the Riken BRC Cell Bank . The human epidermoid carcinoma-derived A431 cells and Chinese hamster ovary (CHO)-K1 cells were purchased from the American Type Culture Collection . Each cell was cultured in α-MEM  (HeLa cells), minimum essential medium (MEM)  (A431 cells), and F-12 nutrient mixture (Ham’s F-12) (CHO-K1)  containing 10% heat-inactivated FBS . Each cell was grown on 100-mm dishes and incubated at 37 °C under 5% CO5 cells) were plated on a 24-well microplate  and incubated for 1 day. They were transfected with CD63-GFP plasmid  (800 ng) complexed with Lipofectamine LTX reagent (2 μl) and PLUS reagent (1 μl)  in α-MEM containing 10% FBS (200 μl). The cells were also treated with puromycin (3 μg/ml)  for the antibiotic selection of HeLa cells stably expressing CD63-GFP (CD63-GFP-HeLa).CD63 is a membrane marker tetraspanin protein of EVs, and we prepared HeLa cells stably expressing GFP-fused CD63 to secrete CD63-GFP-containing EVs (CD63-GFP-EVs) as previously reported96 cells) were seeded onto 100-mm dishes in α-MEM containing 10% exosome-free FBS  for 3 days. The cell culture medium was collected, and the secreted EVs were isolated using ultracentrifugation as previously reportedg) for 10 min at 4 °C. The supernatant was centrifuged  for 10 min at 4 °C and again centrifuged  for 30 min at 4 °C to remove cell debris. The supernatant was then centrifuged  for 70 min at 4 °C  in duplicate, and the pellet was collected in PBS. The concentrations of isolated EVs were described in terms of their protein concentrations, which were determined using a Pierce BCA protein assay kit .CD63-GFP-HeLa cells or HeLa cells (without expression of CD63-GFP) (each 3 × 10Isolated EVs were added to lysis buffer (62.5 mM Tris-HCl (pH = 6.8), 2% SDS, 10% glycerol, 0.002% bromophenol blue, 5% 2-mercaptoethanol). The boiled samples were separated via 10% SDS-PAGE, transferred onto polyvinylidene fluoride (PVDF) membranes , and treated with anti-CD63 antibody . A secondary antibody labeled with horseradish peroxidase  was used, and immunoreactive species were detected using the Enhanced Chemiluminescence (ECL) Plus Western Blotting Detection System  with the Amersham Imager 600 . Immunoreactive species were detected at a position of approximately 50 kDa in SDS-PAGE analysis using the anti-CD63 antibody.Synthesized stearyl-r8 peptide  diluted with phosphate-buffered saline (PBS) was added to a solution of EVs (100 μg) in PBS  and incubated for 1 h at 37 °C. Removal of unattached stearyl-r8 peptide was accomplished by washing with PBS and filtration using Amicon Ultra centrifugal filters . The attachment of stearyl-r8-GC(Alexa488) and r8-GC(Alexa488) to EVs was confirmed using a spectrofluorometer .5 cells, 2 ml) were plated onto a 35-mm glass dish  and incubated in cell culture medium  containing 10% FBS for 24 h at 37 °C under 5% CO2. After complete adhesion, the cells were washed with cell culture medium containing 10% FBS and treated with each EV sample (100 μl/well). The cells were stained with Hoechst 33342 dye  for 15 min at 37 °C prior to cell washing. The cells were then washed with fresh cell culture medium and analyzed using a FV1200 confocal laser scanning microscope  equipped with a 40× or 60× objective without cell fixation. For the detection of macropinocytosis uptake, co-treatment with Texas red-dextran  with EV samples was performed.Cells  modified with or without stearyl-r8 (16 μM) for 1 h at 37 °C. After the removal of the medium, the cells were fixed with 4% paraformaldehyde at room temperature for 30 min and washed with PBS. The cells were then treated with 0.1% Triton X-100 (100 μl/well in PBS) at room temperature for 5 min and again washed with PBS. The expression of syndecan-4 was visualized by treatment with syndecan-4 antibody , and secondary antibody labeled with fluorescence (Alexa Fluor 488 goat anti-mouse IgG (H + L), Invitrogen) was then used (each 30 min at room temperature) prior to analysis using a FV1200 confocal laser scanning microscope (Olympus).4 cells, 1 ml) were plated onto a 24-well microplate (Iwaki) and incubated in cell culture medium  containing 10% FBS for 24 h at 37 °C under 5% CO2. After complete adhesion, the cells were washed with cell culture medium containing 10% FBS and treated with each EV sample or macropinocytosis marker, FITC-labeled dextran  (Sigma-Aldrich) (600 μl/well) prior to washing with 0.5 mg/ml heparin in PBS . The cells were then treated with 0.01% trypsin at 37 °C for 10 min prior to the addition of PBS (200 μl) and then centrifuged at 1,500 rpm (200 × g) for 5 min at 4 °C. After the removal of the supernatant, the cells were washed with PBS (400 μl) and centrifuged at 1,500 rpm for 3 min at 4 °C. This washing cycle was repeated, and the cells were suspended in PBS (400 μl) and subjected to fluorescence analysis with a Guava easyCyte  flow cytometer using 488-nm laser excitation and a 525-nm emission filter. Live cells  for the detection of cellular fluorescence intensity were quantified based on forward-scattering and side-scattering analyses. For induction of macropinocytosis in Cells  amiloride16Suspended EVs in PBS (30 μg/ml) were dropped onto a carbon-coated grid (400 mesh) and washed with distilled water. Uranyl acetate was applied to the grid and left for 10 s at room temperature. Next, the reagent was removed with filter paper and dried prior to imaging with a transmission electron microscope (TEM) .The zeta-potential and particle size of the EVs diluted in PBS (58.2 μg/ml) were determined using the zeta-potential and particle size analyzer ELSZ-DN2  according to the manufacturer’s instructions.5 cells, 2 ml) were plated onto a 35-mm glass dish (Iwaki) and incubated in cell culture medium (α-MEM) containing 10% FBS for 24 h at 37 °C under 5% CO2. After complete adhesion, the cells were washed with cell culture medium containing 10% FBS and treated with each EV sample (100 μl/well) for 20 min at 37 °C. After removal of the medium, the cells were fixed with 4% paraformaldehyde at room temperature for 30 min and washed with PBS. The cells were then treated with 0.1% Triton X-100 (100 μl/well in PBS) at room temperature for 5 min and again washed with PBS. Cellular F-actin was stained with rhodamine-phalloidin (Molecular Probes) for 20 min at 4 °C, and the cells were washed with PBS prior to analysis using a FV1200 confocal laser scanning microscope (Olympus) equipped with a 40× objective.HeLa cells  dissolved in H2O (100 μl) was reacted with 2 equivalents of FITC (Sigma-Aldrich) dissolved in dimethyl sulfoxide (10 μl) and N,N-diisopropylethylamine (0.5 μl) at 30 °C for 2 h as previously reported9For the preparation of FITC-labeled saporin, saporin  were mixed with FITC-labeled dextran  (Sigma-Aldrich) or saporin (50 μg) in PBS 100 μl). After electroporation ; transfer pulse: five pulses ) in a 1-cm electroporation cuvette at room temperature using a super electroporater NEPA21 Type II , the unencapsulated FITC-dextran or saporin was removed by washing and filtration using Amicon Ultra centrifugal filters (100 K device), as previously reported90 μl. Aft94 cells, 100 μl) were incubated in 96-well microplates in cell culture medium containing 10% FBS for 24 h at 37 °C under 5% CO2. The cells were then treated with each EV sample (50 μl) at 37 °C under 5% CO2. After the sample treatment, WST-1 reagents (10 μl) were added to each well and the samples were incubated for 40 min at 37 °C. The absorbencies at 450 nm (A450) and 620 nm (A620) were measured, and the value obtained by subtracting A620 from A450 corresponded to the viable cell number.Cell viability was analyzed using the WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) assay, as previously described9t-test was used after verification of the equal variances with an F-test. Welch’s correction was performed when the variances across groups were assumed to be unequal. For multiple comparison analyses, one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test or Dunnett’s post hoc test was used. Differences were considered significant when the calculated p-value was < 0.05.All statistical analyses were performed using GraphPad Prism software . For comparisons of two groups, unpaired Student’s How to cite this article: Nakase, I. et al. Vectorization of biomacromolecules into cells using extracellular vesicles with enhanced internalization induced by macropinocytosis. Sci. Rep.6, 34937; doi: 10.1038/srep34937 (2016).


Me4H) capping ligands with strong magnetic exchange coupling provided by an N23− radical bridging ligand results in a series of dilanthanide complexes exhibiting exceptionally large magnetic hysteresis loops that persist to high temperatures. Significantly, reducing the coordination number of the metal centers appears to increase axial magnetic anisotropy, giving rise to larger magnetic relaxation barriers and 100-s magnetic blocking temperatures of up to 20 K, as observed for the complex [K(crypt-222)][(CpMe4H2Tb)2(μ−Increasing the operating temperatures of single-molecule magnets—molecules that can retain magnetic polarization in the absence of an applied field—has potential implications toward information storage and computing, and may also inform the development of new bulk magnets. Progress toward these goals relies upon the development of synthetic chemistry enabling enhancement of the thermal barrier to reversal of the magnetic moment, while suppressing alternative relaxation processes. Herein, we show that pairing the axial magnetic anisotropy enforced by tetramethylcyclopentadienyl (Cp 23– radical-bridged dilanthanide complexes that exhibit giant coercivities and 100-s magnetic blocking temperatures of up to 20 K.Single-molecule magnets typically only retain information in the presence of an applied magnetic field and at very low temperatures. Here, Demir, Long and co-workers design N U must be overcome to reverse the orientation of the magnetic moment. The potential application of such species in high-density information storage1, as well as quantum computing2 and spin-based electronics3, hinges upon improving not just the defining thermal energy barrier to magnetization reversal, but also the magnetic blocking temperature and coercive field—metrics that determine the ability of the molecules to retain information upon removal of an applied magnetic field 4. The use of selected 4f metal ions, particularly TbIII, DyIII, and ErIII, has recently dominated this area of research due to the advantageous combination of high magnetic moment and intrinsic magnetic anisotropy engendered by large spin–orbit coupling11. Progress in increasing blocking temperatures and coercive fields has been challenging mainly owing to the prevalence of through-barrier relaxation pathways, such as quantum tunneling of the magnetization, though recent reports on a molecule containing a single DyIII ion demonstrate Orbach relaxation proceeding through nearly the entire effective magnetization reversal barrier (Ueff) of greater than 1200 cm−1, with a correspondingly high 100-s blocking temperature13.Single-molecule magnets are molecules in which a strong axial anisotropy, imparted by the ligand field surrounding one or more metal centers, acts to create a bistable ground state, for which an activation barrier Ueff14. One strategy for suppressing quantum tunneling of the magnetization is to establish rigorously symmetry-protected pure MJ states15, a condition as-yet only experimentally achieved in adatom-surface experiments16, though reports of the high-blocking temperature [Dy(Cpttt)2]+ cyclopentadienide) complex suggest that rigorous symmetry may not be crucial to achieve high performance in single-ion magnetic molecules13. Another promising route is the design of systems with strong intramolecular magnetic coupling between two or more metal centers. In addition to creating a more classical coupled system with large total angular momentum, magnetic coupling has been postulated to generate an exchange bias field that impedes quantum tunneling of the magnetization20. In recent years, a particularly successful strategy to promote strong magnetic coupling between lanthanide centers has been the employment of paramagnetic bridging ligands28. Significantly, the diffuse spin orbitals of anionic radical ligands are better able to penetrate into the core electron density of the deeply buried 4f orbitals. Our strategy toward high-performance single-molecule magnets therefore targets systems in which radical ligands facilitate a direct exchange coupling with lanthanide ions constrained in coordination environments that promote strong axial magnetic anisotropy. Here we show that this can be achieved in two new series of organometallic N23− radical-bridged dilanthanide complex salts: [K(crypt-222)(THF)]2, was purchased from Sigma-Aldrich, dissolved in toluene, filtered through Celite, and recrystallized from toluene at −35 °C before use. The compound 4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo[8.8.8]-hexacosane  was purchased from TCI America and used as received. The compounds, KCpMe4H29 and KC846, were prepared according to literature procedures. Synthesis details for the precursor complexes CpMe4H2Ln(η3-C3H5) and CpMe4H2Ln(BPh4),  or CpMe4H2Sc(BPh4)30. Preparation of the complexes CpMe4H2Ln(µ-Cl2)K(THF)x and (CpMe4H2Ln(THF))(µ-N2), 3-Ln, )2(µ−N2), where Ln = Gd, Dy were published previously, although (CpMe4H2Dy(THF))2(µ−N2) was reported in a different cell, and these compounds were synthesized by reduction of (C5Me4H)3Ln(THF)47. Elemental analyses were performed by the Micro-Mass Facility at the University of California, Berkeley, using a Perkin-Elmer Series 2400 Series II combustion analyzer. IR spectra were recorded on a Perkin-Elmer Avatar Spectrum 400 FTIR Spectrometer equipped with ATR.The manipulations described below were performed under an inert atmosphere with rigorous exclusion of air and water using Schlenk, vacuum line, and glovebox techniques. Solvents were dried using a commercial solvent purification system from JC Meyer Solvent Systems (Me4H2Ln(BPh4) was dissolved in 15 ml of THF to give a colorless solution. Subsequently, potassium graphite was added at once whereby the solution color turned to orange–red. After 5 min of stirring at room temperature, black and white insoluble materials, presumably graphite and KBPh4, were removed by filtration. The orange–red filtrate was pumped down under reduced pressure to yield a solid that was extracted with 5 ml of toluene to afford a dark red solution. Green crystals of (CpMe4H2Ln(THF))2(µ−N2) suitable for X-ray analysis were grown in the freezer over the course of 48 h.In a nitrogen-filled glovebox, Cp3-Gd: Isolated 263 mg in crystalline yield (49%) from the reaction of 790 mg (1.10 mmol) of crystalline CpMe4H2Gd(BPh4) and 149 mg (1.10 mmol) of potassium graphite. IR (neat): 2962 m, 2905 s, 2855 s, 2716 w, 1443 m, 1377 w, 1327 w, 1242 w, 1173 w, 1108 w, 1030 s, 917 w, 890 m, 875 s, 833 w, 777 s, 762 s, 741 m, 613 s cm−1Anal. Calcd for C44H68N2O2Gd2: C, 54.40; H, 7.06; N, 2.88. Found: C, 54.76; H, 7.21; N, 2.77.3-Tb: Isolated 162.9 mg in crystalline yield (52%) from the reaction of 460.4 mg (0.639 mmol) of crystalline CpMe4H2Tb(BPh4) and 86.4 mg (0.639 mmol) of potassium graphite. IR (neat): 2960 m, 2930 m, 2905 s, 2895 s, 2858 s, 1459 m, 1446 m, 1437 m, 1429 m, 1376 m, 1368 m, 1030 s, 917 m, 874 s, 860 s, 780 s, 765 s, 743 s, 663 m, 655 m, 615 s, 600 s cm−1. Anal. Calcd for C44H68N2O2Tb2: C, 54.21; H, 7.03; N, 2.87. Found: C, 54.31; H, 7.03; N, 2.79.3-Dy: Isolated 131.2 mg in crystalline yield (55%) from the reaction of 350.3 mg (0.4837 mmol) of crystalline CpMe4H2Tb(BPh4) and 65.4 mg (0.484 mmol) of potassium graphite. IR (neat): 2954 m, 2936 m, 2901 s, 2857 s, 2858 s, 1461 m, 1448 m, 1441 m, 1432 m, 1426 m, 1379 m, 1370 m, 1202 w, 1030 s, 916 w, 874 s, 862 s, 781 s, 763 s, 742 m, 665 m, 626 m, 616 s, 611 s cm−1. Anal. Calcd for C44H68N2O2Dy2: C, 53.81; H, 6.98; N, 2.85. Found: C, 53.84; H, 7.10; N, 2.82.Me4H2Ln(THF))2(µ−N2) (6 ml in the case of Dy). Subsequently, potassium graphite was added at once to the reaction mixture, whereby the solution color turned to red–brown. After 5 min of stirring at room temperature, black insoluble material, presumably graphite, was removed by filtration. The dark brown filtrate was stored in the freezer for 24 h to afford dark brown crystals of 1-Ln that were suitable for X-ray analysis.In an argon-filled glovebox, a 10 ml THF solution containing crypt-222 (6 ml in the case of Dy) was added to a 10 ml pale green THF solution of crystalline  from the reaction of 113.9 mg (0.1166 mmol) of crystalline (CpMe4H2Gd(THF))2(µ−N2), 43.9 mg (0.117 mmol) of crypt-222, and 15.8 mg (0.117 mmol) potassium graphite. IR (neat): 2939 m, 2881 s, 2844 s, 2711 w, 1473 w, 1458 w, 1444 w, 1353 s, 1329 w, 1314 w, 1297 w, 1283 w, 1261 w, 1244 w, 1172 w, 1132 m, 1102 s, 1076 m, 1062 s, 1042 m, 1029 m, 946 s, 922 m, 901 m, 862 w, 826 w, 809 w, 788 w, 763 m, 746 m, 727 m, 727 w, 716 w, 702 w, 663 w, 616 w, 600 w cm−1. Anal. Calcd for C66H112KN4O9Gd2: C, 54.32; H, 7.74; N, 3.84. Found: C, 54.50; H, 7.89; N, 3.78.1-Tb: Isolated 168 mg in crystalline yield (78%) from the reaction of 144 mg (0.148 mmol) of crystalline (CpMe4H2Tb(THF))2(µ−N2), 55.7 mg (0.148 mmol) of crypt-222, and 20.0 mg (0.148 mmol) of potassium graphite. IR (neat): 2940 mbr, 2879 s, 2846 s, 2712 w, 1474 w, 1455 m, 1443 m, 1352 s, 1297 w, 1283 w, 1260 w, 1243 w, 1171 w, 1132 s, 1102 s, 1077 s, 1062 s, 1044 s, 1030 s, 947 s, 931 m, 922 m, 903 m, 856 w, 831 w, 824 w, 765 m, 746 s, 727 w, 615 w, 557 s cm−1. Anal. Calcd for C66H112KN4O9Tb2: C, 54.20; H, 7.72; N, 3.83. Found: C, 54.13; H, 7.85; N, 3.80.1-Dy: Isolated 63.8 mg in crystalline yield (57%) from the reaction of 74.5 mg (0.0759 mmol) of crystalline (CpMe4H2Dy(THF))2(µ−N2), 28.6 mg (0.0759 mmol) of crypt-222, and 10.3 mg (0.0759 mmol) of potassium graphite. IR (neat): 2938 mbr, 2878 s, 2847 s, 1476 m, 1459 m, 1452 m, 1443 m, 1353 s, 1298 w, 1284 w, 1259 w, 1242 w, 1172 w, 1133 s, 1101 vs, 1062 s, 1043 s, 947 s, 931 m, 922 m, 903 m, 883 w, 853 w, 822 w, 767 m, 746 s, 696 w, 672 w, 641 w, 614 w, 560 s cm−1. Anal. Calcd for C66H112KN4O9Dy2: C, 53.94; H, 7.68; N, 3.81. Found: C, 53.85; H, 7.88; N, 4.00.2-Tb: In an argon-filled glovebox, [K(crypt-222)(THF)][(CpMe4H2Tb(THF))2(µ−N2)]  was dissolved in 12 ml of 2-MeTHF to give a brown–black solution. The solution was filtered and the brown–black filtrate was stored in the freezer for 48 h to afford brown–black crystals of 2-Tb that were suitable for X-ray analysis . Anal. Calcd for C59H98KN4O7Tb2: C, 53.19; H, 7.41; N, 4.21. Found: C, 53.34; H, 7.46; N, 4.12.2-Dy: The same procedure was used as above, with 62.0 mg (0.042 mmol) [K(crypt-222)(THF)][(CpMe4H2Dy(THF))2(µ−N2)], and 10 ml of 2-MeTHF to give a brown–black solution, which was filtered and stored in the freezer for 48 h to afford brown–black crystals of 2-Dy that were suitable for X-ray analysis . IR (neat): 2956 m, 2878 s, 2852 s, 2717 w, 1477 m, 1456 m, 1442 m, 1378 w, 1354 s, 1296 m, 1259 m, 1132 s, 1101 vs, 1079 s, 1022 m, 947 s, 932 s, 828 m, 819 m, 803 w, 765 s, 749 s, 620 m, 567 vs cm−1. Anal. Calcd for C59H98KN4O7Dy2: C, 52.90; H, 7.37; N, 4.18. Found: C, 53.06; H, 7.23; N, 4.28.Me4H2Gd(THF))2(µ−N2.)]  was dissolved in 10 ml of 2-MeTHF to give a brown–black solution. The solution was filtered and the brown–black filtrate was stored in the freezer for 48 h to afford brown–black crystals of 4 that were suitable for X-ray analysis . IR (neat): 2952 m, 2879 s, 2842 s, 2711 w, 1473 m, 1441 m, 1376 w, 1350 m, 1327 w, 1297 m, 1256 m, 1232 w, 1172 w, 1131 s, 1114 s, 1101 vs, 1076 s, 1057 s, 1031 m, 1015 m, 991 w, 966 w, 951 s, 932 s, 896 w, 869 w, 859 w, 833 w, 820 m, 798 w, 751 s, 700 w, 684 w, 654 w, 615 w cm−1. Anal. Calcd for C64H108KN4O8Gd2: C, 54.32; H, 7.69; N, 3.96. Found: C, 54.10; H, 7.80; N, 3.82.In an argon-filled glovebox, [K(crypt-222)(THF)][(Cpλ = 0.71073 Å) and a Bruker APEX-II detector for 2-Tb, 4, 3-Dy, CpMe4H2Dy(BPh4), CpMe4H2Gd(BPh4), CpMe4H2Tb, and CpMe4H2Dy, or at Beamline 11.3.1 at the Advanced Light Source on a Bruker D8 Diffractometer equipped with a Bruker PHOTON100 CMOS detector using synchrotron radiation with λ = 0.6888 Å for 1-Dy and CpMe4H2Gd and λ = 0.7749 Å for 1-Gd, 1-Tb, 2-Dy, 3-Tb, 3-Gd, and CpMe4H2Tb(BPh4) (further details are provided in Supplementary Methods and Supplementary Tables 2-Tb),1547762 (2-Dy),1547763 (4), 1547764 (1-Tb) 1547765 (1-Dy), 1547766 (1-Gd), 1547767 (3-Tb), 1547768 (3-Dy), 1547769 (3-Gd), 1547770 (CpMe4H2Tb(BPh4)), 1547771 (CpMe4H2Dy(BPh4)), 1547772 (CpMe4H2Gd(BPh4)), 1547773 ), 1547774 ), 1547775 ).X-ray diffraction experiments were performed at 100 K on crystals coated with paratone-N oil and mounted on Kapton or MiTeGen loops. Data were collected at the small molecule X-ray crystallography facility at the University of California, Berkeley using a Bruker QUAZAR diffractometer equipped with a microfocus sealed X-ray source , 2-Ln , and 3-Ln  were prepared by adding crushed crystalline samples to 7 mm quartz tubes. Sufficient liquid eicosane (at 40 °C) was added to saturate and cover the samples to prevent crystallite torqueing and provide good thermal contact between the sample and the bath. Tubes were fitted with sealable adapters, evacuated on a Schlenk line, and flame sealed under vacuum using a H2/O2 flame. Magnetic susceptibility measurements were collected using a Quantum Design MPMSXL SQUID magnetometer. For the hysteresis loops of 2-Tb, magnetic susceptibility measurements were collected using a Quantum Design 14 Tesla Dynacool magnetometer at the facility of Quantum Design in San Diego, CA. High-frequency ac measurements were also performed at the facility of Quantum Design in San Diego, CA, where a 9 T PPMS equipped with the ACMSII measurement option to probe the ac relaxation at frequencies above 1500 Hz was used. Dc susceptibility data measurements were performed at temperatures ranging from 2 to 300 K for 1-Ln , 2-Ln , and 3-Ln  using applied fields of 1000, 5000, and 10,000 Oe. Ac magnetic susceptibility data measurements were performed using a 4 Oe switching field. All data were corrected for diamagnetic contributions from the eicosane and core diamagnetism estimated using Pascal’s constants48. Cole-Cole plots were fitted using formulae describing χ′ and χ″ in terms of frequency, constant temperature susceptibility (χT), adiabatic susceptibility (χS), relaxation time (τ), and a variable representing the distribution of relaxation times (α)33. All data were fitted to α values of ≤0.09.The magnetic samples of 1-Dy, 1-Tb, 2-Dy, and 2-Tb were modeled using the Hamiltonian given in Eq. , spin-flip energies, ΔE, were estimated for comparison with the experimental barriers, Ueff.Magnetic susceptibility data for www.ccdc.cam.ac.uk/data_request/cif. All other data can be obtained from the authors on request.Crystallographic data have been deposited in the Cambridge Structural Database as specified above for each compound under the deposition numbers CCDC 1547761-1547775 that is available free of charge from The Cambridge Crystallographic Data Centre via Supplementary Information


The structures and configurations of these new compounds were elucidated through comprehensive spectroscopic analyses including 1D NMR, 2D NMR  and HRESIMS, and electronic circular dichroism (ECD) data. Neuroinflammation is implicated in the pathogenesis of various neurodegenerative diseases, such as Alzheimers’ disease (AD). All isolated compounds were evaluated for the potential anti-neuroinflammatory activities in BV2 microglia cells. Several compounds showed differential effects on the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in lipopolysaccharide (LPS)-stimulated and Aβ1–42-treated mouse microglia cell line BV-2. Molecular docking revealed that bioactive compounds  could interact with iNOS protein other than COX-2 protein. Collectively, our results suggested that this class of cyathane diterpenoids might serve as important lead compounds for drug discovery against neuroinflammation in AD.Ten new polyoxygenated cyathane diterpenoids, named neocyathins A–J ( Microglia are the resident immune cells in the central nervous system (CNS). It is well-known that microglia are often activated by different stimuli to mediate inflammatory responses in the brain, suggesting the importance of microglial inhibition against neuroinflammation and neurodegeneration3. Lipopolysaccharide (LPS) and amyloid-β (Aβ) could activate microglia and subsequently induce inducible oxide synthase (iNOS) and cyclooxygenase-2 (COX-2)5. Excessive NO production directly causes neuronal injury and even neuronal death3. Thus, neuroinflammation may be a potential therapeutic target for treating AD and other neurodegenerative diseases.Neuroinflammation is implicated in the pathogenesis of various neurodegenerative diseases including Alzheimer’s disease(AD)Cyathus, Hericium, and Sarcodon8. These diterpenes were demonstrated to display a wide range of biological properties, including anti-inflammatory9, antimicrobial10 antitumor11 and antagonism toward the kappa-opioid receptor12. Several cyathane diterpenoids were found to stimulate the synthesis of NGF in human nerve cells14 indicating their potential as therapeutic agents to treat neurodegenerative ailment.Basidiomycetes are known to produce a broad spectrum of secondary metabolites. A number of cyathane diterpenoids with an unusual 5/6/7 tricyclic skeleton, including their xylosides, were isolated from a diverse variety of higher Basidiomycetes of the genera Cyathus is a genus in the family of Nidulariaceae, collectively known as the bird’s nest fungi. These fungi are widely distributed all over the world, and usually grow on decaying wood/woody debris and cow/horse dung. Although generally inedible, Cyathus species is well known as the prolific producers of bioactive cyathane diterpenoids.The fungi 19. The aim of the present study was to identify new cyathane diterpenoids against neuroinflammation. We isolated a total of 14 compounds and elucidated the structures by NMR, HRESIMS, and ECD analyses. All isolated compounds were evaluated for the anti-neuroinflammatory effects in lipopolysaccharide (LPS)- or Aβ1–42-stimulated mouse microglia cell line BV-2.We have been vigorously searching for novel bioactive compounds from basidiomycete fungiCyathus africanus was extracted with EtOAc and subjected to a series of chromatographic separations. Among a total of 14 compounds isolated, there were 10 new diterpenes, named neocyathins A–J (1–10) and four known congeners (11–14)  were identified as cyathin I (11)20 (12R)-11α,14α-epoxy-13α,14β,15-trihydroxycyath-3-ene (12)21 cyathin O (13)22 and allocyafrin B4 (14)23 respectively, by comparing their NMR data with those reported in literatures.The culture broth of 1 was isolated as a white powder. The molecular formula of 1 was determined to be C20H28O5, with seven degrees of unsaturation, on the basis of HRESIMS at m/z 371.1824 [M + Na]+. The 1H NMR spectrum , 1.15 , 1.26 , 1.37 ], two olefinic protons , one oxymethine [δH 4.24 ], and two oxymethylene protons . The 13C NMR data (Table C 64.4 (C-15)), five methines , and seven quaternary carbons , one olefinic proton [δH 5.75 ], two oxymethines , and two oxymethylene protons [δH 4.17 ]. The 13C NMR data (Table C 65.0 (C-15)], five methines , and six quaternary carbons [three olefinic and one carbonyl]. The NMR spectroscopic data of 2 were similar to those of cyathatriol7 except for the presence of a ketonic carbonyl group at C-2 (δC 211.1).Compound 2 was established through further analysis involving 1D NMR, HMBC and COSY spectra. The 1H-1H-COSY and HMBC spectra of 2 allowed the identification of four partial structures  at 206 nm (Δε + 43.4) and the negative CEs at 242 (Δε − 17.7) and 320 nm (Δε − 5.61). Consistently, the time-dependent density functional theory (TDDFT) method (cam-B3LYP/TZVP) provided similar calculated ECD values, thereby implying its absolute configuration to be 5R, 6R, 9S, 11R, 14S. Accordingly, the structure of 2 was established as shown, and named neocyathin B.To confirm the absolute configuration of ile Fig. . The exp3 was isolated as a yellow oil. The molecular formula of 3 was determined to be C20H30O5 based on the HRESIMS at m/z 373.1980 [M + Na]+. The 13C NMR data . The main differences between 3 and 14 were the presence of two hydroxy groups at C-1 (δC 88.5) and C-2 (δC 83.5) and an olefinic bond at C-3 (δC 140.2) and C-4 (δC 141.4) in 3. These observations were supported by the HMBC correlations from H-17 to C-1, C-4, from H-19 and H-20 to C-3, from H-10 to C-4, from H-5 and H-10 to C-4, C-3, from H-1 to C-2, C-3, and from H-2 to C-3  -3 -3 Fig. . Thus, t4 was isolated as a white powder. The molecular formula of 4 was determined to be C20H30O5, with six degrees of unsaturation, based on the HRESIMS at m/z 373.1979 [M + Na]+. The 1H NMR spectrum , 1.18 , 1.32 , 1.42 ], one olefinic proton [δH 6.12 ], two oxymethines , and two oxymethylene protons . The 13C NMR data (Table C 72.3 and 72.8 and one olefinic at δC 123.2), and six quaternary carbons . The 1H-1H COSY spectrum of 4 showed the spin system of CH3-19/CH-18/CH3-20, CH2-1/CH-2, CH-5/CH2-10/CH-11 and CH2-7/CH2-8. In the HMBC spectrum of 4, the HMBC correlations from H-2 to C-4, C-9, from H-5 to C-4, from H-10 to C-4, C-6, C-11 and C-12, from H-13 to C-6, C-11, C-12 and C-15, from H3-16 to C-5, C-6, C-7, and C-14, from H3-17 to C-1, C-4, C-8, and C-9, and from H3-19(20) to C-3, C-18 completed the structure of the cyathane diterpenoid skeleton  and C-4 (δC 78.2), together with the degree of unsaturation requirement, suggested the epoxy bridge between C-3 and C-4. Detailed analysis of the 1D NMR together with the HMBC and COSY data revealed the main differences between compounds 4 and 3 were the presence of an additional epoxy bridge between C-3 and C-4 instead of a double bond, and the absence of the hydroxy group at C-1 (δC 47.0) in 4. This deduction was supported by the HMBC correlations from H-17 to C-1, C-4, from H-19 and H-20 to C-3, from H-5 and H-10 to C-4, and from H-2 to C-4, C-9 , and six quaternary carbons . The chemical shifts for C-11 (δC 80.0) and C-14 (δC 111.2) together with the degree of unsaturation requirement supported the existence of an epoxy bridge between C-11 and C-14, which was implied by HMBC correlation of H-11 (δH 4.68) with C-14.Compound 1H-1H-COSY and HMBC spectra of 5 allowed the identification of four partial structures, CH3-19/CH-18/CH3-20, CH-1/CH2-2, CH-5/CH2-10/CH-11 and CH2-7/CH2-8 , whose absolute configuration had been unambiguously determined26. Furthermore, the experimental ECD spectrum of compound 5 matched well with calculated ECD spectrum of -5 . The NMR spectroscopic data of 6 resembled those of 5 except for the presence of a ketonic carbonyl group at C-2 (δC 209.5), suggesting the α,β-unsaturated ketone unit  present in the molecule. This was supported by HMBC correlations of H-1 to C-2 and C-4, H-19 and H-20 to C-3, and H-5 and H-17 to C-4  experiments. The β-configuration of the hydroxy group at C-1 of 6 was determined by NOESY cross-peaks of H-1/H-7a, H-5/H-17, and H-7b/H-17 , which were supported by the HMBC correlations from H-1 (δH 3.59) to C-2, and from H-2 (δH 4.46) to C-1 (δC 90.5), C-3 (δC 139.6)  NMR data. Thus, the structure of 7 was established as shown, and named neocyathin G.The relative configuration of the hydroxy group at C-2 of ion Fig. . The ass8 was determined to be C20H28O5, based on the HRESIMS at m/z 371.1825 [M + Na]+, with seven degrees of unsaturation. The 1H NMR spectrum , 1.17], 1.18 , 1.27 , one olefinic proton [δH 5.89 ], two oxymethines , and two oxymethylene protons . A comparison of the 13C NMR data between 8 and 14  in the molecule. This was supported by the HMBC correlations of H-17/C-1, C-4, H-2/C-1, C-3, C-4, and C-9  and C-13 (δC 126.6) in 7 were replaced by an epoxy group at C-12 (δC 64.0) and C-13 (δC 57.0) in 8. This deduction was confirmed by the HMBC correlations of H-13/C-12, C-14, and H-15/C-12  experiments  -8  in 9. The location of the OH group was supported by the HMBC correlations from H-15  to C-11 (δC 76.9), C-12 (δC 49.7), and from H-13 (δH 4.28) to C-11  in 9 was both assigned as α-form by NOESY experiments, in which the key correlations of H-5/H-12 and H-5/H-13 . Thus, the structure of 9 was established as shown, and named neocyathin I.The molecular formula of 10 was determined to be C20H30O5 on the basis of the HRESIMS at m/z 373.1985 [M + Na]+, identical to that of cyathin O (13)21. The 13C NMR data of 10 (Table 13 (Table 10 and 13 were attributed to the α-configuration of a hydroxymethyl group at C-12 (δC 49.8) in 10 instead of the β-orientation of the corresponding hydroxymethyl at C-12 (δC 55.8) in 13 /H-12 (δH 2.33) and H-5β/H-13 (δH 4.49)   from L-arginine oxidized upon stimulation31. Indeed, compounds 2, 5, 8, 12 and 13 significantly suppressed LPS-induced NO production in culture medium.COX-2 and iNOS are two major inflammatory mediators in brain neurodegeneration4. Thus, we investigated all compounds for the anti-inflammatory effects on not only iNOS, COX-2 expression but also NO production in Aβ1–42-treated BV2 cells. As shown in Fig. 1, 3, 6, 8, 9, 10, 11, 12 and 13 inhibited COX-2 expression whereas compounds 2, 4, 5, 7, 8, 9, 10, 11, 12, 13 and 14 significantly suppressed iNOS expression in Aβ1–42-stimulated BV2 cells. In addition, we examined the effects of all compounds on the production of NO. Consistent with previous study, compounds 2, 3, 4, 5, 7, 8, 9, 11, 12 and 13 markedly suppressed Aβ1–42-induced NO production. These results indicated that the bioactive compounds might exhibit anti-neuroinflammatory activities through inhibiting the expression and activity of pro-inflammatory enzymes .Neurotoxic peptide Aβ is well-known to induce iNOS and COX-2 expression and stimulate NO production in microglial cells11 and 14 with iNOS and COX-2 proteins as previously described32. Surprisingly, molecular docking studies revealed strong interactions between compounds 11 and 14 with iNOS protein  might exhibit anti-inflammatory activities by binding to the active cavities of iNOS and COX-2.To further elucidate the inhibitory effects of the bioactive diterpenoids on iNOS and COX-2, we performed molecular docking to understand the interaction of the active compounds ein Fig. . Consist1–10) from the liquid culture of C. africanus. Interestingly, our results indicated that bioactive compounds might have anti-neuroinflammatory activities through the inhibition of pro-inflammatory enzymes expression and activities. Especially, compounds 11 and 14 strongly interact with the iNOS and COX-2 proteins by targeting the residues near the active centers of iNOS and COX-2. Thus, we anticipate that the bioactive components in C. africanus may be utilized for the development of anti-inflammatory agents against AD and related neurodegenerative disorders.In this study, we have isolated and fully characterized through extensive NMR spectroscopic analysis ten new cyathane-type diterpenoids . UV and IR spectra were obtained on a Thermo Scientific Evolution-300 UV–visible spectrophotometer and a Bruker Tensor 27 FT-IR spectrometer with KBr pellets. ECD spectra were obtained on a Chirascan spectrometer. NMR spectra were obtained on Bruker Avance 800 MHz and Bruker Avance III 500 spectrometers with tetramethylsilane as an internal standard at room temperature. High-resolution (HR) ESIMS were recorded on a Thermo Fisher Scientific Q-TOF mass spectrometer. Electrospray ionization mass spectrometry ESIMS were recorded on a Thermo Fisher LTQ Fleet instrument spectrometer. Silica gel  and RP- 18 gel  were used for column chromatography (CC). Semipreparative HPLC was performed on a Waters 1525EF liquid chromatography system equipped with a Hypersil BDS C18 column . Fractions were monitored by TLC. Compounds were visualized by heating silica gel plates immersed in 10% HCyathus africanus was purchased from China General Microbiological Culture Collection Center (CGMCC) with the accession number CGMCC 5.1163. A specimen (No. CA20150728) was deposited at the College of Science, Northwest A&F University, Shaanxi, China. The culture medium consisted of glucose 2%, yeast extract 0.2%, peptone 0.5%, MgSO4 0.05%, KH2PO4 0.1%, with pH 6.5. Fermentation was carried out on a shaker at 130 rpm for 28 days at 28 °C.The fungus 3–MeOH (1:1). The EtOAc layer together with the mycelium extract was concentrated under reduced pressure to give a crude extract (30.2 g), and the latter was applied to a RP-18 column eluted with a gradient of MeOH–H2O (10–100%) to obtain six fractions, A–F. Fraction E was further purified by silica gel CC eluting with a gradient of CHCl3–MeOH (50:1–10:1), to yield five fractions (E1–E5). Fraction E2 was subjected to Sephadex LH-20 (MeOH) and further purified by semi-preparative HPLC  to yield 5 . Fraction E3 was subjected to Sephadex LH-20 (MeOH) and further purified by semi-preparative HPLC  to yield 11 . Fraction E4 was subjected to Sephadex LH-20 (MeOH) and silica gel CC  to afford 12 (5.5 mg) and 10 (7.2 mg). Fraction D was further purified by silica gel CC eluting with a gradient of CHCl3–MeOH (50:1–10:1), to yield five fractions (D1–D5). Fraction D3 was subjected to Sephadex LH-20 (MeOH) and silica gel CC  and further purified by semi-preparative HPLC  to yield 14 . Fraction D4 was subjected to Sephadex LH-20 (MeOH) and further purified by semi-preparative HPLC  to yield 2 . Fraction D5 was subjected to Sephadex LH-20 (MeOH) and silica gel CC  and further purified by semi-preparative HPLC  to yield 4 . Fraction C was further purified by silica gel CC eluting with a gradient of CHCl3–MeOH (50:1–10:1), to yield five fractions (C1–C5). Fraction C2 was subjected to Sephadex LH-20 (MeOH) to obtain two mixtures (C2.1 and C2.2). C2.1 was purified by semi-preparative HPLC  to yield 3  and 7 . C2.2 was applied to silica gel CC  to afford 6 (7.3 mg) and 9 (6.0 mg). Fraction B was further purified by silica gel CC eluting with a gradient of CHCl3–MeOH (50:1–10:1), to yield five fractions (B1–B5). Fraction B3 was subjected to Sephadex LH-20 (MeOH) followed by purification with PTLC  to yield compounds 1 (6.0 mg), 13 (8.3 mg). Fraction B4 was subjected to Sephadex LH-20 (MeOH) and silica gel CC  to afford 8 (5.4 mg).The culture broth (40 L) was filtered, and the filtrate was concentrated to 5 L, then extracted with ethyl acetate (5 L × 3), while the mycelium was extracted three times with CHCl1). White powder; [α]D25 = −49.3 ; UV (MeOH) λmax (log ε): 230 (3.01), 300 (2.23) nm; IR (KBr): νmax = 3436, 2966, 2103, 1690, 1456, 1380 cm−1; for 1H- and 13C- NMR data see Tables m/z 371.1824 [M + Na]+ .Neocyathin A . White p2). White solid; [α]D25 = −34.3 ; UV (MeOH) λmax (log ε): 204 (2.75), 243 (2.92) nm; ECD: λ  (nm) (Δε) = 320 (−5.61), 242 (−17.7), 206 (+43.4); IR (KBr): νmax = 3401, 2926, 2863, 1684, 1374, 1215, 1031 cm−1; for 1H- and 13C- NMR data see Tables m/z 357.2034 [M + Na]+ .Neocyathin B . White s3). Yellow oil; [α]D25 = −107.0 ; UV (MeOH) λmax (log ε): 230 (2.74), 314 (1.88) nm; ECD: λ  (nm) (Δε) = 391 (−0.39), 326 (+1.54), 206 (−22.7); IR (KBr): νmax = 3371, 2942, 2876, 1644, 1455, 1034 cm−1; for 1H- and 13C- NMR data see Tables m/z 373.1980 [M + Na]+ .Neocyathin C (4). White powder; [α]D25 = −23.8 ; UV (MeOH) λmax (log ε): 230 (3.20) nm; IR (KBr): νmax = 3416, 2938, 1641, 1446, 1381, 1028 cm−1; for 1H- and 13C- NMR data see Tables m/z 373.1979 [M + Na]+ .Neocyathin D (5). White powder; [α]D25 = −82.0 ; UV (MeOH) λmax (log ε): 207 (2.68) nm; ECD: λ  (nm) (Δε) = 393 (−0.15), 327 (+0.32), 213 (−5.22); IR (KBr): νmax = 3451, 2958, 2866, 2082, 1640, 1050 cm−1; for 1H- and 13C- NMR data see Tables m/z 357.2032 [M + Na]+ .Neocyathin E . White powder; [α]D25 = −77.9 ; UV (MeOH) λmax (log ε): 206 (2.99), 243 (3.07) nm; IR (KBr): νmax = 3374, 2939, 2872, 1696, 1384, 1031 cm−1; for 1H- and 13C- NMR data see Tables m/z 371.1825 [M + Na]+ .Neocyathin F . White powder; [α]D25 = −66.5 ; UV (MeOH) λmax (log ε): 207 (3.49) nm; IR (KBr): νmax = 3369, 2933, 2870, 1648, 1046, 982 cm−1; for 1H- and 13C- NMR data see Tables m/z 373.1982 [M + Na]+ .Neocyathin G . Yellow oil; [α]D25 = −105.7 ; UV (MeOH) λmax (log ε): 237 (3.05) nm; ECD: λ  (nm) (Δε) = 329 (−2.10), 234 (+10.4), 199 (−21.6); IR (KBr): νmax = 3417, 2962, 2874, 1689, 1593, 1383 cm−1; for 1H- and 13C- NMR data see Tables m/z 371.1825 [M + Na]+, .Neocyathin H (9). White solid; [α]D25 = −47.1 ; UV (MeOH) λmax (log ε): 231 (3.30) nm; IR (KBr): νmax = 2961, 2384, 2310, 1738, 1366, 1218 cm−1; for 1H- and 13C- NMR data see Tables m/z 373.1985 [M + Na]+ .Neocyathin I (10). White powder; [α]D25 = −68.4 ; UV (MeOH) λmax (log ε): 230 (3.46) nm; IR (KBr): νmax = 2935, 1735, 1687, 1606, 1369, 1217 cm−1; for 1H- and 13C- NMR data see Tables m/z 373.1985 [M + Na]+ .Neocyathin J 0. White 34. Conformers were saved and further optimized using the density functional theory (DFT) method and CPCM solvent model at B3LYP/6–31 + G level in Gaussian 09 software package. Frequency was calculated at the same level of theory to check optimized results. The four low-energy conformers with populations greater than 1% and without imaginary frequencies were submitted to ECD calculation by the TDDFT (cam-B3LYP/TZVP) method associated with CPCM solvent model in MeCN. The excitation energies (E), oscillator strength (f), rotatory strength in velocity form (Rvel), and rotatory strength in length form (Rlen) of the lowest 32 excited states were calculated. ECD spectra of different conformers were summated in SpecDis 1.62 according to their Boltzmann-calculated distributions. The summated curve was adjusted +5 nm UV-shift and 0.3 ev.A preliminary conformational search was performed in Conflex6.7 using MMFF94s force field, according to previously reported methods1–42 (GL Biochem (Shanghai) Ltd, China) was dissolved in 1% NH3·H2O at the concentration of 2 mM and incubated at 37 °C for 7 days to allow fibril formation. For biological assays, BV2 cells were pretreated with all compounds at 50 μM for 1 h, and then stimulated with 0.5 μg/mL LPS and 20 μM Aβ1–42 for 24 h in DMEM with 3% FBS.Murine microglia cell line BV2 cells was collected from ATCC and cultured in DMEM including 10% fetal bovine serum (FBS). Lipopolysaccharide (LPS)  were dissolved in PBS at a concentration of 1 mg/mL, while AβCell viability was measured by MTT assay. Briefly, BV2 microglia cells were seeded in 96-well plates overnight and treated with different concentrations of isolated compounds for 24 h or 72 h. After incubation and removal of culture medium, the BV2 cells were incubated with 0.5 mg/ml MTT in phosphate-buffered saline (PBS) for 4 h. Then the MTT was removed and resolved with 150 μL of DMSO. The production of purple formazan was detected for the absorbance at 570 nm on a Bio-Rad microplate reader . The cell viability was presented as a percentage to that of controls.35. Briefly, the cellular proteins were isolated and subjected to electrophoresis on 10% SDS-polyacrylamide gels and transferred onto a polyvinylidenedifluoride (PVDF) membrane. After overnight incubation in 5% BSA in the mixture of Tris-buffered saline and Tween-20 (TBS-T), the membranes were probed with primary antibodies: iNOS , COX-2  and GAPDH  antibodies  for 4 h and subsequently detected by goat anti-rabbit IgG-HRP conjugate secondary antibodies. The activity of peroxidase retained on the blots was measured by enhanced chemiluminescence (ECL) detection reagents from GE Healthcare  according to the manufacturer’s instruction.After drug treatment, the cellular proteins were extracted and analyzed by Western blotting analysis for protein expression as previously describedThe activity of iNOS enzyme was measured by using nitric oxide synthase activity assay kit  following the manufacturer’s instruction. In brief, after drug treatment, the cells were harvested and washed by cold PBS. The cells were resuspended in cold NOS assay buffer containing protease inhibitor on ice. After pipetting up and down, the samples were centrifuged at 4 °C at 10000 × g for 10 min. The supernatants were collected and subjected to the measurement of the protein concentration using a BCA protein assay kit. Sixty microliters of 100 μg protein sample and 45 μL of reaction mix including diluted NOS cofactor 1, NOS cofactor 2 (1X), NOS substrate and nitrate reductase were added into each well, mixed well and incubated for 1 h at 37 °C. Then, 90 μL of NOS assay buffer and 5 μL of enhancer were added into each well, mixed and incubated at RT for 10 min. After that, 100 μL of Griess reagent mixture were added and incubated at RT for 10 min. The absorbance was immediately measured on a microplate reader at OD 540 nm.After drug treatment, nitrite in culture medium was determined to reflect NO production by using Griess reagent kit for nitrite determination . Briefly, 150 μL culture medium, 130 μL milli-Q water and 20 μL Griess reagent ethylenediamine dihydrochloride and sulfanilic acid) were added into 96 well plate, and incubated at room temperature for 30 min. The absorbance of samples was measured on a spectrophotometric microplate reader at 540 nm.30. The three dimensional (3D) crystal structure of iNOS (PDB code: 3E7G) and COX-2 (PDB code: 1CX2) were obtained from the RCSB Protein Data Bank. The standard 3D structure (PDB format) of compounds 11 and 14 were constructed by using the “SKETCH” option function in SYBYL-X, whose configurations were determined by their NOESY spectra and TDDFT ECD calculations. The cubic grid box of 44 Å size  with a spacing of 0.375 Å and grid maps were built. The docking parameters consisted of setting the population size to 150, the number of evaluations to 2,500,000, the number of generations to 270,000, and the number of top individuals that automatically survive to 20, while the number of docking run was set to 40 with other default values during each docking run. The results of the most favorable free energy of binding were chosen as the resultant complex structures.Molecular docking simulations were performed using the software Autodock 4.2 Vina along with AutoDock Tools (ADT 1.5.6) using the hybrid Lamarckian Genetic Algorithm (LGA) as our previous studiesThe results were expressed as means ± SD. The significance was analyzed by one-way ANOVA with Dunnett’s test using Graphpad Prism 5 software and LSD test with IBM SPSS Statistics 19.0 software. A p-value of < 0.05 was considered as statistically significant.Supplementary information


The molecular and crystal structure of a new mononuclear neutral high-spin iron(III) complex with two different tridentate ligands has been studied by an X-ray diffraction analysis. 8H6BrN3OS)(C12H9BrN3O)] (I), crystallizes in the monoclinic space group C2/c and has two different planar tridentate ligands. The central FeIII ion is coordinated to three N, two O and one S atom, forming a distorted octahedral FeN3O2S coordination geometry. In the crystal, the complex molecules are linked by N—H⋯O and N—H⋯N hydrogen bonds and π–π interactions into layers parallel to (100). Magnetic measurements show that the central FeIII ion is in the high-spin state; this is also supported by the bond distances around the FeIII ion.The title neutral mononuclear complex, [1-(5-bromo-2-oxidobenzylidene)thiosemicarbazidato](4-bromo-2-{[2-(pyridin-2-yl)hydrazinylidene]methyl}phenolato)iron(III), [Fe(C III complexes for magnetic materials owing to their interesting thermal- or light-induced spin conversion between the high-spin  and low-spin  states  behavior have been designed and synthesized through the subtle design and combination of different ligands. Among the many organic ligands, Schiff bases are the most common ligands for new FeIII complexes due to their convenient synthesis and regulation. Compared with homo-ligand complexes, the employment of mixed ligands provides more selection and modification strategies for new magnetic complexes. In previous reports, the ligands 5-bromo-salicylaldehyde-2-pyridylhydrazone , 5-bromo-salicylaldehyde-thiosemicarbazone (5-Br-H2thsa) and their derivatives have been explored to assembly FeIII and MnIII complexes with SCO behavior Fe] (I)2thsa ligands. Herein, we report the crystal structure and magnetic property of this iron(III) complex.Much attention have been paid to the design and synthesis of FeC2/c. Compound (I)− and 5-Br-thsa2– – which adopt a meridional coordination mode. The central FeIII ion lies almost within the plane of each ligand [give deviations] and is coordinated to three nitrogen, two oxygen and one sulfur atoms from the two tridentate 5-Br-psal− and 5-Br-thsa2– ligands, forming a distored octahedral FeN3O2S geometry. The Fe—O bond lengths are 1.943 (3) and 1.931 (3) Å, the Fe—N bond lengths range from 2.142 (3) to 2.157 (3) Å, and the Fe1—S1 bond length is 2.4093 (14) Å. All the bond lengths are normal and agree well with those in related high-spin state FeIII compounds  Å  based on g = 2.0. The measurement of the magnetic property shows that the FeIII ion is in the high-spin state, which agrees well with the above-mentioned bond lengths around the FeIII ion. The χmT value keeps nearly constant with decreasing temperature until around 75 K, and then it decreases quickly to a minimum value of 1.12 emu K mol−1 at 2.0 K. This tendency to change of the χmT curve indicates the existence of overall weak antiferromagnetic interactions in (I)C = 4.08 emu K mol−1, which further confirms the presence of overall intermolecular antiferromagnetic interactions between neighboring FeIII ions through intermolecular hydrogen bonds and π–π interactions in complex (I)The magnetic susceptibilities of (I)2thsa ligands were synthesized by refluxing equimolar 5-bromosalicylaldehyde with thiosemicarbazone and 2-pyridylhydrazine, respectively, in an ethanol solvent. All other chemicals were purchased from the Sigma Aldrich Chemical Company and used as received. The precursors [Fe2]Cl and Li[Fe(5-Br-thsa)2] were prepared according to literature methods  and Li[Fe(5-Br-thsa)2] (0.2 mmol) were dissolved in acetonitrile (20 mL). The mixture was filtered and kept at room temperature for two days. Brown block-shaped single crystals were collected with a relatively high yield of 76%. Elemental analysis calculated for C20H15N6O2SBr2Fe: C, 38.80%; H, 2.44%; N, 13.57%; found: C, 38.72%, H, 2.38%; N, 13.62%.All reactions were conducted in air using reagent grade solvents. The 5-Br-Hpsal and 5-Br-H− was found from the difference-Fourier map and refined isotropically. All other hydrogen atoms were placed in calculated positions with C—H = 0.88–0.95 Å and refined in the riding model with fixed isotropic displacement parameters [Uiso(H) = 1.2Ueq].Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989018001263/kq2017sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989018001263/kq2017Isup2.hklStructure factors: contains datablock(s) I. DOI: 1818280CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Flora Republicae Popularis Sinicae 3(1), species identifications of the series Gravesiana have been in disarray, requiring clarification. Two hundred and fifty-nine individuals from 47 different populations were collected for the estimation of morphological characters and phylogenetic analyses. Spores of 26 populations were observed through scanning electron microscope. Our results were different from those of previous research: (1) six identifiable species, rather than five species observed previously, were confirmed in the series Gravesiana, they are A. gravesii, A. juxtapositum, A. mariesii, A. dentatum, A. longzhouensis and A. obovatum, of which the latter three are newly recognized species. (2) Thirteen characters were measured and estimated through the program Mesquite v. 2.71. The character whether the pinna stalks were 1/3-1/2 times longer than the pinna was used to distinguish A. gravesii and A. lianxianense previously and was found to be unreliable here, whereas such characters as the height of the plant (H), pinna aligned forms (FP), number of pinna (NP), pinna margin (M), number of veins flabellate at base (NV), sori number and shape per pinna (NSS), pinna texture (T), and powder-covered or not on the abaxial surface of the pinna (P) are estimated to be stable and reliable characters useful for identification. Descriptions of new species and their retrieve keys are also listed. (3) Surface ornamentations and spore sizes are helpful for us to distinguish species in series Gravesiana.Since the adoption of some ambiguous and quantitative characters in  Adiantum L. is a large genus in the Pteridaceae, with more than 200 species worldwide distributed except in extremely cold or dry regions [Caudata, Flabellulata, Gravesiana, Pedata, Reniformia, Veneri-Capilliformia and Venusta. However, it was only partially consistent with the phylogenetic results because the series Gravesiana was nested within one subgroup of the series Caudata [Gravesiana, containing 5 species, namely A. chienii Ching, A. gravesii Hance, A. juxtapositum Ching, A. lianxianense Ching & Y.X. Lin and A. mariesii Baker [Flora Republicae Popularis Sinicae [Pteridaceae is a large family containing approximately 50 genera and 950 species, with most of them existing in tropical and arid regions . There a regions , 2. In C regions . Lin  [A. gravesii is similar to A. lianxianense but its plant texture is much harder and stronger than A. lianxianense. Besides, whether the pinna stalks were 1/3-1/2 times longer than the pinna is the main difference between A. gravesii and A. lianxianense. A. juxtapositum is considered as an endemic species in Xianxia Ling, Fujian province [A. chienii is an endemic species of the North River, Guangdong province in China. It is similar to A. juxtapositum but can be easily distinguished by its one (rarely two) sori per pinna, while A. juxtapositum has three to many sori per pinna [A. chienii have been collected and kept except for its typus. The morphological characters of these five species are shown in There are few collected specimens of ts typus  when it n 1980)  and Chinn (1980) , A. gravprovince . However80  and Cprovince  found iter pinna , 8. UnfoA. chienii, A. gravesii and A. juxtapositum share a common character—the length of the pinna stalks is less than 1/5 times that of the pinna. However, when we examined each of their specimens  with alternating pinna and 3-to-many kidney-shaped sori per pinna were found during our field investigation. We found some individuals from different populations with similar new characters never encountered before (DT). In addition, many individuals have highly diverse characters in one population. Hence, do these variable individuals belong to one species or to different species? To elucidate the identification of these five species, we investigated a total of 47 populations from Guangxi, Guizhou, Guangdong, Hunan, Jiangxi and Fujian provinces of China  and collected 259 individuals. Such characters as the height of the plant, leaf stalk, pinna stalk, pinna size and shape, veins, sori number per pinna, and scales were measured before DNA extraction. Combined analyses of microscopic spore observations, morphological and molecular data will be carried out to resolve the phylogenetic relationship of the series Gravesiana here.of China  and collA. gravesii, 4 populations were from A. juxtapositum, 2 populations were from A. lianxianense, 8 populations were from A. mariesii, and the other 13 populations were doubtful taxa (A. chienii were collected?). Six plastid genes  of 76 individuals from 42 populations were amplified and sequenced to generate a phylogenetic tree of the series Gravesiana. To construct the phylogenetic tree of Chinese Adiantum, five chloroplast genes  of A. nelumboides X.C. Zhang (2 accessions), A. reniforme L. (11 accessions), A. malesianum Ghatak, and A. induratum Christ were used [Adiantum species were downloaded from GenBank. Vittaria flexuosa Fée was selected as outgroup. Details on the collection of the 78 individuals of the series Gravesiana including collection locality and date, longitude, latitude and altitude are shown in 259 individuals from 49 populations were collected and silica-gel-dried for DNA extraction, of which 22 populations were from ful taxa , 0.25 mmol·L-1 dNTP, and ultrapure water. The PCR products were purified and sequenced with an ABI 3730XL by Majorbio Company.Total DNA was extracted from silica-gel-dried leaf materials using a modified CTAB DNA extraction protocol . Six paiectively , 19, 20.atpA, atpB, rbcL, trnL-F, rps4-trnS and matK) were constructed using maximum parsimony (MP) and Bayesian Markov chain Monte Carlo inference (BI). The maximum parsimony analyses were performed with PAUP* 4.0b10 [The sequences were assembled with Sequencher v. 4.14, aligned using the program Clustal X v. 2.0  and then* 4.0b10 , treatin* 4.0b10 , GTR+I+GGravesiana constructed with six combined chloroplast genes, atpA, atpB, rbcL, trnL-F, rps4-trnS and matK, using the program Mesquite version 2.71 [Thirteen characters of the 78 individuals including the height of the plant (H), the length of pinna stalk (LR), pinna aligned forms (FP), number of pinna (NP), pinna size (S) and shape (SP), pinna margin (M), number of veins flabellate at base (NV), veins tendency near upper margin (TV), sori number and shape per pinna (NSS), pinna texture (T), powder-covered or not on the abaxial surface of the pinna (P), and scale shape (SS) were measured before DNA extraction. The morphologic data were maintained in a spreadsheet and then compiled into a digital matrix. Then, we mapped them onto the phylogenetic tree of the series ion 2.71  under maHeight of the plant (H): (0) H ≤5 cm, (1) 5 cm<H≤13 cm, (2) H>13 cmLength of the pinna stalk (LR): (0) 0 mm<LR≤1 mm, (1) 0 mm<LR≤3 mm, (2) 0 mm<LR≤5 mmPinna aligned forms (FP): (0) alternate, (1) oppositeNumber of pinna (NP): (0) 1<NP≤3, (1) 3<NP≤6, (2) 6<NP≤9, (3) NP>9;Pinna size : (0) 2.0–6.0 × 2.0–5.0, (1) 6.0–15.0 × 5.0–17.0, (2) 6.0–15.0 × 2.0–5.0Pinna shape (SP): (0) obovate, (1) obdeltoid, (2) subround, (3) othersPinna margin (M): (0) entire, (1) dentateNumber of veins flabellate at base (NV): (0) NV≤4(5), (1) NV>4(5)Vein tendency near upper margin (TV): (0) straight up to terminal, (1) curve closed to marginSori number and shape per pinna (NSS): (0) 1, orbicular; (1) 1, reniform; (2) 1 to many, reniform; (3) 1 to many, transversally linear and truncate at false indusia terminal; (4) 1, shape same as (3)Pinna texture (T): (0) membranous, (1) coriaceousPowder-covered or not on abaxial surface of pinna (P): (0) no, (1) yesGravesiana were observed through scanning electron microscopy. Mature spores from the same population were dispersed on stubs directly after being collected and marked with a unique population ID. The spores were gold-coated in a JFC-1600 Auto Fine Coater and observed using a JEOL JSM-6360LV Scanning Electron Microscope at 25 kV at the South China Botanical Garden, Chinese Academy of Sciences. The spore mean sizes were measured by Smile View software (20 spores per population). The descriptive terminology in Spores of Polypodiales  from China [Plant identification terminology: An illustrated glossary [Spores from 26 different populations of series glossary  was follThe electronic version of this article in Portable Document Format (PDF) in a work with an ISSN or ISBN will represent a published work according to the International Code of Nomenclature for algae, fungi, and plants, and hence the new names contained in the electronic publication of a PLOS ONE article are effectively published under that Code from the electronic edition alone, so there is no need to provide printed copies.http://ipni.org, once the paper is published. The online version of this work is archived and available from the following digital repositories: PubMed Central, LOCKSS.IPNI LSIDs (Life Science Identifiers) for new species herein have been resolved and can be available at atpA, atpB, rbcL, trnL-F and rps4-trnS) phylogenetic tree of Chinese Adiantum (in 132 accessions) comprised 6,497 nucleotides, of which 2,364 were variable (36.4%) and 1,772 were phylogenetically informative (27.3%). The MP analysis based on this dataset yielded one maximally parsimonious tree of 5,052 steps with a consistency index (CI) of 0.6033 and a retention index (RI) of 0.9198. The tree obtained from the BI analysis had a similar topology to the MP strict consensus tree ; however, all of its individuals were clustered into nine clades with a high support value  [A. gravesii. In addition, A. mariesii “J. -M. Lu 120” was clustered into clade D together with individuals of doubtful taxa “DT2.”. A. chienii “J. -M. Lu 568” was identical to “FLG1-10” and A. juxtapositum “J. -M. Lu 120.”The topologies derived from the analyses of single dataset and the combined dataset were congruent; thus, we adopted the topology from the combined dataset here. The combined 5-marker  included 78 taxa and were 7,140 nucleotides in length, of which 1,137 were variable (15.9%) and 490 were phylogenetically informative (6.9%). 1,384 steps were run to generate a maximally parsimonious tree; the consistency index (CI) was 0.8779, and the retention index (RI) was 0.9609. Nine main clades were identified in A. gravesii from twelve different populations were clustered into clade A, and 16 individuals of A. mariesii from 7 different populations were clustered into clade B. Seven individuals of the doubtful taxon “DT1” from four populations formed a clade (clade C). Three samples of “DT2” from two populations formed a new clade (clade D), and six individuals of “DT3” from three populations were clustered into clade E. All individuals of A. juxtapositum from four populations were clustered together (clade I).The trees of the series In the 13 characters, scales among all individuals were similar; thus, we abandoned this character. The mapping to the phylogeny tree of the twelve other characters is visualized in Clade B shares common characters: all individuals were no more than 5 cm, with pinna alternate and sub-round and NV were less than 4(5), sori 1 for each pinna, and orbicular. All individuals in clade C can be easily identified by their much shorter height, NV less than 4 and dentate pinna margin. Individuals in clade D were featured by their membranous pinna texture and obovate pinna shape. For clade E, as shown in Figs Gravesiana are similar in polar and equatorial views, but their surface ornamentations and spore sizes are clearly different. All spores are actinomorphic and trilete with polar surface triangle, and the equatorial surface is semicircular or super-semicircular. There are three kinds of surface ornamentations in all: psilate, rugate and verrucate , and IUCN conservation assessments are stated below.Based on above molecular, morphological and spore analyses, clade A was considered to be ens Figs  and 2. CAdiantum dentatum1.  A. H. Wang, F. G. Wang & F. W. Xing sp. nov.urn:lsid:ipni.org:names:77160197–1Type: China. Yang-zi-dong, Fengshan County, Guangxi. 24°23′21″N, 107°03′58"E, 13 June 2014, WAH011 . Fig 8A. dentatum differs from others for its fewer pinnae, obdeltoid or subovate pinna with prominent dentate margin from the broadest middle to apex and its variable orbicular or transversally linear sori.Plants epilithic, small, 1–3 cm tall. Rhizomes short, erect, clothed with linear-lanceolate and black-brown scales, scales margin entire. Stipes densely tufted, 10–30 together, 1.5-9(15) mm long, ca. 0.5 mm thick, dark castaneous, as slender as silk. Lamina 1-pinnate, odd-pinnate or paripinnate, 1.5–2.5 cm long and ca. 2 cm broad; pinnae 1–3 pairs, 3–5, often 1–3, alternate, 2–6 mm apart, obliquely spreading; pinna ca. 2–8 mm long and broad, oval, subovate, obovate, broadly obovate or obdeltoid, cuneate or rounded-cuneate at base, margin entire on both lateral sides but dentate from the broadest middle to apex, upper margin with 1 shallow sinus in middle, texture subcoriaceous; stalk 1–2 mm, articulate, persistent after pinna fall; veins 4–6, flabellate at base, each again 2–4 forked and reached the tooth margin, visible on both surfaces. Sori 1 per pinna, orbicular or transversally linear, false indusia brown, upper margins flat and straight, slightly depressed, persistent, lower margins entire, sometimes with 1 distinct sinus in middle.The plants grow in moist chalk soil covered by black lichen or moss near the entrance to the karst cave Yang-zi-dong in Fengshan county, where drips exist. It has been found in karst caves in Huanjiang, Fengshan, Bama and Daxin in Guangxi Province.WAH013(IBSC); Jing-yang-cun primary school, Baoxu Town, Daxin county, 10 June 2014, WAH012(IBSC); Ming-li-tun, Mulun National Nature Reserve, Huanjiang County, 15 June 2014, WAH014(IBSC).China. Guangxi: Ganhong Dong, Daluo Village, Nashe Township, Bama County, 13 June 2014, A. dentatum should be considered endangered in accordance with the IUCN Red List criteria [Although it grows well in the type locality, because of its restricted distribution region, small population size and low number of individuals in very small or restricted populations, criteria .Adiantum obovatum2.  A. H. Wang, F. G. Wang & F. W. Xing sp. nov.urn:lsid:ipni.org:names:77160198–1Type: China. Yang-zi-dong, Fengshan County, Guangxi. 24°23′21″N, 107°03′58″E, 13 June 2014, WAH016 . Fig 9This species can be easily distinguished from others for its membranous texture, petiole slender as hair, obovate pinna and 2–4 veins flabellate at base, and very small orbicular sori.Plants epilithic, approximately 3.0–4.0 cm tall. Rhizomes short, erect, covered with linear-lanceolate and black-brown scales, scales margin entire. Stipes densely tufted, slender as hair, 0.8–1.8 cm long, ca. 0.5 mm thick, dark castaneous. Lamina 1-pinnate, odd-pinnate or paripinnate, 1.2–3.3 cm long and ca. 0.5–1.0 cm broad; pinnae 3–5, rarely 3, alternate; pinna ca. 2.5–6.0 × 2.0–4.0 mm, obovate, membranous, margin entire, upper margin with 1 shallow sinus in middle; stalk 0.5–1 mm, persistent after pinna fall; veins 2–4, flabellate at base, each again 2 forked and visible on both surfaces. Sori 1, orbicular, small.The plants grow in chalk soil covered by moss near the entrance to the karst cave Yang-zi-dong in Fengshan county. It has also been found to exist in the cave in Si-ming-xia, Yuexu town, Jinxi county.WAH015(IBSC).China. Guangxi: Si-ming-xia, Yuexu Town, Jinxi County, 11 June 2014, A. obovatum should be considered endangered in accordance with the IUCN Red List criteria [Because of its restricted distribution region, small population size and low number of individuals in very small or restricted populations, criteria .Adiantum longzhouensis3.  A. H. Wang, F. G. Wang & F. W. Xing sp. nov.urn:lsid:ipni.org:names:77160199–1Type: China. Jing-yang-cun primary school, Baoxu Town, Daxin county, Guangxi 22°39′12″N, 106°57′27″E, 10 June 2014, WAH017 . Fig 10A. juxtapositum and A. gravesii.The obdeltoid pinna and 1-many kidney-shaped sori per pinna of this species distinguish it from Plants epilithic, 26.5 cm tall. Rhizomes short, erect, covered with lanceolate and black-brown scales, scales margin entire. Stipes tufted, 5–10 together, hard and stong, 5.6–11 cm long, ca. 1.0 mm thick, dark castaneous. Lamina 1-pinnate, odd-pinnate or paripinnate, 9–16 cm long and ca. 3.0–4.0 cm broad; pinnae 9–14, opposite, rarely alternate; ca. 10–18 × 10–21 mm, obdeltoid, coriaceous, margin entire, upper margin with 2–4 shallow sinus; stalk 0.5–1 mm, persistent after pinna fall; veins 7–10, flabellate at base, each again 2–4 forked, visible on both surfaces. Sori 2–4 per pinna, reniform.It grows in moist chalk soil on cliff under-forest in Longzhou county and Daxin county in Guangxi, China.WAH018 and WAH019 (IBSC!); cliffs near the fifth single-log bridge in Longmeng, Sancunshan in the Nonggang Natural Reserve, Longzhou County, 9 June 2014, WAH020(IBSC); Sancunshan in the Nonggang Natural Reserve, Longzhou County, 9 June 2014, WAH021(IBSC); Cliffs near the second single-log bridge in Longmeng, Sancunshan in the Nonggang Natural Reserve, Longzhou County, 9 June 2014, WAH022(IBSC).China. Guangxi: Jing-yang-cun primary school, Baoxu Town, Daxin county, 10 June 2014, A. longzhouensis the endangered scale in accordance with the IUCN Red List criteria [Its restricted distribution region, small population size and low number of individuals in very small or restricted populations make criteria .A. obovatum can be easily misidentified as A. lianxianense based on the description in Lin (1980) if we do not observe the type specimen of A. lianxianense carefully . So it is possible that A. chienii and A. juxtapositum is of the same species. For clade G in A. longzhouensis is that individuals in clade G are covered with white powder on the abaxial surface of the pinna while A. longzhouensis is not. However, we did not treat it as a new species considering its mixed characters: soris reniform or transversally linear with a truncate terminal at false indusium.arefully . A. lianrbicular . Its sim  were much more similar than A. gravesii , pinna shape (SP) and number of veins flabellate at base (NV) of the four individuals “esii see . BesidesGravesiana based on comprehensive analyses results of phylogeny and morphology. The species are A. gravesii (clade A), A. mariesii (clade B), A. dentatum , A. obovatum , A. longzhouensis , and A. juxtapositum (clade I). Of the 13 characters, the height of the plant (H), pinna aligned forms (FP), number of pinna (NP), pinna margin (M), number of veins flabellate at base (NV), sori number and shape per pinna (NSS), pinna texture (T), and powder-covered or not on abaxial surface of pinna (P) are reliable, stable, and discrete characters for us to easily distinguish the six species above. Their retrieval keys are shown below. In addition, A. leveillei “J. -M. Lu 163” and A. lianxianense “J. -M. Lu 441” in Lu et al. (2012) was actually A. gravesii, and A. mariesii “J. -M. Lu 120” was A. obovatum. A. chienii “J. -M. Lu 568” was completely identical to A. juxtapositum.There are six confirmed species in the series 1. plant shorter than 5 cm and number of veins flabellate at base less than 4(5)A. dentatum 2. number of pinna less than or equal to 3, pinna margin dentate·························· 2. number of pinna more than 3, pinna margin entireA. mariesii  3. pinna coriaceous, white powder-covered on abaxial surface, sori 1, large·············A. obovatum  3. pinna membranous, without powder on abaxial surface, sori 1, very small··········1. plant often taller than 5 cm and number of veins flabellate at base more than 4(5)A. gravesii 4. pinna alternate, sori 1, reniform or transversally linear with sinus at indusium terminal······························································································· 4. pinna opposite, rarely alternate, sori 1 to many, reniform or transversally linearA. longzhouensis  5. pinna without white powder on abaxial surface, sori reniform····················A. juxtapositum  5. pinna with white powder on abaxial surface, sori transversally linear, truncate at false indusium terminal······················································································S1 TableatpA, atpB, rbcL, trnL-F, rps4-trnS and matK.Index_ID, sequence_ID, voucher specimen (herbarium), collection locality, longitude and latitude, altitude and GenBank accession number in the order of (DOCX)Click here for additional data file.S2 TablerbcL, atpB, atpA, trnL-F, and rps4-trnS.Taxaname, sequence_ID, voucher specimen (herbarium), collection locality, and GenBank accession number in the order of (DOCX)Click here for additional data file.S3 TableH: Height; LR: Length of pinna stalk; FP: Pinna aligned forms; NP: Number of pinna; S: Pinna size; SP: Pinna shape; M: Pinna margin; NV: Number of veins flabellate at base; TV: Vein tendency near upper margin; NSS: Sori number and shape per pinna; T: Pinna texture; P: Powder-covered or not on the abaxial surface of the pinna.(DOC)Click here for additional data file.


Therefore, this study aimed to assess whether and how γδT cells impact podocytes via the CD28/B7-1 pathway. WT and TCRδ−/− mice were assessed. LPS was used to induce nephropathy. Total γδT and CD28+γδT cells were quantitated in mouse spleen and kidney samples. B7-1 and phosphor-SRC levels in the kidney were detected as well. In vitro, γδT cells from the mouse spleen were cocultured with mouse podocytes, and apoptosis rate and phosphor-SRC expression in podocytes were assessed. Compared with control mice, WT mice with LPS nephropathy showed increased amounts of γδT cells in the kidney. Kidney injury was alleviated in TCRδ−/− mice. Meanwhile, B7-1 and phosphor-SRC levels were increased in the kidney from WT mice with LPS nephropathy. CD28+γδT cells were decreased, indicating CD28 may play a role in LPS nephropathy. Immunofluorescence colocalization analysis revealed a tight association of γδT cells with B7-1 in the kidney. High B7-1 expression was detected in podocytes treated with LPS. Podocytes cocultured with γδT cells showed higher phosphor-SRC and apoptosis rate than other cell groups. Furthermore, CD28/B7-1 blockage with CTLA4-Ig in vitro relieved podocyte injury. γδT cells exacerbate podocyte injury via CD28/B7-1 signaling, with downstream involvement of phosphor-SRC. The CD28/B7-1 blocker CTLA4-Ig prevented progressive podocyte injury, providing a potential therapeutic tool for PNS.Primary nephrotic syndrome (PNS) is a devastating pediatric disorder. However, its mechanism remains unclear. Previous studies detected B7-1 in podocytes; meanwhile,  Primary nephrotic syndrome (PNS), characterized by proteinuria, hypoalbuminemia, hyperlipidemia, and edema, has become a devastating disorder in children. PNS can be divided into minimal change disease (MCD), focal segmental glomerulosclerosis (FSGS), mesangial proliferative glomerulonephritis (MPGN), and membranous nephropathy (MGN). Among them, MCD accounts for nearly 80% of pediatric cases. Most children with MCD respond well to corticosteroid treatment, but relapse and corticosteroid resistance still constitute important clinical challenges. Nevertheless, lack of knowledge regarding PNS mechanism is a barrier in developing efficient therapeutic methods.+T cells have been reported in PNS patients. We also found Th17/Treg subset dysfunction in pediatric PNS  . Western (μg/ml) . Further (μg/ml) . Then, tδ−/− mice . The results of cyto-immunofluorescence showed that podocytes of Group D expressed stronger phosphor-SRC  (P < 0.05)  . Notably < 0.05) . Phalloi γδT cells by flow cytometry. CCR2+γδT cells accounted for more than 95% of all γδT cells in nephrotic WT mouse kidney specimens. However, CCR5+γδT cells accounted for only 70–80% of total γδT cells (∗P < 0.05) (∗∗P < 0.01)   and podo < 0.01)  comparedPodocyte injury is considered the most important pathological feature in MCD. As terminally differentiated cells, podocytes rarely recover from injury. Therefore, effective therapeutic methods should prevent progressive podocyte injury. However, the uncertain mechanism remains a barrier in developing efficient treatments for MCD. Based on previous findings, we assessed MCD from an immunological perspective. Consistent with other studies , 18, exp γδT cells are activated at the early stage of disease generation and development [ γδT cells could constitute the aggravating factor in several kidney diseases [ γδT amounts increase in peripheral blood from children with MCD [ γδT cells could also be a risk factor in MCD. We firstly demonstrated infiltration of γδT cells in kidneys from WT mice with LPS nephropathy by flow cytometry and immunofluorescence. Then, TCRδ−/− mice were evaluated as well. Interestingly, compared with control group mice, TCRδ−/− animals treated with LPS had slight proteinuria. However, proteinuria in TCRδ−/− mice treated with LPS was much less severe than that of WT mice with LPS nephropathy. These preliminarily results supported our speculation.As innate-like immune cells,elopment . Studiesdiseases . Previouwith MCD . Therefo γδT cells and B7-1 was analyzed by immunofluorescence. Interestingly, γδT cell infiltration was closed to and even overlapped with B7-1 positive areas. Then, we detected CD28 loss on γδT cells by flow cytometry. CD28 is a B7-1 ligand. Its disappearance is also a marker for γδT cell activation. Upon binding with B7-1, CD28 is suppressed. As shown above, CD28+γδT cell percentage in the kidney of mice with LPS nephropathy was significantly lower than that of control mice, indicating that CD28 in γδT cells had likely bound B7-1 on podocytes. However, whether γδT cells affect podocyte homeostasis directly remains unclear. Therefore, more in vitro experiments are required.B7-1, also termed as CD80, is considered a marker of immune cells. However, several studies have reported B7-1 expression on podocytes. B7-1 can be induced in LPS nephropathy and has been also detected in kidney tissue and urine samples from PNS patients [μm transwell plates were employed. After addition of γδT cells to the upper chamber with podocytes in the lower chamber, the two cell types were separated. However, cytokines produced by γδT cells could diffuse into the lower chamber. Podocytes were divided into five groups, including Groups A, B, C, D, and E, representing the control group, LPS treatment group without γδT cells, LPS treatment group cultured with γδT cells in the upper well of the transwell, LPS treatment group cocultured with γδT cells, and LPS treatment group cocultured with γδT cells and treated with CTLA4-Ig, respectively. Podocytes were exposed to LPS. After 48 hours of stimulation, the early apoptosis rates of Groups C and D were significantly higher compared with the control value. However, only Group D had higher late apoptosis rate. These findings indicated that despite the presence of both indirect (such as molecule) and direct interactions, direct interactions of γδT cells and podocytes remain the main pathogenic pathway. Notably, the podocyte apoptosis rate of Group E was slightly higher compared with those of Groups A, B, and C, but significantly lower than that of Group D, suggesting CTLA4-Ig could prevent progressive injury in podocytes. CTLA4-Ig is a CD28/B7-1 blocker; it has high affinity for B7-1 and competes with CD28 for its binding. Since CD80 detection in podocytes, several studies have assessed the therapeutic effects of CTLA4-Ig in vivo and in vitro [For in vitro experiments, 0.4 in vitro , 22. Yu in vitro . However γδT cells and B7-1 rather than B7-1 itself. SRC is a nonreceptor tyrosine kinase that affects phosphorylation or dephosphorylation of diverse signaling proteins. It participates in multiple tyrosine signaling pathways, including cytoskeletal dynamics. Buvall and colleagues reported that phosphor-SRC activation could lead to synaptopodin dephosphorylation, promoting synaptopodin binding to the serine/threonine phosphatase calcineurin. This leads to loss of 14–3-3 binding, resulting in synaptopodin degradation and loss of stress fibers ultimately [ γδT cells had higher apoptosis rate and phosphor-SRC expression levels compared with other groups. Moreover, the F-actin detection of podocytes showed F-actin loss in Group D. However, phosphor-SRC detection in Group E revealed that CD28/B7-1 blockage through CTLA4-Ig could inhibit phosphor-SRC activation and progressive injury in podocytes. These results demonstrated that direct interactions between γδT cells and podocytes may activate intracellular phosphor-SRC, leading to podocyte injury.As shown above, phosphor-SRC expression was much higher in Group D and returned to baseline when CTLA4-Ig was added which can block the interaction between CD28 and B7-1. However, the B7-1 expression was induced by LPS treatment and had no significant difference among Groups B-E. The results indicated phosphor-SRC was induced by the interaction betweentimately . As show γδT cells exacerbate podocyte injury via binding to B7-1 with further phosphor-SRC activation, leading to altered cytoskeleton. CTLA4-Ig, a CD28/B7-1 blocker, could prevent progressive injury in podocytes, representing a potential therapeutic tool for pediatric PNS.In conclusion, γδT cells accounted for more than 95% of all γδT cells that infiltrated the kidney. Therefore, γδT cell migration was mainly induced by the CCR2/CCL2 axis.In addition, we assessed chemokines and their receptors in LPS nephropathy. Compared with control mice, WT mice with LPS nephropathy had higher CCL2 gene expression in the kidney. Meanwhile, CCR2+ γδT cells and B7-1 from podocytes induces phosphor-SRC activation, leading to podocyte injury. Blockage of such interaction, for example, by incubation with CTLA4-Ig, may be a desirable treatment, especially in children with PNS who are not sensitive to corticosteroids.We found that the interaction of CD28 from