Accordingly, TCRδ−/− mice have impaired germinal center formation, inefficient Tfh cell differentiation, and reduced serum levels of chicken ovalbumin (OVA)-specific antibodies after CFA/OVA immunization. In a mouse model of lupus, TCRδ−/− mice develop milder glomerulonephritis, consistent with decreased serum levels of lupus-related autoantibodies, when compared with wild type mice. Thus, modulation of the γδ T cell-dependent humoral immune response may provide a novel therapy approach for the treatment of antibody-mediated autoimmunity.γδ T cells have many known functions, including the regulation of antibody responses. However, how γδ T cells control humoral immunity remains elusive. Here we show that complete Freund’s adjuvant (CFA), but not alum, immunization induces a subpopulation of CXCR5-expressing γδ T cells in the draining lymph nodes. TCRγδ + γδ T cells release Wnt ligands to initiate the T follicular helper cell differentiation program and promote antibody production.Many immune functions have been reported for γδ T cells, including the regulation of antibody responses. Here the authors show that CXCR5 Recently, the transcription factor achaete-scute homologue-2 (Ascl2) was shown to initiate the Tfh development7. In a mechanism involving the β-catenin pathway, naïve CD4+ T cells upregulate Ascl2, thus initiating the Tfh program that involves CXCR5 upregulation, CCR7 downregulation, and Th1 and Th17 gene signature inhibition7. However, the source of endogenous β-catenin activation molecules (Wnt agonists) is not known. The Tfh cell program is then maintained by expression of transcription factor B cell lymphoma 6 (Bcl6)1.Naïve CD42.Once differentiated, Tfh cells migrate to the B:T cell border of a lymphoid organ, where they encounter cognate antigen-activated B cells. This Tfh–B cell interaction results in B cell proliferation and differentiation. B cells then migrate to the center of the follicle and give rise to the germinal center where isotype switching and antibody affinity maturation take place8. Thus, “non-αβ” T cells can mediate immunoglobulin class switching and antigen-dependent antibody production, suggesting that γδ T cells play an important role in these processes. In fact, it has been shown that γδ T cell deficient (TCRδ−/−) mice, either immunized or not, have reduced serum antibody levels, including IgG1, IgG2b, and IgE10. Importantly, some of these antibody subclasses, such as IgG2b and IgG2c were αβ T cell independent whereas IgG1 and IgE were αβ T cell dependent. Interestingly, the hypogammaglobulinemia observed in TCRδ−/− mice depends on the specific γ gene deletion. For example, Vγ1 knockout mice have hypogammaglobulinemia, whereas Vγ4 and Vγ6 double-knockout mice have increased serum antibody levels, particularly IgE, compared to wild-type (WT) mice, an effect likely to be dependent on IL-410. This suggests that γδ T cell-dependent antibody production involves both αβ T cell dependent and independent pathways and that this effect is controlled by the cross-talk between γδ T cell subsets.In the absence of αβ T cells, B cells are able to expand and secrete copious amounts of T cell-dependent antibodies, which react to self-antigens, mimicking the pathogenesis of systemic lupus erythematosus (SLE)11, a chemokine receptor that allows migration toward CXCL13 in the B cell follicle; (2) CD40 ligand (CD40L)12, crucial for B cell activation, and (3) IL-4 and IL-10 cytokine secretion11, involved in immunoglobulin class switch. Consistent with this, γδ T cells have been implicated in antibody-mediated autoimmune diseases such as SLE. Notably, pathogenic anti-DNA autoantibody-inducing γδ T cell lines were isolated from patients with active lupus nephritis13. Moreover, a subgroup of patients with SLE and Sjogren’s syndrome displayed a marked increase in γδ T cell numbers that were normalized by immunosuppressant treatment14. Thus, these studies suggest the involvement of γδ T cells in antibody-mediated autoimmune conditions. However, the mechanisms underlying γδ T cell-dependent humoral immunity remain elusive. For example, whether γδ “Tfh-like” cells exist or whether γδ T cells communicate directly with B cells or interfere with Tfh cell development.In humans, γδ T cells promote B cell somatic hypermutation and isotype switching by expressing several factors: (1) CXCR5+CXCR5+ cells secrete Wnt ligands that induce CXCR5 expression on CD4+ T cells, leading to their differentiation into Tfh cells. Consistent with this, TCRδ−/− mice show reduced Tfh cell frequencies and germinal center formation and have decreased production of both OVA-specific antibodies and self-reactive antibodies compared to WT mice. Moreover, in a murine model of lupus, TCRδ−/− mice develop milder glomerulonephritis compared to WT mice. These data advance our understanding of the mechanisms by which γδ T cells control humoral immune responses and promote antibody-mediated diseases.Here, we show that upon immunization with CFA, but not Alum, CXCR5 expression is induced on γδ T cells in a γδTCR activation-dependent fashion. TCRγδ−/−, and TCRβ−/− mice were immunized with either CFA/OVA or Alum/OVA at day 0, received an OVA boost at day 14 and sera and feces (for secretory (s)IgA analysis) for OVA-specific antibody measurement were collected at day 21. We found that, upon CFA immunization, serum levels of OVA-specific immunoglobulin levels were reduced in TCRδ−/− mice , complement factors, fibrinogen S, aquaporin 4 (AQP4), myelin-associated glycoprotein (MAG), and muscarinic receptor , we measured B220+FAS+GL7+ expression in WT and TCRδ−/− mice 7 days after immunization. The frequencies of B220+FAS+GL7+ cells in TCRδ−/− mice were comparable to those in non-immunized mice and significantly lower than in immunized WT mice  and spleen of WT vs. TCRδ−/− mice 1, 3, 5, 7, and 21 days after immunization. In dLNs, we found that TCRδ−/− mice had 50% less CXCR5+ expression on CD4+ T cells 1, 3, 5 and 7 days post-immunization , TCRγδ+CXCR5+ cells did not upregulate CD40L nor IL-21  and this effect was neither blocked by the TLR1/2 antagonist CU-CPT22 nor potentiated by the addition of anti-TCRγδ . Among them were those related to antigen presentation, such as MHC class II molecules , CD40 and CD86  with Alexa Fluor 488-conjugated ovalbumin (OVA). TCRγδ+CXCR5+ cells took up OVA, but to a lesser extent than CD11c+ DCs . No OVA uptake was observed in TCRγδ+CXCR5− cells  mice, responder T cells proliferated in the presence of TCRγδ+CXCR5+ cells and to a greater extent in the presence of CD11c+ DCs . However, consistent with results from the uptake assay, no proliferation was observed when responder T cells were co-cultured with TCRγδ+CXCR5− cells, when TCRγδ+CXCR5+ cells or CD11c+ DCs were not pulsed with OVA323–339 peptide or when TCRγδ+CXCR5+ and CD11c+ DCs were sorted from MHC-II–/– mice , including Wnt1, Wnt2, Wnt3a, Wnt4, Wnt6, Wnt7a, Wnt7b, Wnt8a, Wnt8b; and non-canonical (β-catenin independent) pathways, such as Wnt5a and Wnt11. We found that Wnt6 was highly expressed in CD11c+ DCs from non-immunized mice, but was downregulated from day 1 to 3 after immunization and returned to the basal level at day 4. Wnt6 was not expressed in γδ T cells  and Cxcr5 (72 h in co-culture) mRNA expression in CD4+ T cells . Of note, we could not sort enough TCRγδ+CXCR5+ and TCRγδ+CXCR5− cells to be separately transferred. We then immunized the mice with CFA and measured Tfh cells in the dLNs and OVA-specific antibodies in the sera 7 and 21 days later, respectively. We found that TCRδ−/− mice transferred with γδ T cells from Wnt-C59-treated mice had reduced frequencies of Tfh cells  in vivo. This effect was mediated by Wnt ligand secretion because inhibiting the enzyme responsible for Wnt release, PORCN, abrogated both Ascl2 and CXCR5 expression in CD4+ T cells. Notably, Wnt8b plays a major role in CXCR5 expression on CD4 T cells since blocking Wnt8b with a monoclonal antibody prevented this effect. Thus, TCRγδ+CXCR5+ cells, by functioning as APCs and releasing Wnt ligands, induce Ascl2 in CD4+ T cells that in turn initiates the Tfh cell program. The fact that γδ T cells control antibody production by presenting antigen and inducing Tfh cell differentiation does not exclude the crucial role of DCs in Tfh cell development. Because TCRγδ+CXCR5+ cells do not express ICOSL, they cannot induce Bcl6 in CD4+CXCR5+ T cells that, in the absence of DCs, would abort Tfh cell differentiation5.Ascl2 can be induced by Wnt agonists37. However, the presence of γδ T cells within the follicles may be related to their ability to secrete Wnt ligands, which have been shown to protect germinal center B cells from rapid apoptosis38. The frequency of TCRγδ+CXCR5+ cells increased 1 day after immunization, which is in contrast with Tfh cells that gradually upregulated CXCR5. This suggests that γδ T cells have a rapid mechanism for CXCR5 induction. Consistent with this, we found that γδTCR activation, but not toll-like receptor (TLR) 1/2, played a crucial role in CXCR5 upregulation on γδ T cells. CFA, but not Alum, induced CXCR5-expressing γδ T cells in vivo, particularly on the Vγ1+ γδ T cell subset, and this effect was likely mediated by the M. tuberculosis present in the CFA. Accordingly, MT contains heat shock protein 65 (Hsp65), that can directly activate the γδTCR from Vγ1+ γδ T cells39. Notably, pristane injection also induced CXCR5 upregulation on γδ T cells, again demonstrating that pathogen recognition receptors (PRRs) other than TLR1/2 may also induce CXCR5 expression on γδ T cells.CXCR5-expressing γδ T cells could be observed even in non-immunized mice, and immunization increased the number of γδ T cells in the T cell zone, but also in the follicles of dLNs, which is consistent with the upregulation of CXCR5 on γδ T cells. The role of γδ T cells in the follicle is not known, particularly taking into consideration our findings that γδ T cells do not express CD40L nor produce IL-21, critical factors for B cell activation and maturation+CXCR5+ cells migrate toward the follicle and present antigens to naïve CD4+ T cells via MHC-II. Wnt ligands are released during this process to induce Ascl2 and initiate the Tfh cell program. Ascl2 leads to CXCR5 upregulation on CD4+ T cells7. DCs, which also present antigens to CD4+ T cells, provide co-stimulatory signals that lead to Bcl6 expression, which terminates Tfh cell differentiation. These new generated Tfh cells activate cognate antigen-expressing B cells at the B:T cell border by providing CD40L and IL-21 signals, resulting in the generation of high-affinity antibody-secreting plasma cells. Our results raise the possibility that patients affected by antibody-mediated autoimmune conditions may benefit from γδ T cell modulation as a component for treatment of their disease.In summary, our findings help explain the poorly understood mechanism by which γδ T cells participate in the humoral immunity. We propose the following: upon arrival in secondary lymphoid organs, antigens directly activate γδ T cells by binding to TCR and/or PRRs, which induce CXCR5 expression on a subset of γδ T cells. TCRγδ−/−, CXCR5−/−, TCRβ−/−, TCRγδ-GFP, and TCRδ−/− mice were purchased from the Jackson Laboratory. Tcf7L (TCF-1) Tg mice were kindly provided by Dr. Ana Anderson from Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA. OT-II-Foxp3-GFP mice were housed in a conventional specific pathogen-free facility at the Harvard Institutes of Medicine and/or at the Building for Transformative Medicine according to the animal protocol with the full knowledge and permission of the Standing Committee on Animals at Harvard Medical School and Brigham and Women’s Hospital. Three to ten mice were used per group and every experiment was reproduced at least twice, which is commonly used for in vivo studies to alleviate unnecessary animal suffering. No animal was excluded from analysis and no randomization was performed.Male and female, 6–10 week-old on B6 genetic background mice were used in this study. C57BL/6J wild type, MHC-II−/− or OT-II-Foxp3-GFP mice were first enriched using CD4 microbeads or TCRγδ isolation kit . Then, naïve (CD4+CD62L+CD44−Foxp3−) or memory (CD4+CD62L−CD44+CD25−) cells were sorted  with peridinin chlorophyll protein (PerCP)–conjugated anti-CD4 , allophycocyanin (APC)-conjugated anti-CD62L  and phycoerythrin (PE)-conjugated anti-CD44 . TCRγδ+CXCR5+ and TCRγδ+CXCR5− cells were sorted with Alexa Fluor 700 (AF700)-conjugated anti-CD3ε , APC-conjugated anti-TCRγδ  and PE-Cy7-conjugated anti-CXCR5 . For TCRγδ cell sorting, AF488-conjugated anti-CD11c  was used to exclude any remaining CD11c+ cells to prevent dendritic cell contamination. Dead cells were also excluded based on 7-AAD (BD Bioscience) staining. In some experiments, antigen-presenting cells were first enriched using CD11c microbeads (Miltenyi Biotec) and sorted for CD11c+ cells with exclusion of dead cells based on 7-AAD staining. Gating strategies for cell sorting is shown in Supplementary Fig. −1; Sigma-Aldrich) and ionomycin  and a protein-transport inhibitor containing monensin  before detection by staining with antibodies. Surface markers were stained for 25 min at 4 °C in Mg2+ and Ca2+ free HBSS with 2% FCS, 0.4% EDTA (0.5 M) and 2.5% HEPES (1 M) then were fixed in Cytoperm/Cytofix (eBioscience), permeabilized with Perm/Wash Buffer (eBiosciences). Flow-cytometric acquisition was performed on a Fortessa (BD Biosciences) by using DIVA software (BD Biosciences) and data were analyzed with FlowJo software versions 9.9 or 10.1 (TreeStar Inc.). Intracellular staining antibodies used: BV-421-anti-IFN-γ , PE-Cy7-anti-IFN-γ , FITC-anti-IL-17A , PE-anti-IL-21 . Other antibodies included: BV605-anti-CD4 , BV421-anti-CD4 , BV421-anti-TCRγδ , PE-anti-TCRγδ ; AF700-anti-CD19 , AF488-anti-B220 , AF647-anti-B220 , PE-Cy5-anti-B220 , PE-Cy7-anti-IgM , APC-anti-IgD , PE-anti-IgD , BV605-anti-CD138 , BV421-anti-CD43 , PE-anti-BP-1 , AF700-anti-CD24 , PE-Cy5-anti-IL-21R , APC-anti-ICOS , PE-anti-BCL6 , PerCP-efluor710-anti-BCL6 , APC-anti-CD40L , APC-anti-GL7 , AF488-anti-CD95 , PE-Cy7-anti-I-A/I-E , AF647-anti-I-A/I-E , BV650-anti-I-A/I-E , APC-anti-CD11c , AF488-anti-CD11c , PE-Cy7-anti-CD11c , AF700-anti-CD86 , AF488-anti-CD86 , PE-anti-CD40 , PE-anti-TCR Vβ5.1/5.2 , APC-anti-Vγ1.1/Cr4 , PE-anti-Vγ2 .In the experiments where sorted cells were required, a pool of cells from inguinal lymph nodes (ILN) of C57BL/6, TCRδ−1 of M. tuberculosis H37RA (Difco) plus PBS or CFA enriched with 4 mg ml−1 of M. tuberculosis H37RA plus OVA . In some experiments mice were also immunized (s.c.) in the flanks (100 μl per flank) or i.p. (200 μl) with OVA (50 μg per mouse) adsorbed in 1 mg of Alum . To investigate the involvement of different subtypes of γδ T cells on their ability to control the humoral immune response, 200 μg of anti-Vγ2 mAb  or isotype control (BioXCell) were injected i.p. three days before either CFA or Alum immunization and three days before OVA booster dose.Mice were immunized subcutaneously (s.c.) in the flanks with 100 μl (per flank) of either Complete Freud Adjuvant (CFA) enriched with 4 mg ml−/− mice received a single i.p. injection of 0.5 ml of pristane (Sigma). Sera were collected before pristane injection and 3 months thereafter for autoantibody analysis. Five months after pristane injection, mice were sacrificed and kidneys removed for histopathology investigation as well as spleens taken for FACS analysis.Eight-week-old C57BL/6 and TCRδ−/−, and TCRδ−/− mice were first immunized with OVA emulsified in CFA containing 4 mg ml−1 of M. tuberculosis H37RA (Difco), or adsorbed in Alum as described above. Fourteen days later, mice received an i.p. injection of OVA (50 μg) in PBS and were sacrificed 7 days thereafter for sera and intestinal lavage fluids collection. Ready-SET-Go! ELISAs (eBioscience) for detecting total IgG antibodies and subclasses were performed according to the manufacturer’s instructions. Sera were added at starting dilution 1:10,000, followed by 2-fold serial dilutions. For secretory IgA (sIgA) detection, intestinal lavage fluids were collected by flushing the colon with cold PBS, spun down for 20 min, 2000 rpm at 4 °C, and supernatants used for sIgA detection with starting dilution 1:100, followed by 2-fold serial dilutions. For anti-OVA antibody titers, 96-well plates  were coated overnight with 20 μg ml−1 OVA solution in coating solution (eBioscience) at 4 °C. Ready-SET-Go! ELISAs (eBioscience) for detecting total IgG antibodies and subclasses were then performed according to the manufacturer’s instructions. Sera or intestinal lavage fluids were added at starting dilution 1:100, followed by 2-fold serial dilutions. Samples were read using a Tecan microplate reader  and processed using i-Control microplate reader software (Tecan).Eight-week-old C57BL/6, TCRβ−/− mice were sent to Genomics and Microarray Core Facility at UT Southwestern Medical Center  for analysis, using the autoantigen microarray super panel (128 autoantigens). Briefly, samples were treated with DNAse I, diluted 1:50, and incubated with autoantigen array. The autoantibodies binding to the antigens on the array were detected with Cy3 labeled anti-IgG and Cy5 labeled anti-IgM and the arrays were scanned with GenePix® 4400A Microarray Scanner. The images were analyzed using GenePix 7.0 software to generate GPR files. The averaged net fluorescent intensity (NFI) of each autoantigen was normalized to internal controls (IgG or IgM).Sera collected from both naïve wild type and TCRδγδ T cells were depleted by injecting a single dose of 250 μg i.p. of anti-TCRγδ depleting antibody .4 sorted CXCR5− γδ T cells from a pool of spleen and lymph nodes of 15 naïve WT mice were cultured for 48 h at 37 °C in the presence of medium only (unstimulated); anti-TCRγδ monoclonal antibody ; anti-TCRγδ (2 μg ml−1) + anti-CD28  monoclonal antibodies; TLR1/2 agonist (Pam3CSK4 (Invivogen), 500 ng ml−1); anti-TCRγδ (2 μg ml−1) + TLR1/2 agonist (500 ng ml−1); M. tuberculosis ; anti-TCRγδ (2 μg ml−1) + MT (10 μg ml−1); TLR1/2 antagonist (CU-CPT22 (Millipore Sigma), 0.5 μM); TLR1/2 agonist (500 ng ml−1) + TLR1/2 antagonist  or MT (10 μg ml−1) + TLR1/2 antagonist (0.5 μM). CXCR5 expression on γδ T cells was analyzed by flow cytometry.5 × 10For in vivo study of CXCR5 expression on γδ T cells, 500 μg per mouse of anti-TCRγδ monoclonal antibody  was administered i.p. and 24 h later CXCR5 expression on γδ T cells was analyzed by flow cytometry.+ dendritic cells and TCRγδ+CXCR5+, TCRγδ+CXCR5− cells were first enriched using CD11c microbeads or TCRγδ isolation kit  and sorted. For TCRγδ sorting, AF488-conjugated anti-CD11c  was used to exclude any remaining CD11c+ cells in order to prevent dendritic cell contamination. For uptake assay, TCRγδ+CXCR5−, TCRγδ+CXCR5+ and CD11c+ dendritic cells were incubated for 3 h at 37 °C with 50 μg ml−1 of ovalbumin (OVA) coupled to Alexa Fluor 488 (Invitrogen) in a 96-well round-bottom plate. After incubation, cells were collected, thoroughly washed and analyzed by flow cytometry. For in vitro presentation assay, cells were first incubated overnight at 37 °C with 50 μg ml−1 of either OVA323–339 peptide (Invivogen), OVA protein (Sigma) or medium only (unloaded cells as control) in a 96-well round-bottom plate. On the next day, cells were thoroughly washed and incubated at 1:2 ratio (antigen-presenting cells:responder cells) with sorted naïve (CD4+CD62L+CD44−Foxp3−) cells from OT-II-Foxp3-GFP mice previously stained with CellTrace Violet dye (Invitrogen) for 4 days. Proliferation was then analyzed by flow cytometry.CD11c+ dendritic cells, TCRγδ+CXCR5+ and TCRγδ+CXCR5− cells from WT mice were first enriched using CD11c microbeads or TCRγδ isolation kit  and sorted. For TCRγδ sorting, AF488-conjugated anti-CD11c  was used to exclude any remaining CD11c+ cells in order to prevent dendritic cell contamination. Cells were then incubated overnight at 37 °C with 50 μg ml−1 of either OVA323–339 peptide, OVA protein or medium only (unloaded cells as control) or 50 μg ml−1 of OVA323–339 peptide plus either 1 μM of the porcupine (PORCN) inhibitor  or 20 μg ml−1 of anti-Wnt8b monoclonal antibody (LSBio) in a 96-well round-bottom plate. On the next day, cells were thoroughly washed and incubated at 1:2 ratio (antigen-presenting cells:responder cells) with sorted naïve (CD4+CD62L+CD44−Foxp3−) cells from OT-II-Foxp3-GFP mice previously stained with CellTrace Violet dye (Invitrogen) for 4 days. CXCR5 expression on CD4 T cells was then analyzed by flow cytometry.CD11c+CXCR5+ and TCRγδ+CXCR5− cells from WT mice were first enriched using TCRγδ isolation kit  and sorted. AF488-conjugated anti-CD11c  was used to exclude any remaining CD11c+ cells in order to prevent dendritic cell contamination. Cells were then stimulated with plate-bound anti-CD3  and anti-CD28 , both at 2 μg ml−1, for 3 days at 37 °C in a 96-well round-bottom plate. Wnt-C59 at 1 μM was added to some wells to prevent Wnt ligand release. At the 3rd day of culture, supernatants were collected and 100 μl added to naïve (CD4+CD62L+CD44−Foxp3−) cells in a 96-well round-bottom plate and stimulated with plate-bound anti-CD3 and anti-CD28 (both at 2 μg ml−1) at 37 °C. Three days later, CXCR5 expression on CD4 T cells was analyzed by flow cytometry.TCRγδ−/− and MHC-II−/− mice were first enriched using TCRγδ isolation kit  and 5 × 105 sorted γδ T cells were intravenously transferred into TCRδ−/− mice. Three days after transfer, mice were immunized with CFA and 7 days later draining  lymph nodes were collected for FACS analysis. To investigate the involvement of Wnt ligands secretion by γδ T cells on their ability to induce Tfh cell differentiation, WT C57BL/6J mice were treated with either 10 mg/kg of Wnt-C59 (Tocris) or vehicle (3.5% DMSO in PBS) intraperitoneally and 1 day later, animals were sacrificed and spleens and lymph nodes collected. γδ T cells were first enriched using TCRγδ isolation kit  and 5 × 105 sorted γδ T cells were intravenously transferred into TCRδ−/− mice. One day after transfer, mice were immunized with CFA and 7 days later draining  lymph nodes were collected for FACS analysis.To investigate whether CXCR5 expression and antigen presentation function of γδ T cells were involved in the ability of γδ T cells to induce Tfh cell differentiation in vivo, γδ T cells from spleen and lymph nodes of WT C57BL/6J, CXCR5−/− mice were injected s.c. in the inner thigh with a mix of 20 μl of BV421-anti-CD4 , 20 μl of AF647-anti-B220  and PBS in a total volume of 100 μl/thigh. In some experiments, either 20 μl of PE-anti-CXCR5  or 20 μl of PE-anti-GL7  were also injected in combination with BV421-anti-CD4 and AF647-anti-B220. Three hours later, mice were sacrificed and inguinal lymph nodes removed for confocal microscopy. Lymph nodes were placed on a glass microscopy slide with PBS and imaged at 10× or 20× with Zeiss 710 confocal microscope.TCRγδ-GFP and TCRγδKidneys were excised from animals 5 months after pristane treatment and immediately fixed in 10% neutral buffered formalin. Samples were then embedded in paraffin and 5 μm were cut and stained with hematoxylin and eosin. Slides were loaded into an Aperio ScanScope XT (Aperio), scanned via the semi-automated method and checked for image quality using visual inspection. By using the eSlide Manager (Aperio), sections were evaluated for histopathological changes, such as glomerulus enlargement (diameter of 50 glomeruli per mouse were measured) and cellularity .+CD44+CD25− cells, CD11c+ dendritic cells, total γδ T cells, TCRγδ+CXCR5+ and TCRγδ+CXCR5− cells were sorted and RNA was extracted with a RNeasy Plus micro kit (Qiagen), then was reverse-transcribed with a high capacity cDNA reverse transcription kit (Applied Biosystems) and analyzed by quantitative RT-PCR with a Vii 7 Real-time PCR system (Applied Biosystems) with the following primers and probes : Ascl2 (Mm01268891_g1) Ifng (Mm00801778_m1), Il17a (Mm00439619_m1), Il21 (Mm00517640_m1), Il4 (Mm00445259_m1), Il21r (Mm00600317_m1), Il6 (Mm00446191_m1), Il10 (Mm00439616_m1), Wnt1 (Mm01300555_g1), Wnt2 (Mm00470018_m1), Wnt3a (Mm00437337_m1), Wnt4 (Mm01194003_m1), Wnt5a (Mm00437347_m1), Wnt6 (Mm00437353_m1), Wnt7a (Mm00437356_m1), Wnt7b (Mm01301717_m1), Wnt8a (Mm01157914_g1), Wnt8b (Mm00442108_g1), Wnt11 (Mm00437328_m1). The comparative threshold cycle method and the internal control Gapdh (Mm99999915_g1) was used for normalization of the target genes.Memory CD4+CXCR5+ and TCRγδ+CXCR5− following the manufacturer’s protocol. Assay and spike-in controls were used for normalization based on identical amounts of input RNA (60 ng/sample). The correlation of gene expression among cell types were calculated and plotted as heatmap using the Multi Experimental Viewer (MeV) software. Differentially expressed genes were identified by Student’s t-test and statically relevant results consisted in p-value < 0.05.nCounter mouse mRNA Assay Kit (NanoString Technologies) were used to detect mRNA from sorted TCRγδt-test or one-way ANOVA, followed by Tukey multiple comparisons). Differences were considered statistically significant with a p value of less than 0.05.GraphPad Prism 7.0 was used for statistical analysis (unpaired, two-tailed Student’s The datasets included in this study are available as Supplementary Data Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2


Ka > 106 M–1). Isothermal titration calorimetry (ITC) studies reveal that enthalpic stabilization is a dominant driving force for both of the complexations through multiple host–guest CH-π and hydrogen-bonding interactions. Furthermore, long oligomers with an average molecular weight of 1000 Da  are also threaded into the capsules to give pseudo-rotaxane-shaped 2:1 host–guest complexes in water, selectively.Oligo(ethylene oxide)s are known as widely useable yet not very interactive amphiphilic compounds. Here we report that the long amphiphilic oligomers are bound by a polyaromatic capsule in two different manners, depending on the chain length. For instance, the shorter pentamer is crammed into the isolated cavity of the capsule, whereas the longer decamer is threaded into the capsule to form a 1:1 host–guest complex in a pseudo-rotaxane fashion. These unusual bindings occur instantly, spontaneously, and quantitatively even in water at room temperature, with relatively high binding constants ( Molecular capsules typically bind only guests with volumes smaller than their cavities. Here, the authors find that a polyaromatic capsule accommodates linear amphiphilic oligomers in a length-dependent manner, whereas short chains are fully crammed into the cavity, long chains can be incorporated into the capsule in a threaded fashion. Similar behavior is observed in artificial host–guest systems. For example, Rebek’s hydrogen-bonded capsules take up one molecule of long-chain alkanes in a helical fashion in their cylindrical cavities5. One of Gibb’s hydrophobic effect-driven capsules also encapsulates long alkanes in a helical or folded fashion in its spherical cavity8. However, as observed for other molecular capsules reported previously, longer alkanes as well as other compounds with volumes larger than the host cavities are hardly bound by the capsules due to the limited spaces available15.To bind or not to bind substrate molecules is ubiquitous in biological receptors and enzymes under aqueous ambient conditions. Such host–guest events depend strictly on the size and shape complementarity between the guest structure and the host cavity, surrounded by the protein assemblies, and on their mutual interactions18. However, owing to the weak interactions with aliphatic and small aromatic frameworks, the encapsulation of OEOs within molecular cages and capsules in solution has been seldom achieved19, except for aliphatic cyclodextrin tubes21. Therefore, thermodynamic insights into the host–guest interactions remained obscure to date. We thus focused our attention on polyaromatic panels as promising frameworks to interact with OEO chains23 and employed polyaromatic capsule 1  ions and four bent bispyridine ligands, provides a spherical hydrophobic cavity (~ 1 nm in diameter and 480 Å3 in free volume) encircled by the eight anthracene panels. Owing to the 12 exterior hydrophilic groups, the polyaromatic framework can interact with guest molecules only from the inside. The isolated cavity of 1 has been shown to fully accommodate not only hydrophobic molecules 29 but also hydrophilic ones s)31 in aqueous media. However, the binding of large and long molecules, particularly whose sizes and volumes are larger than the cavity, had not been achieved so far by the capsule.Oligo(ethylene oxide)s (OEOs) are very useful amphiphilic molecules and components that find broad application in materials chemistry and biochemistry, because of the high solubility in both organic solvents and water as well as the low biological toxicity and chemical reactivitye 1 Fig. 24,25 to 6 M–1 for the 1:1 host–guest complexes). The obtained, threaded products are uncommon pseudo-rotaxane-shaped capsules, which would be a new promising building block for functional interlocked nanostructures such as molecular machines and soft materials34.In contrast to such common complexation behavior, herein we report that a molecular capsule binds one molecule of linear amphiphilic oligomers in two different manners, a cramming or threading fashion, depending on the chain length. Namely, the shorter oligomers are fully accommodated in the closed cavity, yet the longer ones stick the ends out from the cavity Fig. . These unEO  and pentamer 5EO (0.39 μmol) were combined in D2O (0.5 mL) at room temperature, 1:1 host–guest complex 1•5EO was exclusively formed within 5 min  upon full encapsulation in the polyaromatic cavity of 1 . The observed, sharp aromatic signals of 1 and the upfield-shifted proton Ha indicate full encapsulation of the amphiphilic chain of 5EO within 1 in water. Besides the NMR integration, a 1:1 host–guest ratio of the product was evidenced by the Electrospray ionization time-of-flight mass spectrometry (ESI-TOF MS) spectrum, where prominent peaks assignable to [1•5EO – n•NO3–]n+ (n = 4–2) species were found  in a quantitative fashion , owing to their volume and shape complementarity, demonstrated by the 1H NMR competitive binding experiments  with the five anthracene panels of 1, indicating the presence of effective host–guest CH-π interactions  and the ether oxygen atoms as well as three intramolecular hydrogen bonds of the guest were observed in the cavity36. The optimized structures of 1•nEO (n = 6 and 8) also suggested that the spherical framework of 1 forces the flexible OEO chains into coiled conformations in the cavity , by 1H NMR and ITC analyses. In a manner similar to 1•5EO, 1:1 host–guest complex 1•CE formed quantitatively upon mixing 1 with CE in water at room temperature  ion and the bispyridine ligand is inert at ambient temperature so that the encapsulation of the linear as well as cyclic oligomers by 1 should occur without any dissociation step of the capsular frameworks. A 1H NMR competitive binding experiment of capsule 1 with 5EO and CE (1.0 equiv. each) in water resulted in the quantitative formation of a mixture of 1•5EO (90%) and 1•CE (10%) at room temperature  were measured for 1•5EO (Table 1•CE (ΔΔH = –4.9 kJ mol–1) but the entropic contribution is smaller than that for 1•CE (TΔΔS = –5.0 kJ mol–1), owing to the flexible structure of acyclic 5EO. The large enthalpic gain most probably arises from multiple host–guest CH-π and hydrogen-bonding interactions by cramming of the guest into the polyaromatic host cavity  is relatively high even in water and comparable to that for 1•CE  were bound by capsule 1 to form pseudo-rotaxane-shaped host–guest complexes in water. For example, methyl-capped decamer 10EO is composed of a long amphiphilic chain with an extended length of ~4 nm and a volume of ~ 520 Å3  in water at room temperature led to the quantitative formation of 1:1 host–guest complex 1•10EO within 5 min  regions, respectively, at room temperature . The optimized structure of 1•10EO also suggests the existence of multiple host–guest CH-π and hydrogen-bonding interactions in the cavity and at the openings of 1  as compared with that for 1•5EO (–16.6 kJ mol–1) is most likely derived from the large restriction of the molecular motion of long 10EO threaded through 1. The binding constant of 1 for 10EO is still relatively high (Ka = 1.1 × 106 M–1), which is approximately half of those for 5EO and CE.Thermodynamic parameters for the formation of f 1 Fig. . The larOEO1000 and OEO2000 with average molecular weights of 1000 and 2000 Da, respectively. Oligomers OEO1000 and OEO2000 contain a mixture of CH3O(CH2CH2O)nCH3 . When capsule 1 (0.39 μmol) and OEO1000 (0.19 μmol) were mixed in water (0.5 mL) at room temperature, to our surprise, 2:1 host–guest complexes (1)2•OEO1000 were predominantly generated within 5 min 2•OEO1000 showed relatively broadened peaks derived from the threaded OEO1000 as well as sharp and split peaks derived from the polyaromatic framework of 1 2•OEO1000 closely resembles that of 1:1 pseudo-rotaxane complex 1•10EO at 5 °C  on the NMR timescale at room temperature. The ESI-TOF MS spectrum showed molecular ion peaks assignable to a 1:1 host–guest complex 2•OEO1000 was estimated to be a large negative value (ΔH = –131.3 kJ mol–1), which is approximately twice of that for 1•10EO (Table 1)2•OEO1000 (TΔS = –99.6 kJ mol–1) is approximately four times smaller than that for 1•10EO. The large entropy loss is most probably caused by the restricted motion of the long and flexible chains of OEO1000 upon their threading into the polyaromatic cavities. The optimized structure of (1)2•OEO1000 exhibits that the amphiphilic chain of OEO1000 (q = 1) is long enough to penetrate two molecules of capsule 1 separately 2•OEO2000 were selectively formed upon simple mixing of 1 and OEO2000 in water 2•OEO2000 is more favorable than that of (1)2•OEO1000 on the basis of the estimated Gibbs free energy (ΔG) and binding constant (Ka) by the ITC studies s, in water at room temperature. The isolated polyaromatic cavity of the capsule quantitatively binds one molecule of the tetramer to the octamer in a cramming fashion. On the other hand, the longer oligomers such as the decamer and docosamer (22-mer) are quantitatively bound by the capsule in a threading fashion. The observed, straightforward, and chain length-dependent complexation is driven by effective enthalpic stabilization through multiple host–guest CH-π and hydrogen-bonding interactions in the cavity of the capsule. Although both oligo(ethylene oxide) chains and polyaromatic panels are well-studied and have been applied as building blocks for wide-ranging functional molecules, we, for the first time, revealed effective intermolecular interactions between the chains and the panels, which could prove to be useful chemical tools to construct new interlocked supramolecular structures and materials with controllable functions.1 and methyl-capped oligo(ethylene oxide)s 5EO-10EO were synthesized according to previously reported procedures41. The cavity volume estimated from the crystal structure of 1  & ASCEND-500 (500 MHz), ESI-TOF MS: Bruker micrOTOF II, Single crystal XRD: Rigaku XtaLAB Pro MM007 HyPix-6000HE, ITC: MicroCal system, VP-ITC model, Theoretical calculation: Fujitsu Limited SCIGRESS program (version FJ 2.6). Solvents and reagents: TCI Co., Ltd., Wako Pure Chemical Industries Ltd., Kanto Chemical Co., Inc., Sigma-Aldrich Co., and Cambridge Isotope Laboratories, Inc. Ultra-pure water (Milli-Q) was used for the ITC analysis. Capsule 1 , 5EO , and D2O (0.5 ml) were added to a glass test tube. The mixture was stirred at room temperature (or 60 °C) for 5 min. The quantitative formation of 1•5EO was confirmed by NMR, ESI-TOF MS, and ITC analyses : δ –0.23 , –0.08–0.18 , 0.28 , 2.49 , 3.13 , 3.49 , 3.95 , 3.99 , 4.09 , 4.49 , 4.63 , 5.98 , 6.54 , 6.79 , 6.97 , 7.27 , 7.70 , 7.79 , 7.97 , 8.01 , 8.34 , 8.57 , 9.22 . ESI-TOF MS (H2O): m/z 2003.7 [1•5EO – 2•NO3–]2+, 1315.2 [1•5EO – 3•NO3–]3+, 970.9 [1•5EO – 4•NO3–]4+.1•5EO’: 1H NMR : δ –0.36 , –0.14 , –0.02–0.15 , 0.21 , 2.48 , 3.12 , 3.48 , 3.95 , 3.99 , 4.08 , 4.48 , 4.62 , 6.01 , 6.47 , 6.73 , 6.96 , 7.25 , 7.51 , 7.70 , 7.78 , 8.01 , 8.02 , 8.33 , 8.54 , 9.22 . ESI-TOF MS (H2O): m/z 1989.8 [1•5EO’ – 2•NO3–]2+, 1305.9 [1•5EO’ – 3•NO3–]3+, 963.9 [1•5EO’ – 4•NO3–]4+.1 , 18-crown-6 , and D2O (0.5 ml) were added to a glass test tube. The mixture was stirred at room temperature (or 60 °C) for 5 min. The quantitative formation of 1•CE was confirmed by NMR, ESI-TOF MS, and ITC analyses : δ –0.09 , 0.03 , 2.53 , 3.19 , 3.51 , 3.97 , 4.06 , 4.13 , 4.52 , 4.64 , 5.92 , 6.58 , 6.86 , 6.94 , 7.35 , 7.50 , 7.72 , 7.80 , 7.98 , 8.01 , 8.34 , 8.58 , 9.20 . ESI-TOF MS (H2O): m/z 2002.6 [1•CE – 2•NO3–]2+, 1314.4 [1•CE – 3•NO3–]3+, 970.3 [1•CE – 4•NO3–]4+.1 , 10EO , and D2O (0.5 ml) were added to a glass test tube. The mixture was stirred at room temperature (or 60 °C) for 5 min. The quantitative formation of 1•10EO was confirmed by NMR, ESI-TOF MS, and ITC analyses : δ –0.52–1.80 , 2.15–2.67 , 2.96–3.30 , 3.50 , 3.96 , 4.07 , 4.52 , 4.62 , 5.96 , 6.01 , 6.09–7.25 , 7.25–8.89 , 9.01–9.39 . ESI-TOF MS (H2O): m/z 2113.4 [1•10EO – 2•NO3–]2+, 1388.3 [1•10EO – 3•NO3–]3+, 1025.7 [1•10EO – 4•NO3–]4+.1 , OEO1000 , and D2O (0.5 ml) were added to a glass test tube. The mixture was stirred at room temperature for 5 min. The quantitative formation of (1)2•OEO1000 was confirmed by NMR and ITC analyses 2•OEO2000 in water 2•OEO1000: 1H NMR : δ –0.48–1.89 , 2.16–2.68 , 2.86–3.31 , 3.51 , 3.82–4.27 , 4.52 , 4.62 , 5.95 , 6.02 , 6.10–7.25 , 7.25–8.94 , 8.99–9.41 .(1)2•OEO2000: 1H NMR : δ –0.34–1.84 , 2.17–2.69 , 2.89–3.29 , 3.48 , 3.81–4.25 , 4.49 , 4.62 , 5.94 , 6.00 , 6.08–6.57 , 6.57–7.25 , 7.25–8.86 , 9.01–9.44 .2O solutions (1.77–3.78 mM) of 5EO, CE, 10EO, OEO1000, or OEO2000 to the H2O solutions (0.11–0.14 mM) of capsule 1 at 25 °C  Å, b = 21.4245(5) Å, c = 27.3518(5) Å, V = 10003.1(4) Å3, Z = 2, ρcalcd = 1.201 g cm–3, F(000) = 3720.0, T = 93 K, reflections collected/unique 80903/34070 (Rint = 0.0393), R1 = 0.0834 (I > 2σ(I)), wR2 = 0.2714, GOF 1.049. All the diffraction data were collected on a Rigaku XtaLAB Pro MM007 HyPix-6000HE diffractometer (λ(CuKα) = 1.54184 Å). The contribution of the electron density associated with greatly disordered counterions and solvent molecules, which could not be modeled with discrete atomic positions, were handled using the solvent mask in the Olex2 program  Å, b = 20.4314(14) Å, c = 28.8216(18) Å, V = 9820.3(12) Å3, Z = 2, ρcalcd = 1.251 g cm–3, F(000) = 3809.0, T = 93 K, reflections collected/unique 81517/20534 (Rint = 0.1983), R1 = 0.1273 (I > 2σ(I)), wR2 = 0.4023, GOF 1.017. All the diffraction data were collected on a Rigaku XtaLAB Pro MM007 HyPix-6000HE diffractometer (λ(CuKα) = 1.54184 Å). The contribution of the electron density associated with greatly disordered counterions and solvent molecules, which could not be modeled with discrete atomic positions, were handled using the solvent mask in the Olex2 program (see Supplementary Methods and Supplementary Table CSupplementary InformationPeer Review File


P21/n, with Z = 8. The new Piedfort unit is chiral and its core structure closely approximates to D2 symmetry, with both enantiomers present in the crystal. Rather than being superposed with a staggered arrangement of nitrogen atoms, the rings are rotated by an angle of approximately 45° with respect to each other.The title host compound, designed to self-assemble to form a new type of extended core Piedfort unit reminiscent of an eight-legged spider host, forms a number of crystalline inclusion compounds favouring oxygen-containing guest molecules. We have established the presence of this unit in the unsolvated molecular crystal at 100 K, which is monoclinic, space group  62H84N4O4, designed to self-assemble to form a new type of extended core Piedfort unit reminiscent of an eight-legged spider host, forms a number of crystalline inclusion compounds favouring oxygen-containing guest molecules. We have established the presence of this unit in the unsolvated molecular crystal at 100 K, which is monoclinic, space group P21/n, with Z = 8. The new Piedfort unit is chiral and its core structure closely approximates to D2 symmetry, with both enantiomers present in the crystal. Rather than being superposed with a staggered arrangement of nitrogen atoms, the rings are rotated by an angle of approximately 45° with respect to each other, and the shortest contact between them is 3.181 (2) Å. The compound’s significant inclusion properties may be taken to suggest the participation of an extended Piedfort unit in the microcrystalline adducts formed. The presence of such a dimeric host unit in the clathrates has, however, not yet been established because of the current lack of suitable single crystals for X-ray analysis.The title host compound, C Subsequently, Henderson et al. phenoxy]-1,3,5-triazine 1b, also featuring a back-to-back arrangement of two trisubstituted 6π-electron aromatic rings. X-ray analysis revealed three types of Piedfort unit with respective symmetries Ci3, C3 and D3, now designated as C3i-PU, C3-PU and D3-PU . This assembly mode with opposite enantiotopic faces pointing outwards is also achiral by virtue of an improper axis of rotation, for a 90° component rotation (not shown) when the assembly has idealized S4 pyrimidine core have been omitted for clarity. The new extended Piedfort unit, comprised of the two molecules in the asymmetric unit, is chiral and its core structure closely approximates to D2 symmetry, with both enantiomers present in the crystal. This is exemplified by the values of the pseudo torsion angles N4A—C3A—N1B—C4B, −47.9 (1)°, N4B—C3B—N1A—C4A, −49.9 (1)°, N2A—C1A—N3B—C2B, −56.9 (1)°, N2B—C1B—N3A—C2A, −58.1 (1)°. The pyrimidopyrimidine core units defined by the ten atoms N1, C1, N2, C5, C2, N3, C3, N4, C6 and C4 are approximately planar. A calculated least-squares plane through the ten atoms of the core gave r.m.s. deviations from planarity of 0.0333 and 0.0693 Å for molecule A and molecule B, respectively, and a calculated dihedral angle between them of 4.96 (3)°, showing them to be almost coplanar. The oxygen-atom displacements from the mean plane of the core of molecule A are as follows: −0.127 (1), 0.017 (2), −0.125 (1) and 0.118 (2) Å for atoms O1A to O4A respectively. For the mean plane of the core of molecule B the oxygen-atom displacements are −0.241 (1), 0.286 (2), −0.141 (2) and 0.339 (2) Å for atoms O1B to O4B, respectively. The core of molecule B is markedly less planar than that of molecule A and exhibits a twist about the central C—C bond C5B—C6B of 6.61 (6)°. When viewed down the overlapping centroids of the central C—C bonds, C5A—C6A and C5B—C6B, it can be seen that the two pyrimidopyrimidine cores are rotated approximately 45° with respect to one another and that the shortest contact between the two cores is 3.181 (2) Å, see Fig. 3a and 3b.Colourless block crystals of d]pyrimidine with four uniform homochiral side chains could produce two geometrically distinct (flexible) Piedfort D2 forms. Since these forms would not be enantiomerically related, they would differ in stability and solubility, and preferential crystallization of just one of these two D2 forms might yield a novel chiral host lattice featuring potential amplification of chirality. Also, the successful production of benzene-based Piedfort units pyrimidopyrimidines, which have been shown to exhibit novel anti-tumour behaviour. Structure KETTAE and its s-anomer KETSUX pyrimidopyrimidine with both structures existing as monohydrate solvates. Structure RPPYPY20 pyrimido-pyrimidines, the substitutions being 2,4,6-trichloro and 4-amino-6-chloro, respectively. It is clear that the present structure is a unique example of the use of the pyrimidopyrimidine core in the formation of a new class of potential host–guest compounds.A search of the Cambridge Structural Database , 3,5-di-t-butylphenol  and sodium hydride  in dry THF (15 mL) gave, following column chromatography on silicic acid (eluting with DCM), solvent removal under vacuum, and recrystallization from acetone gave the product as a solid gave the product as a crystalline solid, m.p. > 567 K, : 1H NMR  δ 7.14 , 7.07 , 7.01 , 6.96 , 1.16 , 1.15 . 13C NMR  δ 167.2, 159.9, 152.6, 152.3, 151.9, 151.8, 135.8, 120.0, 118.9, 115.8, 115.7, 35.3, 35.2, 31.7, 31.1; FAB MS, m/z 949.6 [M+ + H], C62H85N4O4, calculated 949.7. Crystals suitable for X-ray diffraction studies were obtained by recrystallization from CH2Br2/ethyl benzoate (ca 1:5).Preparation of 2a–c were prepared analogously in yields of 67, 33 and 72%, respectively; all had m.p. > 567 K, and 1H NMR, 13C NMR and MS data corresponding to their formulated structures. It is worthy of recording that the nature of the bulky substituents on the two meta positions is important since no evidence of host behaviour has yet been found for the phenoxy counterpart of 2d, nor for 3,5-substitution by the smaller methoxy group  = 1.2–1.5Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989019002470/hb7803sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989019002470/hb7803Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989019002470/hb7803Isup3.cmlSupporting information file. DOI: 1897649CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Mentha haplocalyx (Mentha canadensis) is widely used as a medicinal plant in traditional Chinese medicine, and the extracts of its aerial parts are found to significantly inhibit the activity of α-glucosidase with an IC50 value of 21.0 μg/mL. Bioactivity-guided isolation of the extracts afforded two new compounds (1 and 2), together with 23 known ones (3–25). Their structures were established by extensive spectroscopic analyses . Compounds 1–17 and 21–25 were evaluated for their α-glucosidase inhibitory activities. Compound 11 was the most active ones with an IC50 values of 83.4 μM. These results verify the α-glucosidase inhibitory activity of M. haplocalyx (M. canadensis) and specify its active compounds for the first time.The online version of this article (10.1007/s13659-019-0207-0) contains supplementary material, which is available to authorized users. Diabetes mellitus is a chronic disease caused by inherited or acquired deficiency in insulin secretion and by decreased responsiveness of the organs to secreted insulin . Such a Mentha haplocalyx Briq. (Mentha canadensis L.), a perennial herbaceous plant of the family Lamiaceae, is widely distributed in southwest of China and popularly used in food, cosmetics and medicines. As a traditional Chinese medicine, it is clinically used to treat diseases in the nerve center, breath, procreation and digestive systems +. The IR absorptions suggested the existence of a hydroxyl group (3427 cm−1) and an α,β-unsaturated carbonyl group (1635 cm−1). The 13C NMR (DEPT) data  and an α,β-unsaturated carbonyl group  were observed. 1H NMR data , a trisubstituted olefinic proton , and one terminal double bond proton . The glucose unit protons  were also present. All the above information suggested that compound 1 was a menthane-monoterpene glycoside. Apart from the glucose unit, the NMR data of 1 closely resembled those of the reported compound (R)-7-hydroxycarvone  suggested that 7-OH in 1 might be glycosylated, which was further confirmed by the HMBC correlation of H-7 (δH 4.99 and 4.28) with C-1ʹ (δC 104.1) -7-hydroxyisopiperitenone7-O-β-d-glucopyranoside (S)-7-hydroxyisopiperitenone 7-O-β-d-glucopyranoside (S)-configuration. Therefore, compound 1 was elucidated as (4S)-7-hydroxy-carvone 7-O-β-d-glucopyranoside.Compound ycarvone . Whereasmentclasspt{minima2, white powder, had a molecular formula of C47H82O3 from its HREIMS (m/z 694.6258 [M]+). The 13C NMR (DEPT) data (Table δC 53.4), two oxy-methines , a trisubstituted double bond  were showed. A palmitoyl group  was also present. The 1H NMR data (Table δH 0.85–1.21), a triplet methyl , a methoxyl (δH 3.25), and a trisubstituted olefinic proton . These NMR data suggested that 2 was an oleanane-type triterpene fused with a palmitoyl group, and closely resembled those of the reported compound -11-hydroxy-olean-12-en-3-yl palmitate + .Colorless caramelized solid, C47H82O3; 1H NMR  and 13C NMR  see Table m/z: 694.6258 [M]+ .Colorless powder, Cd-glucopyranoside  as a substrate following the reported method with slight modifications [2CO3 (40 μL). The absorbance was recorded at 405 nm by a Bio-Rad 680 microplate reader . The negative control was set by adding PB instead of the sample using the same procedure for the tests. Acarbose (Bayer) dissolved in PB was utilized as the positive control. The blank was set by adding phosphate buffer instead of the α-glucosidase using the same method. Inhibition rate (%) = [(ODnegative control − ODblank) − (ODtest − ODtest blank)]/(ODnegative blank − ODblank) × 100%. All data were subjected to an analysis of variance using SPSS 18.0. The significant differences in inhibition rates between the treatment and blank control were calculated using one-way analysis of variance (ANOVA).The α-glucosidase inhibitory activity was measured in a 96-well microtiter plate based on p-nitrophenyl-α-ications . In brieM. haplocalyx (M. canadensis) were firstly found to exhibit significantly inhibitory activity against α-glucosidase. Two new compounds (1 and 2) and 23 known ones were isolated and identified through bioactivity-guided fractionation. Among them, compounds 3–9 and 24 were reported from M. haplocalyx (M. canadensis) for the first time, while compounds 10–20, 22–23 and 25 were firstly isolated from the genus Mentha. Bioactivity assay further traced the active compounds , whose inhibitory activity against α-glucosidase had not been reported before. It was noted that the monoterpene glucosides and the ionone glycosides endowed this plant with the α-glucosidase inhibitory activity.In this study, the extracts of the aerial parts of 1 and 2; Structures of the known compounds 3–25.Supplementary material 1 (DOC 1878 kb). HRMS, IR, UV, 1D and 2D NMR spectra of compounds Below is the link to the electronic supplementary material.


Scientific Reports 10.1038/s41598-018-32615-8, published online 26 September 2018Correction to: In the original version of this Article, Matthew W. Keller and Benjamin L. Rambo-Martin were omitted as equally contributing authors.The original version of this Article also contained typographical errors.In the legend of Table 1 where:“Influenza A/Florida/20/2018 (H1N1pdm09), A/Texas/50/2012 (H3N2), A/chicken Ghana/20/2017 (HPAI H5N1) and A/British Columbia/1/2015 (LPAI H7N9) viruses were used to demonstrate this method’s broad utility across contemporary influenza A viruses of current clinical significance”.now reads:“Influenza A/Florida/20/2018 (H1N1pdm09), A/Texas/50/2012 (H3N2), A/chicken Ghana/20/2015 (HPAI H5N1) and A/British Columbia/1/2015 (LPAI H7N9) viruses were used to demonstrate this method’s broad utility across contemporary influenza A viruses of current clinical significance”.Additionally, in the Results section under subheading ‘Sequencing RNA from crude versus purified influenza rA/Puerto Rico/8/1934 (H1N1) virus’ where:15,16, Nextera, and MiSeq approach (Tables 2 and S3)”.“The read level accuracy was 86.3 ± 0.3%, and the consensus sequence was 98.97 ± 0.01% in concordance with consensus sequence generated using our standardized multi-segment reverse transcriptase polymerase chain reaction (M-RTPCR)now reads:15,16, Nextera, and MiSeq approach (Tables 2 and S3)”.“The read level accuracy was 86.2 ± 0.3%, and the consensus sequence was 98.97 ± 0.01% in concordance with consensus sequence generated using our standardized multi-segment reverse transcriptase polymerase chain reaction (M-RTPCR)These errors have now been corrected in the PDF and HTML versions of the paper.


Backbone 1H–15N correlations have been assigned for all non-proline residues, except for the first methionine residue.K-Ras is a key driver of oncogenesis, accounting for approximately 80% of Ras-driven human cancers. The small GTPase cycles between an inactive, GDP-bound and an active, GTP-bound state, regulated by guanine nucleotide exchange factors and GTPase activating proteins, respectively. Activated K-Ras regulates cell proliferation, differentiation and survival by signaling through several effector pathways, including Raf-MAPK. Oncogenic mutations that impair the GTPase activity of K-Ras result in a hyperactivated state, leading to uncontrolled cellular proliferation and tumorogenesis. A cysteine mutation at glycine 12 is commonly found in K-Ras associated cancers, and has become a recent focus for therapeutic intervention. We report here  Ras GTPases play a critical role in regulating pathways involved in cell growth, proliferation, differentiation and apoptosis .K-Ras contains a guanosine nucleotide-binding domain (G domain of ~ 20 kDa) at the N-terminus and a short hypervariable region at the C-terminus. Several regions within the G domain are particularly important for nucleotide exchange and downstream signalling, including the P-loop (residues 10–17) that binds nucleotide phosphate, as well as the Switch I (25–40) and Switch II (57–75) regions that interact directly with GEFs , encompassing residues 1–169, at pH 7.0. Under these solution conditions all the backbone amide signals were observed. Chemical shift differences in the 2D 1H–15N HSQC spectra of the GDP-bound forms of WT K-Ras  tag followed by the TEV protease cleavage site. Protein was expressed in E. coli BL21(DE3) cells (Novagen), and induced by IPTG at the optical cell density between 0.5 and 0.7. IPTG-induced cells were grown at 30 °C for 16 h. Proteins uniformly labeled with 15N or 13C/15N were prepared by growing E. coli cells in minimal media containing 15NH4Cl (1 g/l) or 13C6 glucose (2 g/l)/15NH4Cl (1 g/l)  supplemented with Bio-Express 1000 (CIL) using the identical protocol used for LB media. Harvested cells were suspended in Buffer A  supplemented with complete protease inhibitor cocktail tablet (Roche) and lysonase (Novagen) (10 μl/l cell culture), agitated 30 min at 4 °C, and sonicated  at 4 °C, 7 × 7 s cycles at 35% amplitude with 1 min on ice between cycles. Supernatant lysate was loaded onto pre-equilibrated (in Buffer A) Ni-NTA column (Qiagen). After 1 h of mixing the lysate, beads were washed 20 column volume (cv) in Buffer B . Protein was eluted in Buffer C . Ni-NTA purified protein was dialyzed in Buffer D  and digested with TEV protease at 4 °C overnight. Tagless protein incorporating a Ser residue at the N-terminus (due to cloning exigencies) was recovered by passing through a Ni-NTA column. Purified protein, as detected by Coomassie Blue Staining after SDS-PAGE, was a single band of ~ 19 kDa with apparent purity of > 96%. Protein was further purified by size exclusion chromatography on a Superdex 75 column  pre-equilibrated in buffer E . The GDP-bound state of the purified protein was ascertained by HPLC.DNA encoding residues 1–169 of K-Ras4B13C/15N- and 15N labeled K-RasG12C-GDP (0.5–0.6 mM) were prepared in a 93% H2O/7% D2O solvent composition containing 50 mM TRIS-d11, 1 mM TCEP-d16, 1 mM MgCl2, 100 µM 2,2-dimethyl-2-silapentanesulfonic acid (DSS) as internal standard, and 0.05% (w/v) NaN3 to avoid any unwanted bacterial growth over time. All NMR experiments were performed at 298 K on a Bruker Avance 800 MHz spectrometer equipped with a 5-mm TCI cryoprobe. NMR data were acquired in the gradient-selected sensitivity-enhanced mode. Backbone 1H, 15N, 13Cα, 13Cβ, and 13CO assignments were carried out using double and triple resonance experiments of 2D 1H–15N HSQC, 2D 1H–15N TROSY, HNCACB, HN(CO)CACB, HNCA, HN(CO)CA, CC(CO)NH, and HNCO . All NMR spectra were visualized and analyzed using CCPNMR analysis  with accession number 27387. Crosspeaks in the 2D 1H–15N HSQC spectrum demonstrates that the protein is well-folded in solution under the chosen condition  have been identified and assigned. The amide signal belonging to the first methionine residue could not be visualized presumably due to the exchange of 1HN protons with the bulk solvent. The chemical shifts of 13Cα, 13Cβ, and 13C′ for all residues (including the Met1 and the 4 proline residues) are assigned using the triple resonance data. Residues Gly13 and Lys16 show the most downfield-shifted 1HN chemical shifts, a pattern also seen for GDP-bound WT K-Ras   uses a construct which is 3 residues longer at C-terminus than WT K-Ras (1–166). Second, while the NMR assignment reported for WT K-Ras used a perdeuterated sample, non-deuterated NMR samples were used in this study to yield high-resolution NMR data and completion of unambiguous NMR assignments.The study presented herein has two experimental differences compared to the study of WT protein . Side-chain 13C assignments were achieved for 224 out of possible 269 aliphatic carbon resonances (including 95.9% of 13Cβ assignments). 100% of 13Cα, 98.8% of 13Cβ (crosspeaks not seen for Ser17 and Thr158), and 96.5% of 13CO resonances were assigned unambiguously. A chemical shift index (CSI) v 3.0 (Hafsa et al. 13Cα, 13Cβ, and 13C′ atoms suggest that the K-RASG12C-GDP conformation comprises a mixed distribution of α helix and β sheet secondary structures (Fig. 1–α1–β2–β3–α2–β4–α3–β5–α4–β6–α5, and match well with the secondary structure order observed in the GDP-bound crystal structure of K-Ras G12C (Hunter et al. In total, assignments were made for 95.5% of the backbone resonances, including G12C-GDP.In conclusion, we present the backbone NMR assignment of GDP-bound K-Ras harboring an oncogenic cysteine mutation at site 12. Efforts are underway to collect additional NMR data to complete all the side-chain resonances of KRas


Lactobacillus plantarum WCFS1 possesses eleven proteins included in GH13 family. Among these, proteins annotated as maltose-forming α-amylase (Lp_0179) and maltogenic α-amylase (Lp_2757) were included.α-Amylases specifically catalyse the hydrolysis of the internal α-1, 4-glucosidic linkages of starch. Glycoside hydrolase (GH) family 13 is the main α-amylase family in the carbohydrate-active database. L. plantarum α-amylases were structurally and biochemically characterized. Lp_2757 displayed structural features typical of GH13_20 subfamily which were absent in Lp_0179. Genes encoding Lp_0179 (Amy2) and Lp_2757 were cloned and overexpressed in Escherichia coli BL21(DE3). Purified proteins showed high hydrolytic activity on pNP-α-D-maltopyranoside, being the catalytic efficiency of Lp_0179 remarkably higher. In relation to the hydrolysis of starch-related carbohydrates, Lp_0179 only hydrolysed maltopentaose and dextrin, demonstrating that is an exotype glucan hydrolase. However, Lp_2757 was also able to hydrolyze cyclodextrins and other non-cyclic oligo- and polysaccharides, revealing a great preference towards α-1,4-linkages typical of maltogenic amylases.In this study, Lp_0179 and Lp_2757 The substrate range as well as the biochemical properties exhibited by Lp_2757 maltogenic α-amylase suggest that this enzyme could be a very promising enzyme for the hydrolysis of α-1,4 glycosidic linkages present in a broad number of starch-carbohydrates, as well as for the investigation of an hypothetical transglucosylation activity under appropriate reaction conditions. These enzymes are distinguished from typical α-amylases by containing a N-terminal domain and by exhibiting preferential substrate specificities for cyclomaltodextrins over starch, as well as high transglycosylation and hydrolytic activities. Concretely, these enzymes have shown to be capable of hydrolysing the potent inhibitor acarbose, and transfer the product to a sugar acceptor molecule . A greatd-Glc-(1 → 6)-α-d-Glc-(1 → 4)-d-Glc) was hydrolysed at a much lower extent and the polysaccharide dextran  was not hydrolysed at all . Escherichia coli DH10B and E. coli BL21 (DE3) were used as transformation and expression hosts in the pURI3-Cter vector [E. coli strains were cultured in Luria–Bertani (LB) medium at 37 °C and shaking at 140 rpm. When required, ampicillin was added to the medium at a concentration of 100 μg/mL.r vector . The E. L. plantarum WCFS1 were PCR-amplified by Advantage HD DNA polymerase (TaKaRa). Gene lp_0179 (amy2) of 1320 bp encoding a protein annotated as α-amylase, maltodextrins and cyclodextrins (UniProtKB code: F9USZ1) was amplified by using primers 1750 (5´-TAACTTTAAGAAGGAGATATACATtggcacgcgatacgcaaacgcaat) and 1751 (5´-GCTATTAATGATGATGATGATGatgattggactggtcagcaactttagt) . The gene lp_2757  encoding a protein annotated as maltogenic α-amylase (UniProtKB code: F9URM8) was amplified by using primers 1800 (5´-TAACTTTAAGAAGGAGATATACATatgcaactagctggaattaggcacc) and 1801 (GCTATTAATGATGATGATGATGATGagccacggttaattcaaagcctcc). Purified PCR products were inserted into the pURI3-Cter vector using a restriction enzyme- and ligation-free cloning strategy [E. coli DH10B cells were transformed, recombinant plasmids were isolated, and those containing the correct insert were identified by DNA sequencing, and then transformed into E. coli BL21 (DE3) cells for expression.Two genes encoding for putative α-amylases in strategy . This veEscherichia coli BL21(DE3) was transformed with pURI3-Cter-0179 and pURI3-Cter-2757 recombinant plasmids. E. coli cells were grown in LB medium containing 100 μg/mL ampicillin until an optical density at 600 nm of 0.4 was reached and then induced by adding isopropyl-β-d-thiogalactopyranoside (IPTG) at 0.4 mM final concentration. Following induction, the cells were grown at 22 °C for 18 h and collected by centrifugation . The cells were resuspended in phosphate buffer  containing 300 mM NaCl. Crude extracts were prepared by French press lysis of the cell suspension (three times at 1100 psi). The insoluble fraction of the lysate was removed by centrifugation at 47,000g for 40 min at 4 °C, and the supernatant was filtered through a 0.45 μm pore-size filter and then applied to a Talon Superflow resin (Clontech) equilibrated in phosphate buffer  containing 300 mM NaCl and 10 mM imidazole. The bound enzyme was eluted using 150 mM imidazole in the same buffer. The purity of the enzymes was determined by SDS-PAGE in Tris–glycine buffer. Fractions containing the His6-tagged Lp_0179 or Lp_2757 proteins were pooled and dialyzed against 50 mM sodium phosphate buffer, 300 mM NaCl, pH 7 at 4 °C using dialysis membranes  of 3.5 kDa pore diameter. Dialyzed proteins were analyzed for glycosyl hydrolase activity.p-nitrophenyl α-d-maltopyranoside (Sigma-Aldrich) as the substrate. The rate of hydrolysis of p-nitrophenyl (pNP) α-d-maltopyranoside for 10 min at 30 °C was measured in 50 mM MOPS buffer pH 7.0 containing 20 mM NaCl and 1 mM DTT at 420 nm in a microplate spectrophotometer PowerWave HT . The reaction was stopped by addition of sodium carbonate 1 M at pH 9.0. In order to carry out the reaction (75 µL), a stock solution of 25 mM of p-nitrophenyl-α-D-maltopyranoside was prepared in water and mixed with 50 mM MOPS buffer pH 7.0 containing 20 mM NaCl and 1 mM DTT to obtain a 1 mM substrate final concentration. Control reactions containing no enzyme were utilized to detect any spontaneous hydrolysis of the substrates tested. Enzyme assays were performed in triplicate.Hydrolase activity was determined by a spectrophotometric method using L. plantarum α-amylases was determined by using a library of 25 p-nitrophenyl glycoside derivatives: pNP-α-d-galactopyranoside, pNP-α-d-glucopyranoside, pNP-α-d-manopyranoside, pNP-α-d-xylopyranoside, pNP-α-L-arabinofuranoside, pNP-α-L-arabinopyranoside, pNP-α-L-fucopyranoside, pNP-α-L-rhamnopyranoside, pNP-β-d-fucopyranoside, pNP-β-d-galactopyranoside, pNP-6-P-β-d-galactopyranoside, pNP-β-d-glucopyranoside, pNP-6-P-β-d-glucopyranoside, pNP-β-d-glucuronide, pNP-β-d-maltoside, pNP-β-d-mannopyranoside, pNP-β-d-ribofuranoside, pNP-β-d-xylopyranoside, pNP-β-L-fucopyranoside, pNP-N-acetyl-α-d-glucosaminide, pNP-N-acetyl-β-d-glucosaminide, pNP-α-d-maltopentaoside, pNP-α-d-maltopyranoside, pNP-β-d-cellobiose and pNP-β-d-lactopyranoside. Most of them were purchased from Sigma-Aldrich ; pNP-6-P-β-d-galactopyranoside was purchased from BioGold , and pNP-6-P-β-d-glucopyranoside was synthesized by J. Cumella [p-nitrophenyl glycoside was prepared in water. The reaction mix consisted of 75 μL of mixed substrate and 4 μg of enzyme solution (in 50 mM MOPS buffer pH 7.0 containing 20 mM NaCl and 1 mM DTT). Reactions were carried out at 30 °C in a microplate spectrophotometer PowerWave HT  as described above.The substrate specificity of Lp_0179 and Lp_2757  Cumella . A stockpNP-glycoside library to colorimetrically monitor hydrolysis measuring the liberation of p-nitrophenolate at 420 nm. The screening was performed in a 96-well Flat Bottom plate (Sarstedt) where each well contains a different substrate (1 mM). Amylase solution 4 μg (in 50 mM MOPS buffer pH 7.0 containing 20 mM NaCl and 1 mM DTT) was added to each well and reactions were followed by measuring the increase in absorbance at 420 nm for 10 min at 30 °C in a Synergy HT BioTek microplate spectrophotometer. Blanks without enzyme were carried out for each substrate and data were collected in triplicate and the average activities were quantified. Results are shown as means ± standard deviations.The enzymatic substrate profile of purified α-amylases was determined on the d-panose and maltopentaose, all purchased from Sigma-Aldrich, . α-Amylases (20 µg) were incubated in 50 mM MOPS buffer pH 7.0 containing 20 mM NaCl, 1 mM DTT and 5% (w/v) of substrate with the exception of potato starch, amylose, amylopectin and pullulan that were added at 1% (w/v). The reactions were carried out at 30 °C for 24 or 48 h. GC-FID analyses were performed to determine the reaction products generated from the assayed substrates. The samples were analysed as trimethylsilylated oximes (TMSO) prepared following the method of Brobst and Lott (1966) [In addition, the hydrolytic activity of Lp_0179 and Lp_2757 was also assayed by using putative α-amylase substrates such as potato starch, amylose, amylopectin, pullulan, dextrin 20 (dextrin from maize starch), dextran 20 kDa, α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, acarbose, t (1966) . Brieflyt (1966) . The quapNP-α-d-maltopyranoside as substrate. In addition, the Lp_2757 kinetics on pNP-α-d-maltopentaoside were also determined. Kinetic values of Km and Vmax were determined by nonlinear regression analysis fitting to Michaelis–Menten curves of formation rates of pNP as a function of the concentration of substrates from 0 to 10 mM .The enzyme kinetics of Lp_0179 and Lp_2757 were studied using pNP-α-d-maltopyranoside hydrolytic activity of Lp_0179 and Lp_2757 were studied by using buffers of different pH ranging from 3 to 8. The buffers (50 mM) used were citrate buffer (pH 3), acetic acid-sodium acetate (pH 4–6), MOPS (pH 6.5 and 7) and Tris–HCl (pH 8). The optimal temperature was assayed by incubating purified α-amylases in 50 mM MOPS buffer pH 7.0 containing 20 mM NaCl, 1 mM DTT at different temperatures . For temperature stability measurements, recombinant α-amylases were incubated in 50 mM MOPS buffer pH 7.0 containing 20 mM NaCl, 1 mM DTT at 22, 30, 37, 55 and 65 °C for 30 min and 1, 2, 4, 6, and 20 h. Aliquots were withdrawn at these incubation times to test the remaining activity at standard conditions. The non-heated enzymes were considered as control (100%). The analyses were performed in triplicate.The effects of pH and temperature on pNP-α-d-maltopyranoside was added, and the reaction mixture was incubated 10 min at 30 °C. The residual hydrolytic activity was measured after the incubation of purified enzymes with each additive. The analyzed additives were, KCl, CaCl2, HgCl2, ZnCl2, CuCl2, NiCl2, FeCl2, MnCl2, Triton-X-100, Tween 20, Tween 80, SDS, urea, DMSO, cysteine, β-mercaptoethanol, PMSF, DEPC and EDTA. Hydrolytic activity measured in the absence of any additive was taken as control (100%). Experiments were done in triplicate.To study the effect of metals and ions on α-amylase activity, the enzymes were incubated in the presence of the different additives at a final concentration of 1 mM for 5 min at room temperature. Then, Additional file 1: Figure S1. Sequence alignment of the α-amylase representatives of thirteen different GH13 subfamilies with focus on the intermediary group with MPDLN as CSR V [Neisseria polysaccharea, accession Q9ZEU2), GH13_16 , GH13_17 , GH13_18 , GH13_20 , GH13_21 , GH13_23 , GH_29 , GH13_30 , GH13_31 , GH13_34 , GH13_35 , GH13_36  subfamilies and Lp_0179  are represented. Aligment was done using the program ClustalOmega. Residues that are identical (*), conserved (:) or semiconserved (.) in all sequences are indicated. Dashes indicated gaps introduced to maximize similarities. The GH13 three domains are highlighted in blue (domain A), green (domain B), and yellow (domain C). The catalytic triad is highlighted in pink. The seven GH13 CSRs are boxed. Conserved residues in amylolytic enzymes are highlighted in red [as CSR V . The seqAdditional file 2: Figure S2. Sequence alignment of the α-amylase family GH13_20 representatives from Alicyclobacillus acidocaldarius (AAC) (accession Q9WX32), Thermoactinomyces vulgaris (TVU) (Q08751), Klebsiella oxytoca (KOX) (Q48398), Bacillus sp. I-5 (BSP) (Q59226), Lysinibacillus sphaericus (LSP) (Q08341), Anoxybacillus flavithermus (AFL) (Q5BLZ6), Geobacillus caldoxylosilyticus (GCA) (CoLZ63), Thermus sp. IM6501 (O69007), Bacillus stearothermophilus (BST) (P38940), and Lactobacillus plantarum WCFS1 (LPL) (F9URM8). Aligment was done using the program ClustalOmega. Residues that are identical (*), conserved (:) or semiconserved (.) in all sequences are indicated. Dashes indicated gaps introduced to maximize similarities. The GH13 three domains are highlighted in blue (domain A), green (domain B), and yellow (domain C). The N-terminal domains are indicated in dark blue. The seven conserved sequence regions (CSR) found in GH13_20 subfamily α-amylases are also indicated. The residues of the GH13 catalytic triad  are highlighted in pink colour. Conserved residues in amylolytic enzymes are highlighted in red [SP) Q5922, LysinibAdditional file 3: Table S1. Concentrations (mg/mL) of the products after the enzymatic hydrolysis with Lp_2757.


H-indole-2-one ring of compound (II). There is also a significant difference in the conformation of the five-membered thiazolidine ring in the two compounds.The title compounds, (I) and (II), differ by the presence of a methyl group in position 5 on the 1 20H16N2O3S, (I), and C21H18N2O3S, (II), differ by the presence of a methyl group in position 5 on the 1H-indole-2-one ring of compound (II). The two compounds have a structural overlap r.m.s. deviation of 0.48 Å. There is a significant difference in the conformation of the thiazolidine ring: it has a twisted conformation on the fused N—C bond in (I), but an envelope conformation in compound (II) with the S atom as the flap. The planar pyrrolidine ring of the indole ring system is normal to the mean plane of the five-membered pyrrolidine ring of the pyrrolothiazole unit in both compounds, with dihedral angles of 88.71 (9) and 84.59 (8)°. The pyran rings in both structures have envelope conformations with the methylene C atom adjacent to the C=O group as the flap. In both compounds, there is a short intramolecular C—H⋯O contact present. In the crystal of (I), molecules are linked by C—H⋯O hydrogen bonds forming chains propagating along the b-axis direction. The chains are linked by N—H⋯π interactions, forming layers parallel to (10The title compounds, C In (II)q2 = 0.529 (2) Å and φ = 105.8 (1)°] with atom S1 as the flap, deviating by 0.896 (1) Å from the mean plane through the remaining four atoms.There is a significant difference in the conformation of the five-membered thiazolidine ring in the two compounds. In compound (I)q2 = 0.335 (2) Å and φ = 39.4 (1)°] with atom C9 as the flap, deviating by 0.518 (2) Å from the mean plane through the remaining four atoms. In (II)C2(C10) asymmetry parameter is 0.005 (1)].In compound (I)H-indol-2-one ring is planar in both compounds, with a maximum deviation of 0.054 (1) and 0.080 (1) Å from the mean plane for atom C14 in (I)The 2,3-dihydro-1Cs(C2) asymmetry parameters  in both structures have distorted sofa conformations, with ΔIn both compounds, the planar pyrrolidine ring (N2/C13–C15/C20) of the indole ring system is normal to the mean plane of the pyrrolidine ring (N1/C8–C10/C13) of the pyrrolothiazole unit, with a dihedral angle of 88.71 (9)° for (I)via two C—H⋯O intermolecular interactions ; see Fig. 4In the crystal of compound (I)via pairs of N—H⋯O hydrogen bonds  chains propagating along [010] in an anti-parallel manner. As a result of the various N—H⋯O and C—H⋯O hydrogen bonds, a three-dimensional structure is formed if Fig. 5. There ad Table 2et al., 2016S1 . They include: 2,4-dichloro-5′-methyl-6a,6b,7,8,9,11a-hexahydro-6H-spiropyrrolizine-11,3′-indole]-2′,6(1′H)-dione monohydrate -dione -dione . In these structures, the dihedral angle between the planar pyrrolidine ring of the indole ring system and the mean plane of the pyrrolidine ring of the pyrrolothiazole unit varies from 77.60° in 1′-phenyl-6′-thiacycloheptane-1-spiro-2′-perhydropyrrolizine-3′-spiro-3′′-indoline-2,2′′-dione -dione -one ethanol solvate -2-oxo-1,1′,2,6′,7′,7a′-hexahydrospirothiazole]-6′-carboxylate -1,1′,2,6′,7′,7a′-hexahydrospirothiazole]-6′-carboxylate monohydrate -one  and 1,3-thiazolane-4-carboxylic acid (1 mmol) dissolved in methanol (7 ml) was added. The irradiation was continued until the completion of the reaction (ca 50 min), during which time the product precipitated from the reaction mixture. It was then filtered and dried to obtain the pure product. The compound was further recrystallized from an ethanol–ethyl acetate mixture (1:1) to obtain colourless block-like crystals.Compound (I)ca 45 min), during which time the product precipitated from the reaction mixture. It was then filtered and dried to obtain the pure product. The compound was further recrystallized from ethyl acetate to obtain colourless block-like crystals.Compound (II)Uiso(H) = 1.5Ueq(C-methyl) and 1.2Ueq for other H atoms.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989019000045/su5467sup1.cifCrystal structure: contains datablock(s) I, II, global. DOI: 10.1107/S2056989019000045/su5467Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989019000045/su5467IIsup3.hklStructure factors: contains datablock(s) II. DOI: 10.1107/S2056989019000045/su5467sup4.pdfCSD search S1. DOI: 10.1107/S2056989019000045/su5467sup5.pdfCSD search S2. DOI: 1888373, 1888372CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


In the crystal, N—H⋯Br hydrogen bonds link the components into a three-dimensional network. Weak π–π stacking interactions between the phenyl rings of adjacent cations also contribute to the molecular packing. A Hirshfeld surface analysis was conducted to quantify the contributions of the different intermolecular interactions and contacts.In the cation of the title salt, C The biological activities of compounds containing a thiazolidine core, such as 1,3-thiazolidines, 2,4-dione-, 4-oxo-thiazolidine, etc. were summarized in a recent review As well as their synthetic utility, thiazolidine derivatives possess a broad spectrum of biological activities such as antimalarial, antibacterial, antimicrobial, anti-inflammatory, anticancer, Q(2) = 0.317 (2) Å and φ(2) = 225.2 (4)°: the flap atom is C1. In the arbitrarily chosen asymmetric unit, C1 has an R configuration, but symmetry generates a racemic mixture in the crystal. The dihedral angle between the mean plane of the thiazolidine ring  and the phenyl ring (C4–C9) is 89.27 (13)°.In the cation of (I)Cg2⋯Cg2iv = 3.7758 (16) Å; symmetry code: (iv) 1 − x, 1 − y, 2 − z; where Cg2 is the centroid of the phenyl ring of the cation]: chains of cations form along the [101] direction  shows the two-dimensional fingerprint of the sum of the contacts contributing to the Hirshfeld surface represented in normal mode while those delineated into H⋯H (41.5%), Br⋯N/N⋯Br (24.1%), C⋯H/H⋯C (13.8%) and S⋯H/H⋯S (11.7%) contacts, respectively, are shown in Fig. 3b–e. All contacts are listed in Table 2Hirshfeld surface analysis  contacts. In the other structures, the 3-N atom carries a C substituent: the first three crystal structures were determined for racemic : 0.179 (3). The system of hydrogen bonds connecting two cations and two anions into 12-membered rings is identical in the racemic and in the optically active crystals. YITCEJ  : 4.16 ; 4.45 ; 5.25 ; 7.32–7.50 ; 9.12 ; 9,78 . 13C NMR : 44.42, 62.06, 127.59, 128.76, 129.17, 138.85, 168.53. MS (ESI), m/z: 194.28 [C9H12N3S]+ and 79.88 Br−.To a solution of 2.2 mmol (0.6 g) benzene in 20 ml of ethanol were added 2.3 mmol (0.3 g) of thiosemicarbazide hydrochloride; 3-4 drops of piperidine were added and the mixture was refluxed for 7 h. The reaction mixture was cooled to room temperature and the solid product was precipitated from solution, collected by filtration and recrystallized from ethanol solution to give colourless crystals of (I)Uiso(H) = 1.2Ueq(carrier) was applied in all cases. One outlier  I. DOI: 10.1107/S2056989019013069/hb7855Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989019013069/hb7855sup3.docxHirshfeld surface analysis figures. DOI: Click here for additional data file.10.1107/S2056989019013069/hb7855Isup4.cmlSupporting information file. DOI: 1955268CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Research over the past decade has indicated that melanocortin peptides are potent inhibitors of inflammation and a promising source of new anti-inflammatory and cytoprotective therapies. The purpose of the present paper is to compare protective effects of α-, β-, and γ-melanocyte stimulating hormone on acetaminophen induced liver lesions in male CBA mice. Acetaminophen was applied intragastrically in a dose of 150 mg/kg, and tested substances were applied intraperitoneally 1 hour before acetaminophen. Mice were sacrificed after 24 hours and intensity of liver injury was estimated by measurement of plasma transaminase activity (AST and ALT) and histopathological grading of lesions. It was found that α-, β-, and γ-MSH decrease intensity of lesions by both criteria in a dose-dependent manner.  Acute and chronic inflammatory disorders of the liver are among the greatest challenges of hepatology . Despitescreening procedure for potential hepatoprotective drugs. Beneficial effects of α-MSH have been observed in models of liver fibrosis and necroinflammation [via its reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI) [Melanocortins exert their effects by activating melanocortin receptors, leading to adenyl cyclase activation and subsequent increase of intracellular cAMP concentration ,4. This ammation ,14,15,16 (NAPQI) . Covalen (NAPQI) . The los (NAPQI) . The mos (NAPQI) . Also, t (NAPQI) . The fin (NAPQI) . The effects of α-, β-, and γ-MSH in APAP-mediated liver injury were estimated by measurement of plasma transaminase activity and histopathological grading of liver lesions ,21,22,23The results of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activity in plasma 24 hours after APAP administration suggest that all tested substances  have protective effects .-6 mol/kg, for β-MSH 1 × 10-7 mol/kg, and 1 × 10-7 mol/kg for γ-MSH. The increase in dose was followed by diminishing of protective effects. This finding is usually explained as a result of non-specific binding of tested substance to other receptors and molecules.It must be observed that the protective effect of tested substances has the U-shaped dose-response, a common finding in the field of peptide research . The optet al. [Histopathological analysis of liver lesions is considered as the gold standard when defining protective or toxical effects . Two scaet al. . The secet al. ,22. Resu-6 mol/kg, for β-MSH 1 × 10-7 mol/kg, and 1 × 10-7 for γ-MSH. This data confirm the hepatoprotective effects of α-MSH observed in previously published papers [The optimal protective dose of α-MSH was 1.5 × 10d papers ,14,15,16in vivo tests determine whether a compound has the desired physiological effects [In vivo order of β-MSH and γ-MSH potencies may be modified by several confounding factors including differences in metabolic degradation, and action via non-MC receptor mechanisms [α-MSH is the principal agonist with high affinity for all melanocortins receptors (except MC-2R) ,4. Affin effects . We obse effects ,27. In vchanisms . MC-3R ichanisms , but itsMale CBA mice, bred at Ruđer Bošković Institute, aged 12–16 weeks, were used in the experiment. They were maintained under standard laboratory conditions, with free access to water and commercially available murine food pellets Pure acetaminophen (APAP) from the Krka pharmaceutical company  was used. APAP was dissolved in a warm saline (37 °C) under mild magnetic stirring.2, GenScript, USA, purity > 95%) was used in five doses: 6 × 10-8 mol/kg (0.1 mg/kg), 3 × 10-7 mol/kg (0.5 mg/kg), 6 × 10-7 mol/kg (1 mg/kg), 1.5 × 10-6 mol/kg (2.5 mg/kg) and 3 × 10-6 mol/kg (5 mg/kg).α-MSH (Ac-SYSMEHFRWGKPV-NH-8 mol/kg (0.125 mg/kg), 1 × 10-7 mol/kg (0.25 mg/kg), 2 × 10-7 mol/kg (0.5 mg/kg) and 4 × 10-7 mol/kg (1 mg/kg).β-MSH  was used in four doses: 5 × 101-MSH  was used in four doses: 5 × 10-8 mol/kg (0.075 mg/kg), 1 × 10-7 mol/kg (0.15 mg/kg), 2 × 10-7 mol/kg (0.3 mg/kg) and 4 × 10-7 mol/kg (0.6 mg/kg).γTested substances were dissolved in a warm (37 °C) saline solution.et al., with slight modifications [Hepatitis was induced following the procedure described by Guarner ications ,20,23. Tications ,20,23. TMice were sacrificed by decapitation 24 hours after APAP application. Heparin (250 U) was given intraperitoneally (i.p.) to each animal 15 minutes before sacrifice, and trunk blood was collected into heparinized tubes. Plasma was separated by centrifugation for 5 min at 8,000 g, and was stored at -20 °C for 24 h before transaminase activity determination. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity was determined by standard laboratory techniques. High standard deviations observed in the measurements of liver enzymes are typical for the experimental model of acetaminophen induced hepatotoxicity in mice, even in highly inbred animals ,30. DiffSections of the liver were fixed in 10% phosphate buffered formalin, embedded in paraffin, sectioned at 4 µm, and stained with hematoxilin and eosin (H&E). Sections were examined by using light microscope at magnification ×100. Two scales were used to grade presence and intensity of lesions ,22,23. 0.no lesions1.minimal lesions 2.mild lesions (10–25% necrotic cells or mild diffuse degenerative lesions)3.moderate lesions (25–40% necrotic or degenerative cells)4.marked lesions (40–50% necrotic or degenerative cells)5.severe lesions (more than 50% necrotic or degenerative cells)The first scale is based on grades from 0–5:The second scale is based on the first scale, but simply considers sections with scores ≥3 as significant liver lesions .P-values were ≤ 0.05 [Plasma AST and ALT activity, as well as grade of lesions, are expressed as means, medians, and standard deviations. Difference between control and treated groups was determined using Steel's test. Difference between the groups in number of animals with histopathology score ≥ 3 was tested by using Fisher exact probability test. All applied tests were two-tailed. The results were considered significant if e ≤ 0.05 . Statiste ≤ 0.05 , and dose ≤ 0.05 .Collected results of transaminase activity and histopathological grading suggest that α-, β, and γ-melanocyte stimulating hormones prevent APAP-induced liver lesions. The hepatoprotective effect of α-MSH observed in previously published papers was confirmed ,14,15,16


The thiazine ring is in a conformation that is between half-chair and envelope [θ = 52.51 (17)°]. The phenyl ring on the 2-carbon atom of the thiazine ring is pseudo-axial. The central phenyl ring of the molecule is close to orthogonal to the phenyl rings on either side with an angle of 76.85 (11)° between those planes. In the crystal, pairwise, weak C—H⋯O hydrogen bonds between the central phenyl ring and the oxygen atoms of neighboring molecules result in continuous strips propagating along the a-axis direction. Hydrophobic interactions of the C—H⋯π type are also observed.The crystal structure of the title compound –  There is a report of 3,3′-bis(2-(4-methylphenyl)-2,3,5,6-tetrahydro-4H-1,3-thiazin-4-one), but the data supporting the assigned structure are questionable . In previous work, we have reported the synthesis and crystal structures of several mono-heterocyclic 2,3-diaryl-2,3,5,6-tetrahydro-4H-1,3-thiazin-4-ones bis, (I)meso structure, i.e. the stereocenters have opposite configurations.Bis-heterocyclic compounds are of interest because of their potential biological activity  up Fig. 1. The thia-axis direction  and . In addition, a C—H⋯π interaction [C⋯π-ring = 3.457 (3) Å] between the carbon atom of the thiazine ring and the 2-phenyl ring is observed.A very weak C—H⋯O hydrogen bond between the central phenyl ring and the oxygen atom of the neighboring molecule is detailed in Table 1on Fig. 3, definedH-1,3-thiazin-4-one bis: A two-necked 25-ml round-bottom flask was oven-dried, cooled under N2, and charged with a stir bar, N,N′-bis(1-phenylmethanimine)  and 3-mercaptopropionic acid . 2-Methyltetrahydrofuran (2.3 ml) was added and the solution was stirred. Pyridine  and finally, 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphorinane-2,4,6-trioxide (T3P) in 2-methyltetrahydrofuran  were added. The reaction was stirred at room temperature and followed by TLC (80% ethyl acetate/hexanes). The mixture was poured into a separatory funnel with dichloromethane and distilled water. The layers were separated and the aqueous layer was then extracted twice with dichloromethane. The organics were combined and washed with saturated sodium bicarbonate and then saturated sodium chloride. The organic was dried over sodium sulfate and concentrated under vacuum to give crude product. The crude was recrystallized from CH2Cl2/acetone solution to give white powder. Yield: 0.3108 g 1st crop, 0.0318 g 2nd crop , m.p. 523 K (decomp.). Crystals suitable for X-ray diffraction studies were grown by slow evaporation from CH2Cl2/acetone.Uiso(H) = 1.2Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989018013397/hb7772sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989018013397/hb7772Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989018013397/hb7772Isup3.molSupporting information file. DOI: 1868690CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Gynura bicolor aqueous extract (GAE) at three concentrations upon nerve growth factor (NGF) differentiated-PC12 cells against H2O2 induced injury were examined.Background: Protective effects of 2O2 was used to treat cells with GAE pre-treatments. After incubating at 37 °C for 12 hr, experimental analyses were processed.Methods: NGF differentiated-PC12 cells were treated with GAE at 0.25%, 0.5% or 1%. 100 μM H2O2 exposure decreased cell viability, increased plasma membrane damage, suppressed Bcl-2 mRNA expression and enhanced Bax mRNA expression. GAE pre-treatments reversed these changes. H2O2 exposure reduced mitochondrial membrane potential, lowered Na+-K+-ATPase activity, and increased DNA fragmentation and Ca2+ release. GAE pre-treatments attenuated these alterations. H2O2 stimulated the production of reactive oxygen species (ROS), interleukin (IL)-1beta, IL-6 and tumor necrosis factor-alpha, lowered glutathione content, and reduced glutathione peroxidase (GPX) and catalase activities. GAE pretreatments maintained GPX and catalase activities; and concentration-dependently diminished the generation of ROS and inflammatory cytokines. H2O2 enhanced mRNA expression of nuclear factor kappa (NF-κ) B and p38. GAE pre-treatments decreased mRNA expression of NF-κB and p38. Conclusion: These findings suggested that GAE might be a potent neuronal protective agent.Results: H The oveGynura bicolor DC. (G. bicolor) is a plant food, and available in several Asian countries such as China, Taiwan, Japan and Malaysia. Its leaf part is an edible vegetable. G. bicolor has been applied in folk medicine for diabetes treatment in China southern area  = Kd × [(R–Rmin)/(Rmax–R)] × FD/FS. Kd was 224 nM, R was the ratio of fluorescence values at 340 and 380, Rmax was measured by using triton X-100 to treat cells, Rmin was measured by using ethylene glycol tetraacetic acid to treat cells. FD was the fluorescence value of Ca2+-free form, and FS was the fluorescence value of Ca2+-bound form at 380 and 340 nm.A Cantensity . In brie2.7.ml DCFH-DA. After incubating at 37 °C for 30 min, fluorescence value was recorded by a Gemini XS fluorescence plate reader . Emission and excitation wavelengths were 525 nm and 488 nm, respectively. Result was expressed as relative fluorescence unit (RFU) per mg protein. The level of glutathione (GSH), and the activity of glutathione peroxidase (GPX) or catalase were measured via assay kits purchased from OxisResearch Co.  according to manufacturer’s instructions. The levels (pg/mg protein) of IL-1beta, IL-6 and TNF-alpha were quantified by cytoscreen assay kits obtained from BioSource International Co. . The detection limit was 5 pg/mg protein.ROS level was determined by 2’,7’-Dichlorofluorescein diacetate (DCFH-DA). In brief, 100 μL cell homogenate was mixed with 100 μL 2 mg/2.8.via reverse-transcription procedure. Then, cDNA was further used for PCR process. The primers of target genes were as follow: Bcl-2: forward, 5’-CGT TTG GCA GTG CAA TGG T-3’, reverse, 5’-TTC TTG ATT GAG CGA GCC TT-3’; Bax: forward, 5’-TGG CAG CTG ACA TGT TTT CTG AC-3’, reverse, 5’-TCA CCC AAC CAC CCT GGT CTT-3’; NF-κB, forward, 5’-GAG GTC TCT GGG GGT ACA GTC-3’, reverse, 5’-GGA CAA CGC AGT GGA ATT TTA-3’; p38: forward, 5’-TCC AAG GGC TAC ACC AAA TC-3’, reverse, 5’-TGT TCC AGG TAA GGG TGA GC-3’; glyceraldehyde-3-phosphate dehydrogenase : forward, 5’-AGA GGC AGG GAT GTT CTG-3’, reverse, 5’-GAC TCA TGA CCA CAG TCC ATG C-3’. PCR amplification condition was 3 min denaturation at 95 °C, 10 s annealing at 60 °C and 20 s extension at 72 °C. For Bcl-2, Bax, NF-κB or p38, 35 cycles were performed; for GADPH, 28 cycles were processed. A Sequence Detection System  was used to quantify PCR products.Total mRNA of cells was extracted by reagents obtained from Invitrogen Trizol . RNA concentration was quantified by monitoring the absorbance at 260 nm. Subsequently, 5 μg RNA was applied for generating cDNA 2.9.t-test was applied for Post-hoc comparison. P value lower than 0.05 was defined as significant.Data were obtained from 7 different preparations, and expressed as mean ± standard deviation (SD) (n = 7). Statistical analyses were processed by using one-way analysis of variance. Dunnett’s 3.3.1.2O2 stimulation, GAE at test concentrations did not affect viability and plasma membrane stability in NGF differentiated-PC12 cells (P < 0.05). As shown in 2O2 exposure decreased cell viability (2a) and increased plasma membrane damage (2b), determined by LDH activity, in NGF differentiated-PC12 cells when compared with normal groups (P < 0.05). GAE pre-treatments concentration-dependently increased cell viability and diminished LDH activity (P < 0.05). H2O2 reduced Bcl-2 mRNA expression and enhanced Bax mRNA expression in NGF differentiated-PC12 cells (P < 0.05). GAE pre-treatments at test concentrations raised Bcl-2 mRNA expression (P < 0.05); and concentration-dependently lowered Bax mRNA expression (P < 0.05).Without H3.2.2O2 exposure reduced MMP, increased DNA fragmentation and Ca2+ release in NGF differentiated-PC12 cells (P < 0.05). GAE pre-treatments reversed these changes (P < 0.05), in which concentration-dependent effects were presented in increasing MMP and reducing DNA fragmentation (P < 0.05). H2O2 exposure enhanced caspase-3 and caspase-8 activities; and lowered Na+-K+-ATPase activity in NGF differentiated-PC12 cells (+-K+-ATPase activity (P < 0.05). However, GAE pre-treatment only at 1% reduced caspase-8 activity (P < 0.05).As presented in 3.3.2O2 stimulated ROS generation, decreased GSH content, and reduced GPX and catalase activities in NGF differentiated-PC12 cells (P < 0.05). GAE pre-treatments reversed GSH content and maintained GPX activity (P < 0.05). GAE pre-treatments concentration-dependently lowered ROS level and raised catalase activity (P < 0.05). As presented in 2O2 stimulated the release of inflammatory cytokines, IL-1beta, IL-6 and TNF-alpha in NGF differentiated-PC12 cells (P < 0.05). GAE pre-treatments decreased the production of these inflammatory cytokines (P < 0.05), and concentration-dependent manner was found in lowering TNF-alpha generation (P < 0.05). H2O2 up-regulated mRNA expression of NF-κB and p38 in NGF differentiated-PC12 cells (P < 0.05). GAE pre-treatments at test concentrations down-regulated NF-κB mRNA expression (P < 0.05), and GAE pre-treatments at 0.5% and 1% suppressed p38 mRNA expression (P < 0.05).As presented in 4.2O2 stimulation, GAE treatments at test concentrations did not affect viability and plasma membrane integrity of NGF differentiated-PC12 cells. These findings implied that GAE might not have adverse impact for neuronal cells. Our previous animal study reported that dietary GAE intake at 0.5% markedly attenuated ethanol-induced hepatic glycative damage and lipid accumulation . Our data revealed that GAE pre-treatments markedly overcame the impact from H2O2 and reversed these alterations. These findings indicated that GAE could benefit DNA stability and maintain the integrity of plasma membranes, which consequently favored cell survival. In addition, GAE pre-treatments diminished H2O2 induced Ca2+ release in NGF differentiated-PC12 cells. The less Ca2+ release observed in GAE treated NGF differentiated-PC12 cells could be partially ascribed to the improvement from GAE upon plasma membrane integrity. It is reported that released Ca2+ facilitates nerve impulse transmission and stimulates neuronal excitability, which might promote the development and progression of seizure [2+ release. Further study regarding the antiseizure effect of GAE is worthy to be investigated.Bcl-2 is an anti-apoptotic molecule, and Bax is a pro-apoptotic molecule . Both Bc seizure , 21. Our+-K+-ATPase, a transmembrane protein, is in charge of intracellular Na+ exchange for extracellular K+. The loss of MMP caused the reduction in Na+-K+-ATPase activity, which subsequently impaired ion homeostasis and promoted apoptotic insult [et al. [+-K+-ATPase activity contributed to neuronal swelling and even brain edema. In our present study, H2O2 exposure disturbed mitochondrial membrane, which was evidenced by greater caspase-3 and caspase-8 activities, as well as lower Na+-K+-ATPase activity. However, GAE pre-treatments attenuated mitochondrial membrane injury caused by H2O2. One possibility was that GAE enhanced the defensive capability of mitochondrial membrane against H2O2, which consequently diminished the impact from H2O2 upon caspase-3, caspase-8 and Na+-K+-ATPase. The other possibility was that GAE directly affected caspases and Na+-K+- ATPase activities, which finally mitigated apoptotic stress and benefited Na+/K+ ion homeostasis. These data once again suggest that GAE could maintain mitochondrial membrane stability and alleviated apoptotic stress in H2O2-treated NGF differentiated- PC12 cells.Collapse of MMP activates apoptotic executors such as caspase-3 and caspase-8 . The raic insult . Unterbe [et al.  indicateIt is reported that GAE contained many phytochemicals with anti-oxidative and anti-inflammatory activities such as ferulic acid, chlorogenic acid, quercetin and apigenin . Actuall2O2 stimulation facilitated the generation of oxidants and inflammatory factors such as ROS and TNF-alpha [2O2 was a promotor responsible for neuronal cell oxidative and inflammatory injury. However, our findings indicated that GAE pre-treatments limited the mRNA expression of NF-κB and p38. It is highly possible that the pre-treatments of GAE led to some phytochemical components penetrate plasma membrane of NGF differentiated-PC12 cells, where these component compounds exert their protective actions against subsequent H2O2 assault. Since these signaling pathways have been suppressed, the lower production of downstream factors such as ROS and inflammatory cytokines could be explained. These finding also suggest that GAE was able to protect NGF differentiated-PC12 cells at molecular levels.We believe that the mitigated oxidative and inflammatory stress also contributed to stabilize DNA and mitochondrial membrane integrity in GAE treated NGF differentiated-PC12 cells, which in turn improved cell survival. As observed by others, the activation of NF-κB and p38 signaling pathways due to HNF-alpha , 29. OurG. bicolor is a vegetable. Its aqueous extract is easily prepared and should be safe. Moreover, our previous animal study reported that dietary GAE protected liver against ethanol induced injury [d injury . This an2O2 through maintaining mitochondrial membrane potential, decreasing oxidative and inflammatory injury, and regulating the mRNA expression of Bcl-2, Bax, NF-κB and p38. These findings suggested that this aqueous extract might possess neuronal protective potent.In conclusion, aqueous extract of G. bicolor leaf part enhanced NGF differentiated-PC12 cells survival against H