Tumor necrosis factor-α (TNF-α), one of potent inflammatory cytokines, plays a dominant role in host defense reaction. However, the concrete effect of TNF-α on acute liver injury is totally unclear. Here we reported the concrete effect and possible mechanisms of TNF-α on acute liver injury induced by carbon tetrachloride (CCl4 (1 ml/kg) was subcutaneously injected into the rats. Enbrel, a TNF-α inhibitor, were intraperitoneally injected at dose of 0, 0.25, 0.5, 1, 2, 4 or 8 mg/kg 15 min before the CCl4 injection. 24 h later, rats were sacrificed. Serum ALT and AST were measured with an autoanalyzer. Serum TNF-α were measured by ELISA. HE staining was used to observe the liver tissue morphology. Hepatocellular apoptosis were tested by immunochemistry and Tunnel kit. Inflammatory factors, involve IL-4, IL-6, IL-8, IL-β and IFN-γ were detected by RT-PCR. The NF-κB signal pathway and anti-apoptotic genes include Bcl-XL, FHC, XIAP and Bcl-2 were measured by western-blotting and RT-PCR.SD male rats were equally divided into nine groups. CClThe change of liver function presented an obvious “V” shape in the whole process of persistently increased Enbrel. As Enbrel was increased gradually from 0 to 1 mg/kg, serum TNF-α were blocked, ALT and AST were gradually decreased as TNF-α as well as the numbers of hepatocellular apoptosis, and were declined to the minimum at 1 mg/kg Enbrel. As Enbrel was increased gradually from 1 to 8 mg/kg, ALT, AST and hepatocellular apoptosis were increased instead, and reached to the maximum at 8 mg/kg Enbrel. HE showed that the seriousness of hepatocellular steatosis was the most at 8 mg/kg Enbrel, and second at 0 mg/kg, the weakest at 1 mg/kg in the acute liver injury. Western-blotting and RT-PCR showed NF-κB, p-IκBα and antiapoptotic genes include Bcl-XL, FHC, XIAP, Bcl-2 were decreased as TNF-α was blocked by increased Enbrel.Our results suggested that TNF-α had a dual role in acute liver injury. It was regulated might via the corporate effect of NF-κB signal pawahway and anti-apoptosis. Meanwhile, our findings provide a reference for clinical treatment of acute liver injury.The online version of this article (doi:10.1186/s13578-016-0074-x) contains supplementary material, which is available to authorized users. It is a vulnerable organ as various noxious agents can be assimilated by intestine and transported into liver, inducing inflammation, necrosis, fibrosis, cirrhosis  and evenabolites , 11. It However, in the pathological process of liver injury, numerous inflammatory cytokines are significantly upregulated, such as tumor necrosis factor-α (TNF-α), which mediate inflammation and repair in physiological conditions . Tumor n4-induced acute liver injury remains largely unknown. Our present study is designed to determine the concrete effect of TNF-α on acute liver injured model induced by CCl4 and possible mechanisms associated with it.Despite current knowledge of TNF-α, litter is reported the concrete role of TNF-α in acute liver injury. Moreover, the influence of different concentration of TNF-α mediated by Enbrel (Etanercept), which is a genetically engineered fusion protein that can bind and inactivate TNF-α , on CCl44 administered. Serum alanine aminotransferase (ALT) and aspartate transaminase (AST) were significantly elevated after subcutaneous injection of CCl4. The ALT and AST levels were all decreased pre-treated with Enbrel of 0.25, 0.5 and 1 mg/kg when the serum TNF-α was slightly blocked  and NF-κB-p65 production were activated in CCl4 model. But they were inhibited as pretreatment with Enbrel, while the expression of IκBα had an opposite result .4 (1 ml/kg) was subcutaneously injected into Sprague–Dawley rats. Enbrel at dose of 0, 0.25, 0.5, 1, 2, 4 or 8 mg/kg was intraperitoneally administered to the CCl4-injected rats 15 min before the CCl4 injection. 24 h later, all the animals were weighted, sacrificed, collected the blood while livers were removed, weighted and perfuse in ice-cold saline solution. Liver samples were treated with liquid nitrogen and stored at −80 °C for further studies.100 six weeks old Sprague–Dawley male rats (200–210 g) were purchased from Shanghai Experimental Animal Center of the Chinese Academy of Sciences, Shanghai, China. They were kept in ordinary cages at room temperature of 25 ± 3 °C with a 12 h dark/light cycles, and have free access to standard laboratory feed and water. To study the effects of TNF-α in acute injured rats, they were equally divided into nine groups (10 rats per group). CClSerum activities of ALT and aspartate aminotransferase (AST) were measured with an autoanalyzer . Serum levels of TNF-α in rats were measured by enzyme-linked immunosorbent assay (ELISA) using the Immunotech Human TNF-α ELISA kit . The results are expressed as U/L.Liver tissues for histopathological examination were fixed with 10 % neutral buffered formalin, processed and trimmed, embedded in paraffin, sectioned to a thickness of approximately 5 μm, liver sections were stained with HE following standard protocol, then it was performed for light microscopic examination. TUNEL assay was performed using the In Situ Cell Death Detection kit .Quantitatively measure the expression of cleaved caspase-3 by immunohistochemical staining. Liver tissues were fixed in Carnoy, embedded in paraffin, and sectioned at 5 μm. The sections of tissues were dewaxed and rehydrated with freshly distilled water. Samples underwent inactivation of endogenous peroxidase for 10 min and were washed with distilled water for 5 min (three times), then immersed in 0.1 mol/L citrate buffer solution (pH = 6.0) and heated in a microwave oven until boiling . After washed with phosphate buffered saline, antigen-retrieval buffers were added for 10 min and tissues were then flushed three times with PBS. At room temperature, samples were non-specifically blocked with normal goat serum for 20 min. Sections were incubated overnight at 4 °C with rabbit anti-human cleaved caspase-3 antibody. Sections were washed in PBS and incubated at room temperature for 10 min with biotinylated goat anti-rabbit IgG. After washing with PBS, sections were incubated with streptavidin-peroxidase for 10 min and then stained with diaminobenzidine for 10 min and washed for 5 min. Samples were stained with hematoxylin, and were dehydrated and mounted for microscopic examination. Images of the sections were obtained using the Image-Pro Plus 4.5 software  with brown staining under light microscopy indicating a positive reaction of cleaved caspase-3.Total soluble proteins extraction from liver tissue and western-blot analysis were performed as described. Antibodies used in western-blot experiments were specific for cleaved caspase-3, IκBα (inhibitor of NF-kappa B-α), p-IκBα (Phospho-IκBα), BCL-2 (B cell leukemia/lymphoma 2), BCL-XL (B-cell lymphoma-extra large), XIAP (X-linked Inhibitor of Apoptosis Protein), FHC (Ferritin heavy chain), β-actin and goat anti-rabbit secondary antibody were obtained from Cell Signaling Technology. NFκB-p65 antibody was obtained from Abcam.The transcript levels of FHC, Bcl-XL, Bcl-2, XIAP, TNF-α, IL-1β, IFN-, IL-6, IL-8 and IL-4 in liver tissues of rats were measured using RT-PCR technique. Total RNA was isolated using the TriPure reagent (Roche Diagnostics), RNA concentrations and purity were determined by measuring the absorbance A260–A280 nm ratio. The relative quantities of mRNAs were obtained by using the comparative Ct method and were normalized with glyceraldehydes-3-phosphate dehydrogenase (GAPDH). The primer sequences were as following: FHC: Forward primer 5′-CCACGTGACCAACTTACGC-3′, Reverse primer 5′-AGTCAGCTTATCTCTCATCACCG-3′; Bcl-XL: Forward primer 5′-TCCCTTCAGAACCTTATCTTGG-3′, Reverse primer 5′-TCCCGGAAGAGTTCATTCAC-3′; Bcl-2: Forward primer 5′-ACAGCCAGGAGAAATCAAA-3′, Reverse primer 5′-GTCGCTACCGTCGTGACTTC-3′; XIAP: Forward primer 5′-CTGCATTGCATTCCATTAGC-3′, Reverse primer 5′-TGCTTCTCTGTCTAAGGTTTCAA-3′; TNF-α: Forward primer 5′-TTCTGTCTACTGAACTTGGGGGTGATCGGTCC-3′, Reverse primer 5′-GTATGAGATAGCAAATCGGCTGACGGTGTGGG-3′; IL-1β: Forward primer 5′-GGCTGCTTCCAAACCTTTGA-3′, Reverse primer 5′-GAAGACACGGATTCCATGGT-3′; IL-6: Forward primer 5′-ATGAAGTTCCTCTCTGCAAGAGAC-3′, Reverse primer 5′-CACTAGGTTTGCCGAGTAGATCTC-3′; IL-8: Forward primer 5′-AATTCTCGAGTCGCGAATGGCTGCTCAAGGCTG-3′, Reverse primer 5′-ATTACGGCCGTCGCGATTAGGCATCACTGCCTG-3′.All data are presented as mean ± SEM. Groups were compared by analysis of variance (ANOVA) with a posteriori contrast by least significant difference. For all analyses, p < 0.05 was considered statistically significant.


In the crystal of this dichlorinated 3-formylchromone derivative, molecules are linked through stacking interactions, C—H⋯O hydrogen bonds and short C⋯O contacts. Halogen bonds between the formyl O and Cl atoms and type II halogen–halogen contacts between the Cl atoms are also formed. 10H4Cl2O3, a dichlorinated 3-formylchromone derivative, the fused-ring system is slightly puckered [dihedral angle between the benzene and pyran rings = 3.66 (10)°]. The dihedral angle between the pyran ring and the formyl plane is 8.64 (7)°. In the crystal, molecules are linked through π–π stacking interactions [centroid–centroid distance between the benzene and pyran rings = 3.727 (2) Å], C—H⋯O hydrogen bonds and short C⋯O contacts [2.838 (4) Å]. Halogen bonds between the formyl O atoms and the Cl atoms at the 7-position  are also formed along the a axis, resulting in helical structures constructed by C—H⋯O hydrogen bonds and Cl⋯O halogen bonds along the b axis. In addition, type II halogen–halogen contacts between the chlorine atoms at the 7- and 8-positions  are observed.In the title compound, C The type I contact is considered to be van der Waals, and the type II is halogen bonding, i.e., an electrostatic interaction , and a van der Waals contact is observed between the formyl oxygen atom and the chlorine atom in 8-chloro-4-oxo-4H-chromene-3-carbaldehyde . On the other hand, as for the dichlorinated 3-formylchromone, halogen bonding between the formyl oxygen atom and the chlorine atom at the 8-position and a type I short halogen–halogen contact between the chlorine atoms at the 6-position are observed in 6,8-dichloro-4-oxochromene-3-carbaldehyde . As part of our investigation into these types of chemical bonding, I herein report the crystal structure of a dichlorinated 3-formylchromone, 7,8-dichloro-4-oxo-4H-chromene-3-carbaldehyde. The main objective of this study is to reveal the interaction modes of the chlorine substituents of the title compound in the solid state.I have recently reported the crystal structures of chlorinated 3-formylchromone derivatives 7-chloro-4-oxo-4de Fig. 1a, and ade Fig. 1b. On thde Fig. 1c. As paThe molecular structure of the title compound is shown in Fig. 2i , and through C—H⋯O hydrogen bonds that involve C1/O2 and C4/O3 atoms, as shown in Fig. 3In the crystal, the molecules are linked through π–π stacking interactions between molecules related by translation-symmetryii along the a-axis direction , resulting in helical structures constructed by C—H⋯O hydrogen bonds and Cl⋯O halogen bonds along the b-axis direction, as shown in Figs. 3iii . This interesting feature is possibly caused by a dipole–dipole interaction between the O atom and the C—H group that is enhanced by the polarizing effect of the two chlorine atoms at the 7- and 8-positions of the chromone ring. These observations should be helpful in understanding interactions of halogenated ligands with proteins, and thus valuable for rational drug design.In addition to the C—H⋯O hydrogen bonds, halogen bonds and type II halogen–halogen contacts, an unusually short contact is revealed between the α,β-unsaturated carbonyl O2 and the C—H group of C1N,N-dimethylformamide (20 ml) was added dropwise POCl3 (11.7 mmol) at 273 K. After the mixture had been stirred for 14 h at room temperature, water (100 ml) was added. The precipitates were collected, washed with water, and dried in vacuo (yield: 64%). 1H NMR : δ = 7.82 , 8.08 , 9.05 , 10.10 . Single crystals suitable for X-ray diffraction were obtained by slow evaporation of a tetrahydrofuran solution of the title compound at room temperature.3′,4′-Dichloro-2′-hydroxyacetophenone was prepared from 2,3-dichlorophenol by a Fries rearrangement reaction. To a solution of 3′,4′-dichloro-2′-hydroxyacetophenone (5.9 mmol) in Uiso(H) = 1.2Ueq(C)].Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S205698901501275X/zl2631sup1.cifCrystal structure: contains datablock(s) General, I. DOI: 10.1107/S205698901501275X/zl2631Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S205698901501275X/zl2631Isup3.cmlSupporting information file. DOI: 1410048CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


In the crystal structure of 1-[(6-chloropyridin-3-yl)sulfonyl]-1,2,3,4-tetrahydroquinoline, the tetrahydropyridine ring of the quinoline system adopts a half-chair conformation and the bond-angle sum at the N atom is 350.0°. 14H13ClN2O2S, adopts a half-chair conformation with the bond-angle sum at the N atom being 350.0°. The dihedral angle between the least-squares planes of the two aromatic rings is 50.13 (11)°. In the crystal, inversion dimers linked by pairs of C—H⋯O hydrogen bonds generate R22(10) loops. Additional intermolecular C—H⋯O hydrogen bonds generate C(7) chains along [100].The tetrahydropyridine ring of the quinoline system in the title compound, C In a continuation of our work on the derivatives of 1,2,3,4-tetrahydroquinolines A⋯O1 hydrogen bonds, generating C(7) chains along [100], as shown in Fig. 2In the crystal, inversion dimers linked by pairs of C11—H11⋯O2 hydrogen bonds generate 3 solution and brine, and dried over anhydrous Na2SO4. The solvent was evaporated under reduced pressure to give 1-[(6-chloropyridin-3-yl)sulfonyl]-1,2,3,4-tetrahydroquinoline, (I)n-hexane (1:1 v/v) to obtain crystals suitable for X-ray diffraction studies.To an ice-cold solution of 1,2,3,4-tetrahydroquinoline  and triethylamine  in dichloromethane (50 ml), a solution of 6-chloropyridine-3-sulfonyl chloride  in dichloromethane (20 ml) was added dropwise and stirred for 30 min. The reaction mixture was diluted with dichloromethane (150 ml), the organic layer washed with aqueous 5% NaHCOUiso(H) = 1.2Ueq(C) for aromatic H atoms and with C—H = 0.97 Å and Uiso(H) = 1.2Ueq(C) for methylene H atoms.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989015008099/wm5147sup1.cifCrystal structure: contains datablock(s) I, New_Global_Publ_Block. DOI: 10.1107/S2056989015008099/wm5147Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989015008099/wm5147Isup3.cmlSupporting information file. DOI: 1061311CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


A) and 2′,4′-dihydroxy-3′,6′-dimethoxychalcone (B), previously isolated from Polygonum limbatum Meisn. (Polygonaceae). The structures were elucidated by spectroscopic means and comparison with published data. The cytotoxicity of compounds was determined by using the resazurin assay in the parental drug-sensitive CCRF-CEM cell line and its multidrug-resistant P-glycoprotein-over-expressing subline, CEM/ADR5000.The present report describes the semi-synthesis of a few O-prenylated phenolic derivatives and their in vitro antitumor activities. These compounds were prepared by modifying two naturally occurring antitumor phenols, 5,7-dihydroxy-3-(1′-hydroxy-1′-phenyl-methyl)-6-methoxy-chroman-4-one (A and B: 5-hydroxy-6-methoxy-7-O-(3′-methylbut-2′-enyl)chroman-4-one (1), trivially named metapchromone, 5-acetoxy-6-methoxy-7-O-[3′-methylbut-2′enyl]chroman-4-one (2), trivially named sargisin, 2′-hydroxy-3′,6′-dimethoxy-4′-O-(3″-methylbut-2″-enyl)chalcone (3) trivially named limbachalcone A, and 2′-acetoxy-3′,6′-dimethoxy-4′-O-(3″-methylbut-2″-enyl)chalcone (4) trivially named tsedengchalcone. Their preliminary cytotoxic activities have been determined. We also report herein the isolation of 1-methylhydantoin (C) and betulinic acid (D) from Polygonum limbatum for the first time.We describe in the present paper four new semi-synthetic derivatives of The study clearly suggests that semi-synthesis involving O-prenylation and acetylation of chalcones or other chromanones should be avoided in a search for anticancer drugs. This conclusion should be helpful when selecting substituents for the synthesis of potential anticancer drugs. Polygonum belongs to the family Polygonaceae, comprising 300 species growing all over the world, although most of them are found in temperate and tropical regions chroman-4-one (2) trivially named sargisin, 2′-hydroxy-3′,6′-dimethoxy-4′-O-(3″-methylbut-2″-enyl)chalcone (3) trivially named limbachalcone A, and 2′-acetoxy-3′,6′-dimethoxy-4′-O-(3″-methylbut-2″-enyl)chalcone (4), trivially named tsedengchalcone. Their preliminary antitumor activities have been determined. We also report herein the isolation of 1-methylhydantoin (C) as a natural product from the Polygonum genus for the first time.The genus  regions , 2. PolyPolygonum genus and the Polygonaceae family could be an important chemotaxonomic finding.The isolation of 1-methylhydantoin and betulinic acid from the A, B and C isolated from P. limbatum were elucidated on the basis of spectroscopic data such as IR, 1D and 2D NMR spectra. Comparison of the data with those reported in the literature led to the identification of compounds such as betulinic acid (D) +; 301.09 [M+Na]+; 233.00.1H NMR  and 13C NMR : see Table Yellow powder; mp 75–77 °C; IR (KBr): ν1  was dissolved in pyridine (0.5 mL); Ac2O (8 mL) and 4-dimethylaminopyridine (DMAP) (1 mg) were added, and the mixture was stirred at room temperature for 24 h. H2O (8 mL) was then added to the mixture, which was stirred for 30 min  as isocratic eluent gave a new derivative, 5-acetoxy-6-methoxy-7-O-(3′-methylbut-2′-enyl)chroman-4-one (2) , which was crystallized from n-hexane-EtOAc (7:3) and trivially named sargisin.Compound m/z 663.06 [2M+Na]+, 603.33 [2M-AcOH+Na]+, 543.40 [2M-2AcOH+Na]+, 343.34  and13C NMR  see Table Brownish powder, mp 87–89 °C, ESIMS:B  was dissolved in 3 mL of acetone (0.1 M); prenyl bromide (2 mL) and K2CO3 (3 mg) were added successively. The mixture was heated at 40 °C for 3 h  was then added to the mixture, which was stirred for 40 min. Extraction with CH2Cl2 (3 × 10 mL),dryingover Na2SO4, and chromatographic purification on a silica gel column with mixtures of n-hexane-EtOAc yielded, after crystallization, 2′-hydroxy-3′,6′-dimethoxy-4′-O-(3″-methylbut-2″-enyl)chalcone (3) , trivially named limbachalcone A.Compound 3 h Fig. . Distillmax = 2931, 1629, 1560, 1334, 1119, 792 cm−1; 1H  and 13C NMR ; ESIMS: m/z1126.92 [3M+Na]+, 759.83 [2M+Na]+, 391.25 [M+Na]+.Yellowish powder; mp 98–100 °C;IR (KBr): ν1  was dissolved in pyridine (0.4 mL); Ac2O (5 mL) and DMAP (1 mg) were added, and the mixture was stirred at room temperature for 24 h  was then added to the mixture, which was stirred for 30 min. Extraction with CH2Cl2 and chromatographic purification on a Sephadex LH-20 gel column with CH2Cl2/MeOH (1:1) as isocratic eluent, yielded 2′-acetoxy-3′,6′-dimethoxy-4′-O-(3″-methylbut-2″-enyl)chalcone (4) , which was crystallized from n-hexane-EtOAc (6:4) and trivially named tsedengchromone.Compound 4 h Fig. . Distillm/z 843.13 [2M+Na]+, 433.42 [M+Na]+; 1H NMR  see Table Brownish powder; mp 102-104 °C; ESIMS: 1H NMR ; δ = 3.95 ; 2.90 ; 13C NMR ; see Table m/z (rel. int.): 114 , 86 (6), 42 (72).Brownish powder; mp 155-157 °C, 4 cells per well were seeded in 96-well-plates in a total volume of 100 μL. The studied compound was immediately added at varying concentrations to an additional 100 μL of culture medium to obtain a total volume of 200 μL/well. After 72 h, resazurin   in distilled H2O was added to each well and the plates were incubated at 37 °C for 4 h. Fluorescence was measured on an Infinite M2000 ProTM plate reader  using an excitation wavelength of 544 nm and an emission wavelength of 590 nm. A preliminary assay was done with all samples on leukaemia CCRF-CEM cells at 125 μM. Each assay was done at least twice with six replicates each. The viability was evaluated by comparison with untreated cells. IC50 values represent the compound concentrations required to inhibit 50 % of cell proliferation and were calculated from a calibration curve by linear regression with the aid of Microsoft Excel [The resazurin reduction assay  was perfft Excel .


The contributions of γδ T-cells to immunity to infection or tumors critically depend on their activation and differentiation into effectors capable of secreting cytokines and killing infected or transformed cells. These processes are molecularly controlled by surface receptors that capture key extracellular cues and convey downstream intracellular signals that regulate γδ T-cell physiology. The understanding of how environmental signals are integrated by γδ T-cells is critical for their manipulation in clinical settings. Here, we discuss how different classes of surface receptors impact on human and murine γδ T-cell differentiation, activation, and expansion. In particular, we review the role of five receptor types: the T-cell receptor (TCR), costimulatory receptors, cytokine receptors, NK receptors, and inhibitory receptors. Some of the key players are the costimulatory receptors CD27 and CD28, which differentially impact on pro-inflammatory subsets of γδ T-cells; the cytokine receptors IL-2R, IL-7R, and IL-15R, which drive functional differentiation and expansion of γδ T-cells; the NK receptor NKG2D and its contribution to γδ T-cell cytotoxicity; and the inhibitory receptors PD-1 and BTLA that control γδ T-cell homeostasis. We discuss these and other receptors in the context of a five-step model of receptor signaling in γδ T-cell differentiation and activation, and discuss its implications for the manipulation of γδ T-cells in immunotherapy. Both human γδ T-cell subsets are highly prone to secrete IFNγ, but IL-17 can be induced in highly inflammatory conditions triggered by infections  agonists , 17. Fol2γδζ2 in humans and TCRγδCD3δ2γ2ζ2 in mice  in the CD3 cytoplasmic domains by the Src-family kinases (SFKs) Lck and Fyn . The recSFK-mediated phosphorylation of the ITAMs on CD3 chains allows the recruitment, phospholylation, and activation of Zap70 that facilitates phosphorylation of the scaffolding proteins SLP-76 and LAT. This lead to the formation of a supramolecular signalosome that recruits the phospholipase PLCγ1, resulting in propagation of downstream signaling events . Here agIn humans, the major γδ T-cell subset in the peripheral blood, Vγ9Vδ2 T-cells, are uniquely and specifically reactive to self- and foreign non-peptidic phosphorylated intermediates of isoprenoid synthesis – “phosphoantigens” or “phosphoagonists” (P-Ags) –28. Thes+ CD122+ γδ T-cells producing IFN-γ or CD27− CCR6+ γδ T-cells making IL-17  or tumor necrosis factor receptor (TNFR) superfamilies. Ig superfamily members have a variable Ig-like extracellular domain and a short cytoplasmic tail, whereas TNFR family members present extracellular domains rich in six cysteine repeats (which form disulfide bridges) and a more complex cytoplasmic tail [reviewed in Ref. ]. These Plasmodium berghei infection, and failed to expand both their IFN-γ+ and IL-17+ subsets  and IL-17+ (CD27−) γδ T-cells. Most interestingly, these phenotypes are established in the thymus, and since embryonic stages. Based on the results from our  antibodies reduced, Vγ9Vδ2 T-cell expansion in vitro . Moreove T-cells , our worListeria monocytogenes in CD30-sufficient versus deficient mice. They demonstrated a selective depletion of IL-17-producing Vγ6+ T-cells in mucosal tissues in the steady-state and upon infection  antibodies promoted the expansion of IFN-γ+ Vγ1+ T-cells, which protected (in an IFN-γ-dependent manner) also from Listeria infection  , which bInterleukins are key determinants of T-cell survival, proliferation, and differentiation. IL-7, IL-15, and IL-2 are essential for lymphocyte development and homeostasis; upon inflammation, other cytokines, namely, IL-1β, IL-12, IL-18, IL-21, and IL-23, take a central role in determining T-cell functions. Here, we review the main contributions of homeostatic and inflammatory cytokines specifically to γδ T-cell physiology.+) thymic epithelial cells showed that γδ T-cells were significantly reduced in the adult thymus and in the gut, whereas they were completely absent in the fetal thymus and epidermis (IL-7 and IL-15 are seemingly the key determinants of murine γδ T-cell development –90 and hpidermis . In the pidermis . Converspidermis .− γδ T-cells to resist activation-induced cell death (AICD) and undergo proliferative responses to TCR agonists. Such an IL-7/IL-17 axis was also reported to be required for the γδ T-cell response to viral hepatitis infection in vivo  program in functionally immature γδ thymocytes . This waMycobacterium tuberculosis infection in macaques required a phosphoantigen/IL-2 combination  over type 2 (IL-4+) effector fates , including natural cytotoxicity receptors (NCRs).The C-type lectin-like NK receptor group 2 member D (NKG2D) is the best studied NKR in γδ T-cells. NKG2D binds extracellularly to multiple ligands of the MIC(A–B) and ULBP –6 familiIntracellularly, NKG2D binds to DNAX-activating protein of 10 kDa (DAP10), which carries an YXNM motif that after tyrosine phosphorylation recruits PI3K or a Grb2–Vav1–SOS1 signaling complex Table . HoweverThe controversy on a primary stimulatory versus costimulatory role of NKG2D in γδ T-cells has been discussed elsewhere , 114. BrAnother NKR implicated in tumor cell recognition by Vγ9Vδ2 T-cells is DNAX accessory molecule-1 (DNAM-1). DNAM-1 is an Ig-like family glycoprotein composed of a cytoplasmic domain containing three putative sites of phosphorylation by intracellular kinases. The phosphorylation of the Ser329 by protein kinase C (PKC) was shown to be critical for the association between DNAM-1 and LFA-1, which recruits the Fyn Src kinase to phosphorylate the Tyr322 of DNAM-1, thus initiating downstream signaling leading to SLP-76 and Vav1 phosphorylation Table . Antibod+ T-cell population capable of targeting hematological tumors resistant to fully activated Vγ9Vδ2 T-cells  and TCR stimulation. Once expressed on the cell surface, NKp30 and NKp44 can signal via CD3ζ and DAP12, respectively  is absent or low expressed on circulating Vγ9Vδ2 T-cells but is rapidly induced upon activation . The cytB- and T-lymphocyte attenuator (BTLA) is another inhibitory receptor, member of the CD28 family and structurally related to PD-1 and CTLA-4. Binding to its ligand, herpesvirus entry mediator (HVEM), induces phosphorylation of the ITIM domain and association with SH2 domain-containing protein tyrosine phosphatase 1 (SHP-1) and SHP-2, which leads to attenuation of cellular activation and growth . Recent − γδ T-cell subset (+ CD27− γδ T-cells and were more susceptible (than wild-type controls) to dermatitis, which could be reversed by agonist BTLA antibodies. Thus, BTLA may be an important target for controlling pathogenic γδ T-cells in inflammatory and autoimmune diseases.A detailed study on BTLA function in murine γδ T-cells has revealed a selective involvement in the homeostasis of the IL-17-producing CD27l subset . Althougin vivo pre-clinical models and hopefully next in the clinic.A multitude of surface receptors has been shown to participate in γδ T-cell differentiation and activation. However, some crucial aspects remain to be elucidated, such as the identity of most γδTCR ligands. Most importantly, we must improve the transfer of past and current basic research into future protocols for γδ T-cell-based immunotherapy. In this context, some key questions are: how to balance γδTCR activation with “exhaustion” due to chronic stimulation? What can be achieved by manipulating the NK-like activation mode of γδ T-cells? Which costimulatory receptors should be modulated, and at what stages, to boost the desired γδ T-cell responses? Which combinations of cytokines enable the best effector γδ T-cells for each therapeutic application? Which receptors are most useful to tune down or switch off pathogenic effector γδ T-cells? The answers to these questions must be obtained in appropriate For now, we would like to propose that the five types of receptor signals reviewed here define five distinct layers of regulation of γδ T-cell differentiation, activation, and function. The γδTCR is critical for the initial stages of differentiation and for proliferative responses; both processes further require cytokine signals that promote cell survival, proliferation, and terminal effector function. Costimulatory and inhibitory receptors control the extent of γδ T-cell expansion, with interesting biases toward specific effector subsets. Finally, NK receptors play a decisive role in tumor cell targeting by γδ T-cells. Thus, we believe that the recognition of “stressed self” can be mediated by the γδTCR but also chiefly by NK receptors like NKG2D. As such, the characterization of both type of ligands on tumors may be critical to design protocols, select and monitor patients, and increase the chances of efficacious γδ T-cell-based cancer immunotherapies.Bruno Silva-Santos is a co-founder and share holder of LymphAct S.A. The other co-authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Mammalian αE-catenin is allosterically regulated: the monomer binds the β-catenin·cadherin complex, whereas the homodimer does not bind β-catenin but interacts with F-actin. As part of the cadherin·catenin complex, αE-catenin requires force to bind F-actin strongly. It is not known whether these properties are conserved across the mammalian α-catenin family. Here we show that αT (testes)-catenin, a protein unique to amniotes that is expressed predominantly in the heart, is a constitutive actin-binding α-catenin. We demonstrate that αT-catenin is primarily a monomer in solution and that αT-catenin monomer binds F-actin in cosedimentation assays as strongly as αE-catenin homodimer. The β-catenin·αT-catenin heterocomplex also binds F-actin with high affinity unlike the β-catenin·αE-catenin complex, indicating that αT-catenin can directly link the cadherin·catenin complex to the actin cytoskeleton. Finally, we show that a mutation in αT-catenin linked to arrhythmogenic right ventricular cardiomyopathy, V94D, promotes homodimerization, blocks β-catenin binding, and in cardiomyocytes disrupts localization at cell-cell contacts. Together, our data demonstrate that αT-catenin is a constitutively active actin-binding protein that can physically couple the cadherin·catenin complex to F-actin in the absence of tension. We speculate that these properties are optimized to meet the demands of cardiomyocyte adhesion. The adherens junction (AJ)1–9–In mammals, αE -catenin is allosterically regulated: the monomer binds the β-catenin·cadherin complex, whereas the homodimer does not bind β-catenin but interacts with F-actin , 10. β-C14–α-Catenin functions in adhesion and mechanical signaling must be integrated in all tissues. In cardiomyocytes, the AJ functions with the desmosome to physically link opposing cells in a specialized adhesive structure called the intercalated disc (ICD) . Contrac21–Here we show that αT-catenin is a constitutive actin-binding α-catenin that can directly couple the AJ to the actin cytoskeleton. Our data also reveal that the V94D mutation linked to ARVC alters αT-catenin dimerization potential to disrupt β-catenin binding and cellular localization. We postulate that αT-catenin protein conformation and ligand binding properties are tuned to meet the specific demands of cardiomyocyte adhesion.A, N1 and N2), binds β-catenin, and mediates homodimerization  and binds vinculin in response to mechanical force . We compared the amino acid sequence of Mus musculus αT-catenin with M. musculus αE-catenin and M. musculus αN-catenin. αT-Catenin is 58% identical and 77% similar to αE-catenin; likewise, it is 59% identical and 77% similar to αN-catenin . αE-Catenin and αN-catenin are 81% identical and 91% similar, making αT-catenin the most divergent of the mammalian family. We then analyzed sequence homology across domains between αT-catenin and αE-catenin . The region with the lowest degree of homology is N2 , whereas the region with the highest degree of homology is M2 .Structural studies of αE-catenin have revealed that the protein is a series of helical bundles , 27, 28.Fig. 1force 14–, 29, 30.M. musculus αT-catenin and M. musculus αE-catenin from Escherichia coli and used limited trypsin proteolysis to examine domain organization. As shown previously (D). Tryptic digestion of αT-catenin revealed three stable fragments at 30, 25, and 18 kDa (D). N-terminal sequencing revealed that the 30-kDa fragment started at aa 379 and contained bundles M2 and M3 (D). The entire M2-M3 region forms a protease-resistant fragment in mouse αE-catenin (D)   and fishE. coli was first purified by Mono Q ion exchange chromatography (A). Two peaks were routinely observed during elution off a Mono Q column , and SDS-PAGE analysis of peak fractions revealed they both contained full-length αT-catenin . A similar ion exchange chromatography profile is observed with M. musculus αE-catenin (data not shown), and the two peaks correspond to the monomer (peak 1) and homodimer (peak 2) species. Both αT-catenin peak fractions were subsequently purified over a Superdex 200 (S200) size exclusion chromatography (SEC) column. The Mono Q peak 1 fraction eluted in a single, discrete peak , consistent with it being a single, likely monomeric, species. The S200 elution profile of Mono Q peak 2 was similar to peak 1, although a second, small peak was sometimes observed where a dimer species would be expected to elute .We assessed the oligomerization state of αT-catenin by chromatography. Recombinant αT-catenin protein prepared from tography A. Two pem) of αT-catenin with αE-catenin monomer and homodimer by analytical SEC. At all concentrations tested (25–50 μm), αT-catenin eluted in a single peak after both αE-catenin homodimer and monomer, suggesting that αT-catenin is a monomer (C). We then used SEC and sucrose density gradient centrifugation to determine the molecular mass of αT-catenin, αE-catenin monomer, and αE-catenin homodimer (C) were compared with known standard proteins to calculate the Stokes radius (D). The calculated Stokes radius of αE-catenin homodimer was similar to past observations .We then compared the primary S200 peak . The Svedberg coefficients were determined to be 7.0S for αE-catenin homodimer . The molecular mass of αT-catenin was calculated to be 109 kDa, similar to that of αE-catenin monomer (106 kDa). Finally, αT-catenin migrated as a single band by native PAGE, faster than either αE-catenin monomer or dimer, consistent with the SEC analysis (H). We conclude that αT-catenin is primarily a monomer in solution.We then used sucrose density gradient centrifugation to determine the sedimentation coefficients of αT-catenin, αE-catenin monomer, and αE-catenin homodimer. Proteins were separated on 5–20% sucrose gradients, and the fraction peak was determined and compared with a standard curve to calculate the sedimentation coefficient , E and Fculation ), 5.2S fculation G. The moanalysis H. We conA) and the minor peak in the peak 2 SEC elution  suggest that αT-catenin might exist as a homodimer. However, if the Mono Q peak 2 elution represented a homodimer species of αT-catenin, then the majority of these dimers dissociated during SEC . We were never able to purify a sufficient quantity of the potential dimer species for analysis by SEC or native-PAGE. Also, attempts to promote dimerization by incubation of the monomer at physiological (37 °C) temperatures caused the protein to aggregate and fall out of solution. Although we were unable to analyze the dimerization kinetics of wild-type (WT) αT-catenin, our analysis of the V94D mutant revealed that αT-catenin, similar to αE-catenin and αN-catenin, has dimerization potential (described below). Nonetheless, we took advantage of the lack of a stable dimer in solution to study the behavior of αT-catenin monomer binding to F-actin.Dimerization kinetics differ significantly between mouse αE-catenin and αN-catenin at physiological temperatures . αE-CateFig. 2m F-actin, the samples were centrifuged, and the resulting pellets were analyzed. αT-Catenin cosedimented with F-actin above background (A), and the bound protein was quantified and plotted over free protein to calculate the affinity of the interaction (B). Bovine serum albumin (BSA) and αE-catenin were run as negative and positive controls, respectively . Plotted data were fit to a hyperbolic function . αT-Catenin bound to F-actin with a dK of 0.4 ± 0.2 μm, similar to αE-catenin dimer . In contrast, robust bundling of 2 μm F-actin was observed with 4 μm αE-catenin homodimer (C and 5D). The weak bundling observed with αT-catenin could result from either the dimer species being stabilized on the actin filament or activation of a cryptic dimerization domain as observed in the vinculin tail , we were able to calculate the affinity of the complex for F-actin. The β-catenin·αT-catenin complex bound to F-actin with a dK of 1.1 ± 0.2 μm . Although β-catenin lowers the affinity of αT-catenin for F-actin slightly, the interaction strength is considerably stronger than that of the Danio rerio β-catenin·αE-catenin complex (>10 μm) and similar to the strength of αE-catenin homodimer association with F-actin . We then ran the V94D peak over an S200 SEC column where it eluted as a single peak before WT αT-catenin and similar to the possible homodimer peak . We then compared the concentrated V94D protein  with WT αT-catenin by analytical SEC. The V94D mutant eluted as a single species before WT αT-catenin with a larger Stokes radius . The V94D mutant also displayed a higher sedimentation coefficient than WT αT-catenin . The Stokes radius and sedimentation coefficient produced a molecular mass of 183 kDa , roughly double that of WT αT-catenin. We conclude that the V94D mutation creates a stable αT-catenin homodimer.Two mutations in αT-catenin have been linked to ARVC: replacement of a valine for an aspartic acid at aa 94 (V94D) in the N1 domain and deletion of a leucine at aa 765 (L765del) in the ABD . Yeast tFig. 4E–G). Similar to the full-length construct, the V94D mutation altered the elution and sedimentation profiles of the ΔABD construct (E and F). The calculated molecular mass of αT-catenin V94D ΔABD was 146 kDa compared with 90 kDa for αT-catenin ΔABD, consistent with it forming a homodimer.Because full-length αT-catenin V94D is difficult to purify, we deleted the ABD (aa 660–895) in both WT and V94D αT-catenin to improve protein yield. We analyzed the SEC and sedimentation properties of the ΔABD constructs , E–G. Sionstruct , E and FH). In the presence of BS3, however, V94D migrated as a 150-kDa protein at all concentrations tested, indicating a cross-linked dimer. Incubation with BS3 did not affect αT-catenin ΔABD migration at low concentrations, although at higher concentrations (2 and 4 μm), a 150-kDa species was detected. We speculate that this could reflect a transient homodimer species. We conclude that the V94D mutation promotes dimerization of αT-catenin.We analyzed the oligomeric state of the αT-catenin ΔABD proteins by cross-linking. Increasing concentrations of αT-catenin ΔABD and αT-catenin V94D ΔABD were incubated with or without the cross-linker bis(sulfosuccinimidyl)suberate (BS3), and the resulting products were analyzed by SDS-PAGE. As expected, αT-catenin ΔABD and αT-catenin V94D ΔABD ran as 75-kDa proteins in the absence of cross-linker H. In theI). However, the β-catenin/homodimerization domain  was protected relative to WT , consistent with this domain being stabilized in the homodimer state.We used limited proteolysis to determine whether the V94D mutation affected domain organization. Like WT αT-catenin, three fragments were resistant to trypsin cleavage in V94D I. HoweveJ). Thus, the V94D mutation creates an obligate αT-catenin homodimer that cannot bind β-catenin.We then questioned whether the V94D homodimer could interact with β-catenin. We mixed increasing concentrations of WT or V94D αT-catenin with GST-tagged β-catenin, pulled down the β-catenin, and assessed binding. Wild-type αT-catenin bound GST-β-catenin at stoichiometric levels; however, little to no V94D bound J. Thus, A) with an affinity similar to that of WT αT-catenin , suggesting that homodimerization does not increase the affinity of αT-catenin for F-actin.Dimerization promotes αE-catenin binding to F-actin , 10. We Fig. 5m F-actin with 4 μm αT-catenin V94D relative to 4 μm WT αT-catenin (C and D). Although increased, the level of bundling was still less than that observed with 4 μm αE-catenin homodimer (C and 5D). We conclude that the V94D mutation promotes αT-catenin-mediated F-actin bundling.We then tested whether the V94D homodimer could bundle F-actin. We consistently observed increased bundling of 2 μ-catenin , C and Domodimer C and 5D.A, C, and zoom in E). In contrast, V94D was largely peripheral to cell-cell contacts  and localized to actin fibers . This was confirmed by directly measuring colocalization between N-cadherin and EGFP-αT-catenin or EGFP-αT-catenin V94D signals at AJ clusters in transfected cells using Pearson's r (F). This analysis revealed a significant reduction in colocalization between N-cadherin and EGFP-αT-catenin V94D at AJs (F). Thus, the V94D mutation disrupts αT-catenin subcellular localization in cardiomyocytes.αT-Catenin localizes to the adherens junction at the ICD in cardiomyocytes . To deterson's r F. This aD at AJs F. Thus, in vitro results show that, in solution, αT-catenin binds F-actin as a monomer and in complex with β-catenin, properties that separate it from mammalian αE-catenin. αT-Catenin monomer binds F-actin with a slightly higher affinity than αE-catenin homodimer (0.4 versus 1.0 μm) (m). We conclude that αT-catenin binding to F-actin, unlike mammalian αE-catenin, is not allosterically regulated. This would permit αT-catenin to directly couple the cadherin·catenin complex to the actin cytoskeleton in the absence of tension, although mechanical force could strengthen the αT-catenin-actin interface.Our  1.0 μm) . AlthougM. musculus αE-catenin and αN-catenin homodimerize in solution, although the kinetics of dimerization differ significantly between the two mammalian α-catenins (versus 25 μm). However, the kinetics of dissociation differ markedly: αN-catenin equilibrates quickly, whereas a kinetic block limits αE-catenin dissociation  precluded a detailed analysis of dimerization kinetics. Nonetheless, our results lead us to postulate that αT-catenin has dimerization potential and that the homodimer species, similar to αN-catenin, dissociates quickly (i.e. no kinetic block).Both catenins . At physociation . The αE-in vivo are unclear. The V94D mutation, which drives αT-catenin into the dimer state in vitro, shifted localization from cell-cell contacts and promoted recruitment to F-actin bundles when expressed in cardiomyocytes. Actin filament cross-linking is essential for cardiomyocyte cytoskeletal organization and function. The barbed ends of actin filaments from adjoining sarcomeres interdigitate at the Z-disc where they are cross-linked primarily by α-actinin to form a structural lattice  and plakoglobin,Our limited proteolysis experiments revealed that both the β-catenin/homodimerization domain and middle domain were more protease-sensitive in αT-catenin than in αE-catenin. Notably, the N2 bundle within the β-catenin/homodimerization domain of αT-catenin is the region with the least conservation compared with αE-catenin. αT-Catenin binds β-catenin D and plaThe core M region (M1–M3) of αE-catenin is required for its function as a mechanosensor in which tension alters α-catenin conformation to promote ligand binding , 29, 43.Increased flexibility between the M2 and M3 domains could also promote ligand binding within this region. Notably, αT-catenin, but not αE-catenin, was shown to bind plakophilin-2, a desmosomal protein that links to intermediate filaments, and the binding interface was mapped to M3 . αT-Catein vitro studies showed that αT-catenin could directly couple the actin cytoskeleton to cadherin·catenin in the absence of tension. We speculate that this property of αT-catenin might permit the cadherin·catenin complex to maintain a static linkage to the actomyosin network over a range of forces such as those produced by repeated cycles of contraction and relaxation in cardiomyocytes. Our biochemical analyses also suggest that αT-catenin dimerization properties and M region stability differ from those in αE-catenin. How these differences impact in vivo function is unclear, but we speculate that they could impact molecular interactions and tension sensing. In the mammalian heart, αT-catenin may have evolved to complement αE-catenin functions in adhesion and signaling.α-Catenin functions in adhesion and mechanical signaling must be integrated in all tissues. Contractile forces place physical demands on heart junctional complexes: not only must they withstand repeated cycles of force but tension-sensing proteins within these complexes must be tuned to regulate signaling and maintain homeostasis. Our M. musculus αT-catenin was cloned into pGEX-TEV  and then incubated with tobacco etch virus protease overnight at 4 °C to cleave protein from the GST tag. All proteins were purified by Mono Q anion exchange chromatography followed by S200 gel filtration chromatography in 20 mm Tris, pH 8.0, 150 mm NaCl, 10% glycerol, and 1 mm DTT. Eluted protein was concentrated to 20–50 μm working concentrations using a Millipore column concentrator, flash frozen in liquid nitrogen, and stored at −80 °C.GST-tagged αT-catenin, αE-catenin, and β-catenin were expressed in BL21(DE3) escribed , 36. GSTm Tris, pH 8.0, 150 mm NaCl, and 1 mm DTT. Protein was injected at 25–30 μm.Analytical SEC was performed at 4 °C on a Superdex 200 column in 20 mm Tris, pH 6.8, 150 mm NaCl, 300 mm sucrose, 100 mm DTT, and 0.02% bromphenol blue) and loaded onto a 5% native gel . Gels were run at 80 V for 5 h at 4 °C, stained with Coomassie Blue, and imaged on a LI-COR Biosciences scanner.FPLC-purified αE-catenin and αT-catenin were diluted in cold native gel sample buffer (20 mm αT-catenin was incubated at room temperature in 0.05 mg/ml sequencing grade trypsin (Roche Applied Science) in 20 mm Tris, pH 8.0, 150 mm NaCl, and 1 mm DTT. Reactions were stopped with 2× Laemmli buffer at the indicated times, and samples were analyzed by SDS-PAGE. For N-terminal sequencing, digested peptides were blotted onto PVDF membrane; stained with 0.1% Coomassie Blue R-250, 40% methanol, and 1% acetic acid; destained; and dried. Individual bands were excised and sequenced by Edman degradation (Iowa State University Protein Facility).12 μSR) was determined by analytical size exclusion chromatography using a Superdex 200 column equilibrated with 20 mm Tris, pH 8.0, 150 mm NaCl, and 1 mm DTT. Standard proteins were bovine carbonic anhydrase (SR = 2.4 nm), bovine serum albumin (SR = 3.5 nm), yeast alcohol dehydrogenase (SR = 4.6 nm), sweet potato β-amylase (SR = 5.4 nm), horse spleen apoferritin (SR = 6.7 nm), and bovine thyroglobulin (SR = 8.5 nm). The partition coefficient, Kav, was calculated for all standards and α-catenin proteins used in this study. The Stokes radius was calculated from a standard curve of (−logKav)1/2Sversus R.The Stokes radius  for 4 h at 4 °C. All α-catenin proteins were loaded at concentrations ≥20 μm. After centrifugation, 200-μl fractions were collected and analyzed by SDS-PAGE. Gels were imaged on a LI-COR Biosciences scanner, and the percentage of protein in each fraction was measured in ImageJ. Plotted data were fit to a Gaussian curve to determine the peak fraction in Prism software. Standard proteins were bovine carbonic anhydrase (2.8S), bovine serum albumin (4.3S), yeast alcohol dehydrogenase (7.4S), sweet potato β-amylase (8.9S), and horse spleen apoferritin (16.6S). The sedimentation coefficient of α-catenin proteins was determined from a standard curve of sedimentation coefficient (S) versus fraction.Gradients of sucrose were made by layering sucrose dissolved in 20 mescribed . Each laThe molecular mass of α-catenin proteins used in this study was calculated from the measured Stokes radius and sedimentation coefficient as described , 45.m HEPES, pH 7.5, 100 mm KCl, 2 mm MgCl2, 0.5 mm ATP, and 1 mm EGTA) for 1 h at room temperature to polymerize filaments. Gel-filtered αT-catenin or αT-catenin·β-catenin heterocomplex was diluted to the indicated concentrations in 1× reaction buffer  with and without 2 μm F-actin and incubated for 30 min at room temperature. Samples were centrifuged at 50,000 rpm  for 20 min at 4 °C in an S100-AT3 rotor. Pellets were resuspended in Laemmli sample buffer, separated by SDS-PAGE, and stained with Coomassie Blue. Gels were imaged on a LI-COR Biosciences scanner and measured and quantified in ImageJ. To determine the amount of bound protein, background sedimentation (no F-actin pellet) was first subtracted from cosedimentation (F-actin pellet). Bound protein across samples was then normalized to the F-actin pellet. The amount of bound protein was calculated from a standard curve created from the starting material. All binding data were processed with Prism software.Chicken muscle G-actin  was incubated in 1× actin polymerization buffer  in 20 mm HEPES, pH 7.4, 150 mm NaCl, and 1 mm DTT for 30 min at room temperature, separated by SDS-PAGE, stained with Coomassie dye, and imaged on a LI-COR Biosciences scanner.Purified αT-catenin ΔABD and αT-catenin V94D ΔABD were incubated with or without 1 mm Tris, pH 8, 150 mm NaCl, and 5 mm DTT. Samples were incubated with gentle mixing for >2 h at 4 °C and then washed five times in PBS + 0.05% Tween 20 and 5 mm DTT before elution in Laemmli sample buffer. Samples were separated by SDS-PAGE, stained with Coomassie dye, and imaged on a LI-COR Biosciences scanner.Increasing amounts of αT-catenin or αT-catenin V94D (1–15 μg) were added to 15 μg of GST-β-catenin bound to glutathione-agarose in 20 mm Ara-C, and 1 μm isoproterenol. Transfections were performed 24 h postplating using Lipofectamine 2000 (Life Technologies).All animal work was approved by the University of Pittsburgh Division of Laboratory Animal Resources. Cardiomyocytes were isolated from mouse neonates (P1–P3) as described . Cardiomm 1,4-piperazinediethanesulfonic acid, pH 7.0, 25 mm HEPES, pH 7.0, 10 mm EGTA, pH 8.0, 2 mm MgCl2, and 0.12 m sucrose), washed with PBS, blocked for 1 h at room temperature in PBS + 10% BSA, washed three times in PBS, incubated with primary in PBS + 1% BSA for 1 h at room temperature, washed three times in PBS, incubated with secondary in PBS + 1% BSA for 1 h at room temperature, washed three times in PBS, and mounted in Fluoromount G (Electron Microscopy Sciences). F-actin was stained using Alexa Fluor-phalloidin (Invitrogen) and antibodies against αE-catenin (Enzo Life Sciences) or N-cadherin (Invitrogen). Cells were imaged on a Nikon Eclipse Ti inverted microscope outfitted with a Prairie swept field confocal scanner, Agilent monolithic laser launch, and Andor iXon3 camera using NIS-Elements imaging software. Maximum projections of 4-μm image stacks were created for image analysis and presentation. For Pearson's r calculations, signal colocalization was measured between user-defined N-cadherin-positive AJ clusters and EGFP signals using ImageJ. Colocalization data were plotted and analyzed with Prism software.Cells were fixed in 4% paraformaldehyde in PHEM buffer (60 mA. V. K. conceived and coordinated the study and designed experiments. E. D. W., I. W. D., C. D. M., J. A. H., D. B. S., and A. V. K. performed research. E. D. W., C. D. M., J. A. H., and A. V. K. analyzed data. A. V. K. wrote the paper.


Three coumarin derivatives display intramolecular N—H⋯O and weak C—H⋯O hydrogen bonds, which probably contribute to the approximate planarity of the molecules. The supramolecular structures feature C—H⋯O hydrogen bonds and π–π interactions, as confirmed by Hirshfeld surface analyses. viz. 6-methyl-N-(3-methylphenyl)-2-oxo-2H-chromene-3-carboxamide, C18H15NO3 (1), N-(3-methoxyphenyl)-6-methyl-2-oxo-2H-chromene-3-carboxamide, C18H15NO4 (2), and 6-methoxy-N-(3-methoxyphenyl)-2-oxo-2H-chromene-3-carboxamide, C18H15NO5 (3), were synthesized and structurally characterized. The molecules display intramolecular N—H⋯O and weak C—H⋯O hydrogen bonds, which probably contribute to the approximate planarity of the molecules. The supramolecular structures feature C—H⋯O hydrogen bonds and π–π interactions, as confirmed by Hirshfeld surface analyses.Three coumarin derivatives,  This confers freedom of rotation of the phenylamide substituent around it. Despite that, the molecules are approximately planar, as can be inferred by the set of values of the dihedral angles in Table 1H-chromen-2-one ring, the O31/C31/N32 atoms of the amide residue and the phenyl substituent, which are all less than 11°. This may be correlated with the conformation assumed by the amide group around the C—N rotamer which displays an −anti orientation with respect to the oxo oxygen atom of the coumarin, thus allowing the establishment, in all three structures, of an intramolecular N—H⋯O hydrogen bond between the amino group of the carboxamide and the oxo group at the O2 position of the coumarin and a weak C—H⋯O intramolecular hydrogen bond between an ortho-CH group on the exocyclic phenyl ring and the O atom of the carboxamide. Thus these two interactions, which both form S(6) rings, probably contribute to the overall approximate planarity of the molecules since they may prevent the molecules from adopting some other possible conformations by restraining their geometry.The values for the distances of the C3—C31 bonds [mean value 1.508 (4) Å] connecting the coumarin system to the amide spacer are of the same order as a C1, 2 and 3 are given in Tables 2As mentioned above, the NH group is involved in an intramolecular hydrogen bond. It is not involved in any intermolecular interactions thus only carbon atoms may act as donors for the carbonyl and methoxy-type acceptors. Details of the hydrogen bonding for compounds 1, the molecules are linked by the C5—H5⋯O1 weak hydrogen bond to form a C(6) chain, which runs parallel to the a axis, Fig. 42, the molecules are linked by the C8—H8⋯O1 weak hydrogen bond to form an A⋯O31 contact involving a methyl hydrogen atom. In 3, the molecules are linked by the C4—H4⋯O2, C5—H5⋯O1 and C8—H8⋯O6 bonds to form a chain of a axis, Fig. 6x + 1, y, z) weak hydrogen bond.In et al., 2008Crystal Explorer 3.1  plots . In 3, there are several contacts, three of those involving the oxygen atoms of the coumarin system and those directly connected to it that are acceptors for H atoms of the coumarin residue of another molecule. These multiple contacts result in chains of hydrogen-bonded rings, as described in the previous section, and seem to operate a co-operative effect since the hydrogen bonds in 3 are stronger than in 1 and 2 (see the well-defined sharp spikes in the FP plot of 3).Forgetting the prevalence of the H⋯H contacts on the surface, inherent to organic molecules, the most significant contacts are the H⋯O/O⋯H ones. Those appear as highlighted red spots on the top face of the surfaces Fig. 7 to 9 tha3, the percentage for H⋯C/C⋯H contacts is higher than that for the other compounds. The FP plots also reveal a cluster at de/di ≃ 1.8 Å and di/de ≃ 1.2 Å characteristic of C—H⋯π contacts that seem to assume higher importance in the supramolecular structure in 3. On the other hand, the C⋯C contacts prevail in 1 and 2. In fact, the packing in 1 is built up by several π–π interactions  of centrosymmetrically related molecules.The values for the remaining contacts listed in Table 5s Table 6. Also, ws Table 6 and 11 ▸et al., 2016A search made in the Cambridge Structural Database  and diethyl malonate (1 mmol) and catalytic amounts of piperidine were dissolved in ethanol (10 ml) and refluxed for 4 h. After cooling to room temperature, the suspension was filtered off and ethyl 6-methylcoumarin-3-carboxylate was obtained. This compound was then dissolved in 20 ml of an ethanolic solution with 0.5% NaOH (aq.) and hydrolyzed under reflux for 1h. After reaction, 10% HCl (aq.) was added and the desired carboxylic acid was then filtered and washed with water coumarin-3-carboxamide (1) ; 6-methyl-N-(3′-methoxyphenyl)coumarin-3-carboxamide (2) ; 6-methoxy-N-(3′-methoxyphenyl)coumarin-3-carboxamide (3) .Then, to a solution of 6-methylcoumarin-3-carboxylic acid (1 mmol) in dichloromethane, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (1.10 mmol) and 4-dimethylaminopyridine (DMAP) (1.10 mmol) were added. The mixture was kept under a flux of argon gas at 273 K for five minutes. Shortly after, the aromatic amine (1 mmol) with the intended substitution pattern was added. The reaction mixture was stirred for 4 h at room temperature. The crude product was filtered and purified by column chromatography (hexane/ethyl acetate 9:1) or by recrystallization with ethanol to give the desired product, (Murata Uiso = 1.2Ueq(C), C—H(methyl) 0.98 Å and Uiso = 1.5Ueq(C) The amino H atoms were refined.Crystal data, data collection and structure refinement details are summarized in Table 710.1107/S2056989016008665/hb7589sup1.cifCrystal structure: contains datablock(s) 1, 2, general, 3. DOI: 10.1107/S2056989016008665/hb75891sup2.hklStructure factors: contains datablock(s) 1. DOI: 10.1107/S2056989016008665/hb75892sup3.hklStructure factors: contains datablock(s) 2. DOI: 10.1107/S2056989016008665/hb75893sup4.hklStructure factors: contains datablock(s) 3. DOI: Click here for additional data file.10.1107/S2056989016008665/hb75891sup5.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989016008665/hb75892sup6.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989016008665/hb75893sup7.cmlSupporting information file. DOI: 10.1107/S2056989016008665/hb7589sup8.pdfSupporting information file. DOI: 10.1107/S2056989016008665/hb7589sup9.pdfSupporting information file. DOI: 1482450, 1482449, 1482448CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


They are linked  9H8N2O, crystallized with four independent molecules in the asymmetric unit. The four molecules are linked via one O—H⋯N and two N—H⋯N hydrogen bonds, forming a tetramer-like unit. In the crystal, molecules are further linked by O—H⋯N and N—H⋯O hydrogen bonds forming layers parallel to (001). These layers are linked via C—H⋯O hydrogen bonds and a number of weak C—H⋯π interactions, forming a three-dimensional structure. The crystal was refined as a non-merohedral twin with a minor twin component of 0.319.The title compound, C N-Arylhydroxylamines can be generated in chemical, biochemical and biological systems either by reduction of nitroaromatic compounds or oxidation of arylamines. Interestingly, few aryl hydroxylamines have been crystallographically characterized. In part, this may be due to the instability of these compounds. For example, N-arylhydroxylamines can undergo spontaneous oxidation to generate the nitroso derivatives  in the asymmetric unit , 0.550 (5), 0.971 (6) and 0.293 (5) Å, respectively. The four molecules are linked via one O—H⋯N and two N—H⋯N hydrogen bonds, forming a tetramer-like unit ; see Table 1s Table 1, forming2Cl2  at 273 K was added a slurry of Raney nickel (0.5 ml). To this mixture, hydrazine hydrate  was added dropwise with stirring over the course of 1 h while keeping the solution under an inert atmosphere of nitrogen gas. The solid was removed by filtration and the resulting solution diluted with water (2 ml) and then extracted with ethyl acetate (2 × 10 ml). The combined organic extracts were washed with brine and dried over sodium sulfate. Column chromatography on silica gel, eluted with ethyl acetate and MeOH/CH2Cl2, gave the title compound as a yellow solid . It was found to be unstable upon standing in organic solvents. Crystals of the title compound were obtained by dissolving pure product in warm ethyl acetate followed by rapid cooling to give yellow crystals. 1H NMR : δ 8.53 , 8.07 , 7.82 , 7.33 . 13C NMR  δ 151.40, 147.64, 144.76, 136.72, 131.05, 129.15, 122.54, 121.01, 107.68. HRMS  m/z calculated for C9H9N2O: 160.0715; found: 160.0707.To a stirred solution of 6-nitroquinoline  in EtOH/CHUiso(H) = 1.2Ueq.Crystal data, data collection and structure refinement details are summarized in Table 2CELL_NOW  I. DOI: 10.1107/S160053681402193X/su2793Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S160053681402193X/su2793Isup3.cmlSupporting information file. DOI: 861650CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Plants have evolved complex coordinated regulatory networks to cope with deficiency of phosphate (Pi) in their growth environment; however, the detailed molecular mechanisms that regulate Pi sensing and signaling pathways are not fully understood yet. We report here that the involvement of Arabidopsis BIK1, a plasma membrane-localized receptor-like protein kinase that plays critical role in immunity, in Pi starvation response.BIK1 was induced by Pi starvation and GUS staining indicated that the BIK1 promoter activity was detected in root, stem and leaf tissues of plants grown in Pi starvation condition, demonstrating that BIK1 is responsive to Pi starvation stress. The bik1 plants accumulated higher Pi content in root and leaf tissues and exhibited altered root architecture such as shorter primary roots, longer and more root hairs and lateral roots, as compared with those in the wild type plants, when grown under Pi sufficient and deficient conditions. Increased anthocyanin content and acid phosphatase activity, reduced accumulation of reactive oxygen species and downregulated expression of Pi starvation-induced genes including PHR1, WRKY75, AT4, PHT1;2 and PHT1;4 were observed in bik1 plants grown under Pi deficient condition. Furthermore, the expression of PHO2 was downregulated while the expression of miRNA399a and miRNA399d, which target to PHO2, was upregulated in bik1 plants, compared to the wild type plants, when grown under Pi deficient condition.qRT-PCR analysis revealed that expression of BIK1 is a Pi starvation-responsive gene that functions as a negative regulator of Pi homeostasis in Arabidopsis.Our results demonstrate that  Phosphate (Pi) is one of the indispensable macronutrients to plants for growth, development and reproduction. Pi deficiency (−Pi) is one of the main limiting factors for increasing crop yield and improving quality because of low bioavailability of Pi in soil –4. To coGenetic, physiological and biochemical studies in Arabidopsis have demonstrated that the acquisition, allocation, and metabolism of Pi are highly regulated processes and require the concerted action of multiple membrane Pi transport systems –23. AmonFunctional characterization of genes in a number of mutants with altered response to Pi depletion have led to the identification of several different regulatory mechanisms controlling the adaptive responses to Pi starvation. These regulatory mechanisms include transcriptional regulation by transcription factors such as PHR1, WRKY6, WRKY42, WRKY45, WRKY75, ZAT6, bHLH32, ERF070 and MYB62 –47, postArabidopsis thaliana BOTRYTIS-INDUCED KINASE1 (BIK1) encodes a plasma membrane-localized receptor-like protein kinase and plays critical roles in Botrytis cinerea resistance [bik1 mutant plants showed an altered root architecture [BIK1 in Pi starvation response in Arabidopsis. Therefore, we investigated whether BIK1 functions in Pi starvation response and our results demonstrate that BIK1 plays a role in regulation of Pi homeostasis in Arabidopsis.The sistance . Recent sistance –73 and dsistance . The bikitecture , similaritecture , indicatbik1 plants produced shorter primary roots and longer and significantly more root hairs and lateral roots than WT plants [BIK1 is responsive to Pi starvation stress by analyzing the expression patterns of BIK1 in seedlings grown under + Pi and –Pi conditions. As shown in Fig. BIK1 was detected in roots, shoots and leaves of seedlings grown under + Pi condition and no significant changes in BIK1 expression was observed during the experiment period. However, expression level of BIK1 was markedly induced with similar patterns in roots, shoots and leaves of seedlings after transferring to medium without Pi supplement  and –Pi conditions for 27 days were measured. As shown in Fig. bik1 plants as compared to WT plants under both + Pi and –Pi conditions. When grown under + Pi condition, total Pi contents in roots and leaves of bik1 plants were 0.43 and 1.12 times higher over those in WT plants, respectively  and starvation (−Pi) conditions. When compared with WT seedlings, the bik1 seedlings grown under + Pi condition showed shorter primary root and more lateral roots  or without Pi (−Pi) for 5 days. When grown under + Pi condition, the expression levels of AT4 [WRKY75 [PHT1;2 and PHT1;4 [bik1 seedlings were comparable to those in WT seedlings, while the expression of PHR1 [bik1 seedlings was reduced  is involved in the metabolic adaptations of Pi starvation [The maintenance of cellular Pi homeostasis in plants involves complicated regulatory mechanisms. Several studies have demonstrated that posttranslational modifications such as phosphorylation, sumoylation and ubiquitination of regulatory proteins play critical roles in Pi starvation responses , 55, 57.arvation  and actiarvation . These farvation , plays aarvation –73.BIK1 could be induced by infection with B. cinerea as well as treatments with some well-known defense signaling molecules [BIK1 expression and determination of BIK1 promoter activity in stable BIK1pro::GUS transgenic seedlings demonstrate that BIK1 can be induced by Pi starvation and Pi starvation-induced expression of BIK1 in root and shoot tissues initiated earlier than in leaf tissues  for its full activity in plant immune responses [Several lines of evidence presented in this study support that BIK1 functions in Pi starvation response. Earlier study has shown that expression of olecules . Our qRTues Fig. . The patand ZAT6 , 40, 44.oot Fig.  that is esponses , 70. In bik1 seedlings showed a significant change in root architecture and the primary root length of bik1 plants was significantly decreased  and bik1  mutant [4NO3, 1.88 mM KNO3, 0.31 mM MgSO4, 0.1 mM MnSO4, 0.03 mM ZnSO4, 0.1 mM CuSO4, 0.3 mM CaCl2, 5.0 mM KI, 0.1 mM CoCl2, 0.1 mM FeSO4; 0.1 mM EDTA, 0.1 mM H3BO3, 1 mM Na2MoO4.2H2O, 3 g/L sucrose, 10 g/L agar, pH 5.8. When treated for Pi sufficiency (+Pi) and Pi deficiency (−Pi), the medium was supplemented with 1 mM KH2PO4, 10 μM KH2PO4 and 0.99 mM KCl. For hydroponic cultivation, seedlings at 5 ~ 7-leaf stage were transferred to 1/2 Hoagland solution for short adaption and then transferred to hydroponic solution containing 250 μM Pi or no Pi [−1 · m−2 photons m−2 sec−1 of intensity) and 8 h dark cycle at 22 ± 2 °C with 60 % relative humidity.) mutant  were useSeven-day-old seedlings grown on 1/2 MS medium were transferred to Petri dishes and treated for + Pi or –Pi for another 7 days. Length of main root, number and length of the lateral root were measured. Total numbers of root hairs in a 5 mm region from root tip were recorded. Data were recorded from 15 individual plants from each treatment.2O = 18:1:81, V/V/V) and boiled for 90 s. The mixtures were kept overnight in dark at room temperature, centrifuged for 40 min at 5000× g, and the absorbance (A) was measured at 535 nm and 650 nm. The A535 values were corrected with the A650 values using formula A535 = A535-2.2 × A650. Anthocyanin contents were calculated according to a standard curve prepared with the same protocol.Measurement of anthocyanin was performed as described previously . Briefly4MoO4, 2.5 N H2SO4, and 35 μM ascorbic acid). The reactions were incubated at 45 °C for 20 min and the absorbance at 650 nm was measured spectrophotometrically. Contents of total Pi were calculated according to the Pi standard curve prepared with the same procedure.Total Pi content was quantified according to the U.S. Environmental Protection Agency Method 365.2 with minor modifications . Brieflyp-nitrophenol sodium phosphate and 2.8 mL buffer were kept at 30 °C for 10 min, with shaking occasionally, and terminated by addition of 1 mL 0.5 M NaOH. The absorbance of the reactions was determined spectrophotometrically at 400 nm and the activity of acid phosphatase was calculated from the production of p-nitrophenol. Total protein was estimated using Bradford’s reagent and the total acid phosphatase activity was expressed as mU/mg protein. Staining of acid phosphatase activity was performed as described by Tomscha et al. [Activity of acid phosphatase was quantified using the pNPP hydrolysis assay according to a previously described method . Brieflya et al.  with mina et al. .2O2, 30 mg samples from 4-week-old plants were completely ground, followed by addition of 200 μL 20 mM K2HPO4 (pH6.5) phosphate buffer. Quantification of H2O2 was performed using a commericial kit  according to the manufactruer’s recommendation. Detection of superoxide anion was performed by the nitroblue tetrazolium (NBT) staining [3 and 0.1 % NBT for 30 min, cleared by boiling in 96 % ethanol, remained in 50 % ethanol before taking photos.For quantification of Hstaining . Four-we–△△CT method with three independent biological replicates.Total RNA was extracted using TRIZOL reagent  according to the manufacturer’s instructions. First strand cDNA was synthesized from 500 ng of total RNA using SuperScript III Kit . The qPCR reactions contained 12.5 μL SYBR Premix Ex TaqTM , 0.1 μg cDNA and 7.5 pmol of each of gene-specific primers in 25 μL and were conducted on a CFX96 real-time PCR system . Gene-specific primers used were as followings: BIK1-q-F, 5′-ACT TAT GGG TAC GCC GCG CCT GAG T-3′; BIK1-q-R, 5′-GGC ACG GAC CAC TTG GTC CA-3′; GUS-q-1F, 5′-AGG TGC ACG GGA ATG TTT CG-3′; GUS-q-1R, 5′-TGT GAG AGT CGC AGA ACA TT-3′; PHR1-q-1F, 5′-GTG ATT GGC ATG AAT GGG CTG AC-3′; PHR1-q-1R, 5′-CGC AAT TCC ACA GAC GGA GAA GG-3′; AT4-q-1F, 5′-GAT CGA AGT TGC CCA AAC GA -3′; AT4-q-1R, 5′-GAG CGA TGA AGA TTG CAT GAA G-3′; WRKY75-q-1F, 5′-GAG AAA TCC ACC GAA AAC TTC GAG CAT AT-3′; WRKY75-q-1R, 5′-GCA TGG TTT TTC TTT TCA ACA CAC GTA AAA TGT A-3′; PHT1.2-q-1F, 5′-AGG GCA AGT CCC TCG AAG AAC T-3′; PHT1.2-q-1R, 5′-ATC AAA CAA ACC ACA AAC AAC TCC ACA T-3′; PHT1.4-q-1F, 5′-TTG CTC CTA ATT TTC CTG ATG CT -3′; PHT1.4-q-1R, 5′-TGT GCC GGC CGA AAT CT-3′. Pri-miRNA399a-1F, 5′-TGG CAG GAA ACC ATT ACT TAG ATC T-3′; Pri-miRNA399a-1R, 5′-TCA CTA ATT AAA AGC AAT GCA TAA AGA GA-3′; Pri-miRNA399d-1F, 5′-TTA CTG GGC GAA TAC TCC TAT GG-3′; Pri-miRNA399d-1R, 5′-ATT TTA CTT GCA TAT CTA GCC AAT GC-3′; PHO2-q-1F, 5′-AGG TTT GAA GCT CCA CCC TCA-3′; PHO2-q-1R, 5′-CCC AAG ATG TGA TTG GAG TTC C-3′; UBQ10-q-1F, 5′-GGC CTT GTA TAA TCC CTG ATG AAT AAG-3′; UBQ10-q-1R, 5′-AAA GAG ATA ACA GGA ACG GAA ACA TAG T-3′. Relative gene expression levels were calculated using 2BIK1 start codon was PCR amplified using primers AtBIK1-GUS-1F  and AtBIK1-GUS-1R  and cloned into vector pCAMBIA1301. Transformation of wild-type Col-0 plants was performed using the floral dip infiltration method mediated by Agrobacterium tumefaciens strain GV3101. Plants of T2 generations from kanamycin-resistant transformants were used for GUS histochemical staining [A 2 kb sequence upstream of the staining .B. cinerea, Botrytis cinerea; BCIP, 5-bromo-4-chloro-3-indolyl phosphate; BIK1, Botrytis-induced kinase1; MKK, mitogen-activated protein kinase kinase; MPK, mitogen-activated protein kinase; NBT, nitroblue tetrazolium; PAMP, pathogen associated molecular pattern; Pht1, PHOSPHATE TRANSPORTER1; Pi, phosphate; −Pi, Pi deficiency; pNPP, p-nitrophenol sodium phosphate; PPC1, phosphoenolpyruvate carboxylase 1; qRT-PCR, quantitative reverse transcription PCR; ROS, reactive oxygen species; WT, wild type+Pi, Pi sufficiency;


Caenorhabditis elegans. Tissue-specific biotinylation is achieved by the expression in select tissues of the bacterial biotin ligase BirA, which biotinylates proteins tagged with the Avi peptide.Affinity purification followed by mass spectrometry (AP/MS) is a widely used approach to identify protein interactions and complexes. In multicellular organisms, the accurate identification of protein complexes by AP/MS is complicated by the potential heterogeneity of complexes in different tissues. Here, we present an in vivo biotinylation-based approach for the tissue-specific purification of protein complexes from C. elegans development. MAPH-1.1 co-purified specifically with DLG-1 purified from neurons, and shared limited homology with the microtubule-associated protein MAP1A, a known neuronal interaction partner of mammalian DLG4/PSD95. A CRISPR/Cas9-engineered GFP::MAPH-1.1 fusion was broadly expressed and co-localized with microtubules.We generated N- and C-terminal tags combining GFP with the Avi peptide sequence, as well as four BirA driver lines expressing BirA ubiquitously and specifically in the seam and hyp7 epidermal cells, intestine, or neurons. We validated the ability of our approach to identify bona fide protein interactions by identifying the known LGL-1 interaction partners PAR-6 and PKC-3. Purification of the Discs large protein DLG-1 identified several candidate interaction partners, including the AAA-type ATPase ATAD-3 and the uncharacterized protein MAPH-1.1. We have identified the domains that mediate the DLG-1/ATAD-3 interaction, and show that this interaction contributes to The method we present here is able to purify protein complexes from specific tissues. We uncovered a series of DLG-1 interactors, and conclude that ATAD-3 is a biologically relevant interaction partner of DLG-1. Finally, we conclude that MAPH-1.1 is a microtubule-associated protein of the MAP1 family and a candidate neuron-specific interaction partner of DLG-1.The online version of this article (doi:10.1186/s12915-016-0286-x) contains supplementary material, which is available to authorized users. Drosophila melanogaster cells II; mibIs31V; dlg-1(ok318)XBOX107: mibIs14I; mibIs31V; dlg-1(ok318)XBOX108: mibIs35  II; mibIs31  V ; dlg-1(ok318) XBOX109: mibIs33I; mibIs30XBOX110: mibIs7II; mibIs30XBOX111: mibIs14I; mibIs30XBOX112: mibIs33I; mibIs23V; lgl-1(tm2616)XBOX113: mibIs7II; mibIs23V; lgl-1(tm2616)XBOX114: mibIs14I; mibIs23V; lgl-1(tm2616)XBOX115: mibIs40III; mibIs33IBOX116: mibIs7II; mibIs40IIIBOX117: mibIs14I; mibIs40IIIBOX118: mibIs35  II; mibIs40  IIIBOX119: mibIs7  II; mibIs25  XBOX133: mibIs33  I; mibIs25  XBOX134: mibIs14  I; mibIs25  XBOX135: maph-1.1(mib12[GFP::maph-1.1]) IBOX188: maph-1.1(mib12[GFP::maph-1.1]) I; mibIs35  II; mibIs40  IIIBOX209: maph-1.1(mib12[GFP::maph-1.1]) I; mibIs35  II; mibIs31  VBOX212: atad-3(ok3093) II; hels97 IV (referred to as ATAD-3FL)SV1311: atad-3(ok3093) II; hels98 IV (referred to as ATAD-3ΔETAV).SV1312: 2HPO4, 3 mM NaH2PO4, 140 mM NaCl, 5 mM KCl, 0.05 % Tween-20) for 1 h at room temperature. For detection of GFP, blots were incubated with rabbit polyclonal anti-GFP  or anti-biotin  in PBST + 5 % skim milk for 1 h at room temperature, washed with PBST three times for 10 min each at room temperature, incubated with anti-rabbit IgG antibody conjugated to horseradish peroxidase  for 45 min at room temperature, washed with PBST three times for 10 min each at room temperature, and finally washed once with PBS at room temperature for 10 min. Blots were developed using enhanced chemiluminescent western blotting substrate (Bio-Rad Laboratories).Protein samples were separated on 10 % acrylamide gels, and subjected to western blotting on polyvinylidene difluoride membrane . Blots were blocked with 5 % skim milk in phosphate-buffered saline with Tween  was added as food source. Animals were allowed to develop until the L3/L4 stage in an incubator at 20 °C shaking at 200 rpm. To harvest the animals, the culture was transferred to 50 ml conical tubes and cooled on ice for 20 min. Animals were then pelleted by centrifugation . After aspirating the supernatant, animals were pooled in a single 50 ml tube, and washed twice in ice-cold M9 lacking MgSO4  for 15 min at 4 °C. The lysate was cleared at 13,000 rpm for 15 min at 4 °C. For immunoprecipitations, 1 mg of total protein was used with either 1 μl mouse anti-PSD95 antibody  non-covalently bound to 5 μl protein G Sepharose beads, 1 μl rabbit anti-ATAD-3 antibody , and a Photometrics Evolve 512 EMCCD camera at a final magnification of 110 nm per pixel, including an additional magnification introduced by an extra intermediate lens 2.0X (Edmund Optics). For FRAP experiments we used a similar microscope setup equipped with an ILas system , with the laser set at 100 % laser power and a Plan Apo × 60 NA 1.40 oil lens. Imaging was performed at 1 or 2 frames per second. Microscopy of fixed samples was performed on a Zeiss LSM700 laser scanning confocal microscope equipped with a × 63 Plan-Apochromat 1.4 NA objective; 405 nm, 488 nm, 555 nm, and 633 nm lasers; and the following emission filters: SP490 (400–490 nm), SP555 (400–555 nm), SP640 (400–640 nm), BP490-555 (490–555 nm), LP560 (560–750 nm), LP640 (640–750 nm), and BP592-662 (592–662 nm). The LSM700 was controlled by the Zen software package. Maximum projections were generated from a series of slices of a Z-stack with ImageJ and processed with Adobe Photoshop CS6 and Adobe Illustrator CS6.Images were processed and analyzed in ImageJ. For the FRAP analysis, the average gray value of a 100 × 10 pixel region in the FRAP region or a similar non-bleach area was calculated and background subtracted frame-by-frame by subtracting the average intensity of an empty, non-bleached area. FRAP recovery was calculated as the recovery from the first frame after bleach (set to 0) normalized to the average of the five frames before bleach.Microtubule depolymerization speeds were calculated by making kymographs of the acquired movies using the KymoResliceWide plugin in ImageJ and measuring the distance and time of a depolymerization event. Graphs in Additional file


Complete enzymatic hydrolysis of xylan to xylose requires the action of endoxylanase and β-xylosidase. β-xylosidases play an important part in hydrolyzing xylo-oligosaccharides to xylose. Thermostable β-xylosidases have been a focus of attention as industrially important enzymes due to their long shelf life and role in the relief of end-product inhibition of xylanases caused by xylo-oligosaccharides. Therefore, a highly thermostable β-xylosidase with high specific activity has significant potential in lignocellulose bioconversion.Geobacillus sp. strain WSUCF1 and expressed in Escherichia coli. Recombinant β-xylosidase was active over a wide range of temperatures and pH with optimum temperature of 70°C and pH 6.5. It exhibited very high thermostability, retaining 50% activity at 70°C after 9 days. WSUCF1 β-xylosidase is more thermostable than β-xylosidases reported from other thermophiles (growth temperature ≤ 70°C). Specific activity was 133 U/mg when incubated with p-nitrophenyl xylopyranoside, with Km and Vmax values of 2.38 mM and 147 U/mg, respectively. SDS-PAGE analysis indicated that the recombinant enzyme had a mass of 58 kDa, but omitting heating prior to electrophoresis increased the apparent mass to 230 kDa, suggesting the enzyme exists as a tetramer. Enzyme exhibited high tolerance to xylose, retained approximately 70% of relative activity at 210 mM xylose concentration. Thin layer chromatography showed that the enzyme had potential to convert xylo-oligomers  into fermentable xylose. WSUCF1 β-xylosidase along with WSUCF1 endo-xylanase synergistically converted the xylan into fermentable xylose with more than 90% conversion.A gene encoding a highly thermostable GH39 β-xylosidase was cloned from Properties of the WSUCF1 β-xylosidase i.e. high tolerance to elevated temperatures, high specific activity, conversion of xylo-oligomers to xylose, and resistance to inhibition from xylose, make this enzyme potentially suitable for various biotechnological applications. Xylan represents the second most abundant polysaccharide in the biosphere after cellulose, and therefore is a potential source of sugars for the production of biofuels. Complete degradation of xylan to monomeric sugars requires synergistic action of several xylanolytic enzymes. Endo-1,4-β-xylanases EC 3.2.1.8) hydrolyze the xylan backbone and β-D-xylosidases (EC 3.2.1.37) sunder the resulting xylooligomers to free xylose [.1.8 hydrGeobacillus, Thermoanaerobacter, Thermatoga, and Thermoanaerobacterium [Xylanases have also been suggested to complement the activity of cellulases for the efficient hydrolysis of lignocellulose ,3. Kumaracterium ,7-11. Thacterium .8 structure and a conserved catalytic machinery [Geobacillus sp. strain WSUCF1. This strain was isolated from samples collected from a compost facility and is an aerobic spore forming thermophile [The bacterial β-D-xylosidases mainly belong to glycosyl hydrolase families GH 3, 30, 39, 43, 52, and 54. Family 39 enzymes belong to the largest glycoside hydrolase clan (GH-A), whose members share a TIM-barrel (β/α)achinery . Reportsrmophile .Geobacillus spp. Therefore, the WP_020755811 β-xylosidase was selected for further study.Genome sequence of WSUCF1 revealed several genes encoding glycoside hydrolases. Out of 865 ORFs for carbohydrate metabolism, 70 ORFs were found to be involved in polysaccharide degradation . It was Geobacillus spp. . β-xylosidases from different GH families  made separate clusters on the phylogenetic tree , Geobacillus sp. Y412MC61 (YP_003253769), and Geobacillus sp. G11MC16 (ZP_03147822), respectively. β-xylosidase from Geobacillus thermoleovorans (DQ345777) belonging to GH43 family and Geobacillus stearothermophilus (ABI49956) belonging to GH52 family showed only 29 and 33% identity respectively.Alignment of the amino acid sequences of WSUCF1 β-xylosidase (WP_020755811) revealed 98, 95, and 91% identity with GH39 β-xylosidases from Geobacillus sp. strain WSUCF1 genome. The gene was cloned in the pRham™ N-His SUMO Kan vector, expressed in E. coli, and purified using Ni-NTA affinity chromatography. SDS-PAGE analysis of the purified β-xylosidase showed a prominent band of 58 kDa  with optima at 6.5 Figure A. β-xylo2+ and Hg2+. More than 70% activity was retained in the presence of Zn2+, Co2+, Ni2+, Ca2+, Mn2+, and Mg2+ whereas it lost 73 and 100% activity in the presence of Cu2+ and Hg 2+, respectively.WSUCF1 β-xylosidase was highly thermostable in the range of 50-70°C Figure . The enzm and Vmax values for WSUCF1 β-xylosidase with paranitrophenyl- beta-xylopyranoside (pNP-X) were 2.38 mM and 147.0 U/mg, respectively. The substrate specificity of the purified WSUCF1 β-xylosidase was determined using various substrates. WSUCF1 β-xylosidase was highly active on p-nitrophenyl β-D-xylopyranoside (pNPX). No specificity was observed for other p-NP substrates such as p-nitrophenyl β-D-cellobioside, p-nitrophenyl α-D-xylopyranoside, p-nitrophenyl α-L-arabinofuranoside, and p-nitrophenyl β-D-glucopyranoside (data not shown). In addition, polysaccharides such as oat-spelt xylan, beechwood xylan, and birchwood xylan did not serve as substrates for WSUCF1 β-xylosidase.The kinetic parameters were calculated from Lineweaver-Burke plots of specific activities at various substrate concentrations (data not shown). The KEffect of various xylose concentrations on WSUCF1 β-xylosidase activity was investigated. Results showed that concentrations of up to 50 mM xylose did not have much inhibitory effect. The enzyme retained 85 and 66% of its activity with 50 and 210 mM xylose concentrations, respectively  results showed that WSUCF1 β-xylosidase actively hydrolyzed xylo-oligosaccharides Figure . It was Mixtures of recombinant WSUCF1 endo-xylanase  and WSUCGeobacillus spp. are from GH52 and GH43 family. For example, β-xylosidases of Geobacillus thermodenitrificans TSAA1 [Geobacillus stearothermophilus [, Geobacillus pallidus [Bacillus stearothermophilus 21 [Geobacillus thermoleovorans IT-08 [Geobacillus thermoleovorans IT-08 [Cellulose and hemicellulose components of lignocellulosic biomass are a renewable source of sugars. The main component of hemicellulose is xylan which can be converted into xylose using an effective enzyme system of endo-xylanase and β-xylosidase. β-xylosidase is known to be the key enzyme for converting xylo-oligosaccharides to xylose. Thermostable β-xylosidases produced from thermophiles are of importance due to their prolonged activity during the hydrolysis which may allow a reduced dose of enzyme and cost-efficient conversion . Out of ns TSAA1 , Geobacimophilus , Geobacipallidus , Bacilluhilus 21  belongedns IT-08  belongedThermoanaerobacterium saccharolyticum JW/SL-YS485 [Geobacillus thermoleovorans IT-08 [G. thermodenitrificans TSAA1 [G. pallidus [Paecilomyces thermophila - 43.4 U mg−1 [Aspergillus awamori X-100 - 4.2 U mg−1 [−1.A high specific activity of β-xylosidase enzyme for xylo-oligosaccharides is an important attribute for efficient enzymatic hydrolysis of the hemicellulose component of lignocellulose . WSUCF1 ns IT-08 , G. therns TSAA1  and G. ppallidus  that sho4 U mg−1 ; Aspergi2 U mg−1  and commThermoanaerobacterium sp. has been reported to exist as a tetramer [Omitting heat denaturation prior to SDS-PAGE resulted in an increase in the apparent mass of the β-xylosidase from 58 kDa to 230 kDa. Interestingly, only the 230 kDa protein displayed xylosidase activity in the zymogram. This suggests that the WSUCF1 β-xylosidase is resistant to full denaturation by the detergent alone, and retains at least some of its quaternary structure which is necessary for successful recovery of catalytic activity following SDS-PAGE. Based on the 4-fold increase in apparent mass, it appears that that the WSUCF1 β-xylosidase exists in tetrameric form. GH39 β-xylosidase from tetramer . PropertGeobacillus stearothermophilus T-6 also exhibited maximum activity at 6.5 [Geobacillus thermoleovorans IT-08 [Geobacillus thermodenitrificans TSAA1 [G. pallidus [Geobacillus spp. had a narrow pH range except G. pallidus [Geobacillus thermoleovorans IT-08 [Geobacillus thermodenitrificans TSAA1 [G. pallidus [WSUCF1 β-xylosidase was active across a broad pH range of 4.5-9.5 with maximum activity at pH 6.5. β-xylosidase from y at 6.5  whereas ns IT-08 , Geobacins TSAA1  and G. ppallidus  showed tpallidus . At pH 9ns IT-08  and Geobns TSAA1 , respectpallidus  retainedGeobacillus stearothermophilus T-6 , Geobac8 , Geobac°C, , Geopallidus . Geobacins IT-08  β-xylosiGeobacillus pallidus [Bacillus halodurans XylBH39 and XylBH43 displayed approximate half-life values of 2.40 and 0.05 h, respectively at 60°C, Geobacillus thermoleovorans IT-08 [Thermoanaerobacterium saccharolyticum JW/SL-YS485 [Geobacillus thermoleovorans IT-08 [Geobacillus sp. WSUCF1 appears to be among the most thermostable β-xylosidases from thermophiles having optimum growth temperatures of ≤ 70°C reported to date  were significantly different from those of the other reported β-xylosidases. Compared to WSUCF1 β-xylosidase, Thermoanaerobacterium saccharolyticum JW/SL-YS485 β-xylosidase [Geobacillus thermodenitrificans TSAA1 β-xylosidase [m of 28 mM and 2.8 mM, respectively, which shows WSUCF1 β-xylosidase has more specificity towards substrate.The kinetic properties of WSUCF1 β-xylosidase . Amino acid sequence of Geobacillus sp. strain WSUCF1 β-xylosidase was used as a BLAST query for seeking other homologous amino acid sequences. The sequence alignment was created with ClustalW program. Sequences were aligned manually using the Mega 5.2 software. Phylogenetic relationship was inferred using the Neighbor-Joining (NJ) method as described earlier [Nucleotide sequence from the whole genome of n WSUCF1  was tran earlier .Geobacillus sp. strain WSUCF1 (simply referred to as WSUCF1), PCR primers (F 5’- CGCGAACAGATTGGAGGT AAGGTTGTAAACGTGCCAAGC-3’ and R 5’- GTGGCGGCCGCTCTATTATGAAGAATA CGATGTCATTTC-3’) containing homologous sequences to the ends of the pRham N-His SUMO Kan Vector   were designed to amplify the β-xylosidase gene sequence. PCR product containing the gene of interest is cloned as a fusion to cleavable SUMO  tag under the control of the L-rhamnose-inducible rhaPBAD promoter harbored on the pRham™ N-His SUMO Vector. PCR conditions were as follows: initial denaturation at 94°C for 2 min followed by 25 cycles of 94°C for 15 sec, 55°C for 15 sec and 72°C for 1 min 30 sec in 50 μl reaction with final extension of 10 min at 72°C. The amplicon was cloned into the vector according to the manufacturer’s instructions, and the resulting plasmid transformed into E. coli chemically competent cells . After transformation, E. coli cells were plated on agar plates containing Terrific broth (TB) and 30 μg/ml kanamycin, incubated for 36 hours at 37°C. The positive clones were screened using colony PCR and confirmed by β-xylosidase activity assay as described below. Cycling conditions for colony PCR were: initial denaturation 94°C, 5 sec; 25 cycles of 94°C for 15 sec, 55°C for 15 sec and 72°C for 1 min; extension 72°C 10 min.To amplify the 1509 bp β-xylosidase gene sequence from 2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0). After binding, the column was washed with wash buffer . The bound enzyme was eluted with elution buffer  at a flow rate of 0.5 ml/min collecting 1 ml fractions. Fractions containing β-xylosidase activity were pooled. To remove the His tag from the protein, the protein was cleaved with SUMO specific protease which also contains the His tag (as per manufacturer’s protocol). After the cleavage, the protein was separated from the SUMO protease and the His tag by re-performing metal affinity chromatography with Ni-NTA agarose resin column. His tag and SUMO protease bound to the resin while the protein was eluted. The homogeneity of the purified enzyme was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as described below. Protein concentrations in the samples were determined using 2-D Quant kit .A positive clone was grown in 250-mL flask containing 50 ml Terrific broth (TB), kanamycin (30 μg/ml), and the expression of β-xylosidase was induced by addition of 0.2% rhamnose followed by incubation at 37°C for 48 hours. Cells were centrifuged at 10,000 rpm for 15 minutes. Ten ml of clear culture supernatant was applied to a Ni-NTA agarose resin column (1.5 cm × 5 cm) equilibrated with equilibration buffer , and 20 μL of appropriately diluted enzyme. The reaction was carried out at 70°C for 10 min and then stopped by the addition of 100 μL of sodium carbonate (2% w/v). The p-nitrophenol released was determined by measuring the absorbance of each sample at 410 nm. One unit of enzyme activity was defined as the amount of enzyme that released 1 μmol of p-NP per minute under the standard assay conditions.SDS-PAGE was performed as described by Laemmli (1970) . The molActivity profile of β-xylosidase was determined at different pH 3–10. The enzyme assays were carried in the following discontinuous buffer system under standard assay conditions: 100 mM sodium citrate (pH 3–6), sodium phosphate (pH 6–8), Tris–HCl (pH 7.5-9), or glycine-NaOH (pH 8.5-10). The optimum pH was used to determine the optimum temperature for the β-xylosidase. Experiments were carried out in the temperature range of 40-80°C. In order to assess thermostability of β-xylosidase, 1 ml of enzyme was incubated at 50-80°C for varying time intervals. Subsamples (20 μl) were removed at specific time intervals over the period of incubation. The residual enzyme activity was measured and plotted against time. Initial activity was assumed to be 100% and residual enzyme activity reported as percentages of the initial activity during the incubation period.m and Vmax) for the purified recombinant β-xylosidase were determined by measuring the enzyme activity using 0–15 mM pNP-X as substrate in 50 mM phosphate buffer (pH 6.5) at 70°C. The data was plotted according to the Lineweaver-Burk method to calculate Km and Vmax values.Kinetic parameters (KSubstrate specificity of the purified enzyme was investigated under standard assay conditions as described above. Activities towards p-nitrophenyl derivatives were measured by the rate of p-nitrophenol formed during hydrolysis from 2.5 mg/ml of the substrates in 50 mM phosphate buffer (pH 6.5) at 70°C for 10 min, and detected by spectrophotometry at 410 nm. Substrates used were p-nitrophenyl β-D-xylopyranoside, p-nitrophenyl α-L-arabinofuranoside, p-nitrophenyl β-D-cellobioside, p-nitrophenyl β-D-glucopyranoside.2+, Zn2+, Ba2+, Ni2+, Mn2+,Ca2+, Co2+, Cu2+ and Al3+ to the enzyme reaction mixtures at a final concentration of 1 mM. The enzyme reaction mixtures were incubated for 10 min at 70°C, and then activities were measured as described above. The activity of the enzyme without adding any metal ions was considered as 100%.The effect of metal ions on enzyme activity of β-xylosidase was determined by adding chloride salts of MgThe inhibitory effect of different concentrations of xylose was determined by incubating 20 μl enzyme solution, 80 μl of 2.5 mg/ml pNPX dissolved in 100 mM phosphate buffer (pH 6.5) and varying amounts of xylose with a final concentration of 0–300 mM at 70°C for 10 min. Residual β-xylosidase activities were measured as described above.Four μl of 1.5% (w/v) xylo-oligosaccharides was incubated with 0.05 U of the β-xylosidase in 50 mM phosphate buffer (pH 6.5) at 70°C for 1, 2 and 6 h. Aliquots were withdrawn at 0, 1, and 2 h, the reactions were stopped by boiling for 5 min. Products of enzymatic hydrolyses were analyzed by spotting on silica gel plates 60 F-254 . The plates were developed with butanol-acetic acid-water  solvent system followed by heating for few minutes at 105°C in an oven, after spraying the plates with a methanol-sulfuric acid mixture  . A mixtuGeobacillus sp. WSUCF1, [2SO4 as mobile phase.For enzymatic hydrolysis of xylan, the reaction mixture containing 5 mg purified recombinant endoxylanase/g xylan , 2.5 mg