Their structures were elucidated on the basis of extensive spectroscopic analysis. Antrodols A–C (1–3) are first examples of humulane-type sesquiterpenes isolated from cultures of higher fungi, and antrodol A (1) was the first report of humulane-type sesquiterpene with a methyl rearranged at C-3. All compounds were evaluated in the enzyme inhibition assay against two protein-tyrosine phosphatases (PTPs): MEG2 and PTP1Bc.Three new humulane-type sesquiterpenes, antrodols A–C (The online version of this article (doi:10.1007/s13659-014-0032-4) contains supplementary material, which is available to authorized users. Antrodiella albocinnamomea has resulted in the isolation of three new humulane-type sesquiterpenes, antrodols A–C (1–3). Fungal sesquiterpenes formed via the humulane-protoilludane biosynthetic pathway are characteristic chemical markers for the subdivision Basidiomycota. The largest group of sesquiterpenes belonging to the classes of lactaranes, secolactaranes, marasmanes, isolactaranes, norlactaranes, and caryophyllanes were believed to be derived from humulane +, suggesting four degrees of unsaturation. Its IR data exhibited absorption bands for a hydroxy group (3462 cm−1) and an olefinic bond (1640 cm−1). The 1D NMR data of 2 strongly resemble to those of -2,3-epoxy-6,9-humuladien-5-ol-8-one(4)  were upfield in 2 , suggesting the double bond in 4 was replaced by an epoxide ring in 2 and the carbonyl was changed to a methylene. In the HMBC spectrum . From these data, compound 2 was determined to be 3,4:10,11-diepoxy-6E-humulen-8α-ol, named antrodol B.Compound 3 was assigned the same molecular formula as that of 2 (C15H24O3) by HREIMS at m/z 275.1622 [M]+ . The IR and 1D NMR data were extremely similar to those of 2, indicating that compound 3 was also a humulane-type sesquiterpene. Compared to 2, the main differences were below: (I) the epoxide ring at C-3 and C-4 in 2 was replaced by a double bond in 3; (II) the ∆6 olefinic bond was replaced by an epoxide ring; (III) the hydroxy group was placed at C-5 in 3 rather than at C-8 in 2. Those assignments were deduced by 2D NMR data analysis. In the 1H-1H COSY spectrum, the cross-peaks of H-3/H-4/H-5 indicated a CH=CH–CH moiety in 3; the hydroxy was at C-5 due to the downfield chemical shift at δ 81.8; the epoxide ring at C-6 and C-7 was deduced from the characteristic 13C NMR signals at δ 66.0 and 58.8. The above assignments were corresponded with the HMBC correlations from Me-13 to C-3, from 5-OH to C-4, C-5 and C-6, from Me-14 to C-5, C-6 and C-7 as shown in Fig. 3 was established by an ROESY experiment. In the ROESY spectrum, the ROESY correlations between Me-12 and H-4 and H-11; H-5, H-7, H-11 and Me-14 indicated that H-4, H-5, H-7, H-11, Me-12 and Me-14 were in the same side (assigned as α-orientation) while the ROESY correlations between Me-13 and H-3, H-1β; and H-1β and Me-15 demonstrated that H-3, Me-13 and Me-15 were in the β-orientation. The E-geometry of ∆3-double bond was further determined by the large coupling constant of 3JH-3/H-4 (15.9 Hz). Consequently, compound 3 was determined to be 6,7:10,11-diepoxy-3E-humulen-5β-ol, and named antrodol C.Compound 1–3) were evaluated in the enzyme inhibition assay against three PTPs: MEG2 and PTP1Bc. As summarized in Table 1) was demonstrated to have moderate inhibitory activities against protein-tyrosine phosphatase MEG2 and PTP1Bc with IC50 values of 8.0 and 10.0 μg/mL, respectively. Antrodol C (3) was showed mild inhibitory activities against protein-tyrosine phosphatase PTP1Bc with IC50 values of 15.1 μg/mL.The biological activities of antrodols A–C . ESIMS and HREIMS were recorded with an API QSTAR Pulsar I spectrometer. Preparative MPLC was performed on a Buchi apparatus equipped with Buchi fraction collector C-660, Buchi pump module C-605 and manager C-615. Column chromatography was performed on silica gel  and Sephadex LH-20 for chromatography was purchased from Amersham Biosciences. Fractions were monitored by TLC and spots were visualized by heating silica gel plates sprayed with 10 % HAntrodiella albocinnamomea was provided by Prof. Yu-Cheng Dai, Institute of Microbiology, Beijing Forestry University, and fermented by Mr. Zheng-Hui Li, Kunming Institute of Botany. A voucher specimen is deposited in the Institute of Microbiology, Beijing Forestry University. The culture medium consisted of glucose (5 %), peptone from porcine meat (0.15 %), yeast powder (0.5 %), KH2PO4 (0.05 %), and MgSO4 (0.05 %).The fermentation was carried out at 27 °C for 360 h with aeration at 0.4 vvm (air volume/culture volume/minute) and agitation at 150 rpm on a 100 L fermenter.The fermented whole broth (70 L) was filtered through cheesecloth to separate into supernatant and mycelia. The former was concentrated under reduced pressure to about a quarter of the original volume and then extracted three times with ethyl acetate (20 L×3) at room temperature, and the organic solvent was evaporated to dryness under reduced pressure to afford a brown crude extract. The mycelia was extracted three times with acetone (10 L×3). The acetone solution was concentrated under reduced pressure to afford an aqueous solution. The aqueous solution was extracted three times with ethyl acetate (3 L×3), and the ethyl acetate solution was evaporated to dryness under vacuum to obtain the crude extract of the mycelia. Both of the extracts were combined for further purification (45 g).2O as eluent. Fraction 3B was then separated by Sephadex LH-20 eluting with acetone to give 1 (2.6 mg). Fraction 4 was eluted with petroleum ether–acetone (2:1), was further purified by MPLC using MeOH/H2O as eluent to afford seven subfractions (4A–4G). 4C was then subjected to Sephadex LH-20 (acetone) and silica gel CC  to yield 2 (6.2 mg) and 3 (5.8 mg).The combined crude extract was then fractionated by silica gel column chromatography (CC) eluted with a gradient of petroleum ether–acetone (100:0–0:100) to obtain eight fractions. Fraction 3 was eluted with petroleum ether–acetone (3:1), was then purified into five subfractions (3A–3E) by MPLC using MeOH/Hα]D25  + 16.46 ; UV (MeOH) λmax (log ε) 202.8 (3.73) nm; IR (KBr) νmax 3440, 2925, 1635 cm−1; 1H and 13C NMR spectra data see Table m/z: 277 [M+Na]+; HREIMS: m/z 254.1887 [M]+.Colorless oil; [α]D25 + 75.20, ; UV (MeOH) λmax (log ε) 202.4 (3.53) nm; IR (KBr) νmax 3441, 2958, 2928 1631, 1387 cm−1; 1H and 13C NMR spectra data see Table m/z 275 [M+Na]+; HREIMS: m/z 275.1599 [M]+.An amorphous solid; D25 – 84.18, ; UV (MeOH) λmax (log ε) 201.2 (3.35), 229.2 (2.40) nm; IR (KBr) νmax 3462, 2962, 2928 1640, 1462, 1451, 1392 cm−1; 1H and 13C NMR spectra data see Table m/z 275 [M+Na]+; HREIMS: m/z 275.1622 [M]+.An amorphous solid; [E. coli and purified by Ni–NTA affinity chromatography [p-nitrophenol and the absorbance at 405 nm was recorded as the amount of p-nitrophenol. The IC50 value was determined by the non-linear curve fitting of the percentage inhibition versus inhibitor concentration plot. Ursolic acid  was used for the positive control [Human MEG2 and PIP1Bc with an N-terminal 6× His-tag were recombinantly expressed in tography –14. The  control . All theSupplementary material 1 (DOCX 1607 kb)Below is the link to the electronic supplementary material.


This report describes the crystal structure of highly substituted trifluoroazepane as an important building block of therapeutic interest. Data were collected with synchrotron radiation on a very small crystal. 15H16F6N4O3, was determined using synchrotron radiation on an extremely small crystal (0.015 × 0.01 × 0.01 mm). Although the diffraction was weak, leading to high residuals and a poor data-to-parameter ratio, the data allowed ready solution and refinement to reveal the entire structure. The solid-state structure is in accordance with the absolute configuration assigned based on that of the known starting material. The compound comprises a highly substituted seven-membered N-heterocyclic cation and a trifluoromethanesulfonate counter-anion. The title compound crystallizes with two independent cations (A and B) and anions (C and D) in the asymmetric unit. Two geminal F atoms, a single F atom, a benzyl ether and an azide group are substituted on consecutive C atoms between the NH2 and CH2 units of the azepan-1-ium ring system. The seven-membered rings adopt different conformations with the principal differences occurring in the CF2CHFCH2 segments of the ring systems. The geminal F atoms on the quaternary C atom exhibit essentially identical bond angles [109 (2) and 106 (2)°] in the two independent molecules. The two molecules associate as a dimeric unit via two C—H⋯F interactions. An extensive series of N—H⋯O, N—H⋯F, C—H⋯O, C—H⋯N, C—H⋯F and C—H⋯π contacts generate a three-dimensional network with cations and anions linked into ABCD repeat columns along a.The structure of the title compound, C However, about 20% of pharmaceuticals and 30% of agrochemicals have at least one fluorine atom , directly using synchrotron radiation.We have previously reported stereospecific deoxyfluorination reactions of substituted seven-membered N-heterocycles such as azepanes  with two sets of cations and anions (molecule A and B) in the asymmetric unit. Each cation has the same stereochemistry. An ORTEP view of the cation in molecule A, Fig. 1B cation and anion are labelled similarly but with trailing B characters after the atom numbers. The absolute configuration was assigned based on that of the known starting material.The compound crystallizes in a chiral space group  was carried out using protocols reported earlier  in the unit cell, respectively. Hence the X-ray structure reported here for (1) validates our conformational analysis methodology as reported earlier  Å. Each anion further connects to an A cation with O1C accepting three interactions and N1A as a bifurcated donor, leading to the formation of N1A—H1AA⋯O1C, N1A—H1AB⋯O1C and C4A—H4AA⋯O1C hydrogen bonds and generating et al., 1995A molecules along a. These columns are further supported by weak C7A—H7AA⋯Cg2 contacts (Cg2 is the mid-point of the C10A—C11A bond of the C8A–C13A phenyl ring), Fig. 5B cations are linked to D anions with O2D accepting three interactions and forming N1B—-H1BA⋯O2D, N1B—H1BB⋯O2D and C4B—H4BA⋯O2D hydrogen bonds. Unlike the AC system however, a C4B—H4BA⋯F1D hydrogen bond completes the B⋯D cation–anion contacts. These generate B—H7BA⋯Cg1 contacts (Cg1 is the midpoint of the C10B–C11B bond of the C8B–C13B phenyl ring) link adjacent B molecules, also forming columns of B cations and D anions along the a-axis direction, Fig. 6A and B cations are limited to very weak C12B—H12B⋯N4A hydrogen bonds linking adjacent columns of A and B cations, Fig. 7ABCD repeat unit in the direction of the a axis, Fig. 8c.In the crystal structure, et al. -4,5,6-trihydroxy-3-methyl azepanium chloride is the only one to be reported of a substituted azepanium salt, Li et al.  -6-Azido-5-benzyloxy-3,3,4-trifluoroazepane-1-carboxylic acid-tert-butyl ester  was dissolved in trifluoroacetic acid  at 298 K. The solution was allowed to stir for 5 min before the TFA was evaporated under an N2 flow. The reaction flask was kept under high vacuum  for 3 h to remove traces of TFA. A colorless, oily residue was obtained which was recrystallized from dichloromethane to give colorless needles characterized as (1) . 1H NMR  δ 7.44–7.34 , 4.93 , 14.7 Hz, 1H), 4.80 , 4.73 , 4.08 , 3.89–3.82 , 3.67–3.57 , 3.48 , 3.10 ; 13C NMR  δ 135.7, 129.0, 128.5, 128.5, 118.4 , 90.2 , 79.6 , 73.9, 60.6, 45.8 , 45.6.(4Uiso(H) = 1.2Ueq(N/C). Because of the lower reflections-to-parameter ratio, anisotropic displacement parameters of several atoms in the least-squares refinement had to be restrained using the RIGU command. These were applied to azide groups, atoms in the seven-membered and a few atoms in phenyl rings.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989015019416/sj5470sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989015019416/sj5470Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989015019416/sj5470Isup3.cmlSupporting information file. DOI: 1431203CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The layer stacking sequences are AAA… in the p-methoxy compound and ABAB… in the p-fluoro compound.Despite their different compositions and space groups, the irregular KF +·C7H7BF3O−, (I), and K+·C6H4BF4−, (II), are molecular salts containing para-substituted phenyltrifluoridoborate anions. In each compound, the B atom adopts a distorted tetrahedral BCF3 geometry. Despite their different compositions and space groups, the irregular KF8 coordination polyhedra of the potassium cations in the structures are almost identical. These polyhedra share faces and edges, generating infinite (010) layers in (I) and infinite (001) layers in (II). In (I), adjacent layers are stacked in an AAA… fashion, whereas in (II), they are stacked in an ABAB… sequence.The title compounds, K The metal ion is displaced by 1.00 Å from the geometric centroid of the eight F atoms. In terms of the F atoms in (I)3 tetrahedron, whereas F2 bonds to two K+ ions (mean K—F = 2.755 Å) in an FBK2 distorted T-shape. If the geometry around F3 is not merely deemed to be irregular, it could be described as an FBK3 trigonal-based pyramid, with the long K—F bond (Table 18 polyhedra share faces in the [100] direction and edges in [001]: the shortest K⋯K separation is 4.4523 (4) Å.The potassium ion in (I)ic Fig. 2, with fid Table 1 as the aThe asymmetric unit of compound (II)+ ion in (II)8 coordination polyhedron The extended structure of (II)AAA….In (I)ABAB….When the crystal structure of (II)+.C6H5BF3− 8 coordination geometry as the equivalent species in (I)6H5BF3− anion has found use as a bulky charge-balancing species (I) and (II)Uiso(H) = 1.2Ueq(C) or 1.5Ueq(methyl C). The methyl group in (I)The H atoms were placed in idealized positions (C—H = 0.95–0.98 Å) and refined as riding atoms with 10.1107/S1600536814009684/wm0001sup1.cifCrystal structure: contains datablock(s) I, II, global. DOI: 10.1107/S1600536814009684/wm0001Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S1600536814009684/wm0001IIsup3.hklStructure factors: contains datablock(s) II. DOI: 1004280, 1004281CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Mycobacterium leprae. The polarized clinical presentations in leprosy are associated with differential immune activation. In tuberculoid leprosy, macrophages show a classical activation phenotype (M1), while macrophages in lepromatous disease display characteristics of alternative activation (M2). Bacille Calmette-Guérin (BCG) vaccination, which protects against leprosy, can promote sustained changes in monocyte response to unrelated pathogens and may preferentially direct monocytes towards an M1 protective phenotype. We previously reported that M. leprae can dampen the response of naïve human monocytes to a strong inducer of pro-inflammatory cytokines, such as BCG. Here, we investigated the ability of the pathogen to alter the direction of macrophage polarization and the impact of BCG vaccination on the monocyte response to M. leprae.Macrophages play a central role in the pathogenesis of leprosy, caused by M. leprae interferes with subsequent M1 polarization, indicated by lower levels of M1-associated cytokine/chemokines released and reduced expression of M1 cell surface markers. Exposure to M. leprae phenolic glycolipid (PGL) 1, instead of whole bacteria, demonstrated a similar effect on M1 cytokine/chemokine release. In addition, we found that monocytes from 10-week old BCG-vaccinated infants released higher levels of the pro-inflammatory cytokines TNF-α and IL-1β in response to M. leprae compared to those from unvaccinated infants.We show that in vitro exposure of monocytes from healthy donors to M. leprae has an inhibitory effect on M1 macrophage polarization, likely mediated through PGL-1. By directing monocyte/macrophages preferentially towards M1 activation, BCG vaccination may render the cells more refractory to the inhibitory effects of subsequent M. leprae infection.Exposure to  Mycobacterium leprae. Monocyte/macrophages show a dynamic plasticity, allowing them to respond to environmental stimuli by presenting a classical (M1) or alternative (M2) activation state [Macrophages play a central role in the pathogenesis of leprosy, a chronic debilitating disease caused by assical M or alterrnative M activation state . Leprosyon state , 5.Bacille Calmette-Guérin (BCG) vaccination protects against leprosy and is associated with reduced burden of unrelated diseases, suggesting non-specific protection that may involve shaping innate immunity –8. BCG vM. leprae alone, or M. leprae followed by BCG, induced the release of cytokine/chemokines that are associated with negative regulation of inflammation [M. leprae itself was a poor inducer of the pro-inflammatory cytokine TNF-α, consistent with other reports [M. leprae, the cells produced a pro-inflammatory cytokine profile matching that of BCG alone [M. leprae to interfere with M1 maturation of monocyte induced by exposure to IFN-γ and M2 maturation induced by exposure to IL4/IL13 in vitro. We also tested whether BCG vaccination, by favoring an M1 phenotype, may render the cells resistant to the inhibitory effects of M. leprae.We previously reported that stimulation of naïve monocytes from healthy donors with ammation . M. lepr reports . HoweverCG alone . Here, wMycobacterium leprae Thai-53 from the National Hansen’s Disease Programs Laboratory Research, Louisiana State University, Baton Rouge (American Leprosy Missions and the Society of St. Lazarus) [M. leprae PGL-1, obtained through the NIH Biodefense and Emerging Infections Research Resources Repository NIAID, NIH: NR-19342, was used as described [N = 18) or BCG-vaccinated at birth (N = 20) were provided by Dr. Willem Hanekom, University of Cape Town, South Africa.Lazarus) –15 and BLazarus) . Purifieescribed . PeripheThese studies were approved by the Institutional Review Boards of Rutgers University and the University of Cape Town .M. leprae, cells were exposed to M. leprae at multiplicity of infection (MOI) 5:1 or 20:1 (bacilli:cells) for 5 h prior to IFN-γ priming (M1) or IL-4/IL-13 treatment (M2). Cell viability was confirmed by trypan blue exclusion . Controls included: M1 or M2 polarization alone, M. leprae-stimulation alone, and unstimulated/untreated cells.PBMCs were isolated from buffy coat (New Jersey Blood Center) of healthy donors; monocytes were purified using anti-CD14 antibody-conjugated magnetic beads  and allowed to adhere before treatment . For M1 5 cells/well) in RPMI 1640/20 % human serum, and exposed to M. leprae (MOI 5:1) or culture medium alone.Monocytes were isolated from infant PBMCs by adherence , plated Cell supernatants were removed after 48 h (M1) or 24 h (M2) and probed using a multiplex human cytokine/chemokine panel  according to manufacturer’s instructions. Data/statistical analysis were done as described . Cells wM. leprae (MOI 5:1) prior to M1 polarization significantly reduced levels of IL-6, IL-1β and IL-12p70 by 19 % ± 27, 47 % ± 25, and 46 % ± 20, respectively, compared to M1 controls  led to further reduction; IL-6, IL-1β and IL-12p70 were reduced by 36 % ± 32, 62 % ± 28 and 60 % ± 25, respectively. TNF-α was also significantly lowered by 22.2 % ±5.0 in response to M. leprae pre-stimulation, but only at higher MOI. In contrast, MCP-1 and IP-10 levels were significantly increased  by M. leprae (MOI 5:1) pre-stimulation compared to M1 controls , IP-10 levels were not significantly different at the two MOIs  released high levels of TNF-α (mean 123.4 ± 17.0 ng/ml), IL-6 (152.1 ± 175.3 ng/ml), IL-1β (0.3 ± 0.07 ng/ml), IL-12p70 (2.1 ± 0.8 ng/ml), MCP-1 (6.2 ± 2 ng/ml), IP-10 (121 ± 18.9), Rantes (11.6 ± 2.3 ng/ml), MIP-1α (22.5 ± 3.2 ng/ml) and MIP-1β (88.7 ± 26.7 ng/ml) compared to unstimulated/untreated cells. Exposure of cells to ols Fig. . Pre-stiols Fig. . While MOIs Fig. . No consractions , 23, 24.M. leprae at low MOI, the percentages of CCR7+ (P ≤ 0.001) and CD40+ (P ≤ 0.05) cells were reduced compared to M1 polarization alone . HLA-DR+, CD80+ and CD86+ M1 cell percentages were unaffected by M. leprae pre-stimulation. The mean fluorescence intensities (MFI) of CCR7, CD40 and CD80 in M1 cells were also significantly reduced by M. leprae pre-exposure  over M2 polarization alone (242.7 ± 108), with low levels produced by unstimulated/untreated cells (24.9 ± 12.3), and had no effect on IL-1Ra and IL-10 (data not shown). Thus, the effect of M. leprae on monocytes is primarily due to inhibition of M1 activation and does not appear to significantly affect M2 polarization.In contrast, the impact of M. leprae on monocytes from 10-week old unvaccinated or BCG-vaccinated infants (Fig. M. leprae were significantly higher in monocytes from vaccinated infants than those from unvaccinated infants, while IL-6 and MCP-1 showed trends towards higher levels in vaccinated versus unvaccinated infants. These results demonstrate that in vivo activation of monocytes due to BCG vaccination may render the cells refractory to the inhibitory effects of M. leprae.Finally, we compared the effect of nts Fig. . Levels M. leprae can alter the functional capacity of monocytes, which may diminish the efficacy of the host response to subsequent stimuli. Our results support increasing evidence suggesting that the innate immune response may be shaped by prior history of exposure, which could also explain the protection afforded by BCG vaccination against M. leprae and other unrelated pathogens.Exposure to


Cell surface marker analysis of these cells identified subsets reflecting common lymphoid progenitors, common myeloid progenitors and multipotential progenitors. To assess whether the Lin−Vβ8.2+Cβ− BM subset contains hematopoietic progenitors, cells were sorted and adoptively transferred into sub-lethally irradiated recipients. No T-cell or myeloid progeny were detected following introduction of cells via the intrathymic or intravenous routes. However, B-cell development was detected in spleen. This pattern of restricted in vivo reconstitution disputes Lin−Vβ8.2+Cβ− BM cells as committed T-cell progenitors, but raises the possibility of progenitors with potential for B-cell development.Germline transcription has been described for both immunoglobulin and T-cell receptor (TCR) genes, raising questions of their functional significance during haematopoiesis. Previously, an immature murine T-cell line was shown to bind antibody to TCR-Vβ8.2 in absence of anti-Cβ antibody binding, and an equivalent cell subset was also identified in the mesenteric lymph node. Here, we investigate whether germline transcription and cell surface Vβ8.2 expression could therefore represent a potential marker of T-cell progenitors. Cells with the TCR phenotype of Vβ8.2 This was strain specific with a lower 1.4% found in SJL/J mice Germline transcription is the production of mRNA transcripts from either the unrearranged T-cell receptor (TCR) or immunoglobulin (Ig) loci that are in ‘germline’ configuration. A common view has been that production of germline transcripts is tightly coupled to rearrangement events that take place at these loci to produce a functional TCR or B-cell receptor (BCR). By this scenario, one would expect germline transcripts to be produced at VDJ recombination when the chromatin is open and fully accessible to transcriptional machinery. However, germline TCR-Vβ transcripts have been identified in early murine T cells well before the TCR-Vβ rearrangement event in RAGH proteins have been reported Staphylococcus enterotoxin B have been identified in mice Several unusual TCR and Ig receptor structures have been identified as evidence of germline-encoded proteins and novel TCR structures. For example, germline-encoded IgVin vivo subsets expressing Vβ8 but not Cβ determinants have been identified, there is little known about them. The developmental changes reported to occur in C1-V13D cells following intrathymic passage suggest that this cell line represents immature lymphoid cells that can differentiate along the T-cell lineage. Since germline transcripts occur during early lymphopoiesis +Cβ− cells in mouse thymus, BM, lymph node and spleen. The subset of lineage (Lin)−Vβ8+Cβ− cells in BM has been further analysed for expression of markers which define hematopoietic progenitors, and their capacity to differentiate and produce T-cell progeny upon adoptive transfer in mice. While we found no evidence of T-cell reconstitution, the lymphoid characteristics of this progenitor subset were supported by specific production of mature B cells in spleen.The expression of germline TCR-Vβ8 transcripts has been documented in both early B and T-cell subsets and cell lines like C1-V13D 2 asphyxiation. Spleen, thymus and BM were aseptically removed from 5 to 10 mice for preparation of cell suspensions. For isolation of hematopoietic cells from BM, femur and tibia of hind legs were removed, excess tissue discarded, and the bones crushed in a small volume of medium PBS/2%fetal calf serum. Additional medium was added until all BM cells were released away from bone fragments.C57BL/Ka and C57BL/Ka-Thy1.1 (BA) mice expressing either Ly5.1 or Ly5.2 were bred and maintained in Research Animal Facility at Stanford University according to approved protocols. Male and female mice were used at 4–8 weeks of age. Mice were killed by CO4Cl, 100 mM KHCO3, 0.1 mM Na2EDTA, pH 7.2–7.4) followed by washing in PBS/2%FCS. Cells were stained with antibody either directly with fluorochrome-conjugated antibodies, or indirectly with a purified antibody followed by a second stage conjugate. Antibodies and their specificity are shown: TCR-Vβ8.1/8.2 (MR5.2), TCR-Cβ (H57-597), Thy1.1 (19EX5), NK1.1 (PK136), B220 (RA3-6B2), Ly5.1 (ALI-4A2), Ly5.2 (A20.1.7), CD127 (A7R34), Sca-1 (E13-161-7), c-Kit (2B8), CD4 (GK1.5), CD8 (53-6.7), CD3ε (KT31.1), TCRγδ (GL3), I-Ab (AF6-120.1), CD11c (HL3), CD44 (IM7), CD25 (7D4), CD19 (MB19-1), Mac-1 (M1/70) and Gr-1 (8C5). All antibodies were purified from hybridoma culture supernatants with the exception of antibodies specific for CD11c, CD25, CD44, TCRγδ, I-Ab, NK1.1, TCR-Cβ, TCR-Vβ8.1/8.2 and Ter119 purchased from BD Biosciences Pharmingen . Anti-CD19 antibody was purchased from eBiosciences . Secondary antibody conjugates used included Streptavidin-PE and Streptavidin-Cy7PE from Invitrogen .Spleen, thymus and lymph node cells were dissociated, and the cell suspension filtered through nylon mesh. Red blood cells were removed using lysis buffer (150 mM NH−) were gated using side scatter (SSC), and investigated for marker expression or sorted for cell subset isolation. For isolation of rare cell subsets, sorted cells were checked flow cytometrically and resorted to ensure high purity.Following staining, cells were resuspended in PBS/2%FCS containing 1 μg/ml of propidium iodide (PI) to detect dead cells by flow cytometry. Normal BM cells were stained to set PI gates for sorted and depleted BM subsets. Cells were analysed for up to 5-colour staining using a FACS Vantage SE , and CellQuest Pro software (Becton Dickinson). Viable cells (PI+) from BM involved staining cells with purified antibodies specific for known cell lineage markers. Antibodies used were specific for Thy1.1 (19XE5), CD3ε (KT31.1), CD4 (GK1.5), CD5 (53-7.3), CD8 (53-6.7), B220 (RA3-6B2), Mac-1 (M1/70), Gr-1 (8C5) and Ter119). Following antibody staining, cells were washed and incubated with magnetic Dynabeads (#M-450) Sheep anti-Rat IgG  at 4°C. A magnetic field was applied and the supernatant collected. The procedure was repeated. Cells were resuspended in 400 μl PBS/2%FCS containing goat F(ab’)2 anti-Rat IgG (H+L) conjugated to Cy5PE and incubated on ice for 20 min. Washed cells were resuspended in 400 μl of PBS/2%FCS containing 50 μl of rat IgG as a blocking antibody. Cells were incubated for 10 min on ice and the Lin− cell population then stained with antibody.Depletion of cells of known lineage  mice were injected into C57BL/Ka-Ly5.2 sub-lethally irradiated hosts (4.75 Gy). Mice were given 500 cells either (a) intravenously through the orbital plexus, or (b) intrathymically, into a single thymic lobe +Lin−Sca+ KLS) or Lin−Vβ8−Cβ− cells, also sorted out of BM. Post transplantation, mice were maintained on water containing polymyxin B sulphate (106 U/ml) and neomycin sulphate (1.1 g/l). Mice were killed at 4 weeks, and cells stained with antibodies to detect hematopoietic cell types among donor-derived progeny. This time was chosen as a compromise between time for detection of T-cell progeny of common lymphoid progenitors (CLP) which occurs between 14 and 28 days To test whether Lin+Cβ− cells was analysed flow cytometrically in thymus, BM, mesenteric lymph nodes and spleens of C57BL/6-Ly5.1 (BA) mice. To avoid any steric hindrance, cells were first incubated with anti-Cβ antibody, followed by anti-Vβ8, an isotype control antibody or no antibody. Cells were stained with antibodies specific for lineage and stem cell markers to further define the Vβ8+Cβ− subset. Live cells were gated as PI negative (PI−) cells, and the Vβ8+Cβ−B220− cell population identified  was identified  and spleen (0.6%), not present in thymus.The prevalence of Vβ8+Cβ− subset in BM and other hematopoietic progenitor subsets was investigated flow cytometrically. Subsets in spleen were also analysed since the spleen is the source of the C1-V13D cell line, and contains hematopoietic stem/progenitor cells loSca-1loCD127(IL-7Rα)+ cells hiSca-1hiCD127− cells in BM also reflect a source of T-cell progenitors +Cβ− cells reflect T-cell progenitors, the Vβ8+Cβ− subsets in spleen and BM were investigated primarily for expression of markers which delineate T-cell progenitors from other hematopoietic progenitors.The relationship between the Vβ8− BM cells were prepared and stained with antibodies to Vβ8 and Cβ to gate the Vβ8+Cβ− subset. This population was then analysed for expression of c-Kit, Sca-1 and CD127, and distinct subsets of c-Kit+ hematopoietic progenitors delineated  cells, Sca-1−c-KithiCD127− [common myeloid progenitor (CMP)-like], but almost no Sca-1+/hic-Kit+CD127− (HSC- or MPP-like) cells could be distinguished among the Vβ8+Cβ− subset of BM. The Sca-1+c-Kit−CD127− subset does not resemble progenitors and could reflect lymphoid cells. The phenotypes of CLP, CMP, MPP and HSC with respect to these markers have been described previously − BM, resorted Lin− BM and spleen is shown in Figure+Cβ− subset of spleen and BM, the HSC-like or MPP-like subset was less well represented among the BM subset.Lin+Cβ− subsets of spleen and BM could contain a range of cells reflecting hematopoietic progenitors.This suggests that the Vβ8+Cβ− subset of BM contains hematopoietic progenitors, and possibly T-cell progenitors, that develop into mature cells following adoptive transfer in vivo. A single time-point of 4 weeks was chosen as sufficient time post-transfer to see the rapid development of T cells from CLP, which should appear in thymus and spleen from 2 to 4 weeks. At 4 weeks, it is also possible to detect myeloid cells as the earliest progeny of any HSC and MPP developing through a CMP intermediate. Four weeks is also the earliest point at which lymphoid progeny of HSC and MPP are evident in thymus and spleen from CLP developing within BM On the basis of these results, it was has been suggested that the Vβ8−Vβ8+Cβ− BM cells from BA (Ly5.1) mice prepared as described in Figure+Lin−Sca+) or Lin−Vβ8−Cβ− cells were prepared. Some mice were injected with PBS to provide a control for inoculation and for Ly5.1 and Ly5.2. In a second experiment, the same Lin−Vβ8+Cβ− cells were given intravenously, but anti-Thy1.1 antibody was used in place of anti-CD3ε antibody for depletion of T cells, to preclude any loss of T-cell precursors on the basis of CD3ε expression. In this experiment, the Lin−Vβ8−Cβ− subset containing HSC and MPP was sorted and served as a positive control for reconstitution. At 4 weeks post-transfer, mice were killed and both splenocytes and thymocytes collected and stained with antibodies specific to detect the presence of cells of different hematopoietic lineages.In a first experiment, 500 resorted Lin−) and then assessed for expression of Ly5.1 and lineage markers. Control Ly5.2 mice given PBS showed no staining for Ly5.1 (Tableb) and dendritic cells . FigureAdoptively transferred cells developing in spleen and thymus of the Ly5.2 hosts were first identified as viable  which could be progenitors  has been reported previously −Vβ8+Cβ− cells intrathymically, only 2 out of 3 showed reconstitution with donor cells. No T cells or B cells were detected in the thymus of donor-reconstituted mice given Lin−Vβ8+Cβ− cells  cells showed full reconstitution of every cell lineage common to spleen or thymus as anticipated Figs. Hematoptors Fig.. The precells 0.0% has beefer Table. This reumber 0.0% which c−Vβ8+Cβ− BM cells, the absence of T-cell production precludes the presence of T-cell progenitors among this subset. The absence of myeloid cell development in spleen also precludes the presence of HSC or MPP among this subset. The production of B cells and their localization in spleen but not thymus is also consistent with evidence of Vβ8+B220+ cells in peripheral mesenteric lymph node and spleen but not thymus  were identified in several organs including thymus, mesenteric lymph nodes, BM and spleen of mice. To some extent these subsets resemble C1-V13D, described previously as an immature spleen-derived T-cell line expressing TCR-Vβ8.2 peptides as well as germline Vβ8.2 transcripts +Cβ− subset of BM and spleen was therefore analysed for expression of c-Kit, Sca-1 and CD127 markers, which distinguish CLP from other progenitor populations in BM. An initial study of c-Kit, CD127 and Sca-1 expression on a gated Vβ8+Cβ− subset of whole spleen and BM revealed multiple sub-populations phenotypically resembling HSC, MPP, CMP and CLP.Here, the hypothesis is investigated that Vβ8+Cβ− subset contains lymphoid progenitors was based on transcriptional evidence that both early T and B cells express GL-Vβ8.2 transcripts −Vβ8+Cβ− cells isolated from BM, no development of T cells was detected in thymus or spleen, and further development of Lin−Vβ8+Cβ− BM cells was found to be limited to B lymphopoiesis. While B cells can arise in low numbers as an endogenous thymic cell population +Cβ− B cells were instead detected in spleen in the absence of any thymic B-cell development. It is not yet possible to determine whether development of these B cells occurred in spleen itself, or was dependent on lodgement of progenitors in BM.The hypothesis that the Vβ8+Cβ− cell line, can undergo some T-cell differentiation upon intrathymic transfer showing TCRαβ expression −Vβ8+Cβ− subset from BM contains lymphoid progenitors with capacity to differentiate into only B cells, while a similar or equivalent subset resident in spleen may have T-cell differentiative potential. Such a model invokes a heterogeneous population of progenitors in mice expressing germline Vβ8 peptide. This is supported by evidence for expression of GL-Vβ8.2 transcripts in many cell types including early T cells, early B cells, BM progenitors and early dendritic cells . Indeed, C1-V13D has been shown to express low levels of early T-cell markers including CD3ε −Vβ8+Cβ− BM subset therefore appears to contain a later lymphoid progenitor committed to the B-cell lineage.Results presented here contrast with previous evidence that the C1-V13D cell line, representing a splenic Vβ8−Vβ8+Cβ− BM population does not appear to contain bipotent CLP-like cells with capacity to differentiate into both T and B cells. In terms of bipotent lymphoid potential, thymic DN1 cells are known to be heterogeneous with specific sub-populations retaining ability to give rise to B cells −Vβ8+Cβ− BM subset −Vβ8+Cβ− cell subset does not express B220, it is distinct from the CLP-2 population identified in BM as a B220+ cell The Lin+Cβ− cells +Cβ−B220+ cells exist in BM (0.07%) and mesenteric lymph node (0.12%) of BA mice, but not in spleen and thymus . It is possible that a truncated TCR-β chain with no transmembrane region is expressed only and then is secreted by cells. Such transitional expression could also account for the variable expression reported for TCR-Vβ8.2 on the surface of C1-V13D cells TCR-Vβ8.2 genes into C1-V13D cells was used to overcome this problem in demonstrating cell surface expression Evidence for germline transcription of in vivo. A substantial level of diversity exists within the TCR and BCR repertoire against which developing T and B lymphocytes must be tolerized. Therefore, germline-encoded Ig or TCR proteins may play an antigenic role in this process within thymus for T-cell development, or within BM, spleen and lymph node for B-cell development. The expression of multiple germline TCR-Vβ transcripts in the cloned C1-V13D cell line also supports this model One possible role for germline-encoded TCR-Vβ peptides could be generation of tolerance to T cells


E conformation about the azomethine double bond and an intramolecular O—H⋯N hydrogen bond involving the phenolic hydrogen and the azomethine N atom. In the crystal, molecules are linked by bifurcated N—H⋯O hydrogen bonds involving the same acceptor atom, forming chains propagating along [001].The title compound, has an  21H19N3O3, has an E conformation about the azomethine double bond. The central moiety of the hydrazinecarboxamide moiety [–N—N—C(=O)—N–] has an almost coplanar arrangement [maximum deviation for the C atom = 0.010 (2) Å]. This central moiety is flanked by three aromatic rings and its mean plane makes dihedral angles of 24.7 (1), 72.91 (12) and 34.26 (11) Å, respectively, with the phenolic ring, the phenyl ring attached to the same C atom as the phenolic ring, and the phenylhydrazine ring. The adjacent phenolic and phenyl rings are twisted away from each other to reduce steric hindrance and make a dihedral angle of 80.59 (12)°. The phenolic and phenylhydrazine rings are inclined to one another by 28.89 (11)°. The rigidity of the molecule is increased by an intramolecular O—H⋯N hydrogen bond involving the phenolic hydrogen and the azomethine N atom. In the crystal, the carbonyl O atom forms bifurcated hydrogen bonds with the two NH atoms of the hydrazinic group, leading to the formation of chains propagating along [001]. Within the chains there are also C—H⋯O hydrogen bonds present. The chains are linked via C=O⋯π [3.4316 (18) Å] and parallel slipped π–π interactions, involving inversion-related benzene rings , forming sheets lying parallel to (100).The title compound, C This central moiety is flanked by three aromatic rings  which are inclined to its mean plane by 24.70 (10), 72.91 (12) and 34.26 (11) °, respectively. Rings C1–C6 and C8–C13, attached at the same C atom (C7), are twisted away from each other and make a dihedral angle of 80.59 (12)°. They are inclined to the phenylhydrazine ring (C15–C20) by 28.89 (11) and 52.42 (12)°, respectively. In the crystal structure of the dimethylformamide solvate of the title compound  Å; angle C14=O3⋯Cg = 95.3 (1)°; Cg is the centroid of the C8–C13 ring; symmetry code: (i) x, −y + z + In the crystal, the carbonyl O atom (O3) acts as the acceptor in bifurcated hydrogen bonds with the NH atoms of atoms N2 and N3 of the hydrazinic group, leading to the formation of chains propagating along [001]; Table 1N4-phenylsemicarbazide , a methanolic solution (25 ml) of 2-hydroxy-4-methoxybenzophenone  was added and the resulting solution was boiled under reflux for 2 h, after adding three drops of conc. HCl. On slow evaporation at room temperature, colourless crystals separated out. They were filtered off and washed with methanol and ether. Single crystals of the title compound suitable for X-ray analysis were obtained by slow evaporation of a solution in methanol . FT–IR  νmax: 3316 , 3249 , 3145 , 1662 , 1631 , 1059 . 1H NMR : 12.94 , 9.10 , 9.03 , 3.90 , 6.33–7.672 . ESI mass spectrum, m/z: 362.3 (M+1). Analysis calculated for C21H19N3O3: C, 69.79, H, 5.30, N, 11.63%. Found: C, 69.68, H, 5.72, N, 11.93%.To a warm methanolic solution (25 ml) of Uiso(H) = 1.5Ueq(C) for methyl H atoms and 1.2Ueq(C) for other H atoms.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989015005757/su5098sup1.cifCrystal structure: contains datablock(s) I, Global. DOI: 10.1107/S2056989015005757/su5098Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989015005757/su5098Isup3.cmlSupporting information file. DOI: 1055367CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


In the neutral complex (I), the nickel cation is coordinated by one cyclen ligand and two monodentate N-bound tcm− anions in a distorted octahedral geometry. The tcm− ligands are mutually cis. The CuII atom in (II) displays a distorted tetragonal–pyramidal geometry, with the four N-donor atoms of the cyclen ligand in the equatorial plane, and one tcm− anion bound through a single N atom in an axial site, forming a monocation. The second tcm− molecule acts as a counter-ion not directly coordinating to the copper cation. In both (I) and (II), extensive series of N—H⋯N and C—H⋯N hydrogen bonds generate three-dimensional network structures.The structures of two mononuclear transition-metal complexes with tricyanomethanide (tcm Among the CuI or CdII tcm− complexes with such co-ligands, numerous structural types ranging from doubly interpenetrated  sheets to three-dimensional rutile networks have been observed pyrazine, 2,2′-bipyrazine and 2,3,5,6-tetrakis(2-pyridyl)pyrazine and found them to have interesting magnetic properties. 1,4,7,10-Tetraazacyclododecane (cyclen) is a novel co-ligand with four potential nitrogen donor atoms. However, no tcm− complexes incorporating cyclen as a co-ligand have been reported previously. As part of our systematic investigation of the effect of cyclen as a co-ligand on the structures and properties of tcm− complexes, we have prepared two new tcm− complexes and we report herein the syntheses and crystal structures of Ni(cyclen)(C4N3)2 (I)4N3)]+(C4N3)− (II)Coordination polymers constructed by the tricyanomethanide anion  of the cyclen unit and the N5 and N8 atoms of the coordinating tcm− anions. The apical sites are occupied by N2 and N4 from the cyclen ligand, Fig. 1In (I)− ligand in an apical site, forming a five-coordinate cation with a distorted square-pyramidal coordination geometry. The second tcm− anion does not enter the inner coordination sphere of the metal al Fig. 2, but actal Fig. 2.et al., 2009et al., 2008− ligands  distances in (I)et al., 2014et al., 2013− complexes cis-related basal N atoms and the N—Ni—N angle range from 84.9 (1) to 97.3 (1)° and 81.6 (1) to 101.2 (1)°, respectively. The corresponding values for (II)In (I)− ligand is almost planar, with the mean deviations from the planes through all atoms of the coordinating tcm− anions being 0.0128 and 0.0322 Å, respectively in (I)− anion and the tcm− counter-anion are 0.0211 and 0.0074 Å respectively. Bond lengths and angles within the anions are also in good agreement with those found in other tcm− complexes rk Fig. 3.via N—H⋯N and C—H⋯N hydrogen bonds to two adjacent complex cations and three tcm− anions, forming another extensive three-dimensional network rk Fig. 4.− ligands are quite common with 47 unique compounds recorded in the Cambridge Crystallographic Database amine]bis(tricyanomethanide)nickel(II) [tris(2-aminoethyl)amine-κ4N]nickel(II) ]2[Pt(CN)4]2·6H2O and [Ni(cyclen)]2[(Ni(CN)4)]2·6H2O ]+·[Au(CN)2]− ]+·NO3−  of potassium tricyanomethanide  was then added. After stirring for another 5 min, the purple solution was filtered and the filtrate slowly evaporated in air. After two weeks, purple block-like crystals of (I)16H20N10Ni: C 46.75%, H 4.90%, N 34.07%. Found C 46.91%, H 5.03%, N 34.26%. Using copper(II) nitrate instead of nickel(II) nitrate, blue block-like crystals of (II)16H20N10Cu: C 46.20%, H 4.85%, N 33.67%. Found C 46.42%, H 5.01%, N 33.85%.A 5 ml ethanol solution of 1,4,7,10-tetraazacyclododecane  and 2 ml of a green aqueous solution of nickel(II) nitrate  were mixed and stirred for 5 min; the resulting solution was purple. A 3 ml ethanol–water solution (EtOH:HUiso = 1.2Ueq(C) for CH2. In (II)Crystal data, data collection and structure refinement details are summarized in Table 510.1107/S2056989015009524/sj5461sup1.cifCrystal structure: contains datablock(s) global, I, II. DOI: 10.1107/S2056989015009524/sj5461Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989015009524/sj5461IIsup3.hklStructure factors: contains datablock(s) II. DOI: 10.1107/S2056989015009524/sj5461sup4.pdfSupporting information file. DOI: 10.1107/S2056989015009524/sj5461sup5.pdfSupporting information file. DOI: 10.1107/S2056989015009524/sj5461sup6.pdfSupporting information file. DOI: 1401692, 1401691CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Herein, we have demonstrated a new paradigm for two-photon tracking gene delivery based on a dendritic nano-sized hexanuclear ruthenium(II) polypyridyl complex. Because this metallodendrimer has a multivalent periphery, the complex, which is 6.1 nm, showed high stability and excellent dispersibility and could stepwise condense DNA in vitro. With the outstanding photochemical properties of Ru(II) polypyridyl, this complex could track gene delivery in vivo using one- and two-photon imaging.Fluorescent tracking gene delivery could provide us with a better understanding of the critical steps in the transfection process. However, for  The 1H NMR spectrum of ligand L was not obtained due to its poor solubility in common NMR solvents. Complex {[Ru(bpy)2]6L}(ClO4)12 (Ru6L) was prepared by refluxing Ru(bpy)2Cl2.2H2O and L in ethylene glycol and isolated as a ClO4– salt, with a 62% yield. This complex was characterized by 1H NMR spectroscopy, IR spectroscopy, ESI-MS and elemental analysis  was 612 nm, with a lifetime of 110 ns. The emission quantum yield was 0.027, and the largest two-photon absorption (TPA) cross-section was 175 GM at 830 nm with reference to rhodamine B  DNA in each well. As shown in Ru6L (0-6 μM), pBR 322 DNA gradually diminished and was retained in the well at a +/− ratio  of 1.1. The primary DNA condensation-driving forces were considered electrostatic interactions by the multivalent periphery of Ru6L. The DNA binding ability was determined by a DNA titration approach with CT-DNA . The binding constant was calculated to be 1.75 × 105 M−1 . We used the endocytic inhibitors chloroquine and NH4Cl to examine the role of this pathway in Ru6L uptake. As observed from the relative intensities and location of Ru6L in the cells after treatment with these inhibitors . After another 12 h, EGFP expression was detected, and higher EGFP expression was found after 24 h. Similar behaviors were observed under one-photon and two-photon excitation. In addition, due to the two-photon absorption, the background signal was strongly suppressed, and a higher resolution image was obtained using two-photon confocal laser scanning microscopy.To investigate the intracellular behaviors of  by Ru6L . We staiRu6L with intrinsic two-photon luminescence as DNA carriers is an attractive solution to these problems because this complex exhibits near infrared (NIR) excitation wavelengthsThe most commonly used method for intracellular plasmid trafficking is the fluorescent labeling of non-viral vectors with organic dyes1426Ru6L was used. The luciferase expression was stable when the +/− ratios of the particles ranged from 12 to 24, and the highest luciferase expression was in response to the +/− ratio of 20.We determined the relative transfection efficiency of this nonviral system by luciferase assays, and plasmid pGL3 was used a control vector . As a coRu6L-pEGFP DNA particles  based on polybenzimidazole ligands. This complex has a multivalent periphery and a nanosize of 6.1 nm. Gel retardation assay, TEM, AFM and dynamic light scattering studies show that Ru6L exhibits high stability and excellent dispersibility and could stepwise condense DNA in vitro. More interestingly, Ru6L was applied as a non-viral gene vector for tracking DNA delivery in live cells using one- and two-photon fluorescence microscopes. With two-photon microscopy, a high signal-to-noise contrast was achieved by irradiation with an 830 nm laser. Our work provides new insights into improving real-time tracking during gene delivery and transfection as well as important information for the design of multifunctional non-viral vectors.In the present study, we have designed and synthesized a dendritic nano-sized hexanuclear Ru(II) polypyridyl complex . The UV-Vis spectra were recorded on a Perkin-Elmer Lambda 850 spectrophotometer. Emission spectra were recorded on a Perkin-Elmer LS 55 spectrophotometer at room temperature (25 °C). Time-resolved emission measurements were conducted on an FLS 920 combined fluorescence-lifetime and steady-state spectrometer. Quantum yields of luminescence at room temperature (25 °C) were calculated according to literature procedures using [Ru(bpy)3]2+ (φ = 0.028 in aerated aqueous solution) as the reference emitterMicroanalyses  were performed with a vario EL cube elemental analyzer. Infrared spectra were obtained with a Nicolet 170SX-FTIR spectrophotometer and KBr discs. Electrospray ionization mass spectra (ESI-MS) were recorded on an LCQ system .Fast atom bombardment mass spectrometry (FAB-MS) was recorded using a VG ZAB-HS. Atomic force microscopy (AFM) images were obtained in air at room temperature with an SPA400 atomic force microscope unit and an SPI3800N control station (Seiko Instruments) operated in the tapping mode. Probes were made of a single silicon crystal with a cantilever length of 129 mm and a spring constant of 33–62N/m . Dynamic light scattering and zeta potential experiments were performed using dynamic laser light scattering equipment . Transmission electron micrographs were obtained with a JEM100CX electron microscope.2CO3  in DMF (30 mL) was heated to 80 °C for 24 h under nitrogen. After cooling to room temperature, the solution was diluted with 300 mL of water. The mixture was filtered, and the solid was washed successively with boiling MeOH, CHCl3 and Et2O, affording the desired product as a white solid. Yield: 0.21 g (50%). Anal. Calcd. for C54H42O12: C, 73.46; H, 4.79. Found: C, 73.24; H, 4.80. IR νmax : 1689 (CHO).1H NMR  δ 9.76 , 7.70 , 7.04 , 5.42 . ESI-MS: m/z = 905.4 [M+Na]+.A mixture of 4-hydroxybenzaldehyde , 1,2,3,4,5,6-hexakis(bromomethyl)benzene , and KQ, 1,10-phenanthroline-5,6-dione  and NH4OAc  in glacial acetic acid (20 mL) was heated to 130 °C for 8 h, yielding a suspension. The reaction mixture was filtered while hot, and the solid was washed successively with EtOH, hot DMF and Et2O, affording the desired product as a yellow solid. Yield: 0.20 g (58%). Anal. Calcd. for C126H78N24O6: C, 74.77; H, 3.88; N, 16.61. Found: C, 74.55; H, 4.16; N, 16.43. IR νmax : 3392 (N-H). FAB-MS: m/z = 2025 [M+1]. The 1H NMR spectrum was not obtained due to its poor solubility in common NMR solvents.A mixture of L  and Ru(bpy)2Cl2.2H2O  in ethylene glycol (10 mL) was heated to 180 °C for 14 h under nitrogen to give a clear deep-red solution. The cooled reaction mixture was diluted with water (15 mL). Saturated aqueous sodium perchlorate solution was added under vigorous stirring, and the solution filtered. The dark red solid was collected, washed with small amounts of water and diethyl ether, dried under vacuum, and purified by column chromatography on alumina using acetonitrile-ethanol as the elutant. The solvent was removed under reduced pressure, and red microcrystals were obtained. Yield: 0.13 g (62%). Anal. Calcd. for C246H174Cl12N48O54Ru6: C, 51.85; H, 3.08; N, 11.80. Found: C, 51.64; H, 3.37; N, 11.57.IR νmax : 3360 (N-H) and 1087 (ClO4−). ESI-MS: m/z = 423.6 [M-6ClO4-6H]12+, 438.8 [M-9ClO4-6H]11+, 453.8 [M-7ClO4-6H]11+, 481.9 [M-9ClO4-6H]10+, 500.8 [M-12ClO4+4H]9+, 512.0 [M-11ClO4-5H]9+, 554.5 [M-7ClO4-6H]9+, 574.9 [M-11ClO4-4H]8+, 600.5 [M-9ClO4+2H]8+, 642.7 [M-12ClO4-5H]7+, 657.6 [M-11ClO4]7+, 672.4 [M-10ClO4-3H]7+, 750.5 [M-12ClO4-H]6+, 766.5 [M-11ClO4-4H]6+, 782.8 [M-10ClO4-6H]6+, 799.4 [M-9ClO4-6H]6+, 815.5 [M-8ClO4-6H]6+, 832.8 [M-7ClO4-6H]6+, 901.3 [M-12ClO4+2H]5+, 921.0 [M-11ClO4-H]5+, 940.5 [M-10ClO4]5+, 959.0 [M-9ClO4-6H]5+, 979.7 [M-8ClO4-3H]5+. 1H NMR  δ 9.10 , 8.81 , 8.21 , 8.20 , 8.04 , 7.95 , 7.82 , 7.54–7.59 , 7.28 , 5.56 .A mixture of Ru6L was dissolved in CH3CN at a concentration of 5 μM. The solution was drop cast onto a carbon-coated Cu grid, and the excess solution was absorbed by filter paper to avoid aggregation. The TEM image of the drop cast sample was taken with a JEM100CX electron microscope.The complex Ru6L at a concentration of 5 μM in distilled water that had been passed through a Millipore-Q ultra-purification system. Typically, 5 runs were measured for the solution, and the average of all runs is reported.Dynamic laser light scattering equipment was used to determine the average hydrodynamic diameter and the zeta potential of TM 355II . Two-photon luminescence measurements were performed in fluorometric quartz cuvettes. The experimental luminescence excitation and detection conditions were conducted with negligible reabsorption processes, which can affect TPA measurements. The quadratic dependence of the two-photon induced luminescence intensity on the excitation power was verified at an excitation wavelength of 830 nm. The two-photon absorption cross-section of the complex was calculated at each wavelength according to Equation (1)52:The two-photon absorption (TPA) spectra of complex were determined over a broad spectral region by a two-photon induced luminescence (TPL) method relative to Rhodamine B in methanol as the standard. The two-photon luminescence data were acquired using an OpoletteI is the integrated luminescence intensity, C is the concentration, n is the refractive index, and φ is the quantum yield. The subscript ‘1’ refers to the reference samples, and ‘2’ for the experimental samples.where Ru6L at the given +/− ratios in 50 mM Tris-HCl (Tris = Tris(hydroxymethyl) aminomethane) solution (pH = 7.4) or in cell culture, followed by vortexing for 30 min to allow equilibration at room temperature.The DNA particles were prepared by incubating the mixtures containing DNA and Ru6L in 50 mM Tris-HCl solution (pH = 7.4), and the solutions were analyzed by electrophoresis for 1.5 h at 75 V on a 1% agarose gel in TBE buffer . The gel was stained with 1 μg/mL ethidium bromide (EB) and photographed on an Alpha Innotech IS-5500 fluorescence chemiluminescence and visible imaging system.Negative supercoiled pBR 322 DNA was treated with Ru6L-pBR 322 DNA particles at various +/− ratios in 50 mM Tris-HCl solution (pH = 7.4). Typically, 5 runs were measured for each solution, with the average of all the runs reported.Dynamic laser light scattering equipment was used to determine the average hydrodynamic diameter and the zeta potential of Ru6L-2kb DNA and the morphologies Ru6L-pBR 322 DNA were examined with AFM. Samples were dropwise-added (10 μL) onto a mica substrate, which was freshly cleaved by pulling off the top sheets with tape. One min later, the substrate was spin coated  and rinsed with 20 μL of distilled water. AFM images were obtained in air at room temperature. The images were captured in a 256 × 256 pixels format and analyzed with the software accompanying the imaging module.The 2kb DNA fragment was generated by PCR using pBR 322 plasmid DNA as a template2. Exponentially grown HeLa cells were seeded in triplicate onto 96-well plates at 1 × 104 cells/well. After incubation for 24 h, the cells were treated with increasing concentrations of the tested complex and Ru6L-pEGFP DNA particles for 24 h. To stain the viable cells, 20 μL of MTT (5 mg/mL) was added to each well. The cells were then incubated for 4 h at 37 °C. After the medium was carefully aspirated without disturbing the formed crystals, the dye was dissolved in 200 μL of DMSO. A Tecan Infinite M200 monochromator-based multifunction microplate reader was used to measure the optical density of each well, with background subtraction at 590 nm. The cell survival rate in the control wells without Ru6L solutions were considered as 100% cell survival.HeLa cells were maintained as monolayer cultures in DMEM supplemented with 10% fetal bovine serum (FBS) at 37 °C under 5% CO5 cells mL−1 and 1 mL was added to five 35-mm2 Petri dishes  for laser confocal microscopy. After 24 h, the first dish was incubated with 5 μM Ru6L at 37 °C for 2 h. The second dish was incubated with 5 μM Ru6L at 4 °C for 2 h. The third dish was pretreated with 50 mM 2-deoxy-D-glucose and 5 μM oligomycin in PBS for 1 h at 37 °C and then incubated with 5 μM Ru6L at 37 °C for 2 h. The fourth and the fifth dishes were pretreated with endocytic inhibitors NH4Cl (50 mM), and chloroquine (50 μM) for 30 min respectively, and then incubated with 5 μM Ru6L at 37 °C for 2 h.The cells were trypsinized, counted, and adjusted to 1 × 10Ru6L and the emission spectra were integrated over 580-630 nm (single channel).After being washed with fresh PBS (pH = 7.0) three times, the cells were imaged on a Zeiss LSM 710 NLO confocal microscope (63 × /NA 1.4 oil immersion objective). For two-photon images, the excitation wavelength of the laser was 830 nm for 4 cells mL−1, and 1 mL of the cell solution was added to each plate. After 24 h, the cell culture medium was replaced with 800 μL serum-free DMEM. Ru6L-pEGFP DNA particles at the +/− ratio of 20, containing 1 μg pEGFP DNA in 200 μL serum-free DMEM, was added to the cells, and the cells were incubated at 37 °C for 4 h.The cells were trypsinized, counted, and adjusted to 1 × 10For flow cytometry, the cells were washed with PBS three times, trypsinized and centrifuged in PBS. Cells were harvested, and single cell suspensions in 0.5 mL PBS were prepared and subjected to flow cytometric analysis. A flow cytometer (Coulter Co. USA) was used to measure the fluorescent intensity, with excitation at 488 nm.in situ, without displacement from the culture dish. Cells were fixed in 0.1 M PBS containing 2.5% glutaraldehyde and 4% paraformaldehyde for 1 h, rinsed with distilled water, stained with 0.5% uranyl acetate for 1 h, dehydrated in a graded series of ethanol , and embedded in epoxy resin. The resin was polymerized at 60 °C for 48 h. Ultrathin sections (50–75 nm) obtained with an LKB ultramicrotome were stained with 2% aqueous uranyl acetate and 2% aqueous lead citrate.For TEM imaging analysis, cell processing was performed 5 cells mL−1 and 1 mL was added to five 35-mm2 Petri dishes  for laser confocal microscopy. After 24 h, the first dish was incubated with Ru6L-pEGFP DNA particles at the +/− ratio of 20 at 37 °C for 4 h. The second dish was incubated with Ru6L-pEGFP DNA particles at the +/− ratio of 20 at 4 °C for 4 h. The third dish was pretreated with 50 mM 2-deoxy-D-glucose and 5 μM oligomycin in PBS for 1 h at 37 °C and then incubated with Ru6L-pEGFP DNA particles at the +/− ratio of 20 at 37 °C for 4 h. The fourth and the fifth dishes were pretreated with endocytic inhibitors NH4Cl (50 mM), and chloroquine (50 μM) for 30 min respectively, and then incubated with Ru6L-pEGFP DNA particles at the +/− ratio of 20 at 37 °C for 4 h.The cells were trypsinized, counted, and adjusted to 1 × 10Ru6L and the emission spectra were integrated over 580-630 nm (single channel).After being washed with fresh PBS (pH = 7.0) three times, the cells were imaged on a Zeiss LSM 710 NLO confocal microscope (63 × /NA 1.4 oil immersion objective). For two-photon images, the excitation wavelength of the laser was 830 nm for 5 cells mL−1, and 1 mL of the cell suspension was added to a 35-mm2 Petri dish  for laser confocal microscopy. After 24 h, the cell culture medium was replaced with 800 μL serum-free DMEM. Ru6L-pEGFP DNA particles at the +/− ratio of 20, containing 1 μg pEGFP DNA in 200 μL serum-fee DMEM, was added to the cells, and the cells were incubated at 37 °C for 4 h. Then, the medium was replaced with fresh DMEM containing 10% FBS, and the cells were incubated for various timed .The cells were trypsinized, counted, and adjusted to 1 × 10Ru6L.After being washed with fresh PBS (pH = 7.0) three times, the cells were imaged on a Zeiss LSM 710 NLO confocal microscope (63 × /NA 1.4 oil immersion objective). The excitation wavelength of the laser was 488 nm, and the emission spectra were integrated over 580-630 nm (single channel). For two-photon images, the excitation wavelength of the laser was 830 nm for 4 cells per well and then incubated for 24 h. Cells were washed with PBS three times and then cultured with serum-free DMEM. Ru6L-pGL3 DNA control vector particles with increasing concentrations of the tested complex containing 0.2 μg plasmid were added to the cells, and the cells were incubated at 37 °C for 4 h. The medium was then replaced with fresh DMEM with 10% FBS, and the cells were incubated for an additional 24 h. Cells were washed with PBS, harvested and treated for 30 min at 4 °C with end-over-end rotation in lysis buffer . The luciferase assay was performed according to the manufacturer’s protocol (Promega). Relative light units (RLUs) were measured with Varioskan Flash  and a GloMaxTM 96 microplate luminometer .HeLa cells were seeded onto a 96-well cell-culture plate at a cell density of 1 × 10How to cite this article: Qiu, K. et al. A dendritic nano-sized hexanuclear ruthenium(II) complex as a one- and two-photon luminescent tracking non-viral gene vector. Sci. Rep.5, 10707; doi: 10.1038/srep10707 (2015).


Supramolecular chains arise in the crystal packing as a result of O—H⋯O and N—H⋯N hydrogen bonding.The  17H17F6N2O+·C10H8F3O3−, comprises two piperidin-1-ium cations and two carboxylate anions. The cations, each having an l-shaped conformation owing to the near orthogonal relationship between the quinolinyl and piperidin-1-ium residues, are pseudo-enantiomeric. The anions have the same absolute configuration but differ in the relative orientations of the carboxylate, methoxy and benzene groups. Arguably, the most prominent difference between the anions occurs about the Cq—Om bond as seen in the Cc—Cq—Om—Cm torsion angles of −176.1 (3) and −67.1 (4)°, respectively . The presence of Oh—H⋯Oc and Np—H⋯Oc hydrogen bonds leads to the formation of a supramolecular chain along the a axis (h = hydroxy and p = piperidin-1-ium); weak intramolecular Np—H⋯Oh hydrogen bonds are also noted. Chains are connected into a three-dimensional architecture by C—H⋯F interactions. Based on a literature survey, related molecules/cations adopt a uniform conformation in the solid state based on the letter L.The asymmetric unit of the title salt, C R*,S*)-quinolin-4-yl](hydroxymethyl)piperidin-1-ium chloride salt, usually referred to as racemic erythro-mefloquine hydrochloride, is an anti-malarial drug  at the piperdinyl-N atom, the resulting  and [(−)-erythro-mefloquinium] cations with two independent (+)-3,3,3-trifluoro-2-methoxy-2-phenylpropanoate anions providing the charge balance, Scheme 2. There is a non-crystallographic enantiomeric relationship between the cations so the sample is classified as a kryptoracemate. Surveys of this phenomenon have appeared in recent times for both organic -PhC(CF3)(OMe)CO2H, was carried out. However, as revealed by the X-ray crystal structure determination described herein, the isolated crystalline salt contained both mefloquinium enantiomers and two independent carboxylate anions. It is noticeable that in the 1H NMR spectrum in DMSO solution of the isolated crystals, the proton signals, H5, H6 and H7, of the quinolinyl ring are doubled, e.g. at δ 7.80 and 7.85 (H6), 8.36 and 8.37 (H5) and 8.85 and 8.89 (H7) p.p.m., suggesting that the quinolinyl fragments in the complex salt are experiencing two slightly different magnetic environments. This doubling is not found for racemic mefloquinium salts of non-chiral acids, such as acetic and nitrobenzoic acids.In the present study, the reaction of racemic (±)-erythro-mefloquine with the chiral carboxylic acid, (+)-PhC and 1.255 (5) Å] and C45—O6,O7 [1.239 (5) and 1.257 (6) Å] pairs of bond lengths.The crystallographic asymmetric unit of (I)a, with R- and S-configurations at the C12 and C13 chiral centres, respectively, is assigned as [(+)-erythro-mefloquinium], while with inverted configurations at the C29 and C30 centres, respectively, Fig. 1b, the N3-containing cation is [(−)-erythro-mefloquinium]. The cations are related by a pseudo centre of inversion and indeed the N1-containing molecule is virtually superimposable upon the mirror image of the N3-molecule, Fig. 1c, with the r.m.s. difference for bond distances and angles being 0.0082 Å and 0.550°, respectively  and 162.9 (3)°, respectively, and C35—C36—O5—C38 is −176.1 (3)°], in the second anion, Fig. 2b, these groups do not lie in a plane . In addition, the benzene rings occupy different relative positions to the carboxylate groups as indicated in the C6/CO2 dihedral angles of 89.1 (2) and 77.91 (17)° respectively.The anions in (I)N, H2N and H3N protons of the piperidin-1-ium residues is bifurcated. Two of these interactions are intramolecular N—H⋯Oh (h = hydroxyl) while the remaining N—H⋯O interactions, including that formed by the H4N atom, have a carboxylate-O atom as the acceptor. The result of the hydrogen bonding is the formation of a supramolecular chain along the a axis, Fig. 3a. The chains associate via C—H⋯F contacts to form the three-dimensional crystal structure, Fig. 3b; see Table 1As expected from the chemical composition, the molecular packing is dominated by O—H⋯O and N—H⋯O hydrogen bonding, Table 1et al., 2016i.e. two in (II) and (V), three in (VI) and (XV), four in (IV) and five in (III), a reasonable sample of structures is available for comment. The data confirm the proximity of the hydroxy-O and ammonium-N atoms in these species, and the l-shaped conformation owing to the orthogonal relationship between the quinolinyl and piperidin-1-ium residues. Despite varying compositions in (I)–(XVII), it is apparent that the molecular structures found for mefloquine/mefloquinium cations are robust. Finally, as mentioned above, the phenomenon of kryptoracemates is rare, occurring in just 0.1% of organic crystal structures (OMe)CO2H (1 mmol) in EtOH (10 ml) were mixed. The reaction mixture was maintained at room temperature and crystals slowly formed over a period of days. Crystals were collected in four batches, at suitable time intervals. Only the second batch had crystals suitable for the crystallographic study. The melting points of samples from each batch were similar, in the range 431–436 K. Those used in the X-ray study had m.p. 435–436 K. 1H NMR , 1.64–1.69 , 2.95 , 3.26 , 3.48 , 3.55 , 6.05 , 7.27–7.34 , 7.74 , 7.80 , 7.85 , 8.13 , 8.36 , 8.37 , 8.85 , 8.89 . 13C NMR , 115.43, 121.23 , 123.70 , 125.03 , 127.57, 127.60, 128.07, 128.13, 129.11, 129.86 , 136.02, 142.82, 146.73 , 151.43, 168.18. 19F NMR , −66.75 (cation), −69.79 (anion).Solutions of (±)-erythro-mefloquine base (1 mmol) in EtOH (10 ml) and (+)-PhC(CFUiso(H) = 1.2Ueq and 1.5Ueq(O).Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989016008495/hb7588sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989016008495/hb7588Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989016008495/hb7588Isup3.cmlSupporting information file. DOI: 1481913CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


H-1,3-thiazin-4-one with triphenyltin chloride, the three rings of the triphenyltin group are involved in intramolecular interactions of different types, and all the phenyl rings participate in intermolecular π–π interactions.In the adduct resulting from the reaction of 2,3-diphenyl-3,4,5,6-tetrahydro-2 H-1,3-thiazin-4-one-κO)triphenyltin, [Sn(C6H5)3Cl(C16H15NOS)], resulted from reaction of 2,3-diphenyl-3,4,5,6-tetrahydro-2H-1,3-thiazin-4-one with triphenyltin chloride. The thiazine ring has an envelope conformation with the S atom forming the flap. The molecule has five phenyl rings, two of them attached to the thiazine ring at positions 2 and 3, and three in coordination with the SnIV atom. The three rings of the triphenyltin group are involved in intramolecular interactions of different types, C—H⋯O, edge-to-face (or T-type) π–π interactions with the 3-phenyl ring of the thiazine, T-type interactions with both phenyl rings of the thiazine etc. On the other hand, all the phenyl rings participate in intermolecular π–π interactions. There is one instance of a ‘parallel-displaced’-type interaction extending continuously along the a-axis direction and seven instances of T-type interactions stabilizing the crystal lattice.The title adduct, chlorido(2,3-diphenyl-3,4,5,6-tetrahydro-2 All of the complexes reported by Tahara and Eng bind through the carbonyl oxygen atom to the central tin atom and adopt a distorted trigonal–bipyramidal geometry around the tin atom, with the heterocycle and chlorine in axial positions.Eng and coworkers have reported the synthesis and fungicidal activity of 1:1 complexes of triphenyltin chloride complexes with five-membered 1,3-thiazolidin-4-ones   resulting from reaction of (3) with triphenyltin chloride (Scheme 2), which to the best of our knowledge is the first preparation of a tin complex of any 2,3-disubstituted-1,3-thiazin-4-one heterocycle . The same carbon C18 is at a distance of 3.8287 (7) Å from the centroid of ring-3, resulting in a T-type π–π ring-4 ⋯ ring-3 interaction. Ring-6 has T-type interactions with both (ring-2 and ring-3) phenyl rings of the thiazine with inter-centroid distances of 5.112 (1) with ring-3 and 5.954 (1) Å with ring-2. The C3 atom of the thiazine ring is 3.5235 (6) Å from the centroid of ring-5, resulting in a C—H⋯π interaction. Thus all six rings, aromatic and non-aromatic, participate in influencing the structure of the molecule.Intermolecular interactions – The five phenyl rings interact extensively with the phenyl rings of the neighboring molecules in the lattice. Of the eight such π–π interactions, one belongs to the parallel-displaced type and seven are of the T-type. In the parallel-displaced interaction, ring-3 and ring-5 of a molecule interact respectively with ring-5 and ring-3 of molecules on opposite sides, forming a continuous chain along the a-axis direction. The distance between the centroids of these partially overlapping rings is 3.8627 (7) Å and the dihedral angle is 2° between the ring planes. Seven T-type interactions stabilize the lattice further with centroid distances ranging from 5.1688 (9) to 5.8599 (10) Å and the dihedral angles of 69° to 89°. Rings 2, 5 and 6 participate in three interactions each, ring-4 in two and ring-3 in one. The intra- and intermolecular π–π interactions are listed in Table 2.et al., 1986et al., 1970The crystal structure of triphenyltin chloride has also been reported (Tse 4) was prepared by reacting an equivalent each of triphenyltin chloride (Ph3SnCl) and 2,3-diphenyl-3,4,5,6-tetrahydro-2H-1,3-thiazin-4-one (3) . Ligroin (363–383 K b.p. range) was purchased from Fisher Chemical . Low-water acetone was purchased from J. T. Baker . Melting points were determined with a Thomas Hoover Capillary Melting Point Apparatus .1:1 Adduct (4) of 2,3-Diphenyl-3,4,5,6-tetrahydro-2H-1,3-thiazin-4-one (3) with triphenyltin chloride: A two-neck 10 mL round-bottom flask and a 5 mL round-bottom flask with stir bars were oven-dried, fitted with septa, and cooled under N2. Triphenyltin chloride  was added to the 10 mL flask. 2,3-Diphenyl-3,4,5,6-tetrahydro-2H-1,3-thiazin-4-one 3  was added to the 5 mL flask. 2.5 mL of low-water acetone was added to each flask and each solution was stirred. The contents of the 5 mL flask were transferred to the 10 mL flask dropwise by syringe over a period of 30 minutes. After two h of stirring, the stirrer was turned off. The solution was slightly hazy. After four days, the solution was transferred to a 50 mL round-bottom flask with acetone and concentrated under vacuum to a white solid. Recrystallization from ligroin produced (4) as a white powder , m.p. 405–407 K. Crystals for X-ray crystallography were grown by slow evaporation from cyclohexane.2) and 0.93 Å (–CHarom) and with Uiso(H) = 1.2Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989016001730/bg2579sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989016001730/bg2579Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989016001730/bg2579Isup4.molSupporting information file. DOI: 1450325CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Tau is an axonal protein that binds to and regulates microtubule function. Hyper-phosphorylation of Tau reduces its binding to microtubules and it is associated with β-amyloid deposition in Alzheimer’s disease. Paradoxically, Tau reduction may prevent β-amyloid pathology, raising the possibility that Tau mediates intracellular Aβ clearance. The current studies investigated the role of Tau in autophagic and proteasomal intracellular Aβ1-42 clearance and the subsequent effect on plaque deposition.−/− mice partially restored autophagic clearance of Aβ1-42, suggesting that exogenous Tau expression can support autophagic Aβ1-42 clearance. Tau deletion impaired autophagic flux and resulted in Aβ1-42 accumulation in pre-lysosomal autophagic vacuoles, affecting Aβ1-42 deposition into the lysosome. This autophagic defect was associated with decreased intracellular Aβ1-42 and increased plaque load in Tau−/− mice, which displayed less cell death. Nilotinib, an Abl tyrosine kinase inhibitor that promotes autophagic clearance mechanisms, reduced Aβ1-42 only when exogenous human Tau was expressed in Tau−/− mice.Tau deletion impaired Aβ clearance via autophagy, but not the proteasome, while introduction of wild type human Tau into TauThese studies demonstrate that Tau deletion affects intracellular Aβ1-42 clearance, leading to extracellular plaque.The online version of this article (doi:10.1186/1750-1326-9-46) contains supplementary material, which is available to authorized users. Two major pathologies that are linked to Alzheimer’s disease (AD), include extracellular β-amyloid (Aβ) plaques and intracellular neurofibrillary tangles (NFTs) comprised of hyper-phosphorylated Tau (p-Tau) . Althoug−/− mice. Intracellular Aβ may be cleared via autophagy and/or the proteasome [To evaluate whether Tau function affects intracellular Aβ clearance and alters extracellular plaque formation, we used lentiviral gene transfer models to focus on intracellular Aβ1-42 clearance in wild type and Tauoteasome , 15. We oteasome , 16, 17,oteasome , 16. Herin vitro (DIV) with lentiviral constructs driving the expression of human Aβ1-42 or wild type (WT) human Tau for 24 hrs. We previously showed that Nilotinib promotes autophagic clearance of Aβ1-42 [We previously demonstrated that impaired autophagic clearance of intracellular Aβ leads to more plaque deposition in parkin deficient mice , 16, 17.f Aβ1-42 , 18. To We previously reported that lentiviral Aβ1-42 expression leads to elevation of p-Tau in the rat cortex , 14. To Because of the observed effects of Aβ1-42 on p-Tau, we also measured p-Tau in cell extracts via ELISA in parallel with Aβ1-42 as shown in Figure Proteasome inhibition MG132) increased p-Tau in LacZ infected cells  in cells expressing lentiviral human Aβ1-42 (2nd blot) or human Tau (3rd blot) compared to total Tau levels (4th blot) relative to actin (5th blot). The ELISA results of Tau metabolism were confirmed with WB using AT8 antibody  or in the presence of MG132 (3rd blot) or Bafilomycin-A1 (5th blot) relative to actin. Nilotinib reduced p-Tau relative to actin  when Aβ1-42 and Tau were expressed together or separately in the presence or absence of MG132, while Bafilomycin-A1 blocked the effects of Nilotinib on p-Tau reduction.Bafilomycin-A1 robustly increased p-Tau in Aβ1-42 (+DMSO) infected cells  significantly decreased proteasomal function  and homozygous Tau−/− mice [−/− neurons  and in cells expressing Aβ1-42 and Tau together . No Aβ1-42 was detected in Tau expressing Tau−/− primary neurons. Bafilomycin-A1 increased Aβ1-42 levels (p < 0.05) and Nilotinib did not reverse these effects in Tau−/− neurons, but Tau expression reduced Aβ1-42 back to DMSO levels (p < 0.05), further suggesting that Tau expression enhances amyloid clearance. No p-Tau was detected in Aβ1-42 expressing Tau−/− neurons, but Nilotinib significantly  and Tau−/− mice, which are relevant to our experiments because organelle movement was reported to be impaired in these mice [6 multiplicity of infection (MOI). Animals were treated 3 weeks post-injection with daily 10 mg/kg intraperotineal (I.P) injection of Nilotinib or 30 μL DMSO once a day for 3  consecutive weeks as we previously reported [−/− mice compared to Aβ1-42 expressing WT mice  compared to Aβ1-42 alone. These data suggest that exogenous human Tau facilitates autophagic Aβ1-42 clearance. It is important to note that no changes were observed with mouse Aβ1-42 when lentiviral human Tau or Aβ1-42 were expressed in WT or Tau−/− mice (data not shown), and no changes were detected in APP cleaving enzymes [These results were also conducted ese mice , 21, whireported , 16. To  enzymes , indicat−/− compared to WT mice, and although Aβ1-42 together with Tau significantly increased p-Tau in Tau−/− mice (p < 0.04), p-Tau remained significantly lower (p < 0.05) in Tau−/− compared to WT mice. Nilotinib did not change p-Tau when human Tau was expressed alone in Tau−/− mice, but it significantly reduced it (p < 0.03) when Aβ1-42 and Tau were co-expressed.The effects of Nilotinib on p-Tau clearance were also examined. Nilotinib reduced p-Tau , which were identified by light chain protein-3 (LC3) that indicates pre-lysosomal autophagosome formation  was only detected in lentiviral Tau expressing Tau−/− mice  counterstaining shows endogenous p-Tau in the hippocampus of WT mice injected with a lentivirus driving LacZ expression  p-Tau  also increased the number of silver-positive cells  increased the number of silver-positive cells Figure E and K is Figure J and K be Figure G. Exogene Figure A but Nile Figure I and K, −/− brains restored autophagic Aβ1-42 and p-Tau clearance and reduced plaques. We previously demonstrated that the E3 ubiquitin ligase parkin is essential for Nilotinib-induced autophagic amyloid clearance [These studies demonstrate that autophagic intracellular Aβ1-42 clearance requires Tau, suggesting that normal Tau function modulates plaque deposition via regulation of intracellular Aβ1-42 degradation. Inhibition of either the proteasome or autophagy led to partial Aβ1-42 and p-Tau clearance, suggesting that Aβ1-42 and p-Tau may be degraded via either autophagy and/or the proteasome. Tau deletion impaired intracellular Aβ clearance and increased extracellular plaque formation, while introduction of human Tau into Taulearance . Howeverlearance , 16. Taulearance  recentlylearance  model inlearance . Our mod−/− mouse, which is reported to have impaired organelle movement [Tau maybe required to facilitate autophagosome maturation and retrograde transport from distal axons towards cell bodies to fuse with lysosomes , thus comovement , 21. Themovement . As automovement  as is ofmovement  and the −/− mice partially restores autophagic Aβ1-42 clearance, indicating that exogenous human Tau expression may affect Aβ1-42 levels. Expression of exogenous Tau may restore Aβ1-42 clearance due to possible integration of Tau into microtubules, leading to facilitation of autophagy. However, the relationship between Aβ and Tau is contentious with more recent in vivo reports demonstrating that Tau deletion prevents Aβ toxicity, and previous cell culture studies showing that Aβ triggers Tau pathology . Our daiewed in , 11. Howiewed in  and Tau iewed in , 7. Takeiewed in , which mIn conclusion, loss of normal Tau function may alter microtubule stability and affect the execution of autophagy. Our findings agree with previous studies that basal autophagy is essential and elevated autophagic activity is beneficial for neurons to prevent accumulation of protein aggregates –36. The 4 cells, neurons were infected with 3 μl lentiviruses for 24 hrs and then treated with 10 μM Nilotinib  or 1 μL DMSO for 24 hrs or 100 nM Bafilomycin-A1 or 20 μM MG132  for 6 hrs.Fetal WT hippocampal tissue (gestation E15) were dissected and gently triturated with a fire-polished Pasteur pipette, and the resulting pool of dissociated cells were plated in 35-mm dishes precoated with polyethyleneimine (1 mg/ml)  containing 0.5 ml basal growth medium, which was supplemented with 5% horse serum and 0.5% fetal calf serum and replaced with fresh medium every three days. DIV 14 or when the density of cells reached 3×106 multiplicity of infection (MOI) into the right CA1 hippocampus of 1 year old male C57BL/6 or Tau−/− as we previously explained [Lentiviral constructs driving LacZ, human WT Tau and/or Aβ1-42 were stereotaxically injected at 1×10xplained , 18. AllTo extract the soluble protein fraction, brain tissue or cells were isolated and homogenized in 1× STEN buffer (50 mM Tris (pH 7.6), 150 mM NaCl, 2 mM EDTA, 0.2% NP-40, 0.2% BSA, 20 mM PMSF and protease cocktail inhibitor), centrifuged at 10,000 g for 20 min at 4°C and the supernatant containing the soluble protein fraction was collected. To extract insoluble Aβ1-42, the pellet was re-suspended in 30% formic acid and centrifuged at 10,000 g for 20 min at 4°C and the supernatant was collected. Extracts were analyzed by WB on SDS NuPAGE 4-12% Bis-Tris gel . β-actin was probed (1:1000) with polyclonal antibody . Autophagy antibodies, including Beclin-1 (1:1000) and LC3-B (1:1000) were used to probe according to autophagy antibody sampler kit . Tau antibodies were used as we previously described , 18. WBsAnimals were deeply anesthetized with a mixture of (1:8) Xylazine  and Ketamine , washed with 1× saline for 1 min and then perfused with 4% (PFA) paraformaldehyde  for 15–20 min. Brains were quickly dissected out and immediately stored in 4% PFA for 24 hrs at 4°C and then transferred to 30% sucrose at 4°C for 48 h. Tissues were cut using a cryostat at 4°C into 20 μm thick sections and stored at −20°C. Cupric silver and immuno-staining were performed as we previously described .Stereological methods were applied by a blinded investigator using unbiased stereology analysis  to determine the total positive cell counts in 20 cortical/hippocampal fields on at least 10 brain sections  from each animal.2 radius of 6 randomly selected hippocampal and cortical regions in 6E10 stained slides. The number of plaques was averaged per mm2 and compared between treatment condition, as we previously described [Quantification of plaque load or counting plaque number was performed by a blind investigator using ImageJ by drawing a line around individual plaques within 1 mmescribed , 18.A total of 0.5 g of animal brains were homogenized at low speed  in 1× STEN buffer and centrifuged at 1,000 g for 10 min to isolate the supernatant from the pellet. The pellet was re-suspended in 1× STEN buffer and centrifuged once to increase the recovery of lysosomes. The pooled supernatants were then centrifuged at 100,000 rpm for 1 hr at 4°C to extract the pellet containing autophagic vacuoles (AVs) and lysosomes. The pellet was re-suspended in 10 ml (0 .33 g/ml) 50% Metrizamide  and 10 ml in cellulose nitrate tubes. A discontinuous Metrizamide gradient was constructed in layers from bottom to top as follows: 6 ml of pellet suspension, 10 ml of 26%; 5 ml of 24%; 5 ml of 20%; and 5 ml of 10% Metrizamide . After cHuman M17 neuroblastoma cells were co-transfected with 3 μg Tau, LacZ or Aβ1-42 cDNAs for 24 hrs and then treated with 10 μM Nilotinib for additional 24 hrs . Either 20 μM proteasomal inhibitor MG132 or 100nM autophagy inhibitor Bafilomycin-A1 were applied for 6 hrs prior to the beginning of proteasome activity assays. Cell extracts (100 μg) were incubated with 250 μM of the fluorescent 20S proteasome specific substrate Succinyl-LLVY-AMC  at 37°C for 2 hrs. The medium was discarded and proteasome activity was measured in tissue homogenates1–42  ELISA were performed according to manufacturer’s protocol as described [Specific p-Tau ser396  and Aβescribed , 19. Casescribed , 18.Data were analyzed with GraphPad software  using two-way ANOVA. The number of experiments and p values are indicated in the text.qRT-PCR in primary hippocampal neuronal culture was performed on Real-time OCR system with Fast SYBR-Green PCR master Mix  in triplicate from reverse-transcribed cDNA from mouse primary cortical neurons injected with lentiviral LacZ, Tau and/or Aβ1-42 (24 hrs) treated with DMSO or Nilotinib (24 hrs) using the same primers that were utilized to clone human Aβ1-42  or Tau  into theA) Aβ1-42 and B) V5 and C) merged figure showing that both LacZ and Aβ1-42 were co-expressed. Endogenous phosphorylated Tau using D) AT8 and E) V5 and I) merged figure showing LacZ expression. Lentiviral epitope G) V5 and H) AT8 and I) merged figure showing that both LacZ and Tau were co-expressed. Human specific J) Aβ1-42, K) AT8 and L) merged figure shows that Tau and of Aβ1-42 were co-expressed. Histograms represent M) densitometry of Beclin-1 relative to actin and LC3-II relative to LC3-I in WT and Tau−/− mice. Asterisk is significantly different to Aβ1-42 + DMSO, bars are mean ± SEM, two-way ANOVA. (TIFF 11 MB)Additional file 1: Figure S1: To verify equal expression of lentiviral clones, 20 μm thick coronal brain sections were stained human specific