The molecule of the neutral title compound and the cation in the title salt both exhibit a similar kind of disorder, even though their overall conformations are different. There are no direction-specific interactions between the molecules of the neutral compound, but the ions in the salt are linked into hydrogen-bonded sheets. N,N-dimethylethylamminium unit in N,N-dimethyl-[2--2-prop-2-ynyloxyacetoxy]ethylamine, C21H23NO3 (I), is disordered over two sets of atomic sites having occupancies of 0.880 (3) and 0. 120 (3), but there are no direction-specific interactions between the molecules of (I). The cation in N,N-dimethyl-[2--2-prop-2-ynyloxyacetoxy]ethylammonium 2,4,6-trinitrophenolate (picrate), C21H24NO3+·C6H2N3O7− (II), shows a similar type of disorder, with occupancies of 0.654 (11) and 0.346 (11), although the overall conformation of the cation in (II) is different from that in the neutral (I). The component ions are are linked by an almost planar three-centre N—H⋯(O)2 hydrogen bond, and the ion pairs are further linked by a combination of three C—H⋯O hydrogen bonds to form sheets. Comparisons are made with some related structures.The  N,N-dimethyl-[2--2-prop-2-ynyloxyacetoxy]ethylamine (pargeverine) is an established anti-spasmodic drug  Fig. 1 the meth2COCH2CCH et al., 1987et al., 2017The C—O distance in the picrate anion in (II)Despite the abundance of potential hydrogen-bond donors and acceptors in (I)via a near planar, but markedly asymmetric three-centre N-H⋯(O)2 charge-assisted in Fig. 3. In the gs Fig. 4. The comR,3R)-(hydrogentartrate) salt (III)et al., 2017In the (2i 2.461 (7) Å; O—H⋯Oi 167 (9)°, O—H 1.12 (1) Å, H⋯Oi 1.35 (10) Å . The cations are linked to this sheet by a three-centre N—O⋯(O)2 hydrogen bond and they are disposed to either side of the sheet  and containing equal numbers of gs Fig. 5. Within et Fig. 6.N-methyl piperidinium unit in place of the N,N-dimethylethylammonium unit in (II)et al., 20094--1-methylpiperidin-1-ium picrate (propiverinium picrate) (IV) is closely related to compound (II)A sample of compound (I)3) or 0.97 Å (CH2) and N—H 0.98 Å, with Uiso(H) = kUeq(carrier), where k = 1.5 for the methyl groups, which were permitted to rotate but not to tilt, and 1.2 for all other H atoms. The H atoms in the minor disorder components were included in calculated positions using the same procedure. When the refinement of the atomic coordinates for the H atoms bonded to N atoms in (II)K = [mean(Fo2)/mean(Fc2)] for the group of 518 very weak reflections having Fc/Fc(max) in the range 0.000 < Fc/Fc(max) < 0.004, and for (II)K = 9.509 for the group of 789 very weak reflections having Fc/Fc(max) in the range 0.000 < Fc/Fc(max) < 0.006.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989017012968/bq2403sup1.cifCrystal structure: contains datablock(s) global, I, II. DOI: 10.1107/S2056989017012968/bq2403Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989017012968/bq2403IIsup3.hklStructure factors: contains datablock(s) II. DOI: 1562193, 1562192CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


In the crystal, corrugated supramolecular layers in the bc plane are mediated by amine-N—H⋯N(pyridyl) and thioamide-N—H⋯S(thione) hydrogen bonds.The title molecule is twisted, with the dihedral angles between the imine core  core flanked by thiourea (CN2S) and methylpyridyl (C5N) residues (each plane has a r.m.s. deviation of the respective fitted atoms of 0.0066 Å). The dihedral angles between the core and the thiourea and pyridyl residues are 20.25 (8) and 7.60 (9)°, respectively, indicating twists in the molecule; the dihedral angle between the outer planes is 13.62 (7)°. There is an anti-disposition of the amine-N—H atoms which allows for the formation of an intramolecular amine-N—H⋯N(imine) hydrogen bond that closes an S(5) loop. In the crystal, amine-N—H⋯N(pyridyl) hydrogen bonds lead to zigzag (glide symmetry) supramolecular chains along the c-axis direction. These are connected into a supramolecular layer propagating in the bc plane by thioamide-N—H⋯S(thione) hydrogen bonds via eight-membered thioamide {⋯HNCS}2 synthons.The title disubstituted thiourea derivative, C TSC and its derivatives have attracted considerable attention due to their promising biological applications, especially in the realm of anti-tumour (Hussein E. The molecule in (I)anti-disposition of the amine-N—H atoms, which facilitates the formation of an intramolecular amine-N1—H⋯N3(imine) hydrogen bond to close an S(5) loop, Table 1anti to one another.The molecular structure of (I)2 synthons owing to the formation of thioamide-N2—H⋯S1(thione) hydrogen bonds between centrosymmetrically related molecules, Table 1c-axis direction and sustained by amine-N1—H⋯N4(pyridyl) hydrogen bonds, into a supramolecular layer propagating in the bc plane, Fig. 2a. Additional stabilization of the layers is afforded by methyl-C—H⋯π(pyridyl) interactions, Table 1a axis without directional interactions between them, Fig. 2b.The most prominent feature of the molecular packing is the formation of eight-membered thioamide {⋯HNCS}et al., 2017dnorm shown in Fig. 3The Hirshfeld surface calculations were performed according to recent work on a related organic molecule  due to the presence of N—H⋯S hydrogen bonds. The contribution of 0.6% from C⋯S/S⋯C contacts to the Hirshfeld surfaces of (I)The overall two-dimensional fingerprint plot for (I)t Table 2 and intet Table 2, respectt Table 2 and C—H⋯t Table 2, viewed 7 Table 2, are mer7 Table 2d due toet al., 2016i.e. that are neutral and feature N1-bound alkyl or aryl group and a C3-bound pyridyl ring; the C4-bound methyl group is common to all structures. The most closely related structure to (I)i.e. with an unsubstituted 2-pyridyl ring at the C3-position, has been described three times, being originally reported in 1999 −1): 3274 ν(N—H), 1589 ν(C=N), 1118 ν(N—N), 1045 ν(C=S). MS (m/z): 222.All chemicals were of analytical grade and were used without any further purification. 2-Acetyl-4-methyl pyridine  in absolute ethanol (40 ml) was dissolved and added to 4-methyl-3-thiosemicarbazide  dissolved in absolute ethanol (40 ml). The mixture was then heated in a water bath for 10 mins with constant and vigorous stirring until the volume reduced to 30 ml. The product that formed was filtered off, washed with cold ethanol and dried in a desiccator over anhydrous silica gel. Brown platy crystals suitable for single crystal X-ray diffraction analysis were obtained by recrystallization with absolute ethanol as solvent. M.pt: 468.8–470.1 K. IR (cmUiso(H) set to 1.2–1.5Ueq(C). The nitrogen-bound H atoms were located in a difference-Fourier map but were refined with a distance restraint of N—H = 0.88±0.01 Å, and with Uiso(H) set to 1.2Ueq(N).Crystal data, data collection and structure refinement details are summarized in Table 410.1107/S2056989018001305/hb7729sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989018001305/hb7729Isup2.hklStructure factors: contains datablock(s) I. DOI: 1818317CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The crystal structure features two O—H⋯O hydrogen bonds, forming chains along the [010] direction. 20H26O3  was isolated from Taxodium distichum (L.) Rich. The compound crystallizes in the space group P21. The crystal structure features two O—H⋯O hydrogen bonds, forming chains along the [010] direction.The title compound, 6,7-dehydroroyleanone, C Taxodium distichum (L.) Rich. is a tree native to North America that can grow to 25 m in height  Rich. led to the isolation of the title compound 6,7-dehydroroyleanone. Herein we present the crystal structure of 6,7-dehydroroyleanone, which was undertaken in order to establish unambiguously the stereochemical features of this natural compound.A⋯O1 hydrogen bond  Rich. collected in Xining, China, in April 2015 (SC0185). The air-dried seeds of Taxodium distichum (1.1 kg) were extracted with 95% EtOH and then partitioned successively with petroleum ether (PE), ethyl acetate (EtOAc) and n-butyl alcohol (n-BuOH) to give a PE extract (30 g), an EtOAc extract (50 g) and an n-BuOH extract (68 g). The PE extract (30 g) was subjected to normal-phase silica-gel column chromatography (300–400 mesh) with a gradient solvent system of petroleum ether–ethyl acetate  to give ten major fractions, denoted F1–F10. F7 (2.8 g) was sequentially subjected to Sephadex-LH20 gel column chromatography  to give four major fractions F7.1–F7.4. F7.3 was purified by semi-preparative HPLC  to give an orange solid, which was recrystallized from a solvent mix of CH2Cl2–MeOH (5:1) affording orange block-like crystals suitable for X-ray diffraction analysis. For the 1H and 13C NMR data of 6,7-dehydroroyleanone, see Chang et al.  = 1.5Ueq(C) for methyl H atoms and 1.2Ueq(C) for all others. The OH hydrogen atom was refined freely with Uiso(H) = 1.5Ueq(O).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989017017935/qm2118sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989017017935/qm2118Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989017017935/qm2118Isup3.cdxSupporting information file. DOI: Click here for additional data file.10.1107/S2056989017017935/qm2118Isup4.cmlSupporting information file. DOI: 1551127CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Radix Glycyrrhizae is the rhizome of Glycyrrhiza inflata Bat., Glycyrrhiza uralensis Fisch. or Glycyrrhiza glabra L. The present paper describes the isolation and the structural elucidation of three new dihydroisocoumarins obtained from the 70% EtOH extract of Radix Glycyrrhizae. And the cytotoxic activities of these new compounds were also evaluated using four cell lines, subsequently.R,4S)-4,8-dihydroxy-3-methyl-1-oxoisochroman-5-yl)methyl acetate (1) and -4,8-dihydroxy-3-methyl-1-oxoisochroman-5-yl)methyl acetate (2) along with a new dihydroisocoumarin -4,8-dihydroxy-3,5-dimethylisochroman-1-one (3) were isolated from Radix Glycyrrhizae. Their structures were elucidated on the basis of chemical and spectral analysis, including 1D, 2D NMR analyses, HR–ESI–MSand ECD calculation comparing with those of experimental CD spectra. Cytotoxic activities of the three compounds were evaluated using the HepG2, A549, LoVo and Hela cell lines, respectively. IC50 values indicated compounds 1–3 exhibited moderate or less cytotoxic activity in vitro.A pair of new dihydroisocoumarin epimers  contains supplementary material, which is available to authorized users. Radix Glycyrrhizae is the rhizome of Glycyrrhiza inflata Bat., Glycyrrhiza uralensis Fisch. or Glycyrrhiza glabra L. They are widely distributed in the northwest and northeast of China +, indicating the presence of seven degrees of unsaturation. The 1H-NMR spectrum of compound 1 , two methyl signals at δH1.45  and 2.04 , two aromatic proton signals at δH6.99  and 7.64 , along with some other methylene and methine proton signals . The 13C-NMR spectrum of compound 1 , three oxygenated sp3 carbons , and two methyl carbons (δC16.6 and 21.2). All the NMR data and the degree of unsaturation revealed the presence of a 1,2,3,4-substituted benzyl group and a lactone ring. Signal assignments were specified by Heteronuclear Single Quantum Coherence (HSQC) experiment. The Heteronuclear Multiple-Bond Correlation (HMBC) spectrum -4,8-dihydroxy-5-(hydroxymethyl)-3-methylisochroman-1-one [1 was determined as shown in Fig. Compound 2 was also obtained as yellow crystal. The molecular formula was determined to be C13H14O6 by HR–ESI–MS at m/z 289.0670 [M + Na]+. The 1H and 13C NMR signals of 2 were almost identical to those of 1 with slight difference at C-1, C-3, C-4, C-5, and C-4a. The CD spectrum of 2 gave an exactly opposite absorption band at 250 nm compared with that of 1, and thus 2 was suggested to be the epimer of 1 at C-3. HMBC correlations of 2 shown in Fig. 2, which was the same as that of 1. The relative configurations of 1 and 2 were established by NOESY analysis methyl acetate as shown in Fig. 1 and 2 were also expressed in Fig. 1 and 2, the absolute configurations of C-3 and C-4 were determined to be (R), (S) and (R), (R) for 1 and 2, respectively.The ECD (Electronic Circular Dichroism Spectroscopy) calculating study of 3 was obtained as yellow crystal. The molecular formula was determined to be C11H12O4 by HR–ESI–MS at m/z 231.0637 [M + Na]+. The 1H and 13C NMR spectral data of 3 were similar to those of 2, expect for the disappearance of an acetoxy group at C-10. The absolute configuration of 3 was established by the analysis of its CD spectrum. A positive Cotton effect at 250 nm was shown in the CD spectrum -configuration same as 2.Compound 50 values of these compounds were shown in Table The cytotoxic activities of compounds 1-3 were evaluated using the HepG2, A549, LoVo and Hela cell lines, respectively. The IC1H chemical shifts (δH) were measured in ppm, relative to TMS, and 13C chemical shifts (δC) were measured relative to DMSO-d6 and converted to TMS scale. Column chromatography (CC) was performed on Silica gel  and Sephadex LH-20  columns. HPLC was performed on a Shimadzu LC-10AVP liquid chromatograph with a YMC-pack C18 (ODS) column  and a Shimadzu LC-8AVP liquid chromatograph with a Diamonsil C18 (ODS) column . All reagents for isolation were HPLC or analytical grade and were purchased from Tianjin Damao Chemical Company . Fetal bovine serum and Dulbecco’s modified eagle medium (DMEM) were from Thermo Fisher Scientific, 96-well flat bottom plate were purchased from Corning Inc. , 3--2,5 diphenyltetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich Corporation .The UV spectrum was recorded on a Shimadzu UV-2201 spectrophotometer . The IR spectrum was obtained from a Bruker IFS-55 spectrophotometer using a KBr pellet . The HR-ESI–MS data were obtained on a microTOF-Q Bruker mass instrument . CD spectra were recorded with a Biologic MOS-450 spectrometer using MeOH as solvent. 1D and 2D NMR spectra were run on a Bruker AVANCE 600 spectrometer . Radix Glycyrrhizae was purchased from Anhui Yishengyuan Traditional Chinese Medicine Pellets Co., Ltd., P. R. China, and all the materials were identified by Dr. Xiao Fu, Department of Traditional Chinese Medicine, First Affiliated Hospital of Jinzhou Medical University. The voucher specimen (20150610) has been deposited at First Affiliated Hospital of Jinzhou Medical University.Radix Glycyrrhizae (25 kg) was cut and extracted with 70% EtOH for two times. The combined extracts were concentrated in vacuo to yield a residue, and the residue was then suspended in H2O and successively partitioned with petroleum ether, dichloromethane (CH2Cl2), ethyl acetate (EtOAc). The EtOAc crude extracts (2.3 kg) were applied on a silica gel column and eluted with petroleum ether-acetone gradient (from 500:0 to 0:100) to afford nine fractions. Fr. 6 was subjected to Sephadex LH-20, semi-preparative HPLC to yield compound 1 (12.0 mg) and 2 (9.2 mg). Fr. 7 was subjected to Sephadex LH-20, semi-preparative HPLC to yield compound 3 (15 mg).3OH); UV (MeOH) λmax(log ε) 214, 315 nm; IR (KBr) νmax 3408.9, 2920.2, 2850.0, 1674.3, 1446.1,1384.2, 1207.2, 1138.8 cm−1; CD (mdeg): Δε212 nm + 25.0,Δε250 nm − 14.6, Δε290 nm + 5.6; 1H and 13C-NMR spectral data, see Table m/z 289.0681 [M + Na]+ .Yellow needle crystal (CH3OH); UV (MeOH) λmax(log ε) 218, 316 nm; IR (KBr) νmax 3418.5, 2920.1, 2850.8, 1675.2, 1477.9,1383.7, 1208.1, 1171.9 cm−1; CD (mdeg): Δε198 nm − 12.5,Δε212 nm + 5.0,Δε250 nm + 25.0, Δε316 nm − 3.7;1H and 13C-NMR spectral data, see Table m/z 289.0670 [M + Na]+ .Yellow needle crystal (CH3OH); CD (mdeg): Δε198 nm − 6.5,Δε212 nm + 7.0,Δε250 nm + 13.8, Δε316 nm − 1.1;1H and 13C-NMR spectral data, see Table m/z 231.0637 [M + Na]+ .Yellow needle crystal . Inhibitory concentrations of compounds were calculated and half maximal inhibitory concentrations (IC50) values were confirmed. 5-Fluorouracil and dimethyl sulfoxide  were used as positive control and negative control, respectively.Four cell lines including HepG2, A549, LoVo and Hela cell lines were purchased from the American Type Culture Collection. All the cell lines were used to evaluate the cytotoxic activities of compounds 1-3 in vitro by the method of MTT. Briefly, HepG2, A549, LoVo and Hela cells were seeded in 96-well flat bottom plates at a density of about 1 × 10Radix Glycyrrhizae. Triterpenoids including glycyrrhizic acid, glycyrrhetinic acid and flavonoids including isoliquiritigenin, liquiritigenin, isoliquiritin, Licochalcone A, B and E are considered as the main characteristic constituents of the herb. And the anticancer bioactivities of these characteristic compounds were assayed frequently. Compared to triterpenoids, flavonoids possessed stronger anticancer bioactivities. In this study, three dihydroisocoumarins (1–3) showed the less toxicities on A549 and HepG2 cell lines than that of flavonoids constituents reported previously. IC50 value of isoliquiritigenin, licochalcone A and E on A549 cell lines were 18.5, 14.3 and 17.3 μM, respectively. Licochalcone A possessed almost the same toxicity on HepG2 cell lines . But the dihydroisocoumarins (1–3) showed more toxicity on A549 cell lines than that of Glycyrrhizic acid [A mount of chemical constituents have been isolated and identified from Additional file 1. NMR and MS spectrum of compound 1–3.


Transforming growth factor-β (TGF-β) plays a paradoxical role in cancer: it suppresses proliferation at early stages but promotes metastasis at late stages. This cytokine is upregulated in cholangiocarcinoma and is implicated in cholangiocarcinoma invasion and metastasis. Here we investigated the roles of non-Smad pathway (ERK1/2) and Smad in TGF-β tumor promoting and suppressing activities in intrahepatic cholangiocarcinoma (ICC) cells.TGF-β1 effects on proliferation, invasion and migration of ICC cells, KKU-M213 and/or HuCCA-1, were investigated using MTT, colony formation, in vitro Transwell and wound healing assays. Levels of mRNAs and proteins/phospho-proteins were measured by quantitative (q)RT-PCR and Western blotting respectively. E-cadherin localization was examined by immunofluorescence and secreted MMP-9 activity was assayed by gelatin zymography. The role of ERK1/2 signaling was evaluated by treating cells with TGF-β1 in combination with MEK1/2 inhibitor U0126, and that of Smad2/3 and Slug using siSmad2/3- and siSlug-transfected cells.h-TGF-β1 enhanced KKU-M213 cell invasion and migration and induced epithelial-mesenchymal transition as shown by an increase in vimentin, Slug and secreted MMP-9 levels and by a change in E-cadherin localization from membrane to cytosol, while retaining the cytokine’s ability to attenuate cell proliferation. h-TGF-β1 stimulated Smad2/3 and ERK1/2 phosphorylation, and the MEK1/2 inhibitor U0126 attenuated TGF-β1-induced KKU-M213 cell invasion and MMP-9 production but moderately enhanced the cytokine growth inhibitory activity. The latter effect was more noticeable in HuCCA-1 cells, which resisted TGF-β-anti-proliferative activity. Smad2/3 knock-down suppressed TGF-β1 ability to induce ERK1/2 phosphorylation, Slug expression and cell invasion, whereas Slug knock-down suppressed cell invasion and vimentin expression but marginally affected ERK1/2 activation and MMP-9 secretion. These results indicate that TGF-β1 activated ERK1/2 through Smad2/3 but not Slug pathway, and that ERK1/2 enhanced TGF-β1 tumor promoting but repressed its tumor suppressing functions.Inhibiting ERK1/2 activation attenuates TGF-β1 tumor promoting effect (invasion) but retains its tumor suppressing role, thereby highlighting the importance of ERK1/2 in resolving the TGF-β paradox switch.The online version of this article (doi:10.1186/s12935-017-0454-2) contains supplementary material, which is available to authorized users. Cholangiocarcinoma (CCA), a bile duct malignancy, is one of the most severe forms of cancer with a 5-year survival rate < 5% . AlthougOpisthorchis viverrini or Clonorchis sinensis) infection, depending on geographical location [O. viverrini infection is the major risk factor for CCA [O. viverrini-infected hamsters to N-nitrosodimethylamine promotes liver fibrosis and cholangiocarcinogenesis [Risk factors of ICC include hepatolithiasis, primary sclerosis cholangitis, cirrhosis, and liver fluke  or cyclin-dependent kinase inhibitors, or suppressing those of Myc, Id1/2 or survivin , 13. HowTGF-β1 is overexpressed in biliary dysplasia, hyperplasia and ICC  and is iERK and its upstream regulators, namely, Ras, B-Raf and growth factor receptors, frequently are mutated and participate in cancer progression . UpregulO. viverrini-associated ICC cell lines, HuCCA-1 and KKU-M213, derived from cells of Thai CCA patients, were generously provided by Professor Stitaya Sirisinha  and Professor Banchob Sripa  respectively. Cells were maintained at 37 °C in HAM’s F-12 media  consisting of 15 mM HEPES, 14 mM NaHCO3, 100 U/mL penicillin G, 100 μg/mL streptomycin, and 10% heat-inactivated fetal bovine serum (FBS) under a humidified 5% CO2 atmosphere.Human 3) were plated onto a 96-well plate in 10% FBS media and cultured for 24 h, then treated with 5 ng/mL h-TGF-β1 , or 10 μM SB431542 (TβRI kinase inhibitor) , or 1 μM U0126 (MEK1/2 inhibitor) , or a combination of h-TGF-β1 and each inhibitor in 10% FBS media for up to 72 h. Control cells were treated with vehicle  and 0.025% dimethyl sulfoxide (DMSO)). At indicated times, the numbers of viable cells were determined by replacing media with 100 μL of 0.5 mg/mL 3--2,5-diphenyltetrazolium bromide  in 10% FBS media. Cells were incubated for 4 h, then supernatant was removed and insoluble formazan dye was dissolved in 200 µL of DMSO. A 540 nm was measured using a Multiskan EX Microplate Reader .For MTT assay, ICC cells  crystal violet in 25% methanol. Colony forming ability was determined by measuring the area of crystal violet stain using an Image J program .Confluent cells in a 24-well plate were incubated for 24 h with 0.1% FBS media containing h-TGF-β1 with and without SB431542. Control cells were treated with vehicle. Wounds were created by scratching with a 1-mL-pipet tip and wells were washed with 0.1% FBS media, then 0.1% FBS media containing h-TGF-β1 with and without inhibitor were added. Cells were incubated for 24 h to allow migration into wound area. Images under phase contrast microscope (40 × magnification) were recorded and analyzed using an Image J program at 0 h (immediately after wound creation) and at 24 h.5 ICC cells  was added to the upper chamber, while the lower chamber was filled with the same media. Following incubation at 37 °C for 6 or 12 h, non-invaded cells in the upper chamber were removed and invaded cells attached underneath the membrane were stained with 5% crystal violet in 25% methanol. The numbers of invaded cells were counted in five random fields under a light microscope (100 × magnification).In vitro invasion and migration abilities of ICC cells were determined using Matrigel-coated and -uncoated Transwells  respectively. In the in vitro invasion assay, the upper surface of a Transwell polycarbonate membrane (6.5-mm diameter) was coated with 30 μg of Matrigel . Then a 200 µL aliquot of 10−ΔΔCt method [ICC cells (80% confluent) were treated with h-TGF-β1 in 0.1% FBS media with or without SB431542 for 6 and 24 h. RNA was extracted using Illustra RNAspin Mini RNA Isolation Kit . Total RNA (1 µg) was reverse transcribed using a random hexamer primer and Improm-II™ reverse transcriptase . QRT-PCR mixture (20-µL) reaction contained 1 × FastStart Universal SYBR Green Master cocktail  and 6 pmol of each specific primer pair , respectively). Thermocycling was performed in Stratagene Mx 3000P instrument  as follows: 95 °C for 10 min; and 40 cycles of 94 °C for 30 s, 58 °C for 1 min and 72 °C for 1 min. Relative mRNA levels of treated compared to untreated control cells were determined using 2t method .Steady-state mRNA levels of TGF-β, activin and nodal were quantified by qRT-PCR as described above using RNA extracted from 80% confluent cells cultured in 10% FBS media. Specific primer pair for TGF-β, activin and nodal was 5′-AACCCACCCGAAATCTATGAC-3′/5′-GCTGAGGTATCGCCAGGAAT-3′, 5′-GGAGCTCAGACAGCTCTTACC-3′/5′-GCAAATTCTCTTTCTGGTCCCC-3′, and 5′-AGACATCATCCGCAGCCTACA-3′/5′-GTCCATCTGAAACCGCTCTAAG-3′, respectively , 28.3VO4, 40 mM β-glycerophosphate, and 1 mM dithiothreitol, then subjected to 10% SDS-PAGE. Proteins were transferred onto nitrocellulose membrane and incubated with primary antibodies against phospho-Smad2, phospho-ERK1/2, total-ERK1/2 , vimentin, Slug, β-actin and GAPDH (loading control) , followed by horse-radish peroxidase-conjugated anti-IgG secondary antibodies (Santa Cruz Biotechnology). Chemiluminescent signals of immunoreactive proteins were visualized using Luminata™ Forte Western HRP substrate  and recorded by a G-Box Chemi XL system .ICC cells (80% confluent) were treated with h-TGF-β1 with or without SB431542 or U0126 in 0.1% FBS media. At indicated times, cells were lysed with a solution of 150 mM Tris–HCl pH 7.4, containing 150 mM NaCl, 5 mM EGTA, 5 mM EDTA, 0.1% SDS 1% sodium deoxycholate, 1% Nonidet P-40, 1X protease inhibitor cocktail (Roche Diagnostics), 50 mM NaF, 2 mM Na5) were treated with h-TGF-β1 with and without SB431542 or U0126 in 0.1% FBS media. At indicated times, 20 μL aliquots of conditioned media were mixed with non-reducing SDS-PAGE loading buffer and subjected to 7.5% SDS-PAGE containing 1 mg/mL gelatin for detection of MMP-2 and -9, or 10% SDS-PAGE containing 1 mg/mL gelatin plus 10 μg/mL plasminogen for uPA detection. After washing with 2.5% Triton X-100, gels were incubated for 18 h in a solution containing 50 mM Tris–HCl pH 7.5, 10 mM CaCl2, 1 μM ZnCl2 and 1% Triton X-100 for gelatinase assay or 100 mM Tris–HCl pH 7.8, 150 mM NaCl and 1% Triton X-100 for uPA assay. Gels were stained with Coomassie blue dye and clear band of 67 and 82kDa in gelatin gel corresponds to MMP-2 and -9 respectively, and of 43 kDa in plasminogen-gelatin gel to that of uPA.Amounts of MMP-2 and MMP-9 in conditioned media were determined by gelatin zymography and uPA amount by plasminogen-gelatin zymography. Cells . Cell nuclei were stained with DAPI (Molecular Probes®). Coverslips were mounted with Prolong® gold antifade reagent (Molecular Probes®) and examined under a confocal microscope .ICC cells (60% confluent) were deposited on coverslips and treated for 24 h with h-TGF-β1 with and without SB431542 or U0126 in 0.1% FBS media, then incubated with phosphate-buffered saline (PBS) containing 4% paraformaldehyde and 2% sucrose, and permeabilized with 0.25% Triton X-100 followed by incubation with 2% BSA in PBS. Cells then were treated with mouse anti-E-cadherin antibody (Santa Cruz Biotechnology), followed by Alexa Fluor5) in 10% FBS media were cultured in a 6-well plate for 24 h, then transfected with 15 nM siRNA targeting mRNA of MMP-9 (siMMP-9), Smad2/3 (siSmad2/3), Slug (siSlug) (Santa Cruz Biotechnology) or 15 nM AllStars siRNA Negative Control (siNeg)  using Lipofectamin® RNAiMAX reagent  following manufacturer’s protocol. After 24 or 48 h, cells were treated with or without h-TGF-β1 in 0.1% FBS media for another 24 h. Invasive ability of treated cells then was determined using the Transwell assay as described above.ICC cells  of three independent experiments conducted at least in duplicate. Statistical comparisons of more than two groups were performed using one-way analysis of variance (one-way ANOVA) with LSD’s post hoc test. Data are considered significantly different when TGF-β1 is involved in invasion and metastasis of a number of cancers including CCA , 23. ThiTGF-β enhances tumor invasion and metastasis through promoting EMT, a process mediated by a group of transcription factors, such as Slug and Snail, via the canonical signaling Smad pathway , 30. TheEMT induced by h-TGF-β1 was demonstrated by determining the increase in levels of Slug, E-cadherin, vimentin and matrix degrading enzymes. Slug level was raised 1.5–2.5 folds following 2–6 h of exposure to 5 ng/mL h-TGF-β1, which was maintained for up to 24 h Fig. a. This sNot only Smad but ERK1/2 has been reported to act as downstream effectors contributing to TGF-β-induced EMT . The notSmad phosphorylation and Slug production are well-known downstream regulators of TGF-β1-induced progression of many types of cancers , 30. ThuIn general, TGF-β1 exhibits anti-proliferative and apoptotic activities during the early stages of tumorigenesis . HoweverColony formation assay was performed to investigate the long-term effect of h-TGF-β1 and U0126 on proliferation of the two ICC cell lines, KKU-M213 and HuCCA-1. The ability of cells to proliferate was quantitated from determination of colony area. KKU-M213 cells were sensitive to TGF-β anti-proliferative activity (74 ± 1% reduction in colony area after 7 days of TGF-β-treatment compared to control), while blocking ERK activation with U0126 alone only slightly reduced colony area (13 ± 1% compared to control), but interestingly, when combined the colony area was further diminished (84 ± 1% compared to control)  cells  and NSCLUsing two human ICC cell lines, KKU-M213 and HuCCA-1, we demonstrate that h-TGF-β1 promotes cell invasion and that inhibition of ERK1/2 pathway suppresses this effect in both ICC cell lines with KKU-M213 being more sensitive to ERK1/2 inhibition. Focusing on the more responsive KKU-M213 cell line, h-TGF-β1 activates ERK1/2 signaling through the Smad2/3 but not Slug pathway. This ERK1/2 activation is partly responsible for the role of h-TGF-β1-induced ICC cell migration, EMT and MMP-9 expression. As regards TGF-β tumor suppression role, both ICC cell lines partially retain the cytokine anti-proliferative ability with KKU-M213 cells being more sensitive. Unlike in the case of cell invasion, blocking ERK pathway activation failed to attenuate TGF-β anti-proliferative activity and even exerted an additional effect on inhibiting cell proliferation. Taken together, these data indicate distinct roles of ERK1/2 in TGF-β1 tumor promoting and tumor suppressing effects in that ERK enhances TGF-β-role in promoting invasiveness but attenuates its growth inhibitory function in ICC cells Fig. .Ras/ERK pathway contributes to both TGF-β tumor suppressing (anti-proliferation and apoptosis) and tumor promoting (migration and invasion) activities . For theThe effect of ERK signaling on TGF-β anti-proliferative activity depends on cell types. In p53-reconstituted lung cancer H1299 cells, ERK promotes anti-proliferation by inducing p21 expression through p53 (Ser 9) phosphorylation . HoweverIt is worth noting that the KKU-M213 cell line used in this study has basal migratory and invasive properties that are sensitive to SB431542, not only an inhibitor of TβRI (ALK-5) but also of ALK-4 and ALK-7, suggesting that the cells secrete and are responsive to TGF-β or its superfamily member(s), such as activin or nodal, which relay signals through their cognate receptors ALK-4 or ALK-7 . This noThe mechanisms and kinetics of TGF-β-stimulated ERK1/2 activation are diverse depending on cell types and context. It may act through ShcA induction or Src phosphorylation, independent of Smad, leading to a rapid and transient (within minutes) activation of Ras/ERK pathway , 48; or This study shows that in ICC cell lines, TGF-β1 stimulates cell migration and invasion (tumor promoting effects) and suppresses cell proliferation (tumor suppressing effect). The former function of TGF-β depends on ERK1/2 activation, whereas the anti-proliferative function is weakened by this pathway. This opens the door to target ERK-signaling pathway for selective suppression of ICC TGF-β-induced invasion and metastasis while enhancing the cytokine anti-proliferation function.Additional file 1. Relative quantitative immunoblot of U0126 effect on total ERK, phospho-ERK1/2 and vimentin levels in h-TGF-β 1-induced ICC cells. Cells were treated with 5 ng/mL h-TGF- β 1 and/or 10 µM SB431542 or 1 µM U0126 in 0.1% FBS media for 24 h before analyzing for total ERK, phospho-ERK1/2 (a) and vimentin (b) by immunoblotting. Relative protein levels were analyzed from protein band intensity normalized relative to GAPDH and compared to those of untreated controls. Data are presented as mean ± SEM of fold change in protein levels relative to control. *P value < 0.05.Additional file 2. Effects of U0126 on h-TGF-β1-induced HuCCA-1 cell invasion. Cells (105) pre-treated with 5 ng/mL h-TGF-β1 and/or 1 µM U0126 in 0.1% FBS media for 24 h were plated onto in vitro invasion Transwell chamber and allowed to invade for 12 h. Invasion ability are presented as mean ± SEM of percent change in numbers of invaded cells compared to h-TGF-β-treated condition obtained from three independent experiments. *P value < 0.05, **P value < 0.001.Additional file 3. Relative quantitative immunoblot of the effect of Smad and Slug silencing on ERK and Smad phosphorylation and Slug and vimentin expression in ICC cells. Smad2/3 and Slug expression in the ICC cells were suppressed using specific siRNA. After 48 h of transfection, cells were treated with or without 5 ng/mL h-TGF- β 1 in 0.1% FBS media for 24 h, followed by analysis for total and phospho-ERK1/2 (a), total and phospho-Smad2/3 (b), Slug (c) vimentin (d) and GAPDH levels by immunoblotting. Relative protein levels were analyzed from protein band intensity normalized relative to total ERK (a), total Smad 2/3 (b) or GAPDH  and compared to siNeg-transfected control. Data are presented as mean ± SEM of fold change in protein levels relative to control. *P value < 0.05, **P value < 0.001 compared to siNeg. #P value < 0.05, ##P value < 0.001 compared to siNeg treated with TGF-β.Additional file 4. Role of ERK1/2 activation in h-TGF-β1 anti-proliferative activity of HuCCA-1 cell line. Cells were treated with 5 ng/mL and 200 cells seeded on 24-well plate were treated with h-TGF-β1 with or without U0126 in 10% FBS media for 7 days. Colony formation ability was quantified as percent ± SEM of crystal violet-stained area compared to control. *P value < 0.05, **P value < 0.001.Additional file 5. Steady state levels of TGF-β1, activin and nodal expression in KKU-M213 cells. Levels of TGF-β1, activin and nodal mRNA were determined by SYBR-green-based qRT-PCR using RNA extracted from 80% confluent cells cultured in 10% FBS media. Relative mRNA levels were calculated using 2−ΔCt formula compared to that of 18 s rRNA.


H-chromene-3-carbaldehyde, molecules are linked through stacking interactions and C—H⋯O hydrogen bonds. Halogen bonds between the formyl O and I atoms are also formed.In the crystal of 7-iodo-4-oxo-4 10H5IO3, an iodinated 3-formylchromone derivative, the non-H atoms are essentially coplanar (r.m.s. deviation = 0.0344 Å), with the largest deviation from the least-squares plane [0.101 (3) Å] being found for the formyl O atom. In the crystal, molecules are linked through stacking interactions [centroid–centroid distance between the benzene rings = 3.700 (3) Å] and C—H⋯O hydrogen bonds. Halogen bonds between the I atoms at 7-position and the formyl O atoms  are also formed along [110], resulting in sheets perpendicular to the c axis, constructed by C—H⋯O hydrogen bonds and I⋯O halogen bonds.In the title compound, C The main objective of this study is to reveal the interaction mode of the iodine substituent at the 7-position of the chromone ring in the solid state.As part of an investigation of halogenated 3-formylchromones relevant to urease inhibitory activity and halogen bonding, I herein report the crystal structure of 7-iodo-4-oxo-4The mean deviation of the least-square planes for the non-hydrogen atoms is 0.0344 Å, and the largest deviation is 0.101 (3) Å for O3, indicating that these atoms are essentially coplanar Fig. 1. All boni molecules , and through C—H⋯O hydrogen bonds (Table 1ii molecules  along [1 1 0], resulting in sheets perpendicular to the c-axis, constructed by C—H⋯O hydrogen bonds and I⋯O halogen bonds . In the crystals of 7-chloro- and 7-bromo-3-formylchromones, type I and type II halogen⋯halogen contacts are found, respectively , and these halogen⋯halogen contacts are commonly found for Cl and Br atoms , but not in 7-fluoro-, 7-chloro-, and 7-bromo-3-formylchromones. This is in agreement with an assumption that the iodine atom should have the largest σ-hole  was added dropwise POCl3 (13.2 mmol) at 273 K. After the mixture was stirred for 14 h at room temperature, water (100 ml) was added. The precipitates were collected, washed with water, and dried in vacuo at 333 K (yield 86%). 1H NMR : δ 7.84 , 7.96 , 7.99 , 8.49 , 10.36 . Single crystals suitable for X-ray diffraction were obtained by slow evaporation of a 1,2-dichloroethane solution of the title compound at room temperature.2′-Hydroxy-4′-iodoacetophenone was prepared from 3-acetoxyiodobenzene by a Fries rearrangement reaction. To a solution of 2′-hydroxy-4′-iodoacetophenone (4.4 mmol) in Uiso(H) = 1.2Ueq(C)].Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989016016972/zl2682sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2056989016016972/zl2682Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989016016972/zl2682Isup3.cmlSupporting information file. DOI: 1511202CCDC reference: crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Specifically, the reduction in lymphocyte numbers due to in vivo activation of macrophages was ameliorated in Icam-1-deficient mice. In addition, overexpression of ICAM-1 or VCAM-1 in non-phagocytic NIH3T3 cells led to active phagocytosis of live cells. These data indicate molecular mechanisms underlying live cell phagocytosis induced by hyper-inflammation, and this experimental model will be useful to clarify the pathophysiological mechanisms of hemophagocytosis and to indicate therapeutic targets.Conditions of severe hyper-inflammation can lead to uncontrolled activation of macrophages, and the ensuing phagocytosis of live cells. However, relationships between inflammatory stimuli and uncontrolled phagocytosis of live cells by macrophages are poorly understood. To identify mediators of this process, we established phagocytosis assays of live cells by stimulating macrophages with CpG DNA, interferon-γ, and anti-interleukin-10 receptor antibody. In this model, various cell surface receptors were upregulated on macrophages, and phagocytosis of live cells was induced in a Rac1-dependent manner. Subsequent inhibition of the ICAM-1, VCAM-1, and both of these receptors abolished  •A specific combination of inflammatory stimuli induce phagocytosis of live cells•ICAM-1 and VCAM-1 induced on macrophages mediate live cell phagocytosis•Icam-1-deficient miceHemophagocytosis was ameliorated in in vitro cell culture model of hemophagocytosis and found that ICAM-1 and VCAM-1 were induced on macrophages by a specific combination of inflammatory stimuli to mediate hemophagocytosis. We expect that our findings will facilitate the development of therapeutic approaches against the hemophagocytic syndromes.Under severe inflammatory conditions, macrophages are hyper-activated and start to eat live blood cells and their precursors, a process known as “hemophagocytosis”. Hemophagocytosis accompanies with inflammatory diseases such as infections, malignancies, or autoimmune diseases, but molecular mechanisms that cause hemophagocytosis remain unclear. We established  This process is known as efferocytosis and is dependent on recognition and selective phagocytosis of dead cells by phagocytes . Among pIn contrast, phagocytosis of live cells can be induced by uncontrolled activation of macrophages by strong immunologic conditions such as systemic infection, autoimmunity, and malignancy. Specifically, phagocytosis of live blood cells and their precursors by macrophages is known as hemophagocytosis. It is often observed in hemophagocytic lymphohistiocytosis (HLH) and macrophage activation syndrome (MAS), which are characterized by overwhelming immune activation and excessive production of inflammatory cytokines . Severalin vitro cell culture model of live cell phagocytosis will be useful not only for identifying phagocytic receptors of live cells but also for clarifying the pathogenesis of hemophagocytosis to indicate therapeutic targets. However, specific in vitro stimuli inducing phagocytosis of live cells by macrophages remain elusive. Recently, repeated injections of Toll-like receptor (TLR) 9 ligand, CpG DNA into mice have been reported to induce MAS-like diseases and hemophagocytosis (We therefore considered that establishment of an ocytosis . Accordi22.1Icam-1 KO mice were generated by pronuclear injection of CRISPR/Cas9 pX330 vectors targeting mouse Icam-1 exon-4 (CGCTGCGTTTTGGAGCTAGCGG) and exon-6 (TCCTAAGATGACCTGCAGACGG) to introduce frameshift mutations (PAM sequences are underlined). All mice were housed in a pathogen-free facility and all animal experiments were performed according to protocols that were approved by the Animal Research Committee of Kanazawa University, Japan. Bone marrow-derived macrophages (BMDMs) were generated by culturing bone marrow cells from femurs and tibias of mice for 4–6 days in high glucose DMEM  supplemented with 10% FBS (Biowest), 1% penicillin/streptomycin, and 10 units/ml of macrophage colony-stimulating factor (M-CSF), which was prepared using conditioned medium from human M-CSF overexpressing mouse L929 cells (AB_312996), CD3 , CD4 , CD8a , CD11b , CD68 , Gr-1 , ICAM-1 , IL-10R , Integrin αV , Integrin β3 , LAMP-1 , LFA-1α , PECAM-1 , VCAM-1 , VLA-4α , and VLA-5α , and rat IgG1κ , IgG2a κ (RTK2758), IgG2b κ , and Armenian hamster IgG (HTK888) isotype control antibodies, and mouse IL-10 ELISA Kit were purchased from BioLegend. The D89E mutant of mouse MFG-E8 was prepared as described previously  mice  were a k29 cells . Primary29 cells . All celeviously .2.2Icam-1 and Vcam-1 were prepared using RT-PCR after RNA extraction from CpG, IFN-γ, and αIL-10 cotreated BMDMs using the following primers: ICAM-1-Fw, 5′-CCCGGATCCCTACCATGGCTTCAACCCGT-3′ and ICAM-1-Rv, 5′-AAAGCGGCCGCTCAGGGAGGTGGGGC-3′ (BamHI and NotI sites are underlined), and VCAM-1-Fw, 5′-ATTAATTAAGCCACCATGCCTGTGAAGAGGTC-3′ and VCAM-1-Rv, 5′-AAAGCGGCCGCCTACACTTTGGATTTCTGTGC -3′ (PacI and NotI sites are underlined). PCR fragments were digested using BamHI/PacI and NotI restriction endonucleases, and were inserted into pMXs retrovirus vectors (Vβ3 were generated as described previously (DNA fragments for full length coding sequences of mouse  vectors . The pMX vectors  was perfeviously .2.35 cells) were cultured on 24-well plates (Corning) for flow cytometric analyses or on NUNC Lab-Tek II 8-well chamber glass slides (Thermofisher) for microscope analyses, and were then activated using various combinations of IFN-γ (100 U/ml), CpG ODN-1826 (0.5 μg/ml), and/or αIL-10R (1.25 μg/ml) in the absence or presence of cycloheximide (1 μg/ml) or the Rac1 inhibitor NSC23766 at 50, 100, or 200 μM for 20 h. Prey cells such as thymocytes, splenocytes, and myeloid cells were freshly prepared from 4 to 6 week old C57BL/6 mice. Myeloid cells were prepared from bone marrow by depleting T and B cells using a FACSAria cell sorter (BD Biosciences) with αCD3 and αB220 antibodies. Apoptosis was induced in thymocytes using 10 μM dexamethasone treatments for 4 h, and in myeloid cells by UV irradiation at 200 J/cm2 and incubation for 2 h. For flow cytometric analyses, prey cells were washed twice with PBS and were incubated for 30 min with 1 μM CellTracker green dye (CMFDA) (Thermofisher). Reactions were stopped by adding 1 ml of FBS and the cells were then washed twice with DMEM containing 10% FBS. The labeled cells (1 × 106 cells) were added to BMDMs that had been pre-treated with or without 4 μg/ml antibodies, 10 or 20 μM BAPTA-AM, or 7 μg/ml MFG-E8 D89E for 30 min. Phagocytosis proceeded for 2.5 h at 37 °C and prey cells that remained free were removed by washing twice with PBS. Phagocytes were collected from the plates by trypsinization, and were washed and suspended in PBS containing 2% FBS and 0.02% NaN3. Percentage phagocytosis was determined in triplicates by quantifying the percentage of CMFDA-positive phagocytes using FACSVerse (BD Biosciences). For microscope analyses, prey cells (1 × 106 cells) were added to GFP-labeled phagocytes on glass slides that had been pre-treated with or without antibodies at 4 (for BMDMs) or 10 μg/ml  for 30 min. In some experiments, prey cells were pre-labeled with 0.1 μg/ml pHrodo Red (Thermofisher). After co-culturing for 2.5 h, cells were washed twice with PBS and fixed in 4% paraformaldehyde (PFA)/PBS. When co-cultured with splenocytes, the phagocytes were permeabilized with ice-cold acetone and were stained with APC αB220, αCD4, or αCD8a antibodies in PBS containing 1% BSA to identify cell types of the engulfed cells. The cells were then mounted with ProLong Gold Antifade (Thermofisher) containing 1% DAPI , and were observed using an FV10i confocal microscope . Engulfed cells can be detected as dark spots in the cytoplasm and total numbers of engulfed cells of 200 phagocytes (50 cells/field × 4 fields) were counted blindly, and phagocytosis indexes were determined as average numbers of engulfed cells per 100 phagocytes.Phagocytes (1 × 102.4In Situ Apoptosis Detection Kits (Merck Millipore), MitoPT JC-1 Assay Kit (ImmunoChemistry Technologies), or FITC-Annexin V (BioLegend), respectively. For electron microscope analyses, BMDMs were cultured on Cell Desk  polystyrene cover slips. After phagocytosis, cells were fixed using 2% formaldehyde and 2.5% glutaraldehyde in 0.1 M sodium-phosphate buffer (pH 7.4) and were washed three times for 5 min in the same buffer. Cells were then post-fixed for 1 h in 0.1 M sodium-phosphate buffer (pH 7.4) containing 1% osmium tetroxide and 1% potassium ferrocyanide, and were then dehydrated in a graded series of ethanol and embedded in Epon812 . Subsequently, 80 nm ultra-thin sections were stained with a saturated solution of uranyl acetate and lead citrate. Electron micrographs were obtained using a JEM-1011 transmission electron microscope .TUNEL, JC-1, or Annexin V staining was performed using ApopTag Fluorescein Direct 2.5β-actin RNA expression in each sample. The original microarray data were deposited in GEO database under accession number GSE90881. The primers for real-time PCR were as follows: β-actin-Fw, 5′-CTTTGCAGCTCCTTCGTTG-3′ and β-actin-Rv, 5′-CGATGGAGGGGAATACAGC-3′; ICAM-1-Fw, 5′-GTCCGCTGTGCTTTGAGAAC-3′ and ICAM-1-Rv, 5′-TGAGGTCCTTGCCTACTTGC-3′; VCAM-1-Fw, 5′-GAAATGCCACCCTCACCTTA-3′ and VCAM-1-Rv, 5′-CGGGGGAGATGTCAACAATA-3′; LFA-1α-Fw, 5′-CAGAACAAGAACCCCGATGT-3′ and LFA-1α-Rv, 5′-CCTGGCACCAGACTCTTCTT-3′; and VLA-4α-Fw, 5′-AATGCCTCAGTGGTCAATCC-3′ and VLA-4α-Rv, 5′- TCTCCTCCAGGCATGTCTTC-3′.BMDMs were stimulated with CpG (0.063 μg/ml), IFN-γ (100 U/ml) + αIL-10R (1.25 μg/ml), or with all three agents at 37 °C for 20 h. Total RNA from each BMDM sample was then extracted using RNeasy Plus Mini Kits (QIAGEN) according to the manufacturer's instructions. Microarray analyses were performed by MBL, Japan, using SurePrint G3 Mouse Gene Expression 8 × 60 K Microarrays (Agilent). For qPCR analyses, RNA was reverse transcribed using ReverTra Ace qPCR master mix , and cDNA products were amplified using a LightCycler 96 (Roche) with Universal SYBR Select master mix (Thermofisher). Data were analyzed using the delta Ct method and were normalized to 2.6E. coli DH5α  cells were grown at 37 °C in 100 ml of LB medium until the mid-exponential phase (OD600 = 0.8–1.0). Cells were then collected by centrifugation at 4000 × g for 10 min and were inactivated by mixing with 50 ml of 70% (v/v) ethanol for 30 min. Mixtures were then centrifuged and cells were washed and suspended in PBS, and were then treated with UV irradiation for 30 min. Subsequently, 200 μg aliquots of dead E. coli were injected into peritoneal cavities of 8–10 week old male mice, and after 2 days, 4 × 107 CMFDA-labeled freshly-isolated live cells were injected into peritoneal cavities with blocking antibodies or corresponding isotype controls (10 μg). After 2.5 h, peritoneal cells were collected and plated on glass slides for 1 h to allow attachment of macrophages. Attached macrophages were then fixed and stained with antibodies against CD11b and Gr-1, and were observed using a confocal microscope. Total numbers of engulfed cells in 50 CD11b+/Gr-1− peritoneal macrophages (10 cells/field × 5 fields) were counted, and phagocytosis indexes were determined as the number of engulfed cells per 100 macrophages. To induce in vivo hemophagocytosis and a peripheral pancytopenia, male mice were injected i.p. on days 0, 2, 4, 6, and 8 with PBS or dead E. coli (20 mg). On day 7, peripheral blood was sampled by cheek bleed, and blood cell counting was performed with FACSCanto II (BD Biosciences). The number of T or B cells was counted by staining with αCD3 or αB220 antibody after RBC lysis. On day 10, mice were euthanized, and spleens were taken for immunohistochemical analysis with antibody against CD68.33.1To reconstitute phagocytosis of live cells in cell culture, BMDMs were co-cultured with CMFDA-labeled freshly-isolated live thymocytes for 2.5 h and percentages of macrophages containing live thymocytes were quantified using flow cytometry. Unstimulated macrophages did not engulf live cells, whereas 4.8% of macrophages engulfed live cells following 20 h treatments with CpG DNA a. Althou3.2To identify mechanism that is associated with the presence of live cells inside phagosomes, we investigated phagosome maturation using pHrodo-labeled thymocytes, which emit red fluorescence following acidification due to phagosome–lysosome (P–L) fusion . GFP-labPreviously, P–L fusion was associated with elevated cytosolic free calcium levels . Thus, t3.3via ICAM-1, we performed experiments using heterogeneous splenocytes as prey instead of thymocytes and identified phagocytosed cell types using immunocytochemical analyses of cell-specific markers. These analyses showed that the αICAM-1 antibody specifically inhibited the phagocytosis of live CD4+ and CD8+ T cells but not B cells . ICAM-1 reportedly binds LFA-1 , as reflected by sufficient expression of endogenous VCAM-1 in NIH3T3 cells (data not shown). However, enhanced phagocytosis of live myeloid cells by NIH3T3 transformants expressing integrin αVβ3 was inhibited by antibodies against VCAM-1, integrin αVβ3, and VLA-4, whereas antibodies against other molecules had no effect . Taken together, these results indicate that phagocytosis of live myeloid cells is mediated by VCAM-1 and integrin αVβ3.We next examined whether ICAM-1 and/or VCAM-1 are involved in the phagocytosis of live cells other than lymphocytes. For this purpose, we prepared myeloid cells from bone marrow by depleting T and B cells, and confirmed that they were Annexin V negative-viable cells . As with live T and B cells, cotreatment of BMDMs with CpG, IFN-γ, and αIL-10R induced phagocytosis of freshly-isolated live myeloid cells, and this phagocytosis was not inhibited by the addition of the D89E mutant of MFG-E8 . Following injection into peritoneal cavities, freshly-isolated live cells were efficiently engulfed by peritoneal macrophages that had been stimulated with non-viable E.coli , but reductions in T and B cell numbers was ameliorated in Icam-1 KO mice , indicating the presence of other integrins or integrin-like proteins that mediate live cell phagocytosis. Rac1 is reportedly activated following stimulation of ligands with integrins, and facilitates actin-remodeling for the engulfment .This research was supported by grants from


MS) exploiting multistage fragmentation (MSn). We found increased concentrations of bile acid precursors in CSF from each of the disease states and that patients with inflammatory CNS disease classified as suspected autoimmune disease or of unknown aetiology also showed elevated concentrations of 25-hydroxycholestertol  in CSF. Cholesterol concentrations in CSF were not changed except for patients diagnosed with amyotrophic lateral sclerosis (P < 0.01) or pathogen-based infections of the CNS (P < 0.05) where they were elevated. In plasma, we found that 25-HC (P < 0.01), (25R)26-hydroxycholesterol ((25R)26-HC, P < 0.05) and 7α-hydroxy-3-oxocholest-4-enoic acid  were reduced in relapsing-remitting MS (RRMS) patients compared to controls. The pattern of reduced plasma levels of 25-HC, (25R)26-HC and 7αH,3O-CA was unique to RRMS. In summary, in plasma, we find that the concentration of 25-HC in RRMS patients is significantly lower than in controls. This is consistent with the hypothesis that a lower propensity of macrophages to synthesise 25-HC will result in reduced negative feedback by 25-HC on IL-1 family cytokine production and exacerbated MS. In CSF, we find that the dominating metabolites reflect the acidic pathway of bile acid biosynthesis and the elevated levels of these in CNS disease is likely to reflect cholesterol release as a result of demyelination or neuronal death. 25-HC is elevated in patients with inflammatory CNS disease probably as a consequence of up-regulation of the type 1 interferon-stimulated gene cholesterol 25-hydroxylase in macrophages.Multiple sclerosis (MS) is an autoimmune, inflammatory disease of the central nervous system (CNS). We have measured the levels of over 20 non-esterified sterols in plasma and cerebrospinal fluid (CSF) from patients suffering from MS, inflammatory CNS disease, neurodegenerative disease and control patients. Analysis was performed following enzyme-assisted derivatisation by liquid chromatography–mass spectrometry (LC–The online version of this article (doi:10.1007/s12035-016-0281-9) contains supplementary material, which is available to authorized users. The size of the final C18 column used for CSF was 50 mg while that used for plasma was 200 mg. Exact details are given in Online Resource Non-esterified sterols in plasma were assayed by LC–t tests were performed against the CP group, *P < 0.05; **P < 0.01. Concentrations given in the text are mean ± standard deviation (SD). The boxplots in Figs. P values for the significance of the correlations are listed in the Tables in Online Resources P values that are below 0.05/((21*20)/2) = 0.000238 for CSF and below 0.05/((22*21)/2) = 0.000216 for plasma are highlighted in the Online Resource Tables, these are significant after a Bonferronni correction at 5 %.An ANOVA test was run for each sterol on linear and logarithmic scales. The log scale used a transformation log2+1) to avoid issues with zero and small numbers. Uni-variant  to avoidP values) against log2 fold change between RRMS and CP shown in Figs. t test was made using the modification by Welch that accommodates for different variances in the two groups.The volcano plots of statistical significance (3 (25-D3), cholestenoic and cholenoic acids in plasma. Table P < 0.01). We also see a reduction in the concentration of (25R)26-hydroxycholesterol ((25R)26-HC, 22.63 ± 5.03 ng/mL cf. 27.97 ± 7.89 ng/mL, P < 0.05) and 7α-hydroxy-3-oxocholest-4-enoic acid  in RRMS patients. It is important to note that 25-HC is generated by macrophages [CH25H or reduced activity of the enzyme and that RRMS patients have a reduced capacity to produce 25-HC.Shown in Fig. rophages , while trophages . The levP < 0.01) [P < 0.05), as it is in patients diagnosed with PBI , compared to CP (1.54 ± 0.42 ng/mL).In agreement with some, but not all, earlier studies, the level of 24S-hydroxycholesterol (24S-HC) is reduced in plasma from the group of patients with either AD or PD compared to CP  , 26. TheP < 0.05) compared to CP  compared to CP (17.40 ± 4.63 ng/mL), as is the level of its precursor 3β-hydroxycholest-5-enoic acid . The intermediary metabolite between these two acids, 3β,7α-dihydroxycholest-5-enoic acid , is also elevated (3.71 ± 4.07 ng/mL cf. 2.12 ± 1.65 ng/mL) but not to significance. MS is a demyelinating disease and it likely that cholesterol is released in the CNS is metabolised through (25R)26-HC and 3β-HCA to 7αH,3O-CA, although the actual concentration of non-esterified cholesterol in CSF does not differ from controls (1.24 ± 0.33 μg/mL). 3β,7β-Dihydroxycholest-5-enoic acid  which may be a metabolic product of 3β-HCA, 3β,7α-diHCA or 7-oxocholesterol (7O-C) is also elevated in CSF from RRMS patients .It is generally considered that in healthy individuals CSF levels of 25R)26-HC correlate with levels of this oxysterol in the circulation . However5R26-HC cP < 0.01). The concentration of 7α,(25R)26-dihydroxycholest-4-en-3-one 26-diHCO, 0.03 ± 0.01 ng/mL, cf. 0.02 ± 0.01 ng/mL, P < 0.01) is increased as is its downstream metabolite 7αH,3O-CA, although not quite to significance . Other neurodegenerative diseases including AD and PD are also believed to result in increased cholesterol release in brain as neurons die [P < 0.05), 7αH,3O-CA  and 3β,7β-diHCA  are elevated in this disease group.The CSF from the ALS group shows differences in cholesterol metabolite concentrations compared to controls and also in the concentration of cholesterol itself  in CSF while those diagnosed with SA/UA show increased concentrations of (25R)26-HC , 7α,(25R)26-dihydroxycholesterol 26-diHC, 0.01 ± 0.01 ng/mL cf. 0.00 ± 0.00 ng/mL, P < 0.05) and 7α,(25R)26-diHCO .Patients diagnosed with PBI show an increase in concentration of 7α,(25R)26-diHCO  did any of the disease states show any statistical differences in the level of 25-HC in CSF; this is also true for the 25-HC metabolite 7α,25-diHCO  .The limit of quantification of our analytical method in CSF for most analytes is 0.01 ng/mL ; however, for 25-HC, it is somewhat higher at 0.03 ng/mL, although detection can be made at 0.01 ng/mL. Only in the patient group of SA/UA  in plasma from patients with RRMS, falling from 1.54 ± 0.42 ng/mL in CP to 1.19 ± 0.26 ng/mL in RRMS plasma , in plasma from patients with PBI and AD or PD. Non-esterified oxysterols are present in CSF at pg/mL levels [P < 0.05). The plasma data for RRMS patients, indicating a reduced capacity of macrophages to synthesise 25-HC, is in-line with the recent study by Reboldi et al. which shows that in mouse macrophages 25-HC is a mediator of negative feedback towards interleukin 1 (IL-1) family cytokine production and inflammasome activity, through binding to INSIG (insulin-induced gene) and antagonising the sterol response element-binding protein-2 (SREBP-2) driven mevalonate pathway, thereby, reducing Il1b transcription and repressing IL-1-activating inflammasomes [Ch25h knockout mice show exacerbated experimental autoimmune encephalomyelitis (EAE), an IL-17-driven inflammatory disease model of MS, and increased susceptibility to septic shock. Ch25h-deficient macrophages produce more proinflammatory IL-17A+ T cells following lipopolysaccharide (LPS) stimulation and cytokines that cooperate with transforming growth factor-β (TGFβ) to induce Th17 cells include IL-1β. Notably, after LPS stimulation Ch25h-deficient macrophages overproduce Il1b [.In the current study, we have measured the levels of non-esterified sterols, oxysterols, 25-Dd P < 0.0 in plasmL levels , and in mmasomes . Thus, ruce Il1b .Il1b transcription is not known. Reboldi et al. suggested that this may be through reducing the cellular content of sterols generated via the mevalonate pathway [Ch25h gene significantly attenuates EAE by limiting trafficking of pathogenic CD4+ T lymphocyte to the CNS [P < 0.05) in RRMS plasma  and of its metabolites 3β-HCA (P < 0.05) and 7αH,3O-CA (P < 0.01) in CSF of RRMS patients , formed in the peroxisome as a thioester is also found to be elevated in RRMS CSF  but not its downstream metabolites. Interestingly, both 3β-HCA and 3β,7β-diHCA have been shown to be neurotoxic [In contrast to plasma, there is a significant elevation in 25R)26-HC (5R26-HC (urotoxic .P < 0.05) and 7αH,3O–CA (P < 0.01) of the “acidic pathway” are elevated in the AD and PD group, while in the ALS group 7α,(25R)26-diHCO (P < 0.01) is increased  as are (25R)26-HC (P < 0.05), 7α,(25R)26-diHC (P = 0.05) and 7α,(25R)26-diHCO (P = 0.01). When plasma was analysed from these patients, none of the metabolites showed statistical differences compared to controls except 7α,(25R)26-diHCO, which unlike the situation in CSF was reduced in concentration in plasma (P < 0.05). Little is known of the regulation of enzymes of the acidic pathway of bile acid biosynthesis [CH25H in macrophages of the inflamed CNS.Patients with inflammatory CNS disease include those diagnosed with SA/UA and those diagnosed with PBI. The patients with SA/UA show elevated levels of 25-HC in CSF  excluding and (B) including the inflammatory CNS disease group). As might be expected (25R)26-HC is highly correlated with 7α,(25R)26-diHC and 3β-HCA corresponding to the first metabolites of the two branches of the “acidic pathway” of bile acid biosynthesis . A fascinating feature of the pair-wise correlations is the tight cluster indicated by the “red box” incorporating 7αH,3O-CA, two dihydroxy-3-oxocholest-4-enoic acid isomers  and 7αH,3,24-diO-CA. We do not have an authentic standard for the latter compound whose identification is based on exact mass, retention time and multistage fragmentation (MSn) spectrum and the location of the hydroxy groups on the two dihydroxy-3-oxocholest-4-enoic acid isomers is unclear. In both isomers, one hydroxy group is located at the 7α position while the second hydroxy group is most likely on the side chain. The pair-wise correlations between successive members of the “acidic pathway” provide strong evidence for the activity of this pathway in the CNS. The absence of correlations with 7α-hydroxycholesterol (7α-HC) confirms that the “neutral pathway” of bile acid biosynthesis is not operative in the CNS. The rate limiting enzyme of this pathway, CYP7A1, is liver specific [4-BA, 7αH,3,24-diO-CA; 7αH,3O-CA; 7α,x-diH,3O-CA; and 7α,y-diH,3O-CA cluster together, as do 3β-HCA, (25R)26-HC, 3β,7β-diHCA, 3β,7α-diHCA and 7α,(25R)26-diHC. The clustering patterns evident in the dendrogram in Online Resource It is of interest to study pair-wise correlations between CSF concentrations and specific sterol analytes which are arranged in metabolic order in Fig. sis Fig. . (25R)26specific . The corsis Fig. -BA. A faP values for the significance of correlation). As expected from the pathway outlined in Fig. 4-BA, while (25R)26-HC correlates with 3β-HCA and 7αH,3O-CA. Unsurprisingly, 3β-HCA correlates strongly with 3β,7α-diHCA, while 7αH,3O-CA correlates strongly with 7αH,3O-Δ4-BA. As evident from the dendrogram in the Fig. in Online Resources While the metabolite correlations in CSF are dominated by the “acidic pathway” of bile acid biosynthesis, the correlations in plasma are reflective of both the “acidic pathway” and of the “neutral pathway” starting with 7α-HC followed by 7α-hydroxycholest-4-en-3-one ESM 2(PPTX 109 kb)ESM 3(DOC 109 kb)ESM 4(PPTX 597 kb)ESM 5(PPTX 550 kb)ESM 6(XLSX 21 kb)ESM 7(XLSX 23 kb)ESM 8(XLSX 31 kb)ESM 9(XLSX 32 kb)ESM 10(PDF 84 kb)ESM 11(PDF 64 kb)ESM 12(PDF 54 kb)


SYNE1 gene (23560 G>T) encoding Nesprin-1 [The nucleus is the main microtubule-organizing center (MTOC) in muscle cells due to the accumulation of centrosomal proteins and microtubule (MT) nucleation activity at the nuclear envelope (NE) , 2, 3, 4esprin-1 , 16. Fin  •BioID of Nesprin-1α identifies centrosomal proteins at myotube nuclear envelope•Nesprin-1α-containing LINC complexes recruit Akap450 to myotube nuclear envelope•Akap450 is required for microtubule nucleation at the nuclear envelope•Microtubule nucleation at the nuclear envelope is involved in nuclear positioning in vitro and in computer simulations.Gimpel et al. find that LINC complexes comprising Nesprin-1α and Sun1/2 are required for the recruitment of centrosomal proteins and Akap450-mediated microtubule nucleation activity to the myotube nuclear envelope. Absence of microtubule nucleation from the nucleus affects nuclear positioning in muscle cells  These results indicate that the aggregates of centrosomal proteins observed in Nesprin-1-depleted cells are retained at perinuclear Golgi complex fragments.Several NE proteins, including LINC complex components, are mutated in striated muscle diseases, like Emery-Dreifuss muscular dystrophy (EDMD) , 32, 33. patient , resulti patient . We aske control G. Next, myotubes A and 2B.Pericentrin-Akap450 centrosomal targeting (PACT) domain [∗-Nesprin-1α construct used in the BioID experiments  domain . Non-diferiments B and exa5–7 days J. These ∗-tagged Nesprin-1α or Nesprin-1α (WD/AA) constructs  patient cells. Wild-type myotubes predominantly nucleated MTs from the NE but also from minor PCM1- or Akap450-positive seeds in the cytoplasm (SYNE1 (23560 G>T) patient myotubes, all MT nucleation involved PCM1- or Akap450-positive seeds within the cytoplasm with little or no MT regrowth occurring from the NE. Nevertheless, myotubes from SYNE1 (23560 G>T) patient cells continued to maintain longitudinal MT arrays, comparable to those observed in wild-type myotubes (SYNE1 (23560 G>T) cells with nocodazole completely depolymerized MTs with little effect on PCM1 distribution, be it NE-associated or cytoplasmic (SYNE1 (23560 G>T) patient myotubes only displayed MT nucleation defects but no obvious disorganization of the actin cytoskeleton, consistent with Nesprin-1α lacking the N-terminal actin-binding domain of Nesprin-1 giant  , 38. We To elucidate the role of centrosomal proteins on the recruitment of other centrosomal proteins, we depleted Akap450, Pericentrin, Pcm1, and Cdk5rap2   using spin vivo and in vitro [-targeting siRNAs and determined the nuclear distribution along myotubes by calculating the nuclear spreading factor (SF), defined as the ratio between the observed and the maximal theoretical average distance between nuclei according to the myotube length. Nuclei in Nesprin-1-depleted myotubes were less spread throughout the myotube when compared to control myotubes (SYNE1 (23560 G>T) patient myotubes when compared to wild-type myotubes .Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Edgar R. Gomes (2 in Dulbecco’s modified Eagle’s medium (DMEM) containing 4.5 g/L D-glucose, 4 mM L-glutamine, 1 mM sodium pyruvate,10% fetal calf serum, and 100 U/ml penicillin; 100 μg/ml streptomycin. C2C12 cells are female. Cells were not authenticated. For immunofluorescence, C2C12 myoblasts were seeded on coverslips, 8-well μ-slides or 96-well μ-plates coated with 0.1% gelatin or Matrigel diluted 1:100 in DMEM. When C2C12 cells reached ∼90% confluence, they were switched to DMEM supplemented with 2% horse serum and 100 U/ml penicillin; 100 μg/ml streptomycin to induce myogenic differentiation for the indicated time points.Mouse C2C12 myogenic precursor cells were cultivated at 37°C/5% COFor generating Nesprin-1 CRISPR cell lines, wild-type C2C12 were transfected overnight using Lipofectamine 3000 according to manufacturer’s instructions with pX330 encoding Nesprin-1 N- and C-termini-directed sgRNAs and pEGFP-N1 in a 9:9:2 ratio. Cells with high levels of EGFP expression, representing high levels of pX330 transfection, were sorted as single cells into 96-well plates at the Singapore Immunology Network FACS facility. Loss of Nesprin-1 NE localization in clonal lines was assessed by immunofluorescence microscopy. Nesprin-1 CRISPR cell lines were cultivated and maintained as wild-type C2C12 cells as described above.SYNE1 gene (nucleotide 23560 G>T) were immortalized by Kamel Mamchaoui and Vincent Mouly  via transduction with retrovirus vectors expressing hTERT and Cdk4 as described previously [Human myoblasts from a healthy control or from a congenital muscular dystrophy patient carrying a homozygous nonsense mutation within the eviously , 16. Hum−/−  and Sun2−/−  mice were previously described [∗STAR Biological Resource Centre facility in accordance with the guidelines of the Institutional Animal Care and Use Committee. Primary myoblasts were obtained from E18.5 mouse embryos as Sun1−/−Sun2−/− double mutant mice die shortly after birth. Limb muscles were dissected away from skin and bones. Muscle tissue was digested in enzyme solution  for 30 min at 37°C [2 to allow contaminating fibroblasts to adhere. The myoblast enriched fraction was then plated onto 0.1% gelatin-coated tissue culture plates and maintained in Ham’s F10 containing 20% fetal bovine serum and 10 μg/ml bFGF in 37°C/5%CO2. Myoblast enrichment by preplating of fibroblasts was repeated every 2-3 days until most fibroblasts were removed from the myoblast culture. Primary myoblasts were differentiated into myotubes by changing to DMEM supplemented with 2% horse serum and 100 U/ml penicillin; 100 μg/ml streptomycin. Due to the difficulty of sexing mouse embryos visually, sex of primary myoblasts derived from E18.5 embryos was not determined.The Sun1escribed , 54 and  at 37°C  with genThe following primary antibodies were used for immunofluorescence and Western Blot analysis: rabbit anti-Pericentrin, mouse anti-Pericentrin, mouse anti-myosin, sarcomere MHC, mouse anti-GAPDH, mouse anti-Nesprin-1 (clone 9F10), mouse anti-Myc (clone 9E10), rabbit anti-Akap9, rabbit anti-PCM1, mouse anti-Myogenin (clone F5D), rat anti-tyrosinated-α-tubulin (clone YL1/2), rabbit anti-Sun1, rabbit anti-Sun2, rabbit anti-Kif5b, mouse anti-Cep170, mouse anti-GM130, and rabbit anti-CDK5RAP2. The mouse anti-Nesprin-1 , anti-Klc1/2, and anti-Pericentrin antibodies were kind gifts from Glenn E. Morris, Scott T. Brady, and Kunsoo Rhee, respectively. Details of secondary antibodies are in the C2C12 cells were transfected at approximately 30% confluency with siRNA  by addition of transfection complexes pre-formed for 20 min, containing 0.3 μL Lipofectamine RNAiMAX per pmol of siRNA in Opti-MEM medium. The following siRNAs were used: non-targeting control (NC) siRNA#1, NC siRNA#2, NC siRNA#3, Nesprin-1 siRNA#1, Nesprin-1 siRNA#2, Nesprin-1 siRNA#3, Sun1 siRNA#1, Sun1 siRNA#2, Sun2 siRNA#1, Sun2 siRNA#2, Pericentrin (Pcnt) siRNA#1, Pcnt siRNA#2 or 1:1 mix of Pcnt siRNA#1 and #2, Akap450 siRNA#1, Akap450 siRNA#2, Pcm1 siRNA#1 and Pcm1 siRNA#2, Cdk5rap2 siRNA#1, and Cdk5rap2 siRNA#2, and their sequences are provided in the ∗-Nesprin-1α was obtained by PCR amplification of Nesprin-1α from pcDNA3.1 containing Nesprin-1α using 5′-AAGCTCGAGATGGTGGTGGCAGAGGACTTGC-3′ and 5′- AGGCCACGCGTCCTAGGTCAGAGTGGAGGAGG-3′ followed by restriction digestion with XhoI and MluI and ligation into pTripZ-mycBirA∗ [∗-Nesprin-2β was obtained by PCR of Nesprin-2β coding sequence from HeLa cell cDNA using 5′- GCGCCTCGAGATGTCCATGGAGCGGCGCATG-3′ and 5′-GCGCACGCGTCCTAGGTCATGTGGGGGGTGGCCCATTG-3′, restriction digest with XhoI and MluI and ligation into pTripZ-mycBirA∗. Site-directed mutagenesis of Nesprin-1α to obtain Nesprin-1α (WD/AA) was performed using the megaprimer method – the primers 5′-AAGCTCGAGATGGTGGTGGCAGAGGACTTGC-3′ and 5′-TCGTAATCGTGGGCCGCTTCCAGGGGGATGG-3′ were used to generate a forward megaprimer, which was used with reverse primer 5′-AGGCCACGCGTCCTAGGTCAGAGTGGAGGAGG-3′ to PCR amplify mutant Nesprin-1α. Nesprin-1α (WD/AA) was then cloned into pTripZ-mycBirA∗ by restriction digestion with XhoI and MluI and ligation. For CRISPR-mediated gene editing, oligos encoding single guide RNA sequences targeting Nesprin-1  were annealed by heating to 95°C then cooled gradually to room temperature, then ligated into BbsI-digested pX330-U6-Chimeric BB-CBh-hSpCas9  [Mouse cDNA was obtained from mouse hindlimb skeletal muscle RNA using ThermoScript RT-PCR System for First-Strand cDNA Synthesis with random hexamers. Human cDNA was obtained from HeLa cell RNA using the same kit. Nesprin-1α cDNA was obtained by PCR from mouse skeletal muscle cDNA using the following primers 5′-GCGCCTCGAGATGGTGGTGGCAGAGGACTTGC-3′ and 5′-GCGCCTTAAGTCAGAGTGGAGGAGGACCGTT-3′ and ligated into pcDNA3.1 with an EGFP tag following restriction digest with XhoI and AflII. pTripZ-mycBirAmycBirA∗ . GST-Nest#42230) .2O for 20 min at room temperature. One day prior to transfection, 9.000 C2C12 cells were seeded per well in 200 μL growth medium. When cells reached approximately 80% confluency, cell transfection was carried out using jetPrime. In brief, a total amount of 1 μg DNA was diluted in 50 μL jetPrime buffer and mixed by vortexing vigorously for 10 s. 2 μL jetPrime reagent were added, the mixture was vortexed again for 10 s and shortly spun down. After incubation for 10 min at room temperature, the transfection mix was added drop wise onto the cells which were kept in 200 μL freshly added growth medium containing serum and antibiotics. The following day, the growth medium was replaced to circumvent toxic side effects of the transfection reagent. The cells were kept for 36 hr in growth medium to ensure proper expression, fixed in 4% PFA and stained for Myogenin and DAPI.For co-transfection of dsRed-PACT and GFP-Nesprin-1α or GFP, respectively, 8-well μ-slides were coated with sterile-filtered 0.1% gelatin/ddHE. coli using 0.1 mM IPTG at 30°C for 2 hr. Pelleted bacteria were resuspended in 4°C phosphate buffered saline with 0.1 mg/ml lysozyme then lysed by several freeze-thaw cycles using liquid nitrogen, followed by sonication to shear DNA. Lysates were cleared by centrifugation at 35000 g for 30 min and filtering the supernatant through a 0.45 μm filter. GST-Nesprin-1α-326-634 was purified from cleared lysate by incubation on Glutathione Sepharose 4B beads for 2 hr at room temperature, washed with 3 × 15 column volumes phosphate buffered saline, and eluted in 10 mM reduced glutathione in 50 mM Tris, pH 8. For monoclonal antibody production mice were immunized intraperitoneally with 50 μg of fusion protein emulsified with Freund’s complete adjuvant. After three weeks a 50 μg boost was administered by the same route in incomplete adjuvant. After additional three weeks, a further 50 μg was administered in PBS. Three days later, the spleen was harvested, minced finely, and passed through a 100 μm cell strainer. SP2/0 myeloma cells were maintained in growth medium . Spleen cells and SP2/0 myeloma cells were washed in GKN saline solution . Spleen cells were fused with 1-2.5 × 107 myeloma cells by gradual addition to cell pellet at 37°C of 1 mL 50% (w/v) polyethylene glycol (Mw ∼1500) over 1 min, incubation for 1 min, addition over 1 min each of 1 mL, 2 mL, 8 mL, then 30 mL GKN saline solution (modified from [Expression of GST-Nesprin-1α-326-634 was induced in mid-log phase BL21 (DE3) ied from ). Cells ∗-Nesprin-1α and stable integration of the transgene was selected for using 0.67 μg/ml puromycin. For each BioID sample, stably transduced cells were expanded in 5-layer flasks and either harvested for myoblast samples, or switched to differentiation medium for 4 days before harvesting for myotube samples. Prior to harvesting, cells were incubated with 50 μM biotin and 1 μg/ml doxycycline for 1-2 days. For control samples, cells were incubated without biotin and doxycycline, or with biotin only. Affinity purification of biotinylated proteins was performed as previously described [C2C12 cells were transduced with lentivirus expressing myc-BirAescribed , 57, by Mass spectrometry samples were in-solution digested. After streptavidin affinity purification, beads were denatured using 50% 2,2,2-trifluoroethanol (TFE) in 100 mM triethylammonium bicarbonate (TEAB). Proteins were reduced in 25 mM Tris(2-carboxyethyl)phosphine and alkylated with 55 mM 2-chloroacetamide. Prior digestion sample was further diluted with 100 mM TEAB to achieve TFE final concentration below 5%. Digestion with Lys-C for 4 hr (1:100 enzyme/protein ratio) and Trypsin for 18 hr (1:100) was performed. Following acidification with 1% trifluoroacetic acid, samples were desalted with Sep-Pak C-18 cartridges. Samples were labeled with a TMT (tandem mass tag) isobaric labeling kit in 100 mM TEAB with 10 μL label for 25 μg peptides as suggested in the manufacturer’s instructions. Specifically, each sample was re-suspended in 25 μL TEAB; to tag the samples for multiplex measurement, 10 μL of specific TMT label was added to each sample separately . Reaction was incubated at room temperature for 1 hr and quenched with 30 μL of 10 mM ammonia formate. Following quenching, samples were mixed and fractionated. Samples were desalted on C18 Basic Resins 10 μm and eluted using a step gradient (4 fractions) containing 17.5%; 25%; 30%; 50% acetonitrile further optimized to (3 fractions) 15%, 22%, 50% acetonitrile in 7.5% ammonium hydroxide. Each fraction was vacuum centrifuged and submitted for analysis. An Orbitrap Fusion mass spectrometer coupled to nanoUPLC Easy LC 1000 system was used for analysis. Samples were injected and separated in EasySpray column 50 cm x 75 μm  in a 120 min gradient  in data dependent mode using Orbitrap analyzer (speed mode −3 s cycle) with ion targets and resolution . Peak lists were generated with Proteome Discoverer 2.1 software and searches were done with Mascot 2.5.1 against concatenated forward/decoy Uniprot/Swiss-Prot with BirA sequences/contaminants database with the following parameters: precursor mass tolerance (MS) 30 ppm, OT-MS/MS 0.06 Da, 3 missed cleavages; static modifications: Carboamidomethyl (C); variable modifications: Oxidation (M), Deamidated (NQ), Acetyl N-terminal protein Phospho (STY), Biotin(K). Forward/decoy searches were used for false discovery rate estimation for PSM and peptides matching high confidence (FDR 1%) and medium confidence (FDR 5%).∗ specific peptides, correcting factors were derived and used for normalization of specific Nesprin-1α interacting proteins. Proteins identified in at least two biological replicates were used for further analysis.Following protein identification, extracted TMT reporter abundances were used for protein quantitation. For protein normalization, the bait (BirA-Nesprin-1α) expression was assessed in both myoblasts and myotubes. Using BirA2. Cells were then pre-extracted for 30 s with 1% Triton X-100 in PHEM buffer , fixed with 4% paraformaldehyde (PFA) and stained as indicated below.48 hr differentiated C2C12 or human immortalized cells were treated with 5 μg/ml nocodazole for 2 hr in the corresponding differentiation medium. After a quick wash, cells were let to recover in fresh differentiation medium for 4 min  or 5 min (C2C12 cells) at 37°C/5% COCells were fixed either in methanol for 20 min at −20°C or in 4% PFA for 15 min at room temperature (RT) and then permeabilized with 0.5% Triton X-100/PBS for 5 min at RT. For detection of Klc1/2 at the NE, cells were pre-extracted for 20 s prior to fixation. After three washes in PBS, cells were blocked in 3% bovine serum albumin or 10% goat serum in PBS for 30 min at RT. Primary antibodies were incubated in blocking buffer for 1-2 hr at RT or at 4°C overnight. Following primary antibody incubation, cells were washed with PBS, incubated with fluorophore-conjugated secondary antibodies and DAPI in blocking buffer for 1 hr at RT, washed with PBS, and mounted in Fluoromount-G or Prolong Diamond or 1% DABCO  in 10% PBS / 90% glycerol.2 using a 10x 0.3 NA PL Fluo objective. Confocal images of human immortalized cells after MT re-growth assay and staining were acquired on a Leica SPE confocal microscope using the 63x 1.3 NA Apo objective or on a Nikon Spinning disk confocal with EMCCD camera using a 60x (oil) 1.4 NA objective. Confocal images of C2C12 cells were also obtained on a Zeiss LSM510 confocal microscope using a 40x 1.3 NA objective.Wide-field epi-fluorescence imaging was performed on an inverted Nikon Ti microscope equipped with a XY-motorized stage using 20x 0.45 NA S Plan Fluor air or 40x 1.3 NA Plan Fluor oil objectives with a 1.5x magnifier. Digital images were acquired with a CoolSNAP HQ2 camera using Metamorph Software and processed in Fiji. For analyses of C2C12 cells after MT re-growth assay, a maximum projection of four 0.25 μm z sections was directly created during imaging using Metamorph. Images were adjusted using the unsharp mask plugin of Fiji. Wide-field epi-fluorescence images were also acquired on a DeltaVision CORE equipped with a xenon light source and bandpass filters with a Plan Apochromat 40 × /1.35 NA oil-immersion objective lens and a CCD camera . For deconvolved images, z-spacing was fixed at 0.2 μm on the DeltaVision CORE and deconvolution then completed using the SoftWorX program. Live-cell imaging of differentiating human immortalized cells was performed for 63 hr in an environmental chamber at 37°C and 5% COdSTORM [dSTORM, coverslips were mounted in imaging buffer  containing 10 mM β-mercaptoethylamine (MEA), 0.5 mg/ml glucose oxidase, 40 μg/ml catalase, and 10% (w/v) glucose on slides with molds. Images were acquired on a custom-build SD-dSTORM system using a 700-DCXXR dichroic mirror and a F76-635 emission bandpass filter and reconstructed as described previously [Wild-type C2C12 cells were differentiated for 48 hr, fixed and stained for Pericentrin and Nesprin-1 (MANNES1E antibody) as described above. Alexa Fluor 647 and CF 680 labeled secondary antibodies were used. This dye pair has previously been shown to be suitable for spectral demixing (SD)-dSTORM . For SD-eviously , 26.Wild-type C2C12 cells were differentiated for 48 hr, subjected to MT re-growth assay, fixed and stained as described above. Coverslips were mounted using Vectashield and sealed with nail polish. 3D-SIM images were generated using the 488 nm, 568 nm, and 643 nm laser lines and standard filter sets on the OMX V4 Blaze. Precise registration (within ∼40 nm) of the three-color channels was verified using TetraSpeck multicolor beads mounted on coverslips.C2C12 cells were lysed in 1% SDS/PBS and homogenized by full-speed centrifugation for one minute using a QIAshredder. Total protein concentration was determined using the Pierce bicinchoninic acid (BCA) protein assay kit. Equal protein amounts were mixed with 4x Laemmli sample buffer, heated for 5 min at 95°C, loaded on 4%–15% Mini-PROTEAN TGX protein gels and transferred onto a nitrocellulose membrane using the Trans-Blot Turbo Transfer system. For analysis of high molecular weight proteins, samples were loaded on 4%–12% NuPAGE Novex Bis-Tris gels and transferred over night at 30 V in 1x Tris/Glycine buffer containing 10% ethanol and 0.025% SDS. Membranes were blocked in 5% milk/0.05% Tween-TBS and incubated with primary antibodies over night at 4°C. After incubation with HRP-conjugated secondary antibodies for 1 hr at room temperature, proteins were detected by chemiluminescence using SuperSignal West Pico Chemiluminescence or Luminata Forte Western HRP Substrate and Amersham Hyperfilms. For western blots scanned on an Odyssey imaging system, IRDye 800 or IRDye 680 conjugated secondary antibody or streptavidin was used.in vitro [Computer simulations were performed using Cytosim . In briein vitro . MTs areAll experiments were performed at least twice, typically 3-4 times. To ensure randomization, for quantification of cellular phenotypes, all cells meeting particular criteria  in a field of view were counted. While blinding was not used in this study, where possible, automated image analysis was used. Sample-size estimation and statistical method of computation was not performed as it is relatively easy to obtain large numbers (N > 100) in cell microscopy-based experiments. Inclusion and exclusion criteria of any data or subjects is described in the methods and include quantification of only Myogenin or myosin heavy chain positive nuclei to identify differentiated myotubes, and nuclear spreading analysis on myotubes with at least 3 nuclei.We counted the number of nuclei with more than 50% of Pericentrin or Akap450, respectively, at the NE in Myogenin-(MYOG)- or myosin heavy chain-(MHC)-positive cells.MT re-growth was quantified by counting the number of Myogenin-positive nuclei that showed at least 50% of MT seeds at the NE.We counted the number of Myogenin-negative cells that showed NE distribution of the dsRed-PACT construct in cells co-expressing GFP or GFP-Nesprin-1α, respectively.∗∗∗ p < 0.001; ∗∗ p < 0.01, ∗p < 0.05; n.s., not statistically significant.Statistical tests were performed using GraphPad Prism and are described in each figure legend. The exact value of n is indicated within the respective figure. Statistical significance is represented as follows: Mass spectrometry data is provided in supplemental material as P.G., Y.L.L., B.B., B.C., and E.R.G. conceived and designed experiments; P.G., Y.L.L., R.M.S., V.K., and R.P. performed experiments and analyzed data; B.C. performed computational analysis; A.C., K.M., F.N., and J.S. provided resources; P.G. wrote the manuscript; P.G., Y.L.L., B.B., B.C., and E.R.G. revised the manuscript; and S.S., J.S., B.B., B.C., and E.R.G. supervised the project.


The title compounds, (I) and (II), are 2-[(diaminopyrimidin-2-yl)sulfanyl]acetamides. The molecules have a folded conformation, with the pyrimidine ring being inclined to the benzene ring by 42.25 (14)° in (I), and by 59.70 (16) and 62.18 (15)° in the two independent molecules of compound (II). 12H12ClN5OS, (I), and C12H12ClN5OS, (II), are 2-[(diaminopyrimidin-2-yl)sulfanyl]acetamides. Compound (II), crystallizes with two independent molecules (A and B) in the asymmetric unit. In each of the molecules, in both (I) and (II), an intramolecular N—H⋯N hydrogen bond forms an S(7) ring motif. The pyrimidine ring is inclined to the benzene ring by 42.25 (14)° in (I), and by 59.70 (16) and 62.18 (15)° in molecules A and B, respectively, of compound (II). In the crystal of (I), molecules are linked by pairs of N—H⋯N hydrogen bonds, forming inversion dimers with an R22(8) ring motif. The dimers are linked via bifurcated N—H⋯O and C—H⋯O hydrogen bonds, forming corrugated layers parallel to the ac plane. In the crystal of (II), the A molecules are linked through N—H⋯O and N—H⋯Cl hydrogen bonds, forming layers parallel to (100). The B molecules are also linked by N—H⋯O and N—H⋯Cl hydrogen bonds, also forming layers parallel to (100). The parallel layers of A and B molecules are linked via N—H⋯N hydrogen bonds, forming a three-dimensional structure.The title compounds, C The molA⋯N2 hydrogen bond (Table 1S(7) ring motif, as shown in Fig. 1In compound (I)d Table 1 generateA and B, respectively, also generate S(7) ring motifs. In molecule A, the amine group atoms N1 and N2 attached to the pyrimidine ring deviate by 0.006 (3) and 0.004 (3) Å, respectively. The chlorine atom Cl1 attached to the benzene ring deviates by 0.013 (1) Å. In molecule B, the amine group atoms N6 and N7 attached to the pyrimidine ring deviate by −0.003 (3) and 0.050 (3) Å, respectively. Atom Cl2 attached to the benzene ring deviates by 0.074 (1) Å.In compound (II)via bifurcated N—H⋯O and C—H⋯O hydrogen bonds, forming corrugated layers parallel to the ac plane f Table 1. The dime Table 1.A molecules are linked through N—H⋯O and N—H⋯Cl hydrogen bonds, forming layers parallel to (100). Likewise the B molecules are also linked by N—H⋯O and N—H⋯Cl hydrogen bonds, forming layers parallel to (100). The parallel layers of A and layers of B molecules are linked via N—H⋯N hydrogen bonds, forming a three-dimensional structure e Table 2.et al., 2016N-phenylacetamide yielded five hits. Three of these involve  groups. They include the 2-chlorophenyl analogue, N-(2-chlorophenyl)-2-[sulfanyl]acetamide A search of the Cambridge Structural Database N-(4-chlorophenyl)acetamide derivative was added and refluxed for 4 h. At the end of the reaction (monitored by TLC), the ethanol was evaporated in vacuo and cold water was added; the precipitate formed was filtered and dried to give compound (I)To a solution of 4,6-diamino-pyrimidine-2-sulfanyl  in 25 ml of ethanol, was added potassium hydroxide  and the mixture was refluxed for 30 min, after which 3.52 mmol of 2-chloro-Compound (II)N-(3-chlorophenyl)acetamide was added and refluxed for 3 h. At the end of the reaction (monitored by TLC), the ethanol was evaporated in vacuo and cold water was added and the precipitate formed was filtered and dried to give compound (II)To a solution of 4,6-diamino-pyrimidine-2-thiol  in 25 ml of ethanol was added potassium hydroxide  and the mixture was refluxed for 30 min. Then 3.52 mmol of 2-chloro-Uiso(H) = 1.2Ueq.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989017003243/su5348sup1.cifCrystal structure: contains datablock(s) global, I, II. DOI: 10.1107/S2056989017003243/su5348Isup4.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989017003243/su5348IIsup3.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S2056989017003243/su5348Isup4.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989017003243/su5348IIsup5.cmlSupporting information file. DOI: 1534990, 1534989CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: