We report that bacterial RNA polymerase (RNAP) is the functional cellular target of the depsipeptide antibiotic salinamide A , and we report that Sal inhibits RNAP through a novel binding site and mechanism. We show that Sal inhibits RNA synthesis in cells and that mutations that confer Sal-resistance map to RNAP genes. We show that Sal interacts with the RNAP active-center ‘bridge-helix cap’ comprising the ‘bridge-helix N-terminal hinge’, ‘F-loop’, and ‘link region’. We show that Sal inhibits nucleotide addition in transcription initiation and elongation. We present a crystal structure that defines interactions between Sal and RNAP and effects of Sal on RNAP conformation. We propose that Sal functions by binding to the RNAP bridge-helix cap and preventing conformational changes of the bridge-helix N-terminal hinge necessary for nucleotide addition. The results provide a target for antibacterial drug discovery and a reagent to probe conformation and function of the bridge-helix N-terminal hinge.DOI:http://dx.doi.org/10.7554/eLife.02451.001 The need for new antibiotics is becoming increasingly critical, as more and more bacteria become resistant to existing drugs. To develop new treatments, researchers need to understand how antibiotics work. One way antibiotics can kill bacteria is by targeting an enzyme called bacterial RNA polymerase. This enzyme builds chains of RNA that bacteria need to survive.Sal is an antibiotic produced by a marine bacterium found on the surface of a species of jellyfish. Degen, Feng et al. show that Sal kills bacteria by inhibiting bacterial RNA polymerase and explain how Sal inhibits RNA polymerase. Sal binds to a rod-like structural element within RNA polymerase known as the ‘bridge helix’. The bridge helix has been proposed by others to contain two ‘hinges’ that open and close—allowing the bridge helix to bend and unbend—at specific steps in the cycle through which RNA polymerase builds an RNA chain. Degen, Feng et al. show that Sal binds directly to one of the two hinges and show that Sal binds to the hinge in the unbent state. Therefore, Degen, Feng et al. propose that Sal inhibits the enzyme by preventing the hinge from bending.The binding site on RNA polymerase for Sal is different from, and does not overlap, the binding sites of current antibacterial drugs. As a result, Sal is able to kill bacteria that are resistant to current antibacterial drugs. When Degen, Feng et al. administered Sal in combination with a current antibacterial drug that targets RNA polymerase, bacteria did not detectably develop resistance to either Sal or the current antibacterial drug.The structure of the complex between Sal and RNA polymerase suggests several ways that Sal could be modified to improve its ability to interact with RNA polymerase, thereby potentially increasing Sal's antibacterial activity. Future research could develop a range of new drugs based on Sal that could kill bacteria more effectively.DOI:http://dx.doi.org/10.7554/eLife.02451.002 Streptomyces sp. CNB-091, a marine bacterium isolated from the surface of the jellyfish Cassiopeia xamachana  and salinamide B  are structurally related bicyclic depsipeptide antibiotics, each consisting of seven amino acids and two non-amino-acid residues . SalA anamachana , and Salacterium . SalA anan cells . SalA anan cells . A totalan cells .10.7554/Although previous work had established that Sal exhibits RNAP-inhibitory activity in a purified system in vitro and antibacterial activity in culture , previouIn this work, we show that RNAP is the functional cellular target of Sal, we show that Sal inhibits RNAP through a novel binding site and novel mechanism, we determine crystal structures that define RNAP–Sal interactions, and we set the stage for structure-based design and semi-synthesis of Sal analogs with improved properties.14C]-thymidine into DNA, [14C]-uracil into RNA, and [14C]-amino acids into protein. The results in As a first step to determine whether the RNAP-inhibitory activity of Sal is responsible for the antibacterial activity of Sal in culture, we assessed whether Sal inhibits RNAP in bacterial cells in culture. To do this, we assayed macromolecular synthesis by bacterial cells in culture, monitoring incorporation of UTP (0.2 Bq/fmol), 100 μM ATP, and 100 μM GTP in TB . Reaction components except DNA, ApA, and NTPs were pre-incubated 10 min at 24°C; DNA was added and reaction mixtures were incubated 10 min at 37°C; ApA, 0.15 μl 7 μM [α32P]UTP (200 Bq/fmol), ATP, and GTP were added and reaction mixtures were incubated 5 min at 37°C; and 0.5 μl 2 mM UTP was added and reaction mixtures were incubated 5 min at 37°C. Reactions were terminated by adding 10 μl loading buffer  and heating 2 min at 95°C. Products were applied to 7 M urea 15% polyacrylamide (19:1 acrylamide:bisacrylamide) slab gels , electrophoresed in TBE , and analyzed by storage-phosphor scanning . Data shown are means of at least two determinations.Reaction mixtures contained (10 μl): 0-100 μM test compound, bacterial RNAP holoenzyme (75 nM promoter  followed32P]UTP (0.11 Bq/fmol), 400 μM CTP, 400 μM GTP, 50 mM Tris–HCl, pH 8.0, 7 mM HEPES-NaOH, 70 mM (NH4)2SO4, 50 mM KCl, 12 mM MgCl2, 5 mM DTT, 0.1 mM EDTA, 0.08 mM phenylmethylsulfonyl fluoride, 5% methanol, and 16% glycerol. Reaction components other than DNA and NTPs were pre-incubated 10 min at 30°C, DNA was added and reaction mixtures were incubated 15 min at 30°C, NTPs were added and reaction mixtures were incubated 60 min at 30°C. Reaction mixtures were spotted on DE81 filter discs  and incubated 1 min at room temperature. Filters were washed with 3 × 3 ml Na2HPO4, 2 × 3 ml water, and 3 ml ethanol, using a filter manifold . Filters were placed in scintillation vials containing 10 ml Scintiverse BD Cocktail , and radioactivity was quantified by scintillation counting .Radiochemical assays with human RNAP I/II/III were performed essentially as in Half-maximal inhibitory concentrations (IC50s) were calculated by non-linear regression in SigmaPlot .E. coli D21f2tolC  were dispensed into wells of a 96-well plate; were supplemented with 7 µl of pre-warmed 4 µCi/ml [14C]-thymidine, 10 µCi/ml [14C]-uracil, or 30 µCi/ml [14C]-amino acid mix ; were incubated 10 min at 37°C with shaking; were supplemented with 3 µl 3.3 µg/ml SalA in methanol , 3 µl 13 µg/ml Rif in methanol , or 3 µl solvent blank; and incubated at 37°C with shaking. At time points 0, 10, 20, and 30 min after the addition of SalA, Rif, or solvent blank, rows of samples were transferred to a second 96-well plate, containing 100 µl ice-cold 10% trichloroacetic acid (TCA) in each well, and the second plate was incubated on ice. 1 hr after the final time point, TCA precipitates were collected by filtration onto glass-fiber filters , washed with 2 × ∼300 µl 5% TCA, washed with 3 × ∼300 µl water, and washed with 2 × ∼300 µl 10% ethanol, using a Packard FilterMate 196 Cell Harvester with Matrix Filter upper head assembly (PerkinElmer). Filters were dried under a heat lamp, wrapped in a single layer of plastic wrap, and exposed to a storage phosphor screen for 18–19 hr, and analyzed by storage-phosphor scanning .Macromolecular synthesis assays were performed essentially as in 21f2tolC  was cult21f2tolC  at 37°C E. coli D21f2tolC was cultured to saturation in 5 ml LB broth at 37°C, cultures were centrifuged, and cell pellets (∼2 × 109 cells) were re-suspended in 50 μl LB broth and plated on LB agar , 5 U Taq DNA polymerase , and 800 µM dNTP mix  . PCR products containing the rpoC gene (4.2 kB) or the rpoB gene (4.0 kB) were isolated by electrophoresis on 0.8% agarose  and was quantified by measurement of UV-absorbance (procedures as in rpoB plasmid pRL706 (rpoC plasmid pRL663 (lacI (5′-GTTCCGGCGTTATTTCTTGA-3′ and 5′-TCAAGAAATAACGCCGGAAC-3′), or with pRL663 as template and oligodeoxyribonucleotide forward and reverse primers corresponding to nucleotides 217-246 of lacI (5′-CTGCACGCGCCGTCGAAAATTGTCGCGGCG-3′ and 5′-CGCCGCGACAATTTTCGACGGCGCGTGCAG-3′) . Mutagenized plasmid DNA was introduced by transformation into E. coli XL1-Blue (Agilent/Stratagene). Transformants (∼5 × 103 cells) were applied to LB-agar plates containing 200 μg/ml ampicillin, plates were incubated 16 hr at 37°C, and plasmid DNA was prepared from the pooled resulting colonies. The resulting passaged random-mutagenesis library was pooled in a 1/1 (wt/wt) ratio with pooled passaged saturation-mutagenesis libraries of E. coli D21f2tolC. Transformants (∼103 cells) were applied to LB-agar plates containing 0.4 μg/ml SalA (for pRL706) or 1 μg/ml SalA (for pRL663), 200 μg/ml ampicillin, and 1 mM IPTG, and plates were incubated 24–48 hr at 37°C. Sal-resistant mutants were identified by the ability to form colonies on this medium, were confirmed by re-streaking on the same medium, and were demonstrated to contain plasmid-linked Sal-resistant mutations by preparing plasmid DNA, transforming E. coli D21f2tolC with plasmid DNA, and plating transformants on the same medium. Nucleotide sequences of rpoB and rpoC were determined by Sanger sequencing (eight primers per gene).Induced Sal-resistant mutants were isolated using procedures analogous to those used for isolation of induced Myx-resistant mutants in d pRL706  and rpoCd pRL663  was perfrpoC derivatives, temperature-sensitive E. coli strain 397c  were applied to LB-agar plates containing 200 μg/ml ampicillin and 1 mM IPTG, plates were incubated 22 hr at 43°C, and bacterial growth was scored. For complementation assays with rpoB derivatives, temperature-sensitive E. coli strain RL585 306 lacZam2110 galEKamleuamtrpamsueA rpsL tsx srl301::Tn10-84; 3–104 cells) were applied to LB-agar plates containing 200 μg/ml ampicillin, 1 mM IPTG, and 10 μg/ml tetracycline, plates were incubated 22 hr at 43°C, and bacterial growth was scored.For complementation assays with 600 = 0.4–0.8.  Diluted aliquots  were dispensed into wells of a 96-well plate, were supplemented with 3 μl of a twofold dilution series of SalA or SalB in methanol , or 3 μl of a solvent blank, and were incubated 16 hr at 37°C with shaking. The MIC was defined as the lowest tested concentration of SalA that inhibited bacterial growth by ≥90%.Resistance levels of Sal-resistant mutants were quantified by performing broth microdilution assays. Single colonies were inoculated into 5 ml LB broth (LB broth containing 200 μg/ml ampicillin for induced mutants and wild-type controls for induced mutants) and incubated at 37°C with shaking until ODCross-resistance levels of Sal-resistant mutants were determined analogously to resistance levels, using 0.003–200 μg/ml Rif (Sigma–Aldrich), Stl , CBR703 , MyxB  were plated on LB agar containing 0.6 μg/ml (2 × MIC) SalA, 1 μg/ml (2 × MIC) Rif, 6 μg/ml (2 × MIC) MyxB, both 0.6 μg/ml SalA and 1 μg/ml Rif, or both 0.6 μg/ml SalA and 6 μg/ml MyxB, and numbers of colonies were counted after 24 hr at 37°C (at least five independent determinations each). Resistance rates and 95% confidence intervals were calculated using the Ma-Sandri-Sarkar Maximum Likelihood Estimator (http://www.keshavsingh.org/protocols/FALCOR.html)  .E. coli RNAP holoenzyme, 10 nM DNA fragment containing positions −42 to +426 of the lacUV5(ICAP) promoter (Reaction mixtures contained (20 μl): test compound , 40 nM promoter , and 100promoter . ProductE. coli RNAP holoenzyme, 10 nM DNA fragment containing positions −42 to +426 of the lacUV5(ICAP) promoter (32P]UTP (1.5 Bq/fmol) in TB. Reaction components except DNA, ApA, and [α-32P]UTP were pre-incubated 10 min at 37°C, DNA was added and reaction mixtures were incubated 10 min at 37°C, ApA and [α32P]UTP were added and reaction mixtures were incubated 10 min at 37°C. Reactions were terminated by adding 10 μl loading buffer and heating 4 min at 95°C. Products were applied to 7 M urea 15% polyacrylamide (19:1 acrylamide:bisacrylamide) slab gels, electrophoresed in TBE, and analyzed by storage-phosphor scanning .Reaction mixtures contained (10 μl): test compound , 40 nM promoter , 0.5 mM E. coli RNAP holoenzyme, 100 nM DNA fragment containing positions −42 to +426 of the lacUV5(ICAP) promoter (32P]ATP (0.07 Bq/fmol) in TB. Reaction components other than DNA and ATP were pre-incubated 10 min at 37°C; DNA was added and reaction mixtures were incubated 10 min at 37°C; and ATP was added and reaction mixtures were incubated 10 min at 37°C. Reactions were terminated by adding 5 μl loading buffer and heating 3 min at 95°C. Products were applied to 7 M urea 24% polyacrylamide (19:1 acrylamide:bisacrylamide) slab gels, electrophoresed in TBE, and analyzed by storage-phosphor scanning .Reaction mixtures contained (10 μl): 0 or 10 μM SalA, 400 nM promoter , 100 μM E. coli RNAP holoenzyme, 10 nM DNA fragment N25-100-tR2 (32P]ATP (5.5 Bq/fmol), 5 μM UTP, and 5 μM GTP in TB. Reaction components except heparin and NTPs were pre-incubated 10 min at 37°C, heparin was added and reaction mixtures were incubated 3 min at 37°C, and NTPs were added and reaction mixtures were incubated 5 min at 37°C. The resulting halted transcription elongation complexes were exposed to test compounds by addition of 0.75 μl 200 μM SalA, 0.75 μl 4 μM Rif, 0.75 μl 8 mM Stl, 0.75 μl 2 mM CBR703, 0.75 μl 400 μM MyxB, or 0.75 μl 2 mM Lpm and incubation 5 min at 37°C, and were re-started by addition of 0.75 μl 1 mM CTP and incubation 5 min at 37°C. Reactions were terminated by adding 10 μl loading buffer and heating 4 min at 95°C. Products were applied to 7 M urea 15% polyacrylamide (19:1 acrylamide:bisacrylamide) slab gels, electrophoresed in TBE, and analyzed by storage-phosphor scanning .Halted transcription elongation complexes  were prepared essentially as in -100-tR2 , 100 μg/E. coli RNAP holoenzyme , 5 nM DNA fragment T7A1 (positions −65 to +35 of the bacteriophage T7 A1 promoter [32P]CTP (0.44 Bq/fmol) in TB. Reaction components other than DNA, heparin, and NTPs were pre-incubated 30 min at 37°C. DNA was added and reaction mixtures were incubated 15 min at 37°C; heparin was added and reaction mixtures were incubated 2 min at 37°C; and NTPs were added and reactions mixtures were incubated 10 min at 37°C. Reactions were terminated by addition of 10 µl 80% formamide, 10 mM EDTA, 0.04% bromophenol blue, 0.04% xylene cyanol, and 0.08% amaranth red. Products were heated 5 min at 90°C, cooled 5 min on ice, resolved by urea-PAGE . Fits were ranked based on the AICc statistic , the Sy.x statistic , and the number of parameters .Reaction mixtures contained (20 µl): 0–0.4 µM SalA, 10 nM promoter ; preparerea-PAGE , and ana32P-5′-end-labeled oligoribonucleotide  in 25 μl 5 mM Tris–HCl, pH 7.7, 200 mM NaCl, and 10 mM MgCl2, were heated 5 min at 95°C and cooled to 4°C in 2°C steps with 1 min per step using a thermal cycler  and then were stored at −20°C.Nucleic-acid scaffolds for assays were prepared as follows: nontemplate-strand oligodeoxyribonucleotide , template-strand oligodeoxyribonucleotide , and E. coli RNAP core enzyme, 10 nM 32P-labeled nucleic-acid scaffold (200 Bq/fmol), and 20 μM ATP in TB. Reaction components except SalA and ATP were pre-incubated 5 min at 37°C, SalA was added and reaction mixtures were incubated 5 min at 37°C, and ATP was added and reaction mixtures were incubated 0.4 min (wild-type RNAP) or 10 min (ΔTL RNAP) at 37°C. Reactions were terminated by adding 10 μl loading buffer and heating 2 min at 95°C. Products were applied to 7 M urea 15% polyacrylamide (19:1 acrylamide:bisacrylamide) slab gels, electrophoresed in TBE, and analyzed by storage-phosphor scanning .Reaction mixtures for assays contained (10 μl): 0–64 μM SalA 40 nM wild-type or ΔTL E. coli RNA polymerase holoenzyme, 40 nM DNA fragment PT7A1-his-ThisL or PT7A1-opspheP-ThisL (prepared by PCR using plasmid pIA171 or pIA251 [32P]CTP (4 Bq/fmol), 2.5 μM ATP, 2.5 μM GTP, 20 mM Tris–HCl, pH 7.9, 20 mM NaCl, 3 mM MgCl2, 14 mM 2-mercaptoethanol, and 0.1 mM EDTA. Reaction components except ApU and NTPs were pre-incubated 5 min at 37°C, and ApU and NTPs were added and the reaction mixture was incubated 15 min at 37°C. The resulting halted transcription elongation complexes were exposed to SalA (or buffer blank) by addition of 4 μl 40 μM SalA (or buffer blank) and incubation for 5 min at 37°C, and were re-started by addition of 1.2 μl 10 mM ATP, 1.2 μl 10 mM CTP, 1.2 μM 10 mM UTP, 0.8 μl 10 mM GTP, and 2 μl 2 mg/ml heparin. Aliquots (10 μl) were removed at time points  and after a subsequent ‘chase’ , and aliquots were combined with 10 μl loading buffer, and heated 4 min at 95°C. Products were applied to 7 M urea 15% polyacrylamide gels (19:1 acrylamide:bisacrylamide) slab gels, electrophoresed in TBE, and analyzed by storage-phosphor scanning . Pause half-lives and efficiencies were calculated as in Pausing assays were performed essentially as in r pIA251  as templ32P-5′-end-labeled oligoribonucleotide  in 50 μl 10 mM Tris–HCl, pH 7.9, 40 mM KCl, 5 mM MgCl2, and then heating 2 min at 95°C, cooling to 45°C in 30 s, and cooling to 25°C in 2°C steps with 120 s per step, in a thermal cycler (Applied Biosystems). Transcription elongation complexes were reconstituted by mixing 0 or 10 μM Sal and 100 nM E. coli RNAP core enzyme in 90 μl 25 mM HEPES-KOH, pH 8.0, 130 mM KCl, 5 mM MgCl2, 1 mM DTT, 0.15 mM EDTA, 5% glycerol, and 25 μg/ml acetylated bovine serum albumin; incubating 10 min at 24°C; adding 10 µl 500 nM nucleic-acid scaffold; and incubating 15 min at 37°C. Pyrophosphorolysis was initiated by addition of 1 μl 0.05 U/μl apyrase (New England Biolabs) and 1 μl 50 mM sodium pyrophosphate; reaction mixtures were incubated at 37°C, and 10 μl aliquots were withdrawn after 0, 1, 2.5, 5, 10, 20, and 30 min and quenched by mixing with 10 μl 98% formamide, 10 mM EDTA, 0.02% bromophenol blue, and 0.02% xylene cyanol. To confirm that transcription elongation complexes were catalytically active, a ‘chase’ reaction was performed after the last time point, adding 11.4 μl reaction mixture to 0.6 μl 20 mM GTP . Under one condition, small crystals appeared within 2 days. Conditions were optimized using the hanging-drop vapor-diffusion technique at 22°C. The optimized conditions  yielded large crystals with dimensions of 0.2 mm × 0.2 mm × 0.2 mm in one week. SalA and Sal–Br were soaked into RNAP crystals, yielding RNAP–Sal and RNAP–Sal–Br crystals, by addition of 0.2 μl 20 mM SalA or Sal–Br in (±)-2-methyl-2,4-pentanediol  to the crystallization drop and incubation 30 min at 22°C. RNAP, RNAP-Sal, and RNAP–Sal–Br crystals were transferred to reservoir solutions containing 15% (vol/vol) -(−)-2, 3-butanediol (Sigma–Aldrich) and then flash-cooled with liquid nitrogen.Crystallization trials were performed using Crystal Former microfluidic chips  and SmartScreen solutions (Microlytic)  difference map. A structural model of SalA derived from the crystal structure of SalB (CSD 50962; E. coli RNAP–SalA complex model, refined to Rwork and Rfree of 0.286 and 0.325, respectively, has been deposited in the PDB with accession code 4MEX (The structures of the ode 4MEX .


Their structures were assigned on the basis of spectroscopic data analysis. Compounds 1 and 4 showed moderate anti-microbial activities against Bacillus subtilis . Compound 6 exhibited moderate anti-microbial activities against two strains of Helicobacter pylori (Hp-SS1 and ATCC 43504) with MIC values of 50 and 50 μg/mL, respectively.Three new halimane-type diterpenoids formosins A–C (The online version of this article (doi:10.1007/s13659-016-0086-6) contains supplementary material, which is available to authorized users. Excoecaria (Euphorbiaceae) compromising 40 species, are widely distributed in Africa and East Asia +  requiring seven degrees of unsaturation. The IR absorptions  and (1695 cm−1) showed the presence of a carboxylic group. The diagnostic NMR data , a persubstituted double bond, and a carboxylic group (δC 181.5). These functionalities accounted for five out of the seven indices of hydrogen deficiency, requiring the presence of two additional rings in the structure of 1. The aforementioned data suggested that compound 1 is a halimane-type diterpenoid and is structurally related to crotohalimaneic acid + , which is 16 mass units more than that of 1. Comparison of its NMR spectroscopic data , as well as the key HMBC correlations from H-12 to C-13, C-14, and C-16 . Thus, the structure of 2 (formosin B) was determined as shown.Compound 3 displayed a molecular formula of C21H30O5 as established by the (+)-HRESIMS at m/z 385.1991 [M + Na]+  and the 13C NMR data. Analysis of the NMR data , which was located at C-15 to form the acetal motif, which was confirmed by the downfield shifted C-15 (ΔδC 32.3) as compared to that of 2. Compound 3 was obtained as a pair of inseparable C-15 epimers, which exhibited several pairs of very close carbon resonances in the 13C NMR spectrum . Therefore, the structure of 3 (formosin C) with its relative configuration was confirmed as depicted by the HMBC and ROESY spectra .Compound 4 was obtained as a white powder with a molecular formula of C21H30O4 as established by the 13C NMR data and the (+)-HRESIMS ion at m/z 369.1970 [M + Na]+ , demanding seven degrees of unsaturation. The IR absorption bands  showed the presence of a carboxylic group. The characteristic NMR signals for a β-furan ring, a trisubstituted double bond, a methoxyl and a carboxylic groups were observed from the 1H and 13C NMR spectroscopic data analysis  to C-2 (δC 74.3). The carboxylic group was attached to C-9 via the HMBC correlations from H-8, H-10 and H-11 to C-20 (δC 182.6). The key HMBC cross-peaks from H3-18 to C-3 (δC 120.6), C-4 (δC 149.2) and C-5, and from H3-19 to C-4 revealed the presence of Δ3 double bond. The relative configuration of 4 was established by the ROESY experiment  was established as depicted.Compound eic acid  with a c5 possessed a molecular formula C20H28O4 based on the 13C NMR data and the (+)-HRESIMS ion at m/z 355.1885 [M + Na]+ , which is 14 mass units less than that of 4. Detailed analysis of the NMR data  with the relative configuration was further confirmed by HMBC and ROESY spectra .Compound 6, named formosin F, exhibited a sodiated molecular ion at m/z 353.1724 [M + Na]+  in the (+)-HRESIMS, consistent with a molecular formula of C20H26O4, which was supported by the 13C NMR data. Comparison of the NMR data  from H-3 and H-10 to C-2 (δC 199.9). The structure of 6 was thus determined as shown.Compound in vitro by the microdilution method +; (+)-HRESIMS m/z 339.1937 [M + Na]+ .White powder; mentclass2pt{minimc 0.10, MeOH); UV (MeOH) λmax (log ε) 207 (3.92); IR (KBr) νmax 3435, 3000–2500, 1753, 1695, 1456, 1205, 1070, 756 cm−1; 1H NMR (CDCl3), see Table 13C NMR (CDCl3) see Table m/z 355.2 [M + Na]+; (+)-HRESIMS m/z 355.1839 [M + Na]+ .White powder; mentclass2pt{minimc 0.10, MeOH); UV (MeOH) λmax (log ε) 211 (3.58); IR (KBr) νmax 3425, 3000–2500, 1726, 1693, 1659, 1468, 1178, 1065, 600 cm−1; 1H NMR (CDCl3), see Table 13C NMR (CDCl3) see Table m/z 385.2 [M + Na]+; (+)-HRESIMS m/z 385.1991 [M + Na]+ .White powder; mentclass2pt{minimc 0.025, MeOH); UV (MeOH) λmax (log ε) 204 (4.21); IR (KBr) νmax 3427, 3000–2500, 1695, 1452, 1254, 1082 cm−1; 1H NMR (CDCl3), see Table 13C NMR (CDCl3) see Table m/z 369.2 [M + Na]+; (+)-HRESIMS m/z 369.1970 [M + Na]+ .White powder; mentclass2pt{minimc 0.05, MeOH); UV (MeOH) λmax (log ε) 199 (3.89); IR (KBr) νmax 3419, 3000–2500, 1695, 1452, 1383, 1244, 1026, 874, 600 cm−1; 1H NMR (CDCl3), see Table 13C NMR (CDCl3) see Table m/z 355.2 [M + Na]+; (+)-HRESIMS m/z 355.1885 [M + Na]+ .White powder; mentclass2pt{minimc 0.10, MeOH); UV (MeOH) λmax (log ε) 237 (4.86); IR (KBr) νmax 3000–2500, 1703, 1633, 1435, 1381, 1219, 1026, 874 cm−1; 1H NMR (CDCl3), see Table 13C NMR (CDCl3) see Table m/z 331 [M + H]+; (+)-HRESIMS m/z 353.1724 [M + Na]+ .White powder; mentclass2pt{minimBacillus subtilis ATCC 6633 were tested by applying the protocols described in our previous research [The in vitro antibacterial activities against research .Helicobacter pylori strains (Hp-SS1 or ATCC 43504 strain) were carried out in vitro according to the protocols described previously [Antibacterial tests against eviously .Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized usersSupplementary material 1 (PDF 3792 kb)


In the crystals of both compounds, pairs of N—H⋯Nnitrile hydrogen bonds link the molecules, forming inversion dimers with In two cyclohepta by 62.47 (17) and 72.51 (14)°, respectively, in (I), and by 71.44 (9) and 54.90 (8)°, respectively, in (II). The planes of the aromatic rings are inclined to one another by 53.82 (17)° in (I) and by 58.04 (9)° in (II). In the crystals of both (I) and (II), pairs of N—H⋯Nnitrile hydrogen bonds link the molecules, forming inversion dimers with R22(12) ring motifs. In (I), the resulting dimers are connected through C—H⋯Br hydrogen bonds, forming sheets parallel to (10-1), and π–π interactions [inter-centroid distance = 3.7821 (16) Å] involving inversion-related pyridine rings, forming a three-dimensional network. In (II), the resulting dimers are connected through π–π interactions [inter-centroid distance = 3.771 (2) Å] involving inversion-related pyridine rings, forming a two-dimensional network lying parallel to (001).In the title compounds, C Molecules containing heterocyclic substructures continue to be attractive targets for synthesis since they often exhibit diverse and important biological properties. Pyridine is used in the pharmaceutical industry as a raw material for various drugs, vitamins and fungicides, and as a solvent (Shinkai P21/n while the chloro derivative (II)PThe molecular structures of the title compounds, (I)2—NH2– chain, as found in a similar structure N6-(4-fluorobenzyl)-3-nitropyridine-2,6-diamine  Å, ϕ2 = 310.1 (4)° and Q3 = 0.637 (3) Å and ϕ3 = 283.4 (3)° for compound (I)Q2 = 0.475 (2) Å, ϕ2 = 310.3 (2)° and Q3 = 0.635 (2) Å and ϕ3 = 283.58 (17)° for compound (II)In both compounds, the pyridine ring is connected to a benzene ring by a –CHnitrile hydrogen bonds, forming inversion dimers with In the crystal of (I)s Table 1. The resnitrile hydrogen bonds, forming inversion dimers with In the crystal of (II)s Table 2. The dims Table 2. The resp-TSA (p-toluenesulfonic acid) (1.0 mmol) was added. The reaction mixture was heated under reflux for 2–3 h. On completion of the reaction, verified by thin-layer chromatography (TLC), the mixture was poured into crushed ice and extracted with ethyl acetate. The excess solvent was removed under vacuum and the residue was subjected to column chromatography using a petroleum ether/ethyl acetate mixture (97:3 v/v) as eluent to afford the pure products. They were recrystallized from ethyl acetate, giving colourless crystals of compounds (I)Compounds (I)H-cyclohepta[b]pyridine  and 1.441 (3) Å, respectively, in (I)N6-(4-fluorobenzyl)-3-nitropyridine-2,6-diamine  and 1.454 (3) Å, respectively.A similar structure reported in the literature, 2-(4-bromophenyl)-4-(4-methoxyphenyl)-6,7,8,9-tetrahydro-5Uiso(H) = 1.5Ueq(C) for methyl H atoms and = 1.2Ueq for other H atoms.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989014025936/su5027sup1.cifCrystal structure: contains datablock(s) global, I, II. DOI: 10.1107/S2056989014025936/su5027Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989014025936/su5027IIsup3.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S2056989014025936/su5027Isup4.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989014025936/su5027IIsup5.cmlSupporting information file. DOI: 1036150, 1036149CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Upon coordination, the hydrazinylidenepyridine ligand is non-planar, an angle of 54.42 (7)° being observed between the pyridine ring and the aromatic ring of the [2-(4-chlorophenyl)hydrazin-1-ylidene]ethyl group.The title compound, [Rh(η 5-C5Me5)Cl(C13H12ClN3)]Cl, adopts a typical piano-stool geometry. The complex is chiral at the metal and crystallizes as a racemate. Upon coordination, the hydrazinylidenepyridine ligand is non-planar, an angle of 54.42 (7)° being observed between the pyridine ring and the aromatic ring of the [2-(4-chlorophenyl)hydrazin-1-ylidene]ethyl group. In the crystal, a weak interionic N—H⋯Cl hydrogen bond is observed.The cation of the title compound, [Rh(η Upon coordination, the hydrazinylidenepyridine ligand is non-planar, an angle of 54.42 (7)° being observed between the planes of pyridine and the benzene ring of the [(4-chlorophenyl)hydrazono]ethyl group. Otherwise, all geometrical data around the rhodium(III) atom are similar to those found in analogous N,N-chelated pentamethylcyclopentadienyl rhodium complexes 2Rh2(μ-Cl)2Cl2]  with two equivalents of 2-{1-[2-(4-chlorophenyl)hydrazono]ethyl}pyridine . Crystals suitable for X-ray structure analysis were obtained by slow evaporation of a dichloromethane/n-pentane solution (1:1 v/v) containing the title compound. Yield: 80%. IR : 1592 . 1H NMR : δ (p.p.m.) = 9.21 , 8.76 , 8.16 , 8.01 , 7.77 , 7.45 , 7.36 , 2.58 , 1.43 . MS (ESI positive mode): m/z 518.0 [M − Cl]+.The title compound was synthesized by reacting one equivalent of [ = 1.2 Ueq(C) or 1.5 Ueq(C) for methyl H atoms.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S205698901500184X/rz5146sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S205698901500184X/rz5146Isup2.hklStructure factors: contains datablock(s) I. DOI: 1045840CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Based on a previously published highly specific PSMA ligand , pH 3.5 to 4.5, 5 min, 95°C) and 177Lu labelling  were almost quantitative, resulting in specific activities of 250 to 300 GBq/μmol for the 68Ga analogues and 38 GBq/μmol for 177Lu complexes. Due to metabolic instability of l-amino acid spacers, d-amino acids were implemented resulting in a metabolically stable DOTAGA ligand. Compared to the DOTA ligand, the DOTAGA derivatives showed higher hydrophilicity  and improved affinity to PSMA resulting in an about twofold increased specific internalization of the 68Ga- and 177Lu-labelled DOTAGA analogue. Especially, [68Ga]DOTAGA-ffk(Sub-KuE) exhibits favourable pharmacokinetics, low unspecific uptake and high tumour accumulation in LNCaP-tumour-bearing mice.Radiochemical yields for 68Ga/177Lu]DOTAGA-ffk(Sub-KuE) possess remarkable potential for the management of prostate cancer.The pair of diagnostic/therapeutic PSMA-ligands [ N-acetyl-l-aspartyl-l-glutamate (NAAG) to N-acetyl-l-aspartate and glutamate HBED-CC-Ahx-KuE [68Ga]DOTA-FFK(Sub-KuE), [68Ga]HBED-CC-Ahx-KuE showed higher tumour accumulation and improved imaging contrast  ethylenediamine-N,N′-diacetic acid) is not suitable for radiolabelling with therapeutic radiometals, whereas 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and its analogues form stable complexes with a broad range of radiometals, for therapy most importantly 90Y and 177Lu. Given the high expression of PSMA on the majority of prostate cancers, the availability of high-affinity PSMA-targeted probes, labelled with therapeutic radioisotopes, offers promising perspectives for PSMA-targeted endoradiotherapy.These findings were recently integrated into the design of s Figure , such asSub-KuE)  and MIP-1375 [131I]MIP-1466 [131I]MIP-1375 inhibited tumour growth in a time- and dose-dependent manner. First, human applications of [131I]MIP-1095/1466 revealed promising therapy response with moderate side effects [123I-labelled analogues [123I]MIP-1072 and [123I]MIP-1095, were shown to possess suitable characteristics for SPECT imaging. However, these compounds are comparably lipophilic , and th68Ga for PET imaging and 177Lu/90Y for therapy after conjugation of DOTA. However, in order to further facilitate the labelling procedure for 177Lu and 90Y, improve ligand pharmacokinetics, and potentially obtain radiometalated derivatives with higher affinity, we substituted DOTA in DOTA-FFK(Sub-KuE) by 1,4,7,10-tetraazacyclodocecane,1-(glutaric acid)-4,7,10-triacetic acid (DOTAGA) HBED-CC-Ahx-KuE +.(S)eviously . HPLC (1-(OtBu)KuE(OtBu)2 (2): A solution of 3.40 g  1 in 45 mL 1,2-dichloroethane (DCE) was cooled to 0°C, and 2.69 mL  of triethylamine (TEA), and 3.59 g  of Cbz-Lys-OtBu · HCl was added under vigorous stirring. The reaction mixture was heated to 40°C overnight. The solvent was removed in vacuo, and the crude product was purified via silica gel flash-chromatography using an eluent mixture of ethyl acetate/hexane/TEA (500/500/0.8 (v/v/v)). Upon solvent evaporation, 4.80 g of 2 were obtained as a colourless, sticky oil (yield: 80% based on l-di-tert-butyl glutamate · HCl). HPLC (40% to 100% B in 15 min): tR = 14.3 min; K′ = 8.53. Calculated monoisotopic mass for 2 (C32H51N3O9): 621.8; found: m/z = 622.2 [M + H]+, 644.3 [M + Na]+.Cbz2 (3): For Cbz deprotection, 6.037 g  of 2 was dissolved in 150 mL of ethanol (EtOH), and 0.6 g  of Palladium on activated charcoal (10%) was added. After purging the flask with H2, the solution was stirred overnight under light H2-pressure . The crude product was filtered through Celite, the solvent was evaporated in vacuo, and the desired product was obtained as a waxy solid . HPLC (10% to 90% B in 15 min): tR = 12.6 min; K′ = 6.41. Calculated monoisotopic mass for 3 (C24H45N3O7): 487.6; found: m/z = 488.3 [M + H]+, 510.3 [M + Na]+.(OtBu)KuE(OtBu)-Sub-(OtBu)KuE(OtBu)2 (4): 3  was dissolved in 500 μL N,N-dimethylformamide (DMF), and 57 μL  of TEA was added. This solution was added dropwise (within 30 min) to a solution of 33.2 mg  of disuccinimidyl suberate (DSS). After stirring for an additional 2 h at room temperature (RT), the reaction mixture was concentrated in vacuo, diluted with ethyl acetate and extracted with water (twice). The organic phase was dried over Na2SO4, filtered and evaporated to dryness. Due to sufficient purity of the crude 4, it was used for the following reaction step without further purification. HPLC (10% to 90% B in 15 min): tR = 16.9 min; K′ = 8.39. Calculated monoisotopic mass for 4 (C36H60N4O12): 740.4; found: m/z = 741.2 [M + H]+, 763.4 [M + Na]+.NHS-l-Phe-l-Phe-l-Lys(Boc)  and Fmoc-d-Phe-d-Phe-d-Lys(Boc) : Fmoc-Lys(Boc)-OH was coupled to TCP resin according to a previously published method +, 849.3 [M + Na]+, 414.2 [M +2H]2+.DOTA-FFK, 7) : For 38 -Phe-Phe-Lys : For 0.27 mmol peptide-bound resin, 190 mg DOTAGA-anhydride  and 470 μL DIPEA  in NMP were added to the resin. After 18 h of shaking, the resin was washed with NMP and DCM. HPLC (10% to 90% B in 15 min): tR = 10.6 min; K′ = 5.63. Calculated monoisotopic mass for 8 and 9 (C43H62N8O13): 898.4; found: m/z = 899.4 [M + H]+, 921.4 [M + Na]+, 450.2 [M +2H]2+.DOTAGA-FFK(Sub-KuE) (10) +, 1,306.7 [M + Na]+, 642.8 [M +2H]2+.DOTAuE) (10) : To a so-FFK(Sub-KuE) (11) and DOTAGA-ffk(Sub-KuE) (12): Either 8 or 9  was added to TEA  and 27 mg of 4  as described for 10. HPLC (10% to 90% B in 15 min): tR = 9.7 min; K′ = 4.11. Calculated monoisotopic mass for 11 and 12 (C63H93N11O22): 1,355.7; found: m/z = 1,356.2 [M + H]+, 1,378.2 [M + Na]+, 679.2 [M +2H]2+.DOTAGAThe synthesis was performed according to previously published methods ,31,32.-4-iodobenzoate : Under a nitrogen atmosphere, 500 mg  4-iodobenzoic acid was dissolved in 10 mL DCM, and after addition of 278 mg  N-hydroxysuccinimide and 374 mg  dicyclohexyl carbodiimide, the suspension was stirred overnight. The precipitate was filtered off, and the filtrate was evaporated to dryness. The resulting solid was washed with a 1:1-mixture of DCM and hexane to yield the desired product  as a white solid. Due to the limited detectability of the product in ESI-mass spectrometry (MS), a representative conjugate with H-Phe-OtBu (1 eq) was prepared in DMF in the presence of DIPEA (3 eq) and characterized via MS. HPLC (40% to 100% B in 15 min): tR = 10.6 min; K' = 5.63. Calculated monoisotopic mass for I-BA-Phe(OtBu) (C20H22INO3): 451.1; found: m/z = 396.1 [M + H-tBu]+.Succinimidyl-4-tributylstannyl-benzoate : To a solution of 100 mg  14 in 5 mL anhydrous toluene were added 234 μL  of hexabutylditin and 10.7 mg  of the catalyst tetrakis(triphenylphosphine)palladium under a nitrogen atmosphere. The mixture was heated under reflux until the solution turned black (overnight). After cooling, the toluene was removed in vacuo, and the resulting oil was purified using silica gel flash chromatography (ethyl acetate/hexane: 3/7 (v/v)) to yield 15  as a colourless oil. TLC (ethyl acetate/hexane: 3/7): Rf = 0.46Succinimidyl3-BA)(OtBu)KuE(OtBu)2 (13): To a solution of 19.0 mg  3 in 2 mL DCM were added 26.3 μL  of TEA and 19.8 mg  of 15. The mixture was stirred at RT for 4 h and was then diluted with DCM. After washing with water, the organic phase was dried over Na2SO4, filtered and evaporated to dryness. 13  was obtained as a colourless oil. HPLC (10% to 90% B in 15 min): tR = 23.8 min; K' = 13.88. Calculated monoisotopic mass for 13 (C43H75N3O8Sn): 880.8; found: m/z = 902.2/903.3/904.3 [M + Na]+.(SnBu(I-BA)KuE: To a solution of 15 mg  3 in 2 mL DMF was added 11.4 mg  of 4-iodo-benzoic acid, 6.3 mg  of 1-Hydroxy-7-azabenzotriazole (HOAt), 7.2 μL  diisopropyl carbodiimide (DIC) and 23.7 μL  DIPEA. The yellow solution was stirred for 20 h, diluted with ethyl acetate and extracted with water. The organic layer was dried over Na2SO4, filtered and evaporated to dryness. For tBu-deprotection, the crude product was dissolved in 200 μL TFA. After 30 min, the solvent was evaporated and the crude product was purified using preparative RP-HPLC (isocratic eluent: 18% B). HPLC (10% to 90% B in 15 min): tR = 10.5 min; K′ = 5.56 calculated monoisotopic mass for (I-BA)KuE (C19H24IN3O8): 549.3; found: m/z = 550.0 [M + H]+, 571.9 [M + Na]+natGa compounds: For the preparation of the natGa complexes, equal volumes of a 2 mM solution of Ga(NO3)3 in water and a 2 mM solution of the respective PSMA ligand in water were mixed and heated to 40°C for 30 min. After cooling, the natGaIII-chelate formation was confirmed using RP-HPLC and MS. The resulting 1 mM aqueous solutions of the respective natGa-complexes were then further diluted and used in the in vitro IC50 studies without further processing.natGa]DOTA-FFK(Sub-KuE) (natGa-10) HPLC (20% to 60% B in 15 min): tR = 11.6 min; K′ = 6.3. Ccalculated monoisotopic mass (C60H86N11O20Ga): 1,349.5; found: m/z = 1,350.3 [M + H]+, 1,372.1 [M + Na]+, 675.7 [M +2H]2+.[natGa]DOTAGA-FFK(Sub-KuE) (natGa-11) HPLC (25% to 45% B in 15 min): tR = 16.0 min; K′ = 9.0. Calculated monoisotopic mass (C63H90N11O22Ga): 1,421.7, 1,423.7; found: m/z = 1,422.1/1,424.1 [M + H]+, 710.6/712.6 [M +2H]2+.[natGa]DOTAGA-ffk(Sub-KuE) (natGa-12) HPLC (25% to 55% B in 15 min): tR = 12.1 min; K′ = 7.6. Calculated monoisotopic mass (C63H90N11O22Ga): 1,421.7, 1,423.7; found: m/z = 1,422.6/1,424.5 [M + H]+, 1,444.4/1,446.4 [M + Na]+.[natGa]HBED-CC-Ahx-KuE HPLC (25% to 43% B in 15 min): tR = 9.0 min; K′ = 5.0. Calculated monoisotopic mass (C44H59N6O17Ga): 1,012.3, 1,014.3; found: m/z = 1,013.0/1,015.0 [M + H]+, 1,035.0/1,037.0 [M + Na]+.DOTA-FFK(Sub-KuE) (natLu-10) HPLC (25% to 45% B in 16 min): tR = 14.1 min; K′ = 9.1. Calculated monoisotopic mass (C60H86N11O20Lu): 1,455.6; found: m/z = 1,456.4 [M + H]+, 1,478.5 [M + Na]+.[natLu]DOTAGA-FFK(Sub-KuE) (natLu-11) HPLC (25% to 45% B in 16 min): tR = 14.4 min; K′ = 9.3. Calculated monoisotopic mass (C63H90N11O22Lu): 1,527.6; found: m/z = 1,528.4 [M + H]+, 1,550.3 [M + Na]+, 764.2 [M +2H]2+.[natLu]DOTAGA-ffk(Sub-KuE) (natLu-12) HPLC (25% to 55% B in 15 min): tR = 10.4 min; K′ = 6.4. Calculated monoisotopic mass (C63H90N11O22Lu): 1,527.6; found: m/z = 1,528.1 [M + H]+, 764.5 [M +2H]2+.[125I]I-BA)KuE: The radioiodination was performed according to previously published methods NaI  was added to a solution of approximately 0.1 mg 15 in 20 μL MeCN/acetic acid (1/1) and incubated at RT for 10 min. The product was diluted with 10 mL of water and loaded onto a C18 Sep Pak Plus cartridge, which had been preconditioned with 10 mL of MeOH and rinsed with 10 mL of water. The cartridge was then washed with 10 mL water, and the product was eluted in 300 to 500 μL fractions with a 1:1 mix (v/v) of EtOH/MeCN (2 mL). The radioactive fractions were evaporated to dryness, and the residue was dissolved in 200 μL TFA. After 30 min, the solvent was evaporated in vacuo. The crude product was isolated from unlabelled tributyltin precursor by RP-HPLC (20% to 40% B in 20 min) to afford the desired product (10.9 MBq). HPLC : tR = 13.0 min, K′ = 6.22.(5II-BA)Ku methods ,32. Pera68Ga-labelling: A 1.25 mL fraction of 68Ge/68Ga generator  eluate (1 M HCl) was loaded onto a self-filled cartridge containing 300 mg SCX material . The cartridge was then washed with water (1.0 mL) and purged with air. The 68Ga3+ was eluted with aq. NaCl  and HEPES  was added . That solution was mixed with the precursor (3 nmol in 10 μL) and heated at 95°C for 5 min. After cooling, labelling efficiency and radiochemical purity were determined using Radio-TLC and Radio-HPLC. Radiochemical purity of all 68Ga-labelled conjugates was ≥95%. Therefore, the tracers were diluted and used in vitro experiments without further purification. Tracers for in vivo use were prepared in GMP-compliant procedure using a fully automated synthesis module  similar to the procedure described previously [in vacuo.eviously . For ani177Lu-labelling: A 0.1 mM aqueous solution of chelator-coupled peptide (0.66 nmol) was added to 25 MBq [177Lu]LuCl3 . The pH was adjusted to pH 5 by the addition of approximately 150 μL 0.1 M NH4OAc solution. After 45 min at 95°C, the labelling efficiency was examined by TLC and RP-HPLC.n = 6). Vials were vortexed vigorously for 3 min. To achieve quantitative phase separation, the vials were centrifuged at 6,000 g for 5 min in a Biofuge 15 . The activity concentrations in 100 μl-samples of both the aqueous and the organic phase were measured in a γ-counter. Both the partition coefficient P(o/w), which is defined as the molar concentration ratio of a single species A between n-octanol and water at equilibrium, and logP(o/w), which is an important parameter used to characterize lipophilicity of a compound, were calculated.To a solution of 0.5 to 1 MBq of radiolabelled peptide in 500 μl of phosphate-buffered saline , 500 μl of n-octanol was added (177Lu-10 and 177Lu-11 were incubated in 80 μL of human serum (37°C) and 80 μL 0.4 M DTPA solution (RT), respectively and after 1, 24 and 48 h, the stability was determined by TLC analysis. The 68Ga-labelled tracers (40 to 45 MBq) were injected into the tail vein of CD-1 nu/nu mice. The animals were sacrificed after 30 min, and urine, blood and kidney samples were taken. The kidney was frozen in liquid nitrogen, homogenized with a ball mill and extracted with 0.2 to 1 mL PBS containing 200 nmol 2-(phosphonomethyl)pentane-1,5-dioic acid (PMPA). The suspension was first centrifuged , and after ultrafiltration, the extracts were analysed by RP-HPLC. The blood samples were centrifuged to separate the plasma from the blood cells. Plasma proteins were removed by precipitation with acetonitrile  and subsequent centrifugation and ultrafiltration. The blood extract was also analysed by RP-HPLC.Cell culture: PSMA+ LNCaP cells (CLS: 300265) were grown in DMEM/Nutrition Mix F-12 with Glutamax-I (1:1)  supplemented with 10% FCS. Cultures were maintained at 37°C in a 5% CO2/humidified air atmosphere. One day prior to the experiment, cells were harvested using Trypsin/EDTA (0.05% and 0.02%) in PBS, centrifuged and resuspended with culture medium. For cell counting, a Countesse automated cell counter  was used. All in vitro binding and internalization studies were performed using live cells seeded 1 day prior to the experiment. For IC50 determination, 150,000 cells/mL were transferred to 24-well plates (1 mL/well), and for internalization studies, 125,000 cells/mL were transferred into PLL-coated 24-well plates.Determination of IC50: The culture medium was removed, and the cells were washed once with 500 μL of HBSS ), before being left to equilibrate in 200 μL of HBSS (1% BSA) on ice for 15 min. Then, 25 μL/well of either HBSS  or of solutions containing the respective unlabelled ligand of interest in increasing concentrations (10-10-10-4 M in HBSS (1% BSA)) was added, followed by the addition of 25 μL of ([125I]I-BA)KuE in HBSS (1% BSA). Experiments were carried out in triplicate for each concentration. The final radioligand concentration was 0.2 nM in all binding assays. Cells were incubated on ice for 60 min. Incubation was terminated by the removal of the incubation medium. Cells were thoroughly rinsed with 250 μL of HBSS. The wash medium was combined with the supernatant of the previous step. This fraction represents the amount of free radioligand. Cells were then lysed using 250 μL of 1 N NaOH, the lysate was transferred to vials and combined with 250 μL of HBSS used for rinsing the wells. Quantification of the amount of free and bound activity was performed in a γ-counter.Internalization studies: The culture medium was removed, and the cells were washed once with 500 μL DMEM-F12 (5% BSA) before being left to equilibrate in 200 μL DMEM-F12 (5% BSA) at 37°C for a minimum of 15 min. Then, 25 μL (per well) of either DMEM-F12 (5% BSA) or of a 100-μM PMPA solution (blocking) was added, followed by the addition of 25 μL of 68Ga/177Lu-labelled PSMA ligand. The final radioligand concentration was 0.2/0.5 nM in all internalization assays. To determine internalization kinetics, cells were then incubated at 37°C for 5, 15, 30 and 60 min, respectively. Experiments were carried out in triplicate for each time point (control and blocking). Incubation was terminated by placing the plate on ice for approximately 1 min and by subsequent removal of the incubation medium. Cells were thoroughly rinsed with 250 μL of PBS. The wash medium was combined with the supernatant of the previous step. This fraction represents the amount of free radioligand. To remove receptor surface bound radioactivity, the cells were then incubated for 10 min with 250 μL of ice cold PMPA solution (10 μM in PBS). After removal of the supernatant, the cells were thoroughly rinsed with another 250 μL of ice cold PBS. Both fractions were combined. The internalized activity was released by incubation with 250 μL of 1 N NaOH, transferred to vials and combined with 250 μL of 1 N NaOH used for rinsing the wells. Quantification of the amount of free, PMPA-releasable and internalized activity was performed in a γ-counter.7 cells in 200 μL were inoculated subcutaneously on the right shoulder of 6 to 8 weeks old CD-1 nu/nu mice (Charles River Laboratories). Tumours were grown for 2 to 4 weeks  and 4 to 6 weeks  to reach 4 to 8 mm in diameter.All animal experiments were conducted in accordance with general animal welfare regulations in Germany . The prostate cancer cell line LNCaP was suspended 1/1 in serum-free medium and Matrigel  and approximately 10Biodistribution: The 68Ga-labelled PSMA ligands  were injected into the tail vein of isoflurane anesthetized animals. The organ distribution was examined 1 h p.i. and quantified in a γ-counter.μPET imaging: Imaging studies were performed at a Siemens Inveon small animal PET, followed by data analysis using the Inveon Research Workplace software. The animals were anesthesized with isoflurane and injected via tail vein with 14 to 18 MBq (0.2 nmol) of tracer. Dynamic imaging was performed after on-bed injection for 1.5 h. Static images were recorded at 1 h p.i. with an acquisition time of 15 min. For the blockade image, animals were coinjected with 8 mg/kg of PMPA. Images were reconstructed using 3D ordered-subsets expectation maximum (OSEM3D) algorithm without scanner and attenuation correction.tBu-protected PSMA binding motif ((OtBu)KuE(OtBu)2, 3) was synthesized in 73% yield via three steps of solution phase chemistry as previously described with minor modifications , DOTAGA-FFK(Sub-KuE) (11) and DOTAGA-ffk(Sub-KuE) (12) were prepared according to Figure l-Phe-l-Phe-l-Lys(Boc)-OH (FFK(Boc), 5) and H-d-Phe-d-Phe-d-Lys(Boc)-OH (ffk(Boc), 6) were synthesized via solid phase peptide synthesis using a standard Fmoc protocol. The prochelators DOTA-tris-tBu-ester and DOTAGA-anhydride were coupled to the resin-bound peptides in almost quantitative yields. Subsequently, the chelator-conjugated peptides were cleaved from the resin (TFA/TIBS/H2O) and simultaneously deprotected to yield 7, 8 and 9. The tBu-protected PSMA binding motif 3 was first reacted with DSS in solution and then coupled with the chelator-functionalized peptides 7, 8 and 9. After acidic cleavage of the remaining tBu-protecting groups, the final products were purified by RP-HPLC. The products were obtained in approximately 15% yield (based on tripeptide). The identity of 10, 11 and 12 was confirmed by MS.The ligands DOTA-FFK(Sub-KuE) 3 at 40°C for 30 min and an 2.5-molar excess of LuCl3 at 95°C for 30 min, respectively. Quantitative metal complex formation was confirmed by RP-HPLC and MS.The 125I]I-BA)KuE, the reference radioligand in all PSMA binding assays carried out in this study, was synthesized in solution starting from 4-iodo-benzoic acid according to methods described in the literature [3Sn-precursor 13 was destannylated with [125I]NaI using peracetic acid as the oxidizing agent within 10 min at RT. After cartridge purification subsequent tBu-deprotection and RP-HPLC purification, the final product was obtained in a radiochemical yield of 44 ± 5% and radiochemical purity of >99%. The reference material for chromatographic analyses (cold) (I-BA)KuE was synthesized by coupling 4-iodo-benzoic acid to 3, subsequent tBu-deprotection and RP-HPLC purification.(5II-BA)Kuterature ,32. The 68Ga for labelling of 10, 11 and 12 was performed by combination of previously described methods LuCl3, 0.66 nmol of precursor were reacted with 25 MBq [177Lu]LuCl3 (AS = 170 GBq/μmol) at pH 5  resulting in specific activities of AS ≥38 GBq/μmol.To obtain RCY >95% for complexation of 125I]I-BA)KuE [c = 0.2 nM) as the radioligand. The IC50 values for the metal-free PSMA-inhibiting compounds and their respective natGa and natLu complexes are summarized in Table natGa]HBED-CC-Ahx-KuE I-BA)KuE, that was always assayed in parallel as an external reference. All DOTA and DOTAGA-coupled ligands showed a lower internalization efficiency compared to the radioiodinated reference compound, whereas [68Ga]HBED-CC-Ahx-KuE was internalized almost to the same degree as ([125I]I-BA)KuE.Variations in cell count and/or cell viability between experiments can significantly affect the cellular tracer uptake but cannot be fully avoided. Therefore, internalization data Table  are normP between n-octanol and PBS was determined for the compounds listed in Table 68Ga]HBED-CC-Ahx-KuE was the most hydrophilic compound.The partition coefficient log177Lu-10 and 177Lu-11 was investigated by incubation in human serum for 48 h at 37°C and by DTPA challenge  and subsequent TLC analysis. Release of 177LuIII from the complexes was not observed under the respective experimental conditions in the examined time frame.The stability of in vivo, tissue homogenates and body fluids of mice injected with 68Ga-11 and 68Ga-12 were analysed by radio-HPLC. Representative HPLC profiles of extracts and body fluids are shown in Figure 68Ga-11 and 61% and 62% for 68Ga-12, respectively. Rapid in vivo degradation was observed for 68Ga-11 consisting of the l-amino acid tripeptide FFK, resulting in only 21% intact tracer in blood at 0.5 h. 68Ga-12 that consists of the d-amino acid tripeptide ffk, was found to be stable in blood (100% intact tracer) at 0.5 h p.i..To investigate the influence of the amino acid composition of the spacer (KFF vs kff) on the metabolic stability 68Ga-11, 68Ga-12 and the reference [68Ga]HBED-CC-Ahx-KuE in LNCaP-tumour-bearing CD-1 nu/nu mice (1 h p.i.) is shown in Figure A comparison of the biodistribution of compounds 68Ga-10, 68Ga-11, 68Ga-12 and [68Ga]HBED-CC-Ahx-KuE in the same animal model for 15 min at 1 h p.i.. The maximum intensity projections depicted in Figure 68Ga-11 (c) compared to the respective DOTA analogue 68Ga-10 (b). In agreement with the results obtained from the biodistribution  exhibits increased tumour accumulation and a lower unspecific whole body uptake and tumour-to-background ratios even higher than those of the reference compound [68Ga]HBED-CC-Ahx-KuE (a). That tracer uptake into tumour and kidneys is specific and PSMA mediated HBED-CC-Ahx-KuE were investigated by carrying out dynamic μPET scans over a period of 1.5 h in CD-1 nu/nu mice bearing LNCaP tumour xenografts  the DOTA ligand 68Ga-10 exhibited unfavourable tumour targeting compared to [68Ga]HBED-CC-Ahx-KuE. Further, we observed significant inter-individual differences in metabolization kinetics for 68Ga-11 (with its l-amino acid spacer) in mice, which might explain inconsistent in vivo results obtained with PSMA ligands with l-amino acid spacers HBED-CC-Ahx-KuE (IC50 = 6.1 ± 0.8 nM).In addition to the improvement in metabolic stability for terature , a signi68Ga/177Lu-11 and 68Ga/177Lu-12 showed up to twofold increased internalization compared to the corresponding DOTA ligands 68Ga/177Lu-10. Interestingly, 68Ga/177Lu-12 with the ffk spacer showed a higher internalization rate than ligand 11 with the FFK spacer. Blocking experiments with PMPA highlight the high PSMA-specificity of these tracers.As expected from their enhanced PSMA affinities, the DOTAGA ligands 68Ga-12 in LNCaP-tumour bearing CD-1 nu/nu mice could be enhanced  and high kidney accumulation of 96 ± 45% ID/g. The kidney uptake is PSMA-specific, as shown in the blocking experiment . Further, [68Ga]HBED-CC-Ahx-KuE showed enhanced tracer uptake into the lung (1.9 ± 0.4% vs. 1.1 ± 0.3% ID/g), liver (0.5 ± 0.1 vs. 0.2 ± 0.04% ID/g), spleen (14.7 ± 2.6 vs. 5.5 ± 0.2% ID/g) and pancreas (0.9 ± 0.2 vs. 0.4 ± 0.1% ID/g). Therefore, the lower unspecific uptake of 68Ga-12 compared to [68Ga]HBED-CC-Ahx-KuE resulted in higher tumour-to-background ratios, i.e. tumour-to-blood (17.0 ± 0.5 vs. 12.5 ± 0.7) and tumour-to-skeletal muscle (19.8 ± 0.7 vs. 16.2 ± 0.1).Accordingly, the pharmacokinetics and tumour uptake of the new ligands, i.e. d Figure . The metn Figure  and μPET68Ga-12, whereas all other tracers (or their radiolabelled metabolites) exhibit unspecific retention or redistribution effects, resulting in non-linear kinetics (e.g. between 10 and 60 min) in the semilogarithmic plot. Considering the time-activity curves, it is worth noting that based on the kinetics of the reference compound [68Ga]HBED-CC-Ahx-KuE redistribution from tissues and compartments with unspecific uptake in the early phase seems likely and might explain the discontinuous clearance and increasing tumour uptake at later time points. The uptake of DOTA conjugate 68Ga-10 in the tumour is continuously increasing over time, but surprisingly without a corresponding decline of activity in muscle or blood (as measured over the heart). In contrast, a slow washout from the target and non-target tissues was observed for 68Ga-11. Most likely as a result of its metabolic instability, 68Ga-11 exhibited discontinuous and only moderate clearance kinetics. However, 68Ga-12 shows typical and nearly ideal in vivo pharmacokinetics, both for the target tissue (tumour and kidney) as well as the non-target tissue muscle and the blood. Equivalent to our DOTAGA ligand 12, the biodistribution of a naphthyl-containing [177Lu]DOTA-PSMA ligand was described recently [177Lu-12 also reveals favourable properties in vivo.After the initial distribution phase of a radiopharmaceutical, straightforward clearance kinetics is typically characterized by a linear decrease in a semilogarithmic plot (two-compartment model). Interestingly, this kinetic profile Figure  was onlyrecently , therefo10 and DOTAGA-conjugate 11, 68Ga- and 177Lu-12 were found to be rapidly internalized into PSMA expressing cells, to have a favourable pharmacokinetic profile in vivo and only negligible unspecific uptake with no redistribution in vivo. Based on the DOTAGA chelator and the use of a d-amino acid spacer, compound 12 possesses remarkable potential for a PSMA based theranostics concept and is a suitable lead structure for the continuing development of PSMA ligands with further improved affinity.Compared to the metabolically unstable DOTA conjugate


The crystal structures of two salts of unsymmetrical dimethyl hydrazine show different protonation patterns of the cation. 2H9N2+·Br−, (I), and 2,2-dimethylhydrazin-1-ium dihydrogen phosphite, C2H9N2+·H2PO3−, (II). In (I), the cation is protonated at the methylated N atom and N—H⋯Br hydrogen bonds generate [010] chains in the crystal. In (II), the cation is protonated at the terminal N atom and cation-to-anion N—H⋯O and anion-to-anion O—H⋯O hydrogen bonds generate (001) sheets.We describe the syntheses and crystal structures of two molecular salts containing the 1,1-dimethylhydrazinium cation, namely 1,1-dimethylhydrazin-1-ium bromide, C The best known application of this compound is the fuel (reducing agent) in hypergolic rocket fuels 2H9N2+·H2PO3− (II)As an extension of these studies, we now describe the syntheses and crystal structures of 1,1-dimethylhydrazin-1-ium bromide, CI2/a (non-standard setting of C2/c) with one cation and one bromide anion in the asymmetric unit ; C2—N2—N1—H1n = 178.0 (2)°] and the N2—H3n bond bisects the N1H2 group [H3n—N2—N1—H1n = 61 (2)°].Compound (I)it Fig. 1. The catPna21 with one cation and one dihydrogen phosphite anion in the asymmetric unit  Å is typical it Fig. 2. In this− ion accepts three N—H⋯Br bonds and alternating, centrosymmetric et al., 2001In the crystal of (I)2H9N2+ cation: the three H atoms attached to N1 each partake in a strong, near-linear N—H⋯O hydrogen bond to nearby H2PO3− anions  sheets s Table 2. The anits Fig. 5 As usualts Fig. 5, the P—Het al., 2016A search of the Cambridge Structural Database  and hydrobromic acid  were mixed at room temperature to yield a colourless solution and colourless rods (to ∼1 mm in length) of (I)To prepare (I)M) and phosphorus acid  were mixed at room temperature to yield a colourless solution and yellowish slabs of (II)−1 (P—H stretch).To prepare (II)The IR spectra of UDMH, (I)3 groups were allowed to rotate, but not to tip, to best fit the electron density. The constraint Uiso(H) = 1.2Ueq(carrier) or 1.5Ueq(methyl carrier) was applied in all cases.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989016011993/su5314sup1.cifCrystal structure: contains datablock(s) I, II, global. DOI: 10.1107/S2056989016011993/su5314Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989016011993/su5314IIsup3.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S2056989016011993/su5314Isup4.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989016011993/su5314IIsup5.cmlSupporting information file. DOI: 1495475, 1495474CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Colorectal cancer (CRC) is a leading cause of cancer death globally and new biomarkers and treatments are severely needed.t-test, as relevant.Here, we employed HCT116 and LoVo human CRC cells made resistant to either SN38 or oxaliplatin, to investigate whether altered expression of the high affinity glutamate transporters Solute Carrier (SLC)-1A1 and -1A3  is associated with the resistant phenotypes. Analyses included real-time quantitative PCR, immunoblotting and immunofluorescence analyses, radioactive tracer flux measurements, and biochemical analyses of cell viability and glutathione content. Results were evaluated using one- and two-way ANOVA and Students two-tailed 3H]-D-Aspartate uptake, consistent with increased activity of SLC1A1 (or other family members), yet not of SLC1A3. DL-TBOA co-treatment concentration-dependently augmented loss of cell viability induced by SN38, while strongly counteracting that induced by oxaliplatin, in both HCT116 and LoVo cells. This reflected neither altered expression of the oxaliplatin transporter Cu2+-transporter-1 (CTR1), nor changes in cellular reduced glutathione (GSH), although HCT116 cell resistance per se correlated with increased cellular GSH. DL-TBOA did not significantly alter cellular levels of p21, cleaved PARP-1, or phospho-Retinoblastoma protein, yet altered SLC1A1 subcellular localization, and reduced chemotherapy-induced p53 induction.In SN38-resistant HCT116 and LoVo cells, SLC1A1 expression was down-regulated ~60 % and up-regulated ~4-fold, respectively, at both mRNA and protein level, whereas SLC1A3 protein was undetectable. The changes in SLC1A1 expression were accompanied by parallel changes in DL-Threo-β-Benzyloxyaspartic acid (TBOA)-sensitive, UCPH101-insensitive [SLC1A1 expression and glutamate transporter activity are altered in SN38-resistant CRC cells. Importantly, the non-selective glutamate transporter inhibitor DL-TBOA reduces chemotherapy-induced p53 induction and augments CRC cell death induced by SN38, while attenuating that induced by oxaliplatin. These findings may point to novel treatment options in treatment-resistant CRC.The online version of this article (doi:10.1186/s12885-015-1405-8) contains supplementary material, which is available to authorized users. WAF1/Cip1, cell cycle arrest, and cell death -D-Asp following a 6-min incubation in buffer supplemented with a tracer concentration of 100 nM [3H]-D-Asp). Data are shown in Fig. 3H]-D-Asp uptake was competitively inhibited by the substrate L-glutamate, with IC50 values of 20–30 μM. To determine which transporter(s) was responsible for the [3H]-D-Asp uptake, we assessed the effect of DL-TBOA, a nonselective inhibitor of EAATs, and UCPH-101, a specific SLC1A3 inhibitor -D-Asp uptake was inhibited by DL-TBOA with IC50 values around 2 μM, whereas it was essentially unaffected by UCPH-101 at concentrations up to 100 μM. Basal [3H]-D-Asp uptake was decreased by about 60 % in SN38-resistant compared to parental HCT116 cells, whereas that in SN38-resistant LoVo cells was nearly tripled compared to parental LoVo cells. In the oxaliplatin-resistant cell lines, [3H]-D-Asp uptake was slightly decreased in the HCT116 model, and unaltered in the LoVo model.As glutamate transporter activity and membrane localization are heavily posttranslationally regulated , expressnhibitor , 28, 29.al setup , whereas3H]-D-Asp uptake are decreased in SN38-resistant HCT116 cells and increased in SN38-resistant LoVo cells, compared to their parental controls, while neither SLC1A3 protein or activity could be detected in any of the cell lines.Collectively, these data show that SLC1A1 mRNA and protein expression and DL-TBOA-sensitive, UCPH-101-insensitive -D-Asp uptake upon SN38 resistance development were quantitatively more pronounced than the changes in SLC1A1 protein level, and in oxaliplatin-resistant HCT116 cells, SLC1A1 expression was modestly increased, yet [3H]-D-Asp uptake modestly decreased. While not further addressed here, this suggests an additional role for posttranslational regulation of SLC1A1 activity, and/or, contributions from other SLC1A isoforms.The SLC1A1 protein levels paralleled its mRNA levels, whereas SLC1A3 protein expression was not detectable. Basal glutamate transporter activity largely, but not completely, paralleled SLC1A1 expression and was inhibited by L-glutamate and by the broad glutamate transporter inhibitor DL-TBOA, but not by UCPH-101, a specific SLC1A3 inhibitor. Collectively, this suggests that SLC1A1 is at least partially responsible for the observed glutamate transporter activity. Although this is, to our knowledge, the first study to demonstrate SLC1A1 protein and activity changes in drug-resistant cancer cells, altered SLC1A1 mRNA expression was also reported in ovarian cancer cells and in the NCI-60 cancer cell line panel , 15, sugl-cysteine and plays a major role in supporting GSH production [2+]i and consequent cell death in CRC cells -D-Aspartic acid ([3H]-D-Asp) was from PerkinElmer.SN38 was from Sigma-Aldrich, oxaliplatin from Sanofi-Aventis, and DL-Threo-β-Benzyloxyaspartic acid (DL-TBOA) from Tocris. UCPH101 was synthesized as described . Primary2, 37 °C, and propagated by gentle trypsination every 3–4 days. Unless otherwise specified, experiments were carried out at 37 °C.HCT116 human CRC cells originate from a primary colon carcinoma , and LoVTotal RNA was purified using the Machery-Nagel NucleoSpin® RNA II kit. cDNA was synthesized using SuperScript II RT (Invitrogen) and random primers. qPCR was carried out in triplicate in an ABI7900 Real Time PCR machine, using FastStart universal SYBR Green master mix (Roche Applied Bioscience), and 0.2 μM forward and reverse primers. Thermal profile was: 96 °C 10 min  × 40. Normalization was done to GAPDH, PBGD and β-actin for LoVo, and PBGD and β-actin for HCT116. Primers were: SLC1A1:fw:5'-GGATGTCACCCTGATCATTGC-3', rv:5'-CCAAGGACGTTGACCATGGT-3; SLC1A3: fw:5'-CGAAGCCATCATGAGACTGGTA-3',rv:5'-TCCCAGCAATCAGGAAGAGAA-3'; β-actin: fw:5'-AGCGAGCATCCCCCAAAGT-3', rv:5'-GGGCACGAAGGCTCATCAT-3'; PBGD: fw:5'-TCCAAGCGGGAGCCATGTCTG-3', rv:5'-AGAATCTTGTCCCCTGTGGTGGA-3'; GAPDH: fw:5'-GAAGGTGAAGGTCGGAGTC-3, rv:5'-GAAGATGGTGATGGGATTTC-3'. Melting curves confirmed the presence of only one amplicon. Relative expression ratios were calculated as in .3VO4), sonicated, and protein content determined . Lysates were mixed 2:1with NuPage LDS sample buffer (Invitrogen). SDS-PAGE was carried out in NOVEX chambers with NuPAGE 10 % Bis-Tris gels under reducing and denaturing conditions, using BenchMark protein ladder (Invitrogen), and 15 μg protein per lane. Proteins were transferred to PVDF membranes, which were Ponceau S stained, blocked for 1 h at 37 °C in 5 % nonfat dry milk in TBST , incubated overnight at 4 °C with primary antibodies diluted in blocking buffer, washed in TBST, incubated with secondary antibodies for 1 h, washed in TBST, and developed using BCIP/NBT (KPL). Bands were scanned and quantified using UN-SCAN-IT (Silk Scientific).Cells were grown to a confluence of 60-80 % and treated with chemotherapy, siRNA- or plasmid transfection as indicated. Cells were washed once in PBS, lysed in lysis buffer -2,5-diphenyltetrazolium bromide (MTT) in growth medium. The reaction was stopped 1.5-2.5 h later by addition of 100 μl 20 % SDS in 0.02 M HCl, and incubation overnight to allow complete lysis of cells and formazan crystals. Absorbance was measured in an ELISA plate reader (Thermo Scientific MultiScan FC) at 550 nm. Data were background subtracted and % viability relative to controls calculated.Cells were seeded at appropriate density  in growth medium in 96-well plates. Next day, cells were treated with inhibitors and/or chemotherapy in a total volume of 200 μl in growth medium and incubated for 48 h at 37 °C, 5 % COCells were seeded in 96-well plates (Greiner Bio) one day prior to experiments. Cells were treated with chemotherapy and/or inhibitors as indicated and incubated for 48 h. Medium was aspirated, and cells were washed gently in ice cold PBS, fixed in 2 % paraformaldehyde, washed in PBS, and nuclei stained by 5 min incubation with DAPI. Plates were scanned using an OPERA high-throughput microscope (PerkinElmer). Nucleus counting was performed using proprietary OPERA software, based on 100 points per well, and cell counts from non-treated cells as controls.50 nM of non-specific siRNA  or SLC1A1 siRNA  was transfected using Lipofectamine 2000, into 40-50 % confluent LoVo and HCT116 cell lines. For overexpression, cells were transfected with full-length SLC1A1 in pcDNA3.1  using 1 3H]-D-Asp uptake assay was performed essentially as in -D-Asp and test compounds as indicated was added, and the plate was incubated at 37 °C for 6 min. Non-specific [3H]-D-Asp uptake was determined in wells with 3 mM L-glutamate. The assay mixture was quickly removed, and wells were washed with 2 × 100 μl ice-cold assay buffer, followed by 150 μl Microscint™20 scintillation fluid (PerkinElmer). The plate was shaken for 1 h and counted in a Wallac 1450 MicroBeta Trilux scintillation counter .The [ly as in . Briefly4 cells per well), treated the next day with chemotherapy and/or DL-TBOA and incubated for 24 h. Medium was removed and cells were washed twice with ice-cold PBS, which was removed and 500 μl ice-cold 1 % Sulfosalicylic acid was added per well. Cells were incubated on ice for at least 10 min. After centrifugation , 10 μl lysate was used to determine total GSx content. To measure GSSG content, 130 μl sample was mixed with 55 μl 0.2 M Tris (pH 9) and 5 μl 2-Vinylpyridine. Tubes were vortexed carefully and incubated for at least 1 h at room temperature. 10 μl of this mix was mixed first with 90 μl of water in a 96-well plate and then with a reaction mix . Measurements were taken every 30 s for 10 min in an ELISA plate reader (Thermo Scientific MultiScan FC) at 405 nm absorbance. GSH values were obtained by subtraction of GSSG values from GSx values.Cells were seeded in 24-well plates  Microarray data showing the fold change in expression of SLC1A1 and SLC1A3 in SN38- and oxaliplatin (Oxa)-resistant HCT116 and LoVo cell lines compared to respective parental cell line. Data are from . (B) Theine.org; .Additional file 2: Figure S2.Nucleus counting after treatment of parental and drug-resistant HCT116 and LoVo cells with DL-TBOA. Parental and drug-resistant HCT116 and LoVo cell lines seeded in 96-well dishes were exposed to SN38 (0.1 or 0.8 μM) or oxaliplatin (0.8 or 20 μM), alone or in combination with 70 or 350 μM DL-TBOA as indicated, for 48 h. Cells were washed in PBS, fixed in 2 % paraformaldehyde and nuclei were stained with DAPI. The number of adherent cells was determined by automated counting using an OPERA confocal microscope. (A-B) Parental HCT116 cells. (C) SN38 resistant HCT116 cells. (D) Oxaliplatin-resistant HCT116 cells. (E-F) Parental LoVo cells. (G) SN38 resistant LoVo cells. (H) Oxaliplatin-resistant LoVo cells. Data are means with S.E.M. error bars of 3 independent experiments. Values are normalized to those of untreated cells.Additional file 3: Figure S3.p < 0.05, **) p < 0.01, ***) p < 0.001,****) p < 0.0001 compared to the control group without drug or TBOA treatment; #) p < 0.05 compared to controls without TBOA treatment. Two-way ANOVA with Tukey post-test.Effects of DL-TBOA on cell death and survival parameters after chemotherapy treatment of HCT116 cells. Parental and drug-resistant HCT116 cell lines seeded in 6-well dishes were exposed to SN38 (0.8 μM) or oxaliplatin (20 μM), alone or in combination with 350 μM DL-TBOA as indicated, for 24 h. Equal amounts of protein per lane were separated by SDS-PAGE and the protein levels of p21, and PARP-1  were determined by Western blotting. Top: Representative Western blots, with p150 as loading control. Bottom: Densitometric quantifications based on 3 independent experiments per condition. Data are means with S.E.M. error bars of 3 independent experiments. *) Additional file 4: Figure S4.p < 0.05, **) p < 0.01, ***) p < 0.001,****) p < 0.0001 compared to the control group without drug or TBOA treatment; Two-way ANOVA with Tukey post-test.Effects of DL-TBOA on cell death and survival parameters after chemotherapy treatment of LoVo cells. Parental and drug-resistant LoVo cell lines seeded in 6-well dishes were exposed to SN38 (0.8 μM) or oxaliplatin (20 μM), alone or in combination with 350 μM DL-TBOA as indicated, for 24 h. Equal amounts of protein per lane were separated by SDS-PAGE and the protein levels of p21, and PARP-1  were determined by Western blotting. Top: Representative Western blots, with p150 as loading control. Bottom: Densitometric quantifications based on 3 independent experiments per condition. Data are means with S.E.M. error bars of 3 independent experiments. *) Additional file 5: Figure S5.Effects of DL-TBOA on proliferation after chemotherapy treatment. Parental and drug-resistant HCT116 (A) and LoVo (B) cell lines seeded in 6-well dishes were exposed to SN38 (0.8 μM) or oxaliplatin (20 μM) alone or in combination with 350 μM DL-TBOA as indicated, for 24 h. Equal amounts of protein per lane were separated by SDS-PAGE and the protein levels of phosphorylation of retinoblastoma protein on Ser 807/811 (pRb) were determined by Western blotting. Top: Representative Western blots, with β-actin as loading control. Bottom: Densitometric quantifications based on 3 independent experiments per condition. Data are means with S.E.M. error bars of 3 independent experiments. Two-way ANOVA with Tukey post- test.Additional file 6: Figure S6.SLC1A1 overexpression has no detectable effect on cell death- and survival parameters tested. Representative blot data of 3 independent experiments of HCT116 (A) or LoVo (B) cell lines, in absence or presence of transient overexpression of wild type SLC1A1 (pSLC1A1) or corresponding empty vector (pcDNA3.1), followed by 24 h of chemotherapeutic treatment (0.8 μM SN38 or 20 μM Oxa). The protein levels of SLC1A1, p53, p21, and PARP-1  were determined by Western blotting. p150 is shown as a loading control.Additional file 7: Figure S7.Subcellular localization of SLC1A1, nuclei, and F-actin in parental and resistant CRC cells-effects of chemotherapy and DL-TBOA. (A) Immunofluorescence images of parental (PAR), SN38 resistant and oxaliplatin resistant HCT116 cells treated or not for 48 h with 350 μM DL-TBOA, and stained with antibody against SLC1A1 (green) and with DAPI (blue) and Rhodamine-conjugated phalloidin (red) to visualize localization of nuclei and F-actin, respectively. (B) Parental and SN38-resistant HCT116 cells treated for 48 h with 0.8 μM SN38 in the absence or presence of 350 μM DL-TBOA, and stained as in A. (C) Parental and oxaliplatin-resistant HCT116 cells treated for 48 h with 20 μM oxaliplatin in the absence or presence of 350 μM DL-TBOA, and stained as in A. All conditions are representative of 2 or 3 independent biological replicates in duplicate. Scale bar: 10 μm.Below is the link to the electronic supplementary material.


Compounds 1, 2, 4, 8, 15, 17, and 18 significantly inhibited TNFα-induced NF-κB transcriptional activity in HepG2 cells in a dose-dependent manner, with IC50 values ranging from 15.81 ± 2.29 to 29.10 ± 1.54 μM. Furthermore, the transcriptional inhibitory function of these compounds was confirmed by a decrease in intercellular adhesion molecule-1 and inducible nitric oxide synthase gene expression levels in HepG2 cells. These results provide a scientific rationale for the use of the soft coral S. maxima warrant further studies to develop new agents for the prevention and treatment of inflammatory.Anti-inflammatory transcriptional effects of nineteen compounds ( Activation of nuclear factor kappa B (NF-κB) represents a family of Rel domain-containing proteins including five NF-κB units that can form 15 transcription factors through homo- and heterodimerization Baldwin . NF-κB pSinularia is one of the most widely distributed soft coral genera, constituting a dominant portion of the biomass in the tropical reef environment , sinumaximol B (2), sinumaximol C (3), sethukarailin (4), sinumaximol D (5), sinumaximol E (6), sinumaximol F (7), sinumaximol G (8), sinumaximol H (9), -2,6,12(20)-cembratriene-4,8,11-triol (10), isomandapamate (11), and sinumaximol I (12)] , 12-hydroxy-scabrolide A (14), yonarolide (15), ineleganolide (16), 5-epinorcembrene (17), 13-epi-scabrolide C (18), and norcembrene 5 (19)] . A voucher specimen (SM112010_01) was deposited at the Institute of Marine Biochemistry and Institute of Marine Environment and Resources, VAST.The sample of soft coral 2. Human TNFα was purchased from ATgen . Cells were counted with a hemocytometer, and the number of viable cells was determined through trypan blue dye exclusion.Human hepatocarcinoma HepG2 cells were maintained in Dulbecco’s modified Eagle’s medium  containing 10 % heat-inactivated fetal bovine serum, 100 units/mL penicillin, and 10 μg/mL streptomycin at 37 °C and 5 % COH-tetrazolium, inner salt (MTS) assay  was performed to analyze the effect of the different compounds on cell viability. Cells were cultured overnight in 96-well plates (1 × 104 cells per well). Cell viability was assessed after the incubation with the compounds at a concentration of 10 μM for 24 h. The number of viable cells was determined by measuring the absorbance at 490 nm of the dissolved formazan product after addition of MTS for 30 min as described by the manufacturer.A 3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-22+ and an excess of ATP. Under these conditions, luciferase enzymes expressed by the reporter vector could catalyze the oxidative carboxylation of luciferin. Cells were seeded at 1.5 × 105 cells per well in a 12-well plate and grown for 24 h. All cells were transfected using Lipofectamine™ LTX (Invitrogen) according to the manufacturer’s protocol. Luciferase (Luc) activity was assayed using an LB 953 Autolumat  as described previously  . Approximately 2 μg of total RNA was reverse-transcribed using Moloney murine leukemia virus reverse transcriptase and oligo (dT) primers (Promega) for 1 h at 42 °C. The resulting cDNA was polymerase chain reaction-amplified using Taq polymerase premixture . Polymerase chain reaction products were subjected to electrophoresis on 1 % agarose gels and stained with ethidium bromide. Polymerase chain reaction was conducted with the following primer pairs: iNOS sense 5′-TCATCCGCTATGCTGGCTAC-3′, iNOS antisense 5′-CTCAGGGTCACGGCCATTG-3′, ICAM-1 sense 5′-GCCCAGCACTTCACGCATCAG-3′, ICAM-1 antisense 5′-GACCAGGCACCAGACCAAAGACC-3′, glyceraldehyde 3-phosphate dehydrogenase sense 5′-TGTTGCCATCAATGACCCCTT-3′, and glyceraldehyde 3-phosphate dehydrogenase antisense 5′CTCCACGACGTACTCAGCG-3′. β-actin sense 5′-TCACCCACACTGTGCCCATCTACG-3′, and β-actin antisense 5′-CAGCGGAACCGCTCATTGCCAATG-3′.P < 0.5 was considered to be significant.All results are expressed as mean±SD values. Data were analyzed by one-factor analysis of variance. Quantification of polymerase chain reaction products was performed using Image Lab™ software (Bio-Rad). If a statistically significant effect was found, the Newman–Keuls test was performed to isolate the difference between the groups. 1–19, they were applied at various concentrations to HepG2 cells for 24 h, after which cell viability was measured in an MTS assay as described in Materials and methods. None of the compounds displayed any cellular toxicity at the concentration of 10 μM (data not shown). They were therefore used in subsequent experiments for further evaluation of their effects on NF-κB activation, iNOS and ICAM-1 expressions at concentrations of 0.1, 1.0 and 10 μM  values ranging from 15.81 ± 2.29 to 29.10 ± 1.54 μM. Other compounds exhibited moderate, weak or not determined activities (Table To evaluate the anti-inflammatory activity of nineteen compounds listed above, we first examined their inhibitory effects on NF-κB transcriptional activation in HepG2 cells (see Figs. NF-κB was first described in 1986 as a nuclear transcription factor required for immunoglobulin kappa light chain transcription in B-cells. Since its discovery, it has been demonstrated that NF-κB is constitutively expressed in all cell types and plays a central role as a regulator of the cellular stress response. The NF-κB-mediated signaling pathway has been considered both pro-inflammatory and anti-apoptotic in character, and therefore, has been implicated in the pathogenesis of a wide variety of diseases, including inflammatory disorders and tumor development (Robinson and Mann S. maxima on NF-κB transcriptional activation has not been evaluated. In this study, the effects of compounds 1–19 on TNFα-induced NF-κB transcriptional activity in HepG2 cells were evaluated using a NF-κB luciferase assay. To confirm their inhibitory effects of the compounds on NF-κB transcriptional activity, the effects of the isolated compounds on the upregulation of the pro-inflammatory proteins iNOS and ICAM-1 were assessed in TNFα-stimulated HepG2 cells by RT-PCR.To date, the inhibition of components from 1, 2, 4, 8, 15, 17, and 18 significantly inhibited TNFα-induced NF-κB transcriptional activation in a dose-dependent manner with IC50 values of 21.35 ± 3.21, 29.10 ± 1.54, 25.81 ± 1.38, 15.81 ± 2.29, 25.1 ± 2.58, 28.19 ± 2.65, 20.13 ± 0.29 μM, respectively (see Figs. 3, 10–14, and 19 exhibited moderate or weak inhibitory effects with IC50 values ranging from 40.42 ± 2.11 to 325.71 ± 4.77 μM, whereas other compounds were inactive at the indicated concentrations, compared with the positive control (see Table HepG2 cells were first transfected with NF-κB luciferase reporter plasmids. After treatment with 10 ng/mL TNF-α, luciferase activity increased fivefold, demonstrating an increase in transcriptional activity compared to untreated cells. Compounds were pretreated with transfected HepG2 cells at various concentrations, followed by stimulation with TNFα. The results showed that compounds NF-κB activation is known to be involved in the upregulation of inflammatory NF-κB target gene expression, including iNOS and ICAM-1, which play important roles in the inflammatory response. iNOS is highly expressed in macrophages, leading to organ destruction in some inflammatory and autoimmune diseases (Kleinert et al. 1, 2, 4, 8, 15, 17, and 18; we further investigated their effects on ICAM-1 and iNOS gene expression in TNFα-stimulated HepG2 cells using RT-PCR. Consistent with their inhibitory activity toward NF-κB, compounds 1, 2, 4, 8, 15, 17, and 18 significantly inhibited the induction of ICAM-1 and iNOS mRNA in a dose-dependent manner (see Fig. 1, 2, 4, 8, 15, 17, and 18 at the same concentration (see Fig. 1, 2, 4, 8, 15, 17, and 18 isolated from the soft coral S. maxima have therapeutic potential as anti-inflammatory, anti-atherosclerotic, and anti-arthritic substances. However, elucidation of the detailed mechanisms underlying the inhibition of the TNFα-induced NF-κB pathway and subsequent decreases in ICAM-1 and iNOS gene expression by compounds 1, 2, 4, 8, 15, 17, and 18 requires further investigation.To confirm the transcriptional inhibitory function of compounds 1, 2, 4, 8, 15, 17, and 18, therefore this information may facilitate identification of anti-inflammatory lead compounds from diterpenoids and norditerpenoids. This primary finding provides support for further studies on these compounds for the development of anti-inflammatory agents.Consideration of the structure–activity relationship of these compounds indicated that the ketone groups at C-3 and/or C-6, the hydroxyl group at C-7 and/or C-8 are necessary for the anti-inflammatory activity of diterpenoids and norditerpenoids. This finding was confirmed by comparing the structure and activity of compounds


Colorectal cancer is strongly associated with lipid metabolism. NPC1L1, a sterol transporter, plays a key role in modulating lipid homeostasis in vivo. Its inhibitor, ezetimibe, began to be used clinically to lower cholesterol and this caused the great debate on its role in causing carcinogenesis. Here we explored the role of NPC1L1 in colorectal tumorigenesis.−/− (NPC1L1 knockout) mice were treated with azoxymethane (AOM)-dextran sodium sulfate (DSS) to induce colitis-associated colorectal tumorigenesis. Mice were sacrificed 10, 15, 18 or 20 weeks after AOM treatment, respectively. Colorectal tumors were counted and analyzed. Plasma lipid concentrations were measured using enzymatic reagent kit. Protein expression level was assayed by western blot.Wild-type mice and NPC1L1−/− mice significantly had fewer tumors than wild-type. The ratio of malignant/tumor in NPC1L1−/− mice was significantly lower than in wild-type 20 weeks after AOM-DSS treatment. NPC1L1 was highly expressed in the small intestine of wild-type mice but its expression was undetectable in colorectal mucous membranes or tumors in either group. NPC1L1 knockout decreased plasma total cholesterol and phospholipid. NPC1L1−/− mice had significant lower intestinal inflammation scores and expressed inflammatory markers p-c-Jun, p-ERK and Caspase-1 p20 lower than wild-type. NPC1L1 knockout also reduced lymphadenectasis what may be caused by inflammation. NPC1L1 knockout in mice decreased β-catenin in tumors and regulated TGF-β and p-gp in adjacent colons or tumors. There was not detectable change of p53 by NPC1L1 knockout.NPC1L1Our results provide the first evidence that NPC1L1 knockout protects against colitis-associated tumorigenesis. NPC1L1 knockout decreasing plasma lipid, especially cholesterol, to reduce inflammation and decreasing β-catenin, p-c-Jun and p-ERK may be involved in the mechanism.The online version of this article (doi:10.1186/s12885-015-1230-0) contains supplementary material, which is available to authorized users. Cancer is a major public health problem worldwide, causing 1 in 4 deaths in the United States [Evidence is accumulating that lipid metabolism is strongly associated with cancer, including CRC -4. AboutNiemann-Pick C1 like 1 (NPC1L1) has been proven to be one of the most important sterol transporters [NPC1L1 genotype was found to be significantly associated with plasma lipid concentration, especially TC and LDL-C [Serum lipid is modulated by many factors, including some genes, such as leptin, adipose triglyceride lipase and so on. Among these genes, sporters . It was sporters -8. In clnd LDL-C . The NPCnd LDL-C . Dr. Petnd LDL-C .Here, we explored the role of NPC1L1 in colorectal tumorigenesis in vivo by using transgenic mice. Our results testify that NPC1L1 knockout in mice protects against colitis-associated tumorigenesis.−/− mice were described previously [−/− mice were derived from NPC1L1+/− mice with a pure C57BL/6 background and housed in a specific pathogen-free animal facility in plastic cages in a temperature controlled room (22°C) with a daylight cycle from 6 AM to 6 PM. Mice had free access to a standard laboratory rodent chow diet  and water. All animal procedures were approved by the Institutional Animal Care and Use Committee at University of Maryland Health Sciences.NPC1L1eviously ,9. Wild-AOM-DSS induced colorectal tumorigenesis was described previously ,11. Six Mice were sacrificed at different time points and colorectums were excised. Colorectums were opened longitudinally, flushed with ice-cold PBS and fixed in 10% formalin/PBS. Then macroscopic tumors were counted and measured with a caliper. Enlarged abdominal  lymph nodes were counted, collected and fixed in 10% formalin/PBS.10% formalin/PBS fixed colorectums and enlarged lymph nodes were sent to Histoserv INC.  for paraffin embedding and hematoxylin and eosin (H&E) staining. In brief, paraffin embedded samples were cut into 6 μM and stained with H&E. A pathologist counted the malignant tumors  and benign tumors in each slide. The malignant/tumor ratio of each slide was calculated and the average ratio of each group was presented. Inflammation was scored on a 0–4 scale ; 2, mild inflammation ; 3, moderate inflammation (scattered granulocytes with patchy moderate infiltrates); and 4, severe inflammation (multiple extensive areas with abundant granulocytes and marked infiltrates)).Mice were sacrificed with isoflurane. Blood was collected by inferior vena cava injection in an anticoagulant tube and the tube was centrifuged at 800 g for 15 min at 4°C. Supernatant was collected as plasma. Plasma free cholesterol (FC), PL were measured using enzymatic reagent kit from Wako Diagnostics (USA), TC was measured using enzymatic reagent kit from Pointe Scientific (USA). TG was measured using enzymatic reagent kit from Sigma-Aldrich (USA).3VO4, 10 mM β-Glycerophosphate disodium salt pentahydrate) were homogenized for 60 seconds on ice. Tissue homogenates were centrifuged at 13,000 rpm for 20 minutes at 4°C and the supernatant was collected as total cell protein.To obtain total protein, multiple tissues in RIPA Buffer  with protease inhibitors  and phosphatase inhibitors  with protease inhibitors and phosphatase inhibitors were homogenized for 60 seconds on ice. Tissue homogenates were centrifuged at 2,000 rpm for 10 minutes at 4°C. The supernatant was transferred to a new tube and the tube was centrifuged at 110,000 g for 30 min at 4°C. The supernatant was removed and left the tube upside down to drain for 1 minute. Then, the pellet was resuspended in Sample Buffer  Triton X-100) with protease inhibitors and phosphatase inhibitors. This was membrane protein.To obtain membrane protein, colon mucous membrane or colorectal tumors in Membrane Buffer  as previously described. NitroceE-cadherin antibody, β-catenin antibody, TGF-β antibody, p-c-Jun (Ser63) antibody, p-Erk1/2 (Thr202/Tyr204) antibody, β-actin antibody, α-tubulin antibody, HRP-linked secondary antibody were from Cell signal Technology (USA). Caspase-1 p20 antibody, p-gp antibody, p53 antibody was from Santa Cruz Biotechnology, Inc. (USA).P < 0.05 was considered statistically significant.The data shown represent the mean ± standard error. Statistical differences between groups were analyzed by Student’s t-test, one-way ANOVA or chi-square test. To explore colorectal tumorigenesis in vivo, the widely used AOM-DSS model of colitis-associated colorectal tumorigenesis was employed ,11. MiceThe liver, lung, spleen and kidneys of each mouse were examined and neither tumor nor metastatic cancer was found  mice were administrated with AOM-DSS. NPC1L1−/− mice significantly had fewer tumors than WT mice  without any treatment were sacrificed and did not show tumors in the colorectum, cecum, small intestine, lung, liver, spleen or kidneys. This suggests NPC1L1 knockout alone should not cause tumorigenesis in those organs within 24 months.Mice at 12 weeks and 24 months of age  . At week 10, plasma TC concentration of NPC1L1−/− group (1047 ± 75 ug/ml) was significantly lower than that of WT group (1377 ± 68 ug/ml) (p < 0.01). At week 20, NPC1L1−/− mice had a lower concentration of TC (1113 ± 59 ug/ml) compared to WT mice (1239 ± 91 ug/ml), but without significance (p > 0.05).Plasma TC concentration of NPC1L1−/− mice had a lower FC than WT mice only at week 10, where there was a significant difference (p < 0.05). Plasma FC concentrations of NPC1L1−/− mice without treatment, at week 10 and week 20 were 416 ± 41 ug/ml, 359 ± 35 ug/ml and 373 ± 19 ug/ml, respectively. FC concentrations of WT mice were 534 ± 35 ug/ml, 547 ± 59 ug/ml and 422 ± 39 ug/ml, respectively.NPC1L1−/− mice and mice treated at week 20 had a similar TG to WT mice (p > 0.05). TG concentrations of WT mice without treatment, at week 20 were 1123 ± 79 ug/ml and 921 ± 49 ug/ml, respectively. TG concentrations of NPC1L1−/− mice were 976 ± 69 ug/ml and 932 ± 64 ug/ml, respectively. At week 10, NPC1L1−/− mice had a lower TG (799 ± 48 ug/ml) than WT mice (1210 ± 95 ug/ml) (p < 0.01).Untreated NPC1L1−/− mice had lower plasma PL than WT mice. The plasma PL concentration of NPC1L1−/− group without any treatment was 3379 ± 322 ug/ml while that of WT mice was 4892 ± 294 ug/ml (p < 0.05). At week 10, plasma PL concentration of NPC1L1−/− group was 3798 ± 163 ug/ml while plasma PL concentration of WT group was 4801 ± 243 ug/ml (p < 0.01). At week 20, plasma PL concentration of NPC1L1−/− group was 4081 ± 116 ug/ml while that of WT group was 4380 ± 327 ug/ml (p > 0.05).NPC1L1−/−, respectively (P < 0.05)  Figure C.Protein p-c-Jun, p-ERK and Caspase-1 are thought as inflammatory markers and are involved in carcinogenesis -22. Ther−/− mice . At week 20, all WT mice had enlarged lymph node and 9 of the 10 mice had more than one while 8 of the 10 NPC1L1−/− mice had enlarged lymph nodes and only 2 mice had more than one. The WT group had 2.6 ± 0.3 enlarged lymph nodes while the NPC1L1−/− group only had 1.0 ± 0.2 (p < 0.01).Inflammation and cancer metastasis are two main causes in lymphadenectasis ,24. In tTo explore the possible involved pathways, western blot was employed to assay β-catenin/p53/TGF-β/p-gp. The specific protein expression level in adjacent colons and tumors was measured on the same membrane.β-catenin was much higher in tumors than in adjacent colons in both groups. NPC1L1 knockout significantly decreased β-catenin expression level in tumors but no changes were detected in adjacent colons Figures A,B.FigurNPC1L1 knockout in mice didn’t detectably change p53 expression in tumors or adjacent colons Figures A,B.Interestingly, NPC1L1 knockout dramatically increased active TGF-β in tumors but dramatically decreased it in adjacent colons. On the contrary, NPC1L1 knockout decreased TGF-β precursor in tumors and increased it in adjacent colons Figures  C, D.NPC1L1 knockout in mice dramatically increased p-gp in tumors and the opposite in adjacent colons Figures C,D.NPC1L1 protein, a protein of more than 1,300 amino acids, has 13 transmembrane domains and 5 of them constitute a sterol-sensing domain . Its maj−/− mice were resistant to colitis-associated tumorigenesis. At different time points, NPC1L1−/− mice consistently had much fewer colorectal tumors than WT mice (Figures −/− at week 20 was also significantly lower than in WT (Figure Here, we testified that NPC1L1To explore clues to possible mechanisms of NPC1L1 knockout protecting mice against colitis-associated tumorigenesis, NPC1L1 protein in colorectal mucous membranes and tumors was assayed. Western blot showed that NPC1L1 protein level was high in small intestines while it was undetectable in colorectal mucous membranes or colorectal tumors Figure A. It is NPC1L1 major function is a sterol transporter in small intestines and livers to modulate lipid homeostasis ,15. PlasLipid, especially cholesterol, promoted colitis-associated tumorigenesis through inflammation . To expl−/− mice having fewer enlarged lymph nodes (Figure −/− mice may be hyposensitive to inflammatory agent.Lymphadenectasis also indicates inflammatory activity, somehow, because inflammation and cancer are main causes of lymphadenectasis ,23,24. HTo explore clues to the detail mechanisms of NPC1L1 knockout reducing colitis-associated tumorigenesis, β-catenin, p-c-Jun, p-ERK, TGF-β, p-gp and p53 were assayed.NPC1L1 knockout significantly decreased β-catenin, p-c-Jun and p-ERK in tumors. Increasing β-catenin, c-Jun phosphorylation and ERK activation were reported to promote colon tumorigenesis ,11,21,22TGF-β and p-gp also play a positive role during intestinal tumorigenesis ,27. InteProtein p53, the most famous tumor suppressor protein, is involved in colorectal tumorigenesis and lipid metabolism ,29. BeyoNPC1L1 genotype is different amongst people [NPC1L1 knockout in mice protects mice against colitis-associate colorectal tumorigenesis. This indicates NPC1L1 inhibitor might be used to protect against CRC as well as to lower cholesterol. Since t people , our rest people . Therefo


Empedobacter (formerly Wautersiella) falsenii comb. nov. strain Wf282 was isolated from a cervical neck abscess sample from an 18-year-old female patient. The isolate was resistant to many antibiotics, including meropenem and colistin. The total DNA from the multidrug-resistant E. falsenii comb. nov. Wf282 clinical isolate was sequenced. Empedobacter (formerly Wautersiella) falsenii comb. nov. was first described in 2006 and renamed in 2014  , with a score of 2.074 (probable species-level identification). Antibiotic susceptibility testing was performed using the Vitek 2 system, employing the panel AST-079 (Gram-negative susceptibility [GNS] card). The MIC results were interpreted using the CLSI breakpoints , ampicillin-sulbactam (≥32 µg/ml), cephalothin (≥32 µg/ml), meropenem (≥16 µg/ml), and colistin (8 µg/ml); intermediate to piperacillin-tazobactam (64 µg/ml), cefotaxime (16 µg/ml), ceftazidime (16 µg/ml), imipenem (8 µg/ml), and ciprofloxacin (2 µg/ml); and susceptible to cefepime (4 µg/ml), amikacin (8 µg/ml), gentamicin (2 µg/ml), and trimethoprim-sulfamethoxazole (2 µg/ml).http://denovoassembler.sourceforge.net). A total of 1,076,192 high-quality paired-end reads were produced, with an average insertion size of 825 bp. De novo assembly was performed with the SPAdes assembler version 3.1.0 (k-mer coverage of 29.9). The corrected reads showed an average length of 230 bp. The assembled contigs sum 3,738,626 base pairs, with an N50 contig size of 113,944  and a G+C content of 32.24%.Whole-genome shotgun sequencing was performed using Illumina MiSeq-I, using Nextera XT libraries for sample preparation. The reads were assembled with the Ray assembler  might lead us to find novel antibiotic resistance traits that may explain its phenotype, as well as its potential role as a reservoir of antibiotic resistance genes.JSYQ00000000. The version described in this paper is version JSYQ01000000.This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession no.