The relative expression of ELF3, chondrocyte degeneration-related molecules, matrix metalloproteinase, and inflammatory molecules were detected in chondrocytes stimulated with interleukin (IL)-1β or co-incubated with SMSC-212-5p-Exos or SMSCs-derived exosomes (SMSC-Exos). IL-1β induced up-regulation of ELF3, down-regulation of degeneration molecules , up-regulation of matrix metalloproteinase , and up-regulation of inflammatory molecules  could be inhibited by SMSC-212-5p-Exos or SMSC-Exos administration. When compared with the SMSC-Exos, SMSC-212-5p-Exos showed more treatment benefits. All of these indicate that SMSC-212-5p-Exos could suppress chondrocyte degeneration and inflammation by targeting ELF3, which can be considered as a disease-modifying strategy.Excessive chondrocyte degeneration and inflammation are the pathological features of osteoarthritis (OA), and altered miR-212-5p may contribute to meniscus and cartilage degeneration. Whether exosomes derived from miR-212-5p overexpressed synovial mesenchymal stem cells (SMSC-212-5p-Exos) could be utilized to treat degenerative chondrocytes is investigated in this study. Down-regulated miR-212-5p and up-regulated E74 Like ETS Transcription Factor 3 (ELF3) expression were detected in OA synovial tissues, which showed a negative correlation ( Osteoarthritis (OA) is a disease that comprises progressive cartilage degenerative and synovial membrane inflammation, having an increased frequency of osteophyte formation and subchondral bone sclerosis in the aging population . No curaExosomes are small size (30–100 nm), single-membrane organelles released by various cells to shunt loading bioactive molecules to target cells . ResearcMicroRNAs (miRNAs) can post-transcriptionally regulate OA-associated genes in chondrocytes . ExosomaBased on the criteria of the American College of Rheumatology, thirty OA patients  underwent total knee arthroplasty were enrolled. Meanwhile, the synovial tissues were collected from OA patients and 20 donors with accidental deaths (excluding those with OA-related diseases) or normal knee-joint synovium who were subjected to lower limb amputation after acute trauma or open reduction and internal fixation after fracturing of the tibial plateau . The study was approved by the Xijing Hospital, and informed written consent was derived from each participant or close relatives.® easyCyte™ flow cytometer . At passage 3, SMSCs were switched to osteogenic differentiation medium  for 2 weeks or StemPro Adipogenesis Differentiation Kit (Gibco) for 4 weeks. The miR-212-5p mimic or mimic-negative control (NC) (Sigma-Aldrich) were transfected with Lipofectamine® 3,000 (Thermo Fisher) at the concentration of 100 nM according to the manufacturer’s instructions. The following sequences were used: miR-212-5p mimic , mimic-negative control miRNA .Synovial membrane specimens were digested with 0.2% type I collagenase (Thermo Fisher) at 37°C overnight, which were further collected by centrifugation and seeded in a high-glucose DMEM medium  supplemented with 10% FBS for 4 days to allow cell attachment. The medium was refreshed every 3 days, and at day 14, SMSCs were obtained. After blocking with human BD Fc Block™, SMSCs were stained with the following antibodies (Becton Dickinson): anti-CD34, anti-CD44, anti-CD45, anti-Sca-1, and anti-CD105 antibodies to confirm the phenotype using GuavaConditioned medium of SMSCs or miR-212-5p overexpressing SMSCs was filtered with 0.22 μM filters (Merck-Millipore) and centrifuged  to concentrate the volume into approximately 200 μL, which was further ultracentrifuged  to obtain the exosomal particles. A transmission electron microscope (TEM) was utilized to observe the morphology of exosomes, and the size and distribution of exosomes were measured using dynamic light scattering (DLS) analysis.7) were seeded into the 6-well plates 1 day before treatment. When cell confluence reached 80%, 2 µg exosomes (EXO miR-NC and EXO-miR-212-5p) were introduced into the chondrocytes culture medium supplemented with 10 ng/ml IL-1β. Chondrocytes treated with PBS were regarded as the blank controls. After 48 h of treatment, cells were collected for subsequent use.Primary chondrocytes were detached from cartilages dissected from the subchondral bone with 0.25 mg/ml collagenase P and 4 mg/ml protease, and the isolated chondrocytes  was applied to detect the amplification. The primers were listed as following: ELF3, forward 5′- CATGACCTACGAGAAGCTGAGC-3′, reverse 5′- GACTCTGGAGAACCTCTTCCTC-3’; miR-212-5p, forward 5′- CAGTCTCCAGTCACGG-3′, reverse 5′- GAACATGTCTGCGTATCTC-3’; Collagen II (COL2A1), forward 5′- CCTGGCAAAGATGGTGAGACAG-3′, reverse 5′- CCTGGTTTTCCACCTTCACCTG-3’; Aggrecan, forward 5′-CAGGCTATGAGCAGTGTGATGC-3′, reverse 5′- GCTGCTGTCTTTGTCACCCACA-3’; Sox9, forward 5′- AGGAAGCTCGCGGACCAGTAC-3′, reverse 5′- GGTGGTCCTTCTTGTGCTGCAC-3’; MMP-1, forward 5′- ATGAAGCAGCCCAGATGTGGAG -3′, 5′- TGGTCCACATCTGCTCTTGGCA -3’; MMP-3, forward 5′- CACTCACAGACCTGACTCGGTT-3′, reverse 5′- AAGCAGGATCACAGTTGGCTGG-3’; MMP-13, forward 5′- CCTTGATGCCATTACCAGTCTCC-3′, reverse 5′- AAACAGCTCCGCATCAACCTGC-3’; IL-6, forward 5′ AGACAGCCACTCACCTCTTCAG-3′, reverse 5′- TTCTGCCAGTGCCTCTTTGCTG-3; COX-2, forward 5′- CGGTGAAACTCTGGCTAGACAG-3′, reverse 5′- GCAAACCGTAGATGCTCAGGGA-3’; iNOS, forward 5′- GCTCTACACCTCCAATGTGACC-3′, reverse 5′- CTGCCGAGATTTGAGCCTCATG-3’; GAPDH, forward 5′- CTGTGCCGTTGAATTTGCCG-3′, forward 5′- CGGGTTCCTATAAATACGGACTG-3’.TRIzol (Invitrogen) was utilized to extract total RNA from chondrocytes or SMSCs. Total Exosome RNA & Protein Isolation Kit  was used to extract RNA and protein from exosomes for further analysis. TaqMan™ Advanced miRNA cDNA Synthesis Kit (Thermofisher) was utilized to reverse-transcript miRNA into cDNA, and RNU6B was utilized as an internal reference. TransscriptThe QuikChange Lightning Site-Directed Mutagenesis kit  was utilized to mutate the binding site of ELF3 with miR-212-5p. 3′-UTR fragment of ELF3 mRNA (mutant or wide type) was subcloned into pGL3 luciferase vector , which was further co-transfected with miR-212-5p mimic or mimic-NC into HEK-293T cells for 36 h with Lipofectamine 3,000. Subsequently, the luciferase activity was assayed with Luciferase Assay System (Promega).The cellular lysates were quantified using the BCA protein concentration kit , and 20 µg lysates were separated with 10% SDS-PAGE electrophoresis and transferred to PVDF membranes, which were further blocked in Tris-buffered saline with Tween 20 (TBST) containing 0.1% Tween 20 and 5% skimmed milk powder and followed by incubation with the primary antibodies  specific for CD63, CD9, Alix, ELF3, and GAPDH, and then incubated in peroxidase-conjugated secondary antibody (Sigma-Aldrich) at room temperature for 1 h. The dilution used for the primary antibodies against CD63, CD9, Alix, and ELF3 was 1:1,000, the dilution used for the primary antibody against GAPDH was 1:2000, and the dilution used for the second antibody was 1:2000. The signal was developed with an ECL system , and the relative intensity was normalized with GAPDH expression with NIH-Image J1.51p 22.According to the manufacturer’s instructions, the concentrations of IL-6, MCP-1, MMP-3, MMP-13, and TNF-α were detected with commercial ELISA kits . Separated stock standard solutions and samples were measured with a SpectraMax M5 microplate reader at a wavelength of 450 nm.p-value < 0.05 was considered statistically significant.Statistical analysis were performed with Graph-Pad Prism 6.0. Data were presented as means ± SD. The Mann-Whitney test was performed to determine the statistical significance between any two groups, and Dunn’s multiple comparisons test was used to compare multiple groups. p < 0.001), and up-regulated ELF3 mRNA expression (p < 0.001) and increased protein expression of IL-1β (p < 0.001) were also observed. The expression correlation analysis indicated that both the correlation between ELF3 and miR-212-5p  and the correlation between IL-1β and miR-212-5p  were significantly negatively correlated. As expected, the correlation between ELF3 and IL-1β was positively correlated .The relative miR-212-5p expression in OA synovial tissues was down-regulated compared with healthy control (Spindle-like SMSCs were observed at passage 3 (P3) , which cTargetScan  and miRBUp-regulated miR-212-5p expression could be detected in SMSC-212-5p-Exos , indicatThe relative mRNA expression of Collagen II , AggrecaThe relative content of IL-6 , MCP1 F, and TNFIn addition to inflammation, dysregulation between the synthesis and degradation of extracellular matrix mainly mediated by type-II collagen, aggrecan, and matrix metalloproteinases contributes to the articular cartilage degradation and loss process . In thismiR-132/212 clusters are maintained on down-regulation in mesenchymal stem cells at four different stages of transforming growth factor-β3-induced chondrogenesis differentiation . On the As transcription factors belonging to the ETS family, ELF3 participates in autoimmune and tumor neogenesis . The prein vivo models should be constructed to demonstrate the therapeutic benefit of SMSC-212-5p-Exos. Whether other molecules, except miR-212-5p, could contribute to the treatment advantage in SMSC-212-5p-Exos compared with SMSC-Exos need further detailed analysis.ELF3 is identified to direct target miR-212 to suppress nasopharyngeal carcinoma cells proliferation . In thisin vitro model is utilized in the study, and the OA in vivo model should be constructed to demonstrate the treatment benefit of SMSC-212-5p-Exos. All in all, this study indicates that SMSC-212-5p-Exos could target ELF3-mediated OA pathology in chondrocytes, which can be considered as a future treatment option.Accumulating investigations have indicated that exosomes will pave a new therapeutic paradigm for osteoarthritis treatment. Our study testifies that SMSC-212-5p-Exos could alleviate IL-1β induced chondrocytes degradation, degradation, and inflammation process involved in OA. It is worth noting that only


The molecule of the title compound contains an essentially planar indole ring system and a phenyl ring. In the crystal, the molecules are linked by a weak intermolecular C—H⋯O hydrogen bond and C—H⋯π interactions, forming a two-dimensional network structure. 16H14N2O, contains an essentially planar indole ring system and a phenyl ring. In the crystal, the molecules are linked by a weak intermolecular C—H⋯O hydrogen bond and C—H⋯π interactions, forming a one-dimensional column structure along the b-axis direction. These columns are linked by other C—H⋯π interactions, forming a two-dimensional network structure.The molecule of the title compound, C Similar Schiff bases such as salicylideneamines often function as bidentate ligands  Å. The bond lengths and angles in the title molecule are normal and agree with those in other indole imine compounds .The molecular structure of the title compound is shown in Fig. 1cis-conformation of the C—C single bond between the N atoms often form dimers by intermolecular N—H⋯N hydrogen bonds (see Database survey). However, in the crystal the molecules of the title compound are linked by a weak intermolecular C10—H10⋯O1i hydrogen bond and C—H⋯π interactions , forming columns along the b-axis direction  x, −y, z] methanimine skeleton gave 86 hits. Among these, the imino N atom bonded to an H atom gave one hit, to an N atom gave 24 hits, and to a C atom gave 61 hits. A search for the indol-2-ylmethylidene-aniline fragment gave 30 hits, and those containing a (1H-indol-2-yl)methylidene-aniline fragment with a cis-conformation of the C—C single bond gave seven hits. These seven compounds include five examples of dimers linked by complementary N—H⋯N hydrogen bonds -1-(1H-indol-2-yl)methanimine -3-phenylaminoindole  were dissolved in toluene (20 mL), and the solution was refluxed under inert gas for 6 h, followed by evaporation. The residue was purified by recrystallization from a solvent mixture of acetone and n-hexane (1:1), and the title compound was then obtained  as a pale-red powder. The recrystallization of the title compound from a mixture of acetone and methanol afforded single crystals suitable for X-ray structure analysis. 1H NMR  δ = 3.84 , 6.93–6.97 , 7.13 , 7.25–7.31 , 7.40 , 7.66 , 8.48 , 9.25 . HR–MS (m/z): calculated for [C16H15N2O]+, m/z = 251.1179; found, 251.1192.Indole-2-carbaldehyde  and Uiso(H) = 1.2Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989022002973/yk2166sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2056989022002973/yk2166Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989022002973/yk2166Isup3.cmlSupporting information file. DOI: 2159620CCDC reference:  crystallographicinformation; 3D view; checkCIF reportAdditional supporting information:


E)-1-(4-Methoxyphenyl)-3-[4-(prop-2-ynyloxy)phenyl]prop-2-en-1-one undergoes a cyclocondensation reaction with thiosemicarbazide to form the corresponding 4,5-dihydropyrazole-1-arbothioamide, which in turn undergoes further cycloaddition with phenacyl bromides to form 4,5-dihydro-1-(thiazol-2-yl)pyrazoles. The molecules adopt an overall T-shape structure. Different combinations of hydrogen bonds link the molecules into ribbons or sheets.( E)-1-(4-methoxyphenyl)-3-[4-(prop-2-ynyloxy)phenyl]prop-2-en-1-one, C19H16O3 (I), are linked by a combination of C—H⋯O and C—H⋯π(arene) hydrogen bonds to form ribbons. The products (RS)-5-(4-methoxyphenyl)-1-(4-phenythiazol-2-yl)-3-[4-(prop-2-ynyloxy)phenyl]-4,5-dihydro-1H-pyrazole, C28H23N3O2S (II), and (RS)-5-(4-methoxyphenyl)-1-[4-(4-methylphenyl)thiazol-2-yl]-3-[4-(prop-2-ynyloxy)phenyl]-4,5-dihydro-1H-pyrazole, C29H25N3O2S (III), are closely related – differing only by presence or absence of a methyl group at the arylthiazolyl substituent – and crystallize in an isomorphous setting. Both molecules contain an effectively planar dihydro-pyrazole ring, and possess an overall T-shaped structure, which is a characteristic of triaryl-substituted 4,5-dihydro-1-(thiazol-2-yl)pyrazole compounds. The crystal packing is characterized by intermolecular C—H⋯S and C—H⋯π  interactions. A combination of two C—H⋯π(arene) hydrogen bonds links the product molecules into sheets.Two new 1-(thiazol-2-yl)-4,5-dihydropyrazoles have been synthesized from simple precursors, and characterized both spectroscopically and structurally. In addition, two intermediates in the reaction pathway have been isolated and characterized, one of them structurally. The molecules of the intermediate ( N-heterocyclic compounds with a wide spectrum of biological activities including antibacterial  and a substituted acetophenone (B) yields the chalcone intermediate (I)et al., 2020C), which in turn undergoes a further cyclocondensation reaction with a phenacyl bromide to give the thiazolyl-dihydropyrazoles (II)et al., 2019Accordingly, we have sought to combine pyrazole and thiazole pharmacophores in a single molecular skeleton and synthesized triaryl-substituted (thiazol-2-yl)pyrazole compounds . We report here the synthesis of 1-(thiazolol-2-yl)-4,5-dihydropyrazoles from simple precursors. The reaction sequence is summarized in Fig. 1et al., 2012et al., 2017et al., 2015H-pyrazol-1-yl]-4-phenyl-1,3-thiazole pyrazoles have previously been synthesized and characterized. The synthesis and crystal structure of a new thiazolyl-pyrazoline derivative bearing the 1,2,4-triazole moiety has been reported  have been characterized spectroscopically. Chalcone intermediate (I)The proposed synthetic route, as also applied to synthesize many of the aforementioned related compounds, was selected because in some cases, we have introduced mesoionic moieties like sydnone as a part of the triaryl. These sydnones are somewhat sensitive towards vigorous reaction conditions. Under the present conditions selected, the products are stable and the reactions gave reasonably good yields. The chosen synthetic routes of the reported compounds in this study are straightforward with limited steps and readily accessible, cheap starting materials, and yields are reasonably high  . In particular, the spectra of each of (I)For the thiazolylpyrazole products (II)C) Fig. 1, the 1H In the structure of the chalcone intermediate (I)R-configuration at atom C5. However, the space group confirms that both compounds have crystallized as racemic mixtures: this is as expected, as the synthesis of (II)Chemical context and Database survey for references).Compounds (II)In each of (I)–(III), the methoxy C atom is coplanar with the adjacent aryl ring  distance being 2.74 Å and a C5—H5⋯C38 angle of 159°. In (II)A⋯C39i and H5⋯C38i distances of 2.80 and 2.81 Å, respectively, which are only 0.10 and 0.09 Å shorter than the sum of of corresponding van der Waals radii.In (III)Additional short intramolecular C—H⋯O and C—H⋯N contacts are observed in (I)–(III). The packing is devoid of C—H⋯O hydrogen bonding, and no noticeable π–π interactions are observed.et al., 2016Chemical context). BAKLOQ, and PUVVAG have fused thiazol and phenyl rings. All these structures are characterized by a T-shaped structure with pyrazole C5-aryl substituents forming its blade and the remaining part of the molecule, the thiazol-2-yl-pyrazole ring and its substituents, forming a more or less planar structure, which constitutes the stock of the T-shape. Classical hydrogen bonding is not observed in any of these compounds. The dihydropyrazole rings are effectively planar in all these compounds.We briefly compare the structures reported here with those of some related compounds. A search for triaryl-substituted (thiazol-2-yl)pyrazoles in the Cambridge Structural Database ,   and potassium hydroxide  in ethanol (10 ml), and this resulting mixture was stirred at ambient temperature for 5 h. When the reaction was complete, as judged from TLC, the mixture was poured into an excess of ice-cold water and the resulting solid product (I)N,N-dimethylformamide   2180 , 1667 (C=O), 1620 (C=C). NMR (CDCl3) δ(1H) 2.79 , 6.67  (H-2) and 7.54  (H-3), 7.06  and 7.16  (–C6H4–), 7.12–7.24 .All reagents were obtained commercially, and all were used as received. For the synthesis of compound (I)C): thiosemicarbazide  was added to a suspension of (I)C) was collected by filtration and crystallized from a mixture of ethanol and N,N-dimethylformamide . Yield 79%, m.p. 422–423 K. Analysis: found C 65.8, H 5.2, N 11.5%; C20H15N3O2S requires C 65.7, H 5.2, N 11.5%. IR (cm−1) 3339 (NH2), 2120 . 1H NMR (DMSO-d6) δ 3.09  and 3.71  (pyrazole CH2), 3.69 , 3.78 , 4.52 , 5.76 , 6.75  and 7.02  (–C6H4–), 7.13  and 7.64  (–C6H4–). Mixtures of this intermediate  and either phenacyl bromide  for (II)N,N-dimethylformamide  , 1618 (C=N), 1600 (C=C). 1H NMR (CDCl3) δ 2.41 , H-39), 3.46  and 4.10  (pyrazole CH2), 3.90 , 4.56 , 5.43 , 6.95  and 7.20  7.26–7.63 , 7.90 . Compound (III)−1) 2210 , 1620 (C=N), 1605 (C=C). 1H NMR (CDCl3) δ 2,32 , 2.54 , H-39), 3.28  and 3.84  (pyrazole CH2), 3.77 , 4.75 , 5.69 , 6.86 , 7.01 , 7.11 , 7.34 , 7.57  and 7.72  (3 × –C6CH4–), 8.00 . Crystals of compounds (I)–(III) that were suitable for single-crystal X-ray diffraction were selected directly from the prepared samples.For the synthesis of compounds (II)2), 0.96 Å (CH3) or 0.93 Å for all other H atoms, and with Uiso(H) = kUeq(C), where k = 1.5 for the methyl groups, which were permitted to rotate but not to tilt, and k = 1.2 for all other H atoms. For compounds (II)x parameter  - (I−)]/[(I+) + (I−)] , calculated using 1715 quotients, and for (III)x = −0.01 (3), calculated using 1613 quotients.Crystal data, data collection and refinement details are summarized in Table 210.1107/S2056989021009312/zl5017sup1.cifCrystal structure: contains datablock(s) global, I, II, III. DOI: 10.1107/S2056989021009312/zl5017Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989021009312/zl5017IIsup3.hklStructure factors: contains datablock(s) II. DOI: 10.1107/S2056989021009312/zl5017IIIsup4.hklStructure factors: contains datablock(s) III. DOI: Click here for additional data file.10.1107/S2056989021009312/zl5017Isup5.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989021009312/zl5017IIIsup6.cmlSupporting information file. DOI: 2108209, 2108208, 2108207CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Their structures, including absolute configurations, were elucidated by NMR spectroscopic analyses, X-ray crystallography, 13C chemical shifts calculations, and ECD calculations. This is the first report of diterpenoids from Biscogniauxia sp. Furthermore, short-term memory enhancement against Alzheimer’s disease (AD), anti-inflammatory, and cytotoxic activities of 1–2 were also evaluated. The results showed that compound 1 exhibited short-term memory enhancement activity against AD.Two new diterpenoids, including a  Biscogniauxia sp. belongs to the Xylariaceae family, growing on the bark of trees and shrubs, preferably on dead or dying branches. Since the first report on the secondary metabolites in 2005 , structurally diverse active meroterpenoids (diisoprenyl-cyclohexene/ane-type) and their dimers with new skeleton were identified (seco-isopimarane type (1) and an abietane type (2). Their structures, including absolute configurations, were elucidated by NMR spectroscopic analyses, X-ray crystallography, 13C chemical shifts calculations, and ECD calculations. Details of the structure elucidations for 1–2 are reported herein  by HRESIMS indicated the molecular formula of 1 was C23H38O6 (5 degrees of unsaturation). The 13C NMR spectrum showed 23 carbon signals  were revealed by the analysis of 1H-1H COSY data of 1  was obtained as colorless needle crystals. The cationic molecule peak at  signals . Combineata of 1 . Combineelations  from H-1β-Hydroxyrickitin A (2) was obtained as a yellowish oil. In HR-ESI-MS, the protonated molecule peak at m/z 331.1915 [M + H]+  indicated the molecular formula of C20H26O4 (8 degrees of unsaturation). Twenty carbon signals were observed in 13C NMR and DEPT 135 experiment, including eight sp2 carbons , three sp3 quaternary carbons , two sp3 methine carbons , two sp3 methylene carbons , and five methyl carbons . All the proton resonances were associated to those of the directly attached carbon atoms through the HSQC experiment  suggested that H-3 was on the axial orientation in the cyclohexane ring, adopting the opposite orientation as H-2b and H3-20. Meanwhile, the observed ROESY correlation between H-3 and H-1a in cyclohexane ring confirmed the above deduction -2 and -2 were calculated by a quantum chemical method at the B3LYP/6-311 + g  -2 were similar to the experimental one  level. The predicted ECD curve of -2 was similar to the experimental one   level  and an abietane type (2) diterpenoids were obtained from Biscogniauxia sp. (No.71-10-1-1). This is the first report about diterpenoids from Biscogniauxia sp., which has enriched the structural diversity of this genus secondary metabolites. Furthermore, the bioassays  of 1–2 have been carried out. The anti-Alzheimer’s disease (AD) activities of 1–2 were evaluated by AD fly model with memantine as the positive control. The result showed 1 exhibited short-term memory enhancement activity in AD flies (50 > 40 μM).In our work, a AD flies . In addi3: δH 7.26/ δC 77.0) as internal standards. Column chromatography (CC) was carried out on silica gel (200–300 mesh) , and ODS . TLC was performed on precoated silica gel plate . Analytical HPLC was performed on a Dionex HPLC system equipped with an Ultimate 3000 pump, an Ultimate 3000 diode array detector, an Ultimate 3000 column compartment, an Ultimate 3000 autosampler , and an Alltech (Grace) 2000ES evaporative light scattering detector  using a Phenomenex Gemini C18 column . Semi-preparative HPLC was carried out on Shimadzu LC-6AD system equipped with UV detectors, using a YMC-Pack ODS-A column . Medium pressure liquid chromatography (MPLC) was equipped with a dual pump gradient system, a UV preparative detector, and a Dr Flash II fraction collector system . Methanol (MeOH) was purchased from Yuwang Industrial Co. Ltd. . Acetonitrile (CH3CN) was obtained from Oceanpak Alexative Chemical Co. Ltd. . Cyclohexane, ethyl acetate (EtOAc), and chloroform (CHCl3) were analytical grade from Fine Chemical Co. Ltd. .The melting points were determined on an X-5 micro melting point apparatus  without corrected. UV data were recorded using a JASCO V-550 UV/vis spectrometer . IR data were recorded on a JASCO FT/IR-480 plus spectrometer . Optical rotations were measured on a JASCO P1020 digital polarimeter . CD spectra were recorded in MeOH using a JASCO J-810 spectrophotometer  at room temperature. ESI-MS spectra were performed on a Bruker amaZon SL mass spectrometer . HRESIMS spectra were obtained on Waters Synapt G2 TOF mass spectrometer . 1D and 2D NMR spectra were acquired with Bruker AV 400 spectrometer  using the solvent signals  and the 5.8S rRNA gene sequences (ITS1-5.8S-ITS2) of the strain have been deposited at GenBank as KX977400.The strain numbered as 71-10-1-1 was isolated from the lichen 2O (105 ml) was added to each flask, and the rice was soaked overnight before autoclaving at 120°C for 30 min. After cooling to room temperature, each flask was inoculated with 5.0 ml of the spore inoculum and incubated at room temperature for 50 days.The fungal strain was cultured on slants of potato dextrose agar (PDA) at 25°C for 5 days. Agar plugs were used to inoculate nine Erlenmeyer flasks (250 ml), each containing 100 ml of potato dextrose broth (PDB). Fermentation was carried out in 60 Erlenmeyer flasks (500 ml), each containing 70 g of rice. Distilled H2O elution  to afford 4 fractions (w1–w4). The fraction w3 (8.5 g) was further subjected to MPLC on ODS CC (4 × 45 cm) eluted with a gradient of MeOH-H2O  with flow rate 20 ml/min for 470 min to afford 13 major fractions (Fr. w3-1–w3-13). Subfraction w3-8 (1457.6 mg) was subjected to silica gel CC using cyclohexane-ethyl acetate  to afford subfractions w3-8-1–w3-8-12. Subfraction w3-8-7 (78.1 mg) was subjected to semi-preparative HPLC, using MeCN–H2O  at a flow rate of 3 ml/min to yield 1 . Subfraction w3-8-6 (103.5 mg) was subjected to semi-preparative HPLC, using MeCN–H2O  at a flow rate of 3 ml/min to yield 2 .The culture was extracted thrice with EtOAc, and the organic solvent was evaporated to dryness under vacuum to afford a crude extract (109.5 g). Then the crude extract was subjected to silica gel CC (4 × 15 cm) using cyclohexane-MeOH  to afford cyclohexane extract  and MeOH extract . The MeOH extract  was separated by ODS CC (4 × 30 cm) eluting with MeOH–H27.5 D 52.2 ; UV (MeOH) λmax (logε) 207 (3.78), 252 (1.87) nm; IR (KBr) νmax 3448, 2872, 1730, 1708, 1651, 1457, 1264, 1034 cm−1; ESI-MS (positive) m/z 433 [M + Na]+; HRESIMS (positive) m/z 433.2563 [M + Na]+ ; 1H and 13C NMR see Colorless needle crystal; mp 164–166°C; [α]27.5 D 130.1 ; UV (MeOH) λmax (log ε) 214 (4.05), 284 (3.71) nm; CD λnm (∆ε)  284 (–30.70), 336 (+21.13), 453 (+6.03) nm; IR (KBr) νmax 3449, 2955, 2929, 1734, 1691, 1612, 1461, 1382, 1307, 1065 cm−1; ESI-MS (positive) m/z 331 [M + H]+, m/z 353 [M + Na]+; HRESIMS (positive) m/z 331.1915 [M + H]+ ; 1H and 13C NMR data see Yellowish oil; [α]λ = 1.54184 Åat 173.00 (10) K. Crystal data: C23H38O6, M = 410.53, space group monoclinic, P 21; unit cell dimensions were determined to be a = 12.2840 (2) Å, b = 6.04757 (14) Å, c = 15.0642 (3) Å, α = 90.00 °, β = 95.7465 (18) °, γ = 90.00 °, V = 1113.47 (4) Å3, Z = 2, Dx = 1.224 g/cm3, F (000) = 448.0, μ (Cu Kα) = 0.703 mm−1. 17244 reflections were collected to θmax = 62.84°, in which 3365 independent unique reflections  were used in all calculations. The structure was solved by direct methods using the SHELXS program, and refined by the SHELXL program and full-matrix least-squares calculations.5 In the structure refinements, hydrogen atoms were placed on the geometrically ideal positions by the “ride on” method. The final refinement gave R1 = 0.0300 (I > 2σ(I)), WR2 = 0.0768 , S = 1.058, Flack = 0.10 (16) and Hooft = 0.09 (7). Crystallographic data for 1 have been deposited in the Cambridge Crystallographic Data Center as supplementary publication no. CCDC 2099938. Copies of the data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, United Kingdom (fax: +44-(0)1223-336033, or e-mail: deposit@ccdc.cam.ac.uk).Crystallographic data of 1: Upon crystallization from MeOH using the vapor diffusion method, colorless needle-like crystals of 1 were obtained. Data were collected using a Sapphire CCD with graphite monochromated Cu Kα radiation, β42  was driven by a widespread neuronal-expressing Gal4 line, elav-GAL4c155 (P35). A behavioral assay was made from the first generation of the cross between H29.3*P35 (AD flies).Fly Stock. w1118 (isoCJ1) is an isogenic line used as a control in all of the experiments. In the experiment, we named this stock “2U” for convenience. Expression of AFly Culture. All flies were fed at 24°C, 40–60% RH (relative humidity). On day 2, newly hatched H29.3*2U male flies and AD male flies were selected and were put into vials . These flies were placed at 29°C, 40 ± 15% RH during the drug feeding process. The flies were transferred to new vials after 4 h of drug feeding from day 2 to day 8. All flies were placed at 29°C, 40 ± 15% RH.Drug Feeding Schedule. Drugs (compounds 1–2 and memantine) were prepared on the first day of eclosion, and the drug feeding was implemented on day 2. The original concentration of compounds was 10 mM, and the final concentration of compounds was 100 μM. These compounds were dissolved in DMSO. For each performance index, 2 vials of flies were fed with 50 μL of the resulting solution for 7 days .Pavlovian Olfactory Learning. Briefly, in the training period, a group of 100 flies was put into a training tube decorated with copper grids. These flies would be orderly exposed to two odors (octanol or methyl cyclohexanol) for 1 min, who were subsequently surrounded by fresh air for 45 s. These flies would be subjected to electric shock for 1 min when they smell the first odor. In the test period, these trained flies would be immediately transported to the choice point, where the T-maze diverged into two containers containing two odors. The flies were allowed to choose one odor between the two for 2 min. Then the number of flies in two containers would be counted. The parameter of PI (performance index) was used for evaluating flies’ memory behavior. PI = 0 represents these flies cannot memorize the link between electric shock and odors. On the contrary, PI = 100% represents all the flies can avoid electric shock according to odors. Note: The experiment was carried out in a room with 25°C, 70% RH.Statistical Analysis. Data were analyzed and graphs were also plotted  with the2. The cells were cultured for 2–3 days to reach the logarithmic phase and then used for experiments. The cells were treated with tested compounds and positive control ) at different concentrations  and then stimulated with LPS  for the incubated time. The stock solutions of tested compounds were prepared in DMSO, and the final concentration of DMSO was less than 0.1%.Cell culture and treatment. RAW264.7 murine macrophage cell line was obtained from American Type Culture Collection . Cells were cultured in DMEM medium  supplemented with 10% heat-inactivated FBS , 2 mM L-glutamine (Gibco), 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco) at 37°C with 5% CO5 cell per well incubated at 37°C for 24 h incubation, cells were treated with the tested agents for the indicated periods of time. Then, 20 µl aliquot of 0.5% MTT  solution was added to each well followed by 4 h incubation. The culture medium was removed, and the formazan precipitates were dissolved with 150 µl of DMSO. The optical densities (OD) at 570 nm were measured with a ELISA reader . Compounds 1–2 showed no cytotoxic effect on RAW264.7 cells at different concentrations .Cell viability assay. The cytotoxic effect of compounds 1–2 on RAW264.7 cells was evaluated by MTT assay. RAW264.7 cells were dispensed in 96-well plate at a density of 1 × 102 at 37 °C. The cytotoxicity assay was performed using the CCK-8  assay according to the manufacturer’s protocol. Briefly, 1 × 104 cells were seeded into each well of the 96 well plate and treated with different concentrations (2.5–40 μM) of the compounds with cisplatin  as the positive control for 48 h. Then, CCK-8 solution (10 μl) was added to each well and incubated for 2 h at 37°C. The absorbance of the samples at 450 nm was measured using a microtiter plate reader . The 50% inhibitory concentration (IC50) of each compound was calculated using a weighted regression of the plot.Five human cancer cell lines  were used in the cytotoxicity assays. All the cells were cultured in DMEM medium , supplemented with 10% fetal bovine serum , in 5% CO


Human artificial chromosomes (HACs) and mouse artificial chromosomes (MACs) are non-integrating chromosomal gene delivery vectors for molecular biology research. Recently, microcell-mediated chromosome transfer (MMCT) of HACs/MACs has been achieved in various human cells that include human immortalised mesenchymal stem cells (hiMSCs) and human induced pluripotent stem cells (hiPSCs). However, the conventional strategy of gene introduction with HACs/MACs requires laborious and time-consuming stepwise isolation of clones for gene loading into HACs/MACs in donor cell lines (CHO and A9) and then transferring the HAC/MAC into cells via MMCT. To overcome these limitations and accelerate chromosome vector-based functional assays in human cells, we established various human cell lines  with HACs/MACs that harbour a gene-loading site via MMCT. Model genes, such as tdTomato, TagBFP2, and ELuc, were introduced into these preprepared HAC/MAC-introduced cell lines via the Cre-loxP system or simultaneous insertion of multiple gene-loading vectors. The model genes on the HACs/MACs were stably expressed and the HACs/MACs were stably maintained in the cell lines. Thus, our strategy using this HAC/MAC-containing cell line panel has dramatically simplified and accelerated gene introduction via HACs/MACs. HAC/MAC technologies have been used for gene and cell therapies of Duchene muscular dystrophy10 and to generate trans-chromosomic (Tc) animals that include a mouse model of Down syndrome12 and humanised drug metabolism18. Furthermore, several types of HACs have been used in cancer research and drug development for cancer therapy21, centromere and telomere function elucidation22, a system of quantitatively tracking epigenetic memory in the field of synthetic biology23, and protein production24. To accelerate the gene loading of multiple genes into HACs/MACs, we have developed several systems for multiple gene insertions, such as the simultaneous or sequential integration of multiple gene-loading vectors (SIM) system26 via hypoxanthine–guanine phosphoribosyltransferase (HPRT)-deficient cells and drug screening with HAT, a multi-integrase (MI) system 30, and homologous recombination with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)31. HACs/MACs are transferrable into desired cells by microcell-mediated chromosome transfer (MMCT)32. Although MMCT traditionally employs polyethylene glycol33, we developed a novel microcell membrane fusion method with the envelope proteins of measles virus (MV)35, amphotropic virus, and ecotropic virus36, which improved the transfer efficiency (1 × 10−4–1 × 10−5). However, specialised equipment and a laborious and time-consuming process are required for MMCT of HACs/MACs. This is because, in accordance with each experimental purpose, HACs/MACs with desired genes are constructed in CHO and A9 cells, and individually transferred to a target cell line via MMCT, and then clones are isolated, which contain the desired HACs/MACs  line40. We further attempted to adapt the Cre-loxP system  and mouse artificial chromosome (MACs) have unique characteristics as vectors for gene delivery, which include stable and independent maintenance without disruption of the host genome and the capacity to carry numerous genes and megabase-sized genomic loci with physiological regulatory elements41, and MACs were derived from mouse chromosome 11, such as MAC2 without EGFP, MAC4 with EGFP 43, and MAC6 with EGFP. These HACs/MACs had a loxP site and partial HPRT gene as an acceptor site for the SIM system, which enabled simultaneous insertion of three circular plasmids. However, conventional gene introduction via HACs/MACs requires transfer of the HACs/MACs with desired gene(s) by MMCT into target cells and there is a major technical difficulty. In this study, we established a human somatic/stem cell line panel  that contained HACs/MACs  with a GOI were inserted into the HAC/MAC via the SIM system for HEK293 cells, HT1080 cells, and hiPSCs or the Cre-loxP system for hiMSCs. Fluorescence in situ hybridisation (FISH) analyses revealed that a single additional HAC or MAC was maintained independently from the host chromosome in each somatic/stem cell line. Specifically, HEK293 cells contained 21HAC1  on 21HAC2 in hiMSCs with a defined (single in this study) copy number. Among the drug-resistant clones obtained by transfection with a plasmid that carried each promoter, 10 clones were picked up in order of the fluorescence intensity of mCherry under a fluorescence microscope and used for subsequent analysis to measure each promoter activity. Fluorescence imaging of mCherry-expressing cells with each promoter indicated that CAG and EF1α promoter activities were higher than the PGK promoter activity   Fig. e. There hiPSCs (201B7) that contained MAC6-ΔNeoR expressed EGFP Fig. a. We perWe generated a novel human cell line panel with HEK293 cell, HT1080 cell, hiMSC, and hiPSC (201B7) lines that contained HACs/MACs, which enabled rapid and precise insertion of GOIs at a defined site on HACs/MACs by a simple transfection method. The GOIs were stably expressed in each cell line, which indicated that the integration site can act as a “safe harbour” to support transgene expression. Thus, our new preprepared cell panel with HACs/MACs may dramatically simplify the construction of HACs/MACs with desired genes and constructed HACs/MACs can be used immediately and directly for functional analyses of genes in desired cell lines.This study was approved by the Institutional Animal Care and Use Committee of Tottori University  and the Recombinant DNA Experiment Safety Committee of Tottori University to perform recombinant DNA experiments. All experiments were carried out in compliance with the ARRIVE guidelines. All methods were performed in accordance with the relevant guidelines and regulations.41, MAC243, or MAC446, were cultured in Ham’s F-12 medium  with 10% FBS, 1% penicillin/streptomycin (FUJIFILM Wako), and 800 µg/mL hygromycin B (FUJIFILM Wako). CHO cells that contained 21HAC2 were cultured in Ham’s F-12 medium with blasticidin S (FUJIFILM Wako). HEK293 cells were purchased from the ATCC (ATCC® CRL-1573™) and cultured in Eagle’s minimum essential medium  with 10% fetal bovine serum , 1% non-essential amino acids (Sigma-Aldrich), 1% L-glutamine (Sigma-Aldrich), and 1% penicillin/streptomycin (FUJIFILM Wako). HT1080 cells47 obtained from the ATCC (ATCC® CCL-121™) were cultured in Dulbecco’s modified Eagle’s medium (FUJIFILM Wako) with 10% FBS and 1% penicillin/streptomycin. The human immortalised mesenchymal stem cell (hiMSC)39 line was kindly provided by Dr. J. Toguchida and was cultured in Dulbecco’s modified Eagle’s medium (FUJIFILM Wako) with 10% FBS and 1% penicillin/streptomycin. HEK293 clones that contained each HAC/MAC were selected with 200 µg/mL hygromycin B (FUJIFILM Wako). HT1080 cell and hiMSC clones that contained HAC2 were selected with 8 and 4 µg/mL blasticidin S, respectively. HT1080 clones that contained MAC2 were selected with 200 µg/mL hygromycin B. For drug selection after transfection using the SIM system, HACs/MACs expressed the HPRT gene for hypoxanthine-aminopterin-thymidine (HAT) resistance following gene insertion. HEK293 and HT1080 clones were selected after transfection in HAT medium (Sigma-Aldrich). hiPSC cell line 201B7 (HPS0063) was provided by the RIKEN BRC and cultured in StemFit AK02N  with Laminin-511 . hiPSCs that contained MAC6 with a neomycin resistance gene were selected with 90 µg/mL G418.CHO cells derived from a HPRT-deficient cell line (JCRB0218) , which contained 21HAC17 chromosome donor CHO cells that contained each artificial chromosome were cotransfected with 12 μg pTNH6-H-αCD9 for HEK293 cells and hiPSCs or pTNH6-H for HT1080 cells and 12 μg pCAG-T7-F for each recipient cell line by Lipofectamine 2000  in accordance with the manufacturer’s instructions34. CHO 4H6.1 M cells stably expressed MV-H and F, which provided microcells that contained 21HAC2 as reported previously35. Twelve flasks of CHO cells were prepared and micronuclei were induced by treatment with 0.1 µg/mL colcemid. The detailed MMCT protocol has been described previously26. The collected microcells were cocultured and fused with 2 × 106 cells of each recipient cell line for 24 h in a 6-cm dish . Then, the fused recipient cells were subcultured into three 10-cm dishes. Drug selection was started with optimal selectable antibiotics after a further 24 h of incubation. After 14–21 days, drug-resistant colonies were picked up and expanded for the following analyses.To prepare microcells that contained 21HAC1, MAC2, or MAC4, 1 × 1048. X3.1-I-EGFP-I that contained the Cre-loxP system was digested with NcoI and SpeI, which removed EGFP. The HS4 insulator on X3.1-I-EGFP-I was amplified with primers 5′- ATCCATGGATCGACTCTAGAGGGACAGCC -3′ and 5′- ATAACTAGTCGACGCGGCCGCCTCACTGACTCCGTCCTGGA -3′, and ligated using NcoI and SpeI sites. mCherry expression vectors with three types of constitutive promoters  were purchased from VectorBuilder . The vector information is available from the database of VectorBuilder with the following vector IDs: pRP-[Exp]-hPGK > mCherry), pRP-[Exp]-EF1A > mCherry, and pRP-[Exp]-CAG > mCherry. These mCherry expression cassettes were prepared by NotI digestion of the mCherry expression vector. Then, each mCherry expression cassette was inserted into the modified X3.1 without EGFP.To construct pBG-V0b1-ins-ELuc-ins, an EcoRV-digested fragment, which included an ELuc expression unit from CAG-ELuc, was ligated into pBG-V0b1 linearised with EcoRV. To construct pBG2-V1a-ins-tdtmt-ins and pBG2-V2a-ins-BFP-ins, each fragment of pCMV-tdTomato (Takara Bio) and pTagBFP2-N (Evrogen) was amplified by PCR with the following primers, F: 5′- GAACCTGCACTAGCCATCATGTTCTTTCCTGCGTTAT -3′, R: 5′- AAAAACGCGTGTCGATCCTGCACTAGCCATTTAAGATACATTGATGAGTT -3′. Then, each PCR product was digested with AflIII and ligated to a fragment of pinsB4ins prepared by HincII and AflII digestion. Then, fragments from pinsB4ins that contained tdTomato or TagBFP2-N were prepared by EcoRI digestion and ligated into pBG2-V1a for pBG2-V1a-ins-tdtmt-ins and pBG2-V2a for TagBFP2-N via MluI sites in each vector. To construct GLV2-EF1a-tdTomato, tDNA-pEF1a-BGHpA, a synthesised DNA, was digested by EcoRI and ligated in an annealed doubled-stranded palindrome oligo DNA, 5′- AATTCTGACTGTCTAGACAGTCA -3′, which included an XbaI site. Then, tDNA-pEF1a-BGHpA-XbaI was digested by XbaI and ligated to a fragment of pCMV-tdTomato digested by NheI and AvrII. tDNA-pEF1a-tdTomato was digested by AscI and NheI, and ligated to a PCR product amplified by PCR from pBG2-Vla using the following primers: BxbI-PhiC31_CL_F: 5′- CGCATGGCGCGCCTGGCCGTGGCCGTGCTCGTC -3′ and BxbI-PhiC31_CL_R: 5′- CTAGTCCTAGGGACCCTACGCCCCCAACTGA -3′ and by digestion with AscI and NheI. To construct GLV3-NeoR-pEF1a-BFP, tDNA-EF1a-BGHpA, a synthesised DNA was digested by EcoRI and ligated to a PCR product amplified from pTagBFP2-N by primers BFP_cl_EcoRI_F: 5′- CGAGTCGAATTCGCCACCATGGTGTCTAAGGGCGAAGAGCTGA -3′ and BFP_cl_EcoRI_R: 5′- TGTAACGAATTCCTATTAATTAAGTTTGTGCCCCAGTTTGC -3′, and digested by EcoRI. Furthermore, tDNA-EF1a-BFP was digested by AscI and NheI, and ligated to a PCR product amplified from pBG2-V2a by primers PhiC31 attB 3’HPRT Asc1 F: 5′- CGCATGGCGCGCCGATGTAGGTCACGGTCTCGAAG -3′ and PhiC31 attB 3'HPRT cl R: 5′- CTAGTCCTAGGAGGCTGGTTCTTTCCGCCT -3′, and digested by AscI and AvrII (GLV3-pEF1a-BFP). Then, GLV3-pEF1a-BFP was digested by AscI and Hind III, and ligated to three DNA fragments amplified by PCR from GLV3-BFP with primers AscI-PhiC31 attB-AvrII F: 5ʹ- GGCCGCATGGCGCGCCGATG -3′ and AscI-PhiC31 attB-AvrII R: 5ʹ- AAAACCTAGGTCATCATGATGGACCAGATG -3′, pBG2-V1b1 with primers AvrII-NeoR-AgeI F: 5′- AAAACCTAGGGCGGCCGCCGTGACCTGCAC -3′ and AvrII-NeoR-AgeI R: 5′- AAAAACCGGTCCCCAGCTGGTTCTTTCCGC -3′, and an annealed double-stranded oligomer DNA with primers AgeI-HindIII oligo F: 5′- AGCTTGATTTCGGCCTATTGA -3′ and AgeI-HindIII oligo R: 5′- CCGGTCAATAGGCCGAAATCA -3′. The mCherry expression vector with a Cre-loxP system was a modified X3.1-I-EGFP-I vector6 cells per 10-cm dish. HEK293 and HT1080 cells were transfected using a previously described method25. Transfected plasmids were 3.5 µg pBG2-V0b-ins-ELuc-ins, 7 µg pBG2-V1a-ins-tdTomato-ins, 10.5 µg pBG2-V2a-ins-BFP-ins, 3 µg pBS185 (pCMV-Cre), 3 µg pCMV-Bxb1 integrase, and 3 µg pCMV-PhiC31 integrase. These plasmids were mixed and transfected with Lipofectamine 2000 (Invitrogen), following the manufacturer’s instructions. Then, the transfected cells were selected in 2% HAT medium. hiPSCs that contained HACs/MACs were prepared at 2 × 106 cells per 10-cm dish. The introduced plasmids were 3.5 µg pBG2-V0b1-ins-ELuc-ins, 7 µg GLV2-tdTomato, 10.5 µg GLV3-NeoR-BFP, 3 µg pEF1a-Cre, 3 µg pCAG-Bxb1 integrase, and 3 µg pCAG-PhiC31 integrase. The mixture of plasmids was introduced into hiPSCs by a NEPA21 electroporator  with the following conditions: pouring pulse, 135 or 175 V; pulse length, 2.5 milliseconds; pulse interval 50 ms; number of pulses, 2; decay rate, 10% and polarity + , and then transfer pulse, 20 V; pulse length, 50 milliseconds; pulse interval, 50 milliseconds, number of pulses, 2, decay rate, 40% and polarity, + /−. Then, the electroporated cells were expanded in the 10-cm dish and 90 µg/mL G418 was added to the culture medium at 48 h after electroporation. We also used another method of electroporation by a Nucleofector 4D . The introduced plasmids were 3.5 µg pBG2-V0b-ins-ELuc-ins, 7 µg GLV2-tdTomato, 10.5 µg GLV3-NeoR-BFP, 3 µg pEF1a-Cre, 3 µg pEF1a-Bxb1 integrase, and 3 µg pEF1a-PhiC31 integrase. A total of 1 × 106 hiPSCs and plasmids were mixed with P3 Primary Cell 4D-Nucleofector™ X Kit L (Lonza), following the manufacturer’s instructions, and pulsed with the CA-137 program. Eight micrograms of X3.1 that contained mCherry with each constitutive promoter and 2 µg of a Cre expression vector (pBS185) were mixed and introduced into hiMSCs by the NEPA21 electroporator with the following conditions: pouring pulse, 175 V; pulse length, 2.5 millisconds; pulse interval, 50 milliseconds; the number of pulses, 2; decay rate, 10% and polarity + , and then transfer pulse, 20 V; pulse length, 50 milliseconds, pulse interval, 50 milliseconds, number of pulses, 2; decay rate 40% and polarity + /−. Then, hiMSCs were selected in 2% HAT medium. pBS185 CMV-Cre was a gift from Brian Sauer 49.HEK293 and HT1080 cells that contained HACs/MACs were prepared at 5 × 1051. The multiplex CRISPR/FokI-dCas9 vector system targeted six sequences for knockout of the HPRT1 gene: HPRT T1: 5′- TAACGGAGCCGGCCGGCGCGCGG -3′, HPRT T2: 5′- TGGCGTCGTGGTGAGCAGCTCGG -3′, HPRT T3: 5′- AAATCCTCAGCATAATGATTAGG -3′, HPRT T4: 5′- CTCATGGACTAATTATGGACAGG -3′, HPRT T5: 5′- CACAGAGGGCTACAATGTGATGG -3′ and HPRT T6: 5′- TAAATTCTTTGCTGACCTGCTGG -3′. The vector system was transfected into HEK293 cells and then HPRT knockout cells were screened by 100 µM 6TG treatment. HT1080 clones with spontaneous mutation of the HPRT1 gene were selected by 6TG treatment. Gene knockout of the neomycin resistance gene was performed with CRISPR/Cas9 targeting 5ʹ- AGGCTATTCGGCTATGACTGGG -3′27.Gene knockout of HPRT1 was performed in HEK293 cells, hiMSCs, and hiPSCs with the multiplex CRISPR/FokI-dCas9 vector systemPCR analyses were performed with KOD Fx , following the manufacturer’s instructions. The primers to detect gene insertion in HACs/MACs via the SIM system were HPRT junc sp F 5′- CGGCTTCCTCCTCCTGAACAA -3′ and HPRT junc sp R 5ʹ- TCCATAAGACAGAATGCTATGCAACC -3′ for HPRT EX1-2 and HPRT EX3-9 reconstitution in HEK293, HT1080 and hiMSCs, and TRANS L1 5ʹ- TGGAGGCCATAAACAAGAAGAC -3ʹ and SIM Neo Rv 5ʹ- CGCCTTGAGCCTGGCGAACA -3′ for HPRT EX1-2 and Neo cDNA reconstitution in human iPSCs.52 to stain the alpha satellite of hChr.13, 21 and HAC, and mouse Cot-1 DNA to stain the MAC. The probes were labelled with digoxigenin  and the inserted plasmid vector targeted to the chromosome fragment was labelled with biotin (Roche). The DNA probes were labelled with a nick translation kit (Roche), following the manufacturer’s instructions. The detailed protocol has been described previously26.Cells were treated with colcemid to induce metaphase arrest, treated with 0.075 M KCl, and then fixed with methanol/acetate (3:1) (FUJIFILM Wako). FISH was performed with the p11-4 alpha satellite probe6) were subcutaneously transplanted into a testis of anaesthetised severe combined immunodeficiency mice . A mixed anaesthetic agent prepared with 0.3 mg/kg medetomidine hydrochloride, 4 mg/kg midazolam, and 5 mg/kg butorphanol tartrate was administered intraperitoneally to the mice. Teratomas appeared after ~ 8 weeks. The anaesthetised mice were sacrificed and the teratomas were collected. Then, the teratomas were fixed with 20% neutral formalin/PBS and processed for paraffin sectioning. The sections were stained with haematoxylin and eosin.Mice were maintained under specific pathogen-free conditions with a 12-h light–dark cycle. Human iPSCs  equipped with 488, 405, and 561 nm lasers to detect EGFP, BFP2, and tdTomato, respectively. The ratio of fluorescence-positive cells of each fluorescent protein was determined by measuring 10,000 cells in each experiment.4 cells in each well of a 96-well plate. Luciferase activity was measured with Emerald Luc Luciferase Assay Reagent Neo (TOYOBO), following the manufacturer’s instructions. Bioluminescence was measured for 1 s by an EnVision 26.The assay was performed with 6 × 10st strand cDNA Synthesis Kit (Takara Bio), following the manufacturer’s instructions. RT-PCR analyses were performed with KOD one, cDNA, and the following primer sets: CXCL1 F 5′- TGTGAAGGCAGGGGAATGTA -3′ and R 5′- GCCCCTTTGTTCTAAGCCAG -3′, CD90 F 5′- ATGAACCTGGCCATCAGCA -3′ and R 5′- GTGTGCTCAGGCACCCC -3′, IL-8 F 5′- ACCGGAAGGAACCATCTCAC -3ʹ and R 5′- ATTTGGGGTGGAAAGGTTTG -3′, and CCL2 F 5′- GCAGCAAGTGTCCCAAAGAA -3′ and R 5′- AACAGGGTGTCTGGGGAAAG -3ʹ.Total RNA was isolated with a Nucleospin RNA plus kit (Takara Bio), following the manufacturer’s instructions. cDNA was synthesised with a PrimeScript™ II 1qRT-PCR analysis was performed with TB Green® Fast qPCR Mix (Takara Bio), following the manufacturer’s instructions. The following primer sets were used: mCherry F 5′- AAGGGCGAGGAGGATAACAT -3′ and R 5′- ACATGAACTGAGGGGACAGG -3′, GAPDH1F 5′- AGCCACATCGCTCAGACAC -3′, R 5ʹ- GCCCAATACGACCAAATCC -3′. OCT 3/4 F 5′- AGAAGGATGTGGTCCGAGTGTG -3′, R 5′- CCACCCTTTGTGTTCCCAATTCC -3′. The enzyme reactions and measurements were performed with the Applied Biosystems 7300 Fast Real-Time PCR System (Thermo Fisher Scientific), following the manufacturer’s instructions. Fold changes in the gene expression level of mCherry were calculated using the ∆∆Ct method and normalised to GAPDH gene expression.iPS cells were fixed with 4% paraformaldehyde/PBS (Sigma Aldrich) in a freezer overnight. The samples were blocked with PBS that contained 5% dry skim milk and 0.1% NP-40 (FUJIFILM Wako) for 15 min at RT. Then, the samples were stained for 1 h at RT with mouse IgG2b against OCT3/4 (1:50)  as the primary antibody and then anti-mouse IgG2b-Alexa 555 (1:500)  for detection. Image were obtained under a Keyence BZ-X800 .The iPS cells were prepared in confluently and fixed with 4% paraformaldehyde/PBS (Sigma Aldrich) in a freezer overnight. The staining was performed with Alkaline Phosphatase Staining Kit . The image were obtained by microscopy for cell culture  and the attached CCD camera DP22 and Software.Supplementary Information.


In the crystal, a helical, supramolecular chain along the b-axis direction features amine-N—H⋯O(methoxy) hydrogen bonding.The C 4H9)2(C15H13N3O2S)], features a penta-coordinated tin centre defined by the N,O,S-donor atoms of the di-anionic Schiff base ligand and two methylene-C atoms of the n-butyl substituents. The resultant C2NOS donor set defines a geometry intermediate between trigonal–bipyramidal and square-pyramidal. In the crystal, amine-N—H⋯O(methoxy) hydrogen bonding is found in a helical, supramolecular chain propagating along the b-axis direction. The chains are assembled into a layer parallel to (The title diorganotin Schiff base derivative, [Sn(C Significant activity was also observed in an in vitro cytotoxic assay of tin(IV) compounds of 2-hydroxy-5-methoxybenzaldehyde-N(4)-methylthiosemicarbazone -phenyl thiosemicarbazone and 2-acetylpyrazine N(4)-phenylthiosemicarbazone (C4H9)2 et al., 2008trans angle is 157.56 (5)°, for S1—Sn—O1, with the next two widest angles being N2—Sn—C16 [126.42 (9)°] and C16—Sn—C20 [124.08 (11)°]. The distortion from the ideal square-pyramidal and trigonal-bipyramidal geometries is quantified by the value of τ, with values of 0.0 and 1.0, respectively )2 Fig. 1, compris6 rings is 19.63 (13)°, highlighting the observation the Schiff base di-anion deviates significantly from co-planarity.The N,O,S mode of coordination of the Schiff base di-anion gives rise to the formation of five- and six-membered chelate rings, the acute chelate angles, Table 1; these are partly responsible for the observed distortions in the coordination environment. The former ring, comprising the Sn, S1, N1, N2 and C1 atoms is almost planar, presenting a r.m.s. deviation of 0.0087 Å: atom N3 lies 0.016 (3) Å out of this plane. By contrast, distortions are evident in the six-membered chelate ring, defined by the Sn, O1, N2, C2–C4 atoms. The simplest description for the conformation is that of an envelope with the tin atom lying 0.519 (3) Å out of the plane defined by the remaining five atoms (r.m.s. deviation = 0.0379 Å). The dihedral angle between the five-membered chelate ring and the best plane through the five approximately co-planar atoms of the six-membered chelate ring is 13.59 (12)°, that between the five-membered and N-bound phenyl ring is 6.92 (12)° and that between the peripheral Cb-axis direction, Fig. 2a). The only other directional interactions based on an analysis of the points of contact between molecules in the crystal . Layers stack without specific interactions between them, Fig. 2c).Conventional hydrogen bonding is noted in the crystal of (I)dnorm  hydrogen bond. This hydrogen bond is also reflected in the Hirshfeld surface mapped over the electrostatic potential, Fig. 4N and methoxy-O2 atoms, respectively. Complementing the methylene-C18—H18A⋯Cg1 contact listed in Table 2B⋯Cg1 contact in the inter-layer region, Table 3The Hirshfeld surface analysis for (I)de and di diagonal axes, respectively, in Fig. 6a). The individual H⋯H, H⋯C/C⋯H, H⋯O/O⋯H, H⋯N/N⋯H and H⋯S/S⋯H contacts are illustrated in the delineated fingerprint plots in Fig. 6b)–(f), respectively. The percentage contributions for the different interatomic contacts to the Hirshfeld surface are included in Fig. 6i.e. 66.2%. The H⋯H contacts appear as a beak-like distribution tipped at de + di ∼2.4 Å in Fig. 6b), with the short value corresponding to the H6⋯H22A and H7⋯H17A contacts, with details listed in Table 3de + di ∼2.7 Å, Fig. 6c). This feature reflects the C—H⋯ π contacts as discussed above. Although H⋯O/O⋯H contacts only contribute 5.2% to the overall Hirshfeld surface, they appear as the shortest contacts at de + di ∼2.1 Å, being 0.6 Å shorter than the sum of their van der Waals radii, Fig. 6d), and reflect the conventional hydrogen bonding leading to the supramolecular chain. The H⋯N/N⋯H and H⋯S/S⋯H contacts contribute 4.6 and 4.3%, respectively, to the overall Hirshfeld surface. These contacts are reflected as pseudo-mirrored features at de + di ∼3.0 Å in each of Fig. 6e) and (f), with the minimum distance being around the sum of their respective van der Waals radii. The other interatomic contacts, i.e. C⋯C and C⋯N/N⋯C, have a negligible effect on the molecular packing and their contributions to the overall Hirshfeld surface are 1.7 and 0.2%, respectively.The overall two-dimensional fingerprint plot for the Hirshfeld surface of (I)Etot) as well as individual energy components, namely electrostatic (Eele), polarization (Epol), dispersion (Edis) and exchange-repulsion (Erep) are collated in Table 4N⋯O2(methoxy) hydrogen bond (Etot = −83.4 kJ mol−1). In addition to the methylene-C18—H18A⋯Cg1 contacts, molecules in the intra-layer region are stabilized by a number of H⋯H contacts, notably H7⋯H17A contacts with a separation of 2.32 Å, Table 3i.e. Edis, makes the major contribution to the overall interaction energy in the intra-layer region.In the present analysis, the pairwise interaction energies between the molecules in the crystal of (I)B⋯Cg1 contact (Etot = −25.7 kJ mol−1) with the remaining intermolecular contacts between molecules being stabilizing H⋯H contacts. The nature of these contacts leads to the dominance of the Edis component in the molecular packing, Table 4Eele and Edis components of all pairwise interactions sum to −127.0 and −329.5 kJ mol−1, respectively.The greatest stabilization energies in the inter-layer region relate to the weak methylene-C22—H22i.e. RR’Sn(L). Each of the literature structures were reported during 2020, i.e. R = R′ = Me (II) and Ph (III)  co-crystallized with one-half mole equivalent of 3-methoxysalicylaldehyde azine . Selected geometric parameters for the four structures are collated in Table 5a) is shown in Fig. 82NOS coordination geometries. With the exception of (II), the molecules have intermediate coordination geometries with τ . The standout molecule is the dimethyltin derivative (II) which, with τ = 0.00, is well described as having a square-pyramidal geometry. The S1—Sn—O1 angles span a range greater than 15°, i.e. 145.67 (9) in (II) to 161.81 (7)° in (III).The crystal structure determination of (I)The hydrogen-bonding patterns formed in the crystals of (I)–(IV) are also distinct. Supramolecular helical chains, sustained by amine-H⋯O(methoxy) hydrogen bonds are found in each of (I)N-phenylhydrazine carbothioamide] was according to the procedure described in the literature -3N  was added dropwise followed by reflux for 2 h. Then, dibutyltin(IV) dichloride  was added to the mixture followed by reflux for 6 h. The mixture was filtered while hot to remove the [Et3NH]Cl salt that formed and the filtrate was kept at room temperature until bright-yellow crystals appeared. Yield 62%, m.p. 384–385 K. FT–IR : 3322 ν(N—H), 1582 ν(C=N), 1076 ν(N—N), 853 ν(C=S). 1H NMR  δ: 8.63 , 7.54 , 6.65–7.32 , 3.85 , n-Bu: 1.68 , 1.61 , 1.34 , 0.87 . 13C NMR  δ: 162.5 (S2C), 159.0 (C=N), 151.3, 139.6, 128.9, 125.4, 123.1, 120.4, 120.0, 116.9, 116.3, 115.7 (Ar—C), 56.3 (O—CH3), n-Bu: 27.5 (Cα), 26.5 (Cβ), 26.0 (Cγ), 13.6 (Cδ).Synthesis of (I)Uiso(H) set to 1.2–1.5Ueq(C). The N-bound H atom was located in a difference-Fourier map, but was refined with a N—H = 0.88±0.01 Å distance restraint, and with Uiso(H) set to 1.2Ueq(N). The maximum and minimum residual electron density peaks of 1.63 and 0.52 e Å−3, respectively, were located 0.97 and 0.53 Å from the Sn atom.Crystal data, data collection and structure refinement details are summarized in Table 610.1107/S2056989021001870/hb7969sup1.cifCrystal structure: contains datablock(s) I, global. DOI: 10.1107/S2056989021001870/hb7969Isup2.hklStructure factors: contains datablock(s) I. DOI: 2063179CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The sympathetic nervous system is composed of an endocrine arm, regulating blood adrenaline and noradrenaline, and a local arm, a network of fibers innervating immune organs. Here, we investigated the impact of the local arm of the SNS in an inflammatory response in the colon. Intra-rectal insertion of an optogenetic probe in mice engineered to express channelrhodopsin-2 in tyrosine hydroxylase cells activated colonic sympathetic fibers. In contrast to systemic application of noradrenaline, local activation of sympathetic fibers attenuated experimental colitis and reduced immune cell abundance. Gene expression profiling showed decreased endothelial expression of the adhesion molecule MAdCAM-1 upon optogenetic stimulation; this decrease was sensitive to adrenergic blockers and 6-hydroxydopamine. Antibody blockade of MAdCAM-1 abrogated the optogenetic effect on immune cell extravasation into the colon and the pathology. Thus, sympathetic fibers control colonic inflammation by regulating immune cell extravasation from circulation, a mechanism likely relevant in multiple organs.  •Optogenetic activation of local sympathetic fibers in the colon attenuates colitis•The local sympathetic activation reduces immune cell abundance•Reduction in immune abundance is mediated by a decrease in endothelial MAdCAM-1•Endothelial MAdCAM-1 is regulated by noradrenaline via the β-adrenergic receptor Optogenetic activation of sympathetic fibers in the colon attenuated colitis and reduced immune cell abundance. This effect was mediated by endothelial cells, which reduced their expression of MAdCAM-1, required for immune extravasation. Thus, Schiller et al. introduce a mechanism whereby sympathetic fibers control the endothelial barrier to regulate local inflammation. As a central regulator of homeostasis, the brain receives multiple layers of information from the body, including feedback regarding metabolism, temperature, inflammation and tissue damage. The brain integrates this information to orchestrate behavioral and physiological functions, including the activity of the organism’s main protective mechanism, the immune system . Thus, fAnatomically, the SNS is comprised of two arms, an endocrine and a local arm. In the endocrine arm, neuronal fibers that innervate the adrenal gland induce adrenaline and noradrenaline (NA) release to the bloodstream, affecting the whole organism. In contrast, the local arm is comprised of sympathetic fibers directly innervating various tissues and immune organs . These sMany inflammatory conditions are affected by the psychological state of the patient, specifically stress , which iIn this study, we used optogenetic manipulations to investigate the effects of local sympathetic innervation to the colon in a murine model of colitis, dextran sulfate sodium (DSS)-induced colitis . To locaCre-dependent manner, with TH-Cre mice. TH is an enzyme required for catecholamine synthesis . To achieve localized activation of the sympathetic fibers only at the colon, we inserted an optic fiber intra-rectally to anesthetized mice E and actHaving established the functionality of the optogenetic system, we next tested the effects of local sympathetic activation in a mouse model of colon inflammation, DSS-induced colitis . In thisIn our experiments, we used two groups of mice: ChR2/TH mice as the experimental group, and negative littermates lacking ChR2 expression undergoing the same experimental procedure served as the control. Both groups were treated with 3% DSS in their drinking water and underwent local optogenetic activation to their colons via an intra-rectal probe daily for 7 days A. To det+ cells . We injected the adrenergic blockers daily to the ChR2/TH mice and their littermate controls, undergoing daily optogenetic activation to their colons during DSS treatment for 7 days. In the presence of the adrenergic blockers, the optogenetic activation did not affect the clinical symptoms of the colitis, further indicating that the optogenetic effect is NA-dependent  was also significantly attenuated in both the LP and the IEL in the DSS-treated ChR2/TH mice  layer and the lamina propria (LP) layer. Immune cells in the IEL are interspersed between epithelial cells and are the first cells to encounter luminal antigens . The LP Our analysis of the various immune subpopulations in the colon did not identify a specific cell subset that could account for the reduction in immune cell abundance following the optogenetic activation . MoreoveThe observed decrease in immune cell abundance could be mediated by either change in cell proliferation, cell death, or migration. The optogenetic activation did not affect the number of dead or proliferating immune cells in the ChR2/TH colons  or 6-OHDA in the DSS-treated ChR2/TH mice and their controls undergoing daily optogenetic activation. We could not detect any change in MAdCAM-1 and ICAM-1 expression levels on endothelial cells under these conditions .Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Asya Rolls (This study did not generate new unique reagents.This study did not generate any datasets or codes.Mice were maintained under Specific-Pathogen-Free (SPF) conditions on a 12:12 h light cycle (lights on at 07:00). Mice were grouped housed throughout the experiments. All mice were more than 8 weeks at the onset of experiments, and the typical age was 8 to 12 weeks. Several transgenic mice were used in the experiments: TH-Cre mice (B6129X1-Th < tm1(Cre)Te > /Kieg; EMMA), DAT-Cre mice (B6.SJL-Slc6a3tm1.1(cre)Bkmn/J; Jackson Laboratory), and SERT-Cre mice (B6.FVB(Cg)-Tg(Slc6a4-cre)ET33Gsat/Mmucd; Jackson Laboratory) cross-bred with ChR2 mice (RCL-ChR2(H134R)/EYFP; Jackson Laboratory), GFP mice (C57BL/6-Tg(UBC-GFP)30Scha/J) and C57BL/6 mice. All mice were on the same C57BL/6 background. Littermates of the same sex were randomly assigned to experimental groups. For controls, littermates negative for TH-Cre, or SERT-Cre (lacking ChR2 expression) were used, except for ChR2/DAT mice which do not have a negative littermate control (the DAT-Cre strain is homozygote). Therefore, in the ChR2/DAT experiments, the control group was ChR2/DAT mice who underwent the same procedures without light stimulation, namely the rectal probe was inserted but not turned on. All experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All procedures and protocols were approved by the Technion Administrative Panel of Laboratory Animal Care.Colon-derived cells were isolated from the colon of mice . Then, the cells were incubated in Dulbecco’s modified Eagle’s medium  supplemented with 10% heat-inactivated fetal bovine serum , 2mM L-glutamine , 1mM sodium pyruvate  and 1% PenStrep  in a humidified atmosphere of 95% air and 5% CO2 at 37°C.Mice were anesthetized with isoflurane, and an optic fiber (with its polymeric outer cover removed) was connected to a blue LED source  and inserted intra-rectally into the colon (as described in Mice were administered 3% DSS (TdB Consultancy) in their drinking water for 7 consecutive days, and their weight, food and water consumption were monitored daily. During the DSS treatment period, the mice underwent daily optogenetic manipulation . Afterward, the mice were sacrificed, and the entire colon was removed from caecum to anus. The colon length was measured as a marker for inflammation, and the tissue was used for histological staining and scoring, mRNA analysis, immunohistochemistry staining, and flow cytometry analysis.For histology staining, mice were sacrificed, and their colons were fixed in 4% paraformaldehyde (PFA) in PBS for 48 h, cryoprotected in 30% sucrose solution for another 48 h, and then frozen on dry ice. The colons were sliced in 10 μM sections and were mounted on super-frost slides (Fisherbrand). The slides were stained with Hematoxylin & Eosin (H&E). All images were taken using an automatic slide scanner (250 Flash III). The histological scoring was then performed by an investigator blinded to the treatment group of each sample. The components of the histological score were inflammatory infiltrate, goblet cell loss, crypt density, crypt hyperplasia, muscle thickening, submucosal inflammation, crypt abscess, and ulceration (as previously described in + cells using IMARIS software (version 8.1).Mice were sacrificed, and their colons were fixed in 4% PFA in PBS for 48 h, cryoprotected in 30% sucrose solution for another 48 h, and then frozen on dry ice. The colons were sectioned at 10 μm or 40 μm thickness. The 10 μm colon slices were mounted on super-frost slides (Fisherbrand). The 40 μm colon slices were rinsed twice in washing solution (0.05% Tween20 in PBS) and permeabilized for 15 min in 0.5% Triton X-100 in PBS. The following antibodies were used: anti-TH , anti-GFP , anti-β3-tubulin , anti-DAT , anti-serotonin , anti-SERT , anti-CD45 , anti-CD31 , anti-Ki67  and TUNEL (Roche diagnostics). All images were acquired using an Axio imager M2 microscope (Carl Zeiss Inc. US), 4-channel Olympus XI81-ZDC confocal microscope, or a laser scanning confocal microscope (Zeiss LSM 880). The percentage of cells with nuclear staining (Ki67 or TUNEL staining) was calculated out of the total number of CD45CLARITY staining: For clearing of the colon, the X-CLARITY technology was used (Logos biosystem) according to the manufacturer’s instructions. The colons were then incubated with the primary antibodies  diluted 1:100 at 37 °C for 1 week. Then, the colons were rinsed in a washing solution (0.05% Tween20 in PBS) for 2 days, and incubated with the secondary antibodies at 37 °C for 1 week. Then, the colons were incubated in DAPI staining solution (Sigma) for 2 days at 37 °C. All images were acquired using a laser scanning confocal microscope (Zeiss LSM 880) and analyzed using IMARIS software (version 8.1).Analysis of NA levels in the colon was performed using a mouse microdialysis apparatus  to collect fluid from the colon tissue before and after the optogenetic stimulation (following 30 min of optogenetic stimulation). Samples were collected using a CMA 71 mm probe and a flow syringe pump . Afterward, each sample was diluted in PBS containing 0.01 N HCl, 1 mM EDTA, and 4 mM sodium metabisulfite. For the analysis of NA levels in the serum, whole blood from anesthetized mice was collected before and after the optogenetic stimulation (following 30 min of optogenetic stimulation), centrifuged for 15 min at 2000xg, and stored at −80°C until analysis. NA levels in the colon and serum were analyzed using a NA ELISA kit .3, 25 mM glucose, 3 mM KCL, 1.25 mM NaH2PO4, 2 mM CaCl2, 1 mM MgCl2 (pH 7.4), with 0.04% Pluronic acid. The colons were freshly removed from ChR2/TH mice and incubated with the dye at 37°C for 1 h, followed by incubation at room temperature for 15 min. The dye solution was replaced with a buffer containing 125 mM NaCl, 25 mM NaHCO3, 25 mM glucose, 3 mm KCL, 1.25 mM NaH2PO4, 2 mM CaCl2, 1 mM MgCl2 (pH 7.4) and the colons were imaged by laser scanning confocal microscopy (Zeiss LSM 880), objective 20x/0.8 M27 at 3.36 Hz full-field scanning mode (image size 512x157 pixels). Image analysis was performed using MATLAB and Igor software (Wavematrics). The statistical significance was calculate using paired t test comparing the baseline and peak florescence intensity.For calcium imaging, an AM form of the red fluorescent calcium indicator, Calbryte 590 (AAT Bioquest) with an excitation peak of 580 nm and an emission peak of 592 nm was used. The dye was dissolved in DMSO for a stock solution, and later diluted to a working solution in a buffer containing 125 mM NaCl, 25 mM NaHCOMice were sacrificed, and their colons were collected. Colon samples were dissociated using the Lamina propria dissociation kit (Miltenyi Biotec). Cells were incubated with antibodies for 30 min at 4 °C then washed with FACS staining buffer . The following antibodies were used : Alexa Flour 700-conjugated and BV605-conjugated anti-CD45 (30-F11), PE-conjugated anti γ-δ (GL3), Pacific blue (PB)-conjugated anti-TCRβ (H57-597), BV510-conjugated anti-IL-17 (TC11-18H10.1), APC-conjugated and PE-Cy7- conjugated anti-CD4 (GK1.5), AF488-conjugated anti-FOXP3 (150D), BV650-conjugated NKp46 (29A1.4), BV605-conjugated and PB-conjugated anti CD3 (145-2C11), PerCP-conjugated anti-CD8 (53-6.7), BV510-conjugated anti-CD127 (A7R34), PB-conjugated anti T-bet (4B10), PerCP-conjugated anti-GATA3 (16E10A23), PE-conjugated anti RORγ , BV605-conjugated anti CD62L (MEL14), BV650-conjugated anti CD44 (IM7), PerCP-Cy5.5-conjugated anti-CD11b (M1/70), PECy7-conjugated CD11c (N418), BV605-conjugated anti-CD103 (2EF), BV510-conjugated anti LY6C (HK1.4), APC-conjugated anti-LY6G (1A8), BV650-conjugated anti CX3CR1 (5A011F11), PECy7-conjugated anti-CD19 (6D5), BV510-conjugated anti-CD27 (LG.3A10), BV650-conjugated anti-CD138 (281-2), PerCP-conjugated anti-IgG (poly4053), APC-conjugated anti-IgM (RMM-1), biotin-conjugated anti-IgA (RMA-1), FITC-conjugated anti-IgD (1126C.29), PE conjugated anti-ICAM-1 (YN1/1.7.4), AF488-conjugated and biotin-conjugated anti-MAdCAM-1 (MECA-367), PECy7-conjugated anti-CD31 (390), biotin-conjugated anti-CD69 (H1.2F3), PE-conjugated anti-TNF-α (MP6-XT22), biotin-conjugated anti-CD86 (GL-1), PE-conjugated anti-CD80 (16-10A1), AF488-conjugated streptavidin , PerCP-conjugated streptavidin , BV605-conjugated anti streptavidin, PE-conjugated phospho-CREB , and Zombie NIR dye. The samples were resuspended in 200 μl of 1% PFA and analyzed by flow cytometry using a LSRFortessa cell analyzer and FlowJo software. Gating strategies were set on the basis of unstained samples.NA hydrochloride (Sigma Aldrich) was dissolved in a saline solution. DSS-treated mice (3% in the drinking water) were injected daily IP with NA  and Phentolamine (Sigma Aldrich) were dissolved in saline solution. DSS-treated ChR2/TH mice (3% in the drinking water) were injected daily IP with the Nadolol (5 mg/kg) and Phentolamine (10 mg/kg) for 7 days . The mice were subjected to optogenetic manipulation, as described in To assess the effects of NA on colon endothelial cells, cells were isolated from the colon of mice . Then, the cells were incubated with 1 μg/mL LPS (Sigma) diluted in DMEM  supplemented with 10% heat-inactivated FBS , 2mM L-glutamine , 1mM sodium pyruvate  and 1% PenStrep  in a humidified atmosphere of 95% air and 5% CO2 at 37°C for 1 h. Afterward, NA was added (0-1000 μM), and the cells were incubated overnight at 37°C. The cells were then stained with Zombie NIR dye, APC-conjugated anti CD45 (30-F11), PECy7-conjugated anti-CD31 (390), AF488-conjugated anti-MAdCAM-1 (MECA-367) and analyzed with a LSRFortessa cell analyzer and FlowJo software.Colon-derived endothelial cell enrichment: To enrich colon endothelial cells, cells were isolated from the colon of mice . Then, endothelial cells were enriched using the CD45 depletion kit , and the CD31 enrichment kit . Then, the enriched colon-derived endothelial cells . Then, the cells were incubated with 1 μg/mL LPS (Sigma) diluted in DMEM  supplemented with 10% heat-inactivated FBS , 2mM L-glutamine , 1mM sodium pyruvate  and 1% PenStrep  in a humidified atmosphere of 95% air and 5% CO2 at 37°C for 1 h. The cells were next incubated with an α adrenergic blocker  or a β adrenergic blocker  for 20 min. Afterward, 100 μM NA was added, and the cells were incubated overnight at 37°C. The cells were then stained with Zombie NIR dye, APC-conjugated anti CD45 (30-F11), PECy7-conjugated anti-CD31 (390), AF488-conjugated anti-MAdCAM-1 (MECA-367) and analyzed with a LSRFortessa cell analyzer and FlowJo software.Phospho-CREB: To assess the role of CREB phosphorylation in the effect of NA on endothelial MAdCAM-1 expression, cells were isolated from the colon of mice . The colon cells were incubated with 100 μM NA diluted in DMEM  supplemented with 10% heat-inactivated FBS , 2mM L-glutamine , 1mM sodium pyruvate  and 1% PenStrep  in a humidified atmosphere of 95% air and 5% CO2 at 37°C for 30 min at 37°C. The cells were then stained with Zombie NIR dye, APC-conjugated anti CD45 (30-F11), PECy7-conjugated anti-CD31 (390), AF488-conjugated anti-MAdCAM-1 (MECA-367), PE-conjugated phospho-CREB (Ser133), and analyzed with a LSRFortessa cell analyzer and FlowJo software.CREB inhibitor: To evaluate the importance of CREB as the intracellular mediator on MAdCAM-1 levels, cells were isolated from the colon of mice . The isolated cells were incubated with 1 μM 666-15 (CREB inhibitor) diluted in DMEM  supplemented with 10% heat-inactivated FBS , 2mM L-glutamine , 1mM sodium pyruvate  and 1% PenStrep  in a humidified atmosphere of 95% air and 5% CO2 at 37°C overnight. The cells were next incubated with 1 μg/mL LPS (Sigma) for 1 h. Afterward, 100 μM NA was added, and the cells were incubated overnight at 37°C. The cells were then stained with Zombie NIR dye, APC-conjugated anti CD45 (30-F11), PECy7-conjugated anti-CD31 (390), AF488-conjugated anti-MAdCAM-1 (MECA-367) and analyzed with a LSRFortessa cell analyzer and FlowJo software.ChR2/TH mice and their controls (negative littermates exposed to the light stimulation) were sympathetically denervated by two IP injections of 6-OHDA  administered at 24 h intervals, as described previously . 5 days ChR2/TH mice and their controls (negative littermates exposed to the light stimulation) were exposed to 3% DSS for 7 days, and 20 min before the daily optogenetic stimulation, they were injected IP with anti-MAdCAM-1 antibody (MECA-367) or an isotype control . Their weights were monitored daily, and at the end of the experiment the mice were sacrificed, and the entire colon was removed from caecum to anus. The colon length was measured as a marker of inflammation, and the tissue was used for histological staining and scoring, and flow cytometry analysis.The locomotor response was tested in ChR2/TH mice and their controls (negative littermates exposed to the light stimulation) during the experimental protocol (7 days of 3% DSS with daily optogenetic activation). The locomotor activity was recorded by a centrally placed overhead camera in an open field arena (30cm X 30cm). Mice were placed into the arena for 1 h, and the distance traveled by each mouse was assessed during the final 30 min using the ‘distance’ parameter in a video tracking system (EthoVision version 11.5 XT).Colons and small intestines were lysed in TRI-Reagent (Sigma) and stored at −80 °C overnight. Total RNA was isolated according to the protocol supplied with the TRI-Reagent. Total RNA (0.1 μg) was reverse transcribed (RT) using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Real-time PCR analysis was performed using an Applied Biosystems StepOnePlus Real-Time PCR System  in two independent experiments in duplicate, using the Fast SYBR Green Master Mix (Applied Biosystems). Dissociation analysis was performed at the end of each run to confirm the specificity of the reaction. The cycle conditions for real-time PCR were 95 °C for 20 s, followed by 40 cycles of 95 °C for 3 s and 60 °C for 30 s, and a melting curve stage . Quantification of relative gene expression was performed according to the ΔΔ-CT method using StepOne Software 2.3 (Applied Biosystems), and the results expressed as fold difference ± SEM. The following primers were used:GAPDH forward: 5¢- TGAAGCAGGCATCTGAGGG-3¢,GAPDH reverse: 5¢- CGAAGGTGGAAGAGTGGGAG-3¢α4 forward: 5¢- CACAGCCACGGGTCGAA −3¢,α4 reverse: 5¢- AGGTCTGGTTTGGATTCTTTCTGA −3¢β7 forward: 5¢- GCTCTCTGTGGAAATCTACGA −3¢,β7 reverse: 5¢- TCACTCTGAAAAATCTCAGCG −3¢CCL20 forward: 5¢- GGTGGCAAGCGTCTGCTC −3¢,CCL20 reverse: 5¢- GCCTGGCTGCAGAGGTGA −3¢CCL25 forward: 5¢-TGAAAGGAAGAAGTCAAACCATATGA −3¢,CCL25 reverse: 5¢- AGGGTGGCACTCCTCACG −3¢P-selectin forward: 5¢- ACGGGTGTTCTGTAGGAGGCAC −3¢,P- selectin reverse: 5¢- GTTGTTGGGCTCGTTGTCGG −3¢E-selectin forward: 5¢- CCAGAATGGCGTCATGGA −3¢,E- selectin reverse: 5¢- TAAAGCCCTCATTGCATTGA −3¢CCR7 forward: 5¢- GCTCCAGGCACGCAACTTT −3¢,CCR7 reverse: 5¢- GACTACCACCACGGCAATGA −3¢CCR9 forward: 5¢- AGGCCAAGAAGTCATCCAAGC −3¢,CCR9 reverse: 5¢- CCTTCGGAATCTCTCGCCAA −3¢VCAM-1 forward: 5¢- AGTTGGGGATTCGGTTGTTC −3¢,VCAM-1 reverse: 5¢- CATTCCTTACCACCCCATTG −3¢MAdCAM-1 forward: 5¢- AGTTACTGTGCGCTGGACCTTGGCTCCTGGCGACCTGG-3¢MAdCAM-1 reverse: 5¢- TCCTGGCGGCACTGGAACCAGCC-3¢ICAM-1 forward: 5¢- GAGAGTGGACCCAACTGGAA-3¢,ICAM-1 reverse: 5¢- GCCACAGTTCTCAAAGCACA-3¢IL-6 forward: 5¢- TAGTCCTTCCTACCCCAATTTCC −3¢Il-6 reverse: 5¢- TTGGTCCTTAGCCACTCCTTC −3¢TNF-α forward: 5¢- CCTTTCACTCACTGGCCCAA −3¢TNF-α reverse: 5¢- AGTGCCTCTTCTGCCAGTTC −3¢IFN-γ forward: 5¢- GAGGTCAACAACCCACAGGTC −3¢IFN-γ reverse: 5¢- CGAATCAGCAGCGACTCCT-3¢IL-17 forward: 5¢- CCTCACACGAGGCACAAGTG −3¢IL-17 reverse: 5¢- CTCTCCCTGGACTCATGTTTGC −3¢IL-12 forward: 5¢- AAGCTGCATCCTGCTTCAC −3¢IL-12 reverse: 5¢- GATAGCCCATCACCCTGTTGA −3¢IL-21 forward: 5¢- TGCTAGCTCCAGCCTCAGTCT −3¢IL-21 reverse: 5¢- TTAAGTGCTGAACTTGTTTGGATTG −3¢TGF-β forward: 5¢- AAACGGAAGCGCATCGAA −3¢TGF-β reverse: 5¢- GGGACTGGCGAGCCTTAGTT −3¢IL-10 forward: 5¢- GCTCTTACTGACTGGCATGAG −3¢IL-10 reverse: 5¢- CGCAGCTCTAGGAGCATGTG −3¢IL-1β forward: 5¢- AACCTGCTGGTGTGTGACGTTC −3¢IL-1β reverse: 5¢- CAGCACGAGGCTTTTTTGTTGT −3¢https://biorender.com).BioRender was used to design some of the illustration throughout the manuscript . The specific statistical tests, exact value of n, what n represents, definition of center, and dispersion and precision of measures are mentioned in the figure legends.