We investigated the antioxidant potential of equine mesenchymal stem cells derived from muscle microbiopsies (mdMSCs), loaded by a water-soluble curcumin lysinate incorporated into hydroxypropyl-β-cyclodextrin (NDS27). The cell loading was rapid and dependent on NDS27 dosage . The immunomodulatory capacity of loaded mdMSCs was evaluated by ROS production, on active and total myeloperoxidase (MPO) degranulation and neutrophil extracellular trap (NET) formation after neutrophil stimulation. The intracellular protection of loaded cells was tested by an oxidative stress induced by cumene hydroperoxide. Results showed that 10 min of mdMSC loading with NDS27 did not affect their viability while reducing their metabolism. NDS27 loaded cells in presence of 14, 7 µM NDS27 inhibited more intensively the ROS production, the activity of the MPO released and bound to the NET after neutrophil stimulation. Furthermore, loaded cells powerfully inhibited intracellular ROS production induced by cumene as compared to control cells or cyclodextrin-loaded cells. Our results showed that the loading of mdMSCs with NDS27 significantly improved their antioxidant potential against the oxidative burst of neutrophil and protected them against intracellular ROS production. The improved antioxidant protective capacity of loaded mdMSCs could be applied to target inflammatory foci involving neutrophils. In response to inflammation, mesenchymal stem cells (MSCs) are known to migrate to tissue injury sites to participate in immune modulation, tissue remodelling and wound healing . MSCs ha2O2 with halides/pseudohalides to catalyse the formation of powerful reactive oxygen intermediates as hypochlorous acid, which causes protein, lipid and DNA oxidation and cellular damage pyridazine-1,4dione (L-012) was purchased from Fujifilm Wako Chemicals Europe GmbH . The bovine serum albumin fraction V (BSA) was obtained from Roche Diagnostics . Phorbol 12-myristate 13-acetate (PMA), cytochalasin B, N-formyl-methionylleucyl-phenylalanine (fMLP), sodium nitrite, Cumene hydroperoxide (C6H5-C(CH3)2-OOH) were purchased from Sigma-Aldrich . The 96-well microtiter plates (Combiplate 8 EB) and 96-well white plates, the fluorogenic substrate, Amplex red  (Invitrogen), trypsin TrypLE Express  and Hank’s balanced salt solution (HBSS) 1  and Fetal Bovine Serum (FBS) were purchased from Fischer Scientific . The Dulbecco’s Modified Eagle Medium Ham’s F12 (DMEM F12) culture medium with Hepes and glutamine, penicillin-streptomycin, amphotericin B and the Dulbecco’s phosphate buffer saline (DPBS) were purchased from Lonza . The equine MPO ELISA kit was purchased from BiopTis . The purified equine neutrophil MPO was obtained as previously described [Analytical-grade phosphate salts, sodium and potassium chloride, sodium hydroxide, sodium acetate, Hescribed  with theNDS27 the lysine salt of curcumin (curcumin lysinate), incorporated into hydroxypropyl-β-cyclodextrin (HPβCD) was provided by BiopTis . The main excipient of NDS27 2-hydroxypropyl-beta-cyclodextrin (HPβCD) was purchased from Roquette SA .2 in Dulbecco’s modified Eagle (DMEM) F-12 culture medium supplemented with 20% heat-inactivated fetal bovine serum (HI-FBS), 100 IU/mL of penicillin-streptomycin and 0.5% of amphotericin B. All experiments have been conducted with mdMSCs between passages five to eight according to the recommendations of Revatis to maintain their capacity for differentiation into adipocytes, osteoblasts, and chondrocytes.The equine skeletal mdMSCs obtained from muscle microinvasive biopsies , were pr2 culture flasks. For each experiment, cells from 4 T-flasks were generally used. At 75–80% confluency, the medium was removed, and cells were rinsed with 5 mL Dulbecco’s phosphate-buffered saline solution (DPBS). Then, 4 mL of synthetic trypsin  was added and incubated 5 min at 37 °C. After incubation, 6 mL of DPBS were added and the detached cells were transferred into 50 mL tube. These were centrifuged . The cell pellet was resuspended in 5 mL of HBSS and the cell count was determined. The volume of cell suspension (1 million cells/mL HBSS) was adapted to the objective of the experiment for the loading with NDS27 or HPβCD.Cells were seeded in 175 cm2O [−3 M in curcumin and serial dilutions were performed in distilled H2O to obtain NDS27 solutions containing 1.4, 0.7, 0.35 or 0.1 × 10−3 M curcumin.NDS27 is a complex of curcumin lysinate and HPβCD in a 1:5 ratio . NDS27 w2O : the sta2O at the starting stock solution of 3.5 × 10−2 M. From this solution, one dilution was performed in H2O to obtain 7 × 10−3 M.HPβCD the main excipient of NDS27 being in a 5-fold stoichiometric excess was tested for the highest corresponding concentration of curcumin. HPβCD was dissolved in distilled H2O. Different loading times at 37 °C were also evaluated: 2, 5, 10, 20 min.The same volume of each of these dilutions was added to the cell suspension in HBSS to be used at final concentrations of 14, 7, 3.5, 1 µM curcumin and 70 µM HPβCD during the loading. Control samples which did not contain the tested molecules were created by adding distilled Hg, 10 min, 37 °C) and the supernatant containing the excess of the drug was removed. The cell pellets were resuspended in the adequate volume of HBSS according to the experimental procedure.After incubation with the drugs, the cell suspensions were centrifuged (330× −3 M HPβCD or NDS27 (0.7 or 1.4 × 10−3 M) was added to the cell suspension and incubated 10 min at 37 °C. After incubation, the cell suspensions were centrifuged , the supernatants were removed, and the cells resuspended in 1 mL HBSS. Before the dead cell counting 50 µL of Trypan blue were added to 200 µL cell suspension and 10 µL were placed in a Bürker counting chamber before light microscopy observation.Cell viability was evaluated by the Trypan blue exclusion test . Cells wThe cell metabolism was evaluated by the MTS assay by using CellTiter 96 AQueous One Solution Cell Proliferation Assay (Promega). Total absorbance was measured using Microplate Reader at 490 nm. Ten µL of the MTS solution were added to each well containing 100 µL cell suspension. A first absorbance measurement was performed at 490 nm  followed by a further measurement 4 h later following plate incubation at 37 °C in darkness. The difference in absorbance was used for cell metabolism expressed in % of control cells without NDS27 loading and without cumene.g, 10 min, 22 °C), the supernatant was collected and 20 μL injected into the HPLC column . Curcumin was eluted at 22 °C with an elution gradient performed with 0.1% formic acid in water (A) and acetonitrile 100% (B) at a flow rate of 1 mL/min (Merck Hitachi L-7100 solvent delivery system). The gradient was set as follows: 0–5 min: 55% B; 6–7 min: 100% B and 8–10 min: 55% B. Eluted compounds were monitored with a Merck Hitachi L-7400 UV detector set at 419 nm, and EZ Chrom ELITE software was used for integration. The data collected by the chromatographic system were analyzed based on the peak area as compared with a standard curve obtained at the concentrations ranging from 0.625 to 10 µM synthetic curcumin.After loading, cell centrifugation, supernatant removal, and pellet suspension in 100 µL HBSS, curcumin was extracted by adding 300 µL methanol. Following incubation for 10 min in ultrasonic bath and centrifugation . The neutrophils were isolated according to the method described by Pycock et al.  and susp6 neutrophils/well of a white microtiter plate) were incubated for 10 min with 0.125 × 106 mdMSCs non-loaded or loaded with 70 µM HPβCD or 1, 3.5, 7 and 14 µM NDS27. Subsequently, 10 µL of L-012 (1.2 mg/mL in distilled water) and 10 µL of phorbol 12-myristate-13-acetate (PMA) (16 µM in 1% DMSO in ultra-pure H2O) were added to obtain with the cells a final concentration of 0.8 µM. After the PMA addition, the CL response was immediately monitored for 30 min and expressed as the integral value of total CL emission. Two controls were performed, one with the PMA activated neutrophils without mdMSCs, and the other with non-stimulated neutrophils and without mdMSCS, where PMA was replaced by its vehicle solution (1% DMSO in H2O).Dilutions of the cell suspension and reagents were performed to work with a final volume of 200 µL. The superoxide anion production was measured by chemiluminescence (CL) using L-012 probe. Neutrophils (0.5 × 106 cells) non-loaded or loaded with HPβCD or NDS27 were mixed in suspension with neutrophils (1 × 106 cells) in a total volume of 1.978 µL of DMEM F-12 medium supplemented with 20% FBS. For control assays, 1 × 106 neutrophils were used without mdMSCs. Cell suspensions were incubated 30 min at 37 °C; 2 µL of a cytochalasin B solution (2 mg/mL in DMSO) were added, except in control assays dedicated to non-activated neutrophils where cytochalasin B was replaced by 2 µL DMSO. Cell suspensions were again incubated 30 min at 37 °C before the addition of 20 µL of a 10−4 M fMLP solution (in 10% DMSO ultra-pure water) except in the controls with non-activated neutrophils where fMLP was replaced by 10% DMSO in ultra-pure water. The samples were let incubated for 24 h at 37 °C, then centrifuged  and the supernatants collected and frozen at −20 °C until MPO assay.MdMSCs  precoated on a microplate .2O2 and 10 mM sodium nitrite. The reaction was followed by fluorimetry. Total fluorescence developed during 30 min (37 °C) was monitored using a Fluoroskan Ascent (Thermo Scientific) set at 544 nm and 590 nm for excitation and emission wavelengths, respectively. Total fluorescence was directly proportional to the amount of active MPO present in the sample.Hundred µL of the MPO containing the sample were loaded on the SIEFED microplate. Sampling was performed in duplicate. After an incubation for 2 h at 37 °C, the samples were carefully removed, and the plate was drained on a paper towel to remove any remaining liquid. The wells were washed four times with a PBS solution containing 0.1% Tween 20. Finally, the peroxidase activity of the MPO captured by the antibodies was monitored by adding 100 μL of a 40 μM Amplex red solution. This was freshly prepared in a 50 mM phosphate buffer, pH 7.4, supplemented with 10 μM HAn ELISA assay kit  was used to measure the total equine MPO concentration in the collected supernatants, which were diluted 200× with 20 mM PBS containing 0.5% BSA and 0.1% Tween 20.6 mdMSCs) were mixed with neutrophils (1 × 106 cells) in 1.978 µL of DMEM F-12 medium supplemented with 20% FBS. In control assays, only 1 × 106 neutrophils were added. Cell suspensions were incubated 30 min at 37 °C, then 2 µL of a cytochalasin B solution (2 mg/mL in DMSO) were added. A control assay was dedicated for non-activated neutrophils, where cytochalasin B was replaced by its vehicle solution. Cell suspensions were again incubated 30 min at 37 °C before an addition of 20 µL of a 10−4 M fMLP solution (in 10% DMSO ultra-pure water) in all assays. In the control with non-activated neutrophils, fMLP was replaced by 10% DMSO in ultra-pure water. The samples were incubated 24 h at 37 °C. Subsequently, the tubes were centrifuged  and the supernatants were collected and frozen at −20 °C before measurement.HPβCD or NDS27 loaded and non-loaded mdMSCs  antibodies based on the method described by Thälin et al.  TransparThe total MPO bond to the NET was detected by using a second anti-MPO antibody  and the revelation technique from the ELISA assay kit see .Cumene hydroperoxide is an intracellular ROS generator : it was −2 M cumene hydroperoxide solution in ethanol. A control assay (Ctrl-) was performed with unloaded mdMSCs where cumene was replaced by ethanol. A further incubation (30 min at 37 °C) was performed, then 100 µL of the cell suspension were distributed in triplicate in the wells of white 96 well-microplates for CL measurement and in 96-well UV-transparent microplates for the cell metabolism assay using MTS.Unloaded mdMSCs and loaded mdMSCS with NDS27  or HPβCD (70 µM) were resuspended in 990 µL HBSS and treated with cumene through the addition of 10 µL of a 10Before measuring ROS production, 100 µL of equine MPO dissolved in DPBS (20 mU/mL) were added to each well. The ROS production was measured by chemiluminescence (CL) with an L-012 probe. Ten µL of a L-012 solution in water (1.2 mg/mL) were added to each well. After 30 min incubation at 37 °C, the CL response was monitored for 30 min at 37 °C  and the data were expressed as the integral value of the total CL emission.p-value < 0.05 was considered significant.Experiments were performed with cell batches from at least three horses for mdMSCs isolation and culture, and from five horses for blood sampling and neutrophil isolation. In some experiments, mdMSCs from the same horse were tested twice with neutrophil batches from two different horses. Each point in the experiment was performed at least in duplicate. Five independent experiments in duplicate were taken into consideration for the figures and statistical analysis. For the figures, data were expressed as mean standard deviation (SD) and given in relative values (%) by reference to control groups taken as 100%. As some populations or data series did not follow a Gaussian distribution, a nonparametric test was used: a two tail Mann–Whitney test was performed . A We succeeded in the obtention and culture of significant quantities of equine MSCs originating from skeletal muscle micro-biopsies  and deveIn recent years, several priming approaches have been proposed to empower MSC properties. This has given variable results ,35. ManyCultured MSCs were generally incubated several hours with native curcumin solubilized in DMSO or encapsulated in nanoparticles or micelles ,42. In oWe loaded mdMSCs with NDS27, a water-soluble form of curcumin consisting of curcumin lysinate encapsulated in hydroxypropyl-beta-cyclodextrin (HPβCD) . NDS27 iFor our further experiments, we adopted a 10 min incubation at 37 °C for mdMSCs loading.Only a slight decrease of cell viability (±6%) was observed after the cell loading for 10 min. . In paraSeveral studies demonstrated an activity of curcumin on mitochondria. In vitro curcumin mitigated the effects of cellular stress by decreasing the mitochondrial functions, including oxygen consumption, ATP production, calcium retention, and membrane potential . Curcumi2O2 [Previous studies demonstrated cytoprotective effects of curcumin pre-treatment (10 μmol/L for 24 h) on rat bone-marrow and adipose tissue-MSCs against an oxidative stress induced by an exposure to H2O2 ,21. Xu e2O2  found thTo our knowledge, the antioxidant capacity of curcumin-loaded cells has never been studied on the oxidant response of neutrophils. We previously observed that mdMSCs inhibited the ROS production by PMA-stimulated neutrophils . Here we2 consumption by the mitochondria [In addition to its protective effects against the oxidative burst of neutrophils, the loading of mdMSCs with NSD27 affords them the capacity to protect themselves against an intracellular oxidant stress  induced chondria . Our reschondria , who shoIn the present study, we detected ROS production following cumene hydroperoxide treatment, with the chemiluminescent probe (L012), a stable derivative of luminol that allows a sensitive measurement of the overall ROS production from both intra- and extracellular origins . OverallWe have developed a quick and efficient method to load, with a good yield (20%), equine mdMSCs suspensions with the water-soluble curcumin derivative NDS27. The loading decreases the cell metabolism but does not induce cytotoxicity;The loading with NDS27 increases the anti-inflammatory activity of mdMSCs against stimulated neutrophils, as measured by the lowering of ROS production and the significant inhibition of the catalytic activity of degranulated or NET- bound MPO;The loaded mdMSCs acquire a major capacity to decrease the intracellular ROS production induced by an oxidant stress while decreasing cell metabolism.From our results, the learn lessons are the followings:In conclusion, loading equine muscle mesenchymal stem cells with water-soluble curcumin NDS27 enhances their inhibitory properties against neutrophil oxidative burst and provides them with protection against oxidative attacks.An outcome of this study should be the potential clinical use of curcumin-loaded MSCs in inflammatory pathologies involving excessive neutrophil activity.


In this work, N-doped carbon quantum dots (CQDs) were prepared and loaded onto SnS2 nanoparticles via an in situ method. The resulting composite samples (NC@SnS2) were characterized, and their photocatalytic performance was discussed. SnS2 nanoparticles were obtained as hexagonal ones with a bandgap of 2.19 eV. The optimal doping level for NC@SnS2 was citric acid: urea:SnS2 = 1.2 mmol:1.8 mmol:3.0 mmol. It showed an average diameter of 40 nm and improved photocatalytic performance, compared to pure SnS2, following a pseudo-first-order reaction with a kinetic rate constant of 0.1144 min−1. Over 97% of Cr(VI) was photo-reduced after 30 min. It was confirmed that modification of SnS2 with CQDs can not only improve the light-harvesting ability but also stimulate the charge separation, which therefore can enhance the photoreactivity of SnS2 toward Cr(VI) reduction. The excellent stability of NC@SnS2 indicates that it is promising to be practically used in industrial wastewater purification.The photoreduction for hazardous Cr(VI) in industrial wastewater has been considered a “green” approach with low-cost and easy-to-go operation. SnS Here, w2O2CO3 and Bi2WO6@Bi2S3 with visible photoreactivity for NO oxidation and brilliant red X-3B dye (X3B) (2) seems attractive due to its perfect spectral matching with solar spectrum, low cost, and more importantly, low bio-toxicity  . These wtoxicity . A problimproved .2/sp3-hybridized C atoms, with a narrow bandgap covering the whole visible region and even the near-infrared region. In addition, their optoelectronic property, including their bandgap, can be conveniently modified by chemical modifications, offering various kinds of CQDs to meet the requirement for photocatalytic application  have drawn much research attention owing to their virtues of chemical stability, bio-compatibility, economic synthesis, and excellent photoactivity, along with their promising photoelectronic and photocatalytic performance . For CQDlication .2 with these N-doped CQDs. Here, the doped N atoms in CQDs are supposed to offer a midgap for the photogenerated charge carriers in SnS2 semiconductors, so that the electron mobility can be improved, which promotes the separation and transportation of photogenerated charge carriers. As a consequence, improved photocatalytic efficiency from the resulting composite structure (denoted as NC@SnS2) is expected. A schematic presentation for the synthetic strategy of NC@SnS2 is shown in 2 is performed, and corresponding photocatalytic performance for Cr(VI) photoreduction is discussed as well.Guided by the aforementioned consideration, in this work, we intend to prepare N-doped CQDs and modify SnS4, thioacetamide (TAA), citric acid (CA), urea (UA), and Nafion. Micromorphology analysis was performed using a Hitachi S4800 (SEM) microscope , an Oxford X-Max80 field emission scanning electron microscope , a Tecnai F20 (HRTEM), and a Talos F200X (FEI). IR, Raman, and XPS spectra were recorded on a Nicolet iS50 spectrophotometer (KBr method), a Renishaw inVia Raman microscope (excitation = 532 nm), and an ESCALAB250 X-ray Photoelectron spectrometer, respectively. Zeta potentials were determined by electrophoretic light scattering with a zeta sizer . XRD diffraction data were collected on a Rigaku MiniFlex Ultima IV diffractometer  with an operating voltage of 40 kV and a current of 15 mA. UV-Vis diffuse reflectance spectra (DRS) were recorded using a Hitachi U-4100 spectrophotometer equipped with an integrating sphere. UV-Vis absorption spectra were obtained using a Hitachi UV-2450 UV-Vis spectrophotometer. Electrochemical parameters were determined by a lock-in-based surface photovoltage (SPV) and an electrochemical workstation (CHI 760E).All chemical reagents used in this work were AR-grade ones, including SnCl2 nanoparticles were synthesized as follows: a homogeneous solution was prepared by mixing SnCl4 and TAA with deionized water (molar ratio = 1:3 in 70 ml). This solution was sealed in a Teflon autoclave and heated at 190oC for 6 h. The solid product was separated by centrifugation  and washed by H2O/EtOH (1:1). Before usage, the as-synthesized SnS2 nanoparticles were activated at 60oC for 12 h.SnS2 was synthesized by an in situ assembly method described as follows. A homogeneous solution was prepared by mixing CA and UA with deionized water (in 25 ml). The previously obtained SnS2 nanoparticles (3 mmol) were added and stirred for 30 min (×600 rpm). This suspension was sealed in a Teflon autoclave and heated at 160oC for 4 h. After natural cooling, EtOH (50 ml) was added. The solid product was separated by centrifugation  and washed by H2O/EtOH (1:1). The obtained solid powder was dried at 80oC for 12 h and denoted as NC2-1@SnS2 (CA:UA = 0.30 mmol:0.45 mmol), NC2-2@SnS2 (CA:UA = 0.50 mmol:0.75 mmol), NC2-3@SnS2 (CA:UA = 0.90 mmol:1.35 mmol), NC2-4@SnS2 (CA:UA = 1.2 mmol:1.8 mmol), and NC2-5@SnS2 (CA:UA = 1.50 mmol:2.25 mmol).NC@SnS2 samples were synthesized for performance comparison and denoted as NC1@SnS2 (CA:UA = 1.2 mmol:3.6 mmol) and NC3@SnS2 (CA:UA = 1.2 mmol:1.2 mmol).By varying CA:UA ratio, two more NC@SnS2 nanoparticles were first prepared as the supporting host; then, a one-step in situ assembly method was used to synthesize NC@SnS2 samples by directly synthesizing/depositing CQDs on SnS2 surface, as shown in 2 nanoparticles. This in situ assembly method simplified synthetic operation. In addition, the ratio of CA:UA was changed to modify the N-doping level in NC@SnS2 . By fixing the ratio of CA:UA but changing the total amount of CA and UA, the CQD account in NC@SnS2 was modified as well (corresponding to the NC2@SnS2 series). For discussion convenience, CQDs (denoted as NC2 CQDs) were prepared as well, following a similar operation. It can be noticed from the following Section 2.4 that NC2 CQDs were N-doped ones, aiming at the desired performance.As mentioned in oC for 4 h. After natural cooling, EtOH (50 ml) was added. The solid product was separated by centrifugation  and washed by H2O/EtOH (1:1). The obtained solid powder was dried in a freeze dryer for 2 days and denoted as NC2 CQDs.For performance comparison, reference CQDs were synthesized. A homogeneous solution was prepared by mixing CA and UA with deionized water (1.2 mmol:1.8 mmol in 25 ml). This solution was sealed in a Teflon autoclave and heated at 1602 samples was performed in a quartz reactor equipped with a cooling accessory via a water bath. The solar spectrum (excitation source) was simulated using a Xe lamp  and a light filter (400 nm cutoff). The reaction temperature was set at 20oC (±2oC). NC@SnS2 (30 mg) was added into K2Cr2O7 aqueous solution (50 mg/L) and treated with an ultrasonic bath for 10 min. Then, this suspension was placed in dark for 2 h to allow full adsorption for photocatalyst. Finally, this mixture was exposed to the solar spectrum. For photoreaction monitoring, 0.3 ml of suspension was extracted each time  and filtered off using a cellulose acetate membrane filter (pore size = 0.22 nm). The resulting solution was treated with diphenylcarbazide  and sent for UV-Vis absorption titration  on the surface of the photocatalyst is negligible (less than 2%), and modification of SnS2 with CODs has little effect on Cr(VI) adsorption.The photocatalytic performance of NC@SnSitration . Cr(VI) 2 samples was evaluated as follows: after photoreaction, NC@SnS2 samples were filtered off and washed with plenty of freshwater. After being dried at 80oC for 12 h, the recycled NC@SnS2 samples were obtained.The recycling performance of NC@SnS2 as the working electrode (surface area = 0.17 cm2), using the electrolyte of Na2SO4, 0.2 M. The working electrode was prepared as follows: A mixture of NC@SnS2 (50 mg), deionized water (450 μL), EtOH (500 μL), and Nafion (5 μL) was treated with an ultrasonic bath for 10 min. Then, this mixture was spin-coated onto an FTO glass electrode (1.5 cm2 × 2.0 cm2) and heated at 400oC for 60 min under a pure N2 atmosphere. Mott–Schottky plots were sampled at 1,000 Hz, 2000 and 3,000 Hz. Electrochemical impedance spectra (EIS) were recorded at −1.4 V (vs. Ag/AgCl) within 0.1 Hz–10 kHz, using an AC sinusoidal perturbation (10 mV).Photo-generated charge transfer behavior was evaluated using an electrochemical workstation (CHI 760E). A three-electrode system was selected, with a Pt wire as the counter electrode, Ag/AgCl (saturated KCl) as the reference electrode, and NC@SnS2 was prepared by a one-step in situ assembly method, where the N atoms in CQDs are supposed to offer a midgap for the photogenerated charge carriers in SnS2. This midgap is supposed to improve electron mobility and thus promote the separation and transportation of photogenerated charge carriers. Simply speaking, here, the active site of NC@SnS2 should be the N-doped CQDs which capture excited electrons. The successful synthesis and assembly of NC2 CQDs, SnS2 nanoparticles, and NC@SnS2 samples are first discussed by comparing their XRD patterns. It is observed from 2 samples are rather similar to those of as-synthesized SnS2 nanoparticles. Sharp diffraction peaks are observed, which confirms the high crystallinity of SnS2. All these diffraction peaks shall be indexed to a hexagonal SnS2 structure (JCPDS Card No. 23-0677). As for NC2 CQDs, a broad diffraction peak with 2θ = ∼27o is observed, coming from the highly disordered C atoms  peak (vs. other diffraction peaks) between NC@SnS2 and as-synthesized SnS2 nanoparticles, which means an interaction between NC2 CQDs and SnS2 that causes variations in crystal size and preferred orientation. It appears that NC@SnS2 samples tend to grow along the c-axis direction, owing to below two reasons. First, there are enough functional groups on the CQD surface, which allows further nucleation. In addition, SnS2 nanoparticles tend to aggregate in the presence of NC2 CQDs, which will be confirmed by the following morphology analysis.As previously mentioned, NC@SnS C atoms . This br2 was finished by a controllable in situ assembly method. Here, NC2 CQDs were synthesized and directly loaded onto SnS2 nanoparticles under hydrothermal conditions through an electrostatic effect. To confirm the successful in situ assembly between SnS2 host and N-doped CQDs, their zeta potential values are determined as +4.30 mV for the SnS2 nanoparticles and −9.40 mV for NC2 CQDs (both dispersed in deionized water). There shall be strong electrostatic attraction between SnS2 host and N-doped CQDs, which leads to an assembly between them. The zeta potential value of a representative NC@SnS2 sample, NC2-4@SnS2, is determined as −4.50 mV, which is close to that of NC2 CQDs (−9.40 mV), compared to that of SnS2 nanoparticles (+4.30 mV). This is because SnS2 nanoparticles are covered by NC2 CQDs whose surface has negative groups such as -COO-. As a consequence, it is concluded that SnS2 nanoparticles have been successfully covered by NC2 CQDs.The synthesis of NC@SnSin situ assembly is further analyzed via morphological analysis, which gives a visual understanding of NC@SnS2 samples. Here, the morphology of a representative NC@SnS2 sample, NC2-4@SnS2, is shown in 2, its SEM image suggests that the hexagonal morphology has been well preserved after loading NC2 CQDs. The average diameter of these NC2-4@SnS2 particles is 40 nm. As depicted by their TEM and HRTEM images, there are obvious lattice fringes in NC2-4@SnS2, with lattice spacing distances of 0.589 and 0.3 nm, respectively. The former one is attributed to the plane (001) from the SnS2 host. After comparing with the TEM details of NC2 CQDs shown in 2 tentatively confirms the successful loading of CQDs on SnS2 nanoparticles. It is still observed that NC2 CQDs tend to stack on the SnS2 surface, showing a growth along the c-axis ; this is because the abundant functional groups of NC2 CQDs endow themselves with high affinity for SnS2 nanoparticles, leading to the stacking along c-axis. This morphological analysis is consistent with the above XRD analysis. The in-situ assembly of NC2 CQDs on SnS2 nanoparticles is thus confirmed.The successful 2 nanoparticles. IR spectra of NC2 CQDs, SnS2 nanoparticles, and NC@SnS2 samples are recorded and compared in 2 can be confirmed. The SnS2 IR spectrum is a simple one with only several peaks. The broad one around 3,414 cm−1 is attributed to the vibration of the –OH group (adsorbed water), while the sharp one at 1,657 cm−1 is attributed to the absorption of unsaturated bonds on the SnS2 surface , N-H, C=N, and COO− groups  and C=N  are still traced. More obviously, the IR band from unsaturated bonds on the SnS2 surface is blue-shifted to 1715 cm−1, compared to that of SnS2 nanoparticles . This spectral shift suggests that the SnS2 surface has been covered by CQDs, which affects SnS2 unsaturated bonds and CQDs C=N bonds. Additionally, the IR absorption from CQDs COO− groups is not observed in NC@SnS2 samples, suggesting that these COO− groups have been grafted onto the SnS2 surface.IR technique shall provide more details when analyzing the loading status of CQDs on SnS surface . As for − groups . After o2 is shown in 2 samples. There are intense vibrational peaks of 215 cm−1, 309 cm−1, and 587 cm−1, which are attributed to first-order Eg, A1g, and second-order Eg of SnS2, while those of 1,347 cm−1 and 1,600 cm−1 are assigned as the Raman-active graphic (G) and defect (D) bands from CQDs  and S  atoms are observed, confirming the elemental composition of NC2-4@SnS2.Raman spectrum is a powerful tool to reveal sample surface chemistry. The Raman spectrum of NC2-4@SnSrom CQDs . In addi1/2 and S 2p3/2 (162.65 and 161.45 eV) in NC2-4@SnS2 are decreased by ∼0.4 eV, compared to those in SnS2 nanoparticles. Similarly, the N 1 s binding energy values of C-N (400.98 eV) and C=N (401.97 eV) in NC2-4@SnS2 are smaller than those in NC2 CQDs. The XPS peaks from C=O, C-OH, and C-O-O groups have been weakened or minimized in NC2-4@SnS2, compared to those in NC2 CQDs  than carbon (2.5), the doped N atoms shall serve as an electron acceptor. This hypothesis is approved by the following analysis on XPS binding energy. As shown by 2 photocatalyst was confirmed. An improved photocatalytic performance was expected from NC@SnS2. Here, the photoreduction performance of NC@SnS2 samples was evaluated via Cr(VI) photoreaction. Photocatalytic performance comparison between NC@SnS2 samples, having different CQD amounts and different N doping levels was performed, with NC2 CQDs and as-synthesized SnS2 nanoparticles as reference groups. Here, diphenylcarbazide was used as a chromogenic agent, forming a purple-red compound in an acidic solution with absorption at 540 nm. Cr(VI) amount was proportional to the absorbance at 540 nm, so its photoreduction percentage (C/C0) was calculated by A/A0, where A0 was the initial absorbance value  solution remains nearly constant (around 1.0) within the reaction time region 0-30 min, which means that Cr(VI) self-reduction is slim and neglectable. The existence of NC2 CQDs leads to a rather slim decrease (C/C0 = 0.97 at 30 min). Although NC2 CQDs have intense and broad absorption in the visible region (400–800 nm), the absorbed photons and their energy are wasted by thermal vibration, instead of participating in Cr(VI) photoreduction, which may be caused by the low efficiency of charge separation and transfer. An obvious Cr(VI) photoreduction is observed for as-synthesized SnS2 nanoparticles (C/C0 = 0.46 at 30 min). As for NC@SnS2 samples, their Cr(VI) photoreduction percentage is further increased, with C/C0 = 0.03 at 30 min for NC2-4@SnS2, for example. It is assumed that the conjugation planes in CQDs may accept the excited electrons from SnS2, which limits the recombination of photo-induced electron-hole pairs and thus improves the charge transfer separation efficiency of SnS2.Based on the above analysis and discussion, the successful synthesis of NC@SnSce value . It is o2 samples with different CQD amounts and different N doping levels, as shown in 2) or less (NC1@SNS2) N-doping level in NC@SnS2, compared to NC2@SNS2, the photocatalytic performance is simultaneously weakened. This is because N atoms in CQDs help the electron capture procedure and accelerate charge carrier separation. On the other hand, the excess N atoms in CQDs may affect the CQDs conjugation plane and compromise charge carrier separation. As a consequence, there will be an optimal N-doping level, with strong enough electron capture and limited influence on the integrality of the CQD conjugation plane.Based on the previoushypothesis, it is assumed that a suitable amount of CQDs with an optimal N doping level, which ensures an efficient electron capture, is required for optimal photocatalytic performance. This statement is supported by the photocatalytic performance comparison between NC@SnS2 samples , their photocatalytic performance is different from each other, which means that the CQD amount plays an important role as well. Generally speaking, the photocatalytic performance of NC2@SnS2 is improved with increasing CQD amount. This observation is consistent with the aforementioned statement that CQDs help the electron capture procedure and accelerate charge carrier separation. Yet, NC2-5@SnS2 shows the worst photocatalytic performance among NC2@SnS2 samples, although its CQD amount is the highest. This observation is explained by the weakened light harvest since the excess CQDs may worsen SnS2 light absorption and thus compromise the generation of photo-induced electron-hole pairs. Apparently, the generation of photo-induced electron–hole pairs is controlled by SnS2 light harvest, not CQD light harvest. Excitation light shall first penetrate CQDs before reaching SnS2 and being absorbed by SnS2. In other words, SnS2 light harvest is limited by the outer CQDs, which denies a further photocatalytic efficiency improvement. This is a defect for the NC@SnS2 composite structure.Even with the same N-doping level in NC2@SnS2 samples, C/C0 against reaction time is analyzed by a pseudo-first-order reaction, as described by k is the kinetic rate constant and t is reaction time:For a better understanding of the photoreduction dynamics of NC@SnS2 is the highest one among NC@SnS2 samples.The detailed fitting parameters are listed in 2 samples, their mechanism is discussed via their UV-Vis diffuse reflectance spectra (DRS), as shown in -, for example). As for the as-synthesized SnS2, a broad but weak absorption band covering the whole UV-Vis region  is observed, which shall be attributed to the intrinsic band-to-band transitions in SnS2. After loading CQDs, NC@SnS2 DRS shows basically the DRS character of NC2 CQDs, with an obvious red shift of ∼25 nm. The close contact between NC2 CQDs and SnS2, as confirmed previously, shall neutralize the quenching centers in CQDs, which makes CQDs a photosensitizer with a midgap for SnS2, showing the aforementioned absorption redshift  is calculated by Tauc plots (Kubelk–Munk method), as described in It has been reported that SnSα is the absorption coefficient, h is the Planck constant, υ is frequency, and B is the fitting constant. With the DRS data on hand, the E value of SnS2 is calculated as 2.19 eV, as shown in 2 has been determined as −0.19 eV (against RHE), the valence band (VB) level can be calculated as 2.00 eV  to finish the photoreduction. As for the hole in VB, it shall be quenched by H2O, with molecular oxygen generated. This proposed mechanism is presented in Here,  2.00 eV . When Sn2, NC2 CQDs, and as-synthesized SnS2 nanoparticles are compared in 2 (1.85 eV) is lower than those of NC2 CQDs (2.30 eV) and as-synthesized SnS2 nanoparticles (2.05 eV), indicating that NC2 CQDs decrease NC2-4@SnS2 excitation energy, which is consistent with the finding of their DRS comparison. Higher SPV values are observed for NC2-4@SnS2 (maximum = 161 μV) within photon energy region 2.25-3.75 eV, compared to those of NC2 CQDs (maximum = 21 μV) and as-synthesized SnS2 nanoparticles (maximum = 128 μV), which means that NC2 CQDs facilitate the charge carrier separation in NC2-4@SnS2. Meanwhile, the smallest radius is observed for the NC2-4@SnS2 EIS Nynquist plot, among the radii of NC2 CQDs and as-synthesized SnS2 nanoparticles, as shown in 2 is thus finally confirmed here.To strengthen the previousstatement, surface photovoltage (SPV) and electrochemical impedance spectra (EIS) of NC2-4@SnS2 is then discussed as follows: 2. It is observed that the photocatalytic performance has been well preserved after the first five cycles, and then the photoreduction efficiency tends to decrease. Nevertheless, as shown in 2 (after 6 cycles) are nearly identical to those of fresh NC2-4@SnS2, with no obvious shifts or intensity variations. As a consequence, the decreased photoreduction efficiency may be explained by the desorption of CQDs from NC2-4@SnS2. Considering that there is only Van der Waals' force, instead of strong chemical bonds, between CQDs and SnS2, it is highly possible that the CQDs are extracted and removed from the SnS2 surface, leading to decreased photoreduction efficiency. A down-bending tendency is observed for the recycled NC2-4@SnS2, especially for the sixth-recycled NC2-4@SnS2, which tentatively confirms the desorption of CQDs from NC2-4@SnS2.The recyclability and durability performance of NC2-4@SnS2 nanoparticles via an in situ method. The resulting composite samples (NC@SnS2) were characterized by means of XRD, zeta potential, SEM/TEM, IR, Raman, and XPS to confirm their composite structure. Various N-doping levels and doping amounts were tried to optimize photocatalytic performance. The optimal NC@SnS2 showed improved photocatalytic performance, compared to pure SnS2, following a pseudo-first-order reaction with a kinetic rate constant of 0.1144 min−1. It was confirmed by diffuse reflectance spectra, surface photovoltage, and electrochemical impedance spectra that the loaded CQDs increased the charge carrier separation efficiency of SnS2. Good durability was observed for NC@SnS2, with at least five full cycles. On the other hand, the charge carrier separation efficiency of NC2-4@SnS2 is still yet to be satisfied. For further efforts, the holes in SnS2 VB shall be neutralized quickly to improve charge carrier separation efficiency. In addition, the loaded CQDs served only as a midgap for SnS2 but showed no contribution to SnS2 absorption. The intrinsic absorption of SnS2 should be enhanced to improve its absorption of excitation light. Finally, the durability of NC@SnS2 should be improved as well. Our primitive suggestion is to replace N-doped CQDs (carbon quantum dots) with N/P-codoped CQDs  were prepared and loaded onto SnSped CQDs . It has ructure) . In this


Vascular smooth muscle cell (VSMC) phenotype switching is critical for neointima formation, which is the major cause of restenosis after stenting or coronary artery bypass grafting. However, the epigenetic mechanisms regulating phenotype switching of VSMCs, especially histone methylation, are not well understood. As a main component of histone lysine demethylases, Jumonji demethylases might be involved in VSMC phenotype switching and neointima formation.A mouse carotid injury model and VSMC proliferation model were constructed to investigate the relationship between histone methylation of H3K36 (downstream target molecule of Jumonji demethylase) and neointima formation. We found that the methylation levels of H3K36 negatively correlated with VSMC proliferation and neointima formation. Next, we revealed that JIB-04 (a pan-inhibitor of the Jumonji demethylase superfamily) could increase the methylation levels of H3K36. Furthermore, we found that JIB-04 obviously inhibited HASMC proliferation, and a cell cycle assay showed that JIB-04 caused G2/M phase arrest in HASMCs by inhibiting the phosphorylation of RB and CDC2 and promoting the phosphorylation of CHK1. Moreover, JIB-04 inhibited the expression of MMP2 to suppress the migration of HASMCs and repressed the expression of contraction-related genes. RNA sequencing analysis showed that the biological processes associated with the cell cycle and autophagy were enriched by using Gene Ontology analysis after HASMCs were treated with JIB-04. Furthermore, we demonstrated that JIB-04 impairs autophagic flux by downregulating STX17 and RAB7 expression to inhibit the fusion of autophagosomes and lysosomes.JIB-04 suppresses the proliferation, migration, and contractile phenotype of HASMCs by inhibiting autophagic flux, which indicates that JIB-04 is a promising reagent for the treatment of neointima formation.The online version contains supplementary material available at 10.1186/s13148-022-01321-8. Cardiovascular diseases (CVDs) are the leading cause of death worldwide, the prevalence and mortality of which were approximately 485.6 million cases and 17.8 million cases in 2017, respectively . AlthougThe proliferation, migration, and phenotypic switching from contractile to synthetic phenotypes of vascular smooth muscle cells (VSMCs) are critical for neointima formation and vascular restenosis . Recentl2 in the hydroxylation reaction, in which unstable hemiaminal products are produced and broken into formaldehyde and a demethylated histone product [The Jumonji C domain-containing protein (JMJD) family, a main component of histone lysine demethylases (KDMs), are oxygenases that achieve demethylation through hydroxylation reactions. JMJD demethylases catalyze α-ketoglutarate (αKG), oxygen, and Fe(II) to produce succinate and CO product , 12. Rec product , and dem product , 13. TheIn the present study, to explore the roles and mechanisms of KDMs in neointima formation, we used JIB-04, a pan-inhibitor of JMJDs, to treat human aortic smooth muscle cells (HASMCs). We found that JIB-04 inhibited the proliferation, migration, and contractile phenotype of HASMCs by regulating H3K36 methylation. More importantly, we revealed that JIB-04 impaired the fusion of autophagosomes and lysosomes by downregulating STX17 and RAB7 expression to affect the phenotypic switching of HASMCs. Thus, our results indicated that JIB-04 might be a promising drug for inhibiting neointima formation and improving restenosis after CABG or stenting.We first established a HASMC proliferation model by stimulating cells with different concentrations of fetal bovine serum (FBS). We further detected the methylation levels of H3K4, H3K9, and H3K36, which were potential targets of JMJDs, in the HASMC proliferation model. The results showed that compared with that of the 0.5% FBS group, the mono-, di-, and trimethylation of H3K36 was decreased in the HASMCs treated with 2% or 10% FBS  and evaluated cell viability by using CCK-8 and LDH assays. The results showed that JIB-04 had a dose-dependent effect on the viability of HASMCs and had little toxicity to HASMCs at concentrations below 1 μmol/L  was significantly downregulated in the HASMCs treated with JIB-04  showed clear clustering of the samples for the two groups Fig. A. AccordIntriguingly, in addition to cell cycle- and mitosis-related pathways, autophagy was also enriched according to Gene Set Enrichment Analysis (GSEA) and Kyoto Encyclopedia of Genes and Genomes (KEGG) Fig. A and B. In recent years, epigenetic modification has emerged as a promising target for cardiovascular disease treatment , 21. As In the primary cultured HASMCs treated with different concentrations of FBS to induce proliferation, we found that the levels of H3K36me3, H3K9me3, and H3K4me3 were downregulated. Furthermore, in the mouse model of neointima formation, lower levels of H3K36me3 and H3K4me3, but a higher level of H3K9me3 in the injured carotid arteries than in normal controls. These results suggested that there is a paradox of H3K9 methylation on proliferation in vivo and in vitro, or the function of H3K9 methylation is stimulation-dependent. In the HASMCs treated with JIB-04, the methylation levels of H3K36 and H3K9, but not H3K4 were upregulated, which indicated that JIB-04 only increases H3K36 and H3K9 methylation levels in HASMCs. Based on these results, we were prudent to conclude that JIB-04 regulates phenotypic switching of VSMCs by upregulating the methylation of H3K36.Neointima formation is mainly caused by excessive proliferation of VSMCs, and cell cycle checkpoints control cell division and proliferation . In geneGiven that inhibition of VSMC proliferation is often accompanied by altered migratory capacity, we demonstrated that JIB-04 also inhibited the migration of HASMCs by downregulating the expression of MMP2 but not MMP9. MMP2 is a zinc-dependent enzyme cleaving components of the extracellular matrix to facilitate cell migration . In addiAutophagy is a highly conserved catabolic process for the preservation of cellular and organismal homeostasis . AutophaIn conclusion, we demonstrated that JIB-04, a pan-inhibitor of JMJD demethylases, inhibited the proliferation and migration of HASMCs by arresting cells at G2/M checkpoints and inhibiting MMP2 expression, respectively. However, JIB-04 also downregulated the expression of STX17 and RAB7 to disrupt the fusion of autophagosomes with lysosomes. Our results suggested that JIB-04 can affect the function of VSMCs by regulating proliferation and autophagy and is expected to be a candidate drug for the prevention or treatment of neointima formation.All animal experiments were performed in accordance with the protocols approved by the Animal Care and Use Committees of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. Male C57BL/6 mice were housed in specific-pathogen-free facilities with a 12-h light/dark cycle and controlled temperature (20–22 °C). The carotid artery injury model was established as reported previously . Briefly2O2. Tissue sections were blocked with 5% BSA after washing with phosphate-buffered saline (PBS). Primary antibodies against H3K36me3 were used to incubate sections at 4 ℃ overnight, followed by incubation with secondary antibodies for 20 min. Afterward, the sections were stained with diaminobenzidine (DAB) and counterstained with hematoxylin. Sections were then observed after dehydration with gradient ethanol and vitrification with xylene.Histological analysis and immunohistochemistry were performed as reported previously . In shor2.HASMCs were extracted from aortic tissue trimmed from heart donors during transplantation surgery, and HASMCs were cultivated as reported previously . In brie3 cells/100 μL per well were plated in 96-well plates and treated with a JIB-04 concentration gradient  for 24 h. CCK-8 reagent (10 μL per well) was added to DMEM per well and incubated at 37 °C for 1.5 h, and the absorbance at 450 nm was measured using a BioTek Synergy HT microplate reader. For measurement of the effect of JIB-04 on proliferation, a total of 4 × 103 cells/100 μL per well were plated in 96-well plates and treated with DMSO or 0.5 μM JIB-04 for 0 h, 24 h, 48 h, or 72 h. Then, CCK-8 reagent was added, and 450 nm absorbance was detected as described above.A CCK-8  assay was performed to measure the toxicity of JIB-04 on HASMCs and the effect of JIB-04 on HASMC proliferation. For determination of the toxicity of JIB-04, a total of 8 × 103 cells/100 μL per well were plated in 96-well plates, which were treated with a JIB-04 concentration gradient  for 24 h. Then, 10 μL of lysis buffer was added to the high contrast well and incubated at 37 °C for 30 min. Then, 100 μL working solution was added to each well and cultivated at room temperature in the dark for 15 min. Next, 50 μL of stop solution was added to each well, and the absorbance at 490 nm was measured using a BioTek Synergy HT microplate reader.A lactate dehydrogenase (LDH) assay was performed to evaluate the toxicity of JIB-04 on HASMCs by a Cytotoxicity LDH Assay Kit-WST  as reported previously . A total4 cells per well and incubated with EdU medium (50 µmol/L) made up of EdU solution and complete medium at a ratio of 1:1000 for 4 h. Cells were fixed with 4% paraformaldehyde for 30 min after washing with by PBS. Then, the cells were incubated with glycine (2 mg/mL) to neutralize paraformaldehyde and were incubated with a penetrant made up of PBS and 0.5% Triton X-100 for 10 min. Cells were colored with 1 × Apollo staining solution and then DNA was stained with DAPI after the incubation with 0.5% Triton X-100 PBS solution again. The cells were washed with PBS and observed by fluorescence microscopy. Images were captured by an Olympus light microscope BX53 system.A Cell-Light™ Edu Apollo 567 In Vitro kit  was used to perform the EdU incorporation assay. HASMCs treated with DMSO or 0.5 μM JIB-04 were plated in 24-well plates at 2 × 10After cultivation and treatment with DMSO or 0.5 μM JIB-04 on cell climbing slices for 24 h, HASMCs were fixed with 4% paraformaldehyde for 30 min, followed by penetrating the cell membrane with 0.5% Triton X-100 for 30 min. Then, the samples were blocked by using 5% BSA for 60 min at room temperature. Subsequently, the primary antibodies against Ki67  and α-SMA  were incubated overnight at 4 °C. The next day, the Alexa Fluor 568 donkey anti-rabbit IgG (H + L)  secondary antibody and Alexa Fluor 488 donkey anti-mouse IgG (H + L)  secondary antibody were incubated in Ki67 and α-SMA cell climbing slices, respectively, for 60 min. DAPI was used to stain nuclei after washing with PBS three times. An Olympus light microscope BX53 system was applied for image capture.HASMCs were collected by trypsin digestion, followed by washing twice with phosphate-buffered saline (PBS). Cells were collected by centrifugation for 10 min. After the cells were resuspended with PBS, 70% ethanol precooled at − 20 °C was added to fix the cells at 4 °C overnight. The next day, the cells were washed with PBS twice after discarding ethanol. Then, 150 μL of Ribonuclease A  and 150 μL of PI  were applied to stain cells for 4 h in darkness. The stained cells were sorted by using a BD FACSAria™ III sorter, and Flow Jo software (version 10.7.2) was selected for cell cycle analysis.4 cells/100 μL per well) were seeded onto transwell inserts with a polyethylene terephthalate membrane pore size of 8 μm  in 24-well plates. Transwell inserts were transferred and submerged in 800 μL of DMEM/F12 with 10% fetal bovine serum after adherence for 2 h. After migration for 12 h, media within the transwell inserts were carefully removed. Cells were fixed with 4% paraformaldehyde and stained with crystal violet (Sigma-Aldrich). Cells that did not migrate across the transwell membrane were then removed by gentle wiping with a cotton swab. Migrated cells were viewed with an inverted microscope (Nikon).After treatment with DMSO or JIB-04 (0.5 μM) for 24 h, HASMCs were collected by trypsin digestion and resuspended in DMEM/F12 with 0.5% fetal bovine serum. HASMCs  or 0.5 μM JIB-04 (JIB-04 group). After treatment for 48 h, the RNA of HASMCs was extracted and processed by using an RNA-seq pipeline by Novogene Co., Ltd. . The RNA integrity was evaluated by the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system . After quantitative processing, the count files were used as input to the R (version 4.1.2) package DEseq2, and the differentially expressed genes were analyzed based on the negative binomial distribution model. To improve accuracy, we adopted Benjamini and Hochberg’s approach to adjust P value. We screened the differentially expressed genes by an absolute value of logfold change greater than 0.7 and an adjusted P value less than 0.05. Based on the differentially expressed genes, KEGG, GO, and GSEA were performed by the clusterProfiler (v3.16.1) R package .+/RFP+), while the red puncta were considered autophagolysosomes (GFP−/RFP+). The autophagic flux was assessed by counting the number of yellow and red puncta.For monitoring autophagic flux, mRFP-GFP-LC3 lentivirus packed with the mRFP-GFP-LC3 plasmid was used to mark and track LC3. HASMCs were transfected with LC3-mRFP-GFP lentivirus for 24 h. For synchronization, HASMCs were cultivated with DMEM/F12 without fetal bovine serum for 12 h. Then, HASMCs were incubated with JIB-04 (0.5 μM) for 48 h, rapamycin (150 nM) for 12 h and CQ (20 μM) for 12 h. Images were observed under an Olympus light microscope BX53 system. GFP was quenched when the pH decreased, while mRFP remained stable. Thus, after merging the green and red fluorescence images, the yellow puncta were considered autophagosomes (GFPThe antibodies used in this study included α-SMA (ab7817), SM22α (ab14106), H3K9me1 (ab9045), H3K9me2 (ab1220), H3K4me1 (ab8895), H3K36me2 (ab9049), H3K36me3 (ab9050), which were bought from Abcam. β-actin (AC026) and RAB7 (A12308) were got from ABclonal. H3K9me3 (GTX121677), MMP2 (GTX634832), MYH10 (GTX634160), MMP9 (GTX100458), PCNA (GTX100539) were purchased from GeneTex. P-H3 (sc-8656-R) was obtained from Santa Cruz. LAMP3 (AP1827A) was bought from Abgent. STX17 (HPA001204) was got from ATLAS. H3K4me2 (#9725), H3K4me3 (#9727), H3K36me1 , p-AKT (#4060), AKT (#4685), p-FOXO3 (#9466), FOXO3A , p-P38 (#4511), P38(#8690), p-CDC2 (#4539), p-Rb (#8516), Rb (#9313), p-CHK1(#2348), P-CHK2 (#2197), LC3A/B , SQSTM1 , p-AMPKα (#5831), AMPKα (#2535), p-mTOR (#5536), mTOR (#2983), LAMP1 (#9091), and COL1A1  were purchased from Cell Signaling Technology.Total proteins derived from HASMCs were extracted by a complete cell lysis solution made up of RIPA and PMSF at a ratio of 100 to 1. Ultrasound and 95 °C high temperature were used to denaturant proteins. After denaturation, equal amounts of protein were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to a polyvinylidene fluoride membrane , which was blocked with 5% skim milk in Tris Tween-buffered saline. After that, the membrane was incubated with the indicated primary antibody overnight at 4 °C and the peroxidase-conjugated secondary antibody  for 1 h at room temperature. Protein bands were detected by using the ChemiDoc ™ XRS + system (Bio-Rad).Total mRNA was extracted from humans by using TRI Reagent® Solution . The precipitated mRNA was dissolved in nuclease-free water, and the RNA concentration was determined by a Nanodrop2000 (Thermo Fisher Scientific). Subsequently, mRNA was transcribed into cDNA by using a Transcriptor First Strand cDNA Synthesis Kit . Then, 2 μL of DNA solution, primers, DEPC water, and Hieff® qPCR SYBR® Green Master Mix  were added to each well for the real-time PCR assay. Furthermore, the relative mRNA levels were detected by a CFX Connect™ Real-Time PCR Detection System (Bio-Rad) using iQ™SYBR ® Green Supermix . The primers used in this study are listed in Additional file t test to compare the means of two groups, while multiple group comparisons were made by using one-way ANOVA with least significant difference  or Tamhane T2  tests in SPSS software (version 23.0). P < 0.05 was considered statistically significant.All the data are represented as the mean ± standard deviation (SD) in the present study. Statistical analysis was carried out by using Student’s two-tailed Additional file 1: Figure S1. The expression of H3K9 and H3K4 correlated with HASMC proliferation and neointima formation.Additional file 2: Figure S2.  JIB-04 treatment regulated the methylation level of H3K9 rather than H3K4 in HASMCs.Additional file 3: Table S1.  Primers for Real-Time PCR detection.


Piezo1 knockout on neural development in vivo and find that cholesterol biosynthesis and lipid membrane composition are regulated by Piezo1.PIEZO1, a mechanically gated ion channel, transduces mechanical cues in neural stem cells. Here, we show the impact of  Piezo1, a mechanically activated ion channel. We find that Piezo1 deletion results in a thinner neuroepithelial layer, disrupts pseudostratification, and reduces neurogenesis in E10.5 mouse embryos. Proliferation and differentiation of Piezo1 knockout (KO) mouse neural stem cells (NSCs) isolated from E10.5 embryos are reduced in vitro compared to littermate WT NSCs. Transcriptome analysis of E10.5 Piezo1 KO brains reveals downregulation of the cholesterol biosynthesis superpathway, in which 16 genes, including Hmgcr, the gene encoding the rate-limiting enzyme of the cholesterol biosynthesis pathway, are downregulated by 1.5-fold or more. Consistent with this finding, membrane lipid composition is altered, and the cholesterol levels are reduced in Piezo1 KO NSCs. Cholesterol supplementation of Piezo1 KO NSCs partially rescues the phenotype in vitro. These findings demonstrate a role for Piezo1 in the neurodevelopmental process that modulates the quantity, quality, and organization of cells by influencing cellular cholesterol metabolism. Our study establishes a direct link in NSCs between PIEZO1, intracellular cholesterol levels, and neural development.Mechanical forces and tissue mechanics influence the morphology of the developing brain, but the underlying molecular mechanisms have been elusive. Here, we examine the role of mechanotransduction in brain development by focusing on  Neural development involves the orchestration of a complex series of steps to generate the brain and spinal cord. While research has primarily focused on the influence of genetic and chemical factors on neural development, recent studies have demonstrated that mechanical forces also determine neural development and function . MechaniPiezo1 knockout (KO) mouse model  were obtained from Jackson Laboratories (stock no. 026948). Piezo1∆/+ mice were bred with C57BL6/J to maintain the colony.All experiments were carried out in accordance with approved Institutional Animal Care and Use Committee protocols and University of California, Irvine guidelines. . Dams were sacrificed at specified time points, and embryos were harvested. Each embryo was genotyped through a commercial vendor (Transnetyx).Heterozygous mice were bred to generate a mixture of WT, heterozygous, and knockout embryos. Vaginal plug observation was considered embryonic day (E) 0.5E10.5 embryos were drop-fixed immediately after harvesting in Bouin’s fixative  for 24 h at 4°C. Fixed embryos were paraffin-embedded, sectioned sagittally at a thickness of 5 µm, and stained with H&E using standard procedures.Piezo1 KO embryonic brain tissue is considerably more fragile than WT, requiring paraffin embedding to maintain tissue structures during processing. Fixed embryos were paraffin-embedded and sectioned sagittally at 5 µm thickness. Sections were deparaffinized with xylene and 100% ethanol and incubated in RNAscope Target Retrieval Reagent  for 15 min at 95°C. ISH was performed using the RNAscope Multiplex Fluorescent Reagent Kit v2  using the manufacturer’s protocol for formalin-fixed paraffin-embedded (FFPE) sections. WT and Piezo1 knockout tissue sections were incubated with a Piezo1 Probe . Sections were then immunolabeled with anti-TUJ1 antibody  to identify early neuronal populations; in separate experiments PFA-fixed, paraffin-embedded sections were also immunolabeled with anti-Nestin  and anti-Sox2  antibodies. For immunofluorescence of tissue slices after deparaffinization, sections were rehydrated in a series of graded ethanol from 95, 80, to 60% for 5 min each at room temperature, washed with water twice for 1 min, boiled in IHC Select Citrate Buffer pH 6  for 20 min, permeabilized with 0.3% Triton X-100 for 15 min at room temperature, blocked with 5% BSA;  for 1 h at room temperature, and incubated with primary antibodies overnight at 4°C. The following day, sections were incubated with secondary antibodies for 1 h at room temperature. Nuclei were labeled with 6 µg/ml Hoechst  for 5 min at room temperature and mounted with Prolong Diamond . See E10.5 embryos were drop-fixed in 4% v/v paraformaldehyde  overnight at 4°C. We found that E10.5 . Fixed samples were dehydrated in methanol /H2O series of 20, 40, 60, 80, and 100%; 1 h each at room temperature. Samples were then incubated overnight in two parts dichloromethane /one part methanol with shaking at room temperature. The following day, samples were washed twice with 100% methanol and then chilled for 1 h at 4°C. Samples were bleached in chilled fresh 5% H2O2  in methanol (1 volume 30% H2O2 to 5 volumes MeOH) overnight at 4°C. Samples were rehydrated the next day with methanol/H2O series of 80, 60, 40, 20%, and phosphate-buffered saline (PBS), 1 h each at room temperature. Finally, samples were washed in PBS/0.2% Triton X-100  for 1 h twice.Embryos were drop-fixed in 4% v/v PFA  overnight at 4°CPretreated samples were incubated in PBS/0.2% Triton X-100/20% dimethylsulfoxide (DMSO) /0.3 M glycine  at 37°C overnight, then blocked in PBS/0.2% Triton X-100/10% DMSO/6% donkey serum  at 37°C for 1 d. Samples were washed in PBS/0.2% Tween-20  with 10 μg/ml heparin  for 1 h twice, then incubated in primary antibody dilutions see  in PTwH/2O series: 20, 40, 60, 80, and 100%; 1 h each at room temperature. Samples were then left fully submerged in 100% methanol overnight, before being incubated for 3 h with shaking in two parts dichloromethane/one part methanol at room temperature. Samples were then washed twice in 100% methanol for 15 min each before being incubated in dibenzyl ether  overnight before imaging.Immunolabeled samples were cleared using the iDisco protocol . Immunolhttps://digitalinsights.qiagen.com/IPA) with the algorithms developed for use in QIAGEN IPA . Total RNA was monitored for quality using the Agilent Bioanalyzer Nano RNA chip and Nanodrop absorbance ratios for 260/280 nm and 260/230 nm and RNA Integrity Number (RIN) scores of 9.5–10. Library construction, sequencing, and post-processing of the run to generate the FASTQ files were performed at the UCI Genomics High Throughput Facility. Library construction was performed according to the Illumina TruSeq mRNA stranded protocol, and mRNA was enriched using oligo dT magnetic beads. The enriched mRNA was chemically fragmented. First-strand synthesis used random primers and reverse transcriptase to make cDNA. After second strand synthesis, the double-stranded cDNA was cleaned using AMPure XP beads and the cDNA was end-repaired and then the 3′ ends were adenylated. Illumina barcoded adapters were ligated on the ends, and the adapter-ligated fragments were enriched by nine cycles of PCR. The resulting libraries were validated by qPCR and sized by Agilent Bioanalyzer DNA high sensitivity chip. The multiplexed libraries were sequenced using paired-end 150 cycles chemistry on NovaSeq 6000. Raw reads were trimmed using a quality threshold of 15 and Truseq adapter sequence, trimmed reads shorter than 20 bp were discarded. Reference genome sequence mm10 and its transcriptome annotation were collected and indexed for mapping. Gene expression level was quantified using featureCount for raw counts. Statistical comparison was made using DESeq2 to obtain differential gene expression data . PrincipAGEN IPA  using on2 onto #1.5 acid-washed coverslips coated with laminin (10 µg/ml).Neural stem cell cultures were isolated from E10.5 brains. For neurosphere cultures, cells were plated onto non-adherent cultureware at 100 K/ml of media. All mouse NSC cultures were maintained in proliferation media: Dulbecco’s modified Eagle’s media , supplemented with 1× N2 , 1× B27 , 1 mM sodium pyruvate , 2 mM Glutamax , 1 mM N-acetylcysteine , 10 ng/ml b-FGF , 20 ng/ml EGF , and 2 µg/ml heparin . To passage neurospheres, cultures were dissociated using the Neurocult Chemical Dissociation Kit . For staining and differentiation experiments, neurospheres were dissociated and cells were seeded at a density of 30 K/cm2, 10 mM EGTA, and 40 mg/ml sucrose in PBS for 10 min and permeabilized with 0.3% Triton X-100 in PBS for 5 min, blocked with 5% BSA in PBS for 1 h at room temperature, and incubated with primary antibodies in 1% BSA overnight at 4°C. The following day, cells were incubated with secondary antibodies for 1 h at room temperature. See Cells were fixed in 4% v/v PFA, 5 mM MgClAdherent mNSC cells plated on 35 mm glass bottom dishes  were fixed with 4% PFA for 10 min and washed three times with PBS. Fixed cells were stained with 1 µg/ml Nile Red  or 50 µg/ml Filipin III . Filipin III was reconstituted with anhydrous DMSO  at 2 mg/ml and used either the same day or within 2 wk (stored immediately after DMSO reconstitution at −80°C). Since Filipin III is very unstable and bleaches rapidly, cells were stained one sample at a time with Filipin III freshly diluted to 50 µg/ml in PBS containing 500 nM nuclear counterstain siR-DNA  for 45 min in the dark at room temperature. Cells were washed once with 1.5 ml PBS and imaged immediately. Alternatively, fixed cells were stained with Nile Red  for 20 min at 37°C in PBS, nuclei counterstained with 6 μg/ml Hoechst-33342 , washed three times with PBS, and imaged in PBS.2 onto #1.5 acid-washed coverslips coated with laminin (10 µg/ml). After 24 h, media was removed and differentiation media was added . Cells were fed every 2 d and fixed after 4 d or fixed after 10 d in differentiation media. For cholesterol rescue experiments, cholesterol–methyl-β-cyclodextrin  at 10 µg/ml  was added to the media at the time of plating and during media changes. Antibodies used for identifying differentiated cell types include astrocytic marker glial fibrillary acidic protein (GFAP), neuronal marker microtubule associated protein (MAP2), and oligodendrocyte marker (O4). GFAP staining of astrocytes is distinguished by a high GFAP signal compared to very low levels found in proliferating NSCs. See Neurospheres were dissociated and cells were seeded at a density of 30 K/cmNeurospheres were dissociated and seeded in triplicate for each biological replicate  in proliferation media at a density of 10,000 cells/well in a 96-well plate  coated with laminin (10 µg/ml) and allowed to recover for 18–22 h. To visualize nuclei, 100 µl proliferation media containing 250 nM siR-DNA Kit  was added per well. Cerivastatin  was added at 0, 20, 50, and 100 nM. Live cells were imaged with a 10× phase objective with both phase contrast and fluorescence (using filter cube Ex 565–605 nm/Em 625–705 nm) at four to five positions per well every 2 h in the IncuCyte S3 Live-Cell Analysis System for 60 h. Fold proliferation was determined by analysis with Sartorius software of the number of nuclei at each time point in each image and normalized to the number of nuclei at time point 0 for each image. Data presented are from three wells for each embryo. A minimum of three embryos for WT and for KO were used per experiment.g for 5 min and washed three times with PBS. Zombie Violet fixable viability kit  was added at 1:2,000 dilution in PBS. A total of 10,000–30,000 events were analyzed with a BD Fortessa X20 flow cytometer using a 488 nm laser with a 505 nm long pass filter (green) and a 561 nm laser with a 600 nm long pass filter (red). FlowJo v10.7 Software (BD Life Sciences) calculated the geometric mean of green  and red  from cells and the ratio of green to red signal was assessed.One million single cells from dissociated neurospheres were stained with 1 µg/ml Nile Red for 20 min at 37°C in proliferation media. Cells were pelleted at 110 × H&E-stained embryos were imaged with Leica Aperio VERSA 200 (40× objective). ISH and immunofluorescence-stained sections were imaged with Olympus FV3000S laser-scanning confocal microscope (40× [NA 1.25] and 60× [NA 1.25] objectives). Whole-mount–stained embryos were imaged by Zeiss Z-1 Lightsheet Microscope (5× [NA 0.16] objective). Immunolabeled embryo sections and cultured cells were imaged with Keyence BZ-X810 Widefield Microscope . Filipin-labeled cultured cells were imaged with Keyence BZ-X810 Widefield Microscope (60× [NA 1.4] oil objective). The proliferation of NSCs was imaged with Incucyte S3 Live Cell Imaging system (10× [NA 0.3] objective) and analyzed with S3 2020A GUI software. The Incucyte system uses a 12-bit CMOS camera (scientific grade CMOS camera) and contrast-based autofocus to acquire images. Nile Red stained cells were imaged with Zeiss Elyra 7 for Lattice SIM Microscopy with 405, 488, and 561 nm lasers, PlanApo 63× oil immersion objective (NA 1.40), and acquired using a dual camera: 2 pco.edge 4.2 camera system with lattice sim leap mode. Subsequently, Nile Red images were processed using Zeiss’s automated sim leap mode pipeline. See d was calculated, and P values were calculated by two-sided permutation t-test . Piezo1 KO mouse model  and Pmvk, encoding phosphomevalonate kinase , Ch25h (2.09-fold), Cyp27a1 (1.44-fold), and Soat2 (19.2-fold), which would further contribute to the reduction of free cholesterol.For an unbiased approach to illuminate the mechanisms leading to the observed egulated . PCA, whegulated . Moreovepathways , tab 2, pathways . Howevermosterol . Sixteenmosterol . FurtherHmgcr, encoding HMG-CoA reductase, the rate-limiting enzyme for the cholesterol biosynthetic pathway, was downregulated by 1.7-fold in the brains of Piezo1 KO mice  in cellular membranes . AnalysiWT cells . This dePiezo1 KO NSCs. Live-cell proliferation assays performed in the presence of 10 µg/ml water-soluble cholesterol-methyl-β-cyclodextrin , did not significantly improve Piezo1 KO proliferation  membrane cholesterol  cause lathosterolosis, a condition resulting in multiple congenital anomalies and intellectual disability  underlie Smith–Lemli–Opitz syndrome (SLOS), which includes symptoms of microcephaly, mental developmental delay, and hearing impairment , cholesterol transporters , and Piezo2 (Piezo1 function to lipid metabolism are not mechanistically obvious, opening up a new line of investigation on how Piezo1 expression and activity modulates cholesterol biosynthesis.The absence of d Piezo2 , tab 1. PIEZO1 channel activation by membrane tension  is consiPiezo1 KO reduces cholesterol synthesis and alters lipid composition suggests the existence of a bidirectional feedback mechanism: PIEZO1 function enhances cholesterol biosynthesis whose presence in the membrane can, in turn, modulate channel activity. More broadly, our finding that Piezo1 KO results in the downregulation of cholesterol biosynthesis uncovers a novel link between cellular mechanotransduction and cholesterol metabolism. This finding is not only relevant to brain development but also opens up new lines of investigation for examining a potential contribution of PIEZO1 in neurodegenerative diseases impacted by cholesterol dysregulation (Our finding that gulation , such asTable S1z-score of ±2 and IPA P value < 0.05contains 1 Differentially expressed genes in Piezo1 KO E10.5 brains compared to WT and an expanded list of canonical pathways with Click here for additional data file.Table S2Piezo1 KO brains identified by IPAlists changes in canonical pathways in E10.5 Click here for additional data file.Table S3provides a list of antibodies used in this studyClick here for additional data file.Table S4lists the microscopes used.Click here for additional data file.


Nevertheless, cyclo[2]carbazole[2]pyrrole 2 binds representative anions with higher affinity in CD2Cl2 than calix[4]pyrrole (1), a well-studied non-conjugated tetrapyrrole macrocycle that binds anions via four pyrrolic NH hydrogen bond interactions. On the basis of computational studies, the higher chloride anion affinity of receptor 2 relative to 1 is rationalized in terms of a larger binding energy and a lower host strain energy associated with anion complexation. In the presence of excess fluoride or bicarbonate anions, compound 2 loses two pyrrolic NH protons to produce a stable dianionic macrocycle [2–2H]2− displaying a quenched fluorescence.A cyclo[2]carbazole[2]pyrrole (2) consisting of two carbazoles and two pyrroles has been synthesized by directly linking the carbazole 1- and 8-carbon atoms to the pyrrole α-carbon atoms. Macrocycle 2 is an extensively conjugated 16-membered macrocyclic ring that is fixed in a pseudo-1,3-alternate conformation. This provides a preorganized anion binding site consisting of two pyrrole subunits.  Less is more: two NH hydrogen bond donors in a preorganized receptor provide greater anion affinity than the four NH moieties present in the classic anion receptor, calix[4]pyrrole. In contrast to porphyrins 4 as a catalyst and K2CO3 as a base in the presence of 1.0 equiv. of tetrabutylammonium fluoride (TBAF) as a template, 3 and 4 were found to undergo 2 : 2 cyclization to give the 2 in 5.2% yield. Compound 2 was characterized by means of 1H and 13C NMR spectroscopy and high-resolution mass spectrometry, as well as a single crystal X-ray diffraction analysis. Compound 2 was found to be resistant to oxidation when treated with MnO2, FeCl3 and DDQ . This stands in stark contrast to what is seen for related compounds lacking ester groups on the β-pyrrolic positions and presumably reflects the electron-withdrawing ester groups that reduce the electron density within the macrocycle.32 The presence of four NH protons in the core in combination with the conjugated structure was expected to limit considerably the mobility of receptor 2 leading to it being locked in the 1,3-alternate conformation. Support for these suppositions came from a combination of structural, spectroscopic, and computational studies (vide infra).The synthesis of macrocycle 2 is shown in Single crystals of the anion-free receptor 2 suitable for an X-ray diffraction analysis were grown by subjecting a chloroform/methanol solution containing receptor 2 to slow evaporation. The resulting X-ray crystal structure revealed that receptor 2 adopts a pseudo-1,3-alternate conformation in the solid state in which the two pyrrole NH and two carbazole NH moieties are facing in opposite directions. The two pyrrole rings deviate by 41° and 36°, respectively, from the mean plane consisting of the two respective carbazole 1- and 8-carbon atoms, while the two carbazoles are tilted by −24° and −25°, respectively, with respect to the mean plane . One met1H NMR spectral changes that take place in the presence of the corresponding tetraalkylammonium salts in CD2Cl2  salts), as well as HCO3− as its tetraethylammonium (TEA+) salt, chemical shift changes in the NH proton signals of both the pyrrole and the carbazole subunits are seen in the corresponding 1H NMR spectra recorded in CD2Cl2 (b) was downfield shifted. i.e., to δ = 12.88 ppm (Δδ = 3.7 ppm) for Cl−, δ = 12.53 ppm (Δδ = 3.35 ppm) for Br−, δ = 11.27 ppm (Δδ = 2.09 ppm) for I−, δ = 11.66 ppm (Δδ = 2.48 ppm) for HSO4−, and δ = 11.56 ppm (Δδ = 2.38 ppm) for SO42− (a) underwent discernible upfield shifts and were found to resonate as a singlet, a singlet signal at δ = 9.13 ppm (Δδ = −0.4 ppm) for Cl−, δ = 8.87 ppm (Δδ = −0.66 ppm) for Br−, δ = 8.85 ppm (Δδ = −0.68 ppm) for I−, δ = 8.83 ppm (Δδ = −0.70 ppm) for HSO4−, and δ = 8.97 ppm (Δδ = −0.56 ppm) for SO42−  and δ = 10.74 ppm (Δδ = 1.21 ppm) for the fluoride and bicarbonate anions, respectively. The pyrrole NH proton resonance was seen to disappear  for 2 in CD2Cl2 were calculated to be (1.20 ± 0.18) × 104 M−1 for chloride, (6.93 ± 0.12) × 102 M−1 for bromide, (1.35 ± 0.08) × 102 M−1 for iodide, (3.10 ± 0.18) × 102 M−1 for hydrogen sulfate, (5.46 ± 1.03) × 102 M−1 for sulfate, and (1.78 ± 0.32) × 103 M−1 for dihydrogen phosphate, respectively , we believe the former determinant represents the dominant contribution.Similar chemical shift changes were observed when receptor 2 was titrated with other anions such as Br HP2O73− , albeit ectively .34 Compaectively . The enh1H NMR spectra were observed when receptor 2 was titrated with the fluoride or bicarbonate anions in CD2Cl2 (δ = 4.09 ppm) and 11.61 ppm (Δδ = 1.38 ppm), respectively. These findings are rationalized in terms of the relatively small fluoride anion binding deep within the receptor cavity forming weak hydrogen bonds to the carbazole NH protons in addition to relatively strong hydrogen bonds to the pyrrole NHs protons (ca. 1.2 fluoride anion equiv. is consistent with receptor 2 binding the fluoride anion with a high affinity and with a 1 : 1 stoichiometry (Different changes in the n CD2Cl2  and S6†. protons . The sathiometry .c) was also seen. In addition, the pyrrole NH proton resonance was seen to disappear in the presence of >7.0 equiv. of TBAF. These chemical shift changes are attributed to deprotonation of the pyrrole protons in the presence of excess fluoride anion, which results in an intramolecular hydrogen bond interaction between the carbazole NHs and the deprotonated pyrrolic nitrogen atoms. The noticeable upfield shift of the carbazole CH proton signal (Hd) is thought to reflect the increased electron density of the macrocycle that results from deprotonation of the pyrrole NHs. Support for the suggestion that deprotonation occurs under these conditions came from an X-ray crystal structural analysis of single crystals of receptor 2 grown in the presence of excess TBAF (vide infra). Unfortunately, an association constant corresponding to the interaction of receptor 2 with the fluoride anion could not be approximated from these 1H NMR spectral titrations due to the disappearance of the pyrrole and carbazole NH proton signals and the absence of appreciable chemical shift changes for the carbazole CH proton signals before saturation was reached upon the addition of 1.2 equiv. of fluoride  was shifted to higher field with saturations being seen upon the addition of only ca. 1.0 anion equiv. in both solvent systems  with two TBA+ counter cations. This dianionic form is flattened relative to the charge neutral receptor (2). While the two pyrrole rings are tilted by 33° and −33°, respectively, with respect to the mean plane of the macrocycle while the carbazole subunits lie in approximately the same plane as the mean plane  were found to fall in the range of 6.8 × 104 M−1 to 4.0 × 105 M−1 with errors under 10%  between receptor 2 and the anions, defined as ΔE = E(receptor–anion complex) − E(receptor) − E(anion), was computed using the energies for the lowest energy forms of each species. The inherent energies of receptor 2, fluoride, chloride, and hydrogen sulfate were calculated to be 143 kcal mol−1, 0.0 kcal mol−1, 0.0 kcal mol−1 and −100.0 kcal mol−1, respectively. Receptor 2 was found to maintain its approximate 1,3-alternate conformation for the main receptor cavity in all simulations. Multiple conformers, arising from rotations involving t-butyl and ethyl ester groups, were considered.The presumed binding modes of receptor 2 for fluoride, chloride and hydrogen sulfate in solution were supported by molecular mechanics computations performed using the Merck Force Field 94 (MMFF94) model E = −39.8 kcal mol−1 and −26.7 kcal mol−1, respectively, for binding via two pyrroles and ΔE = −32.7 kcal mol−1 and −22.3 kcal mol−1, respectively, for binding via two carbazoles, respectively , rather than its two carbazole subunits  (−1), chloride  and hydrogen sulfate , respectively.Consistent with what was seen in the solid-state X-ray crystal structure of the ion-free form, receptor 2 is characterized by two distinct sides, each of which provides two potential NH binding sites, namely the two pyrrole NH hydrogen bond donors or the two carbazole NH donors, respectively. When an anion is added, the anion remains on the same side of the host during each simulation. In other words, the anion does not migrate through the centre of the receptor cavity; nor does it crawl around the edge of the receptor cavity. The binding energies of receptor 2 for fluoride and chloride were computed to be Δectively . The flul mol−1) . These cE) of calix[4]pyrrole 1 for the chloride anion using the MMFF94 model in vacuo and compared it with that for receptor 2.38,39 For calix[4]pyrrole 1 in its ion-free form, only two stable conformers, i.e., the 1,3-alternate conformer with D2d symmetry and the 1,2-alternate conformer with Ci symmetry, were calculated as being appreciably stable in the gas phase  by 6.8 kcal mol−1 , one obtains, ΔUreorg, the total host strain energy increase on going from the preferred form to the bound form. This value provides a measure of how well the host is structurally organized for binding the guest.This suggestion was further supported by strain energy analyses of receptors 1 and 2.45 However, a strain energy analysis reveals that receptor 2 is significantly more complementary with ΔUcomp = 5.5 kcal mol−1 than calix[4]pyrrole 1 with ΔUcomp = 18.1 kcal mol−1  that is enhanced by roughly a factor of 2.46 We postulate that the electronic contribution of the four esters in 2 relative to 1 is likely less than that provided by eight fluorine atoms and that preorganization effects account for the bulk of the ca. 34-fold improvement in chloride affinity seen on moving from 1 to 2.For calix[4]pyrrole 1, the binding conformer is the one obtained when the bound form of the host is optimized . In thisal mol−1  and 14. 1H NMR spectral studies and a single crystal X-ray structural analysis in conjunction with molecular mechanics calculations carried out in vacuo revealed that receptor 2 adopts a 1,3-alternate conformation in which the two alternating pyrrole and carbazole subunits face in opposite directions. This provides a rigid and highly preorganized anion binding motif with two faces and two potential sets of NH hydrogen bond donors. On the basis of 1H NMR spectral analyses, receptor 2 was found to bind most test anions using only the two diagonal opposing pyrrole NH protons but not the carbazole NH protons. Nevertheless, higher anion affinities for 2 relative to calix[4]pyrrole 1 were seen in the same solvent system (CH2Cl2 for most studies), even though the latter well-studied receptor typically allows for four pyrrole NH–anion interactions. In vacuo molecular dynamic computations involving the chloride anion complex provided support for the conclusion that anions energetically favor binding to the pyrrole donor side of receptor 2 over binding to the carbazole donor side. In contrast to most test anions considered in this study, the small basic fluoride anion binds relatively deep within the receptor cavity with the resulting complex being stabilized by hydrogen bonds to both the pyrrole and carbazole NH protons. When treated with an excess of TBAF, receptor 2 loses two protons from its pyrrole subunits giving rise to a stable dianionic macrocyclic salt [2–2H]2−·(TBA+)2. Molecular dynamics computations using the MMFF94 model revealed that the high affinity of receptor 2 for the chloride anion (and by inference other anions) relative to calix[4]pyrrole 1 can be rationalized largely in terms of a larger stabilization energy associated with anion binding and a correspondingly smaller host strain energy associated with complex formation.A conjugated macrocyclic compound 2 having four central NH protons as found in calix[4]pyrrole has been synthesized by means of a 2 : 2 cyclization reaction involving the Suzuki coupling of 1,8-carbazolediboronic acid bis(pinacol) ester with 2,5-dibromopyrrole-3,4-diethyl ester. The datasets supporting this article have been uploaded as part of the ESI.†1H NMR, UV/Vis, and fluorescence spectroscopic studies. J. H. Y., K. L. and V. M. L. carried out the X-ray diffraction analyses. B. P. H. carried out molecular mechanics computations. J. L. S. and S.·K. K. wrote the manuscript. All authors contributed to the editing of the manuscript.J. L. S. and S. K. K. conceived and supervised the project. A. L. and J. H. O. synthesized the compounds. A. L. performed There are no conflicts to declare.SC-014-D2SC06376J-s001SC-014-D2SC06376J-s002