Inhibiting MAPK14 (p38α) diminishes cardiac damage in myocardial ischemia. During myocardial ischemia, p38α interacts with TAB1, a scaffold protein, which promotes p38α autoactivation; active p38α (pp38α) then transphosphorylates TAB1. Previously, we solved the X-ray structure of the p38α-TAB1 (residues 384–412) complex. Here, we further characterize the interaction by solving the structure of the pp38α-TAB1 (residues 1–438) complex in the active state. Based on this information, we created a global knock-in (KI) mouse with substitution of 4 residues on TAB1 that we show are required for docking onto p38α. Whereas ablating p38α or TAB1 resulted in early embryonal lethality, the TAB1-KI mice were viable and had no appreciable alteration in their lymphocyte repertoire or myocardial transcriptional profile; nonetheless, following in vivo regional myocardial ischemia, infarction volume was significantly reduced and the transphosphorylation of TAB1 was disabled. Unexpectedly, the activation of myocardial p38α during ischemia was only mildly attenuated in TAB1-KI hearts. We also identified a group of fragments able to disrupt the interaction between p38α and TAB1. We conclude that the interaction between the 2 proteins can be targeted with small molecules. The data reveal that it is possible to selectively inhibit signaling downstream of p38α to attenuate ischemic injury. Disrupting TAB1-p38α interaction in vivo has a protective effect during myocardial ischemia and can be achieved in vitro with small molecule inhibitors. The MAPK p38α is a serine threonine protein kinase that is activated by stress. The kinase is expressed in all mammalian cell types and is highly conserved in yeast. Ablating p38α in mice results in early embryonal lethality, and in yeast, it results in failures in adaptation to environmental stress and pheromones. Hence, p38α has an essential role in health and homeostasis , 2.Nonetheless, in animal models of acute myocardial infarction and heart failure, inhibition of p38α reduces infarct size, improves left ventricular function, and limits cardiac dilatation. Conversely, cardiac-restricted forced activation of p38α damages the myocardium. In human hearts, p38α is activated by myocardial ischemia. Thus, p38α inhibition makes therapeutic sense in ischemic heart disease .The clinical trials that have been published to date, those that are on-going, and those that are planned all use pharmacological inhibitors of p38 kinase activity that target the ATP binding site and inhibit the catalytic activity of the kinase in all settings. In all cases where information is available, the clinical trials have been suspended due to adverse side effects such as skin and liver toxicity. The adverse effects are shared between inhibitors based on different scaffolds and with diverse modes of binding to the ATP binding site of p38 , 7. A nuS423TAB1), measured their binding affinity, solved the X-ray structure of the complexes, and compared these to our previously published data on nonphosphorylated p38α and a short TAB1 (residues 384–412) 29-mer peptide , and residues 404–412 of TAB1 bound within the groove defined by αD and αE helices and the reverse turn between β7 and β8   . Phosphod MEF2A) . In p38αIn pp38α, residue pThr180 sat at the interface between the 2 domains of the kinase with the Thr180 phosphate group interacting with Arg67, Arg70, and Lys66 from the N-lobe and Arg149 and Arg173 from the C-lobe, whereas the phosphate from pTyr182 interacted with Arg186 and Arg189 on the C-lobe. This loop arrangement is typical of active kinases .The crystallographic and biophysical data both suggest that TAB1 remains bound to p38α after having induced the kinase autoactivation and having been phosphorylated by it; they also show that no other region, beyond residues 384–412 in TAB1, was involved in the interaction with p38α.E.coli, phosphorylation of p38α and TAB1 were analyzed by immunoblotting. WT TAB1 increased the phosphorylation of p38α, and this was reduced by mutations within TAB1. This pattern was reflected in TAB1 phosphorylation, with the lowest phosphorylation evident on the double mutant . The gene is located on mouse chromosome 15; it contains 11 exons with the ATG start codon in exon 1 and TAG stop codon in exon 11. The amino acid residues V390, Y392, V408, and M409 are located in exon 10. The targeting vector 5′ homology arm and 3′ homology arm was amplified from BAC DNA and confirmed by end sequencing; the mutations V390A, Y392A, V408G, and M409A were introduced into exon 10 by site-directed mutagenesis. The constitutive KI allele was obtained after Cre-mediated recombination, and C57BL/6 ES cells were used for gene targeting  . The 4 pargeting . The ES argeting  were intargeting  positiveargeting .The homozygous KI mice were viable, expressed similar amounts of the mutated form of TAB1 as in-colony WT mice, and developed normally . The bas2-fold change detected was 0.75; in fact, only 27 genes out of the approximately 14,000 detected showed a log2-fold difference in either direction of greater than 0.5. All other genes showed fold differences below 0.5. When the list of differentially expressed genes with a P adjusted value below 0.05 is analyzed through the Reactome database pathway (https://reactome.org/); the only biological pathway that shows a significant overrepresentation in the list of genes is the circadian clock system. Finally, none of the known genes involved in p38α signaling were differentially expressed  in mouse left ventricular myocardium. We performed RNA-sequence (RNA-Seq) analysis of KI versus WT heart and observed no major transcriptional differences. The highest logxpressed .Our interpretation is that, at baseline, beyond the circadian clock pathway, there is no significant biological difference in the transcriptome profile of the WT vs. KI hearts .p38α is expressed in, and regulates the function of, several classes of immune cells. Of most relevance, p38α autophosphorylation plays a role in T lymphocyte maturation, including through TAB1 , 17. We Ischemia activates p38α, and pharmacological inhibition of p38α during ischemia reduces heart damage. Our group has previously shown that, during ischemia, p38α is activated through autophosphorylation induced by the interaction with the scaffold protein TAB1 , 12. HerAs previously reported in the literature, ischemia causes p38α activation and TAB1 phosphorylation  18, 19), 1918, 1We also observed that, in the KI hearts, following 30 minutes of regional ischemia and 120 minutes of reperfusion, myocardial infarction volume as a percentage of the risk volume was significantly reduced . We hypoA caveat with our genetic approach is that we stopped TAB1 from docking onto p38α in all circumstances, but by doing so, MAP2K3 and MAP2K6, the archetypical upstream activators of p38α, may have unhindered access to p38α, possibly participating in a compensatory mechanism that could explain the activation of p38α observed in the KI mice. For example, the interaction points of MKK3b, the most abundant splice variant of MAP2K3, on p38α have previously been described and partially overlap with those of TAB1 , 21. We m = 3.1 °C), relative to DMSO control. In addition, this binding was independent of the ATP-binding pocket, since the stabilization caused by 3-amino-1-adamantanol in the presence (ΔTm1 = 3.1 °C) or in the absence (ΔTm2 = 3.3 °C) of SB220025, a high-affinity ATP competitive inhibitor, was similar  molecules.Finally, the genetic model shows that inhibiting the interaction between p38α and TAB1 throughout development is not lethal, suggesting that drugging the interaction will not have the liabilities associated with ATP-competitive inhibitors of p38α kinase activity. The cardioprotection we observed in our KI mouse model and the fact that all of the binding affinity between the 2 proteins was concentrated on a defined surface  encourages the development of PPI molecules to recapitulate the benefits of genetic inhibition.6 tag followed by a TEV-cleavage site and mouse p38α sequence. The vector was transformed in E. coli strain Rosetta II cells (Novagen). To produce milligram quantities for the structural studies of dual phosphorylated p38α, we ran a large-scale in vitro kinase activation using recombinant constitutively active mkk6DD. The activated p38α was them purified from the reaction mixture, and mass spectrometry was used to confirm stochiometrical phosphorylation of p38α. The protein expression, in vitro activation, and purification procedures followed are described in refs. DNA encoding mouse p38α was derived from a pET14b vector donated by Y. Wang  and subcloned into a pETDuet-1 vector. The full coding sequence included an N-terminal His6 tag followed by a TEV-cleavage site and mouse TAB1 (residues 1–438). The vector was transformed in E. coli strain Rosetta II cells. Recombinant TAB1 (residues 1–438) was expressed and purified according to the same protocols used for p38α.Chemically synthesized DNA encoding mouse TAB1 was subcloned into a pETDuet-1 vector. The full coding sequence included an N-terminal HisS423TAB1 (residues 1–438), we followed ref. E. coli cells were transformed with the pCDF vector containing pS423TAB1 (residues 1–438), recovered with 1 ml super optimal broth (SOB) medium, and plated on a streptomycin/chloramphenicol agar plate. The protocols to grow, express, and purify pS423TAB1 (residues 1–438) are the same as the ones used for TAB1 and p38α; the only difference is that the cells were grown in the presence of 2 mM O-phospho-L-Serine.To overexpress and purify p−1), Kb (equilibrium binding constant in M−1), and n  were the fitting parameters. The reaction entropy was calculated with the relationships ΔG = −RT × lnKb  and ΔG = ΔH − TΔS, where ΔH represents molar enthalpy change, ΔS represents molar entropy change, and ΔG represents molar Gibbs free energy change..All ITC experiments were carried out on an iTC200 microcalorimeter from Microcal . The integrated heat data for the titrations corrected for heats of dilution were fitted with a nonlinear least-squares minimization algorithm to a theoretical titration curve, with MicroCal-Origin 7.0 package. ΔH°  added to 10 mM; the final concentration of the complex was 8 mg/ml. Crystallisation screens were set up using a Mosquito dispensing system and a single hit condition obtained . In-house testing showed the crystals were not cryoprotected and diffracted weakly to approximately 3.5 Å. Grid optimizations of PEG/buffer percentage and type were set up together with additive screens; resultant crystals were subjected to various cryoprotection strategies, flash frozen in liquid nitrogen, and tested at Diamond Light SourceFinal, optimized crystals were grown from 20% 1:1 PEG4000/PEG6000, 50 mM MgCl2, 50 mM MgSO4, and 0.1 M Tris pH 8.0 and were cryoprotected by rapid transfer through well solution supplemented with 20% ethylene glycol and diffracted to 2.6 Å.https://www.rcsb.org/structure/5NZZ).Initial screening of crystals was carried out in-house using an Xcalibur Nova rotating copper anode system with Onyx CCD. Final data were collected at the Diamond synchrotron, beamline IO4.1, 25 Hz Pilatus 6M-F detector. Data were integrated and scaled using Aimless  with PDBpETDuet-1 vector was used for bacterial coexpression of p38α and the different TAB1 mutants: TAB1 (residues 1–502), TAB1 (residues 1–502) with noncanonical site mutations V390A and Y392A, TAB1 (residues 1–502) with canonical site mutations V408G and M409A, and p38-TAB1 (residues 1–502) with canonical and noncanonical site mutations V390A, Y392A, V408G, and M409A. In all the bacterial vectors, p38α was subcloned in the first multiple-cloning site, whereas TAB1 was subcloned in the second multiple-cloning site.HEK293 cells were transfected with plasmids expressing WT p38α, WT TAB1, mutant forms of TAB1, WT TAK1, and WT MKK3. Cells were placed into serum free media (SFM) prior to transfection with complexes containing Turbofect reagent  and plasmid DNA. Transfection complexes were prepared and allowed to combine at room temperature for 20 minutes and were then made up to a volume of 1 ml and added drop-wise to cells. Transfection complexes were left on cells overnight and then aspirated and replaced with SFM. Cells were harvested 24 hours after transfection.The targeting vector 5′ homology arm and 3′ homology arm was amplified from BAC DNA and confirmed by end sequencing; the mutations V390A, Y392A, V408G, and M409A were introduced into exon 10 by site-directed mutagenesis with QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies). The targeting construct was electroporated into C57BL/6 ES cells, 375 G418-resistant colonies were picked, and the PCR assays were performed with primers 5PCR_F and 5PCR_R. Thirty-four potential targeted clones were identified. Six of them were expanded and frozen. The Southern blot analysis was conducted with 5′-probe, 3′-probe, and Neo-probe. The genomic DNA of the potential clones were digested with Bg1II for the 5′-probe and analyzed by Southern blot for a 8.0-Kb band from the WT allele and a 10.0 Kb band for the recombinant allele; for the 3′ probe, the genomic DNA was digested with XmaI and analyzed by Southern blot for a 7.8-Kb band from the WT allele and a 5.7-Kb band for the recombinant allele. Regarding the Neo probe, the genomic DNA was digested with NsiI and analyzed by Southern blot for a 6.6-Kb band for the recombinant allele.TAB1  F1 mice were generated from the ES cell lines 2C9. Five  out of 8 pups were identified as positive by PRC screening: the expected 261-bp fragment from WT allele and 405-bp fragment from recombinant allele were represented as positive F1 mice. The primers used for the screening were: Neo_Del_F and Neo_Del_R.The primers sequences used were: 5PCR_F: 5′-GCGGAGTATTAGGAGCCTGAGGGT-3′, 5PCR_R: 5′-GCTGACCGCTTCCTCGTGCTTTA-3′, 5′-probe_F: 5′-CAGCCACTTGTGAGACCAGAGGA-3′, 5′-probe_R: 5′-GGACAAGCCTGTGAGGTGATGACA-3′, 3′-probe_F: 5′-GGTCCCTCACAAGGGTTAAGCAACT-3′, 3′-probe_R: 5′-GAACTTGTCTGTGCTCTGAGCTGGC-3′, Neo-probe_F: 5′-CCTGAATGAACTGCAGGACGAGG-3′, Neo-probe_R: 5′-AGCTCTTCAGCAATATCACGGGTAGC-3′, Neo_Del_F: 5′-GCTGGCCTTGCTCAACTCCAG-3′, Neo_Del_R: 5′-GACCATCTGTCTCATACCTGACCTCAC-3′.RNA isolated from the 10 left ventricles (5 for each condition: WT vs. KI) was quantified using Qubit fluorometric quantification and assessed for quality using Agilent Bioanalyzer. Library preparation was performed using NEBNext Ultra Directional RNA Library Prep Kit for Illumina and the NEBNext rRNA Depletion kit (New England BioLabs). The library was sequenced on an Illumina HiSeq2500 at the Genomic Centre at King’s College London .RNA-Seq data of mouse heart cell populations were obtained as paired-end reads. Each sample contained typically 10–11 million reads. Sequence reads were aligned to the Ensembl GRCm38 genome using TopHat2 ; alignmehttps://www.ebi.ac.uk/fg/, entry code: E-MTAB-6460The RNA-Seq data have been deposited in Annotare 2.0 webserver, 2+/Mg2+, 2% FCS (v/v), 10 mM HEPES, Collagenase (1 mg/ml), and DNAse (0.1 mg/ml) 37°C for 30 minutes. Cells were isolated from mesenteric lymph nodes using manual mechanical disruption followed by enzymatic digestion (as above) for 15 minutes at 37°C. BM immune cells were isolated from the femur by centrifugation . Spleen and BM RBCs were lysed using RBC lysis buffer (eBioscience) for 90 seconds at room temperature. All samples were incubated with FC block  for 10 minutes at 4°C prior to staining with live/dead dye  for 10 minutes at room temperature. Samples were then stained with the following antibody panels at 4°C for 20 minutes. T cell panel; CD45 Qdot 605, CD5 BV510, TCR-d Pe-Cy7, CD161/NK1.1 BV650, CD4 BV786, CD8 AF700, CD25 APC, GITR PE, CD44 FITC, CD62L PerCP-Cy5.5, KLRG1 BV421. Myeloid panel; CD45 Qdot 605, CD11c BV786, CD11b BV510, F4/80 PerCP-Cy5.5, Ly6C AF700, Ly6G APC, CD103 PE, CD317 BV650, MHCII/IA/IE FITC, CD86 PE-Cy7, (lineage) CD3, CD19, NK1.1 BV421. B Cell Panel; CD45 Qdot, IgG1 PE, B220 (CD45R) AF-700, IgM BV786, IgD PerCP-Cy5.5, GL-7 AF647, CD95 PE-Cy7, CD138 BV650, CD5 BV510, CD21/35 FITC, CD23 BV421. NK/myeloid panel; CD19 Pe-Cy7, CD5 APC, Ly6G APC, Ly6C AF700, MHCII/IA/IE FITC, CD11c BV786, CD161/NK1.1 BV650, CD21/35 PE, CD11b BV510, CD23 BV421. Bone marrow panel; CD45 Qdot 605, CD43 PerCP-Cy5.5, GR1 AF700, CD11b BV510, B220/CD45R Pe-Cy7, CD24 APC, CD138 BV650, BP1/Ly51 PE, CD3 BV785, IgD AF488, IgM BV421 (antibody information is detailed in https://doi.org/10.1172/jci.insight.121144DS1). Samples were acquired using a BD Fortessa X20 equipped with 405 nm, 488 nm, 561 nm and 644 nm lasers. Data was analyzed using DIVA software (BD Biosciences), and plots were produced in R using the ggplot2 package.Single cell suspensions from spleen were prepared by mechanical disruption using Miltenyi C-tubes, followed by enzymatic digestion in PBS Ca2HPO4/NaH2PO4 buffer adjusted to pH 7.4. Slices were fixed overnight in 10% formaldehyde and then placed between 2 cover slips and digitally imaged using a high-resolution optical scanner (Epson). Images were analyzed using ImageJ (SciJava), and the size of infarcted area, left ventricle area at risk, and normally perfused left ventricle zone were outlined in each section by identification of their appearance and borders. Areas were quantified on both sides of each slice and averaged. Infarct size (INZ) was calculated as a percentage of RZ for each heart. To produce ischemic hearts for the biochemical analysis, permanent ischemia was performed by directly tightening the LAD with a double knot; heart samples were collected by cutting and snap-freezing the apex 5 and 10 minutes after occlusion.Animal studies used age-matched (12–14 weeks old) WT and TAB1 KI male mice (Cyagen Biosciences). Mice were placed inside an induction chamber, where 4% isoflurane was provided with an oxygen flow rate of 0.5 l/min until loss of righting reflex. Anesthetized mice on a vertical support suspended by its upper incisors were intubated by inserting a 20-G cannula through the trachea. Artificial ventilation of the lungs was provided with a mouse respirator (Hugo Sachs Elektronic MiniVent Type 845) set at 200 μl of stroke volume and at 150 strokes per minute venting 2% isoflurane. A rectal probe was inserted to monitor and to adjust the homeothermic warming pad to maintain the temperature at 37°C ± 1°C. The thorax was exposed and an incision was performed in the third intercostal space. Then, the operating region was exposed by using chest microretractors. Temporary ischemia of the left ventricle was achieved by placing a 0.2 cm PE90 tube parallel to the left anterior descending coronary artery (LAD) and looping an 8-0 nylon suture  around both tube and LAD. The PE90 tube and the knot were released 30 minutes after ischemia, leaving a loose suture in the area where the occlusion took place. Reperfusion was confirmed visually by the return of blood flow back into the myocardium. To assess the area at risk, the loose suture left around the LAD was tightened after 2 hours of reperfusion, and 300 μl of 3% Evans Blue (3% w/v in 0.9% v/w NaCl) was injected in the left ventricle via the apex to delineate the risk zone (RZ). Heart samples were collected by excising and snap-freezing the whole heart, which was then cut into 750-μm sections from apex to base using a custom mouse block. All slices were incubated at 37°C for 20 minutes with 3% w/v 2,3,5-triphenyltetrazolium chloride (MilliporeSigma) dissolved in 0.1 M Na3VO4, 50 mM NaF, 5 mM Na4P2O7, and 1 protease inhibitor cocktail tablet per 50 ml ). Homogenates were centrifuged  at 4°C for 10 minutes, and supernatants were collected and added to 2× SDS buffer  and boiled for 10 minutes. Samples were resolved on 10% SDS PAGE gels under denaturing conditions and transferred onto PVDF membranes. After blocking in 4% nonfat milk and 1% BSA in Tris-buffered saline, pH 7.4, for 1 hour, membranes were exposed to the following primary antibodies overnight at 4°C, with agitation: anti–dual phospho-p38 , total p38 , anti–phospho TAB1Ser423, and total TAB1 . After washing and incubation with HRP-conjugated secondary antibody, antigen-antibody complexes were visualized by enhanced chemiluminescence detection (Pierce Biotechnology).Rapidly snap-frozen apex samples were homogenized in a cold glass pestle and mortar tube containing chilled buffer  or MKK3b (Ac-GKSKRKKDLRISCMSKP-NH2) peptide was dispensed from a concentrated solution in DMSO using a Hewlett Packard D300 digital dispenser directly into a 384-well low-volume assay plate. The DMSO concentration was normalized to 1% of the final assay volume. To this was added a mixture of 2 nM fluorescein-labeled MKK3b peptide (Ac-GKSKRKKDLRISCMSKPK[ε-fluorescein]-NH2) and 10 μM p38α in a buffer of 50 mM HEPES pH 7.4, 10 mM MgCl2, 1 mM Chaps, and 1 mM DTT. The final well volume was 10 μl. After incubation at ambient temperature for 20 minutes, fluorescence polarization was measured on a Perkin Elmer multimode reader . Data were normalized between averaged control wells of DMSO only (0% displacement) and ligand only (100% displacement). Duplicate titrations were averaged and fitted to a 4-parameter IC50 function.A titration of TAB-1 (Ac-RVYPVSVPYSSAQSTSKTSVTLSLVMPSQ-NH1) and SB220025 vs. SB220025 + 3-amino-1-adamantanol (ΔTm2).Thermal shift assays were performed using an Agilent MX3005p quantitative PCR (qPCR) instrument with 70 cycles at 1°C interval from 24°C–94°C. Samples were prepared by mixing recombinant purified p38α with buffer  and ligands ± DMSO to normalize the DMSO level to 2.5% in all samples. Samples were incubated at room temperature for 30 minutes prior to assays. Sypro Orange  was separately prepared at 1:1,000 dilution in the same buffer and then mixed 1:1 with the protein/ligand samples just prior to the thermal shift assays. The final concentration in the assays was 5 μM p38 ± 12.5 mM 3-amino-1-adamantanol (Fluorochem ) ± 50 μM SB220025 (MilliporeSigma). Melting curves were analyzed using PRISM and Tm values calculated using the Boltzmann sigmoidal function. Each experiment was performed 9 times, and mean ΔTm values calculated for DMSO vs. 3-amino-1-adamantanol . ATP (550 μM) was added to the incubation mixture to start the reaction at 37oC for 2 hours. At the end of reaction, the samples were collected with 2× sample buffer , heated to 95°C for 10 minutes, and run on 10% SDS gel under denaturing conditions. The samples were transferred onto a PVDF membrane using semidry technique, blocked with 4% milk + 1% BSA for 1 hour at room temperature, and probed with appropriate primary antibody overnight at 4°C. The blots were probed with appropriate secondary antibody linked with HRP and visualized by enhanced chemiluminescence. Antibodies used included anti–dual-phosphorylated p38 (Thr180/Tyr182)  at 1:5,000, total p38  at 1:10,000, anti–phosphorylated TAB1  at 1:2,000.An in vitro kinase assay was performed with recombinant active p38α and TAB1 (residues 1–438), and 3 μM of pp38α was incubated with 15 μM of TAB1, with or without 12.5 mM 3-amino-1-adamantanol. The reaction was carried out in 1× kinase buffer . All animal protocols were approved both by the local King’s College Animal Welfare and Ethical Review Board and by the UK Government Home Office .This investigation was performed in accordance with the Home Office Guidance on the Operation of the Animals (Scientific Procedures) Act 1986, published by Her Majesty’s Stationery Office . Animals were maintained humanely in compliance with the “Principles of Laboratory Animal Care” formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals (GFDN cowrote the manuscript, supervised the work, designed and performed the biophysical experiments, and expressed and purified the recombinant proteins. RB helped run the mice infarction studies and ran the Western blots of the mice hearts and of the in vitro kinase assay. CN ran the X-rays and thermofluor experiments and expressed and purified the recombinant proteins. MFC ran the mouse infarction studies. PAG ran the cell biology experiments. DT, RA, EDM, and SV helped maintaining the mouse colony. JK ran the computational analysis of the RNA-Seq data. AL ran the immunophenotype experiments. JPH run the fluorescence polarization experiments. PE helped with the Discussion section. JC ran the analysis of the mice infarction data. MSM cowrote the manuscript and supervised the work.


The continuous consumption of chemical energy powers biological systems so that they can operate functional supramolecular structures. A goal of modern science is to understand how simple chemical mixtures may transition from non-living components to truly emergent systems and the production of new lifelike materials and machines. In this work a replicator can be maintained out-of-equilibrium by the continuous consumption of chemical energy. The system is driven by the autocatalytic formation of a metastable surfactant whose breakdown products are converted back into building blocks by a chemical fuel. The consumption of fuel allows the high-energy replicators to persist at a steady state, much like a simple metabolic cycle. Thermodynamically-driven reactions effect a unidirectional substrate flux as the system tries to regain equilibrium. The metastable replicator persists at a higher concentration than achieved even transiently in a closed system, and its concentration is responsive to the rate of fuel supply. Understanding how simple chemical mixtures transition into truly emergent systems is essential to create new lifelike materials. Here, the authors show a self-replicating system that can be maintained out-of-equilibrium by an oxidant fuel in analogy to simple metabolic cycles. The behaviour of out-of-equilibrium biological systems has inspired many synthetic supramolecular materials and networks7 and enabled function such as self-healing and adaptation in materials8, controlled directional motion12, catalysis13, primitive selection14, and oscillatory behaviour17. However, although self-replication19 under out-of-equilibrium regimes is indispensable to life, it is not well developed in synthetic systems21.The structures and functions of even the simplest living organisms seemingly operate counter to the maximisation of entropy and rely on the consumption of chemical energy to maintain dissipative states19, demonstrating emergent traits such as heritability22 and selection23. However, these replicators still invariably form thermodynamic benzoic acid 4 . Two reaction conditions were tested: 1.0 and 8.0 equivalents of octanethiol 1  was confirmed by dynamic light scattering (DLS), with the observation of aggregates of hydrodynamic diameter between 15 and 20 nm depending on concentration 42. The thermodynamic driving force of these reactions makes them effectively irreversible; this irreversibility provides directionality for the metabolic replicator cycle and the system as a whole. The clockwise flux of substrates in the cycle  to selectively oxidize 3 while avoiding oxidation of 1, which would likely otherwise form non-functional 5. The phase separation is therefore important for the observation of self-replication and the establishment of the chemically fuelled substrate cycle.Closure of the substrate cycle in Fig. 4 as well as control the population of 4  under closed system conditions where the starting materials are simply allowed to react , and so 4 reaches a higher concentration before the destruction step can catch up at the steady state.By continuously providing fuel it is possible to establish a steady population of f 4 Fig. . Remarka4 is completely consumed, demonstrating that the observed steady state concentrations of 2, 3, and 4 are not equilibrium nor kinetically trapped positions. Chemical fuel energy consumed by the system is therefore used to drive the metabolic cycle, via the production of components that are thermodynamically and/or kinetically unstable; halting the fuel supply stops regenerating 2 and results in movement towards the equilibrium state.Upon stopping the fuel supply the entire system decays toward equilibrium so that 1 and one equivalent is consumed in each course of the substrate cycle. The system therefore requires an excess of nucleophile 1, which also, importantly, provides the necessary phase separation, although as further reaction cycles are completed phase separation should be maintained by generation of 5. We point out that if 1 were completely consumed then the reaction mixture, after halting of fuel supply and final equilibration, would be expected to contain some 4.The current system does not regenerate octanethiol 4. When fuel is supplied at 5 μL h−1 (instead of 10 μL h−1) the system is no longer able to support as high a concentration of 4 – fewer building blocks are produced in the substrate cycle – so 4 adopts a lower concentration. Here, halting the fuel supply again causes the replicator to die out and the system degrades to equilibrium . All anhydrous reactions were carried out in flame-dried glassware and under an inert atmosphere of argon provided by a balloon or Schlenk line. All reactions were stirred with magnetic followers. CH2Cl2, when used as a reaction solvent, was dried by purification through two activated alumina purification columns. Flash column chromatography was performed using silica gel . TLC analyses were performed on Merck Kiesegel 60 F254 0.25 mm precoated silica plates.Reagents 1H NMR and 13C NMR spectra were recorded on a 400 MHz spectrometer at room temperature in (CD3)2SO, D2O and CDCl3 and referenced to residual solvent peaks. Infrared spectra were recorded as thin films of neat samples on a Bruker Tensor 27 FT-IR spectrometer equipped with Attenuated Total Reflectance sampling accessories. High-resolution mass spectra were recorded on a Bruker MicroTof  under conditions of electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI).4, 2-Nitro-5-(octyldisulfaneyl)benzoic acid: A flame-dried round-bottom flask was charged with 5,5’-Dithiobis-(2-nitrobenzoic acid) (DTNB) , octanethiol  and CH2Cl2 (25 mL) under an argon atmosphere. Et3N  was added and the reaction was stirred for 2 h. The solvent was removed in vacuo before quenching with 1 M HCl (20 mL) and extraction with EtOAc (3 × 20 mL). The organics were dried over MgSO4, filtered and concentrated in vacuo. The crude product was purified by column chromatography  and freeze dried to yield disulfide 4 as an orange solid . 1H NMR 2SO): δ 8.03 , 7.91 , 7.87 , 2.83 , 1.62–1.54 , 1.34–1.19 , 0.82 ; 13C NMR 2SO): δ 165.7, 145.7, 144.6, 128.9, 128.2, 125.7, 124.9, 38.2, 31.2, 28.5, 28.3, 27.6, 22.0, 13.9; IR ṽmax (film)/cm−1: 2925, 2854, 1710, 1610, 1568, 1526; HRMS : [M + Na]+ calcd. for C15H21O4NNaS2, 366.0810; found, 366.0803.Disulfide 3, 5-mercapto-2-nitrobenzoic acid: A two-necked round-bottom flask equipped with a dropping funnel and an exit needle was charged with a suspension of 5,5′-dithiobis-(2-nitrobenzoic acid)  in EtOH . NaBH4  in distilled H2O (2.0 mL) was added dropwise to the suspension at 0 °C. The reaction was stirred at room temperature until gas evolution subsided and was then diluted with EtOH (5 mL) and H2O (5 mL) before acidification with 1 M HCl (10 mL). The mixture was extracted with CH2Cl2 (3 × 20 mL) and the combined organic layers dried over MgSO4, filtered, and concentrated in vacuo to yield thiol 3 as an orange solid . NMR spectra were recorded in (CD3)2SO and in a 500 mM Tris buffer solution in D2O to completely avoid oxidation. This solution was prepared by dissolution of Trizma® base  in D2O (5.0 mL) before adjustment to pH* ~9 with DCl (0.5 M in D2O). Dissolution of 3  in this buffered solution resulted in a solution of pH* ~8. 1H NMR 2SO): δ 8.03 , 7.93 , 7.86 ; 1H NMR : δ 7.81 , 7.38 , 7.26 ; 13C NMR 2SO): δ 165.5, 146.4, 141.9, 129.1, 129.0, 126.7, 125.3; 13C NMR : δ 176.0, 163.4, 137.2, 135.9, 132.5, 130.2, 123.8; IR ṽmax (film)/cm−1: 1704, 1642, 1604, 1570, 1510; HRMS : [M-H]− calcd. for C7H4O4NS, 197.98665; found, 197.98660.Thiol 5, 1,2-dioctyldisulfane: A round-bottom flask was charged with a solution of octanethiol  in Et2O (9.0 mL). To the solution was added an aqueous solution of H2O2  and NaI (tip of spatula). After stirring for 10 min the reaction was quenched with sat. aq. Na2S2O3 (5 mL) and extracted with Et2O (3 × 5 mL). The combined organics were dried over MgSO4, filtered, and concentrated in vacuo to yield disulfide 5 as a clear oil . 1H NMR : δ 2.68 , 1.70–1.63 , 1.41–1.27 , 0.88 ; 13C NMR : δ 39.4, 32.0, 29.4, 29.4, 29.3, 28.7, 22.8, 14.2; IR ṽmax (film)/cm−1: 2955, 2923, 2853; HRMS : [M + H]+ calcd. for C16H35S2, 291.21747; found, 291.21737.Disulfide 4: Per 50 mL Tris buffer: Trizma® base obtained from Sigma-Aldrich  was dissolved in MilliQ H2O (approx. 45 mL). The solution was adjusted to pH ~7.8–8.0 with 12 M HCl (unless otherwise indicated in procedure) and the volume made up to 50 mL with MilliQ H2O. The buffer solution was stored at 4 °C before use. In general, a 25 mM solution of DTNB 2 prepared in this buffer would be pH ~7.5. Any subsequent adjustment of pH is indicated in procedures.Preparation of 500 mM Tris buffer for biphasic reactions and for physical characterisation of 4 in 500 mM Tris buffer at pH 7.5 with variation in concentration of 4 was measured by dynamic light scattering. A 50 mM solution of disulfide 4 was prepared by dissolution of disulfide 4  in Tris buffer . The solution was adjusted to pH 7.5 with a few drops of 1 M HCl. Subsequent twofold serial dilutions were made with a Tris buffer solution  to a concentration of 0.39 mM. Analyses were performed using a Malvern Zetasizer Nano ZEN5600 model system recording particle and molecule size. Instrument control and data processing were performed using Zetasizer software. Disposable plastic cuvettes were used with 1.0 mL of sample solution, which was filtered through a 200 nm filter immediately before measurement. Measurements were performed using an equilibrated heating probe at 25 °C, setting the following parameters for 500 mM Tris buffer : refractive index = 1.339, viscosity = 1.0780 mPa s. Three repetitions of ten measurements were taken for every concentration. Data are shown in Supplementary Figures The size of aggregates of 4 in 500 mM Tris buffer at pH 7.5 was calculated by measuring the surface tension of solutions of different concentration of 4; the critical micelle concentration is the calculated point at which surface tension no longer decreases with increasing concentration of 4. Prior to buffer preparation, Trizma® base was recrystallised twice from 20% EtOH/H2O, and dried over P2O5 overnight. A 40 mM solution of disulfide 4 was prepared by dissolution of disulfide 4  in Tris buffer . The solution was adjusted to pH ~7.5 with a very small quantity of 12 M HCl. Subsequent twofold serial dilutions were made with a Tris buffer solution  to a concentration of 4.9 × 10−3 mM. The surface tension of each solution was measured using a Kruss K10ST tensiometer, calibrated with the surface tension of pure water. Measurements were performed at 25 °C and each solution was measured three times. All measurements were taken on the same day. Data are shown in Supplementary Figure The critical micelle concentration of 2O to a total volume of 500 mL in a volumetric flask, giving an overall concentration of 0.05 mg mL−1 (0.276 mM). To this standard solution was added maleimide (120 mg per 20 mL), which acted as a quench for the reaction. To monitor the reactions (unless otherwise specified) a small volume of the reaction mixture (aqueous phase) was withdrawn at specified time points by plastic syringe. A 25 μL aliquot of this withdrawn solution was then measured by microsyringe and placed into an MS vial, before quenching with 1.0 mL of external standard solution containing maleimide. One microliter of the resultant solution was the standard injection volume for analysis by UPLC. Chromatograms of each resultant sample were obtained using a Waters Acquity ultra performance liquid chromatography (UPLC) H-Class system with photodiode array (PDA) detector. Instrument control and data processing were performed using Empower software. An Acquity UPLC BEH C18 column  was used. A mixture of H2O:MeCN:5% TFA in H2O with a gradient of 93:2:5 → 0:95:5 over 5 min was used as mobile phase. Peak areas were integrated at a wavelength of 215 nm. Calibration of reaction components showing linear fitting was obtained  in DMSO (10 mL). 2.5 mL of this solution was diluted with MilliQ H mL−1 0.26 mM. To 2)  was dissolved by sonication in 12 mL Tris buffer  to reach pH ~7.5. Any necessary adjustment was made with a few drops of 12 M HCl.Control reaction preparation: 5,5′-Dithiobis-(2-nitrobenzoic acid) (DTNB) (2)  was dissolved by sonication in 6 mL Tris buffer . To this solution was added disulfide 4  with sonication for dissolution, to reach a pH of ~7.5 (matching the pH of the control). Any necessary adjustment was made with a few drops of 12 M HCl.Seeded (15 mM) reaction preparation: 5,5′-Dithiobis-(2-nitrobenzoic acid) (DTNB)  was placed dropwise onto the surface of the solution, and the beginning of this addition was considered time zero. The data from these experiments is shown in Fig. Five milliliters of each solution was placed in a 10 mL round bottomed flask and stirring was started at 100 rpm. Octanethiol 4. Seeding reactions conducted with 0.1 mM and 1 mM disulfide 4 were conducted by first preparing a stock solution (2.5 mM) of disulfide 4 in Tris buffer . The data from these experiments is shown in Fig. Reactions at lower concentration of seed were performed analogously to the above control and seeded reactions. For these experiments, however, no additional pH adjustment was made after dissolution of the disulfides in Tris buffer  to avoid significant difference in reaction volume between experiments. Thus, pH is likely to have varied slightly between experiments conducted with different quantity of the seeded product 2)  was dissolved by sonication in 12 mL Tris buffer  to reach pH ~7.5. Any necessary adjustment was made with a few drops of 12 M HCl. Five milliliters of this solution was placed in a 10 mL round bottomed flask and stirring was started at 100 rpm.5,5′-Dithiobis-(2-nitrobenzoic acid) (DTNB) (2O2 (50% wt.) was added to 64 μL of MilliQ H2O. To this was added an additional 0.45 mL of MilliQ H2O. The solution was placed into a 1.0 mL plastic syringe and the solution pushed to the needle tip before placing in the syringe pump assembly and tightening the assembly. Excess solution from the needle tip was wiped away before lowering into the aqueous solution. The syringe pump was then turned on and run at the initial rate indicated. Octanethiol 1 was added dropwise by microsyringe onto the top of the solution momentarily after starting the pump. The beginning of this addition was considered time zero. The fuel addition was stopped or the rate altered at the time points indicated in Fig. Preparation of oxidant solution: 36 μL of an aqueous solution of H2O2 0% wt. waSupplementary Information


We further revealed that the active form of vitamin D 2D3) could inhibit the expression of stemness markers, attenuate acidosis-induced increase of self-renewal ability and mitochondrial respiration in stem cell-like glioma cells. Our study indicates that the acidosis–CYP24A1–vitamin D pathway may be a key regulator of the cancer stem cell phenotype in malignant glioma and point out the potential value for the utilization of vitamin D to target cancer stem cells and to restrain the growth of malignant glioma in the future.Acidosis is a significant feature of the tumor microenvironment in glioma, and it is closely related to multiple biological functions of cancer stem cells. Here, we found that the self-renewal ability, the mitochondrial activity and ATP production were elevated in stem cell-like glioma cells (SLCs) under acidic microenvironment, which promoted and maintained the stemness of SLCs. Under acidosis, 25-hydroxy vitamin D In recent years, evidence shows that these features are closely associated with the existence of glioma stem cells (GSCs). A small percentage of tumor cells in tumor tissue have the character of stem cells, which are referred to as cancer stem cells (CSCs)2, which are known as the root of tumor growth and recurrence. The development and progression of glioma are regulated by various factors, including stem cell pathways, metabolic conversion, epigenetic modification, copy number variation, gene fusion, somatic mutation, and tumor microenvironment3. Tumor microenvironment plays an important role in the stem cell fate decision, and eventually results in the poor treatment outcome4. In addition, it has been reported that normalizing the tumor microenvironment can improve the curative effect5.Due to the character of rapid and infiltrative growth, high recurrence, as well as the resistance to radiation and chemotherapy, the prognosis of glioma remains very poor6. Low pH is thought to be the driver of tumor progression and treatment resistance8. Furthermore, low pH are the determinant factors of tumor cell metabolism phenotype, which can provide the basic requirements of the tumor cells by changing the core cell metabolic phenotype and making cancer cells to reach its groove12.The main features of microenvironment in glioma is low pH value. It has been reported that the pH value is ~7.1 in the normal brain tissue, while in glioma tissues the pH value is about 6.813, further revealed that the acid environment induced the expression of VEGF through activation of the Ras and ERK1/2 MAPK-signaling pathways14. The acidic environments promoted and maintained glioma stem cell phenotype through inducing the expression of HIF2 alpha and HIF target genes15. Furthermore, Filatova et al. found that acidic environment increased the expression of hypoxia inducible factor (HIF) by heat shock protein 90 (HSP90), rather than PDH/VHL dependence pathway, in order to maintain the stemness of glioma cells16. Studies on the metabolism of GSCs also have made great progress. It is reported that GSCs maintain their demands for energy and biological macromolecular materials mainly through oxidative phosphorylation in mitochondria. Compared with the highly differentiated glioma cells, GSCs consume less glucose, maintain high levels of ATP and mitochondrial respiratory reserve capacity17. Likewise, mitochondrial dynamic regulates the biology characteristics of glioma stem cell, loss of Dynein protein 1 (DRP1) inhibits the proliferation, self-renewal, and tumor formation of glioma stem cell18. At the same time, latest study showed that acetyl coenzyme A was mainly provided by the fatty acid oxidation in a variety of solid tumors cells when the cells were in the acidic conditions, which changed the glucose metabolism in general condition and maintained tricarboxylic acid cycle and respiration of tumor cells. These results suggested that tumor cells can sustain their survival by switching their major metabolic pathways when they are in acid condition19.The study of acidic environment in glioma began in 2001, evidence found that acid environment can increase the transcription of vascular endothelial growth factor (VEGF) in brain glioma cells3-24-hydroxylase and degraded the active hormone 1α,25-dihydroxyvitamin D320. CYP24A1 was accounted for the stemness and metabolism changes of SLCs, mainly through the fast degradation of 1α,25-dihydroxyvitamin D3. Our study suggested that it might be a potential treatment strategy to target CYP24A1 for inhibiting the growth of malignant glioma under acidic microenvironment.As an important feature of the microenvironment of glioma, low pH regulates the angiogenesis, invasion, and resistance to chemotherapy of glioma. But the mechanism of adaptation in acidic environment of glioma cells and their metabolic changes induced by acidic environment are still unclear. In this study, we found that the self-renewal ability and mitochondrial respiration were elevated in stem cell-like glioma cells (SLCs) in acidic microenvironment. CYP24A1 is expressed in the inner membrane of mitochondria, served as 25-hydroxyvitamin DFresh glioma samples were obtained from Beijing Tiantan Hospital and Beijing Sanbo Brain Hospital. Human glioma cell lines (U87MG and T98G) were purchased from the American Type Culture Collection (ATCC) . U251 cells were purchased from the Cell Center of Peking Union Medical College. Four human normal astrocyte cell lines  were purchased from ScienCell .The U87MG and T98G cells were cultured in modified Eagle’s medium (MEM), and the U251 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM). Both media were supplemented with 10% fetal bovine serum (FBS) , 100 U/mL penicillin (Life Science), and 100 U/mL streptomycin (Life Science). The normal human astrocyte cells  were cultured in astrocyte medium .21.Neurosphere formation culture and primary stem cell-like glioma cell culture from glioma specimens were performed as described previously22.For different pH treatment, 25 mM HEPES  was added into SFM, 1 M HCl or 1 M NaOH was used to titrate SFM until the pH value reached 7.4, 6.8, 6.7, 6.6, and 6.5. Then incubate for 24 h and retitrate to the corresponding pH value23. In brief, the sphere cells were plated in 96-well plates in 100 μL SFM culture medium. Final cell densities ranged from 500 to 3 cells/well in 100 μL volumes. After 12 days, the percentage of wells that are not containing spheres (diameter ≥ 50 μm) for each cell plating density was calculated.The limiting dilution assay was performed as described previously23. Cells were diluted to 1000–5000 cells per 100 μL. The cells were allowed to grow for 6–8 days, with 50 μL fresh SFM added at day 3 or 4. Then, diameter of the neurospheres ≥ 50 μm in each well were counted under a microscope.The neurosphere-forming assay was performed as described previously24. β-actin  was used as inter control. The antibodies used included anti-CYP24A1 , anti-GFAP , anti-Tuj1 , anti-Sox2 , anti-CD133 , anti-Nestin , anti-Oct4 , anti-CNPase , and anti-β-actin .Immunoblotting was performed as described previouslyFor staining the SLC spheres and differentiated SLCs, the cells were fixed with 4% paraformaldehyde, permeabilized by 0.3% Triton X-100 (Sigma), and incubated with the indicated first antibodies and fluorescence-conjugated secondary antibodies  and stained with 4′,6-diamidino-2-phenylindole  for nuclear staining. The stained cells were observed with a fluorescence microscope . The antibodies used included anti-CYP24A1  and anti-CAIX . Fluorescence-conjugated secondary antibodies were from Life Technologies .d-lysine (10 μg/mL) for 2 h then coated with laminin (10 μg/mL) overnight. The next day washed the wells twice with saline, and 10,000–100,000 cells were plated per well. Three metabolic inhibitors were injected sequentially as specific time points: oligomycin (1 μM), followed by FCCP (0.75 μM), followed by a combination of rotenone and antimycinA (0.5 μM). Basal OCR were measured using the Seahorse XF24 plate reader. Several parameters of oxygen consumption were analyzed as previously reported25.The oxygen-consumption rates (OCR) of glioma cells and SLCs were measured using the Seahorse XF Extracellular Flux Analyzer (Seahorse Bioscience). Twenty-four-well plates (Seahorse Bioscience) were coated with 50 μL poly-3. In brief, total RNA was isolated using TRIzol reagent according to the manufacturer’s instructions . 2.0 μg total RNA was used for cDNA synthesis  and 1/10 of the cDNA volume was used for quantitative PCR . The PCR amplification was carried out using a CFX96 touch system  or an ABI 7500 system . The Ct values were normalized to human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. All samples were run in triplicate in each experiment. Each assay was repeated three to four times. All primers were synthesized by Life Technology  and TSINGKE .Real-time RT-PCR (QRT-PCR) was carried out using SYBR Premix Ex Taq Master Mix and a 2-Step kit  as described previously6 pH 7.4 or pH 6.8 treated GSC5 cells were digested by accutase (Invitrogen). Total RNA from cells were lysed using TRIzol reagent (Invitrogen). The RNA integrity was measured at an Agilent Bioanalyzer 2100 instrument . The qualified total RNA was further purified using NucleoSpin® RNA clean-up Kit. Total RNA was amplified and labeled with a Low Input Quick Amp Labeling Kit (Agilent Technologies), per the manufacturer’s instructions. The labeled cRNA was purified with an NucleoSpin® RNA clean-up Kit, and 10 μg RNA was used for hybridization on the Genechip Agilent Human lncRNA 4*180K array (Agilent Technologies). The slides were scanned in an Agilent Microarray Scanner G2565CA (Agilent Technologies) under the default settings. Quantile algorithm  was used for the raw data normalization.~102D3 in SLCs, 1 μM 25(OH)D3  was added into the cell culture medium. After 12 h, GSC2 cells were collected under yellow light, 5 × 107 cells were quenched by directly adding 500 μL CH3OH, and then extracted by adding 1 mL methyl tert-butyl ether (MTBE). The extraction solvent is equivalent to MTBE–CH3OH . The cells were vortexed for 5 min (2500 rpm) and transferred to a microcentrifuge tube and snap-frozen in liquid nitrogen. The cells were then thawed and vortexed for 60 s (2500 rpm), the freeze–thaw process was repeated for two more cycles, and the cells were then vortexed for 5 min (2500 rpm) and centrifuged at 12,000 rpm for 10 min. The supernatant was transferred to a microcentrifuge tube and dried by using a centrifugal evaporator at 35 °C and 150 μL MeOH–IPA–H2O  was added to the residue. The samples were vortexed for 5 min (2500 rpm) and centrifuged at 12,000 rpm for 10 min, and the supernatant was then transferred to autosampler vials for analysis. 1α,25(OH)2D3 was quantified by a UPLC–ESI/MS/MS MRM system  and QTRAP 5500 , 25(OH)D3 was used as the internal standard.For the quantification of 1α,25(OH)23, 1 × 106 pH 7.4 or pH 6.8 treated GSC2 cells were injected subcutaneously to the left flank of nude mice in 0.1 mL of PBS. After 6 days, 1 μM/kg NaHCO3 in 0.1 mL of sterile water was injected subcutaneously to the left flank of nude mice every other day. After 27 days the tumor volumes of each group (n = 5) were estimated using the formula V = ab2/2 .Subcutaneous xenografts formation was conducted as described previously5 pH 7.4 or pH 6.8 treated GSC2 cells in 5 μL of culture medium by stereotactic injection to the right neostriatum. 1 μg/kg 1α,25(OH)2D3 was administered intraperitoneally 6 days a week from day 5. Groups of mice (n = 5) were undergoing a magnetic resonance imaging  after 17 days. Animal protocol was approved by the Animal Ethics Committee of Peking Union medical college.For intracranial xenografts, BALB/c-nu mice received 1 × 10t-test was used for statistical analysis, and a P < 0.05 was considered statistically significant.Data were expressed as the means ± SDs and were analyzed by SPSS 14.0 software . Student’s Glioma samples were classified according to the Histological Grades of Tumors of the Nervous System . Informed consent was obtained before surgery. The study was approved by the Medical Ethics Committee of Beijing Tiantan Hospital.21 in mediums with five different pH values , and their self-renewal abilities were examined  which we previously establishedned Fig. . Under aned Fig. . This isned Fig. , which iWe further investigated the mitochondrial respiration of SLCs/7.4 and SLCs/6.8 to explore the effect of acidosis on mitochondrial metabolism, results showed that the basal respiration, maximal respiration, and ATP production were increased in acidic-treated SLCs Fig. . MoreoveTo explore the underlying mechanism of the observation that the stemness and mitochondrial respiration were different under acidosis, the mRNA and long noncoding RNA (lncRNA) microarray analysis was used to identify the differences between pH 7.4 and pH 6.8 treated GSC5 cells. And we found that 888 genes and 1826 lncRNAs were upregulated while 70 genes and 83 lncRNAs were downregulated in pH 6.8 treated GSC5 (fold change > 2). Then we further selected seven genes from 958 genes (888 upregulated and 70 downregulated) and seven lncRNAs from 1909 lncRNAs (1826 upregulated and 83 downregulated) that exhibited 10-fold change greater than pH 7.4 treated GSC5 Figures  and S2B.Next, we used relative quantitative real-time PCR to examine the expression of selected genes and lncRNAs in U87MG-SLCs, U251-SLCs, GSC2, and GSC5 Figures , and S2DWe further performed functional screening by knocking down the candidate genes IL22 (interleukin 22), GUCA2B (guanylate cyclase activator 2B), CYP24A1 , and lncRNAs (RP11-149F8.5 and linc-RRP15-1) Fig. . NeurospTo further determine the effect of the five candidates on stemness and mitochondrial respiration in SLCs, we first used neurosphere formation assay to examine the self-renewal ability in U251-SLCs. Results showed that the increased number of neurospheres was blunted under acidosis by knockdown of IL22, GUCA2B, CYP24A1, while it was completely inhibited when silencing lncRNA RP11-149F8.5 and linc-RRP15-1 Figs.  and S3C.3 and 1α,25-(OH)2D3 into 24-hydroxylated metabolites26.To evaluate the correlation between the expression of CYP24A1 and the malignancy of glioma, and the expression pattern of CYP24A1 in acidic microenvironment in vivo is also important. We first analyzed the expression of CYP24A1 in pH 7.4 and pH 6.8 treated U87MG-SLCs, U251-SLCs, GSC2, and GSC5 cells, as well as four normal cell lines, three glioma cell lines, and four SLCs. CYP24A1 was overexpressed in pH 6.8-treated SLCs and showed the highest level in SLCs compared to other cell lines 2D3 in pH 7.4 and pH 6.8 treated GSC2 cells. The quantity of 1α,25-(OH)2D3 decreased significantly in GSC2 cells under acidosis 2D3 in acidic microenvironment. Therefore, we proved that CYP24A1 may be positively related to the malignancy of glioma and the stemness of GSCs.Next, we injected U251, GSC2, and GSC5 cells subcutaneously in nude mice to obtain glioma xenografts, using carbonic anhydrase IX as an acidic indicator, we examined the expression pattern of CYP24A1 in vivo. The hypoxic and acidic microenvironment existed around the necrotic zone and the oxygen and pH were relatively high in tumor expanding area. We found that carbonic anhydrase IX was highly expressed around necrotic zone, which indicated high hydrogen ion concentration in this area, and the expression of CYP24A1 was similar with the expression of carbonic anhydrase IX Figs.  and S6B.sis Fig. . All of 2D3 has a variety of anticancer functions, it can inhibit the proliferation, invasion and metastasis, as well as induce the apoptosis and differentiation of cancer cells. Moreover, some studies have shown that 1α,25(OH)2D3 could target CSCs in prostate and breast tumor27. These drove us to investigate the function of 1α,25(OH)2D3 on SLCs. To confirm the effective concentration of 1α,25(OH)2D3 treatment, we used 10 and 100 nM to treat SLCs for 4, 8, 12, 24, and 48 h. The expression of stemness markers showed that NESTIN, OCT4, and SOX2 were decreased in GSC5 cells when treated with 1α,25(OH)2D3 10 or 100 nM for 4–24 h, while OCT4 and SOX2 were decreased in GSC2 cells with 1α,25(OH)2D3 100 nM treatment for 4–24 h, only OCT4 decreased with 1α,25(OH)2D3 10 nM treatment, and NESTIN was increased in GSC2 cells lls Fig. . Meanwhi3 Figure . In summ2D3 on self-renewal ability. The size and number of neurospheres were reduced with 10 or 100 nM 1α,25-(OH)2D3 under pH 7.4 and pH 6.8 conditions, and the promotion of neurosphere formation was almost eliminated in GSC2, GSC5, and U251-SLCs under acidic 2D3 to weaken the effect of 1α,25-(OH)2D3 on SLCs. It might be the pathway for SLCs to maintain their cancer stem cell phenotypes.Furthermore, we tested the effect of 1α,25(OH)ic Figs.  and S4B.2D3 repressed the stemness and self-renewal of SLCs, it is interesting whether 1α,25(OH)2D3 could affect the mitochondrial metabolism of SLCs. Then, we measured the mitochondrial oxygen consumption rate of SLCs pre-treated with 1α,25(OH)2D3 10 or 100 nM for 8 h under pH 7.4 and pH 6.8 conditions. The basal respiration and maximal respiration of mitochondrial in GSC2 and GSC5 cells were significantly reduced with 1α,25(OH)2D3 treatment, and the increase of mitochondrial respiration was almost completely suppressed under pH 6.8 conditions ns Figs.  and S5A.ent Fig. .2D3 on SLCs in vivo, we used pH 7.4 and pH 6.8 treated GSC2 cells to form xenografts in the brain of nude mice, then treated with 1α,25(OH)2D3 and evaluated tumor size. We found that treatment of 1 μg/kg 1α,25(OH)2D3 reduced the size of tumors derived from GSC2 cells, while the suppression of 1α,25(OH)2D3 was relatively obvious in tumors arising from pH 6.8-treated GSC2 cells 2D3 may be a safe and effective compound to treat malignant glioma.Finally, to validate the effect of 1α,25(OH)lls Fig. . The res28. Therefore, we further investigated the effect of bicarbonate on the tumorigenic capacity of SLCs. pH 7.4-treated and pH 6.8-treated GSC2 cells were used to form subcutaneous xenografts. Then, we used NaHCO3 to treat the tumor tissue and found that the tumor mass arising from pH 6.8-treated GSC2 was significantly decreased 2D3, also named calcitriol) could inhibit the stemness of SLCs  are side population analysis, CD133-labeled cell sorting and neurosphere growth34. However, these methods only enriched a fraction of GSCs, and the isolated cells are still heterogeneous population. The results that acidosis promoted the cancer stem cell properties of SLCs remind us of acidic condition may be a method to further isolate and purify GSCs.Although the generation of acidic microenvironment partly is due to the metabolic phenotypes of cancer cells2D3) could restrain tumor growth mainly through the vitamin D response elements of target genes38. This is correlated with the inhibition of proliferation and angiogenesis, induction of differentiation and apoptosis function of 1α, 25(OH)2D3 in cancer39. Epidemiological studies implied that vitamin D deficiency was related to the increase of multiple cancer incidence in the world40. Evidence showed that in the area of less sunshine the incidence of colon and prostate cancer was increased42. Moreover, 1α,25(OH)2D3 lead to senescence, cell-cycle arrest, and differentiation of prostate stem cells43. A vitamin D derivative could inhibit the tumor growth and the expression of cancer stem cell marker CD44 in human breast cancer44. Additionally, we found that calcitriol repressed the self-renewal and cancer stem cell marker expression in SLCs, these results revealed the function of vitamin D on the suppression of GSCs in malignant glioma.Vitamin D has been thought to play a potential role in cancer therapy. Research showed that the active metabolite of vitamin D 2D345. It has been considered to be a potential oncogene in breast cancer46. The overexpression of it in lung adenocarcinoma was associated with patients’ poor survival because of the rapid clearance of 1α,25(OH)2D3 and the abrogation of antiproliferative effects47. The combination use of CYP24A1 inhibitor and calcitriol exhibited a more effective anticancer function in prostate cancer49. We found a relatively high expression of CYP24A1 in high level glioma tissue 2D3 in glioma tissues. The expression of CYP27A1 is relatively low in high level glioma tissue, the expression of CYP27B1 is elevated in glioma tissues and increased significantly in grade IV glioma tissue ue Figs.  and S6A. Figures  and S7B.50. So we inquired whether there was a related charged structure in CYP24A1 protein, and found that CYP24A1 had a P450 domain51, but whether this structure could detect proton concentration is unknown. Because of the elevated expression of CYP24A1 mRNA in acidic environments, we examined the effect of acidic environment on the CYP24A1 promoter region in SLCs and found no significant changes (data not shown). Whether there is another transcriptional or post-transcriptional regulatory mechanism requires further study. The over expression of CYP24A1 resulted in the reduction of 1α,25(OH)2D3, thus reversed 1α,25(OH)2D3 caused stemness decrease in SLCs and may increase the malignancy, as well as therapy resistence of SLCs. This study suggested that CYP24A1 may play an important role in malignant glioma and provided a new strategy to use CYP24A1 inhibitor and calcitriol for glioma therapy.Here, we discovered that CYP24A1 was highly expressed in SLCs under acidosis. Recent studies have reported that Smad5 can feel the changes in intracellular pH value, and shuttles between nuclear and cytoplasm. The increase in intracellular pH can make protons dissociate from the MH1 domain of Smad5, resulting in its export to cytoplasm and interaction with HK1, thus accelerates glycolysissupplemental material


Triazole compounds 4–19 were synthetized via Click Chemistry strategy by 1,3-dipolar cycloaddition between terminal acetylenes and aryl azides containing methoxy and methylenedioxy groups as substituents. Our results suggest that most derivatives were active against intracellular amastigotes, with IC50 values ranging from 4.4 to 32.7 µM. The index of molecular hydrophobicity (ClogP) ranged from 2.8 to 3.4, reflecting a lipophilicity/hydrosolubility rate suitable for transport across membranes, which may have resulted in the potent antileishmanial activity observed. Regarding structure-activity relationship (SAR), compounds 14 and 19, containing a trimethoxy group, were the most active , with low cytotoxicity on mammalian cells (SI = 14.1 and 10.6). These compounds induced nitric oxide production by the host macrophage cells, which could be suggested as the mechanism involved in the intracellular killing of parasites. These results would be useful for the planning of new derivatives with higher antileishmanial activities.Sixteen 1,4-diaryl-1,2,3-triazole compounds  Leishmania (Leishmania) amazonensis Lainson and Shaw 1972 is one of the etiological agents of CL. It produces the typical localized lesions or the diffuse form of the disease, in which the parasite spreads due to impaired cell-mediated immune response. Thus, the lesions tend not to heal spontaneously and to be more resistant to treatment [Cutaneous leishmaniasis (CL) is a parasitic infectious disease that affects the skin, cartilage and mucosa of the upper respiratory tract. The resulting ulcers can develop into destructive and disabling injuries, making the illness a serious public health problem . There hreatment ,4.The first choice drugs for CL are the pentavalent antimonials, which are associated to hepato, cardio and nephrotoxicity . When thet al. [1, grandisin 2, and machilin G 3. These compounds have been considered important scaffolds in molecular modification studies due to their antileishmanial and antichagasic activities [et al. [L. (L.) amazonensis intracellular amastigotes, searching for structure-activity relationship information to support the development of new drug candidates for CL.Cassamale et al.  have syntivities ,14,15,16 [et al.  demonstr1–3 were classified as active (<20 µM), moderately active (20–50 µM), and potentially inactive (>50 µM) according to Upegui et al. [L. (L.) amazonensis intracellular amastigotes . Compounds 5, 7, 15, 16, 17, and 18 have been moderately active and 13 did not show potential activity on the parasites   .6 was active on intracellular amastigotes , with the highest selectivity index. This compound was 66 times more toxic to amastigotes than to mammalian cells  for grandisin on L. (L.) amazonensis promastigotes [2 , which may have resulted in a better solubility and consequent in vitro activity. Indeed, more soluble grandisin derivatives have been synthetized in order to reduce its lipophilicity, which may limit in vivo studies [Grandisin derivative an cells . The substigotes . Further studies . It is i6 had significantly decreased the infection index at the concentration of 6.25 µg·mL−1, reaching 98.6% reduction (p < 0.0001) at the concentration of 50 µg·mL−1 (6 has induced an increased production of nitric oxide (NO) compared to untreated infected cells (control) (6 does not seem to have a direct action on L. (L.) amazonensis once it was proven inactive on promastigote forms [Treatment with compound  µg·mL−1 A. 6 has control) A. Thus, te forms .11) and moderately active (15 and 18), but less selective, with important cytotoxicity on mammalian cells   were active (an cells . All of  0.0001) A. This cs tested A.5 with a dimethoxy group as substitution pattern on rings A and B (veraguensin 1 derivative) was moderately active on intracellular amastigotes  with relative selectivity (SI = 3.6). Silva Filho et al. [L. donovani promastigote forms . Once again we may educe that the insertion of triazole ring may have improved the antileishmanial activity due to reduction of octanol/water partition coefficient (3.1 (5) versus 4.2 (1)). Compound 5 has significantly reduced the infection index from the concentration of 12.5 μg·mL−1 , excepting 16, which was moderately active but selective . Despite its moderate activity, 16 was able to significantly induce NO production  (p = 0.0003)  , and red 0.0003) B. It’s il action B.50 values from 13.1 to 16.8 μM . As well as 12, this compound was able to induce an increase of NO production at the highest concentration tested (Compounds from methoxy series showed ICn tested C.19) came from methylenedioxy series (IC50 = 4.4 µM). Compound 19 is a hybrid from machilin G 3 and grandisin 2 and was also quite selective (SI = 10.6)  amazonensis promastigote forms (IC50 = 7.2 µM), and this suggests its direct action on the parasite. On the other hand, position isomer 18  . 19 has s tested D. Cassam  was toxic for the macrophages (SI = 0.3) with moderate antileishmanial activity 2/CuI, Et3N provided the acetylene alcohols 22a–c with 81%–86% yields, after 24 h reaction time [Triazole derivatives patterns . The synand SiO2 ,27. Subsion time ,28.22a–c with KOH under reflux in toluene generated the terminal acetylenes 25a–c with 75% to 79% yield [25d was synthesized by the Corey-Fuchs method  and azides 27a–d  in dichloromethane (2 mL) and water (2 mL), were added CuSO4·5H2O  and sodium ascorbate . The reaction mixture was stirred for 24 h. Then it was added a saturated solution of NH4Cl (30 mL) and the product was extracted with dichloromethane (3 × 20 mL). The organic phase was dried over anhydrous MgSO4, and the solvent was evaporated under reduced pressure. The products were purified by recrystallization from ethyl acetate.To a solution of terminal acetylenes 4–19, theoretical values of logP (ClogP) were calculated with ChemAxon software. The program estimated octanol/water partition coefficient based on a modified version of the method of Viswanadhan et al. [In order to estimate the molecular hydrophobicity of triazole derivatives n et al. .In vitro antileishmanial activity was evaluated on peritoneal macrophages infected with. L. (L.) amazonensis intracellular amastigotes. Parasites (IFLA/BR/1967/PH8 strain) were routinely isolated from BALB/c mice and maintained as promastigotes at 25 °C in Schneider’s Insect Medium  supplemented with 20% fetal calf serum  and 140 µg·mL−1 gentamicin (Sigma-Aldrich®). Macrophages were obtained from peritoneal wash of BALB/c mice after euthanasia. 10 mL of RPMI 1640 (Sigma-Aldrich®) supplemented with 2% l-glutamine, 2.8% bicarbonate buffer, 100 U·mL−1 penicillin and 100 mg·mL−1 streptomycin were injected into the peritoneal cavity. After massage area, liquid was aspirated and transferred to tubes on ice. Peritoneal cells were quantified in Neubauer chamber after cellular exclusion with Trypan Blue staining (Sigma-Aldrich®).5 cells/well) were added to 24-well plates containing circular coverslips. Plates were incubated for one hour at 37 °C/5% CO2 to allow cell adhesion and then 1 × 106L. (L.) amazonensis promastigotes were added to each well. Plates were incubated at 35 °C/5% CO2 for four hours and then cells were treated for 24 h with synthetic compounds 4–19 (6.25–50 µg·mL−1). Amphotericin B (Sigma-Aldrich®) was used as the reference drug (0.25 to 2 µg·mL−1) and untreated cells were used as negative control. Coverslips were processed as described by Rizk et al. [50) was calculated using a nonlinear regression curve. Infection index was obtained as described by Paladi et al. [Peritoneal cells  by comparing to a standard curve of known concentrations of sodium nitrite (1–10 µM) in RPMI medium (Sigma-Aldrich®).To evaluate the nitric oxide production (NO) by infected and treated peritoneal cells, 50 µL of culture supernatant from the antileishmanial assay were collected and incubated with equal volume of Griess reagent  for 10 min at room temperature. According to Ding et al. , the abs5 mL−1) and incubated with compounds at 37 °C/5% CO2 for 48 h at concentrations of 0.25–250 µg·mL−1 to estimate IC50. Amphotericin B (Sigma-Aldrich®) was used as the reference drug (0.025–25 µg·mL−1). Cell growth was evaluated according to Skehan et al. [®, Rio de Janeiro, Brazil) was used as negative control at the concentration used to solubilize the highest concentration of compounds. IC50 was calculated by nonlinear regression curve. Selectivity index was calculated according to Medeiros et al. [Murine macrophages  were seeded in 96-well plates  under protocol 503/2013.1, grandisin 2 and machilin G 3, 15 showed high or moderate antileishmanial activity. Compounds 14 and 19, containing a trimethoxy substituent on ring B, were the most active against intracellular amastigotes, with low cytotoxicity on mammalian cells. These compounds induced nitric oxide production by the host macrophage cells, which could be suggested as the mechanism involved in the intracellular killing of parasites. These results would be useful for the planning of new derivatives with higher antileishmanial activities.Among 16 synthetic derivatives of tetrahydrofuran neolignans veraguensin


Single crystals of three isotypical Cu(II) coordination complexes with paddlewheel structures exhibit mechanical motion while undergoing [2+2] cycloaddition under UV light , and also exhibit large positive thermal expansion on heating. L2], where L = 4-styrylpyridine (4spy) (1), 2′-fluoro-4-styrylpyridine (2F-4spy) (2) and 3′-fluoro-4-styrylpyridine (3F-4spy) (3), which show photosalient behavior  while they undergo [2+2] cycloaddition. These crystals also exhibit anisotropic thermal expansion when heated from room temperature to 200°C. The overall thermal expansion of the crystals is impressive, with the largest volumetric thermal expansion coefficients for 1, 2 and 3 of 241.8, 233.1 and 285.7 × 10−6 K−1, respectively, values that are comparable to only a handful of other reported materials known to undergo colossal thermal expansion. As a result of the expansion, their single crystals occasionally move by rolling. Altogether, these materials exhibit unusual and hitherto untapped solid-state properties.Although a plethora of metal complexes have been characterized, those having multifunctional properties are very rare. This article reports three isotypical complexes, namely [Cu(benzoate) It is relatively easy to design composite materials combining two or more solids with different properties into hybrid materials for specific applications  or photosalient (PS) effects 5(NO2)](Cl)(NO3) were shown to bend as well as to jump violently under UV light 4(L)2], where L = 4-styrylpyridine (4spy) (1), 2′-fluoro-4-styrylpyridine (2F-4spy) (2) and 3′-fluoro-4-styrylpyridine (3F-4spy) (3) also pop violently under UV light, and thus they are photosalient. Furthermore, crystals of these materials exhibit very large anisotropic thermal expansion when heated from room temperature to about 200°C.Recently, a great number of organic, inorganic and organometallic crystals showing these properties have been discovered (Commins 2.2.1.1–3 were obtained by slow evaporation of methanol solution of Cu(NO3)2·3H2O, sodium benzoate and the respective pyridyl ligand  , as shown in Fig. 11, 3.765 Å in 2 and 3.810 Å in 3, and thus they are at distances suitable for [2+2] cycloaddition reactions  as the photoproduct in which the [Cu2(benzoate)4] paddlewheels are joined by the product cyclobutane ligands . The disappearance of the olefinic protons at 8.13 p.p.m., the appearance of cyclobutane protons at 4.82 p.p.m., and a shift in the pyridyl protons from 7.65 and 7.93 p.p.m. to 8.38 and 8.65 p.p.m., confirmed the formation of the expected cyclobutane ring photoproduct. The other two compounds 2 and 3 also showed quantitative photoconversion of their C=C bonds to cyclobutane rings . After the photoreaction of 1–3, the respective one-dimensional CPs  were semi-crystalline, as confirmed using powder X-ray diffraction (PXRD) . Crystal structure determination of a recrystallized sample of 5 provided further evidence of the formation of the one-dimensional CP [Cu2(benzoate)4(rctt-2F-ppcb)], where rctt-2F-ppcb = rctt-1,3-bis(4-pyridyl)-2,4-bis(2′-fluorophenyl)cyclobutane, . The single crystals, depending upon their size and shape, display different types of movements under UV light, similar to isotypical Zn(II) complexes  complexes, which posits the existence of both reactants and photoproducts in the single crystals and rapid buildup of stress in the crystal, until they pop out or fragment into smaller pieces.Interestingly, the crystals and powders of 1–6 were measured by the flotation method. A comparison of the densities of 5  and 5A  shows that the molecules in 5A  and these correspond to the increase in unit cell volumes of 15.4, 17.5 and 13.1% on going from 1–3 to 4–6, respectively (Table S2). The mobility of dynamic single crystals is usually triggered by the sudden release of stress in the form of a very fast phase transition or a chemical reaction accompanied a rapid structural change that drives these phenomena . However, this behavior was not consistent and was not reproducible with all batches of crystals. Hence, we concluded that the motion is not a result of TS effects. Instead, it could be due to non-uniform or sudden heating. A recent report of a TS behavior of the organic compound methscopolamine bromide observed motion that was not accompanied by a detectable phase transition, and this effect was attributed to unusually large anisotropic thermal expansion with coefficients of 135 (1) × 10−6 K−1 and 114 (1) × 10−6 K−1 along the a and c axes, respectively . The DSC of 1–3, recorded from either single crystals or powder, did not show a phase transition from room temperature to their decomposition temperature . The VT-PXRD results corroborate the conclusion obtained from the DSC experiments .To obtain a better insight into the thermal behavior of the compounds reported here, we performed thermogravimetry (TG), differential scanning calorimetry (DSC) and variable temperature powder X-ray diffraction (VT-PXRD) measurements. The TG results show that 2.2.1–3 was investigated by VT-PXRD measurements in the temperature range from room temperature to 200°C, just below their decomposition temperature . Since these compounds crystallize in a monoclinic space group, the coefficients of thermal expansion were calculated using the program PASCal  along the principal X3 axis [ × 10−6 K−1]. Although compounds 1 and 2 show a relatively small PTE , compound 3 exhibits a small negative thermal expansion (α1 = −13.8283 × 10−6 K−1) along the principal X1 axis. The details are displayed for 3 in Fig. 41 and 2 in Figs. S32 and S33, respectively. Furthermore, no hysteresis was observed on cooling for any of the crystals, and all expansions are rather linear in the measured temperature range .The thermal expansion coefficients are reported along the principal 1–3 can be explained through a detailed analysis of the fundamental structural motifs. In all compounds the [Cu2(benzoate)2L4] paddlewheel complexes are connected by π—π interactions between the C=C bonds of the styrylpyridine ligands, resulting in one-dimensional chain-like motifs running in the  cycloaddition reaction, s Table 1. Most of1–3 to 4–6, respectively, as determined from the densities by the flotation method. This is corroborated by the unit cell volume measurements of 4–6 from XRPD experiments . The stress generated by the phase heterometry is released suddenly in the form of a very fast chemical reaction accompanied a rapid structural change that appears to drive this PS effect.Complementary π–π interactions in head-to-tail alignment of the 4spy ligands are congenial to make photoreactive crystals and this alignment results in one-dimensional aggregates of the Cu(II) complexes. Furthermore , all these π–π aggregates are packed parallel to each other. Hence, the formation of the cyclobutane rings from olefin pairs promotes anisotropic volume expansion during the photoreaction. This is further supported by the increase of the unit cell volumes of 15.4, 17.5 and 13.1% on going from 3.et al., 2008et al., 2010et al., 2014et al., 2018et al., 2018et al., 2015et al., 2009et al., 2006et al., 2005et al., 2004et al., 2019et al., 2008et al., 20081–3 promotes large thermal expansion from room temperature to 200°C in addition to photoreactivity and the PS effect. The volumetric thermal expansions (VTE) observed for 1, 2 and 3 are 241.8, 233.1 and 285.7 × 10−6 K−1, respectively. Of these, the value observed for 3 is the largest for metal complexes, based on comparison with the previously reported value of 255.5 × 10−6 K−1 for the palladium(II) complex  while undergoing [2+2] cycloaddition. This observation provides strong evidence that the olefin pairs are intact even at higher temperatures, thus satisfying the Schimidt criteria for a [2+2] cycloaddition. Therefore, anisotropic expansion occurs roughly normal to the one-dimensional aggregates. We conclude that the robustness of the π–π interactions in this crystal packing is ultimately the key structural feature for all three properties observed with these materials. This work therefore provides new insights towards the engineering of multifunctional properties in crystals, and favors these and similar compounds as candidates for in-depth studies into the factors that determine the salient effects.Molecular solids in general are expected to have moderate positive thermal expansion (PTE) due to increasing anharmonic vibrational amplitudes of their molecules. In many cases, structural peculiarities may give rise to very large PTE  1, 2, 3, 5a. DOI: 10.1107/S2052252519014581/lq5028sup2.pdfSupporting data. DOI: Click here for additional data file.10.1107/S2052252519014581/lq5028sup3.mp4Video S1: photosalient effect of a single crystal of 1 showing rolling, hopping, breaking and disintegration of a single crystal during [2+2] cycloaddition reaction under UV light . DOI: Click here for additional data file.10.1107/S2052252519014581/lq5028sup4.mp4Video S2: photoalient behavior of crystal clusters of 1 under UV light. Different types of movements including hopping, breaking and disintegration of clusters of crystals are shown under UV light . DOI: Click here for additional data file.10.1107/S2052252519014581/lq5028sup5.mp4Video S3: occasional thermosalient effect exhibited by small single crystals of 1. The crystals were heated on a hot stage from room temperature to 210 degC showing wiggling, hopping and jumping movements. The video shown is four times faster than the normal speed to highlight the effect. DOI: Click here for additional data file.10.1107/S2052252519014581/lq5028sup6.mp4Video S4: occasional thermosalient effect displayed by big crystals of 1. Small movements, rolling, hopping and sudden jumping of crystals leading to the disappearance of crystals from the sight are shown. The video shown is four times faster than the normal speed to dramatize the effect. DOI: Click here for additional data file.10.1107/S2052252519014581/lq5028sup7.mp4Video S5: photosalient effect of 1 during [2+2] cycloaddition reaction under UV light showing the breaking and disintegration of single crystals . DOI: Click here for additional data file.10.1107/S2052252519014581/lq5028sup8.mp4Video S6: photosalient effect showing the breaking of a single crystal of 1 under UV light . DOI: Click here for additional data file.10.1107/S2052252519014581/lq5028sup9.mp4Video S7: photosalient effect showing the hopping and breaking of a single crystal of 1 under UV light . DOI: Click here for additional data file.10.1107/S2052252519014581/lq5028sup10.mp4Video S8: occasional thermosalient effect on big crystals of 1 showing chipping, rolling and hopping of crystals. DOI: Click here for additional data file.10.1107/S2052252519014581/lq5028sup11.mp4Video S9: photosalient effect of a single crystal of 2 showing complete disintegration single crystals during [2+2] cycloaddition reaction under UV light . DOI: Click here for additional data file.10.1107/S2052252519014581/lq5028sup12.mp4Video S10: occasional thermosalient effect on big crystals of 2. Wiggling, rolling and hopping of crystals are demonstrated. DOI: Click here for additional data file.10.1107/S2052252519014581/lq5028sup13.mp4Video S11: photosalient behavior showing breaking, rolling, hopping and jumping out of sight of single crystals of 3 during [2+2] cycloaddition reaction under UV light . DOI: Click here for additional data file.10.1107/S2052252519014581/lq5028sup14.mp4Video S12: occasional thermosalient effect on big crystals of 3 showing wiggling, rolling and hopping of crystals. DOI: 1845040, 1845041, 1845042, 1845043CCDC references:


It is well established that H2S functions in many physiological processes, including the relaxation of vascular smooth muscle, mediation of neurotransmission, regulation of inflammation, and modulation of insulin signaling. In recent years, it has been revealed that polysulfides, substances with a varying number of sulfur atoms (H2Sn), are generated endogenously from H2S in the presence of oxygen. A series of studies describes that sulfane sulfur has the unique ability to bind reversibly to other sulfur atoms to form hydropersulfides and polysulfides, and that polysulfides activate ion channels and promote calcium influx. Furthermore, polysulfides regulate tumor suppressor activity, promote the activation of transcription factors targeting antioxidant genes and regulate blood pressure by vascular smooth muscle relaxation. Insulin secretion from pancreatic β cells plays a critical role in response to increased blood glucose concentration. H2S has emerged as an important regulator of glycemic control and exhibits characteristic regulation of glucose homeostasis. However, the effects of polysulfides on glucose-stimulated insulin secretion (GSIS) are largely unknown. In this study, we demonstrated that pharmacological polysulfide salts including Na2S2, Na2S3, and Na2S4 considerably inhibit GSIS in mouse and rat pancreatic β-cell-derived MIN6 and INS-1 cell lines, and that the effect is dependent on the activation of ATP-sensitive potassium channels. In addition, we demonstrated that a mixture of Na2S and diethylamine NONOate inhibits GSIS in a similar way to the pharmacological administration of polysulfide salts.Hydrogen sulfide (H H2S function is well established in many physiological processes, including the relaxation of vascular smooth muscle, mediation of neurotransmission, regulation of inflammation, and modulation of insulin signaling3. In recent years, it has been revealed that polysulfides, substances with a varying number of sulfur atoms (H2Sn), are generated from H2S in the presence of oxygen5 and by 3-mercaptopyruvate sulfurtransferase (3MTS)6. In addition, polysulfides are also generated by the interaction between H2S and nitric oxide (NO)7. Polysulfides contain sulfane sulfur, which is present in various proteins and functions as a potential intracellular H2S store that releases H2S under reducing conditions. A series of studies describe the unique ability of sulfane sulfur to bind reversibly to other sulfur atoms to form hydropersulfides (R-S-SH) and polysulfides (-S-Sn-S-)1. Polysulfides have been shown to activate ion channels and promote calcium influx, as well as to regulate tumor suppressor activity, promote the activation of transcription factors targeting antioxidant genes, and modulate blood pressure by vascular smooth muscle relaxation8. H2S and sulfane sulfur constitutively coexist, and recent work suggests that sulfane sulfur species may act as signaling molecules in at least some biological processes9. It is also clear that many of the effects of H2S are mediated through reactions with cysteine sulfur on regulatory proteins and most of these are not mediated directly by H2S but require prior oxidation of H2S and the formation of H2Sn10.Hydrogen sulfide  are largely unknown.H2S2), sodium trisulfide (Na2S3), and sodium tetrasulfide (Na2S4) on insulin secretion under low and high glucose conditions using cell biological and electrophysiological methods. We examined the effects of polysulfides at basal conditions and during GSIS and demonstrated that polysulfides activate ATP-sensitive potassium (KATP) channels, suppressing GSIS in mouse insulinoma 6 (MIN6) cells, rat insulinoma 1 (INS-1) cells, and mouse pancreatic β-cells/islets.In this study, we investigated the effect of pharmacological polysulfide salts including sodium disulfide  for 1 h, and the concentration of insulin in the culture media was assayed  was weaker than that of Na2S3 (IC50 = 39.9 µM) and the effect of Na2S3 was weaker than that of Na2S4 (IC50 = 9.5 µM). Finally, the effect of H2S was investigated. In contrast to sulfane sulfur donors, Na2S did not significantly affect GSIS at any concentration up to 50 µM , a donor of NO that generates polysulfides. Neither 50 µM Na2S nor 50 µM DEA/NO inhibited GSIS in MIN6 cells  and H2S (Na2S) on GSIS, and determined that the combination of Na2S4 and Na2S resulted in an additive effect  and then administered to cells. The pretreated Na2S4 did not affect the immunogenicity of insulin  phosphine hydrochloride (TCEP)) that cleaves disulfide bonds was assessed. First, TCEP was added directly into the culture media, and measured insulin levels remained very low Fig. . However2S4 on GSIS was investigated in INS-1 cells. As in the case of MIN6 cells, Na2S4 inhibited GSIS in a dose-dependent manner  or Annexin V staining. However, neither 50 µM nor 100 µM Na1 h Fig. . In the ath Fig. .Figure 4Our findings show that polysulfides at 50 µM did not cause MIN6 cell death and indicate that the observed suppression of GSIS is not due to cell toxicity caused by polysulfides.15. Here, in agreement with previous studies, we observed an Na2S4 dependent effect on intracellular ATP concentaration upon exposure to 2 mM or 20 mM glucose at the timepoints 1 and 4 h. 20 mM glucose increased ATP concentration compared to 2 mM glucose. However, 25 µM Na2S4 did not affect ATP concentration at all  in cells treated with 20 mM glucose  in mitochondriaiod Fig. . HoweverATP) channels, and depolarizes the plasma membrane18. Glibenclamide, a drug that acts as a channel closer, has been reported to facilitate insulin secretion in pancreatic β-cells even under low-glucose conditions, by binding to and inhibiting the KATP channel inhibitory regulatory subunit sulfonylurea receptor 1 (SUR1)14. The effects of Na2S3 and Na2S4 treatment on glibenclamide-induced insulin secretion were examined in MIN6 cells. Here, we found that even upon addition of 2 mM glucose, 10 µM glibenclamide elicited insulin secretion. Such secretion was then suppressed by treatment with more than 6.2 µM Na2S4  of Na2S3 did suppress glibenclamide-induced insulin secretion in a dose-dependent manner in cells treated with 2 mM glucose conditions , Cav1.2 (Cacna1c), Kv2.1 (Kcnb1), Kv2.2 (Kcnb2), Kir6.2 (Kcnj11), SUR1 (Abcc8), insulin (Ins1), and 18 S ribosomal RNA (Rn18s) was examined by RT-PCR  hyperpolarized the membrane potential induced by 10 mM glucose (n = 6). Washing out of Na2S4 (50 µM) promptly restored the membrane potential depolarization induced by 10 mM glucose, to its original level. Following perfusion with standard 2 mM glucose solution, the firing of action potentials induced by 10 mM glucose disappeared. Glibenclamide (100 µM) addition also induced the firing of action potentials. These results indicated that Na2S4 activated KATP channels reversibly.To identify the ion channels affected by Naque Fig. . Na2S4 , which generates polysulfides in cells, also had comparative suppressive effects on GSIS.In this study, we demonstrated that pharmacological polysulfide salts including Na2Sn) is endogenously generated from H2S in the presence of oxygen20. Polysulfides contain sulfane sulfur, which is maintained in various proteins as a potential intracellular H2S store, which releases H2S under reduced conditions. It has also been reported that polysulfides are enzymatically biosynthesized by reacting with cysteine19. A mixture of Na2S  and diethylamine NONOate , has been reported to generate H2S2 and H2S322. In fact, we demonstrated here that Na2S and DEA/NO efficiently inhibited GSIS in MIN6 cells.Polysulfides, a mixture of substances with varying numbers of sulfurs  and sulfonylurea receptor  subunits27. It is sensitive to adenosine triphosphate (ATP), and various subtypes of KATP channel are expressed in a wide range of tissues29. In pancreatic β cells, including MIN6 cells and INS-1 cells, KATP channels are comprised of SUR1 and Kir6.228. Mutations of SUR1 or Kir6.2 genes are known to cause familial hypoglycemia associated with unregulated insulin secretion30. It is reported that H2S stimulates KATP channels in vascular smooth muscle cells and that H2S also functions as an endogenous opener of KATP channels in INS-1E cells. A series of studies has indicated that H2S stimulates a variety of ion channels such as TRPA1, TRPV1, T-type Ca2+ channels, and KATP channels33. The evidence strongly suggests therefore that polysulfides would also affects these ion channels.Treatment with 50 µM NaATP channels in MIN6 cells derived from mouse insulinoma. First, Na2S4 reversibly hyperpolarized the membrane potential after induction by 10 mM glucose. Second, Na2S4 facilitated conductance of inwardly rectifying K+ currents in a dose-dependent manner upon addition of a standard K+ pipette solution that included ATP. Another series of structural studies indicated that Kir6.x subunits sense changes in intracellular ATP concentration35. In this study, we demonstrated that Na2S4 facilitated the inwardly rectifying K+ conductance in a dose-dependent manner with an intracellularly standard K+ pipette solution with 0.01 mM ATP. However, the effect of Na2S4 was suppressed with a K+ pipette solution comprising 1 mM ATP. Conversely, we also demonstrated that 10 µM of glibenclamide significantly decreased inwardly rectifying K+ currents elicited by polysulfides. Taking into account existing evidence that the direct target of glibenclamide is SUR, it is reasonable to suggest that polysulfides affect Kir6.2, SUR1, or the interaction between them.In line with this logic, we demonstrated the polysulfides activate K2S is sequentially oxidized to form polysulfides with a varying number of sulfur atoms, until the number of sulfur atoms reaches eight. At that point, sulfur molecules cyclize and separate from polysulfides. Polysulfides also sulfurate cysteine residues in TRPA1 channels to modify their activity31. Polysulfides sulfurate Cys422 and Cys622 in TRPA1 channels to activate them, and it is reported that vascular KATP channels are inhibited during oxidative stress caused by S-glutathionylation; the Kir6.1 subunit specifically has been reported to be responsible for oxidant sensitivity in these channels36. Systematic mutational analysis revealed three cysteine residues in the N terminal and transmembrane domains of Kir6.1: namely Cys43, Cys120, and Cys176. Among them, Cys176 contributed to >80% of the oxidant sensitivity of the protein36. In contrast to Cys176, Cys43 exhibited only a modest contribution to S-glutathionylation, and Cys120 was modulated by extracellular oxidants but not by intracellular glutathione disulfide36. In Kir6.2 channels, Cys166 (corresponding to Cys176 in Kir6.1) is suggested to be involved in intrinsic channel gating. As in the case of Kir6.1, several cysteine residues are well conserved between species . In addition to Kir6.x, SUR1 also have well-conserved cysteine residues . Notably, the inhibitory effects of polysulfides on GSIS were demonstrated to be reversible in this study. In addition, Na2S4 activated KATP channels in MIN6 cells reversibly. Together, the evidence presented here suggests that polysulfides modulate reactive cysteine residues.Hin vitro, we did not perform in vivo experiments. In vivo experiments using mice may warrant the impact of polysulfides on systemic insulin secretion and glucose metabolism.There is a limitation of this study. Because we focused on the mechanism of the suppression of insulin secretion by pharmacological polysulfides 38.Mouse insulinoma MIN6 cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM)  containing 450 mg/dl glucose. Rat INS-1 cells were cultured in RPMI1640  supplemented with 10% fetal bovine serum (FBS), 50 μM β-mercaptoethanol, 100 U/ml penicillin, and 0.1 mg/ml streptomycin. Culture conditions used replicated those reported in the literature for these cellsDetails of reagents used in this study are described in Table et al.39. The pancreas was removed and digested with collagenase  in a solution containing 2 mM glucose and trypsin inhibitor  at 37 °C for 30 min with vigorous shaking. The pancreatic tissue was triturated with a pipette and washed two times with an enzyme-free solution. Islets were selected with a glass micropipette under a stereomicroscope. Batches of ten islets were used to measure insulin concentration.Male C57BL/6JJcl mice  were sacrificed by cervical dislocation in accordance with protocols approved by the Animal Experimentation Committee, Kansai Medical University (#19–088). Pancreatic islets were isolated from the pancreas by enzymatic digestion of the tissue, using a slight modification to a protocol described by Lacy 40. Detailed protocols are available in the provided supplementary information and at protocols.io (10.17504/protocols.io.v63e9gn).Insulin concentration in the culture medium of MIN6, INS-1 cells, and pancreatic β cells/ islets was assayed using a Mouse/Rat Insulin H-type™ enzyme-linked immunosorbent assay kit , following the manufacturer’s instructions42. Cell viability was determined by comparing the absorbance values of the treated cells with those of the control cells (MIN6 cells at 24 h of incubation) defined as 100%. All experiments were performed in triplicate or quadruplicate. Detailed protocols are available in the supplementary information and at protocols.io (10.17504/protocols.io.v64e9gw).Cell viability was assessed using the CellTiter 96™ AQueous One Solution Cell Proliferation Assay 42. Assays were performed in triplicate and repeated at least twice. Detailed protocols are available in the supplementary information and at protocols.io (10.17504/protocols.io.v7ke9kw).Intracellular ATP content was evaluated using the CellTiter-Glo™ luminescent cell viability assay kit (Promega)43. MIN6 cells were seeded at a density of 1 × 104 cells/well on an XFp Cell Culture microplate. The XF Cell Mito Stress Test™ was performed in glucose-containing XF base medium, following the manufacturer’s protocol with 1 µM oligomycin, 1 µM FCCP, 0.5 µM rotenone, and 0.5 µM antimycin A. Detailed protocols are available at Supplementary information and protocols.io (10.17504/protocols.io.v92e98e).The cellular oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using an XFp Extracellular Flux Analyzer™ 43. PCR primers sequences were demonstrated as Table Actb :, Abcc8 :, Abcc9 , Kcnj11 , Kcnj8 , Slc2a2 (solute carrier family 2 (facilitated glucose transporter), member 2; Glut2), and Cacna1c . Detailed protocols are available at Supplementary information and protocols.io (10.17504/protocols.io.v7ne9me).Total RNA was isolated using RNeasy™ Mini Kit . First-strand cDNA synthesis and real-time PCR were performed as described46. Membrane potential measurements and whole-cell current recordings were performed using the EPC 800 patch-clamp amplifier . Experiments were conducted at 23–30 °C. Detailed protocols are available at Supplementary information and protocols.io (10.17504/protocols.io.v68e9hw).MIN6 cells were incubated in an extracellular bath solution containing 2 mM glucose for 30 min at 37 °C before patch-clamp experimentsP-values < 0.05.Data are presented as means ± SD. Differences between groups were evaluated with one-way analysis of variance (ANOVA) and two-way ANOVA followed by Dunnett’s test for multiple comparisons. Statistical analyses were performed with Prism8™ . Statistical significance was defined by Supplementary Information