Crystallography-guided discovery of carbazole-based retinoic acid-related orphan receptor gamma-t (RORγt) modulators: insights into different protein behaviors with "short" and "long" inverse agonists.

A series of 6-substituted carbazole-based retinoic acid-related orphan receptor gamma-t (RORγt) modulators were discovered through 6-position modification guided by insights from the crystallographic profiles of the "short" inverse agonist 6. With the increase in the size of the 6-position substituents, the "short" inverse agonist 6 first reversed its function to agonists and then to "long" inverse agonists. The cocrystal structures of RORγt complexed with the representative "short" inverse agonist 6 (PDB: 6LOB), the agonist 7d (PDB: 6LOA) and the "long" inverse agonist 7h (PDB: 6LO9) were revealed by X-ray analysis. However, minor differences were found in the binding modes of "short" inverse agonist 6 and "long" inverse agonist 7h. To further reveal the molecular mechanisms of different RORγt inverse agonists, we performed molecular dynamics simulations and found that "short" or "long" inverse agonists led to different behaviors of helixes H11, H11', and H12 of RORγt. The "short" inverse agonist 6 destabilizes H11' and dislocates H12, while the "long" inverse agonist 7h separates H11 and unwinds H12. The results indicate that the two types of inverse agonists may behave differently in downstream signaling, which may help identify novel inverse agonists with different regulatory mechanisms.

Heterogeneity of Outcome Measures Used in Randomized Controlled Trials for the Treatment of Oral Lichen Planus: A Methodological Study.

OBJECTIVE: Oral lichen planus (OLP) is a chronic inflammatory immune disease, recognized as an oral potentially malignant disorder by the World Health Organization. There is considerable controversy over the standardized treatment of OLP, with great diversities in the outcome measures in clinical trials. This methodological study aimed to estimate the degree of consensus on outcome measures in randomized controlled trials (RCTs) for OLP treatment. METHODS: PubMed, Embase, and Cochrane databases were searched to identify RCTs published from 2004 to 2018 about OLP treatment. All the outcome measures and measurement methods mentioned in the trials were extracted and analyzed. RESULTS: After identification of 1087 articles, 88 RCTs were included. A total of 193 single-outcome measures and 119 composite outcome measures were classified into 11 different domains, the chief of which consisted of clinical symptom (78 trials; 88.6%) and clinical score (58 trials; 65.9%). Visual analog scale (65 trials; 73.9%) and Thongprasom scoring system (38 trials; 43.2%) were the predominant measurement methods. Oral health-related quality of life (except for clinical symptoms) accounted for 4.8% of all the outcome measures. CONCLUSIONS: There was high heterogeneity in outcome measures of RCTs for OLP treatment, making it difficult to make valid comparisons between different clinical trials. A core outcome set should be developed and adopted in future trials for OLP treatment.

Discovery, synthesis, biological evaluation and structure-based optimization of novel piperidine derivatives as acetylcholine-binding protein ligands.

The homomeric α7 nicotinic receptor (α7 nAChR) is widely expressed in the human brain that could be activated to suppress neuroinflammation, oxidative stress and neuropathic pain. Consequently, a number of α7 nAChR agonists have entered clinical trials as anti-Alzheimer's or anti-psychotic therapies. However, high-resolution crystal structure of the full-length α7 receptor is thus far unavailable. Since acetylcholine-binding protein (AChBP) from Lymnaea stagnalis is most closely related to the α-subunit of nAChRs, it has been used as a template for the N-terminal domain of α-subunit of nAChR to study the molecular recognition process of nAChR-ligand interactions, and to identify ligands with potential nAChR-like activities.Here we report the discovery and optimization of novel acetylcholine-binding protein ligands through screening, structure-activity relationships and structure-based design. We manually screened in-house CNS-biased compound library in vitro and identified compound 1, a piperidine derivative, as an initial hit with moderate binding affinity against AChBP (17.2% inhibition at 100 nmol/L). During the 1st round of optimization, with compound 2 (21.5% inhibition at 100 nmol/L) as the starting point, 13 piperidine derivatives with different aryl substitutions were synthesized and assayed in vitro. No apparent correlation was demonstrated between the binding affinities and the steric or electrostatic effects of aryl substitutions for most compounds, but compound 14 showed a higher affinity (Ki=105.6 nmol/L) than nicotine (Ki=777 nmol/L). During the 2nd round of optimization, we performed molecular modeling of the putative complex of compound 14 with AChBP, and compared it with the epibatidine-AChBP complex. The results suggested that a different piperidinyl substitution might confer a better fit for epibatidine as the reference compound. Thus, compound 15 was designed and identified as a highly affinitive acetylcholine-binding protein ligand. In this study, through two rounds of optimization, compound 15 (Ki=2.8 nmol/L) has been identified as a novel, piperidine-based acetylcholine-binding protein ligand with a high affinity.

Successive soliton explosions in an ultrafast fiber laser.

Soliton explosions, as one of the most fascinating nonlinear phenomena in dissipative systems, have been investigated in different branches of physics, including the ultrafast laser community. Herein, we reported on the soliton dynamics of an ultrafast fiber laser from steady state to soliton explosions, and to huge explosions by simply adjusting the pump power level. In particular, the huge soliton explosions show that the exploding behavior could operate in a sustained, but periodic, mode from one explosion to another, which we term as "successive soliton explosions." The experimental results will prove to be fruitful to the various communities interested in soliton explosions.

[Quantitative analysis of nuclear proteome in apoptosis hepatoma cells induced by hydroxycamptothecin].

The experimental foundation for investigating the pharmacological action of hydroxycamptothecin at subcellular quantitative proteomic level can be obtained depending on the information of differentially expressed nuclear proteins in hydroxycamptothecin-treated cells and control cells. The apoptosis was induced by hydroxycamptothecin in hepatoma cells, the nucleus of cells were isolated and verified with western blot. Nuclear proteins labelled with cleavable isotope-coded affinity tag (c-ICAT) reagent were digested and purified. The expression ratio of the identical nuclear protein derived from apoptosis cell and control cell can be gained using shotgun proteomic method based on multiple dimensional liquid chromatography-linear ion trap/orbitrap mass spectrometer combined with c-ICAT strategy. A total of 42 nuclear proteins were significantly (P<0.05) altered in hydroxycamptothecin-treated cells, among them, 12 proteins showed significantly down-regulation, and 30 proteins showed up-regulation compared with control cells. The function of these proteins were likely involved in life processes of cells such as proliferation, apoptosis, differentiation, energy metabolism, nucleic acid synthesis and metabolism, structure of cell skeleton.

[A quantitative analysis of mitochondrial protein differential expressions in hydroxycamptothecin-treated hepatoma cells].

OBJECTIVES: To investigate the differentially expressed mitochondrial proteins in hydroxycamptothecin (HCPT)-treated SMMC-7721 cells by using quantitative proteome. METHODS: SMMC-7721 cell apoptosis was induced by HCPT and the mitochondria were isolated with a mitochondria isolation kit. Mitochondrial proteins labeled with a cleavable isotope-coded affinity tag were identified and quantified using two-dimensional liquid chromatography/tandem mass spectrometry. RESULTS: Highly purified mitochondria were obtained. Seventy-four mitochondrial proteins, which were statistically significantly altered (P less than 0.05) in HCPT-treated cells, were identified and analyzed. A total of 42 proteins were significantly down-regulated, and 32 were up-regulated in the cells that responded to apoptosis. The functions of these proteins were likely involved in cell energy metabolism, nucleic acid translation and transcription, cytoskeleton, etc. CONCLUSION: Our results about the information of differentially expressed mitochondrial proteins in HCPT-treated cells and the control cells will help to understand the mechanism by which HCPT induces cell apoptosis. The integrated techniques we used in this study will be helpful to the investigation of subcellular quantitative proteomics.

[Proteomic analysis of mitochondrial proteins in hydroxycamptothecin-treated SMMC-7721 cells].

OBJECTIVE: To investigate the differentially expressed mitochondrial proteins in hydroxycamptothecin (HCPT)-treated SMMC-7721 cells by comparative proteomic analysis. METHODS: Apoptosis of SMMC-7721 cells were induced by using HCPT and their mitochondria were isolated with a mitochondria isolation kit for cultured cells. Three different solubility protein fractions were extracted with ReadyPrep Sequential Extraction Kit and were separated by two-dimensional gel electrophoresis (2-DE). PDQuest software was used to differentiate mitochondrial proteins between control cells and HCPT-treated cells. Matrix assisted laser desorption/ionization time of flying mass spectrometry (MALDI-TOF-MS) was used to identify some of the different proteins. RESULTS: Highly purified mitochondria and high resolution 2-DE patterns of the proteins were obtained. Forty-four mitochondrial protein spots from the HCPT-treated cells showed different expressions compared to those of the control cells. Twenty of the different protein spots were analyzed by MALDI-TOF-MS. CONCLUSION: Differently expressed mitochondrial proteins in HCPT-treated cells and control cells were obtained in this study. This will be of help to understand the mechanism by which HCPT induces cell apoptosis.

[Cloning and expression of recombinant mitochondrial Delta1-120 apoptosis-inducing factor and its enhancive effect on apoptosis of hepatocellular carcinoma cell line SMMC-7721].

BACKGROUND & OBJECTIVE: Mitochondria play a key role in cell apoptosis, and apoptosis-inducing factor (AIF) is a kind of apoptotic protein located in mitochondria. The research on mitochondrial protein can be helpful for elucidating the role of mitochondria in apoptosis. This study was to clone and express recombinant human Delta1-120 AIF and validate its biological activities of binding DNA and inducing nuclear apoptosis. METHODS: A human AIF gene fragment of 1515 bp(mitochondrial localization sequence was deleted) was amplified from SMMC-7721 cells by reverse transcription-polymerase chain reaction (RT-PCR) and cloned into pET32a(+) vector to construct recombinant plasmid pET32a-AIF. The recombinant plasmid was transfected into E.coli BL2l (DE3). AIF expression was induced by isoprophylthio-beta-D-galactoside (IPTG), and detected by SDS-PAGE and Western blot. AIF protein was purified by Ni afinity chromatography and then renatured. The biological activity of renatured AIF protein was detected by electrophoretic mobility shift assay (EMSA) and Hoechst staining. RESULTS: The 1.5 kb AIF gene was successfully isolated, and cloned into pET32a(+) vector. Plasmid pAIF was identified by restrictive enzyme analysis and sequencing. Recombinant E.coli. strains expressing AIF were obtained. AIF protein amounted to 11% of the total bacterial protein when induced with IPTG at 37 degrees Celsius for 4 h. AIF was specially recognizing by anti-AIF and anti-his antibody. The purity of purified protein reached over 95%. After renaturation, AIF protein binded DNA and induced nuclear apoptosis. CONCLUSION: AIF protein with high purity and biological activity was obtained by the method described above.

Changes in the protein spectrum of mitochondria isolated from hydroxycamptothecin-treated hepatoma cells.

As one of the most potent topoisomerase inhibitors, hydroxycamptothecin is more active and less toxic than conventional camptothecin. Recently, we found that hydroxycamptothecin can induce cell apoptosis via the mitochondrial pathway. This study was designed to investigate the mitochondrial protein profile in HCPT-treated cells using high-accuracy and high-sensitivity protein-identification technology. Of the 39 mitochondrial protein spots investigated, 25 displayed elevated and 14 suppressed abundance in hydroxycamptothecin-treated cells. The 25 spots were identified by mass spectrometry and they included proteins involved in many essential cellular functions. The potential role of these proteins in hydroxycamptothecin-mediated apoptosis is also discussed. This study has produced a short list of mitochondrial proteins that might hold the key to the mechanism by which hydroxycamptothecin induces mitochondrial dysfunction and cell apoptosis. It has laid the foundation for further elucidating the role of hydroxycamptothecin during apoptosis. Successful applications of multiple techniques including two-dimensional gel electrophoresis, matrix-assisted laser desorption ionization time-of-flight mass spectrometry and Western blot analysis have demonstrated that proteomic analyses provide appropriate approaches for understanding of the roles of anticancer drugs.

Hydroxycamptothecin-induced apoptosis in hepatoma SMMC-7721 cells and the role of mitochondrial pathway.

The camptothecin (CPT) derivative hydroxycamptothecin (HCPT) containing 10-hydroxy represents one of the most potent topoisomerase I inhibitors described. This anticancer agent, currently undergoing clinical trials on gastric tumours, has been shown more active and less toxic than conventional camptothecins. To shed light on the mechanism of action of HCPT at the cellular level, we examined cell growth, apoptosis, changes of mitochondrial membrane potential, cytochrome c and AIF translocation in cancer cells by exposing these cells to HCPT for indicated time. The effect of HCPT on cell proliferation was measured by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromid) assay and apoptosis was measured using flow cytometry, fluorescence microscopy and electron microscopy. Changes of mitochondrial membrane potential were monitored by fluorescence microscope. Western blot analysis was used to evaluate the release of mitochondrial cytochrome c and AIF; On the other hand, translocation of cytochrome c and AIF from mitochondria to cytosol during apoptosis were confirmed by confocal microscopy. HCPT could noticeably inhibit the proliferation of SMMC-7721cells and the IC(50) dose was about 0.22 microM; SMMC-7721 cells treated with HCPT showed typical characteristics of apoptosis rather than necrotic including phosphatidylserine (PS) exposed from the inner to the outer leaflet of the plasma membrane, abnormal cell morphology, chromatin condensation and nuclear fragmentation; On the other hand, during process of cell apoptosis, mitochondrial transmembrane potential was reduced; Compared with the control group, the mRNA and protein expression of cytochrome c and AIF in treated and untreated SMMC-7721 cells were not significantly changed (not shown). However, when cells were treated with HCPT, the massive translocation of cytochrome c and AIF to the nucleus was evident. Our results indicate that HCPT can inhibit proliferation and induce apoptosis of human hepatoma SMMC-7721 cells. Mitochondrial pathway of apoptosis, especially for cytochrome c and AIF translocation, may play an important role in apoptosis induced by HCPT.

[Effect of hydroxycamptothecin on apoptosis-inducing factor (AIF) expression and on AIF translocation in human hepatocellular cancer cell SMMC-7721].

OBJECTIVE: To study the effect of hydroxycamptothecin (HCPT) on apoptosis-inducing factor (AIF) expression and AIF translocation from mitochondria to the nucleus in human hepatocellular cancer cell SMMC-7721 during apoptosis. METHODS: After treatment with 80 mg/ml of HCPT, the cancer cells were stained with A0/EB to monitor their apoptosis. Their mitochondria was examined with electronmicroscopy and the AIF expression of the cells was tested by RT-PCR and Western blot. The translocation of AIF from mitochondria to the nucleus during apoptosis was analyzed by confocal microscopy. RESULTS: SMMC-7721 cells treated with HCPT showed chromatin condensation, nuclear fragmentation and mitochondria swelling. The mRNA and protein expression of AIF in treated and untreated SMMC-7721 cells were not significantly different. However, cells treated with 80 mg/ml HCPT for 6 h or 12 h showed massive translocation of AIF into the nuclei. CONCLUSION: These results show the important role the mitochondrial pathway of apoptosis plays in HCPT-induced tumor cell death, at least in SMMC-7721 cells.