An NADPH-Dependent Ketoreductase Catalyses the Tetracyclic to Pentacyclic Skeletal Rearrangement in Chartreusin Biosynthesis.

Redox tailoring enzymes play key roles in generating structural complexity and diversity in type II polyketides. In chartreusin biosynthesis, the early 13 C-labeling experiments and bioinformatic analysis suggest the unusual aglycone is originated from a tetracyclic anthracyclic polyketide. Here, we demonstrated that the carbon skeleton rearrangement from a linear anthracyclic polyketide to an angular pentacyclic biosynthetic intermediate requires two redox enzymes. The flavin-dependent monooxygenase ChaZ catalyses a Baeyer-Villiger oxidation on resomycin C to form a seven-membered lactone. Subsequently, a ketoreductase ChaE rearranges the carbon skeleton and affords the α-pyrone containing pentacyclic intermediate in an NADPH-dependent manner via tandem reactions including the reduction of the lactone carbonyl group, Aldol-type reaction, followed by a spontaneous γ-lactone ring formation, oxidation and aromatization. Our work reveals an unprecedented function of a ketoreductase that contributes to generate structural complexity of aromatic polyketide.

Electronic Modulation of Pd-Based Bimetallic Catalysts with Sulfur-Doped Carbon Support for Phenylacetylene Semihydrogenation.

The semihydrogenation of phenylacetylene to styrene represents an important process for optimizing the polystyrene production and also a model reaction for the evaluation of selective hydrogenation catalysts. Although the alloying strategy and surface engineering for noble metal (particularly for Pd) catalysts can effectively inhibit the overhydrogenation of styrene, the selectivity of phenylacetylene semihydrogenation to styrene is generally below 95% near the full conversion. Here, we demonstrate the electronic modulation of Pd-based bimetallic nanocluster catalysts based on the strong metal-support interactions for improving the catalytic selectivity for phenylacetylene semihydrogenation. A series of Pd-M (M = Fe, Co, Ni, Cu, Ga) bimetallic nanoclusters of ∼2 nm are immobilized on mesoporous sulfur-doped carbon (meso_S-C) supports, which exhibit a high selectivity of >97% for the semihydrogenation of phenylacetylene to styrene. The strong interaction between metal and the meso_S-C supports enables the modulation of electronic structure of the bimetallic nanoparticles and thus leads to the selectivity enhancement for the phenylacetylene semihydrogenation.

PCSK9 promotes the secretion of pro-inflammatory cytokines by macrophages to aggravate H/R-induced cardiomyocyte injury via activating NF-κB signalling.

The upregulation of proprotein convertase subtilisin/kexin type 9 (PCSK9) was reported to be involved in regulating the levels of inflammatory markers and apoptosis in macrophages. This study aims to investigate the function and regulation of PCSK9 in myocardial ischaemia. The results of our study showed dramatically increased expression of PCSK9 induced by hypoxia/reoxygenation (H/R) stress rather than by apoptosis in primary murine cardiomyocytes and HL-1 cells. Moreover, PCSK9 promoted H/R-induced pro-inflammatory cytokine release from macrophages, while silencing of PCSK9 inhibited the expression of the pro-inflammatory cytokines TNF-α, IL-6 and IL-1ß. Additionally, PCSK9 facilitated the release of pro-inflammatory cytokines from macrophages under H/R conditions, which decreased cardiomyocyte viability and promoted apoptosis of cardiomyocytes. For the underlying mechanisms, we identified PCSK9-induced NF-κB activation as being involved in the cardiomyocyte apoptosis, which was blocked by the NF-κB inhibitor BAY 11-7082. Collectively, this study provides new insights into the therapeutic possibility of regulating PCSK9 in cardiomyocytes for the treatment of ischaemic cardiomyopathy.

Bialternacins A-F, Aromatic Polyketide Dimers from an Endophytic Alternaria sp.

Six novel aromatic polyketide dimers, bialternacins A-F (1-6), were isolated from a plant endophytic Alternaria sp. The structures of compounds 1-6 were elucidated on the basis of extensive spectroscopic analysis, single-crystal X-ray diffraction, and electronic circular dichroism analysis. Compounds 1, 2, 5, and 6 were characterized as four pairs of racemic mixtures. Compound (+)-5 was demonstrated to show acetylcholinesterase inhibitory activity with an IC50 value of 15.5 µM. A putative biosynthetic pathway for these compounds was proposed.

Molecular Basis for the Final Oxidative Rearrangement Steps in Chartreusin Biosynthesis.

Oxidative rearrangements play key roles in introducing structural complexity and biological activities of natural products biosynthesized by type II polyketide synthases (PKSs). Chartreusin (1) is a potent antitumor polyketide that contains a unique rearranged pentacyclic aromatic bilactone aglycone derived from a type II PKS. Herein, we report an unprecedented dioxygenase, ChaP, that catalyzes the final α-pyrone ring formation in 1 biosynthesis using flavin-activated oxygen as an oxidant. The X-ray crystal structures of ChaP and two homologues, docking studies, and site-directed mutagenesis provided insights into the molecular basis of the oxidative rearrangement that involves two successive C-C bond cleavage steps followed by lactonization. ChaP is the first example of a dioxygenase that requires a flavin-activated oxygen as a substrate despite lacking flavin binding sites, and represents a new class in the vicinal oxygen chelate enzyme superfamily.

Strepchazolins A and B: Two New Alkaloids from a Marine Streptomyces chartreusis NA02069.

Two new alkaloids, strepchazolins A (1) and B (2), together with a previously reported compound, streptazolin (3), were isolated from a marine actinomycete, Streptomyces chartreusis NA02069, collected in the Coast of Hainan Island, China. The structures of new compounds were determined by extensive NMR, mass spectroscopic and X-ray crystallographic analysis, as well as modified Mosher's method. Compound 1 showed weak anti-Bacillus subtilis activity with the MIC value of 64.0 µM, and weak inhibitory activity against acetylcholinesterase (AChE) in vitro with IC50 value of 50.6 µM, while its diastereoisomer, Compound 2, is almost inactive.

Comparative analysis of genetic polymorphisms among Monascus strains by ISSR and RAPD markers.

BACKGROUND: The genus Monascus includes several species of fungi valued across Asia for their culinary uses and diverse medicinal properties. In this study, we evaluated the applicability of random amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR) markers in characterizing the genetic diversity in 41 Monascus strains collected from various regions of Fujian Province, the leading producer of Monascus in China. RESULTS: Seven screened ISSR primers generated 56 polymorphic bands, of which 93.33% were polymorphic. The genetic similarity coefficients (GSC) of the strains ranged from 0.50 to 1.00. Comparative sequence analysis using seven screened RAPD primers amplified a total of 49 polymorphic bands, of which 81.67% were polymorphic; GSC values ranged from 0.62 to 1.00. CONCLUSION: Correlation analysis revealed a significant positive correlation in genetic distances assessed using above two markers, which indicated they were suitable for Monascus species characterization. ISSR markers were more suitable for the classification and determination of Monascus species, while RAPD markers appear to be preferable for analyzing the differences among strains within the same species. Our study revealed that Monascus possesses rich genetic diversity, and that the genetic relationships among the selected strains were, to a very limited extent, correlated to their geographical variation. © 2016 Society of Chemical Industry.

[Depression status of the family member and its influencing factors in the patient with mental disorder caused by cerebral trauma: 245 cases analysis].

OBJECTIVE: To determine the family member's depression status and its influencing factors in the patient with mental disorder caused by cerebral trauma. METHODS: The self-designed information questionnaire and self-rating depression scale (SDS) were used to investigate 245 family members' depression conditions. On the basis of SDS score index, all cases were divided into depression group and non-depression group. The data were analyzed by the univariate analysis and multiple factors regression analysis. RESULTS: In the 245 family members, 117 family members had different degrees of depressive symptoms including 68 with mild depression, 42 with moderate depression, and 7 with severe depression. In the multiple factors regression analysis, genetic relationship, personality characteristics and payment method were the main influencing factors of the depression status. CONCLUSION: Much attention should be paid to the family member's depression condition of the patient with mental disorder caused by cerebral trauma through specific psychological intervention.

[Effects of Fusobacterium nucleus derived outer membrane vesicles on claudin-4 expression in oral epithelial cells].

PURPOSE: To investigate the effect of outer membrane vesicles (OMVs) secreted by Fusobacterium nucleatum (F.n) on Claudin-4 of human oral keratinocytes (HOK) and oral epithelial barrier function. METHODS: Fusobacterium nucleatum was cultured under anaerobic conditions. The OMVs were extracted by dialysis and characterized by nanosight and transmission electron microscopy (TEM). HOK were stimulated with OMVs at different mass concentrations(0-100 µg/mL) for 12 h, and stimulated with 100 µg/mL OMVs for 6 h and 12 h respectively. The expression of Claudin-4 at gene and protein level was analyzed by RT-qPCR and Western blotting. Inverted fluorescence microscope was used to observe co-localization of HOK and OMVs and localization and distribution of Claudin-4 protein. Human oral epithelial barrier was constructed by Transwell apical chamber. Transepithelial electrical resistance(TER) of barrier was measured with a transmembrane resistance measuring instrument(EVOM2), and the permeability of the barrier was evaluated by transmittance of fluorescein isothiocyanate-dextran(FD-4). Statistical analysis was performed with GraphPad Prism 8.0 software package. RESULTS: Compared with the control group, the expression of Claudin-4 at protein and gene level in the HOK of OMVs stimulated group was significantly reduced (P＜0.05), and immunofluorescence showed that the continuity of Claudin-4 fluorescence among cells was destroyed. OMVs stimulation decreased TER value of oral epithelial barrier(P＜0.05) and increased the transmittance of FD-4(P＜0.05). CONCLUSIONS: OMVs derived from Fusobacterium nucleatum may damage oral mucosal epithelial barrier function through inhibiting the expression of Claudin-4.

[Correlation between low expression of Hsp90 protein in keratinocytes and the number of small extracellular vesicles and its potential clinical significance].

PURPOSE: To investigate the correlation between the level of heat shock protein 90(Hsp90) and the amount of small extracellular vesicles(sEVs) in keratinocytes. METHODS: Human keratinocytes(HaCaT) were cultured in vivo and divided into wild-type group, short hairpin RNA interference group (shRNA group, low expression of Hsp90), and 17-Allylamino-17-demethoxygeldanamycin group (17-AAG group, Hsp90 protein inhibitor). sEVs were isolated from culture system by ultracentrifugation, and their morphological characteristics were observed under transmission electron microscopy (TEM). Western blotting was applied to identify the biological characteristics of sEVs. The number of sEVs particles was detected by nanoparticle tracking analysis (NTA). GraphPad Prism8.0 software was used to analyze the difference in the number of sEVs among the groups by t test (non-parametric Mann-Whitney U test). RESULTS: HaCaT-derived sEVs, obtained by ultracentrifugation, were consistent with the criteria of morphological and biological identification. No expression of Hsp90 protein was detected in HaCaT-derived sEVs. When interfered with Hsp90-shRNA, the number of sEVs were significantly increased. On day 5, the sEVs number of shRNA-interfering group was (177.4±4.18)×108(n=3), while that of vector group was (82.34±4.83)×108(n=3), and the difference was statistically significant (P＜0.0001). After 5 days of inhibition with 17-AAG, the sEVs number of 17-AAG group was （652.5±26.73）×108(n=3) and that of control group was (262.22±5.44)×108(n=3), the difference was statistically significant (P＜0.000 1). CONCLUSIONS: Low expression of Hsp90 protein can promote the secretion of sEVs in HaCaT cells. sEVs may be involved in the transfer of molecules between epithelial cells and immune cells.

[Application of WAIS-RC short forms and adult intelligence disability scale in mental impairment assessment].

OBJECTIVE: Study on the application of WAIS-RC short forms and adult intelligence disability scale in mental impairment assessment. METHODS: Mental impairment assessment cases between July 2009 and March 2011 in judicial appraisal institute of Taizhou University were collected. Assessment results obtained with the WAIS-RC short forms and adult intelligence disability scale were compared with the experts assessing conclusions and analyzed using SPSS 11.5 software. RESULTS: Assessment results with the two scales did not fully comply with the expert's conclusions, with reliability coefficient were 0.785 and 0.940 respectively, correlation coefficient were 0.850 and 0.922 respectively. CONCLUSION: The intelligence assessment was influenced by many factors. When the appraised individuals had nerve dysfunction and mild intelligence disability or mental disorders, the two scales should be used together. When the appraised individuals had moderate intelligence disability or mental disorders, adult intelligence disability scale had advantage.

Sulfur-anchoring synthesis of platinum intermetallic nanoparticle catalysts for fuel cells.

Atomically ordered intermetallic nanoparticles are promising for catalytic applications but are difficult to produce because the high-temperature annealing required for atom ordering inevitably accelerates metal sintering that leads to larger crystallites. We prepared platinum intermetallics with an average particle size of <5 nanometers on porous sulfur-doped carbon supports, on which the strong interaction between platinum and sulfur suppresses metal sintering up to 1000°C. We synthesized intermetallic libraries of small nanoparticles consisting of 46 combinations of platinum with 16 other metal elements and used them to study the dependence of electrocatalytic oxygen-reduction reaction activity on alloy composition and platinum skin strain. The intermetallic libraries are highly mass efficient in proton-exchange-membrane fuel cells and could achieve high activities of 1.3 to 1.8 amperes per milligram of platinum at 0.9 volts.

Comprehensive transcriptome analysis of reference genes for fruit development of Euscaphis konishii.

BACKGROUND: Quantitativereal-time reverse transcriptase polymerase chain reaction is the common method to quantify relative gene expression. Normalizating using reliable genes is critical in correctly interpreting expression data from qRT-PCR. Euscaphis konishii is a medicinal plant with a long history in China, which has various chemical compounds in fruit. However, there is no report describing the selection of reference genes in fruit development of Euscaphis konishii. METHODS: We selected eight candidate reference genes based on RNA-seq database analysis, and ranked expression stability using statistical algorithms GeNorm, NormFinder, BestKeeper and ReFinder. Finally, The nine genes related to the anthocyanin synthesis pathway of Euscaphis konishii were used to verify the suitability of reference gene. RESULTS: The results showed that the stability of EkUBC23, EkCYP38 and EkGAPDH2 was better, and the low expression reference genes (EkUBC23 and EkCYP38) were favourable for quantifying low expression target genes, while the high expression reference gene (EkGAPDH2) was beneficial for quantifying high expression genes. In this study, we present the suitable reference genes for fruit development of Euscaphis konishii based on transcriptome data, our study will contribute to further studies in molecular biology and gene function on Euscaphis konishii and other closely related species.

Discovery, Biosynthesis, and Heterologous Production of Loonamycin, a Potent Anticancer Indolocarbazole Alkaloid.

Genome mining of an indolocarbazole-type gene cluster from a marine-derived Nocardiopsis flavescens NA01583 strain led to the discovery of three new indolocarbazole alkaloids (1-3). Heterologous expression of the intact loo gene cluster in a surrogate host Streptomyces lividans K4-114 led to the successful production of 3. Notably, compound 1 showed potent cytotoxic activities toward eight cancer cell lines with IC50 values ranging from 41 to 283 nM.

4-Methyl-3-[4-(3-pyrid-yl)pyrimidin-2-yl-oxy]aniline.

In the title compound, C(16)H(14)N(4)O, there are inter-molecular N-H⋯N hydrogen bonds which may be effective in stabilizing the crystal. The title compound is an important medicament and is used in the synthesis of anti-tumour drugs.

Comparative Proteomics Analysis Between the Short-Term Stress and Long-Term Adaptation of the Blattella germanica (Blattodea: Blattellidae) in Response to Beta-Cypermethrin.

A proteomic method combining two-dimensional polyacrylamide gel electrophoresis and tandem mass spectrometry was used to compare the hemolymph expression profiles of a beta-cypermethrin-resistant Blattella germanica L. strain (R) and a susceptible strain (S) after 24 h of beta-cypermethrin induction. The results showed that there were 42 differentially expressed proteins after induction of the R strain: 4 proteins were upregulated and 38 proteins were downregulated. One hundred one hemolymph proteins were differentially expressed after induction of the S strain: 53 proteins were upregulated and 48 proteins were downregulated. The identified proteins were mainly classified into the following categories: energy metabolism proteins such as arginine kinase and triose phosphate isomerase, detoxification-related proteins such as glutathione S-transferases (GSTs), signal molecule-regulated proteins such as nitric oxide synthase (NOS), and other proteins such as kinetic-related proteins and gene expression-related proteins. Several proteins show significant differences in response to short-term stress and long-term adaptation, and differential expression of these proteins reflects an overall change in cellular structure and metabolism associated with resistance to pyrethroid insecticides. In summary, our research has improved the understanding of the molecular mechanisms of beta-cypermethrin resistance in German cockroaches, which will facilitate the development of rational methods to improve the management of this pest.

Hierarchically porous carbons as supports for fuel cell electrocatalysts with atomically dispersed Fe-N x moieties.

The development of high-performance non-platinum group metal (non-PGM) catalysts for the oxygen reduction reaction (ORR) is still of significance in promoting the commercialization of proton exchange membrane fuel cells (PEMFCs). In this work, a "hierarchically porous carbon (HPC)-supporting" approach was developed to synthesize highly ORR active Fe-phenanthroline (Fe-phen) derived Fe-N x -C catalysts. Compared to commercial carbon black supports, utilizing HPCs as carbon supports can not only prevent the formation of inactive iron nanoparticles during pyrolysis but also optimize the porous morphology of the catalysts, which eventually increases the amount of reactant-accessible and atomically dispersed Fe-N x active sites. The prepared catalyst therefore exhibits a remarkable ORR activity in both half-cells (half-wave potential of 0.80 V in 0.5 M H2SO4) and H2-air PEMFCs (442 mA cm-2 at a working voltage of 0.6 V), making it among the best non-PGM catalysts for PEMFCs.

Dalestones A and B, two anti-inflammatory cyclopentenones from Daldinia eschscholzii with an edited strong promoter for the global regulator LaeA-like gene.

Replacement of the native promoter of theglobal regulator LaeA-like gene of Daldinia eschscholzii by a strong gpdA promoter led to the generation of two novel cyclopentenone metabolites, named dalestones A and B, whose structures were assigned by a combination of spectroscopic analysis, modified Mosher's reaction, and electronic circular dichroism (ECD). Dalestones A and B inhibit the gene expression of TNF-α and IL-6 in LPS-induced RAW264.7 macrophages.