Cadmium exposure induces osteoporosis through cellular senescence, associated with activation of NF-κB pathway and mitochondrial dysfunction.

Cadmium (Cd) is a heavy metal toxicant as a common pollutant derived from many agricultural and industrial sources. The absorption of Cd takes place primarily through Cd-contaminated food and water and, to a significant extent, via inhalation of Cd-contaminated air and cigarette smoking. Epidemiological data suggest that occupational or environmental exposure to Cd increases the health risk for osteoporosis and spontaneous fracture such as itai-itai disease. However, the direct effects and underlying mechanism(s) of Cd exposure on bone damage are largely unknown. We used primary bone marrow-derived mesenchymal stromal cells (BMMSCs) and found that Cd significantly induced BMMSC cellular senescence through over-activation of NF-κB signaling pathway. Increased cell senescence was determined by production of senescence-associated secretory phenotype (SASP), cell cycle arrest and upregulation of p21/p53/p16INK4a protein expression. Additionally, Cd impaired osteogenic differentiation and increased adipogenesis of BMMSCs, and significantly induced cellular senescence-associated defects such as mitochondrial dysfunction and DNA damage. Sprague-Dawley (SD) rats were chronically exposed to Cd to verify that Cd significantly increased adipocyte number, and decreased mineralization tissues of bone marrow in vivo. Interestingly, we observed that Cd exposure remarkably retarded bone repair and regeneration after operation of skull defect. Notably, pretreatment of melatonin is able to partially prevent Cd-induced some senescence-associated defects of BMMSCs including mitochondrial dysfunction and DNA damage. Although Cd activated mammalian target of rapamycin (mTOR) pathway, rapamycin only partially ameliorated Cd-induced cell apoptosis rather than cellular senescence phenotypes of BMMSCs. In addition, a selective NF-κB inhibitor moderately alleviated Cd-caused the senescence-related defects of the BMMSCs. The study shed light on the action and mechanism of Cd on osteoporosis and bone ageing, and may provide a novel option to ameliorate the harmful effects of Cd exposure.

Defects of cohesin loader lead to bone dysplasia associated with transcriptional disturbance.

Cohesin loader nipped-B-like protein (Nipbl) is increasingly recognized for its important role in development and cancer. Cornelia de Lange Syndrome (CdLS), mostly caused by heterozygous mutations of Nipbl, is an autosomal dominant disease characterized by multiorgan malformations. However, the regulatory role and underlying mechanism of Nipbl in skeletal development remain largely elusive. In this study, we constructed a Nipbl-a Cas9-knockout (KO) zebrafish, which displayed severe retardation of global growth and skeletal development. Deficiency of Nipbl remarkably compromised cell growth and survival, and osteogenic differentiation of mammalian osteoblast precursors. Furthermore, Nipbl depletion impaired the cell cycle process, and caused DNA damage accumulation and cellular senescence. In addition, nucleolar fibrillarin expression, global rRNA biogenesis, and protein translation were defective in the Nipbl-depleted osteoblast precursors. Interestingly, an integrated stress response inhibitor (ISRIB), partially rescued Nipbl depletion-induced cellular defects in proliferation and apoptosis, osteogenesis, and nucleolar function. Simultaneously, we performed transcriptome analysis of Nipbl deficiency on human neural crest cells and mouse embryonic fibroblasts in combination with Nipbl ChIP-Seq. We found that Nipbl deficiency caused thousands of differentially expressed genes including some important genes in bone and cartilage development. In conclusion, Nipbl deficiency compromised skeleton development through impairing osteoblast precursor cell proliferation and survival, and osteogenic differentiation, and also disturbing the expression of some osteogenesis-regulatory genes. Our study elucidated that Nipbl played a pivotal role in skeleton development, and supported the fact that treatment of ISRIB may provide an early intervention strategy to alleviate the bone dysplasia of CdLS.

The Cytochrome P450 Catalyzing C-S Bond Formation in S-Heterocyclization of Chuangxinmycin Biosynthesis.

Microbial sulfur-containing secondary metabolites show various biological activities, but the C-S bond-forming in their biosynthetic metabolism has not been thoroughly understood. Here, we present genetic, biochemical and structural characterization of a cytochrome P450 monooxygenase CxnD exhibiting C-S bond forming activity in S-heterocyclization of chuangxinmycin biosynthesis. In vivo and in vitro analyses demonstrated that CxnD generated an indole-fused dihydrothiopyran skeleton from a L-Trp-derived thiol intermediate. Furthermore, X-ray crystal structure of CxnD in complex with a substrate analogue and structure-based mutagenesis revealed intimate details of the substrate binding mode. A radical mechanism initiated by abstraction of the imino hydrogen atom or an electron from indole group of the substrate was proposed for CxnD, which provided valuable insights into the molecular basis for the intra-molecular C(sp2 )-H thiolation by the P450 in chuangxinmycin biosynthesis.

Application of a clustered countercurrent-flow micro-channel reactor in the preparation of KMnF3 perovskite for asymmetric supercapacitors.

A clustered countercurrent-flow micro-channel reactor (C-CFMCR) with adjustable magnification times was constructed for the preparation of KMnF3 perovskite fluoride by a co-precipitation process, in which the concentrations and feed rates of reactants were precisely controlled. Benefitting from the enhanced micromixing efficiency of the microreactor, the KMnF3 particles prepared in C-CFMCR were smaller and less aggregated than those produced with traditional stirred reactors (STR). The prepared KMnF3 was applied as the electrode material in supercapacitors, and the electrochemical measurements showed that the KMnF3 obtained under optimal conditions had a discharge specific capacitance of ∼442 F g-1 at a current density of 1 A g-1, with a decline of ∼5.4% after 5000 charge-discharge cycles in an aqueous electrolyte of 2 M KOH. It was also found that the morphologies and electrochemical performances of the prepared KMnF3 particles changed accordingly with the micromixing efficiencies of C-CFMCR, which can be adjusted by the reactor structure and operating conditions. An asymmetric supercapacitor assembled with the KMnF3 and activated carbon exhibited an energy density of 13.1 W h kg-1 at a power density of 386.3 W kg-1, with eminent capacitance retention of ∼81.2% after 5000 cycles. In addition, only a slight amplification effect of C-CFMCR on the co-precipitation process was noticed, indicating that the C-CFMCR is a promising technology for the massive and controllable production of KMnF3 particles as well as other ultrafine particles.

Exploring novel herbicidin analogues by transcriptional regulator overexpression and MS/MS molecular networking.

BACKGROUND: Herbicidin F has an undecose tricyclic furano-pyrano-pyran structure with post-decorations. It was detected from Streptomyces mobaraensis US-43 fermentation broth as a trace component by HPLC-MS analysis. As herbicidins exhibit herbicidal, antibacterial, antifungal and antiparasitic activities, we are attracted to explore more analogues for further development. RESULTS: The genome of S. mobaraensis US-43 was sequenced and a herbicidin biosynthetic gene cluster (hcd) was localized. The cluster contains structural genes, one transporter and three potential transcription regulatory genes. Overexpression of the three regulators respectively showed that only hcdR2 overexpression significantly improved the production of herbicidin F, and obviously increased the transcripts of 7 structural genes as well as the transporter gene. After performing homology searches using BLASTP in the GenBank database, 14 hcd-like clusters were found with a cluster-situated hcdR2 homologue. These HcdR2 orthologues showed overall structural similarity, especially in the C-terminal DNA binding domain. Based on bioinformatics analysis, a 21-bp consensus binding motif of HcdR2 was detected within 30 promoter regions in these genome-mined clusters. EMSA results verified that HcdR2 bound to the predicted consensus sequence. Additionally, we employed molecular networking to explore novel herbicidin analogues in hcdR2 overexpression strain. As a result, ten herbicidin analogues including six new compounds were identified based on MS/MS fragments. Herbicidin O was further purified and confirmed by 1H NMR spectrum. CONCLUSIONS: A herbicidin biosynthetic gene cluster (hcd) was identified in S. mobaraensis US-43. HcdR2, a member of LuxR family, was identified as the pathway-specific positive regulator, and the production of herbicidin F was dramatically increased by overexpression of hcdR2. Combined with molecular networking, ten herbicidin congeners including six novel herbicidin analogues were picked out from the secondary metabolites of hcdR2 overexpression strain. The orthologues of herbicidin F pathway-specific regulator HcdR2 were present in most of the genome-mined homologous biosynthetic gene clusters, which possessed at least one consensus binding motif with LuxR family characteristic. These results indicated that the combination of overexpression of hcdR2 orthologous regulator and molecular networking might be an effective way to exploit the "cryptic" herbicidin-related biosynthetic gene clusters for discovery of novel herbicidin analogues.

Application of micro-impinging stream reactors in the preparation of Co and Al co-doped Ni(OH)2 nanocomposites for supercapacitors and their modification with reduced graphene oxide.

A micro-impinging stream reactor (MISR) consisting of a commercial T-junction and steel capillaries, which is of intensified micromixing efficiency as compared with traditional stirred reactors (STR), was applied for the preparation of Co and Al co-doped Ni(OH)2 nanocomposites and their modification with reduced graphene oxide (RGO). The co-precipitation preparation process was conducted under precisely controlled proportions and concentrations of reactants in the MISR. Therefore, element analysis showed a higher uniform distribution of metal ions within the nanocomposites obtained through the MISR. The structural characterization and electrochemical measurements also showed that the MISR-prepared metal-doped nanocomposites were of more uniform dispersion and superior electrochemical performance than those prepared with STR. In addition, by modifying with RGO in the MISR, the electrochemical performance of Co and Al co-doped Ni(OH)2 nanocomposites could be further improved. The Co and Al co-doped Ni(OH)2/RGO prepared under optimal conditions achieved an ultrahigh specific capacitance of 2389.5 F g-1 at the current density of 1 A g-1 and displayed an excellent cycling stability with 83.7% retention of the initial capacitance after 1000 charge/discharge cycles in 6 M KOH aqueous solution.

Structure-based manual screening and automatic networking for systematically exploring sansanmycin analogues using high performance liquid chromatography tandem mass spectroscopy.

Sansanmycins (SS), one of several known uridyl peptide antibiotics (UPAs) possessing a unique chemical scaffold, showed a good inhibitory effect on the highly refractory pathogens Pseudomonas aeruginosa and Mycobacterium tuberculosis, especially on the multi-drug resistant M. tuberculosis. This study employed high performance liquid chromatography-mass spectrometry detector (HPLC-MSD) ion trap and LTQ orbitrap tandem mass spectrometry (MS/MS) to explore sansanmycin analogues manually and automatically by re-analysis of the Streptomyces sp. SS fermentation broth. The structure-based manual screening method, based on analysis of the fragmentation pathway of known UPAs and on comparisons of the MS/MS spectra with that of sansanmycin A (SS-A), resulted in identifying twenty sansanmycin analogues, including twelve new structures (1-12). Furthermore, to deeply explore sansanmycin analogues, we utilized a GNPS based molecular networking workflow to re-analyze the HPLC-MS/MS data automatically. As a result, eight more new sansanmycins (13-20) were discovered. Compound 1 was discovered to lose two amino acids of residue 1 (AA1) and (2S, 3S)-N3-methyl-2,3-diamino butyric acid (DABA) from the N-terminus, and compounds 6, 11 and 12 were found to contain a 2',3'-dehydrated 4',5'-enamine-3'-deoxyuridyl moiety, which have not been reported before. Interestingly, three trace components with novel 5,6-dihydro-5'-aminouridyl group (16-18) were detected for the first time in the sansanmycin-producing strain. Their structures were primarily determined by detail analysis of the data from MS/MS. Compounds 8 and 10 were further confirmed by nuclear magnetic resonance (NMR) data, which proved the efficiency and accuracy of the method of HPLC-MS/MS for exploration of novel UPAs. Comparing to manual screening, the networking method can provide systematic visualization results. Manual screening and networking method may complement with each other to facilitate the mining of novel UPAs.

Micromixing Study of a Clustered Countercurrent-Flow Micro-Channel Reactor and Its Application in the Precipitation of Ultrafine Manganese Dioxide.

A clustered countercurrent-flow micro-channel reactor (C-CFMCR) has been assembled by the numbering-up of its single counterpart (S-CFMCR). Its micromixing performance was then studied experimentally using a competitive parallel reaction system, and the micromixing time was calculated as the micromixing performance index. It was found that the micromixing time of C-CFMCR was ranged from 0.34 to 10 ms according to its numbering-up times and the operating conditions of the reactor, and it was close to that of S-CFMCR under the same operating conditions, demonstrating a weak scaling-up effect from S-CFMCR to C-CFMCR. The C-CFMCR was then applied to prepare ultrafine manganese dioxide in a continuous manner at varying micromixing time. It showed that the micromixing time had a major effect on the particle structure. More uniform and smaller MnO2 particles were obtained with intensified micromixing. By building a typical three electrode system to characterize their performance as a supercapacitor material, the MnO2 particles prepared by both S-CFMCR and C-CFMCR under optimal conditions displayed a specific capacitance of ~175 F·g-1 at the current density of 1 A·g-1, with a decline of ~10% after 500 charge-discharge cycles. This work showed that C-CFMCR will have a great potential for the continuous and large-scale preparation of ultrafine particles.