Global analysis of the biosynthetic chemical space of marine prokaryotes.

BACKGROUND: Marine prokaryotes are a rich source of novel bioactive secondary metabolites for drug discovery. Recent genome mining studies have revealed their great potential to bio-synthesize novel secondary metabolites. However, the exact biosynthetic chemical space encoded by the marine prokaryotes has yet to be systematically evaluated. RESULTS: We first investigated the secondary metabolic potential of marine prokaryotes by analyzing the diversity and novelty of the biosynthetic gene clusters (BGCs) in 7541 prokaryotic genomes from cultivated and single cells, along with 26,363 newly assembled medium-to-high-quality genomes from marine environmental samples. To quantitatively evaluate the unexplored biosynthetic chemical space of marine prokaryotes, the clustering thresholds for constructing the biosynthetic gene cluster and molecular networks were optimized to reach a similar level of the chemical similarity between the gene cluster family (GCF)-encoded metabolites and molecular family (MF) scaffolds using the MIBiG database. The global genome mining analysis demonstrated that the predicted 70,011 BGCs were organized into 24,536 mostly new (99.5%) GCFs, while the reported marine prokaryotic natural products were only classified into 778 MFs at the optimized clustering thresholds. The number of MF scaffolds is only 3.2% of the number of GCF-encoded scaffolds, suggesting that at least 96.8% of the secondary metabolic potential in marine prokaryotes is untapped. The unexplored biosynthetic chemical space of marine prokaryotes was illustrated by the 88 potential novel antimicrobial peptides encoded by ribosomally synthesized and post-translationally modified peptide BGCs. Furthermore, a sea-water-derived Aquimarina strain was selected to illustrate the diverse biosynthetic chemical space through untargeted metabolomics and genomics approaches, which identified the potential biosynthetic pathways of a group of novel polyketides and two known compounds (didemnilactone B and macrolactin A 15-ketone). CONCLUSIONS: The present bioinformatics and cheminformatics analyses highlight the promising potential to explore the biosynthetic chemical diversity of marine prokaryotes and provide valuable knowledge for the targeted discovery and biosynthesis of novel marine prokaryotic natural products. Video Abstract.

Secondary Metabolic Potential of Kutzneria.

Kutzneria is a rare genus of Actinobacteria that harbors a variety of secondary metabolite gene clusters and produces several interesting types of bioactive secondary metabolites. Recent efforts have partially elucidated the biosynthetic pathways of some of these bioactive natural products, suggesting the diversity and specificity of secondary metabolism within this genus. Here, we summarized the chemical structures, biosynthetic pathways, and key metabolic enzymes of the secondary metabolites isolated from Kutzneria strains. In-depth comparative genomic analysis of all six available high-quality Kutzneria genomes revealed that the majority (77%) of the biosynthetic gene cluster families of Kutzneria were untapped and identified homologues of key metabolic enzymes in the putative gene clusters, including cytochrome P450s, halogenases, and flavin-dependent N-hydroxylases. The present study suggests that Kutzneria exhibits great potential to synthesize novel secondary metabolites, encodes a variety of valuable metabolic enzymes, and also provides valuable information for the targeted discovery and biosynthesis of novel natural products from Kutzneria.

Non-ribosomal peptide biosynthetic potential of the nematode symbiont Photorhabdus.

Photorhabdus, the symbiotic bacteria of Heterorhabditis nematodes, has been reported to possess many non-ribosomal peptide synthetase (NRPS) biosynthesis gene clusters (BGCs). To provide an in-depth assessment of the non-ribosomal peptide biosynthetic potential of Photorhabdus, we compared the distribution of BGCs in 81 Photorhabdus strains, confirming the predominant presence (44.80%) of NRPS BGCs in Photorhabdus. All 990 NRPS BGCs were clustered into 275 gene cluster families (GCFs) and only 13 GCFs could be annotated with known BGCs, suggesting their great diversity and novelty. These NRPS BGCs encoded 351 novel peptides containing more than four amino acids, and 173 of them showed high sequence similarity to known BGCs encoding bioactive peptides, implying the promising potential of Photorhabdus to produce valuable peptides. Sequence similarity networking of adenylation (A-) domains suggested that the substrate specificity of A-domains was not directly correlated with the sequence similarity. The molecular similarity network of predicted metabolite scaffolds of NRPS BGCs and reported peptides from Photorhabdus and a relevant database demonstrated that the non-ribosomal peptide biosynthetic potential of Photorhabdus was largely untapped and revealed the core peptides deserving intensive studies. Our present study provides valuable information for the targeted discovery of novel non-ribosomal peptides from Photorhabdus.

An atlas of bacterial secondary metabolite biosynthesis gene clusters.

Bacterial secondary metabolites are rich sources of novel drug leads. The diversity of secondary metabolite biosynthetic gene clusters (BGCs) in genome-sequenced bacteria, which will provide crucial information for the efficient discovery of novel natural products, has not been systematically investigated. Here, the distribution and genetic diversity of BGCs in 10 121 prokaryotic genomes (across 68 phyla) were obtained from their PRISM4 outputs using a custom python script. A total of 18 043 BGCs are detected from 5743 genomes with non-ribosomal peptide synthetases (25.4%) and polyketides (15.9%) as the dominant classes of BGCs. Bacterial strains harbouring the largest number of BGCs are revealed and BGC count in strains of some genera vary greatly, suggesting the necessity of individually evaluating the secondary metabolism potential. Additional analysis against 102 strains of discovered bacterial genera with abundant amounts of BGCs confirms that Kutzneria, Kibdelosporangium, Moorea, Saccharothrix, Cystobacter, Archangium, Actinosynnema, Kitasatospora, and Nocardia, may also be important sources of natural products and worthy of priority investigation. Comparative analysis of BGCs within these genera indicates the great diversity and novelty of the BGCs. This study presents an atlas of bacterial secondary metabolite BGCs that provides a lot of key information for the targeted discovery of novel natural products.

[Removal Performance of Antibiotic Resistance Genes and Heavy Metal Resistance Genes in Municipal Wastewater by Magnetic-Coagulation Process].

Antibiotic resistance genes (ARGs) in municipal wastewater pose a potential threat to the environment. In this study, the change in absolute and relative abundance of ARGs, metal resistance genes (MRGs), and mobile genetic elements (MGTs) were investigated during an emergent municipal wastewater treatment by the magnetic separation process. Results indicate that all the concentrations of targeted ARGs, MRGs, and MGTs decreased significantly in the primary and secondary stirring tank. However, the absolute abundance of some ARGs and MRGs increased in the effluent, which is likely caused by the presence of ample MGTs, in the order of int1 (2.00×1010 copies·mL-1) > int2 (1.91×108 copies·mL-1) > Tn 916/1545e(5.38×108 copies·mL-1). The results obtained from network and PCA analysis showed that the removal of ARGs and MRGs were significantly associated with variations in the microbial community and common pollutants in urban wastewater, such as suspended solids, phosphorus, and COD, which are important factors for affecting the removal efficiency of antibiotic resistance genes and metal heavy resistance genes. These results show that magnetic separation can effectively reduce common pollutants in urban wastewater and might further restrict the transmission and transfer of ARGs. Moreover, it is necessary to strengthen the subsequent management of magnetic separation effluent and dehydrated sludge by disinfection technologies to lessen the risk of antimicrobial contamination.

MicroRNA-221 promotes cisplatin resistance in osteosarcoma cells by targeting PPP2R2A.

Osteosarcoma (OS), the most common malignant bone tumor, is the main cause of cancer-related deaths in children and young adults. Despite the combination of surgery and multi-agent chemotherapy, patients with OS who develop resistance to chemotherapy or experience recurrence have a dismal prognosis. MicroRNAs (miRNAs) are a class of small noncoding RNAs that repress their targets by binding to the 3'-UTR and/or coding sequences, leading to the inhibition of gene expression. miR-221 is found to be up-regulated in tumors when compared with their matched normal osteoblast tissues. We also observed significant miR-221 up-regulation in the OS cell lines, MG-63, SaoS-2, and U2OS, when compared with the normal osteoblast cell line, HOb. Overexpression of miR-221 promoted OS cell invasion, migration, proliferation, and cisplatin resistance. MG-63 and SaoS-2 cells transfected with miR-221 mimics were more resistant to cisplatin. The IC50 of MG-63 cells transfected with control mimics was 1.24 µM. However, the IC50 of MG-63 cells overexpressing miR-221 increased to 7.65 µM. Similar results were found in SaoS-2 cells, where the IC50 for cisplatin increased from 3.65 to 8.73 µM. Thus, we report that miR-221 directly targets PP2A subunit B (PPP2R2A) in OS by binding to the 3'-UTR of the PPP2R2A mRNA. Restoration of PPP2R2A in miR-221-overexpressing OS cells recovers the cisplatin sensitivity of OS cells. Therefore, the present study suggests a new therapeutic approach by inhibiting miR-221 for anti-chemoresistance in OS.

Effectiveness of Bi-Needle Technique (Hybrid Yeung Endoscopic Spine System/Transforaminal Endoscopic Spine System) for Percutaneous Endoscopic Lumbar Discectomy.

OBJECTIVE: The bi-needle technique is a new technique for percutaneous endoscopic lumbar discectomy. This technique combines the advantages of Yeung endoscopic spine system (YESS) and transforaminal endoscopic spine system (TESSYS) techniques. The aim of this study was to evaluate effectiveness of the bi-needle technique for percutaneous endoscopic lumbar discectomy and compare it with the TESSYS technique. METHODS: We reviewed 86 patients with single-level lumbar disc herniation treated by percutaneous endoscopic lumbar discectomy in our hospital from June 2013 to December 2015. Bi-needle technique was used in 49 patients (30 men, 19 women; average age 40.4 ± 5.2 years). TESSYS technique was used in 37 patients (19 men, 18 women; average age 42.8 ± 6.4 years). Clinical results were evaluated and compared between the 2 groups. RESULTS: Symptoms in both groups were significantly improved at the last follow-up (P < 0.01). There was no statistical difference in visual analog scale and lumbar Japanese Orthopaedic Association scores between bi-needle and TESSYS groups at last follow-up (P = 0.69 and P = 0.33, respectively). Operative time was shorter in the bi-needle group (P < 0.01). Recurrence rate and reoperation rate were lower in the bi-needle group (P = 0.04 and P = 0.03, respectively). Discitis was diagnosed in 2 patients in the TESSYS group. There were no patients with postoperative discitis in the bi-needle group. CONCLUSIONS: The bi-needle technique is safe and effective for treatment of lumbar disc herniation. Compared with TESSYS technique, operative time is shorter, and recurrence and reoperation rates are lower.

[Downregulation of PTTG1 expression inhibits the proliferation and invasiveness and promotes the apoptosis of human prostate cancer LNCaP-AI cells].

OBJECTIVE: To investigate the effects of down-regulation of PTTG1 expression on the proliferation, invasiveness and apoptosis of androgen-independent human prostate cancer LNCaP-AI cells and their sensitivity to androgen antagonists. METHODS: Human prostate cancer LNCaP-AI cells were transfected with siRNA targeting the PTTG1 gene using the Lipofectamine 2000 transfection reagent. The proliferation, invasiveness and apoptosis of the cells were detected by MTT, Transwell assay and flow cytometry, respectively. The protein expressions of PTTG1, p-Akt, and p-ERK were determined by Western blot and the mRNA expression of PTTG1 measured by agarose gel electrophoresis. RESULTS: The siRNA expression vector markedly down-regulated the expression of PTTG1, which effectively suppressed the proliferation of the LNCaP-AI cells, with the inhibition rates of (19.47 ± 2.12), (24.01 ± 2.13) and (48.02 ± 2.22)% at 24, 48 and 72 hours, respectively, after transfection, with statistically significant differences among the three groups (P <0.05). The number of the cells passing through the polycarbonate film was remarkably decreased at 24, 48 and 72 hours (74.67 ± 9.85, 56.44 ± 8.66 and 37.33 ± 6.14) as compared with the baseline (111.11 ± 13.47) (P <0.01), while the apoptosis rate of the cells was significantly increased at 24, 48 and 72 hours (18.32 ± 0.94), (19.94 ± 1.30) and (21.73 ± 1.88)% in comparison with the baseline (ï¼»2.17 ± 0.49ï¼½%)， (P <0.05). PTTG1 siRNA combined with androgen antagonist flumatide exhibited even more significant effects in inhibiting the proliferation and promoting the apoptosis of the LNCaP-AI cells than either used alone, and in a flumatide dose-dependent manner. The inhibition and apoptosis rates of the LNCaP-AI cells treated with 50 nmol/L flumatide were (27.13 ± 3.52) and (3.94 ± 0.48)%, and those treated with siRNA + 50 nmol/L flumatide were (67.51 ± 5.13) and (19.93 ± 1.72)%, respectively, both with statistically significant differences between the two groups (P <0.05). The inhibition and apoptosis rates of the cells treated with 100 nmol/L flumatide were (43.72 ± 3.90) and (5.33 ± 0.66)%, and those treated with siRNA + 100 nmol/L flumatide were (73.19 ± 4.78) and (23.43 ± 1.76)%, respectively, both with statistically significant differences between the two groups (P <0.05). CONCLUSIONS: The siRNA expression vector can down-regulate the expression of PTTG1, which can inhibit the proliferation and invasiveness of LNCaP-AI cells, promote their apoptosis, and increase their sensibility to androgen antagonists. Suppressing the expression of PTTG1 may enhance the effect of androgen-deprivation therapy on advanced prostate cancer.

Molecular Cloning and Characterization of Full-Length cDNA of Calmodulin Gene from Pacific Oyster Crassostrea gigas.

The shell of the pearl oyster (Pinctada fucata) mainly comprises aragonite whereas that of the Pacific oyster (Crassostrea gigas) is mainly calcite, thereby suggesting the different mechanisms of shell formation between above two mollusks. Calmodulin (CaM) is an important gene for regulating the uptake, transport, and secretion of calcium during the process of shell formation in pearl oyster. It is interesting to characterize the CaM in oysters, which could facilitate the understanding of the different shell formation mechanisms among mollusks. We cloned the full-length cDNA of Pacific oyster CaM (cgCaM) and found that the cgCaM ORF encoded a peptide of 113 amino acids containing three EF-hand calcium-binding domains, its expression level was highest in the mantle, hinting that the cgCaM gene is probably involved in shell formation of Pacific oyster, and the common ancestor of Gastropoda and Bivalvia may possess at least three CaM genes. We also found that the numbers of some EF hand family members in highly calcified species were higher than those in lowly calcified species and the numbers of these motifs in oyster genome were the highest among the mollusk species with whole genome sequence, further hinting the correlation between CaM and biomineralization.

[Expression of pituitary tumor-transforming gene 1 during the development of androgen-independent prostate cancer].

OBJECTIVE: To explore the expression of pituitary tumor transforming gene 1 (PTTG1) during the transformation of prostate cancer from androgen-dependent (ADPC) to androgen-independent (AIPC). METHODS: We established an AIPC cell model LNCaP-AI by culturing the androgen-dependent LNCaP cell line in the hormone-deprived medium for over 3 months. The cell model was verified and the PTTG1 expression in the LNCaP cells was detected by Western blot and RT-PCR during hormone deprivation. RESULTS: The AIPC cell model LNCaP-AI was successfully established. The PTTG1 expression was gradually increased in the LNCaP cells with the prolonged time of hormone deprivation and the expressions of matrix metalloproteinases MMP-2 and -9 were elevated at the same time. CONCLUSIONS: The expression of PTTG1 is increased gradually in AIPC, which may be a target of gene therapy for advanced prostate cancer.