Complete genome sequence analysis of Bacillus velezensis A5, a promising biocontrol agent from the Pacific Ocean.

Tobacco bacterial wilt (TBW) caused by Ralstonia solanacearum is a serious soil-borne disease, which seriously damages the growth of tobacco crops. Bacillus velezensis A5 was isolated from 3000 m deep-sea sediments of the Pacific Ocean, and was found to be antagonistic to TBW. Here, we report the complete genome sequence of strain A5, which has a 4,000,699-bp single circular chromosome with 3827 genes and a G + C content of 46.44%, 87 tRNAs, and 27 rRNAs. A total of 12 gene clusters were identified in the genome of strain A5, which were responsible for the biosynthesis of antibacterial compounds, including surfactin, bacillaene, fengycin, difficidin, bacillibactin, and bacilysin. Additionally, strain A5 was found to contain a series of genes related to the biosynthesis of carbohydrate-active enzymes and secreted proteins. Our results indicate that strain A5 can be considered a promising biocontrol agent against TBW in agricultural fields.

Complete genome sequence of Bacillus cereus Z4, a biocontrol agent against tobacco black shank, isolated from the Western Pacific Ocean.

Bacillus species have been considered as promising biological control agents due to their excellent antimicrobial ability. Bacillus cereus strain Z4 was isolated from 2000 m deep sea sediments of the Western Pacific Ocean, which possesses significant antifungal activity against Phytophthora nicotianae, the pathogenic fungus of tobacco black shank disease. To reveal the underlying antifungal genetic mechanisms, here, we report the complete genomic sequence of the strain Z4. The genome has one circular chromosome of 5,664,309 bp with a G + C content of 35.31%, 109 tRNAs, and 43 rRNAs. Genomic analysis identified 10 gene clusters related to the biosynthesis of biocontrol active compounds, including bacillibactin, petrobactin, fengycin, and molybdenum cofactor. Meanwhile, 6 gene clusters were responsible for the biosynthesis of metabolites with unknown functions. Strain Z4 also contains a large number of genes encoding carbohydrate-active enzymes and secreted proteins, respectively. The whole genomic analysis of Bacillus cereus Z4 may provide a valuable reference for elucidating its biocontrol mechanism against tobacco black shank.

Complete genome analysis of Bacillus subtilis TY-1 reveals its biocontrol potential against tobacco bacterial wilt.

Bacillus subtilis TY-1 was isolated from 2000 m-deep sea sediments of the Western Pacific Ocean, which was found to exhibit strong antagonistic activity against tobacco bacterial wilt caused by Ralstonia solanacearum. Here, we present the annotated complete genomic sequence of the strain Bacillus subtilis TY-1. The genome consists of a 4,030,869-bp circular chromosome with a G + C content of 43.88%, 86 tRNAs, and 30 rRNAs. Genomic analysis identified a large number of gene clusters involved in the biosynthesis of antibacterial metabolites, including lipopeptides(surfactin, bacillibactin, and fengycin) and polyketides(bacillaene). Meanwhile, numerous genes encoding carbohydrate-active enzymes and secreted proteins were found in TY-1. These findings suggest that Bacillus subtilis TY-1 appears to be a potential biocontrol agent against tobacco bacterial wilt in agricultural fields.

Bacteroides ovatus-mediated CD27- MAIT cell activation is associated with obesity-related T2D progression.

Type 2 diabetes (T2D) is highly associated with obesity. However, the factors that drive the transition from excessive weight gain to glucose metabolism disruption are still uncertain and seem to revolve around systemic immune disorder. Mucosal-associated invariant T (MAIT) cells, which are innate-like T cells that recognize bacterial metabolites, have been reported to be altered in obese people and to lead to metabolic dysfunction during obesity. By studying the immunophenotypes of blood MAIT cells from a cross-sectional cohort of obese participants with/without T2D, we found an elevation in CD27-negative (CD27-) MAIT cells producing a high level of IL-17 under T2D obese conditions, which could be positively correlated with impaired glucose metabolism in obese people. We further explored microbial translocation caused by gut barrier dysfunction in obese people as a triggering factor of MAIT cell abnormalities. Specifically, accumulation of the bacterial strain Bacteroides ovatus in the peripheral blood drove IL-17-producing CD27- MAIT cell expansion and could be associated with T2D risk in obese individuals. Overall, these results suggest that an aberrant gut microbiota-immune axis in obese people may drive or exacerbate T2D. Importantly, CD27- MAIT cell subsets and Bacteroides ovatus could represent targets for novel interventional strategies. Our findings extend current knowledge regarding the clinical relevance of body mass index (BMI)-associated variation in circulating MAIT cells to reveal the role of these cells in obesity-related T2D progression and the underlying cellular mechanisms.

Characterization of two novel psychrophilic and piezotolerant strains, Shewanella psychropiezotolerans sp. nov. and Shewanella eurypsychrophilus sp. nov, adapted to an extreme deep-sea environment.

Three marine bacterial strains designated YLB-06T, YLB-08T and YLB-09 were isolated under high hydrostatic pressure from deep-sea sediment samples collected from the Southwest Indian Ocean. They were Gram-stain-negative, oxidase- and catalase-positive, facultative anaerobic and motile. In addition, the strains were capable of growing at 0-20 °C (optimum 4-10 °C) and 0.1-40 MPa (optimum 0.1 MPa), were psychrophiles and piezotolerant, and could use trimethylamine N-oxide (TMAO), DMSO, elemental sulfur and insoluble Fe (III) as terminal electron acceptors during anaerobic growth. Strain YLB-06T could also use nitrate, and strains YLB-08T and YLB-09 could use nitrite as a terminal electron acceptor. Phylogenetic tree analyses based on 16S rRNA gene sequences and 400 optimized universal marker sequences indicated that the strains belonged to the genus Shewanella. The 16S rRNA gene highest similarity, together with the estimated ANI and DDH values for these strains with their related type strains, were below the respective thresholds for species differentiation. The ANI and DDH values between YLB-08T and YLB-09 were 99.9% and 91.8%, respectively, implying that they should belong to the same genospecies. The YLB-06T genome had duplicated genes, and multiple movement modalities, attachment modalities, biofilm synthesis systems, intercellular interactions and a strong antioxidant system, which were all beneficial for survival in an extreme deep-sea environment. The G + C contents of strains YLB-06T, YLB-08T and YLB-09 were 45.1, 43.5 and 43.6 mol%, respectively. Based on polyphasic taxonomic properties, two novel psychropiezotolerant species are proposed, Shewanella psychropiezotolerans sp. nov. with YLB-06T (=MCCC 1A12715T = KCTC 62907T) and S. eurypsychrophilus sp. nov with YLB-08T (=MCCC 1A12718T = KCTC 62909T) as type strains.

Two New Cytotoxic Compounds from a Deep-Sea Penicillum citreonigrum XT20-134.

Penicillum citreonigrum XT20-134 (MCCC 3A00956) is a fungus with cytotoxic activity, derived from deep-sea sediment. Five new compounds, adeninylpyrenocine (1), 2-hydroxyl-3-pyrenocine-thio propanoic acid (2), ozazino-cyclo-(2,3-dihydroxyl-trp-tyr) (3), 5,5-dichloro-1-(3,5-dimethoxyphenyl)-1,4-dihydroxypentan-2-one (4), and 2,3,4-trihydroxybutyl cinnamate (5), together with 19 known compounds (6-24), were isolated from an ethyl acetate (EtOAc) extract of its fermentation. The structures of the new compounds were comprehensively characterized by high-resolution electrospray ionization-mass spectrometry (HR-ESI-MS), 1D and 2D nuclear magnetic resonance (NMR). All isolates were evaluated for their cytotoxic activities. The heteroatom-containing new compounds 2 and 4 showed potent cytotoxicity to the human hepatoma tumor cell Bel7402 with IC50 values of 7.63 ± 1.46, 13.14 ± 1.41 µM and the human fibrosarcoma tumor cell HT1080 with IC50 values of 10.22 ± 1.32, 16.53 ± 1.67 µM, respectively.

Contrast-Enhanced Ultrasonography Versus Contrast-Enhanced Computed Tomography for Assessment of Residual Tumor From Hepatocellular Carcinoma Treated With Transarterial Chemoembolization: A Meta-analysis.

OBJECTIVES: This study reviewed the literature to directly evaluate the diagnostic performance of contrast-enhanced ultrasonography (CEUS) versus contrast-enhanced computed tomography (CECT) for assessing residual tumors of hepatocellular carcinoma treated with transarterial chemoembolization. METHODS: PubMed, Embase, the Cochrane Library, and the China National Knowledge Infrastructure were searched through April 30, 2017. The pooled sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio, negative likelihood ratio, diagnostic odds ratio, and summary receiver operating characteristic curve were calculated and compared to examine the diagnostic performance of CEUS versus CECT. RESULTS: A total of 11 studies, including 421 patients and 491 nodules were analyzed. The pooled diagnostic performances of CEUS versus CECT were as follows: (1) sensitivity (95% confidence interval), 0.97 (0.95-0.99) versus 0.72 (0.67-0.76); (2) specificity, 0.86 (0.74-0.94) versus 0.99 (0.95-1.00); (3) positive predictive value, 0.97 (0.95-0.99) versus 1.00 (0.98-1.00); (4) negative predictive value, 0.90 (0.83-0.95) versus 0.51 (0.44-0.58); (5) positive likelihood ratio, 7.79 (4.73-12.82) versus 12.50 (5.74-27.20); (6) negative likelihood ratio, 0.05 (0.03-0.09) versus 0.35 (0.26-0.48); (7) diagnostic odds ratio, 150.56 (57.03-397.49) versus 35.54 (14.89-84.83); and (8) area under the summary receiver operating characteristic curve, 0.9875 versus 0.9239. The sensitivity and negative predictive value of CEUS were significantly higher than those of CECT (both P < .001). The specificity and positive predictive value of CECT were significantly higher than those of CEUS (both P < .05). CONCLUSIONS: Contrast-enhanced US, with better sensitivity and negative predictive value versus CECT, was an effective method for exclusion of residual tumors after transarterial chemoembolization. Contrast-enhanced CT, with higher specificity than CEUS, is a valid approach for identifying residual tumors.

Macrolactins from Marine-Derived Bacillus subtilis B5 Bacteria as Inhibitors of Inducible Nitric Oxide and Cytokines Expression.

In order to find new natural products with anti-inflammatory activity, chemical investigation of a 3000-meter deep-sea sediment derived bacteria Bacillus subtilis B5 was carried out. A new macrolactin derivative was isolated and identified as 7,13-epoxyl-macrolactin A (1). Owing to the existence of the epoxy ring, 1 exhibited a significant inhibitory effect on the expression of inducible nitric oxide and cytokines, compared with previously isolated known macrolactins (2-5). Real-time Polymerase Chain Reaction (PCR) analysis showed that the new compound significantly inhibited the mRNA expressions of inducible nitric oxide synthase (iNOS), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6) in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Reverse transcription-PCR analysis demonstrated that the new compound reduced the mRNA expression level of IL-1ß in a concentration-dependent manner.

Biosynthetic Functional Gene Analysis of Bis-Indole Metabolites from 25D7, a Clone Derived from a Deep-Sea Sediment Metagenomic Library.

This work investigated the metabolites and their biosynthetic functional hydroxylase genes of the deep-sea sediment metagenomic clone 25D7. 5-Bromoindole was added to the 25D7 clone derived Escherichia coli fermentation broth. The new-generated metabolites and their biosynthetic byproducts were located through LC-MS, in which the isotope peaks of brominated products emerged. Two new brominated bis-indole metabolites, 5-bromometagenediindole B (1), and 5-bromometagenediindole C (2) were separated under the guidance of LC-MS. Their structures were elucidated on the basis of 1D and 2D NMR spectra (COSY, HSQC, and HMBC). The biosynthetic functional genes of the two new compounds were revealed through LC-MS and transposon mutagenesis analysis. 5-Bromometagenediindole B (1) also demonstrated moderately cytotoxic activity against MCF7, B16, CNE2, Bel7402, and HT1080 tumor cell lines in vitro.

Preprocedure and Postprocedure Predictive Values of Serum ß2-Microglobulin for Contrast-Induced Nephropathy in Patients Undergoing Coronary Computed Tomography Angiography: A Comparison With Creatinine-Based Parameters and Cystatin C.

OBJECTIVE: This study aimed to investigate the values of serum ß2-microglobulin to predict contrast-induced nephropathy (CIN) before and early after coronary computed tomography angiography (CCTA), comparing with creatinine-based parameters and cystatin C. METHODS: A total of 424 patients were enrolled. Serum ß2-microglobulin, cystatin C, and creatinine were measured at 0, 24, and 48 hours of CCTA. Contrast-induced nephropathy was defined as an elevation of serum creatinine level by 25% or higher or 0.5 mg/dL or greater from baseline within 48 hours. The estimated glomerular filtration rate (eGFR) was calculated by the Modification of Diet in Renal Disease study equation. Receiver operating characteristic curves and multivariate logistic regression analysis were used to detect the efficiency of biomarkers in predicting CIN. RESULTS: Fifty-two subjects (12.26%) developed CIN. Before CCTA, CIN was predicted by both baseline ß2-microglobulin (area under the receiver operating characteristic curve [AUC], 0.791; P < 0.001) and cystatin C (AUC, 0.781; P < 0.001), whereas creatinine and eGFR were not predictive. After CCTA, CIN was predicted by both the absolute post-CCTA levels of ß2-microglobulin, cystatin C, creatinine, and eGFR (AUC, 0.842 vs 0.961 vs 0.691 vs 0.688 at 24 hours, P < 0.001; and 0.937 vs 1.000 vs 0.908 vs 0.898 at 48 hours, P < 0.001) and their relative changes (Δ) to baseline (AUC, 0.677 vs 0.846 vs 0.850 vs 0.844 at 24 hours, P < 0.001; and 0.731 vs 0.968 vs 0.984 vs 0.966 at 48 hours, P < 0.001). Multivariate regression analysis confirmed that baseline ß2-microglobulin (odds ratio, 2.137; 95% confidence interval, 1.805-3.109; P < 0.001) and cystatin C (odds ratio, 1.873; 95% confidence interval, 1.667-2.341; P = 0.003) were independent predictors for CIN. CONCLUSIONS: Serum ß2-microglobulin, with values superior to creatinine-based parameters and similar with cystatin C, was a useful biomarker for the prediction of CIN at pre-CCTA and early post-CCTA.

A head-to-head comparison of homocysteine and cystatin C as pre-procedure predictors for contrast-induced nephropathy in patients undergoing coronary computed tomography angiography.

BACKGROUND: Homocysteine is a potential predictor for contrast-induced nephropathy (CIN). We aimed to compare homocysteine with cystatin C as pre-procedure predictors for CIN in patients undergoing coronary computed tomography angiography (CCTA). METHODS: A total of 580 consecutive patients were enrolled. Concentrations of plasma homocysteine and serum cystatin C were measured before CCTA. CIN is defined as an elevation of creatinine by ≥ 25% or ≥ 0.5mg/dl from baseline within 48h. Receiver operating characteristic curves, Pearson correlation coefficients and logistic regression analysis were used to evaluate the efficiency of potential predictors. RESULTS: Fifty-seven (9.83%) patients developed CIN. Concentrations of homocysteine (19.35 ± 4.32 µmol/l vs. 13.42 ± 3.96 µmol/l, p<0.001) and cystatin C (1.20 ± 0.21 mg/dl vs. 0.99 ± 0.15 mg/dl, p<0.001) increased significantly in CIN subjects. CIN was predicted by homocysteine (AUC 0.829, p<0.001) and cystatin C (AUC 0.774, p<0.001), while creatinine was not predictive. Both homocysteine and cystatin C had positive correlation with ΔCreatinine48h-0 (p<0.001) and negative correlation with ΔeGFR48h-0 (p<0.001). Regression analysis confirmed that increased baseline homocysteine [OR: 1.262 (1.123, 2.554), p<0.001] and cystatin C [OR: 1.565 (1.380, 1.775), p<0.001] were independent predictors for CIN. CONCLUSIONS: Homocysteine, with similar predictive value compared to cystatin C, was an independent biomarker for predicting CIN before CCTA examination.

Microbacterins A and B, new peptaibols from the deep sea actinomycete Microbacterium sediminis sp. nov. YLB-01(T).

Two new peptaibols, namely microbacterins A (1) and B (2), were isolated from the deep sea inhabited actinomycete Microbacterium sediminis spp. nov. YLB-01(T). The sequences of the amino acid residues were determined on the basis of intensive NMR and ESI-MS/MS spectroscopic analysis, in addition to the Marfey's method and CD and optical rotation data for the configurational assignment. Both 1 and 2 exhibited significant cytotoxic activities against a panel of human tumor cell lines.

Two new cytotoxic indole alkaloids from a deep-sea sediment derived metagenomic clone.

Two new indole alkaloids, metagenetriindole A (1) and metagenebiindole A (2), were identified from deep-sea sediment metagenomic clone derived Escherichia coli fermentation broth. The structures of new compounds were elucidated by spectroscopic methods. The two new indole alkaloids demonstrated moderately cytotoxic activity against CNE2, Bel7402 and HT1080 cancer cell lines in vitro.

Indole-based alkaloids from deep-sea bacterium Shewanella piezotolerans with antitumor activities.

Chromatographic separation of a crude extract obtained from a fermentation broth of a chemically unknown bacterium Shewanella piezotolerans WP3 collected in deep-sea yielded three new indole alkaloids namely shewanellines A (1a), B (1b) and C (2), together with 12 known indole alkaloids. The structures were unambiguously elucidated on the basis of 1D and 2D NMR ((1)H, (13)C, COSY, HMBC, HSQC and NOESY) in association with MS and CD data. Compounds 1-4, 7, 9 and 11-14 were selected for the evaluation of their cytotoxic activities against human tumor cell lines HL-60 and BEL-7402, whereas compounds 2, 4 and 9 exhibited significant inhibition toward HL-60.

Gly482Ser mutation impairs the effects of peroxisome proliferator-activated receptor γ coactivator-1α on decreasing fat deposition and stimulating phosphoenolpyruvate carboxykinase expression in hepatocytes.

Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) is a transcriptional coactivator of nuclear receptor peroxisome proliferator-activated receptor γ that critically regulates glucose and fat metabolism. Although clinical evidence suggests that Gly482Ser polymorphism of PGC-1α is associated with an increased incidence of nonalcoholic fatty liver disease, a direct role for Gly482Ser mutation in altering PGC-1α actions on hepatocyte fat deposition remains to be explored. We hypothesized that Gly482Ser mutation impairs the abilities of PGC-1α in ameliorating overnutrition-induced hepatocyte fat deposition and in stimulating hepatocyte expression of cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C; encoded by a key PGC-1α target gene). In the present study, treatment of cultured hepatocytes with palmitate induced fat deposition, serving as a cell model of hepatic steatosis. Upon overexpression of wild-type PGC-1α, H4IIE cells exhibited a significant decrease in palmitate-induced hepatocyte fat deposition compared with control cells and/or cells upon overexpression of mutant PGC-1α (Gly482Ser). Overexpression of wild-type PGC-1α, but not mutant PGC-1α, also caused a significant increase in hepatocyte expression of carnitine palmitoyl transferase 1a, a rate-determining enzyme that transfers long-chain fatty acids into mitochondria for oxidation. In addition, overexpression of mutant PGC-1α did not stimulate PEPCK-C expression as overexpression of wild-type PGC-1α did, likely due to a decrease in the ability of mutant PGC-1α in increasing PEPCK promoter transcription activity. Together, these results suggest that Gly482Ser mutation impairs the abilities of PGC-1α in decreasing fat deposition and in stimulating PEPCK-C expression in cultured hepatocytes.

Differential proteomic analysis of HL60 cells treated with secalonic acid F reveals caspase 3-induced cleavage of Rho GDP dissociation inhibitor 2.

Secalonic acid F (SAF) has been previously identified, however, little is known about its cytotoxic activity and related cytotoxic mechanism. The aim of this study was to evaluate the cytotoxic activity of SAF isolated from a deep sea originated fungus Penicillium sp. F11 in HL60 cells and to analyze the differences in protein expression of HL60 cells treated with SAF. The CCK-8 assay and Annexin V-FLUOS/PI assay indicated that SAF displayed dose- and time-dependent inhibition of HL60 cell proliferation and induced apoptosis. Two-dimensional gel electrophoresis (2-DE) analysis of HL60 cells treated with SAF (4 µg/ml) revealed 10 differentially expressed protein spots (P<0.05), 5 upregulated and 5 downregulated. Three spots (1 downregulated and 2 upregulated) were identified as Rho GDP dissociation inhibitor 2 (RhoGDI 2) proteins by MALDI-TOF MS. Western blotting further demonstrated the decreased abundance of full-length RhoGDI 2 together with the increased abundance of caspase 3-cleaved product of RhoGDI 2. The caspase 3 inhibitor Ac-DEVD-CHO could suppress the cytotoxic effect of SAF and significantly block the cleavage of RhoGDI 2. RhoGDI 2 is a cytosolic regulator of Rho GTPase and the caspase 3-cleaved product of RhoGDI 2 can advance progression of the apoptotic process. Our data showed that SAF may modulate RhoGDI 2 levels in HL60 cells, thereby potentially disrupting cell signaling pathways important for HL60 cell function.

Two new compounds from marine-derived fungus Penicillium sp. F11.

Two new compounds, penicillone A (1) and penicillactam (2), were isolated together with 17 known compounds from a marine-derived fungus Penicillium sp. F11. The structures of the new compounds as well as a firstly literatural reported known compound (3) were assigned by spectroscopic methods including 1D/2D NMR and MS analysis techniques. Their cytotoxicities against HT1080, Cne2, and Bel7402 cell lines were also evaluated.

Study on relationship between expression level and molecular conformations of gene drugs targeting to hepatoma cells in vitro.

AIM: To increase exogenous gene expression level by modulating molecular conformations of targeting gene drugs. METHODS: The full length cDNAs of both P(40) and P(35) subunits of human interleukin 12 were amplified through polymerase chain reaction (PCR) and cloned into eukaryotic expressing vectors pcDNA3.1(+/-) to construct plasmids of P(+)/IL-12, P(+)/P(40) and P(-)/P(35). These plasmids were combined with ASOR-PLL to form two targeting gene drugs [ASOR-PLL-P(+)/IL-12 and ASOR-PLL-P(+)/P(40) + ASOR-PLL-P(-)/P(35)] in optimal ratios. The conformations of these two drugs at various concentrations adjuvant were examined under electron microscope (EM) and the drugs were transfected into HepG2 (ASGr+) cells. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) was performed with total RNA extracted from the transfected cells to determine the hIL12 mRNA transcript level. The hIL12 protein in the cultured supernatant was measured with enzyme-linked immunosorbent assay (ELISA) 48 hours after transfection. RESULTS: Targeting gene drugs, whose structures were granular and circle-like and diameters ranged from 25 nm to 150 nm, had the highest hIL-12 expression level. The hIL-12 expression level in the group co-transfected with ASOR-PLL-P(+)/P(40) and ASOR-PLL-P(-)/P(35) was higher than that of ASOR-PLL-P(+)/IL-12 transfected group. CONCLUSION: The molecular conformations of targeting gene drugs play an important role in exogenous gene expression level, the best structures are granular and circle-like and their diameters range from 25 nm to 150 nm. The sizes and linking styles of exogenous genes also have some effects on their expression level.