Catalytic site flexibility facilitates the substrate and catalytic promiscuity of Vibrio dual lipase/transferase.

Although enzyme catalysis is typified by high specificity, enzymes can catalyze various substrates (substrate promiscuity) and/or different reaction types (catalytic promiscuity) using a single active site. This interesting phenomenon is widely distributed in enzyme catalysis, with both fundamental and applied importance. To date, the mechanistic understanding of enzyme promiscuity is very limited. Herein, we report the structural mechanism underlying the substrate and catalytic promiscuity of Vibrio dual lipase/transferase (VDLT). Crystal structures of the VDLT from Vibrio alginolyticus (ValDLT) and its fatty acid complexes were solved, revealing prominent structural flexibility. In particular, the "Ser-His-Asp" catalytic triad machinery of ValDLT contains an intrinsically flexible oxyanion hole. Analysis of ligand-bound structures and mutagenesis showed that the flexible oxyanion hole and other binding residues can undergo distinct conformational changes to facilitate substrate and catalytic promiscuity. Our study reveals a previously unknown flexible form of the famous catalytic triad machinery and proposes a "catalytic site tuning" mechanism to expand the mechanistic paradigm of enzyme promiscuity.

The mud-dwelling clam Meretrix petechialis secretes endogenously synthesized erythromycin.

Although lacking an adaptive immune system and often living in habitats with dense and diverse bacterial populations, marine invertebrates thrive in the presence of potentially challenging microbial pathogens. However, the mechanisms underlying this resistance remain largely unexplored and promise to reveal novel strategies of microbial resistance. Here, we provide evidence that a mud-dwelling clam, Meretrix petechialis, synthesizes, stores, and secretes the antibiotic erythromycin. Liquid chromatography coupled with mass spectrometry, immunocytochemistry, fluorescence in situ hybridization, RNA interference, and enzyme-linked immunosorbent assay revealed that this potent macrolide antimicrobial, thought to be synthesized only by microorganisms, is produced by specific mucus-rich cells beneath the clam's mantle epithelium, which interfaces directly with the bacteria-rich environment. The antibacterial activity was confirmed by bacteriostatic assay. Genetic, ontogenetic, phylogenetic and genomic evidence, including genotypic segregation ratios in a family of full siblings, gene expression in clam larvae, phylogenetic tree, and synteny conservation in the related genome region further revealed that the genes responsible for erythromycin production are of animal origin. The detection of this antibiotic in another clam species showed that the production of this macrolide is not exclusive to M. petechialis and may be a common strategy among marine invertebrates. The finding of erythromycin production by a marine invertebrate offers a striking example of convergent evolution in secondary metabolite synthesis between the animal and bacterial domains. These findings open the possibility of engineering-animal tissues for the localized production of an antibacterial secondary metabolite.

Pharmaceutically active compounds (PhACs) in surface sediments of the Jiaozhou Bay, north China.

Pharmaceutically active compounds (PhACs) have attracted increasing attention due to their large consumption volumes, high bioactivity and potential ecotoxicity. In this study, a total of 150 commonly used drugs were investigated in sediments of Jiaozhou Bay (JZB). Twenty-five target compounds were detected, of which ten were discovered for the first time in marine sediments. The range of total PhAC content was 3.62-21.4 ng/g dry weight. Ketoprofen (2.49 ng/g), oxytetracycline (1.00 ng/g) and roxithromycin (0.97 ng/g) were the preponderant PhACs. PhACs gradually decreased from east to west, and the distribution of PhACs in the sediment was controlled by the source channel, seawater dynamic process and sediment composition. The diatom, organic matter, and clay proportions in the sediments and the nutrients in the overlying water were the most important environmental factors affecting the distribution of PhACs. PhAC pollution in the sediments of the JZB exhibited an increasing trend. Coprostanol could be used as a chemical indicator of the PhAC concentration in JZB sediments. PhACs were mainly derived from direct pollution due to human fecal excretion in the eastern region. Ofloxacin, tetracycline and oxytetracycline were found to pose high or medium risks to aquatic organisms. It is necessary and urgent to improve the treatment technology of drug residues in sewage treatment plants to decrease the pollution of PhAC residues. With the continuous aging of the global population, the use of PhACs will increase rapidly, which may cause more unpredictable threats to the marine ecosystem. Therefore, the monitoring of PhACs in the marine environment needs to be strengthened, and studies on PhAC occurrence and effects must be considered a priority in global environmental research.

Biogeochemical characteristics and ecological risk assessment of pharmaceutically active compounds (PhACs) in the surface seawaters of Jiaozhou Bay, North China.

The occurrence and distribution of 168 pharmaceutically active compounds (PhACs) in the surface seawater of Jiaozhou Bay (JZB) were investigated using ultra-high-performance liquid chromatography in tandem with a triple-quadrupole mass spectrometer equipped with an electrospray ionization source (UHPLC-ESI-MS-MS). Thirty-six compounds were detected, and 17 of these compounds were first detected in seawater, including sulfabenzamide, sulphacetamide, cephalonium, desacetyl-cefotaxime, cefminox, cefotaxime, cephradine, cefazolin, carprofen, nabumetone, glibenclamide, glimepiride, glipizide, prednisone, fluoromethalone, diazepam and amantadine. The total concentration of PhACs in the surface seawater ranged from 23.6 ng/L to 217 ng/L. The compounds found at the highest mean concentrations included amantadine (24.7 ng/L), lincomycin (8.55 ng/L), carprofen (8.30 ng/L), and tetracycline (7.48 ng/L). The PhAC concentration was higher in the inner bay than in the outside of the bay. In the inner bay, the eastern district showed higher concentrations of PhACs than the western district. Input from the Licun River may be the primary source of pollution. A statistically significant positive correlation was observed between nutrients and PhACs in seawater. Phosphate can be used to indicate the distribution of PhACs in JZB. Based on the individual risk quotient (RQ) values, lincomycin and ofloxacin posed high risks to the relevant aquatic organisms in JZB, especially in the eastern parts. Regular monitoring is required to evaluate the levels of PhACs as they are constantly released into JZB.

Sorafenib kills liver cancer cells by disrupting SCD1-mediated synthesis of monounsaturated fatty acids via the ATP-AMPK-mTOR-SREBP1 signaling pathway.

Sorafenib is a multikinase inhibitor that is effective in treating advanced liver cancer. Although its mechanism of action through several established cancer-related protein kinase targets is well-characterized, sorafenib induces variable responses among human tumors, and the cause for this variation is yet unknown. To investigate the underlying mechanisms, we applied mass spectrometry-based proteomic analysis to Huh7.5 human liver cancer cells and found that sorafenib significantly affected the expression of the key lipogenic enzymes, especially stearoyl coenzyme A desaturase 1 (SCD1), in these cells. Given that SCD1 catalyzes the most crucial and rate-limiting step in the synthesis of monounsaturated fatty acids (FAs), we performed a lipidomic analysis, which showed a dramatically altered lipid profile in sorafenib-treated cells. Detection and analysis of free FAs showed that the levels of monounsaturated FAs, including oleate, were significantly decreased in those cells treated by sorafenib. Addition of oleate protected liver cancer cells from sorafenib-induced death and alleviated the abnormalities of mitochondrial morphology and function caused by the drug. Treatment with sorafenib suppressed ATP production, resulting in AMPK activation via phosphorylation. Further secondary effects included reduction of the levels of sterol regulatory element-binding protein 1 (SREBP1) and the phosphorylation of mammalian target of rapamycin (mTOR) in liver cancer cells. These effects were partly abolished in the presence of compound C (an AMPK inhibitor) and ATP and adenosine, and SREBP1c overexpression also could be resistant to the effects of sorafenib, suggesting that the sorafenib-induced reduction in cell viability was mediated by the ATP-AMPK-mTOR-SREBP1 signaling pathway. Taken together, our results suggest that sorafenib's anticancer activity in liver cancer cells is based on the inhibition of ATP production, SCD1 expression, and monounsaturated FA synthesis. In addition, the decreased monounsaturated FA synthesis further triggered the more serious reduction of ATP production in sorafenib-treated cells. To our knowledge, this is the first evidence that sorafenib disrupts lipogenesis and triggers liver cancer cell death by targeting SCD1 through the ATP-AMPK-mTOR-SREBP1 pathway.-Liu, G., Kuang, S., Cao, R., Wang, J., Peng, Q., Sun, C. Sorafenib kills liver cancer cells by disrupting SCD1-mediated synthesis of monounsaturated fatty acids via the ATP-AMPK-mTOR- SREBP1 signaling pathway.

Concentrations and distribution of phthalate esters in the seamount area of the Tropical Western Pacific Ocean.

A total of 14 phthalate esters (PAEs) were analysed by gas chromatography-mass spectrometry (GC-MS) to better understand its occurrence and distribution in seawater samples of M2 seamount in the Tropical Western Pacific Ocean (TWPO). The concentrations of ΣPAEs in the seawater ranged from 12.13 ng L-1 to 60.69 ng L-1 (av. 28.86 ng L-1), dominated by dibutyl phthalate (DBP), di(2­ethylhexyl) phthalate (DEHP) and diisobutyl phthalate (DiBP). ΣPAEs concentrations in the southwest of the seamount were lower than those in the northeast, with the minima appearing above the seamount summit. Current-seamount interaction was reckoned to be the principal driving factors in the distribution of PAEs. DEHP posed a medium risk in seawater, suggesting that marine plastic pollution has become an urgent environmental issue that calls for more attention and actions. Microplastics leaching and atmospheric deposition might be the potential sources of PAEs.

Occurrence and origins of biomarker aliphatic hydrocarbons and their indications in surface sediments of the East China Sea.

The composition, distribution, indexes and budget of saturated lipid biomarker aliphatic hydrocarbons (AHs) in sediments of the East China Sea (ESC) were analyzed to identify their indications and sources. The resolved n-alkane (R) contents were 0.70-2.89 µg/g, with long-chain n-alkanes as the dominant composition in the ECS. The high R values mainly appeared at south inner shelf and north outer upwelling area, corresponding to the high mud, total organic carbon (TOC) and Chl a contents there. The composition, distribution pattern, combined with indexes of AHs, suggested no petroleum contamination and predominant biogenic sources in the ECS. The biogenic sources mainly were the mixed terrestrial higher plant, marine plankton and bacteria and aquatic macrophyte origins. Biotic source apportionment suggested that terrestrial higher plants were the dominant source of AHs, followed by marine planktons, with the lowest of submerged/floating macrophytes. Quantitative evaluation of R sources suggested that the Changjiang River input was the primary terrestrial contributor, accounting for 67.9% of total terrestrial input. The burial flux of R was 1.11 × 103 t/yr, with inner shelf and estuary as main accumulation areas. Although there was a huge amount of R influx from terrestrial and marine sources, only 9.8% could be preserved in sediments.

Analysis of sterols in selected bloom-forming algae in China.

Sterols, a group of stable lipid compounds, are often used as biomarkers in marine biogeochemical studies to indicate sources of organic matter. In this study, sterols in 13 species of major bloom-forming algae in China, which belong to Dinophyceae, Bacillariophyceae, Ulvophyceae, and Pelagophyceae, were analyzed with gas chromatography-mass spectrometry (GC-MS) to test their feasibility in representing different types of harmful algal blooms (HABs). It was found that (24Z)-stigmasta-5,24-dien-3ß-ol (28-isofucosterol) was a major sterol component in green-tide forming macroalga Ulva prolifera. In bloom-forming dinoflagellates Alexandrium spp., Prorocentrum micans and Scrippsiella trochoidea, (22E)-4α,23-dimethyl-5α-ergost-22-en-3ß-ol (dinosterol) was detected in addition to cholest-5-en-3ß-ol (cholesterol), (22E)-ergosta-5,22-dien-3ß-ol, (22E)-stigmasta-5,22-dien-3ß-ol and other minor sterol components. In brown-tide forming pelagophyte Aureococcus anophagefferens, (24E)-24-propylcholesta-5,24-dien-3ß-ol ((24E)-24-propylidenecholesterol) and (24Z)-24-propylcholesta-5,24-dien-3ß-ol ((24Z)-24-propylidenecholesterol) were detected together with cholesterol, (22E)-stigmasta-5,22-dien-3ß-ol, stigmast-5-en-3ß-ol and campest-5-en-3ß-ol. Among the selected bloom-forming diatoms, Chaetoceros sp. and Pseudo-nitzschia spp. only produced cholesterol, while Cylindrotheca closterium produced solely (22E)-ergosta-5,22-dien-3ß-ol. Sterol content in four bloom-forming algal species correlates well with their biomass or abundance. It's proposed that 28-isofucosterol could serve as a promising biomarker for green algae in green-tide studies. Dinosterol and (24Z)-24-propylidenecholesterol can be used as potential biomarkers to represent bloom-forming dinoflagellates and pelagophytes, while (22E)-ergosta-5,22-dien-3ß-ol is not a good indicator for diatoms.

Proximate composition, amino acid and fatty acid profiles of marine snail Rapana venosa meat, visceral mass and operculum.

BACKGROUND: Rapana venosa (Rv), an important marine snail, demonstrates an increasing nutritional and economic importance. However, there is still limited information available on their nutritional composition. The present study highlights and provides new information on the proximate composition, amino acid and fatty acid profiles of different body parts of Rv, aiming for its better application and research. RESULTS: The operculum contained a high amount of protein and flavor amino acids. The edible tissues, including meat and visceral mass, were valuable sources of essential amino acids (EAA) apart from methionine and cysteine. In addition, the meat contained high amount of taurine. Fatty acid analysis indicated that the edible tissues contained high amounts of ω3 fatty acids, especially eicosapentaenoic acid (EPA) (C20:5ω3) and docosahexaenoic acid (DHA) (C22:6ω3), and had a low ω6/ω3 fatty acid ratio. Interestingly, significantly higher concentrations of most nutritional elements such as fat, EAA, EPA and DHA, were found in the visceral mass compared to those in the meat. CONCLUSION: The operculum of Rv may became a very interesting source for some protein and flavor peptide development, and the edible parts of Rv may be utilized for special dietary applications requiring high amounts of taurine, EPA, DHA and a lower ω6/ω3 fatty acid ratio. © 2017 Society of Chemical Industry.

[Distribution and Sources of n-alkanes in Surface Seawater of Jiaozhou Bay].

Hydrocarbons emitted by human activities probably constitute the largest class of contaminants that are present in coastal areas, because of widespread use of fossil fuels for power generation and logistics, and accidental spillages. The chemical composition of hydrocarbon mixtures found in the marine environment allowed the identification of relative contributions of different natural and anthropogenic sources. Identification of these hydrocarbons, especially n-alkanes, could act as tracers for the possible sources. To evaluate n-alkanes concentrations with emphasis on source analysis, surface water without visible oil was collected from the cruise in April 2016. Determination and quantification were performed by solvent extraction and gas chromatography-mass spectrometry. Various molecular diagnostic parameters were used to assess the contribution of different sources and origin of n-alkanes in surface seawater of Jiaozhou Bay. Concentrations of total dissolved n-alkanes(C11-C37) were between 1.756-39.09 µg·L-1 with a high carbon number predominance profile without odd-even carbon number preference. The n-alkane concentrations varied spatially depending on the distance away from main input sources and the ability of water exchange. It was at a higher concentration in the northeast and west coastal areas, as well as the mouth of Jiaozhou Bay. And concentrations were relatively lower in the inner area and outside of Jiaozhou Bay. In the sea area with strong water exchange, concentrations of total n-alkanes were around 2.196 µg·L-1, which could be considered as the environmental background level of n-alkanes in Jiaozhou Bay. Those n-alkanes were dominated with C24, with a slightly odd carbon number preference in low carbon n-alkanes and an even carbon number preference in high carbon n-alkanes. The values of CPI for the whole range of n-alkanes series, low carbon n-alkanes, and high carbon n-alkanes were 0.949, 1.026, and 0.980, respectively. Diagnostic indices and curves indicated that the dominant inputs of those n-alkanes were from marine biogenic input such as algae and marine bacteria. The concentration profiles of n-alkanes in other areas were characterized by no odd-even predominance in the range of C21-C33 with peaks center at C24 which were indicative of anthropic contributions as emission sources. N-alkanes dominating with anthropic sources comprised a high proportion(83.7%) of total n-alkanes. Activities of harbors and ships were the main contributors of Jiaozhou Bay n-alkanes. Physical processes, such as evaporation and dilution, were the principal weathering mechanisms. Because of its sufficient environmental capacity, Jiaozhou Bay was still moderately contaminated with petroleum.

Transcriptomic Analyses of Scrippsiella trochoidea Reveals Processes Regulating Encystment and Dormancy in the Life Cycle of a Dinoflagellate, with a Particular Attention to the Role of Abscisic Acid.

Due to the vital importance of resting cysts in the biology and ecology of many dinoflagellates, a transcriptomic investigation on Scrippsiella trochoidea was conducted with the aim to reveal the molecular processes and relevant functional genes regulating encystment and dormancy in dinoflagellates. We identified via RNA-seq 3,874 (out of 166,575) differentially expressed genes (DEGs) between resting cysts and vegetative cells; a pause of photosynthesis (confirmed via direct measurement of photosynthetic efficiency); an active catabolism including ß-oxidation, glycolysis, glyoxylate pathway, and TCA in resting cysts (tested via measurements of respiration rate); 12 DEGs encoding meiotic recombination proteins and members of MEI2-like family potentially involved in sexual reproduction and encystment; elevated expressions in genes encoding enzymes responding to pathogens (chitin deacetylase) and ROS stress in cysts; and 134 unigenes specifically expressed in cysts. We paid particular attention to genes pertaining to phytohormone signaling and identified 4 key genes regulating abscisic acid (ABA) biosynthesis and catabolism, with further characterization based on their full-length cDNA obtained via RACE-PCR. The qPCR results demonstrated elevated biosynthesis and repressed catabolism of ABA during the courses of encystment and cyst dormancy, which was significantly enhanced by lower temperature (4 ± 1°C) and darkness. Direct measurements of ABA using UHPLC-MS/MS and ELISA in vegetative cells and cysts both fully supported qPCR results. These results collectively suggest a vital role of ABA in regulating encystment and maintenance of dormancy, akin to its function in seed dormancy of higher plants. Our results provided a critical advancement in understanding molecular processes in resting cysts of dinoflagellates.