Occurrence and prevalence of per- and polyfluoroalkyl substances in the sediment pore water of mariculture sites: Novel findings of PFASs from the Bohai and Yellow Seas using a newly established analytical method.

A new method for the determination of 26 legacy and emerging per- and polyfluoroalkyl substances (PFASs) in marine sediment pore water was developed using online solid phase extraction coupled with liquid chromatography-tandem mass spectrometry. The proposed method requires only about 1 mL of pore water samples. Satisfactory recoveries of most target PFASs (83.55-125.30 %) were achieved, with good precision (RSD of 1.09-16.53 %), linearity (R2 ≥ 0.990), and sensitivity (MDLs: 0.05 ng/L-5.00 ng/L for most PFASs). Subsequently, the method was applied to determine PFASs in the sediment pore water of five mariculture bays in the Bohai and Yellow Seas of China for the first time. Fifteen PFASs were detected with total concentrations ranging from 150.23 ng/L to 1838.48 ng/L (mean = 636.80 ng/L). The ∑PFASs and PFOA concentrations in sediment pore water were remarkably higher than those in surface seawater (tens of ng/L), indicating that the potential toxic effect of PFASs on benthic organisms may be underestimated. PFPeA was mainly distributed in pore water, and the partition of PFHpA (50.99 %) and PFOA (49.01 %) was almost equal in the solid and liquid phases. The proportions of all other PFASs partitioned in marine sediments were significantly higher than those in pore water.

Occurrence and distribution of phycotoxins in the Antarctic Ocean.

Lipophilic phycotoxins (LPTs) and domoic acid (DA) in Antarctic seawater, as well as parts of the South Pacific and the Southern Indian Oceans were systematically investigated. DA and six LPTs, namely pectenotoxin-2 (PTX2), okadaic acid (OA), yessotoxin (YTX), homo-yessotoxin (h-YTX), 13-desmethyl spirolide C (SPX1), and gymnodimine (GYM), were detected. PTX2, as the dominant LPTs, was widely distributed in seawater surrounding Antarctica, whereas OA, YTX, and h-YTX were irregularly distributed across the region. The total concentration of LPTs in surface seawater ranged from 0.10 to 13.57 ng/L (mean = 2.20 ng/L). ∑LPT levels were relatively higher in the eastern sea areas of Antarctica than in the western sea areas. PTX2 was the main LPT in the vertical profiles, and the PTX2 concentration was significantly higher in the epipelagic zone than water depths below 200 m. The predominant sources of PTX2 and OA in Antarctic sea areas are likely to be Dinophysis.

Occurrence and phase distribution of lipophilic marine algal toxins in the bottom boundary layer and sediment-porewater system of two mariculture sites.

To date, understanding the fate of lipophilic marine algal toxins (LMATs) in benthic environments on which cultivated shellfish depend is still limited. In this work, the occurrence, concentration levels, and phase distributions of LMATs in the benthic environments of two mariculture sites (Sishili and Rongcheng Bays) in China were investigated for the first time. Five LMATs: okadaic acid (OA), pectenotoxin-2 (PTX2), gymnodimine, 13-desmethyl spirolide C, and azaspiracid-2 (AZA2) and three derivatives: dinophysistoxin-1 isomer (DTX1-iso), pectenotoxin-2 seco acid, and 7-epi- pectenotoxin-2 seco acid were detected in different environmental samples. OA and PTX2 were the dominant LMATs in the bottom boundary layer (BBL) and sediment, whereas AZA2 was present in the sediment only. Notably, DTX1-iso was found for the first time to be widely distributed in the benthic environments of the bays. In BBL, the average proportion of LMATs in the dissolved phase (99.20%) was much higher than in the particulate phase (0.80%). Partition of LMATs was more balanced between sediment porewater (57.80% average proportion) and sediment (42.20%). The concentrations of ∑LMATs in the BBL seawater ranged from 19.09 ng/L to 41.57 ng/L (mean of 32.67 ng/L), and the spatial distribution trend was higher in offshore than nearshore. ∑LMATs concentrations in the sediment and porewater of the two bays ranged from 17.04 ng/kg to 150.13 ng/kg (mean of 53.58 ng/kg) and from 8.29 ng/L to 120.58 ng/L (mean of 46.63 ng/L), respectively. Their spatial distributions differed from those in BBL, showing a trend of high concentrations in areas with heavy land-based inputs. ∑LMATs concentrations in porewater were significantly higher than those in BBL seawaters, suggesting that the potential hazards of LMATs to benthic organisms may be underestimated.

Cholesterol alone or in combination is associated with frailty among community-dwelling older adults: A cross-sectional study.

BACKGROUND: Biological markers contribute to the precise intervention across the continuum of frailty severity. Few studies have explored the advantages of biological markers collected as part of primary care data among community-dwelling older adult population and controversy remains regarding the classic biological markers for frailty. METHODS: We recruited a total of 8791 adults with a mean age of 71.95 years who met the inclusion and exclusion criteria in Guancheng District and Dalang Town, Dongguan, China. Frailty was assessed by a Chinese frailty evaluation scale. Frailty status was classified with 33-item modified frailty index and latent class analysis was applied to explore the latent classes (subtypes) of frailty. We measured biological markers on blood samples collected. We identify association between specific biological markers or patterns and frailty by logistic regression and association rule mining (ARM) based on the Apriori algorithm. RESULTS: Multivariable analysis of our data showed that an elevated white blood cell (WBC) count and high cholesterol (CHOL) level were associated with pre-frailty (adjusted odds ratio [aOR] = 1.231, 95 % confidence interval [CI] = 1.009-1.501; aOR = 0.703, 95 % CI = 0.623-0.793) and frailty (aOR = 1.500, 95 % CI = 1.130-1.993; aOR = 0.561, 95 % CI = 0.461-0.684) compared with the normal groups. Importantly, significantly high level of CHOL was associated with a lower risk of four frailty subtypes compared with relatively healthy participants with the most power of association in the multi-frail group (aOR = 0.182, 95 % CI = 0.086-0.386). Based on ARM technique to develop correlation analysis to identify important high-risk clusters among older adult transitions from non-frail to frailty, patterns for normal level of CHOL co-occurred with an elevated creatinine (CREA) level have a significant association with the risk of frailty (aOR = 7.787, 95 % CI = 1.978-30.648) after adjusting for targeted confounders. CONCLUSIONS: Our study highlights the correlation between classic biological markers, especially CHOL and frailty status and subtypes among community-dwelling older adult, in the primary care setting. Further large-scale prospective studies are still needed to confirm the role of classic biological markers in frailty.

Regulation of Cat8 in energy metabolic balance and glucose tolerance in Saccharomyces cerevisiae.

Cat8 is a C6 zinc cluster transcription activator in yeast. It is generally recognized that the transcription of CAT8 is inhibited and that Cat8 is inactive in the presence of high concentrations of glucose. However, our recent study found that constitutively overexpressed Cat8 played a regulatory role in Saccharomyces cerevisiae in the presence of 20 g/L glucose. To explore the regulatory network of Cat8 at high glucose concentrations, CAT8 was both overexpressed and deleted in this study. Cell growth and glucose consumption in different media were significantly accelerated by the deletion of CAT8, while the lag period was greatly shortened. RNA-seq and genetic modification showed that the deletion of CAT8 changed the type of energy metabolism in yeast cells. Many genes related to the mitochondrial respiratory chain were downregulated, resulting in a reduction in aerobic respiration and the tricarboxylic acid cycle. Meanwhile, both the energy supply of anaerobic ethanol fermentation and the Crabtree effect of S. cerevisiae were enhanced by the deletion of CAT8. CAT8 knockout cells show a higher sugar uptake rate, a higher cell growth rate, and higher tolerance to glucose than the wild-type strain YS58. This study expands the understanding of the regulatory network of Cat8 and provides guidance for modulating yeast cell growth. KEY POINTS: • The deletion of CAT8 promoted cell growth of S. cerevisiae. • Transcriptome analysis revealed the regulation network of Cat8 under 1% glucose condition. • CAT8 deletion increases the glucose tolerance of cells by enhancing the Crabtree effect.

Enhancement of ß-Caryophyllene Biosynthesis in Saccharomyces cerevisiae via Synergistic Evolution of ß-Caryophyllene Synthase and Engineering the Chassis.

ß-Caryophyllene is a plant-derived bicyclic sesquiterpene with multiple biological functions. ß-Caryophyllene production by engineered Saccharomyces cerevisiae represents a promising technological route. However, the low catalytic activity of ß-caryophyllene synthase (CPS) is one of the main restrictive factors for ß-caryophyllene production. Here, directed evolution of the Artemisia annua CPS was performed, and variants of CPS enhancing the ß-caryophyllene biosynthesis in S. cerevisiae were obtained, in which an E353D mutant enzyme presented large improvements in Vmax and Kcat. The Kcat/Km of the E353D mutant was 35.5% higher than that of wild-type CPS. Moreover, the E353D variant exhibited higher catalytic activity in much wider pH and temperature ranges. Thus, both the higher catalytic activity and the robustness of the E353D variant contribute to the 73.3% increase in ß-caryophyllene production. Furthermore, the S. cerevisiae chassis was engineered by overexpressing genes related to ß-alanine metabolism and MVA pathway to enhance the synthesis of the precursor, and ATP-binding cassette transporter gene variant STE6T1025N to improve the transmembrane transport of ß-caryophyllene. The combined engineering of CPS and chassis resulted in 70.45 mg/L of ß-caryophyllene after 48 h of cultivation in a test tube, which was 2.93-fold of that of the original strain. Finally, a ß-caryophyllene yield of 594.05 mg/L was obtained by fed-batch fermentation, indicating the potential of ß-caryophyllene production by yeast.

Prevalence of the neurotoxin domoic acid in the aquatic environments of the Bohai and Northern Yellow seas in China.

Domoic acid (DA), a natural marine phytotoxin produced by toxigenic algae, is harmful to fishery organisms and the health of seafood consumers. In this study, we performed a whole-sea area investigation of DA in seawater, suspended particulate matter (SPM), and phytoplankton of the Bohai and Northern Yellow seas to clarify the occurrence, phase partitioning, spatial distribution, potential sources, and environmental influencing factors of DA in the aquatic environment. DA in different environmental media was identified using liquid chromatography-high resolution mass spectrometry and liquid chromatography-tandem mass spectrometry. DA was found to be predominantly in a dissolved phase (99.84 %) in seawater with only 0.16 % in SPM. Dissolved DA (dDA) was widely detected in nearshore and offshore areas of the Bohai Sea, Northern Yellow Sea, and Laizhou Bay with concentrations ranging from < limits of detection (LOD) to 25.21 ng/L (mean: 7.74 ng/L), < LOD to 34.90 ng/L (mean: 16.91 ng/L), and 1.74 ng/L to 38.20 ng/L (mean: 21.28 ng/L), respectively. dDA levels were relatively lower in the northern part than in the southern part of the study area. In particular, the dDA levels in the nearshore areas of Laizhou Bay were significantly higher than in other sea areas. This may be due to seawater temperature and nutrient levels exerting a crucial impact on the distribution of DA-producing marine algae in Laizhou Bay during early spring. Pseudo-nitzschia pungens may be the main source of DA in the study areas. Overall, DA was prevalent in the Bohai and Northern Yellow seas, especially in the nearshore aquaculture zone. Routine monitoring of DA in the mariculture zones of the northern seas and bays of China should be performed to warn shellfish farmers and prevent contamination.

Detection, occurrence, influencing factors and environmental risks of paralytic shellfish toxins in seawater in a typical mariculture bay.

Paralytic shellfish toxins (PSTs) producing algae are widely distributed in the global coastal aquatic environment, posing a threat to coastal ecosystem health and mariculture safety. However, the levels and potential environmental risks of PSTs frequently detected in shellfish remain largely unexplored in seawater of mariculture zones. In this study, a new method for trace detection of 13 common PSTs (<1.0 ng/L) in seawater was established based on off-line solid phase extraction (SPE) and on-line SPE-liquid chromatography-tandem mass spectrometry (on-line SPE-LC-MS/MS), and a systematic investigation of PSTs in seawater of the Laizhou Bay, a typical aquaculture bay in China, was conducted to understand their pollution status, environmental impact factors and ecological risks for the first time. Eleven PSTs were detected in the seawater of Laizhou Bay with total concentrations ranging from 0.75 to 349.47 ng/L (mean, 176.27 ng/L), which indicates the rich diversity of PSTs in the mariculture bay and demonstrates the reliability of the proposed analytical method. C1, C2, GTX2, GTX3, dcGTX2, and dcGTX3 were found to be the predominant PSTs, which refreshed the knowledge of PST contamination in the coastal aquatic environment. PST levels in seawater exhibited the highest levels in the southeastern mouth of Laizhou Bay and decreased toward the inner bay. Correlation analyses showed that climatic factors, nutrient status and hydrological conditions had significant effects on the distribution of PST in mariculture bay. Preliminary environmental risk assessments revealed that aquatic organisms throughout the waters of Laizhou Bay are at risk of chronic PST toxicity. These findings imply that the risk of PST in seawater of mariculture bay has previously been grossly underestimated, and that the coastal aquatic environment in North China and even the world may be at more serious risk of PST pollution, which should be taken seriously.

Trace determination of multiple hydrophilic cyanotoxins in freshwater by off- and on-line solid phase extraction coupled to liquid chromatography-tandem mass spectrometry.

Hydrophilic cyanotoxins (HCTs), such as paralytic shellfish toxins (PSTs), anatoxin-a (ATX-a), and cylindrospermopsin (CYN) are highly toxic and toxin-producing algae are widely distributed worldwide. However, HCTs, especially PSTs, are rarely reported in freshwater due to analytical limitations. This may result in an underestimation of the ecological risks and health risks. This study developed a new method to detect ATX-a, CYN, and thirteen common PSTs in freshwater simultaneously by using off-line solid phase extraction (SPE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The limits of detection (LODs) of all targets were lower than 0.05 µg/L, which could meet the regulatory requirements for monitoring of HCTs in drinking water in different countries and regions. To improve the detection sensitivities for trace PSTs, a method based on off-line SPE and on-line SPE-LC-MS/MS was established with LOD around 0.001 µg/L. GTX1&4, GTX2&3, and GTX5 were detected in freshwater in China for the first time, highlighting that overall communities are facing potential risks of exposure to various PSTs in China. High concentrations of ATX-a and CYN were also detected in freshwater from Northern China. The proposed method helps to understand the pollution status of HCT in water bodies, especially during the non-algal bloom period.

Cat8 Response to Nutritional Changes and Interaction With Ehrlich Pathway Related Factors.

Cat8 is an important transcription factor regulating the utilization of non-fermentative carbon sources in Saccharomyces cerevisiae. However, our previous studies found that Cat8 may play a critical role in nitrogen metabolism, but the regulatory mechanism has not been elucidated. In this study, the nuclear localization and analysis of regulatory activity showed that the Cat8 function relies on Snf1 kinase. In the fermentation with glucose or glycerol as carbon sources under phenylalanine (Phe) induction, by comparing the changes of cellular gene expression and Cat8 target gene binding profiles after Cat8 overexpression, enhanced transcription was shown among key genes involved in the Ehrlich pathway (e.g., ARO9, ARO10, and ADH2) and its upstream and downstream related factors (e.g., GAP1, AGP1, GAT1, PDR12, and ESPB6), indicating that Cat8 participated in the regulation of nitrogen metabolism. Moreover, highly active Cat8 interacts with transcriptional activator Aro80 and GATA activator Gat1 coordinately to regulate the transcription of ARO10. Altogether, our results showed that Cat8 may act as a global transcription factor in response to nutritional changes, regulating both carbon and nitrogen utilization. This provides a new insight for us to explore the regulation of cell nutrient metabolism networks in yeast.

Enhancement of Intracellular Accumulation of Copper by Biogenesis of Lipid Droplets in Saccharomyces cerevisiae Revealed by Transcriptomic Analysis.

Copper is an essential micronutrient for life, whose homeostasis is rigorously regulated to meet the demands of normal biological processes and to minimize the potential toxicity. Copper enriched by yeast is regarded as a safe and bioavailable form of copper supplements. Here, a Saccharomyces cerevisiae mutant strain H247 with expanded storage capability of copper was obtained through atmospheric and room-temperature plasma treatment. Transcriptomic analyses found that transcriptional upregulation of DGA1 might be the major contributor to the enhancement of intracellular copper accumulation in strain H247. The positive correlation between biogenesis of lipid droplets and intracellular accumulation of copper was confirmed by overexpression of the diacylglycerol acyltransferase encoding genes DGA1 and LRO1 or knockout of DGA1. Lipid droplets are not only the storage pool of copper but might prompt the copper trafficking to mitochondria, vacuoles, and Golgi apparatus. These results provide new insights into the sophisticated copper homeostatic mechanisms and the biological functions of lipid droplets.

Spatial distribution, vertical profiles and transport of legacy and emerging per- and polyfluoroalkyl substances in the Indian Ocean.

The contamination status and transport of per- and polyfluoroalkyl substances (PFASs) in the seawater of the Indian Ocean (IO) and an adjacent subregion of the Northwest Pacific Ocean (NWPO) were investigated. Eight legacy PFASs were widely distributed in the surface seawater, and perfluoroheptanoic acid (PFHpA) and perfluorooctanoic acid (PFOA) were the two predominant PFASs. ΣPFAS concentration decreased in the following order: NWPO>Joining area of Asia and Indian-Pacific Oceans (JAIPO)>Northeast Indian Ocean>Southwest Indian Ocean. Hexafluoropropylene oxide-dimer acid, a replacement surfactant for PFOA was extensively detected in the IO (~34.8 pg/L) for the first time, showing an early sign of emerging PFAS spread in global open oceans. Eight depth profiles across the JAIPO (down to 5433 m depth) revealed a "surface-enrichment" and "depth-depletion" pattern for PFASs in the water column, and two noticeable fluctuations were mainly located at depths of 150-200 and 200-500 m. Physical processes, including eddy diffusion, and the origin and trajectory of water mass were crucial factors for structuring PFAS vertical profiles. Mass transport estimates revealed a remarkable PFOA contribution through the JAIPO to IO carried by the Indonesian Throughflow, and a nonnegligible PFHpA contribution from Antarctic Immediate Water to deep water of the JAIPO driven by thermohaline circulation.

Enhancing fluxes through the mevalonate pathway in Saccharomyces cerevisiae by engineering the HMGR and ß-alanine metabolism.

Mevalonate (MVA) pathway is the core for terpene and sterol biosynthesis, whose metabolic flux influences the synthesis efficiency of such compounds. Saccharomyces cerevisiae is an attractive chassis for the native active MVA pathway. Here, the truncated form of Enterococcus faecalis MvaE with only 3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) activity was found to be the most effective enzyme for MVA pathway flux using squalene as the metabolic marker, resulting in 431-fold and 9-fold increases of squalene content in haploid and industrial yeast strains respectively. Furthermore, a positive correlation between MVA metabolic flux and ß-alanine metabolic activity was found based on a metabolomic analysis. An industrial strain SQ3-4 with high MVA metabolic flux was constructed by combined engineering HMGR activity, NADPH regeneration, cytosolic acetyl-CoA supply and ß-alanine metabolism. The strain was further evaluated as the chassis for terpenoids production. Strain SQ3-4-CPS generated from expressing ß-caryophyllene synthase in SQ3-4 produced 11.86 ± 0.09 mg l-1 ß-caryophyllene, while strain SQ3-5 resulted from down-regulation of ERG1 in SQ3-4 produced 408.88 ± 0.09 mg l-1 squalene in shake flask cultivations. Strain SQ3-5 produced 4.94 g l-1 squalene in fed-batch fermentation in cane molasses medium, indicating the promising potential for cost-effective production of squalene.

Rising CO2 will increase toxicity of marine dinoflagellate Alexandrium minutum.

Ocean acidification caused by increasing emission of carbon dioxide (CO2) is expected to have profound impacts on marine ecological processes, including the formation and evolution of harmful algal blooms (HABs). We designed a set of experiments in the laboratory to examine the effects of increasing CO2 on the growth and toxicity of a toxic dinoflagellate Alexandrium minutum producing paralytic shellfish toxins (PSTs). It was found that high levels of CO2 (800 and 1200 ppm) significantly promoted the growth of A. minutum compared to the group (400 ppm) representing the current CO2 level. The total yields of PSTs by A. minutum, including both intracellular and extracellular toxins, were significantly enhanced, probably due to the induction of core enzyme activity and key amino acids synthesis for PST production. More interestingly, high level of CO2 promoted the transformation from gonyautoxin2&3 to gonyautoxin1&4 and depressed the release of PSTs from inside to outside of the cells. All these processes collectively led to an apparent increase of A. minutum toxicity. Our study demonstrated that rising CO2 would increase the risk of toxic A. minutum based on the comprehensive analyses of different processes including algal growth and toxin synthesis, transformation and release.

Whole Genome Sequencing and RNA-seq-Driven Discovery of New Targets That Affect Carotenoid Synthesis in Phaffia rhodozyma.

Carotenoids are unsaturated compounds with terpene groups. Among them, astaxanthin has strong antioxidant properties. It is widely used in aquaculture, food, medicine, and cosmetics with a broad market prospect. Phaffia rhodozyma is an important microorganism that synthesizes astaxanthin, but its wild strains have low pigment content, long growth cycle, and low fermentation temperature. Therefore, it is important to research the genetic improvement of the physiological and biochemical properties of P. rhodozyma. In this study, the atmospheric and room temperature plasma mutagenesis technology was adopted, through the functional evolution of the carotenoid production performance; then, through the comparative analysis of the genomics and transcriptomics of the wild strain and evolved strain, the key factor GST1 gene that affects carotenoid synthesis was discovered.

Enhancement of Copper Uptake of Yeast Through Systematic Optimization of Medium and the Cultivation Process of Saccharomyces cerevisiae.

Copper is an essential trace element for living organisms. Copper enriched by yeast of Saccharomyces cerevisiae is regarded as the biologically available organic copper supplement with great potentiality for application. However, the lower uptake ratio of copper ions makes the production of copper enriched by yeast uneconomically and environmentally unfriendly. In this study, S. cerevisiae Cu-5 with higher copper tolerance and intracellular copper accumulation was obtained by screening of our yeast strains collection. To increase the uptake ratio of copper ions, the medium composition and cultivation conditions for strain Cu-5 were optimized systematically. A medium comprised of glucose, yeast extract, (NH4)2SO4, and inorganic salts was determined, then a novel cultivation strategy including pH control at 5.5 and increasing amounts of yeast extract for a higher concentration of copper ion in the medium was developed. The uptake ratios of copper ions were more than 90% after combining 50 to 100 mg/L copper ions with 3.5 to 5.0 g/L yeast extract, which is the highest until now and is conducive to the cost-effective and environmentally friendly production of bioactive copper in yeast-enriched form.

A Novel Metabolite as a Hapten to Prepare Monoclonal Antibodies for Rapid Screening of Quinoxaline Drug Residues.

Quinoxalines (Qx) are chemically synthesized antibacterial drugs with strong antibacterial and growth-promoting effects. Qx is heavily abused by farmers, resulting in large residues in animal-derived foods, which pose a serious threat to human health. Desoxyquinoxalines (DQx), which have the highest residue levels, have been identified as the major toxicant and have become a new generation of residue markers. In this study, we prepared monoclonal antibodies (mAb) based on a new generation metabolite (desoxymequindox, DMEQ) and establish an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) for the rapid determination of Qx residues in food. The mAb exhibited high sensitivity with half maximal inhibitory concentration (IC50) and a linear range of 2.84 µg/L and 0.8-12.8 µg/L, respectively. Additionally, the cross-reactivity (CR) of the mAb showed that it recognized multiple DQx to varying levels. The limits of detection (LOD), limits of quantification (LOQ), and recoveries for the ic-ELISA assay of pork, swine liver, swine kidney, chicken, and chicken liver were 0.48-0.58 µg/kg, 0.61-0.90 µg/kg, and 73.7-107.8%, respectively, and the coefficients of variation (CV) were less than 11%. The results of the ic-ELISA showed a good correlation with LC-MS/MS in animal-derived foods. This suggests that this analytical method can be used for the rapid screening of QX residues.

Engineering of cis-Element in Saccharomyces cerevisiae for Efficient Accumulation of Value-Added Compound Squalene via Downregulation of the Downstream Metabolic Flux.

Transcriptional downregulation is widely used for metabolic flux control. Here, marO, a cis-element of Escherichia coli mar operator, was explored to engineer promoters of Saccharomyces cerevisiae for downregulation. First, the ADH1 promoter (PADH1) and its enhanced variant PUADH1 were engineered by insertion of marO into different sites, which resulted in decrease in both gfp5 transcription and GFP fluorescence intensity to various degrees. Then, marO was applied to engineer the native ERG1 and ERG11 promoters due to their importance for accumulation of value-added intermediates squalene and lanosterol. Elevated squalene content (4.9-fold) or lanosterol content (4.8-fold) and 91 or 28% decrease in ergosterol content resulted from the marO-engineered promoter PERG1(M5) or PERG11(M3), respectively, indicating the validity of the marO-engineered promoters in metabolic flux control. Furthermore, squalene production of 3.53 g/L from cane molasses, a cheap and bulk substrate, suggested the cost-effective and promising potential for squalene production.

Pollution status, influencing factors and environmental risks of neonicotinoids, fipronil and its metabolites in a typical semi-closed bay in China.

The occurrence, spatiotemporal variations, influence factors and environmental risks of eight common neonicotinoids (NEOs), fipronil, and three fipronil metabolites (fipronil and its metabolites are collectively referred to as FIPs) in different seasons from the estuary to the inner area of Jiaozhou Bay, China were comprehensively investigated. First- and second-generation NEOs were found to be the predominant pesticides in this area. The average contents of ∑NEOs and ∑FIPs from the estuary to the inner bay decreased from 12.99 ng/L to 0.82 ng/L and from 1.10 ng/L to 0.17 ng/L, respectively. NEO and FIP concentrations were higher in summer and autumn. High ∑NEO content is distributed in main inflow rivers, such as Dagu River and Licun River, which are influenced by pesticide application. NEO concentrations in all rivers were high upstream and low downstream because of the influence of heavy rainfall and seawater dilution in summer. NEO concentrations were high along the coast and low at the mouth and center of Jiaozhou Bay in summer and autumn and evenly distributed in winter and spring. Temperature has a great influence on most NEOs and FIPs owing to its effect on their degradation. Nitrogen-containing nutrients have an important influence on the distribution of fipronil and acetamiprid, which may be due to the activity of nitrogen-containing functional groups in their structure. Only Licun River, Dagu River and Haibo river sewage treatment plant in summer posed a certain risk of chronic toxicity for NEOs using the new threshold established by the species sensitive distribution (SSD) method for Chinese native aquatic lives. These findings should arouse people's attention.

[Determination of domoic acid in seawater by solid phase extraction-liquid chromatography-tandem mass spectrometry].

Domoic acid (DA) can poison or even be fatal to marine mammals, and poses a potential risk to human health via transmission through the food chain. The level of DA in seawater will affect the safety of seafood. Therefore, a powerful method for the detection of DA in seawater, especially in the coastal mariculture zone, is needed. In order to identify different concentration levels of DA in real seawater, in this study, a method was established for the determination of trace DA in seawater by SPE-LC-MS/MS. First, the LC-MS/MS instrument and sample pretreatment conditions were optimized. Subsequently, DA was separated on a 5 TC-C18 (2) analytical column (150 mm×4.6 mm, 5 µm), and multiple reaction monitoring (MRM) was conducted in the positive electrospray ionization mode. For off-line SPE, the HLB cartridge could enrich DA in seawater. The best enrichment of DA was obtained after adding 0.32 mL formic acid to an 80.0 mL seawater sample. Four on-line SPE columns from Agilent, namely, 5 TC-C18(2) (12.5 mm×4.6 mm, 5 µm), Zorbax Eclipse Plus-C18 (12.5 mm×2.1 mm, 5 µm), Zorbax Eclipse XDB-C8 (12.5 mm×2.1 mm, 5 µm), and PLRP-S (12.5 mm×2.1 mm, 15-20 µm), were tested to determine their suitability to trap DA from seawater samples. The 5 TC-C18 (2) column offered the best retention ability and good peak shape of DA, and was selected as the on-line SPE column. Validation was then performed to assess the sensitivity, linearity, matrix effects (MEs), recoveries, and precisions of the proposed method. After simple treatment of the seawater samples by filtration and acidification, 0.6 mL of the seawater sample was injected directly for on-line SPE-LC-MS/MS. The linearity was good, and ranged from 10.0 to 500.0 ng/L (correlation coefficient R2=0.9992). The limit of detection (LOD) and limit of quantification (LOQ) of DA were 4.0 and 10.0 ng/L, respectively, with good recovery (≥81.0%) and precision (RSDs≤4.2%) at three spiked levels in the blank seawater samples. After the DA in the 80.0 mL seawater sample was enriched by off-line SPE, a 0.6 mL sample was injected for on-line SPE-LC-MS/MS. The DA in the spiked blank seawater sample showed a good linear relationship in the range of 0.3-50.0 ng/L (R2=0.9990). The LOD and LOQ were 0.1 and 0.3 ng/L, respectively. The recoveries of DA at low, medium, and high spiked levels in the blank seawater samples were all ≥69.2%, and the RSDs were ≤4.4%. The MEs of DA with both methods were 18.3% and 13.7%, respectively, indicating that the ME was mild enough to be negligible. In summary, the proposed method is simple, sensitive, robust, and powerful for the detection of DA in inshore and offshore seawater.