Selective enrichments for color microplastics loading of marine lipophilic phycotoxins.

Microplastics (MPs) and marine lipophilic phycotoxins (MLPs) are two classes of emerging contaminants. Together, they may exacerbate the negative impacts on nearshore marine ecosystems. Herein, the loading of 14 representative MLPs, closely related to toxin-producing algae, on MPs and their relations with colorful MPs have been explored for the first time based on both field and lab data. The objectives of our study are to explore the roles of multiple factors (waterborne MLPs and MP characteristics) in the loading of MLPs by MPs with the applications of various statistical means, and to further explore the role of the color of MP in the loading of specific MLPs through lab simulation experiments. Our results demonstrated that MPs color determined the loading of some specific MLPs on MPs and green MPs can load much more than other colorful fractions (p < 0.05). These interesting phenomena illustrated that the color effects on the loading processes of MLPs on MPs are a dynamic process, and it can be well explained by the shading effect of MP color, which may affect the growth and metabolism of the attached toxic-producing algae on MPs and hence the production of specific MLPs. Furthermore, loading of MLPs on MPs can be considered as the comprehensive physicochemical and biological processes. Our results caution us that special attention should be paid to explore the real-time dynamic color shading effects on all kinds of bio-secreted contaminants loading on MPs, and highlight the necessary to comprehensive investigate the interaction between biota, organic contaminants and MPs.

AcrNET: predicting anti-CRISPR with deep learning.

MOTIVATION: As an important group of proteins discovered in phages, anti-CRISPR inhibits the activity of the immune system of bacteria (i.e. CRISPR-Cas), offering promise for gene editing and phage therapy. However, the prediction and discovery of anti-CRISPR are challenging due to their high variability and fast evolution. Existing biological studies rely on known CRISPR and anti-CRISPR pairs, which may not be practical considering the huge number. Computational methods struggle with prediction performance. To address these issues, we propose a novel deep neural network for anti-CRISPR analysis (AcrNET), which achieves significant performance. RESULTS: On both the cross-fold and cross-dataset validation, our method outperforms the state-of-the-art methods. Notably, AcrNET improves the prediction performance by at least 15% regarding the F1 score for the cross-dataset test problem comparing with state-of-art Deep Learning method. Moreover, AcrNET is the first computational method to predict the detailed anti-CRISPR classes, which may help illustrate the anti-CRISPR mechanism. Taking advantage of a Transformer protein language model ESM-1b, which was pre-trained on 250 million protein sequences, AcrNET overcomes the data scarcity problem. Extensive experiments and analysis suggest that the Transformer model feature, evolutionary feature, and local structure feature complement each other, which indicates the critical properties of anti-CRISPR proteins. AlphaFold prediction, further motif analysis, and docking experiments further demonstrate that AcrNET can capture the evolutionarily conserved pattern and the interaction between anti-CRISPR and the target implicitly. AVAILABILITY AND IMPLEMENTATION: Web server: https://proj.cse.cuhk.edu.hk/aihlab/AcrNET/. Training code and pre-trained model are available at.

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.

Occurrence and distribution of lipophilic marine algal toxins in the coastal seawater of Southeast China and the South China Sea.

The composition, levels, and spatial distribution of dissolved lipophilic marine algal toxins (LMATs) including cyclic imines (CIs), yessotoxins (YTXs), okadaic acid (OA) and its derivatives, pectenotoxins (PTXs), azaspiracids (AZAs), and brevetoxins (BTXs) in the coastal waters of Southeast China (Xiamen) and the South China Sea (Hainan Island and Beibu Gulf) were investigated and compared for the first time. Dissolved AZA3 was firstly detected in the coastal seawater of China. OA and PTX2 were widely distributed in the three areas studied. Gymnodimine (GYM), 13-desmethyl spirolide C (SPX1), YTX, and homo-yessotoxins (h-YTX) were found mainly in the South China Sea. The average ∑LMAT concentrations in the coastal waters of Xiamen, Hainan Island, and Beibu Gulf were 10.02 ng/L, 4.21 ng/L, and 44.27 ng/L, respectively. More groups and much higher concentrations of LMATs occurred in the South China Sea than that in the other sea areas of China.

Deep autoencoder for interpretable tissue-adaptive deconvolution and cell-type-specific gene analysis.

Single-cell RNA-sequencing has become a powerful tool to study biologically significant characteristics at explicitly high resolution. However, its application on emerging data is currently limited by its intrinsic techniques. Here, we introduce Tissue-AdaPtive autoEncoder (TAPE), a deep learning method connecting bulk RNA-seq and single-cell RNA-seq to achieve precise deconvolution in a short time. By constructing an interpretable decoder and training under a unique scheme, TAPE can predict cell-type fractions and cell-type-specific gene expression tissue-adaptively. Compared with popular methods on several datasets, TAPE has a better overall performance and comparable accuracy at cell type level. Additionally, it is more robust among different cell types, faster, and sensitive to provide biologically meaningful predictions. Moreover, through the analysis of clinical data, TAPE shows its ability to predict cell-type-specific gene expression profiles with biological significance. We believe that TAPE will enable and accelerate the precise analysis of high-throughput clinical data in a wide range.

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.

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.

Potent allelopathy and non-PSTs, non-spirolides toxicity of the dinoflagellate Alexandrium leei to phytoplankton, finfish and zooplankton observed from laboratory bioassays.

The dinoflagellate genus Alexandrium has been well known for causing paralytic shellfish poisoning (PSP) worldwide. Several non-PSP toxin-producing species, however, have shown to exhibit fish-killing toxicity. Here, we report the allelopathic activity of Alexandrium leei from Malaysia to other algal species, and its toxicity to finfish and zooplankton, via laboratory bioassays. Thirteen microalgal species that co-cultured with Al. leei revealed large variability in the allelopathic effects of Al. leei on the test algae, with the growth inhibition rates ranging from 0 to 100%. The negative allelopathic effects of Al. leei on microalgae included loss of flagella and thus the motility, damages of chain structure, deformation in cell morphology, and eventually cell lysis. The finfish experienced 100% mortality within 24 h exposed to the live culture (2000-6710 cells·mL-1), while the rotifer and brine shrimp exhibited 96-100% and 90-100% mortalities within 48 h when exposed to 500-6000 cells·mL-1 of Al. leei. The mortality of the test animals depended on the Al. leei cell density exposed, leading to a linear relationship between mortality and cell density for the finfish, and a logarithmic relationship for the two zooplankters. When exposed to the treatments using Al. leei whole live culture, cell-free culture medium, extract of algal cells in the f/2-Si medium, extract of methanol, and the re-suspended freeze-and-thaw algal cells, the test organisms (Ak. sanguinea and rotifers) all died at the cell density of 8100 cells·mL-1 within 24 h. Toxin analyses by HILIC-ESI-TOF/MS and LC-ESI-MS/MS demonstrated that Al. leei did not produce PSP-toxins and 13-desmethyl spirolide C. Overall, our findings demonstrated potent allelopathy and toxicity of Al. leei, which do not only pose threats to the aquaculture industry, fisheries, and marine ecosystems but may also play a part role in the population dynamics and bloom formation of this species.

Simple determination of six groups of lipophilic marine algal toxins in seawater by automated on-line solid phase extraction coupled to liquid chromatography-tandem mass spectrometry.

Lipophilic marine algal toxins (LMATs) are highly toxic secondary metabolites produced by marine microalgae that pose a great threat to marine aquaculture organisms and human health. In this study, a novel and automated method for the simultaneous determination of six groups of LMATs in seawater was developed by on-line solid phase extraction (SPE) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Condition optimization and method validation were performed, and the recoveries of all 14 target LMATs featuring different properties ranged from 83.5% to 96.0%. The limits of detection of most target compounds were within ≤3.000 ng/L with good precision (relative standard deviation ≤ 12.1%) and linearity (R2≥0.9916). Compared with off-line SPE methods, the proposed on-line SPE method has better recovery, sensitivity, repeatability, and throughput; in addition, the volume of seawater sample necessary to conduct determinations is greatly reduced in the present method. Finally, the method was applied to determine LMATs in actual seawater samples collected from the Bohai and South Yellow Seas of China in summer, and okadaic acid and pectenotoxin-2 were detected in all seawater samples. The highest concentration of ∑LMATs (22.23 ng/L) occurred in the coastal mariculture area of Shandong Province. Therefore, routine monitoring of LMATs in seawater of the coastal mariculture zone is necessary to prevent shellfish contamination especially in summer, and the proposed on-line SPE-LC-MS/MS method is a powerful way for direct and automatic detection of various LMATs in coastal mariculture area.

Monitoring and warning of lipophilic marine algal toxins in mariculture zone based on toxin profiles of phytoplankton.

Some toxigenic dinoflagellates can produce lipophilic marine algal toxins (LMATs), which are potent threats to marine breeding industries. In this study, a new method based on the profiling analysis of six LMAT classes in phytoplankton was developed for the monitoring and warning of LMATs in mariculture zones. This method was applied to monitor and evaluate LMATs in the Jiaozhou Bay and the Changjiang estuary in China. Results demonstrated that the occurrence and spatiotemporal variations of LMATs in mariculture zones can be revealed by the toxin profiles of phytoplankton, indicating the method's effectiveness for the comprehensive monitoring of the composition and levels of various LMATs in coastal aquaculture zones. The method was further used as an alarm for potential pollution risk from LMATs in mariculture zones at an early stage. The "alert" thresholds of LMAT pollution in the mariculture zones were preliminarily proposed based on the statistical data analysis of LMATs in phytoplankton in three typical mariculture areas in China. This study is the first to conduct simultaneous monitoring and warning of multi-class LMATs based on toxin profiles of phytoplankton, thereby providing new insight into the monitoring and early warning of natural poisonous pollutants in coastal aquaculture zones around the world.

Occurrence, distribution, source, and influencing factors of lipophilic marine algal toxins in Laizhou Bay, Bohai Sea, China.

The composition, distribution, origin, and influencing factors of lipophilic marine algal toxins (LMATs) in surface seawater and phytoplankton in Laizhou Bay, China, were comprehensively investigated for the first time. Okadaic acid (OA), pectenotoxin-2 (PTX2), dinophysistoxin-1 (DTX1), dinophysistoxin-2 (DTX2), and pectenotoxin-2 seco acid (PTX2 SA) were discovered in surface seawater, whereas PTX2, OA, 7-epi-PTX-2 SA, DTX1, PTX2 SA, PTX11, and DTX2 were found in phytoplankton in a decreasing concentration order. ∑LMAT concentrations in seawater and phytoplankton were 1.08-35.66 ng/L (mean: 7.31 ng/L) and 0-3609.75 ng/L (mean: 191.38 ng/L), respectively. LMAT contents in seawater and phytoplankton exhibited the highest levels in the southeastern mouth of Laizhou Bay and decreased toward the inner and outer bays. Dinophysis fortii, D. acuminata, D. rotundata, Procentrum lima, and P. minimum were identified as the potential origins of LMATs in Laizhou Bay. Moreover, increased nutrient level and decreased pH in seawater could increase LMAT content.

Distribution Characteristics and Environmental Control Factors of Lipophilic Marine Algal Toxins in Changjiang Estuary and the Adjacent East China Sea.

Marine algal toxins, highly toxic secondary metabolites, have significant influences on coastal ecosystem health and mariculture safety. The occurrence and environmental control factors of lipophilic marine algal toxins (LMATs) in the surface seawater of the Changjiang estuary (CJE) and the adjacent East China Sea (ECS) were investigated. Pectenotoxin-2 (PTX2), okadaic acid (OA), dinophysistoxin-1(DTX1), and gymnodimine (GYM) were detected in the CJE surface seawater in summer, with concentration ranges of not detected (ND)-105.54 ng/L, ND-13.24 ng/L, ND-5.48 ng/L, and ND-12.95 ng/L, respectively. DTX1 (ND-316.15 ng/L), OA (ND-16.13 ng/L), and PTX2 (ND-4.97 ng/L) were detected in the ECS during spring. LMATs formed a unique low-concentration band in the Changjiang diluted water (CJDW) coverage area in the typical large river estuary. PTX2, OA, and DTX1 in seawater were mainly derived from Dinophysis caudate and Dinophysis rotundata, while GYM was suspected to be from Karenia selliformis. Correlation analyses showed that LMAT levels in seawater were positively correlated with dissolved oxygen and salinity, but negatively correlated with temperature and nutrients, indicating that the hydrological condition and nutritional status of seawater and climatic factors exert significant effects on the distribution of LMATs.

Distribution, partitioning, and seasonal variation of lipophilic marine algal toxins in aquatic environments of a typical semi-closed mariculture bay.

Lipophilic marine algal toxins (LMATs) pose a potential threat to the health of marine shellfish consumers and marine breeding industries. In this study, LMATs in dissolved phases (DP) and particulate phases (PP) in the seawater of Jiaozhou Bay were accurately determined over four seasons to understand their composition, level, phase partitioning, spatiotemporal variation, and potential sources in aquatic environments of a typical semi-closed mariculture bay. Various LMATs, such as okadaic acid (OA), dinophysistoxin-1 (DTX1), dinophysistoxin-2 (DTX2), gymnodimine (GYM), 13-desmethyl spirolide C (SPX1), pectenotoxin-2 (PTX2), pectenotoxin-2 seco acid (PTX2 SA), and pectenotoxin-11 (PTX11), were detected in DP and PP; of these, OA and PTX2 were the dominant LMATs in DP and PP, respectively. The average proportion of ΣLMATs in DP (97.5%) was significantly higher than that in PP (2.5%), which indicates that LMATs are predominantly partitioned into DP. The total concentrations of LMATs in DP ranged from 4.16 ng/L to 23.19 ng/L (mean, 13.35 ng/L) over four seasons. The highest levels of LMATs in DP and PP were found in summer (mean, 16.71 ng/L) and spring, respectively, while the maximum variety of LMATs was found in autumn. This result suggests that seasonal changes could influence the composition, concentration, and phase partitioning of LMATs in aquatic environments of a coastal semi-closed mariculture bay. ΣLMAT concentrations were higher in the western region than in the eastern region of the bay, where shellfish may have a greater risk of exposure. Dinophysis acuminata, Dinophysis fortii, and Prorocentrum minimum were the potential sources of LMATs in the aquaculture seawater. Overall, various LMATs occurred in the semi-closed mariculture bay, and the persistence and bioavailability of these toxins in aquaculture seawater should be determined in future research.