Shark fishing, driven by the fin trade, is the primary cause of global shark population declines. Here, we present a case study that exemplifies how industrial fisheries are likely depleting shark populations in the Eastern Tropical Pacific Ocean. In August 2017, the vessel Fu Yuan Yu Leng 999, of Chinese flag, was detained while crossing through the Galápagos Marine Reserve without authorization. This vessel contained 7639 sharks, representing one of the largest seizures recorded to date. Based on a sample of 929 individuals (12%), we found 12 shark species: 9 considered as Vulnerable or higher risk by the IUCN and 8 listed in CITES. Four species showed a higher proportion of immature than mature individuals, whereas size-distribution hints that at least some of the fishing ships associated with the operation may have been using purse-seine gear fishing equipment, which, for some species, goes against international conventions. Our data expose the magnitude of the threat that fishing industries and illegal trade represent to sharks in the Eastern Tropical Pacific Ocean. This situation is particularly worrisome in the case of sharks, a group with a high proportion of top predators central to marine food web dynamics3. Some sharks have a high market value and hence are targeted by fisheries for their fins and other products4. Given the economic incentives for exploitation and the difficulties involved in controlling this trade, shark fishing has caused substantial population declines5. Consequently, at least 74 species of sharks (16% of shark diversity) are threatened worldwide6.Amid the current biodiversity crisis, an era of unprecedented species extinctionwww.cms.int/sharks/en/legalinstrument/sharks-mou), the first global instrument for the conservation of migratory species of elasmobranchs. Thus, management to prevent population collapses of some of the most heavily traded and vulnerable shark species has become a global priority . Moreover, sharks benefit from marine protected areas that preserve habitats and populations7, and export biomass to non-protected areas, sustaining the yield of nearby fisheries9.In response to global threats, the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) has identified the international shark fin trade as the primary driver of population declines, protecting 46 shark species under this convention. Further, 26 shark species are listed in Annex 1 of the Memorandum of Understanding on the Conservation of Migratory Sharks 12. Here, we present a case study from the Eastern Tropical Pacific Ocean as an example of how the operation of international fleets may hamper national and international efforts to preserve shark populations worldwide.Unfortunately, fisheries management regulations and benefits derived from marine protected areas become ineffective when sharks move into international waters13, a protected area of about 133,000 km2. This reserve has afforded over 20 years of protection for endemic and native marine species and holds at least 36 species of sharks  expanded to the Galápagos Marine Reserve in 1998On August 13th, 2017, an alert was issued to the Navy of Ecuador from the Monitoring Center of the GNP Directorate regarding a sizeable vessel crossing the Galápagos Marine Reserve. The reefer vessel Fu Yuan Yu Leng 999, of Chinese flag, was detained 12 miles north of Punta Pitt, on San Cristóbal Island. The vessel was escorted to Puerto Baquerizo Moreno, the Galápagos Islands’ political and judicial capital, where an inquiry and subsequent trial began.Here, we combined morphological identification with molecular barcoding to assess the identity of 12% of the carcasses contained in the vessel Fu Yuan Yu Leng 999 and estimated the size distribution of sharks targeted. We argue that the high shark biomass and the high proportion of endangered species transported by the reefer vessel, as well as the recent operation of international fleets around the Ecuadorian Exclusive Economic Zones, reveal a pattern of sustained exploitation of sharks in this region and, more generally, the Eastern Tropical Pacific Ocean. We discuss our results in terms of their implications for the impact of international fishing fleets on the conservation of shark species.Rhincodon typus  matched the morphological and molecular identifications , and 8 are listed as CITES species  , pelagic thresher , and blue shark .From the 12 species of sharks found in the vessel Fu Yuan Yu Leng 999, 11 are found within the Galápagos Marine Reserve, 9 are listed as Vulnerable or higher risk by the IUCN  or received catch from coastal fishing ships. Our finding of two grey reef sharks, a non-pelagic species found in the western Pacific and the Central Pacific Islands18, supports this scenario. These detection anomalies highlight the importance of satellite technologies, such as AIS and vessel monitoring systems (VMS), in inferring true vessel trajectories, enforcing approved routes, and diminishing the probability of vessels trespassing into EEZs and marine protected areas19.There is no certainty about the origin of sharks found in the vessel Fu Yuan Yu Leng 999. However, machine-learning analysis of the data from the Automatic Identification System (AIS) of the vessel detected an anomaly in its trajectory 2735 km northwest of the Galápagos Marine Reserve16 Fig. . EstimatWhether the Fu Yuan Yu Leng 999 vessel would engage in potentially illegal operations around the Galápagos EEZ, remains unclear. Still, in its empty freezer holdings, the vessel had a gross capacity for at least 4.5 times the number of sharks found in this study , which may have been received by the vessel along its south-bound trajectory if not been seized.20. Even more worrisome is the situation of the oceanic whitetip shark (20% of the sample), which is Critically Endangered. In 2011, in response to concerns about declining trends in catches of oceanic whitetip sharks, a ban by the Inter-American Tropical Tuna Commission (IATTC) was placed on the retention, transshipping, landing, storing, or selling of this species (Resolution C-11-1021). Thus, our finding of 188 oceanic whitetip sharks implies that the Fu Yuan Yu Leng 999 crew was violating the ban of the IATTC, of which China is a signatory.The number of individuals analyzed in this study constitutes a representative sample of the Fu Yuan Yu Leng 999 vessel’s total cargo, which was highly biased towards sharks. The most common species found in the sample, the silky shark (28%), has been reported as the second most abundant species in the Hong Kong and Guangzhou markets in China, the largest shark fin retail centers in the world20. This species is not yet been listed by CITES because of its Near Threatened status . However, regionally, the blue shark represents the most common shark species landed in Peru22, and has been reported as by-catch in fisheries from Ecuador23 and Chile24. Other endangered shark species, such as hammerheads (collectively 20% of the sample), are highly vulnerable to non-target fisheries in the Eastern Pacific Ocean26.Another common species in the sample, the blue shark (12%), has been reported as the most abundant species in Asian markets27, suggests that these species may have been caught using purse-seine gear. Consistent with these data, the semi-industrial longline fleet from mainland Ecuador caught mostly adults of silky shark and only a small proportion of oceanic whitetip shark in 2008–201228. Samples of both species of hammerhead shark in the vessel were comprised mostly of adults, which is also consistent with the use of purse-seine gear29. In 2016, the IATTC placed a precautionary ban for 2017–2019 on retention, transshipment, landing, or storing of silky sharks caught by purse seines in the convention area (Resolution C-16-0630). Thus, our data, which suggest fishing a high proportion of silky sharks with purse seines, points to yet another violation of the IATTC by the Fu Yuan Yu Leng 999 crew. These data exemplify how the operation of international fishing fleets may be hampering the efforts of other IATTC members to protect shark species.Additional threads may derive from the high proportion of immature individuals for some species in the sample. A comparison of fork length distribution of the seized silky and oceanic whitetip sharks Fig.  with siz31 and a similar event happened in March 201932. More recently, on 16 July 2020, a fleet of 260 ships (mostly Chinese flagged) was reported along the southern border of Galápagos’ EEZ33. By 6 August 2020, the fleet had grown to 340 ships34, and at least 6 were transmitting false coordinates that located them within New Zealand’s waters35.In recent years, international fleets comprising hundreds of vessels have been reported repeatedly near the Ecuadorian EEZs . In July 2017 and August 2017 (a few days before the seizure of vessel Fu Yuan Yu Leng 999), international fleets of ~ 300 ships (mostly Chinese flagged) were detected in the international waters bordering the Galápagos’ EEZThese events suggest that the presence of international fishing fleets around the Ecuadorian EEZs may be systematic. Given the highly mobile nature of the targeted species identified here, international fleets are likely catching sharks that are demographically linked to populations protected under national legislation, undermining the efforts Ecuador has adopted for the conservation of sharks. Current proposals to enhance the protection of migratory marine life in the region include expanding Ecuador’s EEZ along the Carnegie and Cocos ridges , which are targeted by international fishing fleets, with limited governance from Regional Fisheries Management Organizations (RFMOs). A better understanding of the demographic and genetic connectivity of targeted shark populations across jurisdictional boundaries is urgently needed to inform management and improve the conservation of the biological marine resources of the Eastern Tropical Pacific both within EEZs and in Areas Beyond National Jurisdiction (ABNJs).Here, we provide the largest data set on shark species caught in the region and contribute to a growing data base of genetic information needed to assess the identity of fished sharks in the future. Improved governance of fisheries in ABNJs and their compliance with international treaties, will be paramount to preserve biological and economical marine resources of developing countries in the Eastern Tropical Pacific and tropical seas in general.Sampling was conducted onboard vessel Fu Yuan Yu Leng 999 in September and October 2017, in a collaborative effort between the National Army of Ecuador, the GNP Directorate, and the Galapagos Science Center (GSC) of Universidad San Francisco de Quito and the University of North Carolina at Chapel Hill (research permits PC-38-16 and PC-24-17). The procedure was performed under the time and logistic constraints imposed by the seizure’s legal process, the low temperatures of the vessel holds, and the identifying and measuring of frozen samples—with limited personnel and the vessel in motion—while destruction/disposal of the cargo was taking place.28. Tissue samples and pre-caudal body length were taken for as many individuals as possible. The total vessel cargo was weighed to the nearest 0.1 kg before disposal. The size of sexual maturity was inferred from the literature to estimate the ratio of immature-to-mature individuals in our sample. For pelagic thresher (Alopias pelagicus) and silky shark , size of sexual maturity was inferred from pre-caudal length. For other species, we used conversion factors to calculate total length from pre-caudal length, as follows: for shortfin mako (Isurus oxyrinchus), blue shark (Prionace glauca), and smooth hammerhead (Sphyrna zygaena) we followed Mas et al.36; for oceanic whitetip (Carcharhinus longimanus) D´Alberto et al.37; for bigeye thresher (Alopias superciliosus), grey reef shark (Carcharhinus amblyrhynchos), tiger shark , and smooth-hound (Mustelus mustelus), FishBase (https://www.fishbase.de). For scalloped hammerhead (Sphyrna lewini), we used the conversion factor for smooth hammerhead36. Since we lacked sex ID for all individuals, we defined minimum size at maturity using estimates for males only . When a range was provided, we used the minimum size.We tried to take a representative sample of all species present in the vessel, choosing a random sample of individuals within each species. Morphological identification was based on expert’s knowledge and specialized literature27, we converted pre-caudal length to fork length using Oshitani et al.38 and Joung et al.39 conversion factors, respectively.To compare the size structure of silky and oceanic whitetip sharks found in the vessel with that of sharks fished with long-lines and purse-seines40 (permits MAE-DNB-CM-2016-0041 and MAE-DNB-2018-0759-O). We conducted genomic DNA extractions on samples of subcutaneous muscle tissue (2 × 2 mm). Extraction followed a guanidine thiocyanate protein precipitation plus isopropanol DNA precipitation protocol41. Quality and concentration of genomic DNA were measured with a NanoDrop ND-1000 spectrophotometer v3.0.1  or an Epoch Microplate Spectrophotometer (BioTek).We sequenced the mitochondrial gene Nicotinamide Adenine Dehydrogenase subunit 2 (ND2), a barcode gene for sharks2, 0.50 μl of 10 μM of each dNTP, 0.5 μl of each primer (10 μM), and different amounts of ultrapure water with 1 μl of template DNA (adjusted to 100 ng/mL of DNA). Thermocycling conditions included an initial denaturation at 94 °C/3 min; 35 cycles of denaturation at 93 °C/ 30 s, annealing at 58 °C/1 min, and extension at 72 °C/1 min; and a final extension at 72 °C/10 min. The resulting amplicons were visualized via electrophoresis in a 2% agarose gel (UltraPure Agarose). Excess nucleotides and dNTPs were removed from PCR products using ExoSAP-IT PCR Product Cleanup Reagent (Applied Biosystems). Purified amplicons were sequenced with big-dye chemistry and PCR primers, using capillary electrophoresis in an ABI3730xl sequencer. Inspection of chromatograms, contig assembly of forward and reverse sequences, and final sequence checking and edition were performed in Geneious 11.1.5 (Biomatters Ltd.42).The ND2 gen was amplified by PCR in 25 μl reactions comprised of 0.25 μl Paltinum Taq DNA Polymerase, Master Mix (Invitrogen), 2.50 μl 10X PCR Buffer, 1.5 mM of MgClhttps://www.ncbi.nlm.nih.gov/genbank/), we used BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi). To place the sample sequences in a phylogenetic framework, we consulted the Chondrichthyes Tree of Life (https://sharkrays.org) to assemble a list including all shark species from the Pacific Ocean, and downloaded the sequences for these species from Genbank. Locality information for sequences of the species present in the vessel was gathered, when possible, in an attempt to relate the seized samples to general geographic regions in the Pacific Ocean. Additionally, we sequenced samples from nine individuals of the Galapagos shark, Carcharhinus galapagensis, obtained from the Galápagos Marine Reserve (GNPD permit 065-2013).To evaluate the similarity between sequences from the vessel’s samples and those on GenBank , and trimmed to the size of the ND2 mitochondrial gen using Mesquite 3.644. To reduce the size of our matrix and facilitate phylogenetic analyses, we eliminated duplicates  in all datasets separately, using sRNAtoolbox45. Then, we compiled the unique sequences from each dataset and re-aligned them in MAFFT 7.Sequences of samples from vessel Fu Yuan Yu Leng 999, the Pacific Ocean (gathered from GenBank), and Galapagos shark were aligned using MAFFT 746, as implemented in the IQ-TREE web server47 (http://iqtree.cibiv.univie.ac.at/) to infer the best-fit model of nucleotide substitution, estimate the maximum likelihood tree, and assess nodal support. The best-model fit was estimated using ModelFinder48 in “Auto” function and “FreeRate heterogeneity”; the best-fit model (GTR + F + R7) was chosen using the Bayesian information criterion. Nodal support was obtained using 1000 ultrafast bootstrap replicates, 1000 maximum iterations, and minimum correlation coefficient of 0.99. As outgroup, we used the rabbit fish, Chimaera monstrosa (GenBank accession number JQ518716).We used IQ-TREESupplementary Figure S1.Supplementary Figure S2.Supplementary Table S1.Supplementary Table S2.Supplementary Data S1.Supplementary Data S2.


Background: Gene expression and alternative splicing (AS) can promote cancer development via complex mechanisms. We aimed to identify and verify the hub AS events and splicing factors associated with the progression of colorectal cancer (CRC).Methods: RNA-Seq data, clinical data, and AS events of 590 CRC samples were obtained from the TCGA and TCGASpliceSeq databases. Cox univariable and multivariable analyses, KEGG, and GO pathway analyses were performed to identify hub AS events and splicing factor/spliceosome genes, which were further validated in five CRCs.Results: In this study, we first compared differentially expressed genes and gene AS events between normal and tumor tissues. Differentially expressed genes were different from genes with differentially expressed AS events. Prognostic analysis and co-expression network analysis of gene expression and gene AS events were conducted to screen five hub gene AS events involved in CRC progression: EPB41L2, CELF2, TMEM130, VCL, and SORBS2. Using qRT-PCR, we also verified that the gene AS events SORBS2 were downregulated in tumor tissue, and gene AS events EPB41L2, CELF2, TMEM130, and VCL were upregulated in tumor tissue. The genes whose mRNA levels were significantly related to the five hub gene AS events were significantly enriched in the GO term of cell division and Notch signaling pathway. Further coexpression of gene AS events and alternative splicing factor genes revealed NOVA1 as a crucial factor regulating the hub gene AS event expression in CRC. Through in vitro experiments, we found that NOVA1 inhibited gene AS event SORBS2, which induced the migration of CRC cells via the Notch pathway.Conclusion: Integrated analysis of gene expression and gene AS events and further experiments revealed that NOVA1-mediated SORBS2 promoted the migration of CRC, indicating its potential as a therapeutic target. Colorectal cancer is one of the most frequently diagnosed malignant cancers and the second leading cause of cancer-related deaths worldwide . DespiteAlthough the total number of protein-coding genes in the human genome is nearly 20,000, the transcriptome is tremendously more complex . A singlvia activation of Notch signaling. Notch signaling is a primordial, evolutionarily conserved cell fate determination pathway involved in cancer biology, such as cancer stem cells, angiogenesis, and tumor immunity . Activatin vitro experiments, we further explored the potential mechanisms regulating gene AS events and AS events that induce CRC progression.Due to the complex mutual regulation mediated by gene expression and AS events , integra1 and TCGASpliceSeq2 databases, which included 50 colorectal cancers with matched normal tissue. Sixty-six genes of splicing factor genes were obtained from the SpliceAid 2 database3.The transcriptome, AS events, and the prognostic data of 590 colorectal cancers were downloaded from the TCGAFive consecutive, unselected CRC tissues and paired normal tissues were collected from surgical resections of CRC patients at Ruijin Hospital. None of the patients received adjuvant therapy before the operation. CRC tumors were histologically confirmed by two experienced pathologists independently. Written informed consent was obtained from all the patients with CRC. All aspects of this study were approved by the Research Ethics Committee of Shanghai Jiao Tong University. The UICC TNM classification system was used to determine the tumor stage. All tissue samples were stored in liquid nitrogen. The patient information is illustrated in 2. Cancer cells were grown to 60–70% confluence, and the siRNAs were transfected using Lipofectamine 3000 according to the manufacturer’s protocol . After 48 h of transfer, RNA was collected, and then at 72 h, proteins were collected, and functional experiments were performed. The siRNA sequences used in this study were purchased from Bioegene (Shanghai) and are listed in The CRC cell lines were purchased from the American Type Culture Collection and cultured in RPMI 1640 supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin at 37°C with 5% COThe total protein of cancer cells was extracted using RIPA buffer supplemented with 1 mM phenylmethylsulfonyl fluoride. Protein concentration was measured using the BCA Protein Assay Kit . Western blotting was performed according to our previous protocols . The folAfter digestion with trypsin, 5 × 104 cells were seeded in the top chamber containing a polycarbonate membrane with 8-μm pores . The lower chamber was filled with 10% FBS as a chemoattractant. After 48 h, the cells of the inner membrane were scraped using a cotton swab, and the cells on the lower surface were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet for counting.® Universal SYBR qPCR Master Mix . The sequences of the sense and antisense primers are listed in –ΔΔCT. According to the PSI analysis in the TCGASpliceSeq database, two paired primers were designed for quantitative analysis. One pair of primers was used to amplify the alternative exon (V1) and the other for the common exon (V2). The relative PSI value of every AS event in the CRC tissues was calculated as V1/V2.Total RNA from cancer cells and fresh tissues was extracted using TRIzol Reagent (Invitrogen) according to the protocols of the manufacturer. Then, the RNA molecules were dissolved in RNase-free DNase  and stored at −80°C. cDNA synthesis was performed using HiScript II Q Select RT SuperMix for qPCR . Finally, quantitative real-time PCR (qRT-PCR) was conducted using an Applied Biosystems 7500 Sequence Detection System  with AceQFor these genes and AS events that are associated with poor or good prognosis, we introduced an algorithm called CoExpNetViz (version 2.0.2)  in the C4 , we imported these DEGs into a database called Metascape4  for GeneAfter the gene coexpression network was created, these gene pairs were then imported into CytoHubba, an algorithm used for identifying hub genes from complex interactomes . We rankt-test. Data are displayed as mean ± SD. Survival analysis was performed using the Kaplan–Meier method and compared using the log-rank test. Pearson’s correlation analysis was conducted to explore the correlation between the two variables. A value of p < 0.05 was considered statistically significant.To compare the differentially expressed AS events between normal and tumor tissue, ANOVA analysis was performed using “stats” package in R. The differentially expressed genes between normal and tumor tissue were obtained using the “limma” package in R. Differentially expressed genes and AS events are displayed in a heatmap. Univariate Cox regression analysis was performed to obtain prognostic gene expression and gene AS events using the “survival” package in R and further displayed by forest plot. Other statistical analyses were performed using GraphPad Prism 6.0 . Comparisons between two groups were performed using a two-tailed unpaired Student’s p-value < 0.01, By comparing differentially expressed genes between 50 paired normal and tumor tissues, 2,313 genes were obtained and displayed by heatmap , we identified five hub gene AS events: EPB41L2, CELF2, TMEM130, VCL, and SORBS2 , four of the above five hub gene AS events were significantly related to NOVA1 mRNA expression  can be found in the article/Supplementary Material.The studies involving human participants were reviewed and approved by Ethics Committee of Shanghai Ruijin Hospital affiliated to Shanghai Jiao Tong University School of Medicine. The patients/participants provided their written informed consent to participate in this study.RZ and KL were responsible for the conceptualizing and designing of the study. TZ, SC, and YP analyzed and interpreted the patient data and were major contributors in the writing of the manuscript. TZ, CW, XC, and KL collected and assembled the data. YP and SC performed the experiments. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


MCP generated accurate relative abundance estimates and made substantially fewer false positive predictions than other classifiers while retaining a high recall rate. We further demonstrated that the accuracy of species classification was substantially increased using the Microba Genome Database, which is more comprehensive than reference datasets used by other classifiers and illustrates the importance of including genomes of uncultured taxa in reference databases. Consequently, MCP classifies appreciably more reads than other classifiers when using their recommended reference databases. These results establish MCP as best-in-class with the ability to produce comprehensive and accurate species profiles of human gastrointestinal samples.A fundamental goal of microbial ecology is to accurately determine the species composition in a given microbial ecosystem. In the context of the human microbiome, this is important for establishing links between microbial species and disease states. Here we benchmark the Microba Community Profiler (MCP) against other metagenomic classifiers using 140 moderate to complex  We demonstrate that MCP has superior recall and precision and maps a higher proportion of reads from gut metagenome datasets than the other classifiers evaluated.The Microba Community Profiler (MCP) was developed to be a highly accurate and specific tool to estimate the relative abundance of bacterial, archaeal, eukaryotic, and viral community members by aligning metagenomic reads to a high-quality and comprehensive database of microbial reference MAGs and isolate genomes. Similar to other classifiers, MCP provides per-read classifications along with an estimate of the proportion of reads assigned to a species. MCP also explicitly indicates the species predicted to be present in a community profile, in contrast to the majority of classifiers considered in this study which report thousands of false positive (FP) species if profiles are not manually inspected and appropriately filtered. The community profiles produced by the MCP are based on the rank normalized taxa and comprehensive species clusters defined by the Genome Taxonomy Database GTDB; , 2020 whin silico mock communities. Species represented by > 1 genome  had an average intraspecific ANI of 97.8 ± 0.96%. The standardized reference database and comparison of profilers was limited to bacterial and archaeal species as not all classifiers support the classification of eukaryotic or viral species.We evaluated the performance of MCP and nine publicly available metagenomic classifiers , which uThree parameter settings for the MCP were evaluated: (i) MCP with the standardized reference database and default parameters used to filter out expected FP predictions (referred to as MCP); (ii) MCP without removing expected FPs (referred to as unfiltered MCP or uMCP); and (iii) MCP with default filtering parameters using the Microba Genome Database (MGDB), which comprises 73,646 dereplicated genomes from 28,246 species and is the reference database used by MCP in practice (referred to as MCP-MGDB).in silico mock microbial communities with varying species diversity, intraspecific diversity, and genomic similarity to reference database genomes  can be achieved. As expected, the detection limit increases as community members becoming increasingly divergent from genomes in the reference database  followed by the uMCP profiles (F1 = 0.92), mOTUs (F1 = 0.91), and MetaCache (F1 = 0.88) (F1 = 0.81) despite using a reference database built from nearly six times as many genomes as the standardized reference database illustrating that a comprehensive database is not sufficient in and of itself to provide good performance.The performance of classifiers generally decreased with increasing ANI divergence from the reference database , consist = 0.88) . MetaPhlF1 score as determined independently for each classifier on each mock community (referred to as the optimized F1 score). Notably, the average MCP F1 score without optimization of 0.96 is higher than the optimized F1 score of all other classifiers , the accuracy of relative abundance estimates decreased with increasing ANI divergence from the reference database . CentrifThe high precision of the MCP  resultedin silico samples. We focused on these classifiers as they were the strongest performing classifiers on the in silico mock communities and/or are widely used by the research community. Unlike the in silico mock community analysis, here each classifier was evaluated using its recommended reference database. MCP uses the MGDB which consists of 73,646 dereplicated genomes spanning 28,246 species clusters (see section “Properties of the Microba Genome Database”). MetaCache uses a reference database comprising 16,488 bacterial, 343 archaeal, and 8,999 viral genomes annotated as complete in RefSeq. Kraken and Bracken use a slightly expanded set of 18,871 bacterial, 360 archaeal, and 9,334 viral genomes along with a human reference genome and a collection of known vectors. mOTUs uses a pre-built database of marker genes obtained from ∼25,000 bacterial and archaeal reference genomes which have been supplemented with additional marker genes obtained from public metagenomes. The MetaPhlAn database consists of ∼1.5 million unique clade-specific marker genes obtained from ∼100,000 bacterial, archaeal, and eukaryotic genomes. Species profiles for all classifiers are defined according to the NCBI Taxonomy  than Kraken (43.3% on average), Bracken (51.3% on average), or MetaCache  of the 28,246 species in the MGDB are comprised solely of uncultured genomes  with 625 species being comprised exclusively of MAGs obtained by Microba , which iin silico mock communities using the more comprehensive MGDB than the relatively small standardized reference database. In particular, use of the MGDB results in an improvement in correct identification of species comprising the mock communities and in the accuracy of species abundance estimates  to MGDB reference genomes  and percent alignment length (PA) of reads mapped by the MCP. Mapped reads had a PI and PA of 99.72 and 99.99%, respectively, averaged over the 33 fecal samples. Comparing these similarity values to the PI observed for the  genomes , indicatin silico mock communities with decreasing ANI similarity to genomes in a standardized reference database. To the best of our knowledge, this is the first benchmarking study to explicitly investigate the impact of genomic similarity to reference database genomes on classification performance. Our results show that the MCP has the highest combined precision and recall  among all evaluated classifiers indicating that the optimized trade-off between FP and FN predictions used by MCP provides the most accurate community profiles  – 0.00005 × (no. undetermined bases), with an additional 100 added to the assembly quality if it was annotated as complete as determined by consulting the “assembly level” annotation at NCBI. In order to avoid having highly similar genomes in the reference database, a genome was only included if it had an ANI < 99% to all other intraspecific genomes as determined with Mash v2.1.1 . The 67,299 genomes in RefSeq release 97 not covered by GTDB R04-R89 were assigned GTDB classifications using GTDB-Tk v0.3.3  = 100 and σ (standard deviation in number of species) = 25, or μ = 500 and σ = 100, in order to generate medium and high complexity communities, respectively. Communities were constructed with either a single genome selected from each species, or with 2–10 genomes randomly selected from each species. The relative abundance of genomes comprising mock communities was drawn from a log-normal distribution with a mean of 1 and a standard deviation of 2 as commonly used for modeling microbial communities .ni = N×(aisi/∑jajsj), where si is the size of genome i, ai is the relative abundance of genome i, and N is the total number of paired reads comprising the in silico community. All in silico communities were simulated to a depth of 2.1 Gb by randomly sampling 2 × 150 bp paired-end reads with an insert size of 200 ± 25 bp across each genome in the mock community.The number of paired reads generated for each genome was 2 on November 22 2019 and consist of the files nodes.dmp, names.dmp, merged.dmp, nucl_gb.accession2taxid, and nucl_wgs accession2taxid. DIAMOND and Kaiju require protein sequences which were called for each reference genomes using Prodigal v2.6.3  and concatenated into a single FASTA file  in order to facilitate the requirements of the different metagenomic classifiers. The custom databases were built using the same NCBI Taxonomy data files used while constructing the standardized reference database which were obtained from NCBIl v2.6.3  and the Community profiles were generated for mock communities using each of the metagenomic classifiers run with default parameters . DIAMONDin silico mock communities due to reclassifications at NCBI. To account for this, the NCBI TaxIds produced by MetaPhlAn were used to establish species names as defined in the November 22, 2019 NCBI Taxonomy data files, the same files used to construct the mock communities.MetaPhlAn results were obtained using the v296_CHOCOPhlAn_201901 marker set which may have species assignments that differ from those defined for the The MGDB v2 was built from genomes in GTDB R04-RS89, MAGs obtained from Australian fecal samples, MAGs mined from SRA samples by Microba, and MAGs and isolate genomes from https://microba.com/microbiome-research. MCP is a whole-genome alignment tool, which uses a combination of BWA v0.7.17  required 37.6 ± 9.9 min on average and profiling required 14.5 ± 2.4 min on average when using 64 Intel Xeon 2.00 GHz processors.Microba Community Profiler is propriety software available by contacting Microba Life Sciences at  v0.7.17 , SAMtool v0.7.17 , and cusF1 score is the harmonic mean of precision and recall, (2 × P × R)/(P+R), which weights these terms equally in a single metric.Precision and recall can be defined in terms of the number of species correctly (TP) and incorrectly (FP) identified by a classifier along with the number of unidentified species present in a sample (FN). Precision, P = TP/(TP+FP), is the fraction of species identified by a classifier that are correct, while recall, R = TP/(TP+FN), is the fraction of correctly identified species within a sample. The Absolute and relative percent error for each species within a sample is defined in terms of the true, T, and estimated, E, abundance of a species. Absolute error, A = |T-E|, indicates how close abundances estimates are to the true abundance of a species, while relative percent error, R = 100 × A/T, expresses how large the absolute error is compared to the true abundance which highlights poor estimates of low abundance species. The L1 (Manhattan) distance is the sum of absolute errors across all ground truth and predicted species which provides a measure that incorporates FP predictions . The meaPrevious benchmarking studies have suggested the use of the Euclidean distance and the area under the precision–recall curve (AUPR) for evaluating classifier performance . We elecThe detection limit for classifiers was defined as the lowest abundance species in a sample that achieved a specified FDRs, FDR = FP/(TP+FP). This was determined by ordering identified species in ascending order of abundance and calculating the FDR after filtering species below each abundance level. The detection limit for a sample is the lowest abundance at which the desired FDR could be achieved.in silico samples. Samples were downloaded from the NCBI Sequence Read Archive . Genomes used to build the standardized reference database are given in The DP and DW developed the benchmarking framework with recommendations provided by all others authors. FR developed the computational infrastructure used to carry out this study. PV-W identified and performed initial bioinformatic analyses of the US fecal samples. LK reviewed and provided suggestions on early drafts of the manuscript. DP, DW, GT, and PH wrote the manuscript with constructive suggestions from all other authors. All authors contributed to the article and approved the submitted version.DP, FR, PV-W, LK, and DW are the employees of Microba Life Sciences. GT and PH are the founders of Microba Life Sciences. Microba Life Sciences is a microbial genomics company developing microbiome-based diagnostics and therapeutics and offers metagenomic gut microbiome reports. The Microba Community Profiler (MCP) is proprietary software of Microba Life Sciences, but is available commercially for researchers in order to facilitate replicating the results of this study.


Microorganisms thriving in the lake display specific genotypes that facilitate survival, which include at least a multitude of plasmid-encoded resistance traits. Hence, the genetic information provided by the plasmids essentially contributes to understand adaptation to different stressors. Though plasmids from cultivable organisms have already been analyzed to the sequence level, the impact of the entire plasmid-borne genetic information on such microbial ecosystem is not known. This study aims at assessing the plasmidome from Diamante Lake, which facilitates the identification of potential hosts and prediction of gene functions as well as the ecological impact of mobile genetic elements. The deep-sequencing analysis revealed a large fraction of previously unknown DNA sequences of which the majority encoded putative proteins of unknown function. Remarkably, functions related to the oxidative stress response, DNA repair, as well as arsenic- and antibiotic resistances were annotated. Additionally, all necessary capacities related to plasmid replication, mobilization and maintenance were detected. Sequences characteristic for megaplasmids and other already known plasmid-associated genes were identified as well. The study highlights the potential of the deep-sequencing approach specifically targeting plasmid populations as it allows to evaluate the ecological impact of plasmids from (cultivable and non-cultivable) microorganisms, thereby contributing to the understanding of the distribution of resistance factors within an extremophilic microbial community.Diamante Lake located at 4589 m.a.s.l. in the Andean Puna constitutes an extreme environment. It is exposed to multiple extreme conditions such as an unusually high concentration of arsenic (over 300 mg L Thus, plasmids act as important evolutionary driving force by accelerating genome innovation and allowing acquisition of evolutionary novelty2.Plasmids are self-replicating, extrachromosomal, mobile, genetic elements of ecological importance, as they may confer functions or beneficial traits enabling their hosts to thrive in a given environment and—equally important—they can act as horizontal gene transfer vehicles12. Only quite recently, however, it was recognized that to characterize them as a whole is necessary to understand their ecological impact in microbial ecosystems18. In this regard, the total plasmid DNA present in an ecological niche was defined as the “plasmidome”19. Indeed, plasmidome studies based on independent microbial culture methods substantiated the significance of extrachromosomal genetic elements with respect to different environments, such as the bovine rumen and wastewater treatment plants15. We have recently reported the study of the plasmidome of the Puquio de Campo Naranja, an extreme environment of the Puna Argentina18. However, despite the ecological relevance of plasmidomes such studies were only rarely performed, which is possibly due to the complexity of sample processing and the need of tailor-made bioinformatics tools for analysis.There are myriads of studies addressing bacterial and archaeal plasmids, which eventually revealed the typical functions ensuring plasmid replication, mobilization and maintenance, as well as other accessory characteristics21. The highest concentration of As (up to 347 mg L−1 in the summertime) was reported for the Diamante Lake22, which is located inside the Galán Volcano boiler (40 km diameter) at an altitude of 4589 m above sea level in the Catamarca province, Argentina , strong UV irradiation (84 W m−2 of UV-AB at noon) and vast day–night temperature ranges (− 20 °C to + 20 °C)23. Microbial communities facing such an environment need to develop capacities ensuring survival. As stated above, extrachromosomal genetic elements could procure the genes enabling microbes to withstand harsh environmental conditions24. Rascovan et al.25 reported the discovery of a red biofilm consisting of 94% archaeal representatives, with members belonging to the class of the haloarchaea dominating. In the conventional metagenomic studies already carried out on such microbial communities26, a high abundance of genes related to anaerobic arsenate respiration (arr) as well as arsenite oxidation (aio) was detected, strongly supporting the assumption that arsenic is used in bioenergetic processes. In addition, genes related to detoxifying mechanisms for removal of intracellular arsenic were also found, e.g. the acr3 gene and the arsABCRD operon. However, in those studies, the impact of plasmids is possibly underestimated because it is difficult to discriminate them from the chromosomal DNA, due to their low number of copies and corresponding low proportion. In this study, we assessed the plasmidome of the red biofilm from Diamante Lake  as it concerns an environment similarly loaded with high concentrations of metals15. Potential hosts as well as encoded functions were identified.Andean Puna constitutes a large reservoir of Andean Microbial Ecosystems (AMEs) including biofilms, microbial mats, microbialites and endoevaporites, all of which exposed to multiple extreme conditions such as high UV irradiation, and—due to the high altitude—low oxygen pressure, large thermal fluctuations, high dryness, hypersalinity, alkalinity, high concentrations of heavy metals and metalloids such as arsenicSamples were aseptically taken from red biofilms attached to gaylussite crystals at the bottom of submerged microbialites in the Diamante Lake, Catamarca, Argentina  in September 2019 .In parallel, metagenomic DNA was extracted from red biofilm samples by using the FastDNA Spin kit for soil as recommended by the manufacturer . The extracted metagenomic DNA served as template for 16S rRNA gene amplicon sequencing.27; and for archaea F5′-CCCTAYGGGGYGCASCAG-3′ (Arc_340F) and R5′-ATTAGAKACCCSNGTAGTCC-3′ (Arc_806R)28. The plasmid DNA was purified using the SureClean Plus kit .The plasmid DNA was subjected to overnight digestion at 37 °C with exonuclease V Rec BCD  to remove chromosomal DNA. PCR reactions using universal primers covering the V4 region of 16S rRNA gene were performed to check for chromosomal DNA contamination. The primers used for bacteria were F5′-CCTACGGGNGGCWGCAG-3′ (Bac_341F) and R5′-GGATTAGATACCCBDGTAGTC-3′ (Bac_785R)29. Finally, the reads were de novo assembled by using SPAdes software v3.9.0 with the -meta parameter to call the metaSPAdes module30. Recycler algorithm was used to assemble cyclic sequences, which are likely plasmids, phages and other circular elements from the assembly graphs provided by SPAdes31. The bioinformatic analysis pipeline described by Kothari et al.17 was also used to identify the complete closed circular contigs. The circular elements obtained in both cases were compared to DoriC 10, a database of replication origins in prokaryotic genomes including chromosomes and plasmids32.Illumina shotgun paired-end sequencing libraries were generated from isolated plasmid DNA using the Nextera XT sample preparation kit as recommended by the manufacturer . The MiSeq system together with the MiSeq reagent kit version 3 (600-cycle) was used for the plasmidome sequencing as recommended by the manufacturer (Illumina). The quality control of raw sequence reads was carried out with FastQC v0.11.9 and the reads were quality-filtered using Trimmomatic v0.38.033. Metagenome contigs were assembled using SPAdes v3.9.0 from sequencing data of three independent red biofilm samples taken on another occasion and published by Saona et al.26.The reads generated by sequencing of the plasmid DNA were aligned with the metagenome contigs using the Bowtie2 tool34. The assembled plasmidome dataset was submitted to the MG-RAST server35 for functional and taxonomic analysis. Comparisons with the SEED subsystem database were performed by using a maximum E-value of 10–5. The deduced functional profile of the red biofilm plasmidome was compared with the one derived from the metagenome mentioned above26 by employing the software STAMP 36. Comparisons with other plasmidomes were also performed18.Annotation and labeling of all the relevant genomic characteristics on plasmidome contigs were done with Prokka v1.14.518. In addition, the plasmidome contigs were compared to TADB 2.0 database by blastn in order to identify toxin–antitoxin (TA) systems37. Furthermore, the Prokka annotation file of the plasmidome was subjected to Conditional Reciprocal Best BLAST (crb-blast) against plasmid genes sequences from the ACLAME database with an E-value ≤ 10–338. Hits with an identity ≥ 70% and an alignment coverage ≥ 90% were selected. Similarly, putative genes encoding metal resistance and virulence factors were also searched by using the BacMet39 and VFDB databases40, respectively.Both the known plasmid sequences from NCBI database and the domains related to plasmid replication and mobilization were assessed as described previously by Perez et al.22, arsenic resistance-related genes were separately annotated. For this purpose, the amino acid sequences were downloaded from Uniprot and were subjected to Position-Specific Iterated BLAST (PSI-BLAST)41. CD-HIT v4.8.1 was used for creating non-redundant datasets42 and Clustal Omega v1.2.4 for sequence alignments43. Profiles Hidden Markov model were build and searched for in the plasmidome translated gene sequences identified with Prokka by using HMMER 3.3 44.Due to the high arsenic concentration found in the lake45 as a reference.The Resistance Gene Identifier (RGI) software was employed for prediction of antibiotic-resistance genes using the Comprehensive Antibiotic Resistance database (CARD)46, and the HMM profiles downloaded from TnpPred web47 for prediction of prokaryotic transposases by HMMER 3.3.The ISEScan software pipeline was used to search for mobile elements such as insertion sequences48. First, paired-end reads were joined with PEAR v0.9.649. Quality-filtering was performed using the split_libraries_fastq.py script. Forward and reverse primers were removed by using cutadapt v1.1650. USEARCH v1151 was used for zero-radius operational taxonomic unit (zOTU) determination. Taxonomy was assigned against Silva 132 database52.16S rRNA gene amplicon sequencing was performed using the above-described primers partially covering the 16S rRNA gene sequence. The MiSeq system together with MiSeq reagent kit version 3 (600-cycle) was used for sequencing of the amplicons as recommended by the manufacturer (Illumina). Data quality control and analysis were performed using the QIIME software18.Illumina sequencing from the Diamante Lake plasmidome generated 1,071,941 paired-end reads, of which 941,587 passed quality-filtering. The SPAdes assembler produced 13,492 contigs (> 500 bp) corresponding to roughly 16.9 Mb (largest contig 20.415 bp) . Exclusively hypothetical proteins could be annotated from the open reading frames present in the circular elements.Thirty-nine closed replicons were predicted by the Recycler software (> 1000 bp), the largest consisting of 3313 bp, but none displayed a known plasmid origin of replication when compared to the DoriC 10.0 database. The bioinformatic pipeline used to detect circularity produced 20 circular contigs. The largest comprised 8295 bp and the smallest 2025 bp. Only one of them showed a known plasmid origin of replication , corresponding to pSN found in 18. The difference between the two environments displaying rather similar environmental characteristics is possibly due to the large proportion of archaea present in the Diamante Lake, as archaeal genomes contain typically a higher fraction of “dark matter” when compared to bacterial genomes. The isolation and cultivation of most of the archaea, and accordingly, the experimental characterization of archaeal gene products, is challenging53. Likewise, Sentchilo et al.15 also reported 52 and 66% of coding sequences without assigned function in two wastewater treatment plant plasmidomes.MG-RAST analysis revealed that the Diamante Lake plasmidome contains a large fraction of unknown DNA as 18,314 sequences (41.5%) code for predicted proteins with known functions but 25,800 sequences (58.5%) encode putative proteins of unknown function. Thus, such deep-sequencing approaches are suited to detect novel proteins with so far undiscovered functions. In our previous plasmidome analysis from an AME, 39% of the predicted proteins could not be functionally annotated58.From the functional SEED assignment, only 5196 predicted proteins were annotated 28.4%), most of them covering basic metabolic functions such as DNA, RNA and protein metabolism  , and it was found to dominate most of the thalassic NaCl-saturated environments68.In addition to the above plasmid replication and mobilization entries, sequences harboring genes involved in plasmid maintenance such as loci corresponding to toxin–antitoxin (TA) systems were identified (identity and coverage at least 85%). All of the TA systems belong to type II TA-loci  were annotatedThe bias necessarily introduced by the existing databases and developed from the information currently available, as well as the dominance of the archaea in the microbial community studied interfere with the analysis of the resistome encoded by the Diamante Lake plasmidome. Thus, it cannot to be excluded that the lack of relevant knowledge is the reason for the low number of identifiable ARGs and virulence factors.73. The most common resistance system is encoded by the ars operon for which different genetic organizations were described among prokaryotes74. In addition to the extrusion systems, composed of the gene-products encoded by arsA, arsB, arsC, arsD, arsR, and acr3, another mechanism involving a putative arsenite(III)-methyltransferase (ArsM) was reported in Halobacterium sp. NRC-175. We identified 28 proteins possibly related to arsenic resistance; ten of them had been automatically annotated as "hypothetical proteins" by Prokka (Supplementary Table arr) and arsenite oxidation (aio) were not detectable in the plasmidome, as these are usually encoded by the chromosome.Regarding the resistances to metals, a respective search in the BacMet database produced no hits, possibly also due the above reason as the database consists solely of bacterial entries. However, our manual annotation disclosed the presence of arsenic resistance genes. Arsenic hits various microorganisms, however, several bacteria and archaea possess detoxification systems enabling growth even under high As-concentrationsars operons can vary among diverse microorganisms. In the Diamante Lake plasmidome, arsC (arsenate reductase) and acr3 (arsenite efflux transporter) genes were present twice in close proximity, but the most relevant was the arsADR gene cluster of contig 116 , which was isolated from a freshwater pond (NZ_CP007061.1). Fourteen matches were detected between the plasmidome sequences and the sequences of the plasmid pHLAC01 (431 kb) of Halorubrum lacusprofundi ATCC 49239 that was isolated from the Deep Lake, a hypersaline Antarctic site. It is noteworthy that one of these matches  corresponds to contig 116 of the plasmidome, which harbors the arsDA genes  . In the Puquio de Campo Naranja plasmidome, a much lower number of IS (28) was reported. Again, most of them were assigned to the IS5, IS630 and IS4 families18. The absence of Tn3 family transposases in both of the plasmidomes is conspicuous, as it represents one of the most abundant families in bacterial genomes, and Tn3 elements preferentially transpose into plasmids79.A total of 532 insertion sequences (IS) were identified in the Diamante Lake plasmidome, with IS200/IS605 and IS5 like elements being the most frequent ones followed by members of the IS4, IS6, IS630 and ISH3 families  is dominating.When the phylum distribution of the plasmidome was compared with 16S rRNA sequencing data from the corresponding metagenomic DNA sample, the phylum Euryarchaeota again stood out with the highest relative zOTU abundance Fig. A. In botIt is currently possible to study plasmid elements in the course of a conventional metagenomic analysis, but an approach to specifically target plasmid populations allows to overcome the inherent constraints of the bioinformatic tools applied for the analysis of plasmids from total community DNA. Under this perspective, and from the comparison with the metagenome of the same community, this study showed that part of the plasmid information will not be detected when the experimental plasmid-purification is not carried out prior to sequencing. Furthermore, a large fraction of genes with an unknown function was present in the plasmidome dataset, as at least 58.5% of the predicted proteins were hypothetical. In addition, the percentages of SEED assignments were even lower. The relatively few functional annotations may accord with the peculiarities of the extreme environment, which harbors a microbial community that is dominated by archaea. On the other hand, functions related to the response to oxidative stress and DNA repair were annotated, which agrees with the requirement of adaptive mechanisms enabling the hosts to withstand the exposure to the high UV irradiation in the Andean Puna.Comparison of the Diamante Lake plasmidome to that of Puquio de Campo Naranja, revealed a certain degree of similarity between the predicted functional profiles of both AMEs. However, striking differences with respect to antibiotic and arsenic resistance were detected. Sequences pointing to arsenic resistance are more abundant in the Diamante Lake plasmidome, a fact that also accounts for the plasmidome derived from a wastewater treatment plant that contains large quantities of effluents of the chemical/pharmaceutical industry. Our results reflect the high amount of arsenic present in the environment under investigation. Traits expected to be found in a plasmid pool were detected, such as Pfam domains related to plasmid replication, MOB-type relaxase families related to plasmid mobilization and genes belonging type II toxin–antitoxin systems related to plasmid maintenance. Moreover, there are sequences known from megaplasmids of halophilic archaea isolated from different saline environments, which provides further evidence for known plasmid-associated genes in the obtained dataset.The results presented here along with the detection of numerous IS elements favors the opinion that the plasmidome facilitates the mobility and the transfer of genes within such extreme microbial communities.Supplementary Figures.Supplementary Tables.


Nanoemulsions (NEs) are water-dispersed oil droplets that constitute stealth biocompatible nanomaterials. NEs can reach an impressive degree of fluorescent brightness owing to their oily core that can encapsulate a large number of fluorophores on the condition the latter are sufficiently hydrophobic and oil-soluble. BODIPYs are among the brightest green emitting fluorophores and as neutral molecules possess high lipophilicity. Herein, we synthesized three different natural lipid-BODIPY conjugates by esterification of an acidic BODIPY by natural lipids, namely: α-tocopherol (vitamin E), cholesterol, and stearyl alcohol. The new BODIPY conjugates were characterized in solvents and oils before being encapsulated in NEs at various concentrations. The physical  and photophysical properties  are reported and showed that the nature of the lipid anchor and the nature of the oil used for emulsification greatly influence the properties of the bright NEs. Among nanomaterials, lipid nanoparticles draw a particular attention owing to the biocompatibility of their components ,2. Nanoes-indacene) seems perfectly adapted for this purpose, as it is a noncharged green-emitting fluorophore. Moreover, it has received considerable interest in bioimaging applications owing to its desirable properties, including narrow emission band, high quantum yield and molar extinction coefficient (ε), making it a bright-green fluorophore [8 for oil/polymer hybrid NPs [12 constitutes an interesting approach as it is composed of cholesterol, a natural nontoxic lipid. In bioimaging the “green channel” is generally the most accessible due to the widely spread argon laser line at 488 nm as well as the wide use of GFP-labeled proteins in biological studies. Therefore, bright green emitting NEs would be a valuable tool for studying their potential application in biology and medicine using fluorescence bioimaging. Cationic cyanine DiO ,13 and porophore ,16,17. Sorophore , P-B andorophore ,20, PS-2orophore , and BDPbrid NPs . Althoug18 hydrocarbon chain that was shown to have a very low toxicity (LD50 > 5000 mg/kg in rat) [In this work we thus synthesized three natural lipid BODIPY conjugates bearing a biocleavable ester bond  and stud in rat) . NEs com3) at a concentration of 15 mg.mL−1 on a Bruker Avance III 400 MHz spectrometer . Mass spectra were obtained using an Agilent Q-TOF 6520 mass spectrometer ; the samples were submitted in dichloromethane at a concentration of 0.1 mg.mL−1. The protocol for synthesis of all new compounds as well as NMR and mass spectra can be found in the Supporting Information.All starting materials for synthesis were purchased from Sigma-Aldrich, TCI or Alfa Aesar and were used as received unless stated otherwise. Labrafac WL 1349  was obtained from Gattefossé , Vitamin E Acetate (VEA) was purchased from Tokyo Chemical Industry , Kolliphor ELP was from BASF . NMR spectra were recorded in deuterated chloroform  and a FluoroMax-4 spectrofluorometer  equipped with a thermostated cell compartment, respectively. For standard recording of fluorescence spectra, the emission was collected 10 nm after the excitation wavelength. Concentration used for spectroscopic study was set as 1 μM for each dye. The molecular extinction coefficients were obtained from a stock solution that was obtained by dissolving at least 5 mg of BODIPY conjugate in dioxane. The quantum yields were determined by comparison with fluorescein in 0.1 M NaOH (QY = 0.95) as reference  using thSaturated solutions of dyes in oils were prepared by weighting 12 mg of dye to which was added 100 mg of oil at 80 °C (due to the high viscosity of oil) under stirring. The solution was cooled down to room temperature before being centrifuged at 10,000 rpm for 1 min. 5 μL of the supernatant was taken and diluted in dioxane for an absorbance measurement. Solubility of individual dye was calculated by the following equation:Nanoemulsions (NEs) were formulated by spontaneous emulsification method, as described previously . BrieflyThe obtained NEs were characterized by dynamic light scattering (DLS) Malvern Zetasizer ZSP  with a laser operating at 633 nm, and the temperature was maintained at 25 °C. The average size and polydispersity index (PDI) of the NEs were measured three times from three independent formulations. The formulations were stored for two weeks at room temperature for a stability study, and size and PDI were measured.−1.cm−1), 470 nm was chosen as the excitation wavelength (slit opening was set to 16 nm). The emission signal was monitored at 600 nm for all fluorophores. The measurement was conducted over 1 h. A relative fluorescence intensity (%) was normalized to the maximum intensity at the starting point. For the loading dependency experiment, the same method was used except that the emission wavelength was set to 500 nm to increase the photodamage.NEs with 1% dye loading were chosen for the photostability test. All the NEs were diluted to keep the dye concentration at 1 μM in the final solutions. To make sure that all the NEs were excited at the same extinction coefficient  water immersion objective. Excitation was provided by a cw laser diode  and photons were detected through a bandpass 525/50 nm filter  with a fibered avalanche photodiode  connected to an online hardware correlator . The typical acquisition time was 5 min (10 × 30 s) with an excitation power of 0.05 mW at the sample level. The data were analyzed using PyCorrFit software . The FCSmeso position [1H and 13C NMR as well as mass spectroscopy , probably denoting a difference in solvatation or interaction for the two oils. Compared to MCT, BDPs in VEA systematically displayed lower ε, which was compensated with higher quantum yields in the case of BDP-C18 and BDP-Toco, reaching up to 0.98 quantum yield for the latter (18 and BDP-cholesterol formed relatively bright orange emitting aggregates in water (λEm = 570–576 nm) with quantum yield reaching 0.13 and 0.18, respectively , which is comparable with other dyes used for loading NEs [The solubility of the encapsulated dye in NEs is of prior importance as it determines the maximum loading capacity of the NE droplets, and hence condition the maximum brightness that the NEs can reach. We thus assessed the maximum solubility of our BDP conjugates in MCT and VEA that served for nanoemulsification ,25. As iding NEs . On the 18 and up to 12 wt % for BDP-Toco. To this endeavor, spontaneous nanoemulsification with nonionic PEGylated surfactant (Kolliphor ELP) as a water/oil interface stabilizer was employed , regardless of the used lipid oils and the BODIPY-lipid conjugates  [In a second step, the quantum yields of the different NEs were measured. The results showed that the NEs conserved high quantum yields (~0.9) even at 1 wt % loading  .As discussed in the introduction, the dye encapsulated in NEs should not leak out. Indeed, dye leakage leads to a loss of NEs’ brightness as well as an increase of the background noise in bioimaging and labeling of the hydrophobic compartments such as membranes or proteins . We thusResults presented in Moreover, their size also remained stable, showing the stealth of the NEs, where PEGylated shell probably prevents nonspecific binding of serum proteins. Finally, FCS was performed on the highly loaded BDP-Toco NEs (from 1 to 12 wt %). Surprisingly, at high loadings the size and concentration of fluorescent species only slightly increased in the presence of FBS, denoting a strong retention of the BDP-Toco dye in the NEs even at loading as high as 12 wt % . AdditioEx = 500 nm). The results clearly showed that a higher BDP loading led to NEs with lowered photostability  NEs. However, FCS and photostability assays indicated that highly loaded and bright NEs suffer from an important photo instability, probably due to the tendency of the BODIPY fluorophore to undergo destructive photochemical reactions when concentrated, thus limiting its use in NEs tracking. Previously developed fluorescent NEs generally operate in the red and near-infrared regions, [The development of novel fluorescent nanoprobes with remarkable optical properties, suitable particle size, high water dispersibility and good biocompatibility are highly desirable for applications in the biomedical field. In this work, we introduced three BODIPY conjugates with natural lipid anchors, namely tocopherol (vitamin E), cholesterol and stearyl alcohol. Those three dyes exhibit high molar extinction coefficient, quantum yield and solubility in oils, making them good candidates for encapsulation in NEs. As expected, bright fluorescent nanoemulsions were obtained by spontaneous emulsification with small sizes ranging from 40 to 75 nm, good polydispersity index, and without obvious leakage in the presence of FBS. Among the BODIPY conjugates, BDP-Toco is an oil with high miscibility in MCT and VEA. This specific property allowed to obtain NEs with high dye loading percentage and thus ultrabright NEs due to relatively low aggregation-caused quenching (ACQ) at high concentration of BDP. The combination of tocopherol acetate (VEA) and tocopherol BDP ester (BDP-Toco) constituted an effective match as it led to small (~40 nm), physically stable, hermetic (no leakage) and ultrabright (brightness = 45.10regions, ,10,11 whregions, . In the regions,  and peryregions,  while be


Is prenatal exposure to arsenic and other metals associated with the occurrence of atopic dermatitis in young children?In this cohort study of 586 mother and child pairs, prenatal arsenic exposure was associated with approximately 2.4 times the occurrence of atopic dermatitis in children at age 4 years. In addition, coexposure to arsenic and cadmium was associated with increased odds of developing atopic dermatitis.The results of this study suggest that prevention of inorganic arsenic and cadmium exposure among pregnant women could reduce the risk of atopic dermatitis and other allergic diseases in children. The prevalence of atopic dermatitis has substantially increased in recent decades, and atopic dermatitis could lead to allergic airway inflammation later in life. A previous study found that inorganic arsenic exposure was associated with allergic airway inflammation in children aged 8 to 14 years. However, the association between prenatal exposure to arsenic and other metals and the risk of atopic dermatitis among young children remains unknown.To assess the association between prenatal exposure to arsenic and other metals and the occurrence of atopic dermatitis in children at age 4 years.In total, 1152 pregnant women were enrolled in the original Taiwan Maternal and Infant Cohort Study (TMICS), a multicenter birth cohort study conducted at 9 hospitals in northern, central, southern, and eastern Taiwan from October 2012 to May 2015. Of those, 586 mothers and children aged 4 years participated in follow-up questionnaire interviews from August 2016 to January 2019. After excluding 216 participants with missing data, the final statistical analysis of follow-up data included 370 mother and child pairs from the central and eastern regions of Taiwan. Data were analyzed from February 2 to August 12, 2021.Arsenic, cadmium, lead, cobalt, copper, nickel, thallium, and zinc during pregnancy.The outcome was parent-reported atopic dermatitis history among children aged 4 years. The presence of atopic dermatitis was defined as a positive response to the question, “Has your child ever had atopic dermatitis diagnosed by a physician?” During the initial TMICS study period, concentrations of arsenic, cadmium, lead, cobalt, copper, nickel, thallium, and zinc were measured in maternal urine during the third trimester of pregnancy using an inductively coupled plasma mass spectrometer. Estimated total inorganic arsenic exposure was calculated using a model that included data on both total arsenic and arsenic species  obtained from a previous TMICS cohort.Among 370 children included in the analysis, the mean (SD) age was 3.94 (0.59) years; 208 children (56.2%) were male, and 267 children (72.2%) were from the central region of Taiwan. A total of 110 children (29.7%) had atopic dermatitis at age 4 years. Maternal estimated total inorganic arsenic exposure during pregnancy was associated with increased odds of atopic dermatitis among children at age 4 years  after adjusting for parental allergies, child’s sex, geographic area, maternal educational level, and exposure to tobacco smoke. Every increased unit in the weighted quantile sum index of maternal metal exposure was significantly associated with atopic dermatitis . Arsenic (40.1%) and cadmium (20.5%) accounted for most of the metal mixture index.This cohort study found that prenatal exposure to inorganic arsenic and coexposure to inorganic arsenic and cadmium were associated with a higher risk of atopic dermatitis in young children. These findings suggest that prevention of exposure to inorganic arsenic and cadmium during pregnancy may be helpful for the control of atopic dermatitis and other potential allergies in children. This cohort study examines the association between prenatal exposure to arsenic and other metals and the development of atopic dermatitis among young children in central and eastern Taiwan. A total of 1152 pregnant women were enrolled in the initial TMICS from October 2012 to May 2015. Mothers and children from the central and eastern regions were later invited via telephone to participate in follow-up interviews conducted from August 2016 to January 2019. In total, 566 participants were unavailable for follow-up because they moved away from the area without leaving contact information or had scheduling conflicts, and 586 children were successfully followed up at age 4 years. A total of 216 children were excluded from the final analysis because they were missing data on (1) maternal urinary metal concentrations (n = 137); (2) allergic diseases in children at age 4 years (n = 54); or (3) exposure to environmental tobacco smoke among children at age 4 years, maternal educational level or allergic diseases, or child’s sex (n = 25). The final analysis of follow-up data included 370 mother and child pairs . The stuStructured questionnaires, including items about demographic characteristics, lifestyle, dietary patterns, and residential environment, were administered to all participating women by trained interviewers during the third trimester of pregnancy. Physical measurements  and urine samples were obtained, with urine samples collected in glass bottles and stored at −20 °C until analysis. Follow-up interviews were conducted in person at participants’ homes or participating hospitals. The presence of atopic dermatitis among children was defined as a positive response to the question, “Has your child ever had atopic dermatitis diagnosed by a physician?” during the questionnaire interview at age 4 years.−1 for arsenic, 0.066 μg/L−1 for cadmium, 0.016 μg/L−1 for lead, 0.225 μg/L−1 for cobalt, 0.090 μg/L−1 for copper, 0.022 μg/L−1 for nickel, 0.008 μg/L−1 for thallium, and 0.417 μg/L−1 for zinc. For instances in which the outcome was below the limit of detection, concentrations were estimated as the limit of detection divided by the square root of 2. Metal concentrations in maternal urine were divided by urine creatinine for urine volume correction (μg/g creatinine).Frozen urinary specimens were moved to a 4 °C refrigerator for 2 to 4 hours, then defrosted at room temperature (26 °C-28 °C). Urinary metal concentrations were quantified using an inductively coupled plasma mass spectrometer  with a quadrupole mass filter and autosampler. For quality control, we used standard reference materials, spike recovery rate, and blank and duplicate samples. We performed the quality control regimen for every 10 samples, and the control spike recovery rates ranged from 89.0% to 114.5%. The limits of detection were for 0.399 μg/L8 derived from 336 women for whom both total urinary arsenic and total inorganic arsenic concentrations were available .The urinary concentration of inorganic arsenic represents the toxic form of total arsenic exposure, and the urinary concentration of arsenic species  provides a better estimate of inorganic arsenic exposure than the concentration in blood. We calculated total inorganic arsenic as the sum of arsenite, arsenate, monomethylarsonate, and dimethylarsenate. We estimated total inorganic arsenic using a linear regression analysis of data from a previous TMICS pilot studyr = 0.61; P < .001)  and the risk of atopic dermatitis in children. We calculated Q values to assess the multiple comparison effect and reduce type 1 error probability.17Mean values with SDs were used to summarize continuous variables, and 2-sided unpaired 18 We also calculated propensity scores for potential imbalances in exposure groups, and we reduced bias by using a process resembling randomization.19 Previous studies have reported that parental allergies might be associated with 30% of the risk of asthma in children20 and the risk of atopic dermatitis in infants.21 In addition, maternal allergies may be associated with a higher risk of atopic dermatitis among children compared with paternal allergies.22 Therefore, we stratified groups by maternal history of allergic diseases  and included only participants with complete data on maternal allergies.We adjusted for the child’s sex , the presence of parental allergies (yes or no), geographic area , exposure to tobacco smoke during pregnancy and among children at age 4 years (yes or no), and maternal educational level . In sensitivity analyses, we used a directed acyclic graph to select the minimum sufficient factor adjustment for the model.23 The data were randomly divided into training (40%) and validation (60%) sets. We used a bootstrap method on the training data set to quantify the WQS index. The validation data set was then used to test the accuracy of the WQS index. The possible selection threshold of metals was defined as the weight of each metal that was greater than the quotient (0.125) of the total sum of weights in the WQS index (sum = 1) divided by the number of metals (n = 8). We used SAS software, version 9.4 (SAS Institute), to analyze the results of t tests, Mann-Whitney U tests, χ2 tests, and logistic regression analyses, and we used R software, version 4.0.2 , to calculate generalized WQS. A 2-sided P value of less than .05 was considered statistically significant. Data were analyzed from February 2 to August 12, 2021.In the present study, maternal urinary metal concentrations were highly correlated among metals , maternal educational level , annual household income , the presence of maternal allergies , child’s age , maternal age , child’s gestational age at birth , and maternal urinary concentration of copper   age was 3.94 (0.59) years, and the mean (SD) gestational age at birth was 38.68 (1.14) weeks; 208 children (56.2%) were male, and 162 children (43.8%) were female. In total, 267 children (72.2%) were from the central area, and 103 children (27.8%) were from the eastern area . A totalP < .001) and had higher annual household income , lower exposure to tobacco smoke at age 4 years , a slightly lower gestational age at birth , and higher maternal urinary concentrations of cadmium , lead , nickel , and zinc  (eTable 3 in P = .004) (P = .03), a higher incidence of paternal allergies , and higher maternal urinary concentrations of arsenic  and estimated total inorganic arsenic .Children from the central vs eastern region were older  . ChildreP = .003) higher odds of atopic dermatitis in children at age 4 years . In the sensitivity analysis, a significant association was found between maternal urinary arsenic concentrations and estimated total inorganic arsenic concentrations and atopic dermatitis according to the directed acyclic graph  and propensity scores  . Arsenic (40.1%) and cadmium (20.5%) accounted for most of the WQS index  after adjusting for sex, maternal educational level, geographic area, and exposure to tobacco smoke (P = .01]) and the sensitivity analysis . For those without maternal allergies, no association was found between arsenic concentrations and atopic dermatitis .Among children with mothers who had allergies, every doubled increase in maternal urinary arsenic concentrations and estimated total inorganic arsenic concentrations was significantly associated with increased odds of atopic dermatitis  2 cells in the acute phase and from TH1 cells in the chronic phase, and TH2 cells have been suggested to have important roles in cutaneous allergic inflammation as an integral part of atopic dermatitis.25 A previous study reported that newborns with the specific epigenetic pattern of TH2 cell-skewed immunity may have increased responses to common environmental allergens later in life.26 Exposure to arsenic during pregnancy has been associated with a DNA methylation pattern in infants27 as well as changes in children’s immunoglobulin E levels.28The pathophysiological features of atopic dermatitis include epidermal barrier dysfunction, skin inflammation, interaction between the nervous and immune systems, genetic susceptibility, and the effect of microbiota.29 Kim et al30 observed an association between prenatal cadmium exposure and atopic dermatitis in infants aged 6 months, and elevated cadmium concentrations in cord blood were associated with an increased risk of atopic dermatitis.Although metals other than arsenic  were examined in this study, these metals were not associated with the incidence of atopic dermatitis in children at age 4 years; however, an association was found between prenatal coexposure to these metals (including arsenic) and atopic dermatitis in children. The exposure to cadmium was secondary to arsenic in the WQS index. Cadmium is also a well-established toxic metal that can detrimentally impact many organs, including the bones, kidneys, and pancreas.31 treated pregnant mice with 10 ppm of cadmium chloride as an environmentally relevant dose. They found that cadmium exposure may induce decreases in the production of interferon gamma and CD8+CD223+ T cells in the spleens of their offspring at age 7 weeks. A recent study reported that cadmium concentrations mediated by in-cell viability had differentially functional effects on in vitro murine macrophages and mast cells after cadmium exposure.32 Mast cells and macrophages have been suggested to play a role in atopic dermatitis–associated inflammation and pruritus. For example, interleukin 31 is derived from TH2 cells, macrophages, mast cells, and mast cells in atopic dermatitis lesions with higher interleukin 31 immunoreactivity than normal skin.33 Interleukin 23, secreted by dendritic cells and macrophages, has been documented to be an important cytokine to control inflammation in peripheral tissues. Skin tissues of patients with atopic dermatitis have substantially increased activation of the TH17/interleukin 23 and TH22 axes and keratinocyte production.34 Thus, prenatal exposure to cadmium may be associated with atopic dermatitis in early childhood through disruption of immune cell development in the fetus.Hanson et al35After stratifying the analysis by maternal history of allergic diseases, we also found a significant association between maternal arsenic exposure and atopic dermatitis in children at age 4 years. One plausible explanation for this association may be heritability, including genetic and epigenetic factors. Children with mothers who had allergies had greater odds of developing atopic dermatitis, which suggests a genetic role and an association with the in utero environment, which could shape neonatal immunity and, consequently, the development of atopic dermatitis in early childhood.38 Therefore, improving the environment and reducing contact with these potential sources are important steps to reducing the risk of exposure to metals in this susceptible population.The main sources of exposure to metals are diet, air pollution, or cigarette smoking (active or passive) among the general population, and pregnant women are particularly susceptible to metal exposure.39 Our participants are children aged 4 years, and atopic dermatitis is the most commonly observed allergy that can be well diagnosed. The results were similar to those obtained when the analysis was adjusted for the presence of different urinary metals at age 4 years (data not shown), probably owing to the different exposure scenarios. We used well-designed methods for data collection. We examined the association between coexposure to a mixture of metals and atopic dermatitis because of the complex consequences of metal exposure for human health, and some urinary metal concentrations were highly correlated.40This study has strengths. Its main strength is its birth cohort design, in which urinary samples were collected from pregnant women in the third trimester and used to estimate the likelihood that children would develop atopic dermatitis 3 or 4 years later. The use of urine sampling could have increased the participation rate among pregnant women because urine sampling is less invasive than venous blood sampling. Moreover, blood arsenic concentrations only reflect recent exposure and cannot be used as a reliable estimate of chronic exposure.41This study also has limitations. First, there may be insufficient power in the statistical analyses because of the relatively small sample. However, the bootstrap method used in the generalized WQS model was applied to the training data set to reduce the partial effects of insufficient power. Second, we collected maternal urine samples in glass bottles; thus, we only measured total arsenic concentration and did not separately measure the concentrations of individual arsenic species. Therefore, total inorganic arsenic values were not measured directly but estimated using an equation derived from data on total arsenic and total inorganic arsenic concentrations from a previous birth cohort study among pregnant women residing in the same geographic area. Two basic metabolic processes of inorganic arsenic are oxidation-reduction and methylation reactions. Approximately 70% of absorbed organic and inorganic arsenic is excreted from the kidneys via urine.Third, we used parent-reported atopic dermatitis in this study without obtaining data from a complete clinical examination conducted in parallel by a physician. The reliance on parental reporting might produce underestimation of the results through nondifferential misclassification because the women did not have knowledge of their urinary metal concentrations in the third trimester; thus, they would not have been likely to overreport their children’s atopic dermatitis . Moreover, the parent-reported presence of atopic dermatitis in children was based on the question, “Has your child ever had atopic dermatitis diagnosed by a physician?” This question may be more reliable than, “Has your child ever had atopic dermatitis?”42 In addition, we adjusted the model for the presence of parental allergies and further stratified the analysis by maternal allergic status to reduce partial confounding from genetic factors.Fourth, we reported observations among a birth cohort of children aged 4 years residing in the central and eastern regions of Taiwan; thus, external validity may be low, and results may not be generalizable to all children aged 4 years in Taiwan. Fifth, the possibility of residual or unmeasured confounding from genetic factors and exposure to other environmental chemicals cannot be eliminated. Epigenetic factors, such as modifications to genomic DNA and posttranscriptional micro-RNA regulation, have also been associated with the development of atopic dermatitis.This birth cohort study found an association between prenatal coexposure to metals and atopic dermatitis in children at age 4 years. Arsenic followed by cadmium accounted for most of the metal mixture index associated with atopic dermatitis. Prevention of exposure to inorganic arsenic and cadmium during pregnancy may be helpful for the control of atopic dermatitis and other potential allergic diseases in young children. Environmental improvements and reductions in contact with sources of exposure are important to reduce the risk of metal exposure among this susceptible population.


Moringa oleifera leaves have been described as having high antioxidant content. This work investigates the effect of dietary supplementation with Moringa leaves on the antioxidant activity of seabream. Gilthead seabream specimens were divided into four groups, the control group  and three other groups fed diets enriched with Moringa . The antioxidant capacity was measured by assays of free radical scavenging , Rancimat test and linoleic acid system in muscle and skin of gilthead seabream, commercial diet, enriched diet and Moringa. Finally, the polyphenol content of Moringa and the fatty acid composition of seabream fed diets with and without Moringa were determined. Results showed an increase in antioxidant activity in gilthead seabream fed with diets enriched with a higher percentage of Moringa; therefore, Moringa could be considered a functional ingredient in diets for fish bred in fish farms and. The antioxidant potential of Moringa leaves could be mainly attributed to the presence of polyphenolic compounds.Gilthead seabream is bred mainly in fish farms in the Mediterranean Sea. One important factor responsible for the deterioration of fish quality is lipid oxidation.  The close relationship between food and health has led to changes in consumer habits. There is a current trend of modification in feeding of animals to obtain healthy benefits for the consumer, as well as an increase in the nutritional quality of food, without altering its sensory characteristics. The feeding of animals with plants containing antioxidant compounds might serve to introduce the antioxidants into their body which, in turn will be consumed by the population ,2.There is a growing interest in reducing free radicals present in food that justifies the usefulness of natural antioxidants in order to improve quality, texture, colour, taste, nutritional value and increased shelf life of food. Natural antioxidants are mainly obtained from food such as fruits, vegetables, cereals, spices and traditional medicinal herbs and plants ,4.Moringa oleífera L.) is a tropical plant containing small or medium-sized perennial leaves. This plant is native to northern India and is a widely cultivated spice [The Moringa , the species selected in this study. In addition, moringa has been reported to reduce stress biomarker levels in rabbits [Oreochromis niloticus) and common carp (Cyprinus carpio) [Interest in the study of Moringa has been shown in various regions of the world where the population has nutritional problems. The consumption of the leaves, green pods and seeds of this plant has been promoted as a source of various nutrients ; leaf fl rabbits  and seve carpio) ,16.Taking into account the recent increase in foods enriched with antioxidants and the growing interest in the scientific community of the benefits of the intake of foods with antioxidant properties, the aim of the proposed study is to assess the effect of diets enriched with Moringa, in the antioxidant activity of gilthead seabream, which was selected for being one of the most important aquaculture species in the Mediterranean Sea and, therefore, for human consumption.S. aurata, L.) were obtained from a local farm  and acclimatized for 2 weeks in recirculating seawater aquaria (250 L) at the Marine Fish Facility, University of Murcia. The water temperature was maintained at 20 ± 2 °C with a flow rate of 900 L/h and 28% salinity. The photoperiod was adjusted to 12 h light/12 h dark. Fish were fed with a commercial pellet diet  at a rate of 2% body weight/day. Fish were allowed to acclimatise for 15 days before the start of the experimental trial. All experimental protocols were approved by the Ethical Committee of the University of Murcia and carried out in accordance with EU Directive 2010/63/EU [Eighty (125.7 ± 9.5 g weight) specimens of the marine teleost gilthead seabream (10/63/EU  for anim−1 water).Eighty fish were randomly distributed into four identical tanks (20 fish per group), where the following groups were established: (1) control, non-supplemented diet (0%); (2) 5%, diet supplemented with 5 g/100 g of Moringa leaves; (3) 10%, diet supplemented with 10 g/100 g of Moringa leaves; (4) 15%, diet supplemented with 15 g/100 g of Moringa leaves. The fish were fed twice daily for a four-week period at a rate of 2% body weight per day. Twenty specimens from each group were sampled at the end of the trial. All specimens were sacrificed with an overdose of MS-222  was mixed with a suitable amount of Moringa and compacted into granules, thus obtaining diets supplemented with 5%, 10% and 15% Moringa. The prepared diet was subjected to a drying process and stored at 4 °C. The composition of commercial diet (D-2 Optibream AE 1P) was crude protein (48%), fat (18%), ash (6.3%) and cellulose (3.6%).For sample preparation, seabream muscle was extracted and homogenized with a Moulinex Turbo blender (Model Q48) and stored until analysis at −80 °C in a Thermo Electron Corporation freezer (Model ULT790-5-V34). Using the same process, we proceeded with the skin of the gilthead seabream.In addition, diets supplemented with Moringa, commercial diet, Moringa leaves and an additive propyl gallate (PG), commonly used in the food industry as an antioxidant, were analysed.The deoxyribose assay is used to detect possible scavengers of hydroxyl radicals, formed by a mixture of ascorbate and FeCl3-EDTA. The products resulting from the hydroxyl radical (OH·) attack on deoxyribose were measured with thiobarbituric acid .This method is based on inhibition by antioxidants of the 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic) (ABTS) radical cation. The Trolox Equivalent Antioxidant Capacity (TEAC) assay measures the ability of antioxidants to quench the ABTS cation in both lipophilic and hydrophilic environments by comparing their scavenging capacity to that of Trolox .The ability of samples to inhibit lipid peroxidation was tested by using ox brain phospholipid liposomes. The extent of peroxidation was measured in accordance with Jiménez et al. .w/w) concentration, while the propyl gallate was used at the permitted commercial concentration of 100 µg/g with sunflower oil for 3 h at room temperature before analysis. After 3 h of maceration, the Rancimat apparatus began to measure conductivity. Oxidative stability, evaluated by the Rancimat method at 110 °C , reflects resistance to development of rancidity in oils/fats. Results were expressed by the protection factor (PF), calculated from the induction period , the time until a critical point of oxidation is reached [Sample preparation in the Rancimat test consisted of macerating the different samples  at 10% , measures the inhibition of linoleic acid autoxidation .Moringa oleifera for two and four weeks was analysed. The FAMEs were separated by GC  equipped with a fused silica capillary column , split injector and flame ionization detector fitted with a Galaxie Chromatography Data System (Version 1.9.3.2 software). The result of each fatty acid was expressed as %. The total omega-3 fatty acid (mg/100 g) was determined by CG and total omega-6 fatty acids (mg/100 g) were determined by adding all omega-6 fatty acids.The fatty acid composition was determined by gas chromatography according to the official method AOAC . Methylaw/v) sodium carbonate. Absorbance was measured after 30 min at 765 nm on a Spekol 10 spectrophotometer, Carl Zeiss Jena, Germany. Results are expressed as grams (g) of gallic acid equivalents (GAE) per 100 g of dried Moringa (g GAE/100 g DM).The total polyphenol content of food was determined using the Folin–Ciocalteau method . In summp ≤ 0.05.All measurements were performed on three replicates. Results are expressed as mean ± standard deviation, sd. Data were statistically analysed by one-way analysis of variance (ANOVA) to determine differences between groups. Statistical analyses were conducted using SPSS 27.0 and differences were considered statistically significant when p < 0.05) in activity of muscle was observed (43% inhibition) in sea bream fed with a diet supplemented with 15% Moringa. Results showed a very high hydroxyl radical scavenging capacity in all samples of muscle, values around 80%, but no significant differences were observed as regards seabream fed with a commercial diet; similar behaviour was observed in the TEAC assay. As for antioxidant activity evaluated in the skin, samples were good lipoperoxyl radical scavengers and did not show significant differences (p < 0.05). Lower values of hydroxyl radical scavenging were observed in skin samples with respect to muscle, values around 65%, without significant differences observed. All samples also exhibited antioxidant activity when ascorbate was omitted from the reaction mixture (primary antioxidant) as they were able to directly scavenge the hydroxyl radical generated.Antioxidant activity was analysed in the muscle and skin samples of seabream fed for four weeks with diets supplemented with Moringa  and a commercial diet. The results of the antioxidant activity evaluated by lipid peroxidation, hydroxyl radical scavenging and TEAC are shown in p < 0.05) in the lipid peroxidation assay.The Rancimat assay is used to determine whether the antioxidant activity of samples resists heating at high temperatures .p < 0.05), which protects slightly by increasing induction time even higher than the antioxidant additive (PG) (1.30). Results of the seabream skin samples were lower (p < 0.05) than those obtained for seabream muscle (p < 0.05). However, all samples presented values lower than 1, with samples fed with diets supplemented with Moringa showing a low oxidative protection.p < 0.05).The total antioxidant activity was determined by linoleic acid system. This method is used to determine antioxidant activity during storage at unfavourable temperatures (40 °C) measuring the inhibition of linoleic acid autoxidation during storage, which was measured at 500 nm. p < 0.05) in values of oleic acid (1.20% to 1.75%) and linoleic acid (0.51% to 0.64%), such as total omega 6 fatty acids (0.53% to 0.66%), in the seabream samples fed for two weeks with diets enriched with Moringa at 10%.Moringa has a high content of unsaturated fatty acids, oleic acid above all 70%) and linoleic acid 0% and li,27. Therp < 0.05). In all samples (leaf and seed), the highest content of polyphenols found was carried out by extraction with methanol, the lowest content being in water extracts (p < 0.05). Finally, the extracts with methanol/water (80/20) showed similar polyphenol contents compared to water extracts, without significant differences.Additionally, the content of phenolic compounds in Moringa was analysed, which accounts for a large part of its antioxidant activity. The diet of gilthead seabream is based on molluscs, crustaceans and small fish; therefore, fishmeal-based feed is administered in fish farms. Some studies have shown that ingredients based on vegetable crops are the most promising alternative feed ingredients, particularly if produced by terrestrial agriculture in the local region and could contribute directly to the sustainability and cost-effectiveness of fish farming. Vegetable protein meals with high protein content are those most widely tested as substitutes for fish meal .v/v and ethyl acetate, revealing a large abundance of flavonoids and phenolic acids. Each extraction solvent was found to promote different extraction efficiency, the highest total phenolic content being recorded in methanol 100% leaf extracts . Similar results were observed by Nobossé et al. [Moringa, according to Gopalakrishnanb et al. , is growé et al. , where tWe observed different behaviour when analysing antioxidant activity evaluated in the gilthead seabream samples fed a diet supplemented with Moringa. In the case of lipid peroxidation inhibition, gilthead seabream muscle samples show a significant increase in activity when supplemented with 10 and 15% Moringa. This behaviour is also observed when analysing diets, since antioxidant activity is high in those supplemented with 5%, 10% and 15% Moringa, even higher than the activity of PG; therefore, it could be used for the prevention of lipid peroxidation in diets.Sharmin et al.  found siIn addition, oxidative stability in oils using substances such as antioxidants is important, since it allows delaying degradation time of fats and oils and extends their useful life. In this regard, results obtained in the Rancimat or oxidative stability test, where a longer period of induction of oxidation was observed in the Moringa leaves analysed, suggest they have a high protection factor against oxidation. Nascimento et al. , also stSiddhuraju and Becker  examinedIt must be taken into account that the lipid profile of some fish species is characterized by the presence of long-chain polyunsaturated fatty acids, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), susceptible to oxidative deterioration . VariousMoringa oleifera leaves could be mainly attributed to the presence of polyphenolic compounds. These results are encouraging enough to continue with subsequent studies on the use of Moringa as an antioxidant, although it would be necessary to optimize the appropriate concentration in animal supplementation in order for the antioxidant activity to be more effective.Supplementation in the diet of seabream with Moringa supposes an increase in the antioxidant activity of the muscle; therefore, it could be considered a functional ingredient in fish bred in fish farms, thus providing food with improvement in oxidative stability. The antioxidant potential of