In practice, fresh-cut fruit and fruit salads are currently stored submerged in sugar syrup (approx. 20%) to prevent browning, to slow down physiological processes and to extend shelf life. To minimize browning and microbial spoilage, slices may also be dipped in a citric acid/ascorbic acid solution for 5 min before storage in sugar syrup. To prevent the use of chemicals in organic production, short-term (30 s) hot-water treatment (sHWT) may be an alternative for gentle sanitation. Currently, profound knowledge on the impact of both sugar solution and sHWT on aroma and physiological properties of immersed fresh-cuts is lacking. Aroma is a very important aspect of fruit quality and generated by a great variety of volatile organic compounds (VOCs). Thus, potential interactive effects of sHWT and sugar syrup storage on quality of fresh-cut apple slices were evaluated, focusing on processing-induced changes in VOCs profiles. Intact ’Braeburn’ apples were sHW-treated at 55 °C and 65 °C for 30 s, sliced, partially treated with a commercial ascorbic/citric acid solution and slices stored in sugar syrup at 4 °C up to 13 days. Volatile emission, respiration and ethylene release were measured on storage days 5, 10 and 13. The impact of sHWT on VOCs was low while immersion and storage in sugar syrup had a much higher influence on aroma. sHWT did not negatively affect aroma quality of products and may replace acid dipping. Fresh-cut processing induces a catena of physiological responses ,2 finallIn practice, e.g., apple slices are dipped in a mixture of citric and ascorbic acid solutions for sanitation purposes. To prevent the consumption of these chemicals especially in organic production, gentle physical sanitation methods are demanded.As such, hot-water treatments (HWT) in the temperature range of 40–80 °C were shown to effectively reduce microbial contamination, and are relatively inexpensive and easy to use . In addiIn contrast to analyses of sHWT impacts on visual and internal quality attributes of fresh-cut fruit ,17,18, sThus, the present study focused on the evaluation of the potential effects of sHWT  on the aroma-related quality of ‘Braeburn’ apple slices stored in sugar syrup under strictly simulated practical condition. Some parts of the samples were also pre-treated with a commercial ascorbic/citric acid solution to test the potential synergistic effects of this treatment on aroma. During a 13 days-storage at 4 °C, respiration, ethylene emission and, for the first time, the processing-induced direct and indirect changes in VOC profiles were measured at defined intervals. This will enable comprehension of the respective quality-related physiological processes, to effectively select the optimal process conditions and to verify whether sHWT can safely replace the use of chemicals in processing of ecologically produced fresh-cut fruit salads.Malus domestica Borkh.) were obtained from a commercial fresh-cut salad producer. At the Department of Horticultural Engineering , the apples were stored at 4 °C and 95% relative humidity for up to 3 days until the start of the experiments. Undamaged apples of uniform size (mean fresh mass: 150.7 ± 5.1 g and mean dry matter content: 177 ± 11 g kg−1) were selected.Fresh mature ’Braeburn’ apples  by submerging four apples each for 30 s. Samples of two batches each were hot-water-treated at 55 °C or 65 °C, respectively, according to Kabelitz and Hassenberg  and Kabe−1 invert sugar syrup, 72.7%; Hanseatische Zuckerraffinerie GmbH & Co. KG, Hamburg, Germany plus 10 g L−1 OBSTSERVAL HC-2 browning inhibitor . The browning inhibitor contained ascorbic acid, sodium ascorbate and citric acid. All pails were tightly closed and stored at 4 °C for up to 13 days. Three pails (replicates) of each batch were opened on days 5, 10 (the common maximum shelf-life) and on day 13. For further analyses, 26 apple slices of each pail were removed.After washing and hot-water treatments, all apples were cut under semi-practical hygienic conditions in a cooling room at 4 °C. For this, apples were at first halved equatorially and then each half-segment was cut into 16 pieces by a Parti apple cutter/corer . The controls and slices of one of each 55 °C and 65 °C sHWT batches were additional chemically treated  by immersing in ascorbic/citric acid solution (40 g ascorbic and 20 g citric acid solved in 1 L deionized water) for 5 min . Slices 2 concentrations.To determine VOCs emission, ethylene evolution and respiration rate (RR), 20 apple slices were filled in a glass jar (1 L), which was hermetically closed by a glass lid. VOCs emitted by samples accumulated at 4 °C for 1 h. Then VOCs were 10 min-extracted with 50/30 μm divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) SPME fiber  via septum in glass lid. After VOCs extraction, 0.5 mL sample air was taken from headspace with a 0.5 mL A-2 Series syringe  via the same septum to measure ethylene and CO−1). The following temperature program was used: 35 °C (hold 1 min), ramp to 110 °C at 5 °C min−1 and ramp to 230 °C at 10 °C min−1 (hold 3 min). The mass spectrometer (MS) detector worked in full scan mode (mass-to-charge ratio: 35–250 m/z) and operated in electron impact mode at 70 eV. Measured chromatogram peaks were integrated based on quantifier and qualifier ions of target compound . For this, 5 μL of alkane standard was injected in a 3.9 L glass jar, extracted with SPME after equilibrium time of 1 d and analyzed as described above. The RIs were compared with those given by van Den Dool and Kratz [−1 based on the external standard 2-methylbutyl 3-methylbutanoate (CAS-number: 2245-77-4). A standard curve of 2-methylbutyl-3-methylbutanoate was created in the concentration range from 5 to 1000 nL L−1.VOCs were analyzed with a GCMS-QP2010 SE gas chromatograph-mass spectrometer  on a DB-WAX  column (30 m × 0.25 mm × 0.25 μm). SPME fiber was introduced into the GC injector (set to 250 °C and fitted with a SPME liner (0.75 mm × 5.0 mm × 95 mm); Shimadzu GCs; Restek, Bellefonte, PA, USA) for 30 min for VOC desorption and conditioning before next extraction. Helium was used as carrier gas  column (1.8 m × 1/8 in × 2.1 mm) and a 5605PC mole sieve . As carrier gas, helium was used (constant flow rate: 1.4 mL min−1), oven temperature was kept constant at 60 °C. Measured peaks were integrated using Class-VP chromatography data system software v4.2 . Rates of ethylene release were expressed in µg kg−1 h−1, those of CO2 production in mg kg−1 h−1.A small volume of 0.5 mL air was taken from canning glass jar and analyzed with a GC17A gas chromatograph (Shimadzu) to measure ethylene and COn = 3), while respiration was analyzed on a total of 60 apple slices, i.e., 20 from each pail. Statistical analyses (ANOVA) were carried out with WinSTAT  and results were presented as mean ± standard deviation (SD). The significance of differences between the calculated means was analyzed by Duncan’s multiple range test (p < 0.05). The relationships between VOC evolution and sHWT were additionally analyzed by principal components analysis (PCA) using Latentix Ver. 2.00 .For each treatment, three pails were used as replicates for each sample day. VOCs and ethylene emissions were each determined on 20 apple slices from each pail  forming the major group of compounds . The emiDuring further storage, the emissions of the majority of VOCs were significantly reduced compared to those observed immediately after cutting. Cumulative VOCs concentrations decreased by 37.7% for control and by 18.8%–50.7% for sHWT samples, but were nevertheless 3.5–5.8 times higher than for intact apples , which together represent 98.2% of the total variance in VOCs profiles. The effects of cutting, sHWT temperature and duration of storage can be visualized by a scatter point plot A. DecreaFor both PCs, the impact of each single VOC on the total variance in the VOCs profiles can be identified as visualized in Cutting immediately and significantly intensified ethylene release of untreated apple slices by approx. 30%; however, ethylene emission also rapidly, within 4 h, declined to rates below that of intact apples . Storage2-based respiration rates of untreated ’Braeburn’ apples increased 3.4 times immediately after cutting and then slightly declined again within 4 h . Hot-water treatments without post-processing acid application only slightly enhanced the alteration of the VOCs profile typical for intact apples in comparison to those with additional acid dipping. Interestingly, sHWT at 55 °C without additional acid treatment clearly intensified the storage-induced physiological responses of fresh-cut apple slices, i.e., it slightly enhanced their respiration activity and ethylene release. This may indicate a direct response of the treated apples to moderate but not to excessive heat or to acid pretreatment ,53. AddiStoring fresh-cut apple slices in sugar syrup pronouncedly affected the development of their VOCs profiles, as indicated by the distinct increase in ethyl ester emissions. Following sHWT, head space concentration of ethyl acetate of samples increased 0.5–1.6-fold compared to controls; it, however, increased approx. 100 times due to the sugar syrup.2 release observed after immersion in the syrup, corroborate the findings of Rux et al. [2 availability due to impaired gas exchange between tissues and ambient air [2 and increase in CO2 concentrations within the tissue increasingly inhibits physiological processes [The reduction of ethylene and COx et al. . These aient air ,58. Therrocesses . The incrocesses .2 concentration of 1%.Likewise, the increased emissions of ethyl esters may be explained by the increased ethanol formation under these conditions. Alcohol acetyl-CoA transferase (AAT) catalyzes the esterification of carboxylic acids and alcohols ,61, and However, in this study, the influence of oxygen availability on VOC synthesis during storage in sugar syrup submerged apple pieces cannot be sufficiently clarified. Further studies focusing on oxygen availability are necessary.2 availability under this condition. Solely applying sHWT at both 55 °C and 65 °C enhanced the respiration of apples slices, which potentially indicated marginal heat stress. This, however, only slightly intensified ethylene release but increased ethanol emission of samples, indicating alterations in respiratory pathways. Additionally, sHWT at 65 °C resulted only in minor declines of few esters. Nevertheless, the general impact of sHWT on VOCs profiles of apples slices was low, especially in comparison to storage in sugar syrup and did not negatively affect aroma. The omission of pre-processing acid application did not negatively affect the aroma to a crucial degree. Consequently, sHWT might replace the post-processing acid treatments for fresh-cut products.In a semi-practical approach, this study comprehensively evaluated the potential food quality-related effects of sHWT (at 55 °C and 65 °C), cutting and/or post-processing acid treatment and their interactions on the development of aroma-relevant VOCs as well as on the physiological indicators ethylene synthesis and respiration activity in fresh-cut apple slices stored in sugar syrup. Cutting temporarily enhanced ethylene synthesis, respiration activity and VOCs production. In this context, 2-methylbutyl acetate, butyl acetate and hexyl acetate were identified as most relevant cutting-enhanced VOCs, but their emissions declined again during syrup-storage. Syrup-storage increased ethanol and ethyl-esters, and in particular ethyl-acetate, which might be attributed to reduced O


BRCA1-associated breast cancers, we discover an evolution process through which the tumors initiate from cells with SNVs affecting driver genes in the premalignant stage and malignantly progress later via CNVs acquired in chromosome regions with cancer driver genes. These events occur randomly and hit many putative cancer drivers besides p53 to generate unique genetic and pathological features for each tumor. Upon this, we finally identify a tumor metastasis suppressor Plekha5, whose deficiency promotes cancer metastasis to the liver and/or lung.Single-cell whole-exome sequencing (scWES) is a powerful approach for deciphering intratumor heterogeneity and identifying cancer drivers. So far, however, simultaneous analysis of single nucleotide variants (SNVs) and copy number variations (CNVs) of a single cell has been challenging. By analyzing SNVs and CNVs simultaneously in bulk and single cells of premalignant tissues and tumors from mouse and human  Single-cell whole-exome sequencing is a powerful method for deciphering intratumor heterogeneity and identifying cancer drivers. Here, the authors analyze SNVs and CNVs simultaneously in bulk and single cells of BRCA1-associated breast tissues and cancers and show that Plekha5 deficiency promotes metastasis. BRCA1) is a tumor suppressor gene encoding a large protein that is involved in many essential biological processes, including DNA damage repair1, cell cycle checkpoints1, chromatin remodeling2, transcriptional regulation3, and protein ubiquitination4. The deficiency of BRCA1 induces severe genome instability, eventually leading to tumorigenesis7. BRCA1 mutations account for almost one-quarter of cases of hereditary familial breast cancer in humans, and the estimated lifetime risk of developing breast cancer for women with BRCA1 germline mutations is 40–80%8. The majority of BRCA1-deficient breast cancer cases are classified as triple-negative breast cancer based on the absence of expression of estrogen receptor (ER), progesterone receptor (PR), and HER29, and targeted therapy for this type of cancer is difficult. The poly (ADP-ribose) polymerase inhibitors (PARPi) have been approved by the U.S. Food and Drug Administration for the treatment of BRCA1-mutated tumors11; however, only a portion of patients respond to this treatment12 and drug resistance can still reduce the effectiveness of this therapy13. There is a pressing need to better understand this type of cancer at the genomic level from the early premalignant stage through to tumor progression and metastasis stage in order to develop effective strategies for the prevention and treatment of BRCA1-associated breast cancer.Breast cancer-associated gene 1  and can be accumulated in the specific contexts of individual ecosystems under Darwinian natural selection, promoting tumor initiation, formation, and metastasis. We have previously established a mouse model carrying mammary-specific disruption of the full length of Brca1  (Brca1MKO), which develops mammary tumors through multiple progressive steps involving spontaneous mutations of p53 and/or some other unidentified genes14. Using a genetic approach in which mutant mice carrying targeted disruptions of p53, Atm, Chk2, or 53BP1 are bred with Brca1-deficient mice, we found that these genes also play critical roles in various aspects of tumorigenesis19, yet many other putative tumor suppressors and oncogenes have not been identified.Driver events for each individual person, even different tumors in the same patient might be different and need to be elucidated. At present, the biology governing the initiation of 21. However, microscopic tumors, tumor purity, and intratumor heterogeneity may cast limits on the utilization of bulk DNA sequencing. Recently, single-cell DNA sequencing has been used for revealing clonal evolution, intratumor heterogeneity, and driver mutations in cancer25. Single-cell whole-genome sequencing has been used for copy number variation (CNV) analysis in several studies23; however, confident single nucleotide variant (SNV) calling is limited by the expensive cost of high coverage of the whole genome in single-cell whole-genome sequence studies. Whole-exome sequencing (WES) was previously used to obtain high coverage for confident SNV calling in single-cell analysis25; however, single-cell CNV analysis using whole-exome data is limited and challenged by the use of conventional bulk CNV calling algorithms. The combination of SNV and CNV data of the same single cells from different tumor developmental stages will provide an alternative strategy and a different view for characterizing cancer.Bulk high-throughput DNA sequencing has been widely used for driver mutation identification in many different types of cancersBRCA1-deficient breast cancers and identify their drivers, we perform both bulk tissue WES and single-cell WES (scWES) for BRCA1-deficient mammary glands and tumors, followed by validation with Sanger sequencing or droplet digital PCR (ddPCR) assays, as well as functional validation with clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9)-mediated knockouts in vitro and in vivo. We demonstrate that tumors initiate from cells with random SNVs affecting driver genes in the premalignant stage and malignantly progress later via CNVs acquired in chromosome regions with many cancer driver genes, including Plekha5, which acts as a tumor metastasis suppressor.To understand the genetic features and evolution of p53 accelerates mammary tumor formation in mice carrying mammary-specific disruption of Brca1  mice with p53 mutation  mice with wild-type p53 showed predominance in C>T/G>A   dataset21. Analysis of this dataset revealed a high number of somatic nonsynonymous SNVs, ranging from 19 to 171 , predicted TSGs or oncogenes (PRE-TSGs/oncogenes)31, and others  was found in 153 liver metastatic tumor (LMT) but not in 153 primary tumor (PT) ers Fig. . Our resone Fig. ; Table 1PT) Fig. ; Table 1PT) Fig. , and thep53 (p53 +/△) was found in all Brca1co/co;p53+/co;Wap-Cre mice, and loss of the remaining wild-type allele of p53 (p53 LOH/△) occurred in 11 out of 16 tumors  breast cancer patients carrying nts Fig. . These dTo further tie our sequencing data to the pathology of each individual tumor, we identified the driver genes for each corresponding tumor and found that different tumors from different mice presented distinct histopathology Fig. . Next, wNras mutation  and was positive for Nras, 2313-2L had a Kras mutation  and was positive for Kras, 2313-4R had an Akt3 mutation  and was positive for Akt3, 2307-2R had a Tpr (Translocated Promoter Region) mutation  and was positive for Tpr, 153 had an Arhgef11 mutation , and both 153PT and 153LMT were positive for Arhgef11  using the specific markers for tumors with corresponding driver genes. As expected, different tumors were positive for the specific driver genes; for example, 2305-4L has a f11 Fig. .Arhgef11 and Plekha5, in the 153LMT but failed to identify Plekha5 in the 153PT, suggesting that 153PT and 153LMT have a common origin. Consistently, IHC revealed that 153PT and 153LMT were positive for Arhgef11, suggesting that Arhgef11 might serve as a common driver (oncogene) in these two tumors , as well as on PT and metastatic tumor samples  models were also conducted  and Brca1-mutant (BRCA1-MT) single cells from the same mammary tissue to filter out germline mutations and technique errors introduced by single-cell sequencing. To achieve this, we intercrossed mT/mG mice33 with Brca1co/co;MMTV-Cre mice to generate Brca1co/co;MMTV-Cre;mT/mG (Brca1MKO-mT/mG) mice, which not only helped to trace Cre-loxP-mediated deletion of Brca1 exon 11 but also enabled us to obtain Brca1-WT and BRCA1-MT single cells from the same mammary gland  positive 36, were reported to be involved in tumorigenesis, while the roles of some others have not been reported. Interestingly, Gbp4 was mutated in the early (virgin mutant mammary gland (VMMG)) and late (476MMG) stages of premalignant transformation in the mammary gland, as well as in the malignant primary tumor (476PT)  Fig. , c. Gbp4ing Fig. , but somArhgef11 was the most frequently mutated gene in mouse 153   and most often mutated in metastatic tumor cells , as revealed by single-cell sequencing  also identified mutations of Arhgef11 and Plekha5 in 2.01% and 0.5% of patients, respectively. Intriguingly, a tendency for the co-occurrence of mutations in Arhgef11 and Plekha5 was observed in our single-cell data  Fig. . This geing Fig. , but it ing Fig. ; Table 148. Our results revealed that: (1) 476PT consisted of at least three major subclones and might originate from three different Brca1-MT mammary gland cells  153ion Fig. . Later, 49. We attempted to call CNVs from our scWES data using this principle and investigated the premalignant changes in Brca1-MT mammary epithelial cells and tumor cells. To validate single-cell CNV calling, we first compared the CNV profile of single cells with the CNV profile of paired bulk samples called using a popular tool, CNV kit50. For each mutant mammary gland or tumor, a common pattern of aneuploidy was revealed by both the single cells and the bulk  different tumors from different mice had different CNV profiles; (3) metastatic tumor shared very similar CNV profile with paired PT. When we further examined CNV profile in detail, we found that chromosome 11 was obviously amplified in 476PT cells but not in paired mutant mammary gland cells  from 44 to 65% and CNVs within the same single cells, indicating the contribution of CNVs to the driving force for tumor metastasis  CNVlls Fig. ; and (3)sis Fig. . Based oors Fig. , showing51 of genes in the amplified and deleted regions. We found that many cancer-driving pathways were involved, including the pathways in cancers, the MAPK signaling pathway, the PI3K–Akt signaling pathway, and the focal adhesion pathway, which contain many well-known cancer driver genes , we believed that the amplification of Ppm1d might play a role in suppressing p53 and driving tumor formation in this scenario. The oncogene Cxcr4 was found in the amplified region of chromosomes 1 in 153PT and 153LMT cells  xenograft breast tumors by single-cell and bulk WES. The data showed similar CNV profiles in single-cell and bulk samples 56 using cBioportal47  and revealed amplification of ARHGEF11 and PPM1D in 14.27% and 9.21% of patients, respectively. These results revealed that amplification of chromosome 17 in humans , which contains driver genes  for breast cancer, might be a hot-spot event at least in a portion of BRCA1-deficient breast cancers. In addition, other individual-specific driver events also play important roles in tumor initiation, progression, and metastasis. To further validate the CNV calling in single cells, we performed a highly sensitive and quantitative ddPCR assay, and we confirmed the copy-number gain of Ppm1d  in tumors were driving forces for progression and metastasis in these tumors.To determine if CNVs affect the protein level in these samples, we performed IHC on the tissues harboring corresponding gene amplifications to examine the protein levels. As expected, 476PT but not 476MG was positive for Ppm1d   cells  and implanted these cells into mice  compared to that seen in the control group (1/8)  Fig. . Then, wion Fig. . To testlls Fig. . GFP-posoup Fig. , which houp Fig. . Moreoveoup Fig. , suggest/8) Fig. . These dPLEKHA5 in human breast cancer, we knocked out PLEKHA5 in GFP-labeled BRCA1-WT MDA-MB-231 cells (231-GFP)  Fig. . SimilarFP) Fig. . We thenoup Fig. .Plekha5/PLEKHA5 in mouse and human cells had no obvious effect on tumor growth  We identified a tumor evolution process through which tumors initiate from cells with SNVs affecting putative driver genes at premalignant stages and malignantly progress later via CNVs acquired in chromosomal regions with many cancer driver genes. (2) These are random events that hit many putative cancer drivers besides p53 to generate unique genetic and pathological features for each tumor. (3) Cancer drivers or metastasis drivers, when they are present at low frequency or occur at premalignant stages, could be missed by bulk DNA sequencing due to high heterogeneity but can be identified by analyzing only a small number of single cells. (4) One of the examples is the Plekha5 gene, the mutation of which is present in a small population of cells in the PT but became dominant in LMT. (5) Our scWES results combined with CRISPR-Cas9-mediated knockout studies provide solid evidence that Plekha5 does not affect PT growth; however, it is a metastasis suppressor, whose deficiency promotes cancer metastasis to the liver and/or lung.In this study, we have performed simultaneous SNV and CNV analysis in bulk and single cells of P53 is frequently identified in human breast cancers with germline mutations of BRCA144. By analyzing the WSI dataset, we found that 81% of patients with BRCA1 germline mutations carry P53 mutations , although they were prominent in cancers. Loss of BRCA1 is known to cause genome instability7 because of the essential role of BRCA1 in homologous recombination-directed DNA damage repair62. It is therefore predicted that BRCA1 deficiency should be able to cause both SNVs and CNVs; however, deletion of a fragment of a chromosome (CNVs) might impair the functions of all genes within the deleted fragment and cause lethality, whereas SNVs will certainly yield a much milder impact on the viability of cells. Thus, despite Brca1 deficiency in mammary tissues resulting in apoptosis14, cells with SNVs should have a better opportunity to survive than cells with CNVs. Somatic SNVs in known cancer genes  were previously found in normal tissues; however, the mutation burden (3.5 mutations per sample) in each sample was relatively low, and additional transforming events were required for cancer transformation63. In our study, the Brca1-deficient premalignant mammary gland cells had a much higher mutation burden , which might generate the scenario that multiple transforming events hit one cell. In addition, Brca1 deficiency is no doubt one of the transforming factors in this scenario. Once cells are malignantly transformed, they should have a stronger ability to tolerate the lethal effects associated with CNVs and undergo Darwinian evolution, which may benefit cell proliferation, migration, invasion, etc., to drive tumor mass formation and eventually metastasis.Both SNVs and CNVs were detected by many previous genomics studies on Plekha5 is mutated in PT cells and LMT cells, suggesting its role in tumor metastasis. A previous study reported that PLEKHA5 was associated with decreased brain metastasis-free survival but not associated with other metastatic sites in melanoma patients, and knockdown of PLEKHA5 inhibited blood–brain barrier transmigration and invasion of cells in in vitro models64. To further study the role of Plekha5 in our system, we have conducted a number of experiments to investigate the role of Plekha5 in metastasis and found that CRISPR-Cas9-mediated knockout of Plekha5 enhanced cell migration and invasion in vitro. In vivo orthotopic implantation assays showed that knockout of Plekha5/PLEKHA5 in multiple cell lines promoted tumor cell metastasis to other distant organs, such as the lung and liver. Thus, different from the data shown by Jilaveanu et al.64, our study identified Plekha5/PLEKHA5 as a tumor metastasis suppressor gene through scWES screening and validation using CRISPR-Cas9-mediated functional assays. The exact reason for this discrepancy is currently unclear; however, one previous study identified two forms of Plekha5 mRNA and found that long form of Plekha5 is specifically expressed in the brain and short form of Plekha5 is ubiquitously expressed65, which may provide some hints for this discrepancy. Besides, many other differences were involved in these two studies, such as knockdown versus knockout, in vitro versus in vivo, different contexts, etc., which deserve further investigation. Meanwhile, we also showed that Plekha5/PLEKHA5 deficiency does not affect tumor growth; thus, we intend to believe that Plekha5/PLEKHA5 is a metastasis suppressor in breast cancer, which subjects to further investigation. In addition, it is also important to know the potential role of Plekha5 in normal mammary gland development, which will also be carefully studied in the near future.Brca1+/+;MMTV-Cre;mT/mG, Brca1co/co;MMTV-Cre or Brca1co/co;p53+/co;Wap-Cre (Brca1MKO), and Brca1co/co;MMTV-Cre;mT/mG (Brca1MKO-mT/mG) female mice in a mixed background of FVB/129SvEv/Black Swiss were generated inhouse and mice at different age  were used. The BRCA1-MT PDX models, TM00091 and TM00089, were obtained from the Jackson Laboratory. Female BALB/c and nude mice at age of 4–6 weeks were obtained from the animal facility of the Faculty of Health Science, University of Macau. All experiments were approved by the Animal Ethics Committees of the Faculty of Health Science, University of Macau (Protocol ID: UMARE-AMEND-100).All mouse strains were maintained in the animal facility of the Faculty of Health Science, University of Macau, according to institutional guidelines. A condition with a 12 light/12 dark cycle, temperatures of 18–23 °C with 40–60% humidity was used for housing the mice. Brca1-WT epithelial cell line B477 was derived from the mammary gland of Brca1-WT mice . The mouse Brca1-deficient epithelial cell line G600 was derived from the mammary gland of Brca1-deficient mice . The 4T1 cell line, MDA-MB-231 cell line, and the 293FT-cell line for lentiviral production were obtained from American Tissue Culture Collection (ATCC). The 4T1 cells were cultured in RPMI 1640 medium (Gibco Life Technologies) containing 10% FBS with or without 100 IU ml−1 penicillin, and 100 µg ml−1 streptomycin. All other cells were cultured in Dulbecco’s modified Eagle medium (DMEM) (Gibco Life Technologies) containing 10% FBS with or without 100 IU ml−1 penicillin, and 100 µg ml−1 streptomycin.The immortalized mouse BRCA1 germline mutations from the WSI breast invasive carcinoma dataset21 were downloaded from the web-based International Cancer Genome Consortium (ICGC)66 (https://dcc.icgc.org/). The SNV data were summarized and plotted into oncoplots. Well-known TSGs or oncogenes with SNVs in each tumor were considered as potential driver genes. The CNV data were plotted into a heatmap.The SNV and CNV data of human breast cancer patients with 28 (https://bioinfo.uth.edu/TSGene), Oncogene Database29 (http://ongene.bioinfo-minzhao.org/) and COSMIC Cancer Gene Census30 (https://cancer.sanger.ac.uk/cosmic). Predicted TSGs or oncogenes were determined using a published TSG/oncogene-prediction list31.TSGs and oncogenes were determined using three published data resources, including TSGeneFresh mammary glands and tumor tissues were cut into small pieces. Tissues were digested using a “Digestion I” solution  supplemented with 5% fetal bovine serum (FBS) (Prime) , 5 µg/ml insulin , 500 ng/ml hydrocortisone , 10 ng/ml EGF, 300 U/ml collagenase III , and 100 U/ml hyaluronidase  for 2 h at 37 °C and a “Digestion II” solution , 5 mg/ml Dispase II , and 0.1 mg/ml deoxyribonuclease ) for 5 min at 37 °C, followed by 0.25% trypsin-EDTA  treatment for 1 min and red blood cell lysis buffer  treatment for 5 min at room temperature. Samples were filtered through a 40 µm cell strainer to a 5 ml polystyrene tube. Epithelial cells were enriched using the EasySep Mouse Epithelial Cell Enrichment Kit .67. Briefly, cells were resuspended at a concentration of 150–500 cells/µl. The cell suspension was mixed with C1 Cell Suspension Reagent  at the recommended ratio of 3:2 immediately before loading 5 µl of this final mix on the C1 IFC. Images of captured cells were collected using a Leica DMI 4000B microscope under bright field by Surveyor V7.0.09 MT software. Single-cell DNA extraction and WGA were performed on the C1 Single-Cell Auto Prep Integrated Fluidic Circuit (IFC) following the methods described in the manufacturer’s protocol . Enriched epithelial cells from PDX models were isolated by micro pipetting34. Cells were captured and placed into 0.2 ml of microcentrifuge tubes with 4 µl of PBS. Single-cell DNA extraction and WGA were performed using the REPLI-g Single-Cell Kit  following the methods described in the manufacturer’s protocol.Enriched epithelial cells from mice were isolated using the C1 Single-Cell Auto Prep SystemSingle-cell WGA efficiency was evaluated using chromosome-specific primers. We designed 20 pairs of primers (Integrated DNA Technologies) for mouse cells and 22 pairs of primers for human cells to target 20 or 22 loci on different mouse or human chromosomes for PCR:Pou2f1 , 433 bp; Chr2, Rin2 , 275 bp; Chr3, Acad9 , 287 bp; Chr4, Thrap3 , 281 bp; Chr5, Stard13 , 281 bp; Chr6, Met , 298 bp; Chr7, Myadm , 189 bp; Chr8, Kars , 460 bp; Chr9, Mpzl3 , 242 bp; Chr10, Cfap54 , 208 bp; Chr11, Mpp3 , 264 bp; Chr12, Adam17 , 239 bp; Chr13, Calm4 , 231 bp; Chr14, Otx2 , 130 bp; Chr15, Osr2 , 147 bp; Chr16, Ccdc80 , 350 bp; Chr17, Oct4 , 187 bp; Chr18, Arhgap12 , 204 bp; Chr19, Aip , 293 bp; ChrX, Ccdc22 , 209 bpPrimers for mouse: Chr1, AHDC1 , 199 bp; Chr2, HDAC4 , 148 bp; Chr3, LZTFL1 , 299 bp; Chr4, SPOCK3 , 384 bp; Chr5, CENPK , 328 bp; Chr6, ZFP57 , 310 bp; Chr7, TFR2 , 358 bp; Chr8, CNOT7 , 350 bp; Chr9, WNK2 , 195 bp; Chr10, VCL , 309 bp; Chr11, NCR3LG1 , 399 bp; Chr12, POLE , 190 bp; Chr13, ENOX1 , 260 bp; Chr14, HIF1A , 252 bp; Chr15, SPATA8 , 260 bp; Chr16, TOX3 , 314 bp; Chr17, ELAC2 , 304 bp; Chr18, RAB12 , 231 bp; Chr19, EPOR , 210 bp; Chr20, DHX35 , 253 bp; Chr21, APP , 278 bp; Chr22, MORC2 , 270 bpPrimers for human: Chr1, Brca1co/co and Brca1△exon11/△exon11 allele was confirmed by PCR amplification with primers F1/R1 , F1/R2  using whole genome-amplified single-cell DNA, yielding products of 470 bp and 621 bp for the wild-type and the loxP-flanked alleles, respectively. Cells with the genotype of Brca1 for wild type and Brca1△exon11/△exon11 were used for further experiments. The PCR reaction was performed using MyTaq Red Mix  following the thermocycling conditions: one cycle of enzyme activation at 95 °C for 5 min, 35 cycles of denaturation at 95 °C for 30 s, and annealing at 60 °C for 30 s, extension at 72 °C for 1 min, 1 cycle of final extension at 72 °C for 5 min, following hold at 4 °C.The Before library construction, 100 ng (single cell) or 1 µg (bulk sample) of DNA was acoustically sonicated to 200–500 bp using the Covaris Sonicator S220. A MinElute PCR Purification Kit  was used for the purification of fragmented DNA. Libraries were constructed using the NEBNext Ultra DNA Library Prep Kit for Illumina  following the manufacturer’s protocol. AMPure XP  was used for the purification of libraries. The size and concentration of libraries were measured by a 2100 Bioanalyzer (Agilent). The final concentration was confirmed by quantitative real-time PCR using a KAPA Library Quantification Kit .Exome capture of samples from mice was performed for libraries using the SeqCap EZ Exome Library SR Platform (Roche NimbleGen), including the SeqCap EZ Developer Reagent  and the SeqCap EZ Reagent Kit Plus v2 , following the manufacturer’s protocol. The capture platform targeted an over 54.3 Mb of region, including the exons, promoters, and UTRs. Exome capture of samples from humans was performed for libraries using the TruSeq Rapid Exome Kit  following the manufacturer’s protocol. The capture platform targeted an over 64 Mb of region, including the exons, promoters, and UTRs.The whole-exome libraries were sequenced using an Illumina HiSeq 4000 or X Ten platform. Data were processed by using the CASAVA 1.8.1 pipeline (Illumina Inc.), and sequence reads were converted to FASTQ files for downstream analysis.68. The duplicate reads were marked using PICARD (http://broadinstitute.github.io/picard/) version 2.5.0. Realignment around indels using GATK IndelRealigner was implemented to reduce mismatches and improve the read alignment. The base quality scores were recalibrated to correct the over- or under-estimated scores using GATK BaseRecalibrator. The above tools of GATK69 were executed in GATK version 3.6.The alignment of the sequenced reads to the mouse genome (GRCm38.84) or the human genome (GRCh38.p12) was done by employing the BWA-MEM algorithm from the Burrows–Wheeler Aligner software package version 0.7.12-r103970. The variants were filtered with 20 minimum coverage, 5 minimum variant-supporting reads, and the recommended arguments provided by GATK. Single cells and their paired bulk tissue were put through somatic variant calling using SAMtools71 version 1.3 followed by VarScan72 version 2.3.9. Somatic mutations were classified as high confidence using processSomatic with 10% maximum variant allele frequency in the normal sample. An SNV was filtered by somaticFilter with 10% minimum variant allele frequency and P value < 5e−3. Also, for the bulk samples 20 minimum coverage and 5 minimum variant-supporting reads whereas for single cells 10 minimum coverage, 3 minimum variant-supporting reads, and presence in at least three single cells were set. To further reduce the possibility of germline mutation calling, out of all the SNVs found in single cells, the SNVs which were unique to one mouse were retained for further analysis. To ensure the identified SNVs above were captured in all cells analyzed, the identified mutation sites were checked for all single cells, the SNVs with the same mutation, and 2 minimum variant-supporting reads were retained. The data from variant calling were summarized and plotted into oncoplots using R package ComplexHeatmap 2.0.073.SNVs for bulk samples were called using the MuTect2 program50 was used on aligned paired tumor-normal WES data. The CNVs were inferred applying the standard procedure with default parameters (https://cnvkit.readthedocs.io/en/stable/pipeline.html).To estimate the somatic CNVs of bulk tissue samples, CNVkit version 0.8.549. Combined exonic RPKM values were calculated using samtools version 1.3 and bedtools version 2.19.0 for all genes. The normalized values were sorted firstly by their genomic locations, starting from chromosome 1 to chromosome 19 (for mouse) or chromosome 22 (for human) and ending with chromosome X and then by the gene start position. Using a moving average of 100 analyzed genes chromosomal CNVs in each cell and at each analyzed gene were estimated49. All the values were centrally scaled before plotting. The CNVs with values ranging from −1 to 1 were removed to deemphasize low-amplitude CNVs in cells from mice. For mouse samples, the WT mammary gland bulk was used as a control, the regions that were also covered in WT mammary gland bulk sample with a round-off value of more than 1 or less than −1 were removed from the analysis. The majority of tumor cells have amplification in chromosome 11 in mouse 476. To define large amplification of chromosome 11 (driver events) in each cell . To examine copy number amplification in the mammary gland and tumor tissue, 45 ng of genomic DNA was used for each reaction. PCRs were prepared with ddPCR Supermix for Probes  at a final concentration of 900 nM for the primers and 250 nM for each probe and partitioned into a median of 20,000 droplets per sample via a QX200 droplet generator  following the manufacturer’s protocol. The primers and probes were summarized in Supplementary Data Arhgef11 gene, the oligos  were cloned into the lenti-CRISPR/Cas9v2 vector  following the Zhang lab protocol77. For targeting of murine and hominine Plekha5/PLEKHA5 gene, the oligos  were used. The lentiviral plasmid, envelope plasmid (pMD2.G), and packaging plasmid (psPAX2) were transfected together into 293FT cells with PEI to produce viruses. The culture medium was collected and filtered with a 0.45-μm filter at 72 h after transfection. The viral media was 100× concentrated via PEG precipitation, resuspended with PBS, and saved for infection. Target cells were infected with virus together with 8 μg/ml polybrene. Positive cells were selected for 7 days with puromycin 72 h after infection. All cells used for metastasis experiments were stably labeled with a GFP-expressing vector.For targeting of murine Arhgef11 (F1: CGTAGCGTCCAGTGACTACA and R1: TTGATGACCTCTTGCCGGTC), murine Plekha5 (F: CTGTTCCTTTGTTGCCTGCC and R: TGCCCGTCCTTCTGAAATCC) and hominine PLEKHA5 (F: GTGTCTGCCCCTTCTCTCAC and R: CCGTCTCCAAGTGCTGATGA), PCR products or TA-cloning products using PCR products were sequenced. Allele modifications were determined by using the control cell as a reference sequence. Unipro UGENE 1.30.0 was used for sequence analysis.For validation of target modification, genomic DNA was isolated from cell lines and tumor tissues. Following PCR amplification of murine 5 (for B477) cells or 7.5 × 104 (for MDA-MB-231) cells in DMEM medium without FBS were added to the upper chamber and allowed to migrate (migration assay) or invade collagen-coated membranes (invasion assay) for 22 h at 37 °C, 5% CO2 atmosphere toward DMEM medium with 10% FBS (Prime) , 5 µg/ml insulin , 500 ng/ml hydrocortisone , 10 ng/ml EGF. The cells in the upper chamber were removed with cotton swabs. The cells on the lower surface of the membrane were fixed in 4% paraformaldehyde and stained with crystal violet for photographs under a microscope and quantification using image J 1.51.Transwell migration and invasion assays were conducted using the Corning BioCoat Matrigel Invasion Chamber (# 354480) and Control Insert (#354578) according to the manufacturer’s instruction. Briefly, 1.5 × 106 4T1 cells per mouse were suspended in 60 μl of PBS for mammary fat pad injection. In metastasis experiments, 6–8-week-old nude female mice were used. For B477-GFP cells, G600-GFP cells, and MDA-MB-231-GFP cells, 5 × 105 cells per mouse, respectively, were suspended in 60 μl of PBS for mammary fat pad injection. The tumor volume was measured every 3 days. At the end of the experiment, lungs, livers, and brains were dissected and fixed in a 10% formalin solution, and the GFP-positive spots were counted using Image J 1.51.In tumor growth experiments, 6–8-week-old BALB/c female mice were used, 4 × 10Staining of Kras , Nras , Tpr , Akt3 , Vimentin , Hepatocyte (HepPar1) , Ppm1d , Cxcr4 , Arhgef11 , and Plekha5  were performed on formalin-fixed paraffin-embedded (FFPE) tissue using Histostain-Plus Bulk Kit  according to the manufacturer’s instruction. Briefly. Paraffin-embedded mammary gland, PT, liver, or lung samples were sliced into 4 µm thickness. Next, slides were treated with xylene and followed by 100% alcohol treatment for deparaffination. After treated with peroxidase quenching solution (3% hydrogen peroxidase in methanol) for 10 min to block endogenous peroxidase, the slides were boiled in epitope retrieval buffer at 96–100 °C for 20 min for antigen retrieval. Following washes with PBS and incubation with blocking solution (Reagent A) for 10 min, slides were then incubated with primary antibody (1:100–500 dilution) for 1 h. After washed with PBS, slides were further incubated with the biotinylated second antibody (Reagent B) for 10–20 min and followed by enzyme conjugate (Reagent C) treatment for 10 min. Sections were stained with DAB and then counterstained with hematoxylin.The staining of CK14  was performed on cryosection of liver tissues with metastasis from block embedded in optimal cutting temperature compound (OCT). The sections were fixed for 5 min with 4% paraformaldehyde at room temperature and then washed with PBS and PBST (0.25% TritonX-100/PBS) 5 min for each. After blocking with animal-free blocker  for 1 h, sections were incubated with primary antibody (1:100 dilution) at 4 °C overnight with a parafilm in a humid chamber. Following washes with PBST, sections were incubated with goat anti-mouse IgG secondary antibody  (1:1000) for 2 h and counterstained with 4′,6-diamidino-2-phenylindole (DAPI)  for 5 min at room temperature in a dark humid chamber.78 was used with some modification. Briefly, FFPE tissue sections were deparaffinized and rehydrated with xylene for 10 s and 100, 95, 85, 70, 50, 30% of ethanol and water for 1 min each. After stained with hematoxylin  for 30 s, slides were washed with water for 30 s, destained with acid ethanol for 1–5 s, then treated with Scott’s water. Following staining with eosin Y  for 2 min, slides were dehydrated with 70% alcohol for 10 s, 90% ethanol for 10 s, two change of 100% alcohol for 2 min each. Before mounting with mountant , slides were treated with two changes of xylene to extract alcohol.For the H&E staining of the tissue section, standard protocolGSE989357. The normal expression of PLEKHA5 was defined as log2 expression value ≥ 0 and the low expression of PLEKHA5 was defined as log2 expression value ≤ −0.5. Kaplan–Meier curves were generated using GraphPad Prism 6.0.Microarray gene expression data including clinical outcomes of 155 breast cancer patients bearing PTs with or without metastasis were retrieved from Gene Expression Omnibus (GEO) under accession number t test, unless otherwise indicated. For mouse tumor-free survival and human overall survival analysis, statistical significance was calculated using a two-tailed log-rank test (Mantel–Cox). P values were considered statistically significant if the P value was <0.05. For all figures, ****indicates P value < 0.0001. Error bars represent SD.All representative experiments  were performed in triplicates independently. The results were reported as the mean ± SD, statistical significance was calculated using a two-tailed Student’s Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Reporting Summary


Nannochloris sp. (15.93 mg/L/day). The lipid content in the four species, namely Nannochloropsis gaditana, Nannochloris sp., Phaedactylum tricornutum and Tetraselmis suecica, was carried out by high performance liquid chromatography coupled to mass spectrometry (HPLC-MS ) highlighting the identification of up to 77 compounds.Isolation and identification of novel microalgae strains with high lipid productivity is one of the most important research topics to have emerged recently. However, practical production processes will probably require the use of local strains adapted to commanding climatic conditions. The present manuscript describes the isolation of 96 microalgae strains from seawater located in Bay M’diq, Morocco. Four strains were identified using the 18S rDNA and morphological identification through microscopic examination. The biomass and lipid productivity were compared and showed good results for  Aquatic microalgae are photosynthetic microorganisms that live with a variety of other species and meet different ecological requirements . They reNannochloropsis sp. (EPA), Schizochytrium limacinum (DHA), and Phaeodactylum tricornutum [A large amount of funding has recently been invested to select the best species of microalgae with high bioactive metabolites . Microalcornutum .As a consequence, microalgae have great potential in the human diet as supplements for the treatment of physiological aberrations, prevention management, and used as synthetic dietary supplements to providing sustainable natural resources . The isolation and identification of algal species from a natural environment is a well-established procedure. Each species has different growing conditions, including several regulations and key physic-chemical factors controlling growth and development such as temperature, pH, salinity, and silicate for diatoms ,20.The main objective of the present study was to evaluate the effect of the dilution technique and agar plate to separate and isolate different microalgae from Bay M’diq, Morocco. Biomass productivity, lipid productivity, and lipids content of some isolated microalgae were evaluated. Moreover, the 18SrDNA encoding gene of the microalgal isolates was sequenced to confirm the identification of the isolates. The ultra-high performance liquid chromatography coupled to mass spectrometry (UHPLC-MS) has been successfully used for the analysis of several lipid classes such as triacylglycerols (TGs), glycolipids, and phospholipids .Ahigh chDifferent species of microalgae were collected from seawater at Bay of M’diq, which is located in the north-western part of Morocco, between Sebta  and Capo Negro . Samples were selected from three collection sites: (i) proximity to a fish farm of the bay; (ii) off the coast of Martil, and (iii) at Kabila Port . A volumSeawater samples were taken from December 2017 to February 2018 in a regular manner with constant frequencies with nine samples per month.3 8.82 × 10−4 M; NaH2PO4·H2O 3.62 × 10−5 M; Na2SiO3·9H2O 1.06 × 10−4 M; FeCl3·6H2O 1.17 × 10−5 M; Na2EDTA·2H2O 1.17 × 10−5 M; MnCl2·4H2O 9.10 × 10−7 M; ZnSO4·7H2O 7.65 × 10−8 M; CoCl2·6H2O 4.20 × 10−8 M; CuSO4·5H2O 3.93 × 10−8 M; Na2MoO4·2H2O 2.60 × 10−8 M; Thiamin HCl (Vit. B1) 2.96 × 10−7 M; Biotin (Vit. H) 2.05 × 10−9 M; and Cyanocobalamin (Vit. B12) 3.69 × 10−10 M [Once in the laboratory, the samples are purified using two methods; they were inoculated on an agar plate and in a liquid medium (Guillard F/2) that contained: NaNO 10−10 M .−2 to 10−10 until obtained unicellular tubes medium [For those isolated on an agar plate, the first group of samples was filtered through a series of membranes of decreasing mesh . Each membrane was directly plated on agar plates containing F/2medium solidified with 1.5% of agar and incubated in a light chamber at two temperatures (20 and 26 °C). After growth, different colonies were transferred to tubes of 8 mL . Howevers medium ,31  to evaluate the effect of temperature on cell growth; All cultivations were alimented with atmospheric COAfter cell growth, all tubes were inoculated by transferring to 125 mL flasks add by 70 mL of F/2 medium, then incubated with a photoperiod of 18/6 h.The morphological identification of different isolated strains was carried out by microscopic observation. At the end, for molecular identification, DNA was extracted using 1030 mg of microalgal dried biomass.Samples were homogenized using the FastDNA kit for 40 s at speed setting 5 in the Fastprep FG120 instrument  but using the reagents of the ISOLATE II Genomic DNA Kit (Bioline) kit following the manufacture’s protocols. DNA was quantified spectrophotometrically using the Nanodrop ND8000.PCR amplifications were carried out using the Platinum Multiplex Master Mix (Thermofisher) in a 25 µL final volume containing 20 ng DNA, 300 nM each of specific forward and reverse primers, and 12.5 µL of Enzyme Premix (Thermofisher). The amplification protocol used was as follows: initial 11 min denaturation and enzyme activation at 95 °C, 30 cycles of 20 s at 95 °C, 1 min at 56 °C (60 °C) and 2 min at 72 °C and final extension step of 10 min at 72 °C. The 18S rDNA primers used were 18SF and euk516R ,33 and fThe biomass was harvested at a stationary phase growth (after 15 days of cultivation) by centrifugation (at 4400 rpm for 15 min) and lyophilization for 12 h .v/v) mixture at 25 °C under agitation. The procedure was repeated three times. The organic phase was centrifuged at 3000 RCF for 15 min. The sample was then filtered using Whatman N°1 filter paper in a funnel, collected in a flask, and evaporated at 40 °C.The evaluation of lipids fraction from selected microalgae was carried out using the Folch method : 200 mg The productivity of biomass was calculated from the following equation: PB (mg/L/day) = CB/T; where CB (mg/L) was the concentration of biomass from the beginning until the end of the cultivation, and T was the duration of cultivation (15 days).In addition, the lipid productivity of each sample was determined by Li et al.’s (2008) equation: PL = LT/(V culture × T) and % lipids = (LT/(CB × V culture)) × 100; where PL was the lipid productivity (mg/L/day), LT was the total lipids (mg), T was the duration of the experiment, and V culture was the volume ,37.v/v) (A) and IPA (B) was run at a mobile phase flow rate of 500 µL/min: 0–105 min, 0–50% B (hold for 5 min). MS parameters were as follows: m/z range, 150–1200; ion accumulation time, 0.6 s; nebulizing gas (N2) flow rate, during gas flow rate, 2 L/min; detector voltage, 4.5 kV; interface temperature, 450 °C; CDL temperature, 250 °C; block temperature, 300 °C.HPLC-MS analyses of the lipid contents from the obtained residue dissolved in chloroform were performed on a Shimadzu Nexera LC-30A system , consisting of a CBM-20A controller, two LC-30AD dual-plunger parallel flow pumps, a DGU-20A5 degasser, a CTO-20A oven, and a SIL-30AC autosampler. The HPLC system was coupled with an LCMS-2020 single quadrupole mass spectrometer equipped with an APCI interface . Chromatographic separation was achieved on two serially coupled Ascentis Express C18 columns 100 × 2.1 mm L × I.D., 2.7 µm d.p. , and the injection volume was 10 µL. A linear gradient of ACN/water . The different medians of results were analyzed by one-way ANOVA with a significance level of D method .p > 0.05) between the two isolation processes. The two processes used in this experiment turned out to be reproducible to purify microalgal cells. The use of agar plate for isolation was the preferred method for Coccoid and the most soil algae since it represents an axenic culture for direct established without further treatment [The sampling and isolation procedures used in this experiment were successful in establishing a culture collection of ninety-six local microalgae. The microscopic analysis showed that the majority were green algae (Chlorophyta), followed by diatoms and finally cyanobacteria with the lowest number. Sixty-four microalgae were isolated using successive dilution and thirty-two microalgae by inoculation on agar plate A. Followreatment ,31.p < 0.01). Therefore, the silicate nutrient added to the F/2 medium had a great impact in regulating the cell growth [Seventy and twenty-six species were isolated using F/2 culture medium with and without silicate, respectively. The highest number of species were isolated in the culture medium with silicate. The highest number of isolated species was obtained in December 2017 using F/2 medium added by silicate B. In addl growth .Following previous results, the highest number of species, isolated using a culture medium added by silicate, may be explained by the dominance of diatoms. Moreover, Egge and Aksnes (1992) confirmed that diatom strains represent 70% of the cell numbers by using a concentration of 2 mM silicate .p = 0.823) between the temperatures confirmed the possibility of growth cells at 26 °C and 22 °C.Temperature is one of the key criteria for the growth of microalgae and directly acts on the linear and exponential growth of microalgae species ,42. In tNannochloropsis gaditana Lubián (MN625926), Phaeodactylum tricornutum Bohlin (MN625939), Nannochloris sp. KMMCC161 Naumann (MN625923) and Tetraselmis suecica Butcher (MN625941) deposited in the NCBI database under GenBank with mentioned accession numbers in parentheses.Four species of microalgae collected from different seawater habitats were identified by microscopic morphological examination based on the form of their cells . SubsequN. gaditana then, Nannochloris sp., P. tricornutum, and T. suecica with 97.09, 96.92, 24.47, and 12.54 mg/L/day, respectively. The highest and the lowest levels of lipid productivity were respectively obtained for Nannochloris sp., and T. suecica species that were measured to be 15.93 and 0.92 mg/L/day  had a lipid content of 21%, which confirmed that the lipid productivity was greatly affected by cultivation conditions; salinity, light intensity, temperature, pH of medium, and composition of culture medium used [Chlorella species [The total lipid contents of the microalgae cultured under our experimental growth conditions ranged from 18.28% to 47.43% of their dry weight. ium used . In addi species ,47,48,49https://www.lipidmaps.org/), which allowed the identification of a total of 77 compounds in all the selected microalgae species. The lipid composition was constituted basically by mono-, di- and triglycerides , sulfoquinovosyldiacylglicerols (SQDG), mono- and digalactosyldiacylglycerols (MGDG and DGDG), phospholipids (PLs) and carotenoids (xanthophylls and chlorophylls) in the four microalgal isolates. The lipid fraction of microalgae can also include glycolipids, sterols, carotenoids, chlorophylls, and phospholipids. Algae can synthesize fatty acids to product their membrane lipids in the optimal growth conditions, which constitute phospholipids and glycolipids. However, in conditions of stress, algae alter their biosynthetic pathways and generate neutral lipids to store energy in the form of TGs [N. gaditana and Nannochloris sp. with TGs C17:0LAr/C17:0OEp—OOP/SLnP and in T. suecica with TGs (OOP/PPoG/PoOL), while in P. tricornutum, glycolipids SQDG (34:4) occurred as the highest peaks. However, previous studies have demonstrated that some Tetraselmis sp., Nannochloris sp., Phaeodactylum tricornutum, and Nannochloropsis gaditana species can produce more lipids under certain stressed conditions [LC-MS analysis was applied to separate and identify the lipid fraction in the four isolated microalgae. The separation of lipids classes reported in m of TGs ,53,54. Nnditions ,47,49,50The comprehensive lipid profiles obtained for the four strains showed their higher potential of glycolipids, phospholipids, and triacylglycerols to be an ideal source of food additives, ingredient for functional foods, and nutraceuticals applications ,51.Nannochloris sp., had the highest lipid productivity of 15.93 mg/L/day. Furthermore, the HPLC-MS analysis showed their highest content of lipidic molecules  and might be useful as dietary supplements or biofuels feedstock.This study reports the identification of four strains of microalgae from Moroccan Mediterranean seawater, which might be suitable for lipid production to be potentially used as supplement aquatic and animal feed rich in PUFA and other food products with higher omega-3 fatty acids content. In particular, the data from this study showed that


Carthamus Tinctorius L. (safflower) is extensively used in traditional herbal medicine. (2) The aim of this study was to investigate the bioactive properties of polyphenol extracts from flowers of Carthamus Tinctorius (CT) cultivated in Italy. We also evaluated the properties of two bioactive water-soluble flavonoid compounds, hydroxy safflor yellow A (HSYA) and safflor yellow A (SYA), contained in Carthamus Tinctorius petals. (3) The total polyphenol content was 3.5 ± 0.2 g gallic acid equivalent (GAE)/100 g, flavonoids content was 330 ± 23 mg catechin equivalent (CE)/100 g in the flowers. The extract showed a high antioxidant activity evaluated by oxygen radical absorbance capacity (ORAC) and 2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging assays. In addition, flower extract, SYA, and HSYA were able to reduce the susceptibility of low-density lipoprotein to copper-induced lipid peroxidation. In order to investigate the bioactive properties of flower extract, SYA, and HSYA we also studied their modulatory effect of oxidative stress on human dermal fibroblasts (HuDe) oxidized by tert-butyl hydroperoxide (t-BOOH). The CT extract at concentrations ranging from 0.01–20 μg GAE/mL of polyphenols, exerted a protective effect against t-BOOH triggered oxidative stress. At higher concentration the extract exerted a pro-oxidant effect. Similar results have been obtained using HSYA and SYA. (4) These results demonstrate a biphasic effect exerted by HSYA, SYA, and flower extracts on oxidative stress.(1)  Carthamus tinctorius L. (safflower), a traditional herbal medicine used across Asia [Carthamus tinctorius L. seeds are known as a source of α-linoleic acid and have been used to obtain cooking oil in Europe [Carthamus tinctorius L. flowers, other phenolic compounds are chlorogenic acid, syringic acid, quercetin-3-galactoside, and epicatechin [Previous studies have demonstrated healthy promoting properties of seed of oss Asia ,2,3,4. Cn Europe . Gallic catechin . Moreovecatechin ,6. The bcatechin ,8,9,10. catechin ,11. Morecatechin . HSYA recatechin . Previoucatechin . Carthamus Tinctorius L. cultivated in Italy. We evaluated polyphenol and flavonoid levels and antioxidant properties using different experimental approaches. Recent studies on cells in the culture, demonstrate a biphasic effect exerted by several polyphenols on oxidative stress, acting as antioxidants at low concentrations but pro-oxidant at higher concentrations [The aim of this study was to investigate the functional properties of extracts obtained by the flowers of trations ,16. In o2DCFDA) (C400) was supplied by Invitrogen .All cell culture reagents were obtained by Euroclone . All chemical reagents were obtained by Sigma Aldrich . HSYA and SYA compounds were purchased from Chem Faces . The fluorescent probe 2’,7’-dichlorodihydrofluorescein diacetate (HCarthamus Tinctorius (CT) was kindly supplied by Terra e Vita, Recanati (MC), Italy. The flowers were washed and the petals were dried in an oven for 5 h at 40 °C. Five grams of the powdered sample was incubated with 100 mL ultra-filtered water at 80 °C for 10 min in a water bath shaker.g), the supernatant was filtered with 0.22 μm filters, 30 mm diameter [Subsequently after centrifugation  was evaluated following the Folin–Ciocalteu assay . Briefly2 (5%) was added to tubes. At the end of incubation (10 min) at room temperature, 150 µL of 10% AlCl3 were added and samples were further incubated for 10 min at room temperature. After that, 2 mL of NaOH (4%) were added. After incubation for 15 min, absorbance was evaluated at 415 nm against the blank using a double-beam UV–Vis spectrophotometer. Catechin was used as standard for the calibration curve (0–150 µg/mL). All the experiments were done in triplicate and the results are expressed as milligrams of catechin equivalent (CE) of 100 g of dry weight (mg CE/100 g dw).Total flavonoid content (TFC) was quantified according to the method of Kim et al. . BrieflyThe antioxidant activity of CT extracts or HSYA and SYA was determined using an oxygen radical absorbance capacity (ORAC) assay . This asTM HT . A blank sample containing PBS, Fluorescein, and AAPH was prepared. Trolox (from 5 to 300 µM) was used to calibrate the assay. Samples were analyzed in triplicate. The final ORAC values were calculated using the net area under the decay curves (AUC).Fluorescence emission intensity was recorded every 5 min for 3 h at λ ex 485/λ em 530 nm in a Multi-Mode Microplate Reader SynergyData about CT extract were expressed as Trolox equivalents (mmol TE/100 g dw). For SYA and HYSA we calculated the Trolox index as reported by Martinet et al. : Trolox 50). IC50 is the concentration of an antioxidant which exerts a 50% inhibition of free radical activity. EC50 (effective concentration) values and antiradical power (ARP) have been also evaluated:The antioxidant activity of extracts was also determined using a 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging assay . Aliquot–60 µg/mL) were used. The effects of safflower extracts were evaluated in the range of polyphenols (0–17 µg GAE/mL). The kinetic was studied for 4 h into 96-well plates. The lag time was calculated from the oxidation curve and percentage of inhibition rate was calculated:Human low-density lipoproteins (LDL) were isolated from plasma by ultracentrifugation . Lipid p2.HuDe cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% (v/v) heat inactivated fetal bovine serum (FBS), 2 mM glutamine, 100 U/mL penicillin, 100 µg/ml streptomycin, 10 mM non-essential amino acids. Cells were grown at 37 °C in a humidified atmosphere containing 5% (v/v) CO3 cells/well into a 96-well plate and incubated at 37 °C in an atmosphere of 5% CO2. After plating overnight, 100 µL of CT aqueous extract containing increasing concentrations of polyphenols (0–150 µg GAE/mL), HSYA, or SYA (0–150 µg/mL) were added to the cell media. Cells were then incubated at 37 °C for 48 h. Then, 100 μL of MTT solution (5 mg/mL) was added to each well. The absorbance was measured at 540 nm with a Multi-Mode Microplate Reader SynergyTM HT .The effect of safflower extract on cell viability was evaluated using the methyl thiazolyl tetrazolium (MTT) assay . BrieflyTM HT .Moreover, we evaluated the effect of safflower extract, HSYA, or SYA on Lactate Dehydrogenase (LDH) release. LDH release, a good indicator of cellular damage, was measured with a commercially available LDH assay kit as previously described . Absorba2DCFDA as a probe [2DCFDA was dissolved in dimethyl sulfoxide (DMSO) as stock solution and kept frozen in −20 °C. Cells (25 × 103 cells/well) were seeded 24 h before treatment with CT extract, HSYA, or SYA. After 48 h of treatment with CT extract, HSYA, or SYA at the same concentrations used in the test of viability, the medium was removed, and samples were washed with PBS. Then, cells were pre-treated for 45 min at 37 °C with the fluorescent probe 10 µM as final concentration. At the end of the incubation, H2DCFDA was removed and the fluorescence of the cells from each well was measured and recorded on a fluorescence plate reader at λex/λem (485/535 nm). Multi-Mode Microplate Reader SynergyTM HT . The effect of CT extract, HSYA, or SYA was also studied in cells oxidized using 50 µM tert-butyl hydroperoxide (t-BOOH). The formation of ROS in cells treated in different experimental conditions was evaluated using H a probe . H2DCFDAt-test was applied and differences were considered to be significantly different if p < 0.05 .The data from cell experiments are representative of five independent experiments and the data are shown as the mean ± SD. The Student’s Carthamus Tinctorius (CT) were 3.5 ± 0.2 g GAE/100 g and 330 ± 23 mg CE/100 g, respectively. The total antioxidant activity of Carthamus Tinctorius evaluated by an ORAC assay was 130.2 ± 12.3 mmol TE/100 g. Total polyphenols and total flavonoids in As shown in 50 value of 13.4 ± 1.0 μg GAE/mL for CT extract. IC50 and EC50 values were lower for HSYA compared with SYA  b. The stA significant decrease in ROS levels was observed in cells incubated with safflower extract a at conct-BOOH-induced ROS formation was observed also in cells treated with HSYA at concentrations ranging from 0.3 to 60 μg/mL. On the contrary, at the highest concentrations of safflower extract (150 μg GAE/mL), HSYA, or SYA (150 μg/mL) a pro-oxidant effect was demonstrated with a significant increase in intracellular levels of ROS compared with cells oxidized with 50 μM t-BOOH.ROS levels were higher in cells treated with 50 μM t-BOOH compared with control cells (2.6 ± 0.1 vs. 1.0 ± 0.1). As shown in The medicinal properties of safflower have been recently reviewed ,3. Among50, EC50, and antiradical power (ARP) of DPPH, demonstrates that HSYA exerted a higher scavenging property compared with SYA and CT extract. The values observed for HSYA are comparable to those reported in literature for gallic acid. Using the DPPH assay, we demonstrated that safflower extracts exert scavenging properties. The comparison of the anti-radical activity parameters ICN-(p-coumaroyl) serotonin and N-feruloylserotonin and their glucoside derivatives have been identified as bioactive constituents of safflower extract [To better investigate the modulatory properties of oxidative stress exerted by safflower extract, SYA, and HSYA, ex vivo methods were also used by assessing their ability to inhibit the lipid peroxidation of LDL induced in vitro by incubation with copper ions. The increase of levels of conjugate diene is widely used to investigate lipid peroxidation ,28. A si extract . It has  extract . Other p extract ,32. A relationship between antioxidant activity and structural characteristics of polyphenols has been reported by several authors . Literatt-BOOH). The effect on intracellular ROS formation was dependent on polyphenol concentration. At lower concentrations of polyphenols, a protective effect was observed suggesting that CT polyphenols may also play an antioxidant role at the cellular level. In contrast, higher CT polyphenol concentrations, HSYA or SYA increased intracellular ROS levels.As a by-product of oxidative phosphorylation, a moderate quantity of ROS is necessary for cell survival and proliferation . PreviouThe results of this study support previous studies showing that polyphenols in relation to their concentration can have a biphasic effect. At low concentrations, polyphenols can act as antioxidants by acting as “radical scavengers” or by other mechanisms. Conversely, at higher concentrations, polyphenols can promote generation of ROS at a cellular level as previously suggested . The modIn conclusion, although the phenolics inhibit oxidation in certain systems, this does not mean that they protect against all forms of oxidative damage. The evidence that dietary polyphenols exert protective properties against oxidative stress in some experimental models is strong. However, polyphenols can also exert pro-oxidative properties under certain conditions and in certain tissues . Neither


DICER1 mutations. We describe three highly aggressive sporadic malignant teratoid thyroid tumors in 2 females (17 and 45 years) and one male (17 years). Histology showed triphasic neoplasms composed of solid nests of small primitive monomorphic cells embedded in a cellular stroma with primitive immature rhabdomyosarcoma-like (2) or pleomorphic sarcoma-like (1) phenotype. The third component was represented by TTF1+/PAX8+ primitive teratoid epithelial tubules reminiscent of primitive thyroid follicles and/or Wilms tumor, admixed with scattered respiratory- or enteric-type tubules, neuroepithelial rosettes, and fetal-type squamoid nests. Foci of cartilage were seen in two cases, but none contained mature organoid adult-type tissue or skin adnexa. SALL4 was expressed in the small cell (2) and stromal (1) component. Other germ cell markers were negative. Molecular testing revealed a known “hotspot” pathogenic DICER1 mutation in two cases. In addition, case 1 had a missense TP53 variant. This type of thyroid malignancy is distinct from genuine teratomas. The immunoprofile suggests primitive thyroid- or branchial cleft-like differentiation. Given that “blastoma” is a well-accepted terminology in the spectrum of DICER1-associated malignancies, the term “thyroblastoma” might be more convenient for these malignant teratoid tumors of the thyroid gland. Relationship of thyroblastoma to the DICER1 syndrome remains to be addressed.Primary thyroid teratomas are exceedingly rare. Mature and immature variants recapitulate their gonadal counterparts . On the other hand, the so-called malignant teratomas affect predominantly adults and elderly, are highly aggressive, and, according to a few published cases, harbor The online version of this article (10.1007/s00428-020-02853-1) contains supplementary material, which is available to authorized users. Poorly differentiated malignancies of the thyroid gland are uncommon. They mainly encompass anaplastic thyroid carcinoma in the elderly and less frequently, poorly differentiated thyroid carcinoma. Primary teratomas of the thyroid gland are very rare neoplasms of presumable germ cell origin . They remalignant teratomas, on the other hand, are much rarer and affect almost exclusively adults, with a highly aggressive clinical behavior leading to death of affected patients usually within first 2 years of diagnosis [malignant thyroid teratomas [thyroblastoma to distinguish this highly lethal disease and separate it from conventional thyroid teratoma. If properly classified, the genetic background, possible heredity, biological properties, and therapeutic options of this poorly characterized aggressive malignancy would then be better addressed in the future.So-called iagnosis . Other tiagnosis , the preeratomas . We hereTwo cases (cases 1 and 3) were identified routinely and one (case 2) in the consultation files of the authors. Tissue specimens were formalin-fixed and processed routinely for histopathological evaluation. Immunohistochemistry (IHC) was performed on 3-μm sections cut from paraffin blocks using a fully automated system  and the following antibodies: thyroglobulin , calcitonin , TTF1 , PAX8 , pankeratin , p63 , AFP , CD117 , beta-HCG , SALL4 , gylpican-3 , OCT3/4 , D2–40 , PLAP , CD30 , NSE , CD56 , TP53 , WT1 , WT1-c-terminus , S100 , synaptophysin , CD34 , desmin , myogenin , CDX2 , CK20 , HepPar-1 , NUT , TLE1 , SMARCB1/INI1 , SMARCA4 , Ki-67 , chromogranin A , CD99 , GFAP , and HMB45 .DICER1 sequence analysis was performed using the QIAseq Targeted Human Comprehensive Cancer Panel according to manufacturer’s instructions (the list of the 160 genes is shown in the supplementary file). Bioinformatic evaluation of the sequencing data, including variant calling and annotation, was done with the CLC Genomics Workbench . Low-quality variants with a score under 200 were filtered out, as well as variants in non-protein-coding regions, synonymous variants, and those present in GnomAD with an allele frequency of over 1%. The remaining variants were assessed for pathogenicity according to ACMG/AMP criteria. The DICER1 variants were classified as described previously [After careful manual microdissection, DNA was analyzed from FFPE tumor tissue using the Maxwell© 16 system  according to manufacturer’s instructions. 2VAd, 1xI2VA) 8 months after initial diagnosis.A 17-year-old male presented with a recent history of a rapidly growing mid-cervical mass. Imaging confirmed the presence of a mass diffusely infiltrating both thyroid lobes, mainly the right lobe measuring 8.2 cm. Following frozen section examination, which suggested a malignant germ cell neoplasm, subtotal thyroidectomy was performed with involved margins. This was followed by two cycles of germ cell neoplasm-directed chemotherapy  and then re-excision of the tumor bed. Postoperative chemotherapy was continued with an additional 4 PEI cycles simultaneous to local radiotherapy. The patient is alive under ongoing chemotherapy  UI/ml) and anti-microsomal antibodies (1279.5 (0–35) UI/ml). The ultrasound examination documented a 6.3-cm mass involving both thyroid lobes and isthmus. Preoperative fine-needle aspiration cytology was interpreted as suspicious for medullary carcinoma. The patient underwent total thyroidectomy. The patient received two cycles of chemotherapy. The first of 3 days: Etoposide (150 mg/m2) 250 mg, vincristine 2 mg (first day), ifosfamide 2000 mg, and actinomycin (0.5 mg/m2) 0.8 mg. The second of 4 days, approximately 1 month later: Beomycin 30 ng (first day), etoposide 200 mg, and cisplatin 45 mg. The disease rapidly progressed into the mediastinum even after chemotherapy, and the patient died of the disease 1 year after the diagnosis.A 45-year-old woman with a history of multinodular goiter  was admitted because of recent onset of dysphasia and painful rapid increase in thyroid size over the previous 3 months. Clinical examination showed an enlarged firm right thyroid lobe with no lymphadenopathy. Laboratory investigations were normal. Neck CT showed a 6.1 × 3.8 × 3.7 cm right lobe mass with retrosternal extension. No other manifestations were seen on thoracic and abdominal CT. FNA showed an unclassified high grade sarcomatous neoplasm. The patient received a total thyroidectomy and recovered well postoperatively. Follow-up was not available.The surgical specimen consisted of right thyroid lobe of 4.8 × 3.2 × 2.5 cm and the left lobe of 4.8 × 3.8 × 1.8 cm. On cut-surface, a gray-whitish to tan-yellow extensively necrotic mass was seen replacing most of the right lobe and extending variably into the left lobe Fig. . Areas oHistologically, the tumor was triphasic, composed mainly of primitive small round to oval cells, disposed into variably sized and shaped, occasionally communicating, compact aggregates with foci of necrosis and brisk mitotic activity. The periphery showed frequent palisading of the nuclei Fig. . This coThe surgical specimen disclosed a whitish and heterogeneous 6-cm nodule with infiltrative margins on cut surface Fig. . HistoloThe small cell component expressed TTF1, NSE, glypican3, SALL4 Fig. , and Ki-This case was also composed of three components but differed in some aspects Fig. . The mesDICER1 “hotspot” somatic missense mutation  was found. This mutation occurs in an exon that encodes part of the RNAse IIIB domain, which is critical for the correct cleavage of hairpin precursor microRNAs to their mature products [DICER1 p.Gly1809Arg. This is an established somatic mutation, in same RNAse IIIB domain as p.Asp1709Asn. In both cases, the variant allele frequency was consistent with these variants being present in the heterozygous state  was found in case 1, consistent with the mutation-type reactivity seen on IHC in this tumor. This variant appears to be present at a heterozygote allele frequency.Sufficient tumor tissue was available for molecular testing in case 1 and case 2. In case 1, a known products . Case 2 immature from so-called malignant thyroid teratoma has relied mainly on the distinctive demographic and prognostic differences between the two disease categories. Immature thyroid teratomas are essentially neonatal or pediatric diseases with excellent outcome after complete surgical removal [The tumor type we are describing herein has been likely included in the spectrum of what has been named “malignant thyroid teratoma” in the past. Although this term has been largely limited to a subset of thyroid gland malignancies believed to be of germ cell origin, there has been no convincing rational to distinguish immature from malignant teratoma on the basis of morphology alone. Moreover, the terms “immature” and “malignant” are not established histological categories in the pathology of gonadal germ cell neoplasms. Hence, distinction of  removal , 3. On t removal , 3, 4, 5 removal , 3. Imma removal .The current cases, however, display many significant differences from the reported malignant thyroid teratomas, suggesting it might represent a different entity. In particular, reported thyroid teratomas only rarely show overt sarcomatous (mainly primitive rhabdomyoblastic) stromal overgrowth, similar to our cases . Myo-D1 DICER1 mutation  in a case of malignant thyroid teratoma in a 59-year-old female [DICER1 mutation to the PNET-like transformation [DICER1 mutation [DICER1 mutations in 4 of 4 malignant but in none of 4 mature/immature thyroid teratomas [DICER1 variants identified were somatic in nature [DICER1 missense mutations, occurring in exons encoding the critical RNase IIIB domain of DICER1 in two tumors. No second hits were seen, and the allele frequency of the variants is consistent with retention of heterozygosity.In line with a distinct clinicopathological and molecular entity, malignant teratomas lack isochromosome 12, a genetic hallmark in the majority of germ cell neoplasms , 13–16. eratomas . The agen nature  and theyn nature , 18. Ourthyroid carcinosarcoma have been reported [DICER1 mutation in the tumor, confirming similarity to our cases and to the cases reported by Rooper et al., both histologically and genetically [DICER1 mutations as well [The question may be raised as to whether our current cases (in particular case 3) might represent genuine carcinosarcomas. Indeed, case 3 has been originally diagnosed as such. In the current WHO classification , carcinoreported , 20. Howreported , 20. Moretically , 11. Alt as well . This po as well .DICER1 mutations or consistent molecular findings in SETTLE [Spindle epithelial tumor with thymus-like elements (SETTLE) is another mixed epithelial and stromal thyroid neoplasm with presumed branchial cleft-like differentiation . Indeed,n SETTLE .EWSR1/FLI1 rearrangement [Due to its favorable prognosis, the rare entity “carcinoma of the thyroid with Ewing family tumor elements (CEFTE)”, also called adamantinoma-like Ewing family tumor, should be recognized and distinguished from thyroblastoma and other aggressive thyroid malignancies with monomorphic small basaloid cells , 27. At angement .DICER1 gene mutations and characterized by an array of topographically and phenotypically diverse neoplasms of benign, low-grade, or aggressive nature [DICER1 mutations are very uncommon in germ cell neoplasms [The DICER1 syndrome represents an emerging inherited multineoplastic disorder caused by germline e nature , 17, 18.e nature . Indeed,eoplasms , 31.The list of organ manifestations of the DICER1 syndromes are growing steadily and encompass sinonasal (chondromyxoid hamartomas), thyroid , gonadal , genital , renal (cystic nephroma and anaplastic sarcoma of kidney), thoracic (pleuropulmonary blastoma), intracranial , and others , 21, 32.DICER1 mutations were reported [DICER1-associated neoplasms were diagnosed in one of the two children and a presumed intracranial metastasis in the other [DICER1 mutations in both. The authors discussed the probability these teratoma-like lesions being a novel DICER1-related entity.Recently, malignant teratoid sacrococcygeal tumors occurring in two infants and harboring pathogenic germline reported . The hisreported , 33. AddSMARCA4 mutations [SMARCA4 inactivation and another related to DICER1 mutations.A last molecular pathogenetic point to address is the striking similarity of the tumor we are reporting to sinonasal teratocarcinosarcoma, a similarly aggressive primitive multiphenotypic malignancy reported initially by Heffner and Hyams as “malignant teratoma” and affecting predominantly adults at a mean age of 60 years . The molutations . Taken tBased on the above observations, we believe that the current cases and possibly the majority of what has been called malignant thyroid teratomas in the past are probably distinctive DICER1-related primitive malignant teratoid thyroid tumors that are distinct from genuine mature and immature thyroid teratomas. The frequent presence of TTF1+/PAX8+ follicle-like structures indicates organotypical differentiation or primitive thyroid-like elements. This observation is in line with several DICER1-related primitive neoplasms that recapitulate the organ of origin, a finding reflected in the predominance of the “blastoma” terminologies  for several DICER1-related malignancies , 18, 32.DICER1-mutated “thyroblastoma” cases reported to date (Table Looking at the 8 ESM 1(XLSX 9 kb)