Gemmobacter grows phototrophically, aerobically, or anaerobically, and utilizes methylated amine. Here, we present two high-quality complete genomes of the strains con4 and con5T isolated from a culture of Anabaena. The strains possess sMMO (soluble methane monooxygenase)-oxidizing alkanes to carbon dioxide. Functional genes for methane-oxidation  were identified. The genome of strain con5T contains nirB, nirK, nirQ, norB, norC, and norG genes involved in dissimilatory nitrate reduction. The presence of nitrite reductase gene (nirK) and the nitric-oxide reductase gene (norB) indicates that it could potentially use nitrite as an electron acceptor in anoxic environments. Taxonomic investigations were also performed on two strains through polyphasic methods, proposing two isolates as a novel species of the genus Gemmobacter. The findings obtained through the whole genome analyses provide genome-based evidence of complete oxidation of methane to carbon dioxide. This study provides a genetic blueprint of Gemmobacter fulva con5T and its biochemical characteristics, which help us to understand the evolutionary biology of the genus Gemmobacter.The genus  Gemmobacter is of interest because of its metabolic pathways and its habitats. Members of the Gemmobacter species are able to grow phototrophically, aerobically, and anaerobically [Gemmobacter changlensis, isolated from a snow sample collected in the Indian Himalayas, was proposed as psychrotolerant and phototrophic bacteria [Gemmobacter aestuarii, recovered from estuarine surface water, was described as containing a complete gene cluster for photosynthesis [Gemmobacter have been discovered in a wide range of natural environments, such as freshwater, sulphuric cave waters, estuaries, forest ponds, artificial fountains, tidal flats, activated sludge, a white stork nestling, coastal planktonic seaweed, and conserved forages, indicating that members of this genus are widely distributed in natural and artificial environments [obically ,3,4,5,6.bacteria . Anotherronments ,13,14,15Gemmobacter, including Gemmobacter aquatilis, Gemmobacter lutimaris, Gemmobacter sp. HYN0069, Gemmobacter caeni, and Gemmobacter sp. LW-1, utilize methylated amine. These species have related genes encoding the enzymes trimethylamine (TMA) dehydrogenase, TMA monooxygenase, and TMA demethylase in their genome, indicating metabolic potential of using the TMA oxidation pathway to convert trimethylamine to dimethylamine [T, from an Anabaena culture that contained all genes related to the oxidization of methane to carbon dioxide.Comparative genomics analyses revealed that members of the genus hylamine ,8. In a Gemmobacter were characterized as Gram-negative, oxidase and catalase-positive, non-spore-forming, and rod-shaped containing ubiquinone-10 as the major respiratory quinone, with G + C content in the range of 61.4–69.1 mol% [T, together with a taxonomic proposal based on their phylogenetic, genomic, physiological, and chemotaxonomic characteristics.The species in the genus 9.1 mol% ,4,5. HerT and con4 were recovered from a culture of Anabaena using a serial dilution method [Anabaena culture, Anabaena variabilis FBCC010004 strain was obtained from the Freshwater Bioresources Culture Collection  of the Nakdonggang National Institute of Biological Resources . Anabaena cells were cultured in 500 mL standard cell culture flasks with Blue–Green (BG11) broth  under the conditions: 20 °C, 35% humidity, 12 h:12 h light-dark photoperiod, 20 μmol m−2 s−1 irradiance, and 200 rpm agitation [Anabaena culture, a 100 μL sub-sample of the suspended material was aseptically spread onto modified R2A agar  [T and con4, were isolated after three days and routinely sub-cultivated on R2A agar at 30 °C for 48 h and kept in a glycerol solution  at −70 °C for long-term preservation. For most experiments, all strains were cultivated on R2A agar at 30 °C.Strains con5n method . For thegitation . From thNJ, USA)  and incuw/v; at intervals of 1.0%) NaCl. Anaerobic growth was examined after incubating strains con4 and con5T on R2A agar in an anaerobic glove box  containing an atmosphere of 99% N2 and 1% H2 and equipped with palladium catalyst packs to remove oxygen and a gas analyzer to monitor oxygen concentrations. Cell growth at different media was investigated in different media of R2A agar, tryptic soy agar , nutrient agar , and Luria–Bertani  medium. The antimicrobial susceptibility of the strains was checked on R2A agar medium using the filter-paper disk method [−1), ampicillin/sulbactam , chloramphenicol (30 µg mL−1), erythromycin (30 µg mL−1), gentamicin (30 µg mL−1), kanamycin (30 µg mL−1), lincomycin (15 µg mL−1), nalidixic acid (30 µg mL−1), rifampicin (30 µg mL−1), spectinomycin (25 µg mL−1), streptomycin (25 µg mL−1), teicoplanin (30 µg mL−1), tetracycline (30 µg mL−1), and vancomycin (30 µg mL−1). The haloes for susceptibility were recorded when the diameters were more than 10 mm after incubation at 30 °C for 2 days. Other biochemical activities were examined using API 20NE, API ID 32GN, and API ZYM kits  according to the manufacturer’s instructions.Cell motility and morphology were examined via a phase-contrast microscope  and transmission electron microscopy (Philips CM-20) using cells grown for 48 h in R2A media at 30 °C. The growth temperature was tested at 4, 8, 15, 20, 30, 37, and 45 °C. The pH range for growth was determined using different buffering systems: citrate–phosphate for pH 4–7, phosphate for pH 8, and glycine–NaOH for pH 9–11 . Salt toT and con4 and four Gemmobacter-type strains were collected after being grown at 30 °C for 48 h on the same sectors of R2A agar plates. Cell-harvesting standardization was done following the method described by Jin et al. [254, Merck).The whole-cell fatty acid profile was analyzed by gas chromatography  with the TSBA 6 database using Sherlock software v6.1 at KCTC (Korean Collection for Type Cultures) service center. The cells of the strains con5n et al. . The extn et al. ,22. The T and con4 was extracted using a FastDNATM SPIN kit. Amplification of the 16S rRNA genes was done by PCR with 27F/1492R primer sets [T and con4 were compared with the available sequences in the EzBioCloud server [mega7 software [bioedit [clustal x [Gemmobacter with whole genome sequences on the TYGS (Type Strain Genome Server) [T and con4 were sequenced using both the PacBio RSII platform and Illumina next-generation sequencing technology at Macrogen Inc. . The chromosomes and plasmids were assembled using the software package of SMRT portal (v.3.2.0), Pacific Biosciences,  [T and con4 was performed with the Prokka (v.1.4.6) pipeline [For a 16S rRNA gene analysis, genomic DNA of strains con5mer sets . The 16Sd server . Evolutisoftware . Sequenc[bioedit  and cluslustal x ,29,30. Tlustal x . A phylo Server) . The phy Server)  from GBD Server) . The whoCA, USA) . The de pipeline , and a spipeline . The prepipeline ,39. The pipeline , and thepipeline .T and con4 were Gram-negative, non-motile, aerobic, and rod-shaped . The cells were found to be positive for the following enzyme activities (API ZYM test strip): esterase (C4), esterase lipase (C8), α-glucosidase, leucine arylamidase, and naphthol-AS-BI-phosphohydrolase. However, the cells were found to be negative for N-acetyl-β-glucosaminidase, acid phosphatase, alkaline phosphatase, α-chymotrypsin, cystine arylamidase, α-fucosidase, α-galactosidase, β-galactosidase, β-glucosidase, β-glucuronidase, lipase (C14), α-mannosidase, trypsin, and valine arylamidase (−1), ampicillin/sulbactam , chloramphenicol (30 µg mL−1), erythromycin (30 µg mL−1), gentamicin (30 µg mL−1), kanamycin (30 µg mL−1), nalidixic acid (30 µg mL−1), rifampicin (30 µg mL−1), spectinomycin (25 µg mL−1), streptomycin (25 µg mL−1), teicoplanin (30 µg mL−1), tetracycline (30 µg mL−1), and vancomycin (30 µg mL−1) but resistant to lincomycin (15 µg mL−1).The two strains con5d-shaped . The collamidase . The cel18:1 ω7c and/or C18:1 ω6c) for both strains (data not shown). The major fatty acids in strains con5T and con4 were consistent with the major fatty acid components in species from the genus Gemmobacter. Notably, con5T and con4 differed from the four closest relatives in the proportions of some minor fatty acids. The major predominant respiratory ubiquinone was Q-10. The polar lipids consisted of phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylcholine (PC), two unidentified glycolipids , three phospholipids , and three unidentified aminophospholipids  for the type strain con5T; and phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylcholine (PC), two unidentified glycolipids , three unidentified phospholipids , and two unidentified aminophospholipids  for the strain con4 , G. caeruleus N8T (97.7%), G. lutimaris YJ-T1-11T (97.4%), and G. tilapiae KCTC 23310T (97.3%) and less than 97% with the remaining species within the genus Gemmobacter. Strains con5T and con4 also share high similarities with other species than members of Gemmobacter: 96.7% with Cypionkella collinsensis 4-T-34T, 96.7% with Cypionkella psychrotolerans PAMC 27389T, and 96.4% with Cypionkella aquatica DC2N1-10T. However, it was clear from the topology of the phylogenetic tree  [T and con4 with other available type strains of Gemmobacter were 73.31–80.61 % and 19.03–23.15 %  (The genome of strain con5P076367) . Of 4534P076367) . The DNAP076367) .It is generally understood that methanotrophic bacteria are mostly active at the oxic-anoxic transition zone in stratified lakes, using oxygen to oxidize methane. The methanotrophs produce methane monooxygenase to utilize methane as a carbon source ,45. The T possesses sMMO  but does not have a pMMO gene in its genome sequence. The presence of one PQQ-dependent alcohol dehydrogenase (ADH) , two NAD+-dependent ADH , and another alcohol dehydrogenase (adhB) in strain con5T makes it a good candidate for the conversion of methane to aldehyde (gfa), a GSH-dependent formaldehyde dehydrogenase (fdh), and S-formyl-GSH hydrolase (fgh) convert formaldehyde to formate, and then the formate is oxidized by formate dehydrogenase (fdh) to the final product, carbon dioxide (CO2)  . Those gde (CO2) .T contains nirB, nirK, nirQ, norB, norC, and norG genes involved in dissimilatory nitrate reduction, but no dissimilatory nitrate reductase (narG) or nitrous oxide reductase (nosZ) genes are detected, which is incomplete for the denitrification pathway (T cannot perform nitrate reduction, itself, unless it utilizes unknown or incorrectly classified reduction pathways up to date. The genome of strain con5T contains the nitrite reductase gene, nirK, and the nitric-oxide reductase gene norB, and it could potentially use nitrite as an electron acceptor in anoxic environments.Some methanotrophs comprise genes encoding enzymes for the nitrate reduction pathway, which was confirmed to be related to methane oxidation under anoxic conditions , and the pathway . This suT and con4, representing methane oxidizing species from an Anabaena culture belonging to the genus Gemmobacter, were investigated using genomic and polyphasic methods. The findings obtained through the whole genome analyses provide genome-based evidence of complete oxidation of methane to carbon dioxide. This study provides a genetic blueprint of Gemmobacter fulva con5T and its biochemical characteristics, which help us to understand the evolutionary biology of the genus Gemmobacter. Based on the phylogenetic position and the genotypic, chemotaxonomic, and physiological differences, we propose that strains con5T and con4, Gemmobacter fulva sp. nov., should be assigned as a novel species of the genus Gemmobacter in the family Rhodobacteraceae (In this study, the strains con5teraceae .T and con4 are MZ317888 and MZ317889, respectively. Accession numbers for the genome sequences of strains con5T and con4 are CP076361–CP076367 and JAHHWR000000000, respectively.The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains con5


Infusion of fractalkine into the lateral ventricle of adult NPC lineage-tracing mice leads to increased newborn OPC and oligodendrocyte formation in vivo. We also show that OPCs secrete fractalkine and that inhibition of endogenous fractalkine signaling reduces oligodendrocyte formation in vitro. Finally, we show that fractalkine signaling regulates oligodendrogenesis in cerebellar slices ex vivo. In summary, we demonstrate a novel role for fractalkine signaling in regulating oligodendrocyte genesis from postnatal CNS precursor cells.Neural and oligodendrocyte precursor cells (NPCs and OPCs) in the subventricular zone (SVZ) of the brain contribute to oligodendrogenesis throughout life, in part due to direct regulation by chemokines. The role of the chemokine fractalkine is well established in microglia; however, the effect of fractalkine on SVZ precursor cells is unknown. We show that murine SVZ NPCs and OPCs express the fractalkine receptor (CX3CR1) and bind fractalkine. Exogenous fractalkine directly enhances OPC and oligodendrocyte genesis from SVZ NPCs   •Cx3cr1 and bind fractalkinePostnatal and adult murine SVZ precursor cells express •Fractalkine enhances OPC and oligodendrocyte formation from SVZ NPCs•Inhibition of fractalkine signaling reduces SVZ NPC and OPC oligodendrogenesis•ex vivo cerebellar slicesFractalkine signaling regulates oligodendrogenesis in  Cx3cr1 and bind fractalkine. Exogenous fractalkine increases oligodendrogenesis from SVZ NPCs and OPCs. They also show that OPCs secrete fractalkine and that inhibition of endogenous fractalkine signaling reduces SVZ oligodendrocyte formation. Finally, they show that fractalkine signaling is an important regulator of oligodendrocyte genesis in other brain areas, such as the cerebellum.Watson et al. demonstrate that murine SVZ NPCs and OPCs express  However, the role of oligodendrocytes and myelin extends beyond axonal support. A decrease in adult oligodendrocyte and/or new myelin genesis impairs motor skill learning, axon function, and memory formation . Increasunctions , spatialunctions , fear meunctions , and abnunctions .Resident neural precursor cells (NPCs) in the adult brain can be recruited for increased oligodendrogenesis and myelination . NotablyIntriguingly, NPCs and OPCs express chemokine receptors in the developing and adult CNS  . FKN secCx3cr1 and bind FKN. Exogenous FKN increases oligodendrogenesis from SVZ precursor cells in vitro and in vivo. We also show that OPCs secrete FKN and that inhibition of endogenous FKN signaling impairs oligodendrocyte genesis. Together, our data demonstrate FKN signaling as an important regulator of oligodendrocyte formation from precursor cells in the SVZ niche. Finally, we also show that the role of FKN signaling in oligodendrocyte genesis is preserved in the cerebellum.We demonstrate that postnatal and adult SVZ NPCs and OPCs express Cx3cr1, Sox2, and Olig2 mRNA confirmed that Cx3cr1 mRNA was expressed in Sox2+Olig2− NPCs and Sox2+Olig2+ OPCs in the dorsal and dorsolateral SVZ of postnatal day (P) 2, P7, P15, and P65 mice  into the lateral ventricle of adult wild-type (WT) mice, or mice with a constitutive knockout of CX3CR1 (CX3CR1KO) . This is in line with a previous report which showed that Cx3cr1 is enriched in activated NPCs but not quiescent NPCs (SVZ astrocytes), ependymal cells, or neuroblasts . FKN-647 also associated with IBA1+ microglia (To find out whether FKN can bind SVZ precursors X3CR1KO)   A. As an X3CR1KO) B, but no in NPC medium containing fibroblast growth factor (FGF) and epidermal growth factor (EGF), or for 2 DIV in OPC growth medium (GM) containing FGF and platelet-derived growth factor AA (PDGF-AA) , whereas CX3CR1KO OPC cultures had reduced proportions of OLIG2+ and PDGFRα+ cells   H. WT NPCPDGF-AA) I and 2IiPDGF-AA) Ii. WT OP PDGFRα+ J and 2Ji PDGFRα+ G–S2I. CXl cortex .BRIGHT (56.03% ± 4.80%) and FKN-647DIM (31.37% ± 4.52%) cells   cells I and 2Ii47+ OPCs J and 2Ji47+ OPCs . Importactively) , non-speCx3cr1 and bind FKN. These data support and extend previous reports showing that postnatal NPCs and OPCs express Cx3cr1 as determined via RNA sequencing   F, or SOX and FKN or VC. OPCs incubated with FKN showed a statistically significant 31.52% ± 11.77% increase in MBPot 5 DIV G–5I. Theot 5 DIV J. Thus, +OLIG2+ oligodendrocytes were scored based on their interaction with βIII+ neurons  infusion of VC (BSA) or FKN  or FKN A. We ana) or FKN . FKN treo the WM C.Figure + cells that co-expressed OPC marker PDGFRα, early oligodendrocyte marker BCAS1  M. While lination . To asselination N. FKN di(+ cells O. Howevedrocytes P and 6Q.2+ cells R. FKN tr+NF+ (neurofilament) to total NF+ axons with ten equidistant grids, as previously described  OPCs (RNA-seq data from purified OPCs  show tha.5) OPCs A. Enzyme.5) OPCs B.Figure in vivo and in vitro  or CX3CR1 (anti-CX3CR1), which are known to block FKN signaling in vitro . SVZ OPCin vitro C showed drocytes D and 7E  . NPCs in both niches produce neurons and astrocytes throughout life. However, typically only SVZ NPCs are capable of producing oligodendrocytes . Intriguophrenia , implicaophrenia . Our resin vitro via increased production of primary neurospheres .Further information and requests for resources and reagents should be directed to and fulfilled by the lead contact, Anastassia Voronova  or BSA-647 (Thermo Fisher) for one-time delivery was infused into right lateral ventricle via ICV surgery. Anti-FKN (Torrey Pines Biolabs) and IgG (Jackson ImmunoResearch) controls were used at 20 μg/mL, and anti-CX3CR1 (Torrey Pines Biolabs) and IgG controls were used at 40 μg/mL.Murine soluble FKN (R&D Systems) was added at 100, 250, and 500 ng/mL to secondary neurosphere assay; at 25, 100, 250, and 500 ng/mL to NPC monolayer cultures; and at 250 ng/mL to OPCs or cerebellar slice cultures. ERT2 was obtained from STOP and C57BL/6J from Jackson Laboratories, CD1 from Charles River, and CX3CR1KO from KO mice, human CX3CR1I249/M280 RNA is expressed in murine Cx3cr1 locus, but is not translated until bred with Cre-recombinase-expressing mice  for 5 days. Seventy-two hours later, ICV surgery was performed using coordinates −1.000 mediolateral, −0.300 anterior-posterior, −2.500 dorsoventral relative to bregma. FKN was infused for 7 days using osmotic mini-pumps .Two- to three-month-old WT or CX3CR1SVZ was microdissected from P1–P2 or P7 CD1 pups and cultured at clonal density as primary neurospheres for 6DIV and secondary neurospheres for 7 DIV as described in Dissociated primary neurosphere cells were cultured for 1–5 DIV on coverslips coated with poly-D-lysine (Sigma) and laminin (VWR) in medium supplemented with 2% B27 (Invitrogen), 10 ng/mL FGF, and 20 ng/mL EGF (Peprotech). BrdU  was added for 2 h before fixation.Dissociated primary neurosphere cells were seeded as described above in medium supplemented with 2% B27, 10 ng/mL FGF, and 10 ng/mL PDGF-AA (R&D) and cultured for 2–3 DIV. Medium conditioned by OPCs was collected and subjected to CX3CL1 ELISA (RayBiotech) (n = 3 preparations). Differentiation was induced by replacing with medium supplemented with 2% B27 and 40 ng/mL 3,3′,5-triiodo-L-thyronine .Cells isolated from P1–P2 cortices were seeded in Neurobasal Plus medium supplemented with 2% B27 plus (Invitrogen). Cortical neurons were enriched by wiping out proliferating cells with arabinocytoside C  incubation for 48 h. Neurons were cultured without AraC for an additional 4 days, at which point SVZ OPCs were seeded onto neurons and cultured for 2–6 DIV in the presence of 40 ng/mL T3 and FKN or VC.Cerebellar slices (300 μm) isolated from P10–P11 CD1 pups were cultured using an interface method  whereby 2, and dissected brains were fixed in 4% PFA for 16–24 h. Cryosections (18 μm) were rehydrated and subjected to immunohistochemistry (IHC). Cultured cerebellar slices were fixed with 4% PFA for 1 h, followed by permeabilization and IHC. Detailed ICC and IHC protocols as well as primary and secondary antibodies are listed in Cell cultures were fixed with 4% paraformaldehyde (PFA) and subjected to FKN-647 binding assay without cell permeabilization and/or immunocytochemistry (ICC) with cell permeabilization. Mice over 21 days of age were transcardially perfused with Hank’s balanced salt solution followed by 4% PFA. Mice under 21 days of age were euthanized with COCx3cr1, Olig2, Sox2 mRNA, or negative control probe purchased from ACD according to the manufacturer's instructions. Cells with three or more RNAscope dots were considered to be positive for marker expression.RNAscope was performed using cryosections from P2, P7, P15, and P65 CD1 brains as described in in vivo images were imaged in single plane or with z-stacks and optical slice thickness of 0.5–1 μm.RNAscope sections were imaged with a Zeiss LSM700 confocal microscope with z-stacks and an optical slice thickness of 0.2–0.5 μm. All other images were captured using a Zeiss Axio Imager M2 fluorescence microscope. Image acquisition was performed with Zen software (Zeiss). Cultured cells and In vitro and ex vivo data are from at least three independent biological experiments. At least 500–2,000 cells per treatment and biological experiment were counted. For in vivo experiments, 5–8 anatomically matched sections per brain were analyzed from six mice across three independent litters unless indicated otherwise. All data are presented as mean ± SEM. For two-group comparisons, two-tailed paired (in vitro and ex vivo datasets) or unpaired Student's or multiple t tests (in vivo datasets) were used to assess statistical significance between means. For three or more group comparisons, one-way ANOVA was followed by Dunnett's multiple comparisons test. A p value of <0.05 was considered significant .Conceptualization, A.E.S.W. and A.V.; Methodology, A.E.S.W., M.M.d.A., T.F., A.V., G.V., D.G., and A.E.C.; Formal analysis, A.E.S.W., Y.L., M.M.d.A., N.L.D., P.T., and A.V.; Investigation, A.E.S.W., Y.L., M.M.d.A., N.L.D., P.T., T.F., D.G., and A.V.; Resources, A.V. and S.S.; Writing – original draft, A.E.S.W. and A.V.; Writing – review & editing, A.E.S.W. and A.V.; Supervision, S.S. and A.V.; Funding acquisition, A.V.The authors declare no competing interests.


The binding dynamics of our chemosensors for Cys were revealed to have uncomplicated recognition with a stoichiometric ratio of 1 vs. 1. The addition of cysteine increased the emission intensity of the chemosensors. As a consequence, the luminescence off-on effect with sensing selectivity and linear sensing behavior for Cys was demonstrated. Sulfur modification on our chemosensors was shown to be effective in improving their selectivity and photostability.The present work was devoted to developing rhodamine-like chemosensing systems for cysteine (Cys) optical recognition. Aiming at low background light and minimal photobleaching effect, up-converting nanocrystals were firstly synthesized and latterly coated by α-cyclodextrin, and finally used as an exciting host. An energy transfer procedure from these nanocrystals and rhodamine sensors was established  Their participation in biological activities makes amino acids critical human health factors. Cysteine (Cys) is an attractive amino acid since its abnormal level has been connected to human diseases, including skin damage, growth hypoevolutism, bowel disease, etc. . Optical4 lattice is constantly applied based on its advantages of good quantum yield, prime uniformity, limited biotoxicity, and considerable spectroscopic matching for biological windows (4 lattice during NaYF4 preparation which coated the resultant NaYF4 lattice and made it hydrophobic (4 lattice can be modified by a phase transfer with α-cyclodextrin (α-CD). Its self-assembly reaction allows for a microenvironment with a hydrophilic edge and a hydrophobic cage. The hydrophobic oleic acid is bonded with the α-CD cage. While the α-CD hydrophilic edge is open to the surrounding environment , 1-octadecene, oleic acid (OA), cyclohexane, acetonitrile, n-hexane, and RE salts, were purchased from Souxian Chemical Company (Shanghai). Organic solvents and solvent water were redistilled.Untreated starting chemicals, such as rhodamine reagent, terephthalaldehyde, Lawesson’s reagent (LR), and cyclodextrin, were supplied by Donghu Chemical Company (Tianjin) and used for synthesis. Some common products, including POClComposite sample powder was diluted by phosphate buffer  and treated by ultrasonification for 300 s. The concentration was 5 mg in 10 ml. Micromorphology was provided by a Hitachi S-4800 microscope and a JEM-2010 transmission electron microscope. NMR data were collected using a Varian INOVA 300 spectrometer. MS data were collected by an Agilent 1100 MS spectrometer. IR spectra were collected by a Bruker Vertex 70 FTIR spectrometer (KBr). Photophysical spectroscopy and dynamics were performed on a Shimadzu UV-3101PC spectrophotometer, a Hitachi F-7000 spectrophotometer, and a TEKTRONIX TDS-3052 oscilloscope. A 980 tunable laser was applied as the exciting light.3 (15 ml), and POCl3 (10 ml). Then the mixture was stirred at 25°C for 25 min and at 90°C for 12 h under an N2 atmosphere. Solvent and excess POCl3 were extracted by thermal evaporation under decreased pressure. CH3CN (110 ml) and anhydrous NH2NH2 (11 ml) were mixed into the above product and stirred at 25°C for 1 h and at 91°C for another 12 h under an N2 atmosphere. Solvent and excess NH2NH2 were extracted by thermal evaporation under reduced pressure to give rhodamine B hydrazide. 1H NMR (CDCl3), Δ (ppm): 1.16 , 3.23 , 3.61 , 6.22 , 6.36–6.39 , 7.18–7.19 , 7.46 , 7.89 . EI-MS m/e: calc. for C28H32N4O2, 456.2; found, 456.8 [m]+.The Sensor-1 synthetic strategy is explained as follows . The fol2 atmosphere. Then toluene was extracted by thermal evaporation. The solid product was chromatography-purified to give sulfur-substituted rhodamine B hydrazide. Silica gel column, eluent = petroleum: CH2Cl2 (30:1). 1HNMR (CDCl3), δ (ppm): 1.21–1.23 , 3.29–3.32 , 3.79 , 6.14 , 6.41–6.43 , 7.22–7.24 , 7.59 , 8.05 . MS m/z: calc. for C28H32N4OS, 472.2; found, 472.8 [m]+.Then the obtained product was reacted with LR by the following procedure . A mixtu2H5OH (60 ml) was heated at 80°C for 12 h , δ (ppm): 1.19 , 3.43 , 6.36 , 6.31 , 6.49 , 7.25 , 7.57 , 7.69 , 7.88 , 8.11 , 9.70 . 13C NMR (CDCl3), δ (ppm): 12.34, 44.72, 58.49, 68.81, 97.49, 106.29, 108.71, 121.38, 124.47, 127.82, 128.42, 129.35, 133.64, 136.77, 142.73, 145.38, 148.83, 151.64, 153.29, 171.83, and 191.62. ESI-MS m/e: calc. for C36H36N4O2S, 588.3; found, 588.9 [m]+.Finally, a mixture of the above prepared sulfur-substituted product (4 mmol) and terephthalaldehyde (5 mmol) in Cfor 12 h . C2H5OH 1H NMR (CDCl3), δ (ppm): 1.22 , 3.38 , 6.33 , 6.41 , 6.51 , 7.19 , 7.57 , 7.71 , 7.88 , 8.14 , 9.62 . 13C NMR (CDCl3), δ (ppm): 12.72, 44.38, 58.84, 66.51, 97.58, 106.84, 108.47, 123.62, 124.52, 127.26, 128.74, 129.86, 133.46, 136.63, 141.81, 145.53, 148.24, 151.73, 153.25, 164.47, and 191.22. ESI-MS m/e: calc. for C36H36N4O3, 572.3; found, 572.8 [m]+.Sensor-2 was prepared similarly, but rhodamine B hydrazide was used for this route. 3·6H2O (0.04 mmol) was mixed with YbCl3·6H2O (0.40 mmol). Then YCl3·6H2O (1.56 mmol) was added. 1-octadecene (30 ml) and oleic acid (12 ml) were mixed then finally added. This mixture was heated at 160oC for 35 min. Finally, NaOH (5 mmol) was slowly added, followed by the addition of CH3OH (20 ml) and NH4F (8 mmol). This mixture was heated at 75°C for 35 min, 100°C for 35 min, and 300°C for 60 min. At last, 100 ml of ethanol was mixed together. The powder sample was diluted with hexane. The doping ratio was 5 mg in 10 ml. Then the α-CD solution (5 mM) was added (1:1) and stirred for a whole day. The water phase was collected by centrifuge to yield the up-converting lattice coated in cyclodextrin (denoted as CD-NaYF4).Our up-converting lattice was prepared according to a reported route and then modified with α-CD . ErCl3·64, respectively  and ring-open xanthene geometry (fluorescent) makes rhodamine-like molecules promising chemosensors with fluorescence turn-on characteristics . In thisectively . This Na4 lattice was evaluated using microscopy images. As demonstrated by 4 lattice suggests that it contained eight elements, as shown by the inset of 4, such as cyclodextrin and OA. The Cl element was assigned to the rare earth chloride leftover. The remaining elements matched the elemental composition of the desired up-converting lattice. 4 lattice. There were 15 sharp 2θ peaks which belonged to 2θ peaks of hexagonal NaYF4 (JCPDS 28-1192). Since there were no 2θ peaks coming from impurities or other phases, we concluded that ytterbium and erbium ions were trapped in the NaYF4 crystal cell. As a consequence, the excitation lattice NaYF4:Yb3+/Er3+ was constructed.The micromorphology of the as-synthesized up-converting NaYF−1. They were attributed to coupled stretch vibrations of the C-OH group from oleic acid , our chemosensors take their emissive structure, with emission wavelengths of 580 and 575 nm, respectively. Sulfur modification caused a slight red shift (To check the energy transfer (ET) from CD-NaYF lattice . The twoed shift .0) between CD-NaYF4 and Sensor-1 and Sensor-2 was defined by the below formulas. Q0, J, κ2, nd, NA, λ, fd(λ), and εA(λ) in 4 yield , spectral overlap integral, mutual molecular orientation, solvent refraction index, Avogadro number, wavelength, host emission, and chemosensor absorbance efficiency, respectively  were monitored when exposed to Sensor-1 and Sensor-2 and cysteine so that the energy transfer between them could be further understood. In 4 with no difference. Sensor-1 and Sensor-2 quench lattice emission was very slim, with a decay state of 271 μs for Lattice:Sensor-1 and 286 μs for Lattice:Sensor-2. Corresponding ET efficiency (η) is defined by 4 to pure Sensor-1 and Sensor-2 was inefficient. In other words, Sensor-1 and Sensor-2 incorporated spirolactam geometry and were not open to CD-NaYF4 energy transfer. The presence of cysteine (1 eqv.) made Sensor-1 and Sensor-2 take an emissive geometry and open for lattice emission. Consequently, lattice emissive dynamics were affected, showing lifetimes of 146 and 161 μs, respectively. Their η values were increased to 53.1 and 48.2%, respectively. The improved ET from CD-NaYF4 to Sensor-1 and Sensor-2 was thus confirmed. The sulfur substituent slightly increased chemosensor absorption intensity, so the η value of Lattice:Sensor-1 was higher than that of Lattice:Sensor-2.Emission dynamics of CD-NaYFvs. 1. A schematic presentation is shown by s denotes the association constant. This uncomplicated binding mechanism may give a linear sensing response towards Cys concentration variation, which will be later proved.The Job plot experiment was applied to find the binding stoichiometry between Sensor-1, Sensor-2, and cysteine. Here, their total concentration was fixed (10 μM). By gradually increasing the cysteine molar ratio, their emission spectra are compared in s value was fitted based on an absorption titration experiment, as depicted in T is the absorbance without Cys and A0 is absorbance with 100% Cys  transferred its energy to our chemosensors like 2H11∕2 and 4S3∕2. Taking our above analysis on emission decay dynamics, it was concluded that the dominant ET procedure from CD-NaYF4 to Sensor-1 and Sensor-2 was a Forester procedure.As for the final up-converting emission band (∼650 nm), it is usually reported to be unaffected by the energy quencher and applied as an inner standard for fluorescence titration . But thivs. 1) between Sensor-1 and Sensor-2 and cysteine, the chemosensor emission response against cysteine concentration should be discussed using a Stern–Volmer plot, as described by sv is the SV constant. Linear working curves were fitted, as depicted in Job’s Plot and Binding Stoichiometry. In addition, α-CD modification on our excitation lattice ensured our NaYF4 nanocrystals were uniformly dispersed, resulting in linear sensing behavior of our chemosensors. Linearity of Lattice:Sensor-1 was slightly better than that of Lattice:Sensor-2. It appears that the chemosensor sulfur modification improved the linearity of the working curve as well. Corresponding limit of detection (LOD) values were determined as 2.7 μM for Lattice:Sensor-1 and 2.8 μM for Lattice:Sensor-2. These LOD values were much lower than the normal Cys concentration in human serum  (In virtue of their simple binding mechanism (1 tration) . Considesv values were fitted to 1.26 × 105 M−1 and 1.14 × 105 M−1, respectively. These values improved compared to traditional values  should be minimized. According to 0) was the chemosensor emission intensity in the absence of Cys. The observation of intrinsic chemosensor emission intensity suggests that some chemosensor molecules started their structural transformation from a spirolactam structure (non-emissive) to a delocalized xanthene structure (emissive) without the help of Cys. Considering that both chemosensors suffered from such high intrinsic emission intensity, the high I0 may be connected to excitation source. The oleic acid on the NaYF4:Yb3+/Er3+ surface may be responsible for the chemosensor structural transformation since an acidic environment leads to rhodamine structural transformation as well  remained stable during 5 h of radiation exposure. We attributed its stability to its strong NaYF4 structure  may trigger the emission turn-on structural transformation, causing increased emission intensity. Some transition metal ions, such as Cu(II) and Hg(II), may trigger such emission turn-on structural transformation as well . To get vs. 1. The resulting sensing systems exhibited enhanced emission for cysteine with linear response and selectivity. Sulfur modification on our chemosensors was shown to be effective in improving their selectivity and photostability. Nevertheless, chemosensor emission residue should be decreased aiming at better sensitivity.Briefly, this paper reported two optical sensing platforms for cysteine detection. The up-converting nanoparticles were modified by cyclodextrin and applied as an excitation host. Rhodamine-like molecules were synthesized as probes. Full characterization on these nanocrystals and chemosensors was performed to confirm their identity. An energy transfer procedure from these nanocrystals and rhodamine sensors was established via their spectroscopic analysis and emissive decay dynamics comparison. The binding dynamics of our chemosensors for Cys were revealed to have uncomplicated recognition with a stoichiometric ratio of 1 There is still a disadvantage of these Lattice:Chemosensor systems because of the following two reasons. First, the effective working region of these chemosenosrs (2–14 μM) is much lower than the normal Cys concentration in human serum (15.2 ± 0.2 mM). In this case, human serum samples should be diluted 1000 times to meet the optimal sensitivity of these chemosensors (∼2.5). Second, the working curves of these chemosensors are just linear-like ones, with uncertainties. Theoretically, sample Cys concentration can be determined by these working curves. But, before so doing, serum samples must be diluted, causing uncertainty. As a consequence, we cautiously say that these Lattice:Chemosensor systems in this work are able to detect a fixed amount of Cys in human body, but are not good at doing it.For further research effort, their potential application in biological application should be verified, considering the up-converting excitation nanocrystals and good selectivity of these chemosensors. There are problems to be solved, though. First, as mentioned above, the effective working region of these chemosensors is 2–14 μM, while the normal Cys concentration in human serum is 1000 times higher (15.2 ± 0.2 mM). Thus, the doping ratio of the excitation source and chemosensor should be adjusted to meet the normal Cys concentration in human serum. Second, although the up-converting nanocrystals have been coated and modified by α-CD, there are still oleic acid chains on their surface. These oleic acid chains result in an acidic environment around excitation nanocrystals, leading to high intrinsic chemosensor emission intensity. In addition, this acidic environment may harm bio-samples, compromising bio-imaging. As a consequence, before practical bio-imaging, these oleic acid chains should be completely removed.


Women at increased familial breast cancer risk have been offered screening starting at an earlier age and increased frequency than national Screening Programmes for over 30 years. There are limited data on longer-term largescale implementation of this approach on cancer diagnosis.Women at our institution at ≥ 17% lifetime breast cancer risk have been offered enhanced screening with annual mammography starting at age 35 or 5-years younger than youngest affected relative, with upper age limit 50 for moderate and 60 for high-risk. Breast cancer pathology, stage and receptor status were assessed as well as survival from cancer diagnosis by Kaplan–Meier analysis.p < 0.001). Ten-year survival was particularly good for those diagnosed ≤ 40 at 93.8% . Women with lobular breast cancers had worse 10-year survival at 85.9% (95% CI 66.7–94.5). Breast cancer specific survival was good for 119 BRCA1/2 carriers with 20-year survival in BRCA1:91.2% (95% CI 77.8–96.6) and 83.8% (62.6–93.5) for BRCA2.Overall 14,311 women were seen and assessed for breast cancer risk, with 649 breast cancers occurring in 129,119 years follow up . Of 323/394 invasive breast cancers occurring whilst on enhanced screening, most were lymph-node negative (72.9%), T1  and stage-1 (61.4%), 126/394 stage2–4 (32%). 10-year breast cancer specific survival was 91.3% (95% CI 87.4–94.0) better than the 75.9% (95% CI 74.9–77.0) published for England in 2013–2017. As expected, survival was significantly better for women with screen detected cancers (Targeted breast screening in women aged 30–60 years at increased familial risk is associated with good long-term survival that is substantially better than expected from population data.The online version contains supplementary material available at 10.1007/s10549-021-06333-1. Breast cancer is the most common cancer in females with approximately 54,500 women diagnosed annually in the UK 2016) and remains the leading cause of premature death in women aged 30–60 years , tumour size, lymph node status and oestrogen (ER)/progesterone and HER2 receptor status. HER2 testing was only available from 2005. ER+ HER2− and triple negative breast cancer (TNBC) groups include HER2 untested from < 2005 as only 8.6% (6/70) and 7.3% (10/137) of subsequent tests on invasive ductal cancer for ER− and ER+ respectively were also HER2+ . Vital status was established on all breast cancer cases in April-2020 and causes of death confirmed from cancer records and death certification.Breast cancers were defined as detected ‘on programme’ if they were diagnosed at screening episodes or within 18-months . Most women off programme were discharged to population breast screening on the 3-yearly basis NHS breast screening programme aged 50 if moderate-risk and 60 if high-risk.BRCA1/2 (including CHEK2 c.1100delC) testing where indicated by likelihood of a BRCA1/2 PV, many families received testing of affected members through the Familial Breast Cancer Study (FBCS) and 1300 women (900 without breast cancer) consenting to the FHrisk study had testing of an extended panel of breast cancer genes through the BRIDGES study , BRCA2-3/8 . Only one each BRCA1 and BRCA2 deaths in carriers were breast cancer related in women on MRI screening (2/38). In total 34/54(63%) deaths in women with cancers detected in the enhanced screening programme were breast cancer related. There were two cancer deaths in women undergoing BRRM who had breast cancer  (0.44%) as well four non-cancer deaths for a total of 6/455 (1.3%), compared to 274 total deaths in women not undergoing BRRM (2%).Overall deaths were lower in women screened on the enhanced-programme 13.8%) compared with off-programme (20.8%) (Table .8% compap < 0.001); prevalence screen survival 94.5% (95% CI 79.8–98.6) vs incidence screen (p = 0.052). The pathologies with the highest proportion of breast cancer deaths were lobular  triple negative  and high-grade ER+ HER2− cancers  although numbers in each group were relatively small limiting statistical comparison. As expected, the lowest proportion of breast cancer specific deaths was noted in those with grade-1 tumours  and CIS , although breast cancer specific survival was also excellent in grade-2 ER + HER2- breast cancer [10-year-survival = 95.1% (95% CI 85.3–98.4)]. Nearly all triple negative breast cancer deaths occurred in the first 5-years . Breast cancer deaths, as expected, were more frequent in women with symptomatic interval cancers , breast cancer specific survival was virtually identical for all age groups, with 10-year ≤ 40 years survival 93.8% , 5, 10 and 20-year overall survival was 92.2% (80.5–97.0), 92.2% (80.5–97.0) and 79.9% (59.9–90.6). Survival was not significantly different between BRCA1, BRCA2 and non-BRCA carriers on enhanced screening  compared to 59 BRCA2 at 85.1% (64.1–94.3) and 275 non-BRCA 84.7% (76.5–90.3). The BRCA2 survival curve crossed over BRCA1 after 10 years. Only 51 BRCA carriers were aware of their status at breast cancer diagnosis. Kaplan–Meier curves comparing BRCA PV carriers who knew their status at diagnosis versus those who did not and BRCA carriers who had MRI versus those who only had mammography are shown in Supplementary Figures S2 and S3. Survival in the known carriers and MRI screened  and 90.1% (95% CI 62.6– 97.7%) 10-year survival respectively, but this was not significantly better than the controls who did not know their status  and those not undergoing MRI (87.0%: 95% CI 80.6–94.8%). We also carried out a time dependency analysis and this did not show any advantage to knowing the BRCA status . Overall 20-year cumulative risk of breast cancer was 9.7% .Although overall survival was worse in those diagnosed > 50 years  = 96.1% (95% CI 90.5–98.4%) and BRCA2 (n = 376) = 91.5% (95% CI 78.6–96.8%). The latter should be regarded as an underestimate as not all women had undergone BRCA1/2 testing.Five and 10-year breast cancer specific survival in those with breast cancer detected on programme vs off programme screening was:5-year 94.1% (95% CI 91.0–96.1) vs 94.3% (95% CI 90.5–96.6) and 10-year 91.0% (95% CI 87.2–93.7) vs 90.4% (95% CI 85.7–93.4) respectively. Overall 20-year survival in the cohort for all women for breast cancer specific survival was 98.8% (95% CI 98.3–99.1%) and for known BRCA1 grade-3 ER− HER2−  detected on enhanced screening. The majority of breast cancers were detected at stages 0/1 (270/394–68.5%) with only 94/394 (23.5%) interval cancers, seven of which were asymptomatic at BRRM. There were expected breast cancer associations with BRCA1/2 PV carriers of 1.6%  [BRCA2 carrier among MRI screened women. This continues to provide efficacy evidence for MRI as an alternative to BRRM [BRCA2 carriers. Indeed, nearly half the deaths (44%) in BRCA1/2 PV carriers with breast cancers were due to other cancers (6/16) or old age (1/16). A study of 491 women with a germline BRCA mutation, who were annually screened with MRI and mammography for a median of 12.7 years found incidence breast cancer was 2% annually. There were four breast cancer-related deaths among 91 who developed breast cancer [We have reported a mean annual rate of incident prospective breast cancers in % BRCA1) , consist% BRCA1) , 30 when to BRRM , althougt cancer .BRCA1/2 vs 70·1% (67·7–72·3) for non BRCA breast cancer compared to the lower 95% CI of 86.9% in our population. Indeed, the Kaplan–Meier curves continued to drop towards 50% by 15 years in POSH [Breast cancer specific survival was excellent with 10-year survival rates of 91.3% (95% CI 87.4–94.0) noticeably higher than current 10-year breast cancer survival in England of all women presenting with primary breast cancer of 75.9%  . Of part in POSH  far belo in POSH  survival in POSH .CDH1 pathogenic variants, LCIS or lobular breast cancer family history should be considered for MRI breast screening. A small number of cancers (4%) were detected only on CBE with a minority of the screen detected cancers also detected. We have previously highlighted this, but whether this is a cost-effective strategy despite the small size of most of these cancers will require further work [As expected, women with interval breast cancers had higher mortality. Most presented within 12-months with known poorer survival . Interesher work . We haveher work .ATM, CHEK2, PALB2). We do not have follow up for all women after December-2012, but were able to check vital status and cause of death for all women with breast cancer.There are some limitations to the present study. Genetic testing was only carried out in a minority of the screened population although it was performed on the great majority of enhanced programme breast cancers (91%) and assessment of incidence rates based on gene testing was not the primary study aim. Most women with breast cancers had panel testing, allowing extrapolation of likely frequencies of other common familial genes Supplementary file2 (DOCX 89 KB)Below is the link to the electronic supplementary material.


Cardiovascular disease (CVD) has become an increasingly important topic in the field of medical research due to the steadily increasing rates of mortality caused by this disease. With recent advancements in nanotechnology, a push for new, novel treatments for CVD utilizing these new materials has begun. Carbon Nanodots (CNDs), are a new form of nanoparticles that have been coveted due to the green synthesis method, biocompatibility, fluorescent capabilities and potential anti-antioxidant properties. With much research pouring into CNDs being used as bioimaging and drug delivery tools, few studies have been completed on their anti-inflammatory potential, especially in the cardiovascular system. CVD begins initially by endothelial cell inflammation. The cause of this inflammation can come from many sources; one being tumor necrosis factor (TNF-α), which can not only trigger inflammation but prolong its existence by causing a storm of pro-inflammatory cytokines. This study investigated the ability of CNDs to attenuate TNF-α induced inflammation in human microvascular endothelial cells (HMEC-1). Results show that CNDs at non-cytotoxic concentrations reduce the expression of pro-inflammatory genes, mainly Interleukin-8 (IL-8), and interleukin 1 beta (IL-1β). The uptake of CNDs by HMEC-1s was examined. Results from the studies involving channel blockers and endocytosis disruptors suggest that uptake takes place by endocytosis. These findings provide insights on the interaction CNDs and endothelial cells undergoing TNF-α induced cellular inflammation. Cardiovascular disease (CVD) has become a significant focus of research due to the impact and devastating effects it has within the world, and in the United States. Many risk factors of CVD have been associated with the formation of atherosclerosis, but inflammation and cellular dysfunction within the endothelial cell lining play one of the most significant roles in the initiation process ,2,3,4. TNanomedicine has made many advances within the biomedical community, and the treatment of many ailments using nanoparticles have been the direction many researchers have been taking. Through nanotechnology, these extremely small particles that can be less than 10 nm in size, known as carbon nanodots (CNDs), can be modified to fit the needs of the researcher, allowing to produce unique characteristics for each study . These m2+, H2O2 and glucose [Due to the passivation surface of CNDs, they can be conjugated and doped with different dyes and stains, which enables biological imaging of cancer and immune cells ,13. Coup glucose ,15,16. BWith CNDs biocompatibility, drug delivery is another biomedical application research has been leaning towards. Synthesis of CNDs can be extremely simple, inexpensive materials to make, and the materials utilized to generates these particles can modify their properties and purposes . SynthesCNDs have been noted to be potent antioxidants and scavenging reactive oxygen species (ROS), and studies have been able to demonstrate protection against these free radicals as well as oxidative stress . The mec® CRL-3242™) cells were cultured in GenDepot® MCDB131 media, supplemented with 10 mM L-glutamine, 10 ng/mL Endothelial Growth Factor (EGF), 1 g/mL hydrocortisone, 10% Fetal Bovine Serum (FBS), and 1% Penicillin/Streptomycin. Cells were grown in Cellstar Filtered Cap 75 cm2 cell-culture treated, filter screw cap flasks in an incubator set to 37 °C and 5% CO2. Media was renewed every 2–3 days, and cells were be split into a new passage at 85–90% confluence.Human Microvascular Endothelial cells  containing calcium, magnesium, and glucose. Cells were split into 100 mm × 10 mm Corning® Cell Culture-treated petri-dishes and grown to 85–90% confluence. Petri dishes were decanted of their media and rinsed 2 times with 1× Phosphate Buffer Solution (PBS). Treatment (7 mL) was then pipetted onto adherent HMEC-1s and maintained in an incubated at 37 °C and 5% CO2 during treatment.Human Microvascular Endothelial cells (HMEC-1) were treated with 10 ng/mL of TNF-α for 6 h with a dose-dependent co-treatment of 0.03, 0.1 and 0.3 mg/mL CNDs in Sigma-Aldrich/Millipore Sigma® cell culture-treated Petri dishes. Cells were then washed 2 times with PBS and incubated for 30 min in HBSS and varying concentrations of known inhibitory reagents. After 30 min, cells were washed two times and incubated for 6 h with 0.1 mg/mL CNDs. Once 6 h treatment was completed, treatment was removed, and cells were washed 2 times with PBS to remove any excess CNDs. Cells were collected using the cell scrapping method, centrifuged at 5000 RCF for 5 min, and resuspended in 1 mL PBS. Then, 150 μL of resuspended cells was aliquoted into 4 wells on a black 96-well Costar® plate. The fluorescence of the plate was then read using the Bio-Tek® Synergy 2™ plate reader at 360/40 excitation and 460/40 emission.HMEC-1 cells were cultured to 85% confluency in 100 mm × 10 mm Corning® cell culture-treated plate to 85–90% confluency. Media was removed, and cells were washed 2 times with PBS. Cells were then treated in 150 μL/well of HBSS with 0.001, 0.03, 0.1, 0.6 and 1.2 mg/mL of CNDs for 6 h. After treatment, cells were decanted and washed with 500 μL/well of PBS and then replaced with 0.2 g/mL MTT in 200 μL HBSS and incubated for 2 h. Following incubation, the MTT-mixture was removed, and cells were washed with PBS. Then, 200 μL of dimethyl sulfoxide (DMSO) was added to each well. The plate was covered and then placed on a shaker at low speed for 15 min. After purple formazan crystals that had formed were dissolved, cells were read with the Bio-Tek® Synergy 2™ plate reader at 570 nm wavelength.HMEC-1 cells were grown in 24 well CostarHMEC-1 cells were treated with 10 ng/mL of TNF-α in the presence or absence of 0.03, 0.1 or 0.3 mg/mL of CNDs for 6 h. IL-8 and soluble form of ICAM-1 (sICAM-1) in the cell culture supernatants were measured using Quantikine ELISA Kit  following the manufacturer’s instructions. Samples were plotted against standard curves for the determination of concentrations in the samples.® cell culture-treated Petri dishes to 90% confluency. The media was discarded, and cells were washed with 1× PBS and treated in the TNF-α/CND concentrations previously described, for 6 h. After treatment, cells were decanted, and 1 mL of TRIzol®  was added to cells, and 1 mL of mixture was pipetted into a 1.5 mL Eppendorf tube. Then, 200 μL of Chloroform was added and incubated for 5 min at room temperature. Cells were then spun down in a centrifuge at 12,000 RCF for 15 min. The top aqueous layer was removed and combined with 500 μL of 2-propanol, mixed well, and incubated at room temperature for 15 min. The mixture was then centrifuged at 12,000 RCF for 10 min. Pellet formation was occurred and washed with 1 mL of 75% ethanol, centrifuged again at 7400 RCF for 5 min. The washing step was repeated once more. Pellet was then dried, and RNA was dissolved in 10 μL of Nase-free diethylpyrocarbonate (DEPC)-treated water.HMEC-1 cells were cultured within the 100 mm × 10 mm CorningHMEC-1 cells were cultured, treated, and RNA extraction was performed as listed above. The RNA extracted was quantified using Thermo Scientific NanoDrop™ and diluted to a 500 ng/μL concentration. Then 2 µL of diluted RNA were mixed with 5 μL of 5× First Strand Buffer, 1.25 μL of deoxynucleotide triphosphate (dNTP) solution, 1.25 μL of Random Primer, 0.625 μL of Moloney Murine Leukemia Virus Transcriptase (MMLV-RT), and 14.875 μL of DEPC water. The 25 µL solution was converted to cDNA using the Applied Biosystems™ Veriti™ 96-Well Thermal Cycler.® Green PCR Master Mix, 5 μL of DEPC water, 2 μL of forward primer, and 2 μL of reverse primer for the genes of interest with 1 μL of the diluted (1:9) cDNA. The genes of interest were IL-8, ICAM, MCP-1, IL-1β, GCLC and HO-1 with GAPDH serving as the housekeeping gene. The Applied Biosystem® StepOnePlus™ Real-Time PCR system ran for 40 cycles. Each cycle was as follows: 95 °C for 15 s, 58 °C phase for 1 min and a 60 °C cycle for 10 s. To quantify gene expression, the comparative threshold cycle (CT) values were used.Cells were treated, RNA extracted, and cDNA synthesized as previously described. The cDNA was used to target genes of interest by combining 10 μL of Power SYBRThe primer sequences are listed as followed: GAPDH Forward: 5′ AGA ACG GGA AGC TTG TCA TC-3′, GAPDH Reverse: 5′-GGA GGC ATT GCT GAT GAT CT-3′. IL-8 Forward: 5′ CTC TGT GTG AAG GTG CAG TT-3′, Reverse: 5′ AAA CTT CTC CAC AAC CCT CTG-3′. ICAM Forward: 5′ ACA GTG ACC ATC TAC AGC TTT C-3′, Reverse: 5′-CGG GTC TGG TTC TTG TGT ATA A-3′. MCP-1 Forward: 5′-GCT CAG CCA GAT GCA ATC AA-3′, Reverse: 5′-GGT TGT GGA GTG AGT GGT CAA G-3′. IL-1β Forward: 5′-CCA GCT ATG AAC TCC TTC TC-3′, Reverse: 5′-GCT TGT TCC TCA CAT CTC TC-3′. NQO-1 Forward: 5′-TTACTA TGG GAT GGG GTC CA-3′, NQO-1 Reverse: 5′-TCT CCC ATT TTT CAGGCA AC-3′. GCLC Forward: 5′-ACC ATC ATC AAT GGG AAG GA-3′, GCLCReverse: 5′-GCG ATA AAC TCC CTC ATC CA-3′. AR Forward: 5′-TGG ATG GAT AGC TAC TCC GG-3′, Reverse: 5′-CCC AGA AGC TTC ATC TCC AC-3′.® software, and the data are expressed as mean ± SEM. Student t tests were performed to obtain statistical significance . p < 0.05 was considered different.Statistical analyses were performed using GraphPad Prism® Eclipse™ Fluorescence Spectrophotometer was used for characterizing the CNDs. The photoluminescent characteristics of CNDs can be seen in their excitation wavelength around 360 nm and expressed an emission peak around 460 nm (The Caryd 460 nm . Understp > 0.05) toxic effects when compared to the control, suggesting there is no significant change in mitochondrial function with CND treatments.Before looking into potential anti-inflammatory properties of CNDs, it is important to determine if CNDs display any toxic effects to endothelial cells. A colorimetric assay known as an MTT assay was completed to analyze the mitochondrial function of HMEC-1 cells treated with 0.001, 0.03, 0.1, 0.3, 0.6, and 1.2 mg/mL of CNDs for 6 h . MTT is We next wanted to determine the effect of CND’s on proinflammatory biomarkers in endothelial cells. HMEC-1 cells were treated with 0.03, 0.1, or 0.3 mg/mL of CND in the presence of 10 ng/mL of TNF-α for 6 h. After the 6 h treatment, cells underwent RNA extraction, cDNA synthesis, and qRT-PCR analysis. The dose concentrations were chosen based on our MTT cell viability assay  as well p < 0.05), with cells that were co-treated with TNF-α and either 0.03, 0.1, and 0.3 mg/mL of CNDs, when compared to the cells treated with only TNF-α (p < 0.05), in gene expression when compared to cells treated with TNF-α alone (p < 0.05), in gene expression of IL-1β, when compared to cells only treated with TNF-α (p > 0.05), when compared to cells treated with TNF-α . Cells with a co-treatment of TNF-α and 0.3 mg/mL of CNDs also had a significant decrease (p < 0.05) in sICAM production induced by TNF-α when compared to the cells treated with TNF-α alone H: Quinone Oxidoreductase 1 (NQO1), Nuclear factor erythroid 2 (Nrf2), aldose reductase (AR), Glutathione reductase (GR), Glutamate-Cysteine Ligase Catalytic Subunit (GCLC), and Heme Oxygenase 1 (HO-1). Treatment for HMEC-1 cells were followed as previously described, with a co-treatment of TNF-α and either 0.03, 0.1, or 0.3 mg/mL of CNDs for 6 h. After treatment, RNA extraction, cDNA synthesis, and qRT-PCR were completed.p < 0.05) was seen for cells treated with a co-treatment of TNF-α and 0.3 mg/mL when compared to cells treated with only TNF-α (p > 0.05), when comparing the co-treated TNF-α and CND cells with cells treated only with TNF-α (p > 0.05), with co-treated cells compared to only TNF-α treated cells (p > 0.05) of AR in HMEC-1s (p > 0.05), in gene expression of GR comparing our co-treated HMEC-1s with cells treated only with TNF-α , along with reducing the activation of the transcription factor, Nuclear Factor κ-light-chain enhancer (NF-κB) . During ly TNF-α A. NQO1 ith TNF-α B. Nrf2 ied cells C. We wan HMEC-1s D. GR is th TNF-α E.p < 0.05) in fluorescent intensity when cells were pre-treated with phenylglyoxal, which is a known selective phagocytic inhibitor (p < 0.05).Determining the possible routes of entry of CNDs is important to understand the function and role these nanoparticles have in the cell. A standard curve of CNDs was created . Uptake nhibitor A. Chlorpnhibitor B, which p < 0.05). Further studies were completed using other channel blockers (p > 0.05). Other endocytosis disrupting inhibitors were also tested (p > 0.05).Channel blocking inhibitors were also tested due to CND’s known ability for having a slight negative charge ,34. HMECblockers A–L, whico tested A–C and dTNF-α is a known proinflammatory cytokine and can trigger a positive feedback loop, producing more TNF-α along with other proinflammatory cytokines, chemokines and adhesion molecules . This stIL-8 is a proinflammatory cytokine that can cause an extremely rapid response from the immune system, and it has been shown to be highly expressed during cellular dysfunction of endothelial cells ,51. It hXanthine oxidoreductase (XO) is present in most body tissues and fluids. Using the reaction carried out by XO reduces oxygen into a superoxide and lucigenin as the chemiluminescence probe; we further demonstrated CNDs scavenging ability of superoxide free radicals [The mechanism by which CNDs reduces TNF-α induced inflammation remains unclear and is an area that requires further exploration. However, the ability of ROS scavenging by CNDs could be the mechanism that contributes to the pro-inflammatory properties based on two of the following reasons. Previous studies have demonstrated the capability of CNDs as antioxidants and scavengers of ROS. We have demonstrated that CNDs have the capability to be free radical scavengers through DPPH● testing. In this test, transferring of an electron either through oxidation or reduction can stabilize and reduce the absorbance reading. It was demonstrated that CNDs reduced the absorbance of DPPH in a dose-dependent manner as low as 0.02 mg/mL in the solution, demonstrating antioxidant, free radical scavenging potentials ,57. Xantradicals . By way radicals . Altogetradicals ,59. An iradicals . While Rradicals ,61. NADPradicals . The litROS can also be regulated by NQO1, Nrf2, GCLC, GR, AR, and HO-1 ,29,31,63To further understand the action CNDs have on HMEC-1 cells, we studied the uptake of these nanoparticles. Diffusion, protein channels, or through endocytosis were the routes believed these nanoparticles could take; however, diffusion has been quickly ruled out due to the negative charge CNDs have . PreviouIn summary, CNDs reduce TNF-α-mediated endothelial inflammation in HMEC-1 cells. Our results showed CNDs did not possess cytotoxic characteristics. Furthermore, our study was able to narrow down potential routes of uptake for CNDs into HMEC-1s, showing that endocytosis is indeed a route of entry; however, it might not be the only one. These findings provide insights on the interaction CNDs and endothelial cells undergoing TNF-α induced cellular inflammation.


Ginkgo biloba L. (GB) and Selaginella tamariscina (P. Beauv.) Spring (ST), which displayed very strong inhibitory effects toward CYP3A4. The inhibitory effects of these biflavones on clinical drugs that mainly undergo CYP3A4-dependent metabolism were evaluated. The IC50 of GK toward tamoxifen, gefitinib and ticagrelor were found to be of 0.478 ± 0.003, 0.869 ± 0.001, and 1.61 ± 0.039 μM, respectively. These results suggest the potential pharmacokinetic interactions between the identified biflavones and clinical drugs undergoing CYP3A4-mediated biotransformation. The obtained information is important for guiding the rational use of herbal medicine in combination with synthetic pharmaceuticals.CYP3A4-mediated Phase I biotransformation is the rate-limiting step of elimination for many commonly used clinically agents. The modulatory effects of herbal medicines on CYP3A4 activity are one of the risk factors affecting the safe use of drug and herbal medicine. In the present study, the inhibitory effects of nearly hundred kinds of herbal medicines against CYP3A4 were evaluated based on a visual high-throughput screening method. Furthermore, biflavone components including bilobetin , ginkgetin (GK), isoginkgetin (IGK), and amentoflavone (AMF) were identified as the main inhibitory components of  Cytochrome P450 (CYP) enzymes are a large class of heme-thiolate protein superfamily serving the key enzymes in the first-pass metabolism of many foreign substances in humans . Among mDrugs mainly undergoing CYP3A4 metabolism are more susceptible to adverse drug reactions caused by drug-drug interaction with co-administered pharmaceuticals, and the inhibitory effect of herbal medicines on CYP3A4 can be one of risk factors affecting the safe use of drug and herbal medicine combinations . The comN-ethyl-1,8-naphthalimide (NEN) is a CYP3A4-specific fluorescent probe developed by our group . HydroxyGinkgo biloba L. (GB) and Selaginella tamariscina (P. Beauv.) Spring (ST) display the strongest inhibitory effects toward CYP3A4, and four biflavones were identified as the main inhibitory components of these herbal medicines. Subsequently, the inhibitory effects of these biflavones on clinical drugs mainly undergoing CYP3A4-dependent metabolism were evaluated. Results of these studies suggest the potential interaction between these biflavones and clinical drugs.In the present study, we employed rapid the screening of the inhibitory action of herbal medicines against CYP3A4 based on a visual high-throughput screening method using NEN. Our results indicate that 2, glucose-6-phosphate dehydrogenase, and D-glucose-6-phosphate (G-6-P) were obtained from Sigma-Aldrich . The pooled human liver microsomes (HLM) were purchased from Bioreclamation IVT .The prepared slices of Chinese crude medicines were purchased from Beijing Tongren Tang Co., Ltd. . Ginkgetin, isoginkgetin, bilobetin, and amentoflavone were purchased from Herbest Biotechnology Co., Ltd. . N-ethyl-1,8-naphthalimide (NEN) was synthesized by our lab, and was fully characterized by NMR and MS techniques. Midazolam, NADPNaMedical materials were ground to coarse powder, and then immersed into 10 volumes of 95% ethanol. Subsequently, the powdered materials were extracted twice with ultrasound (600 W) for 30 min. The filtered extracts were enriched by a rotary vacuum evaporator. The extracts were dissolved in DMSO as the stock solution (20 mg/ml) for the subsequent.2 (4 mM), NEN (50 µM), extracts of herbal medicine (20 µg/ml), or components. The incubation duration and HLM protein concentrations were 30 min and 0.05 mg/ml, respectively. The reaction was initiated by the addition of NADP+ and terminated by the addition of 100 µL ice-cold acetonitrile (ACN). NEN 4-hydroxylation were utilized as the probe reaction for CYP3A4, and fluorescence image of 96-well microplates were measured by a fluorescent image analyzer  under the excitation wavelength of 488 nm (Em: 570 ± 15 nm). The formation of NEHN was evaluated by the quantitative analysis of fluorescence signal for individual wells of microplates using the equipment supporting software. The percentage residual activity was calculated from percentage relative value of NEHN formation rate.The primary incubation mixture contains HLM, potassium phosphate buffer , G-6-P (10 mM), G-6-P dehydrogenase (1 U/ml), MgClGinkgo biloba L. (GB) and Selaginella tamariscina (P. Beauv.) Spring (ST) were analyzed based on UPLC with a photodiode array detector.The chemical fingerprints of Fractions were collected with an Acquity UPLC system  equipped with a photodiode array detector (2998), an auto sampler, a quaternary gradient pump, and a column compartment. An AQCUITY UPLC HSS C18  analytical column was used and kept at 30°C. The mobile phase consisted of 0.3% trifluoroacetic acid in water (A) and Methanol (B). The following gradient conditions was used: 0.0–5.0 min, 85% A, 5.0–38.0 min, 85–20% A, 38.0–41.00 min, 20% A, 41.0–44.0 min, 20–85% A, 44.0–47.0 min, 85% A for analysis of GB extract; 0.0–3.0 min, 65% A, 3.0–10.0 min, 65%–10% A, 10.0–15.00 min, 10% A, 15.0–18.0 min, 10%–65% A, 18.0–20.0 min, 65% A for analysis of ST extract. The precision of the fingerprinting method was previously confirmed by comparison of the HPLC spectrum mainly considering the relative retention time and peak area. Then, the LC fractions of crude extract  were collected consecutively at 30 or 60 s intervals, where the flow rate was set at 0.3 ml/min.in vacuum, and redissolved in DMSO. The fractions were evaluated for their activity moderating effect for CYP3A4 based on the high-throughput screening method. Then, the target fractions underwent further preparation and purification by semi-preparative HPLC.All LC fractions were dried Ki) values were determined using the equations for competitive inhibition . The high-throughput screening assay was performed, and the inhibition curve was fitted with GraphPad Prism (Version 8.0). The inhibition type was confirmed based on the R square of the following four equations and the Dixon and Lineweaver–Burk plots. The inhibition constant , tamoxifen (20 μM), ticagrelor (30 μM), respectively . The ExiGinkgo biloba L.  and Selaginella tamariscina (P. Beauv.) Spring  exhibited ultra-strong inhibitory effects toward CYP3A4 resulting in virtual elimination of the fluorescence signal in the corresponding wells of the 96-wells microplate. Quantitative analysis of the inhibitory effects of herbal medicine towards CYP3A4 demonstrate that the residual activities of CYP3A4 after treatment with the extracts of GB and ST were 6.3 and 5.0%, respectively (Tribulus terrestris L. (D2), Areca catechu L. (B10), and Fissistigma glaucescens (Hance) Merr. (F3).The inhibitory effects of 93 herbal medicines toward CYP3A4 were preliminary evaluated using the high-throughput screening system constructed with CYP3A4 selective fluorescent probe NEN . Inhibitectively . The quaWe attempted to reveal the inhibitory components of GB and ST since they exhibited strong inhibitory effect towards CYP3A4. However, the complexity and structural diversity of the two herbal medicines are very high, especially GB. In the present study, a combination of chemical fingerprinting-guided LC fraction collection and screening of the inhibitory potential was used to identify the inhibitory components of herbal medicine. The LC fractions of GB and ST were collected consecutively at appropriate time intervals (30 or 60 s) based on the reliable LC method which was confirmed by reproducible relative retention time of the main components of herbal medicine (RSD < 3%). Subsequently, the inhibitory components of GB and ST toward CYP3A4 were successfully identified by bioassay guided LC fractions studies utilizing high-throughput visualization screening. For GB, three inhibitory components were picked out from a comparison of the UPLC-UV fingerprint with the profile of CYP3A4 inhibition . Then, tIC50 values of DGK, GK, IGK, and AMF against CYP3A4 were evaluated as 0.162 ± 0.022, 0.106 ± 0.004, 0.982 ± 0.006, and 0.186 ± 0.004 μM, respectively. These findings suggested that the four biflavones exhibited a significant inhibition activity towards CYP3A4 and further confirmed the above conclusion that DGK, GK, IGK, and AMF were the main inhibitory components of GB and ST, respectively.The concentration range of DGK, GK, IGK, and AMF was used to depict the inhibitory plot and characterize the inhibitory effects of four identified biflavones toward CYP3A4. As shown in Ki) values of DGK, GK, IGK, and AMF toward CYP3A4 were further determined. As shown in Ki value was determined to be of 0.070 ± 0.018 μM. Correspondingly, the inhibition profiles of GK, IGK, and AMF were all fitted to mixed reversible inhibition equation with the Ki values of 0.034 ± 0.007, 0.278 ± 0.087, and 0.101 ± 0.030 μM , Areca catechu L. (B10), Fissistigma glaucescens (Hance) Merr. (F3) and Isatis indigotica Fort. (F11) displayed a moderate inhibitory effect toward CYP3A4, while the inhibitory effect of the remaining herbal medicines was insignificant. It is noteworthy that GB and Isatis indigotica Fort. are the most frequently used herbal medicines in Asia. Patients with ischemic stroke and neurological disorders used GB as a remedy (Isatis indigotica Fort. is one of the few well-recognized traditional herbs with anti-inflammatory, antibacterial and antiviral effects (Tribulus terrestris L. is currently approved as a dietary supplement due to its pro-sexual and androgen enhancing effects (Isatis indigotica Fort., Tribulus terrestris L. Our results provide important information for guiding the rational combination of herbal medicines and synthetic pharmaceuticals.Among the screened herbal medicines, GB and ST showed strong inhibitory effects on CYP3A4. a remedy , and Isa effects . Additio effects . The aboPrevious research reports on interactions between GB and CYP indicated that flavonol aglycones such as kaempferol, quercetin, and apigenin were the major CYP3A4 inhibitory components in GB . The disIC50 of 3.50 ± 0.034, 1.61 ± 0.039, and 10.2 ± 0.382 μM, respectively. Notably, the GB extract is commonly used for treatment in patients exhibiting angina pectoris, myocardial infarction and ischemic stroke, and for improving cardiac function, myocardium microenvironment and neurological function (IC50 values for the effect of AMF on the oxidation of tamoxifen and gefitinib are equal to 2.72 ± 0.093 and 5.20 ± 0.527 μM, respectively. Our results confirmed a significant inhibitory effects of the diflavonoids on the clinical drugs under study, and also pointed out that more attention should be paid to the herbal-drug interactions caused by the CYP3A4-mediated metabolic interaction between clinical drugs with biflavonoids and the respective herbal medicines.Biflavonoids are a class of compounds with outstanding anti-inflammatory and antioxidant biological activities, and have anti-microbial, anti-tumor and neuroprotective effects . In a vafunction . Therefofunction . The pot


Palmaria palmata (Rhodophyta), Laminaria digitata, Saccharina latissima and Ascophyllum nodosum . Screening of organic extracts against helminths of swine (Ascaris suum) and sheep (Teladorsagia circumcincta) revealed that S. latissima and L. digitata had particularly high biological activity. A combination of molecular networking and bio-guided fractionation led to the isolation of six compounds from extracts of these two species identified in both fermented and non-fermented samples. The six isolated compounds were tentatively identified by using MS-FINDER as five fatty acids and one monoglyceride: Stearidonic acid (1), Eicosapentaenoic acid (2), Alpha-Linolenic acid (3), Docosahexaenoic acid (4), Arachidonic acid (5), and Monoacylglycerol (MG 20:5) (6). Individual compounds showed only modest activity against A. suum, but a clear synergistic effect was apparent when selected compounds were tested in combination. Collectively, our data reveal that fatty acids may have a previously unappreciated role as natural anti-parasitic compounds, which suggests that seaweed products may represent a viable option for control of intestinal helminth infections.Widespread use of antimicrobial drugs has led to high levels of drug-resistance in pathogen populations and a need for novel sources of anti-bacterial and anti-parasitic compounds. Macroalgae (seaweed) are potentially a rich source of bioactive compounds, and several species have traditionally been used as vermifuges. Here, we investigated the anti-parasitic properties of four common cold-water Nordic seaweeds;  Gastrointestinal (GI) parasitic helminths are major pathogens causing reduced health, welfare and productivity in livestock . Due to Digenea simplex (Rhodophyta) has for centuries been used as a deworming agent against the human nematode Ascaris lumbricoides, due to the active compound kainic acid  has also been proven to have in vitro nematocidal effects against two ovine GI nematodes, Haemonchus contortus and Trichostrongylus colubriformis, with the active compounds found to be tetrahydrofurans  and one red macroalgae , which are common in Nordic waters and have been used for centuries as occasional livestock feed when resources were scarce  were selected based on their commercial availability and expected low toxicity, and subsequently harvested from Nordic waters (2O) crude extracts of both fermented (FER) and non-fermented (non-FER) seaweeds. The activity of the extracts was then assessed against third stage larvae (L3) from A. suum. After 24 h exposure, hexane extracts of non-fermented S. latissima and P. palmaria, and DCM:MeOH extracts of non-fermented S. latissima and L. digitata induced a larval mortality of >90%. Hexane and DCM:MeOH extracts from fermented S. latissima and the DCM:MeOH extract of fermented L. digitata also induced more than 90% mortality (2O extracts of S. latissima non-fermented samples of the two different locations (p = 0.009), indicating higher activity of polar compounds in the extract from the Faroe Islands as compared to the extract from Grenå, Denmark , MeOH:H2O extracts of S. latissima from Grenå (p = 0.0003), hexane extracts of L. digitata from Grenå (p = 0.0003), MeOH:H2O extracts P. palmata from Grenå (p = 0.045)  .T. circumcincta, a GI nematode ubiquitous in sheep production systems , DCM:MeOH extraction from L. digitata (p = 0.03) and P. palmata (p = 0.015), MeOH:H2O extraction from L. digitata (p < 0.0001) and P. palmata (p = 0.006). A statistically significant increase in activity was also observed for the fermented DCM:MeOH extraction of S. latissima as compared to non-fermented, however, both samples still exhibited activity above 97% (p = 0.046). In contrast, hexane extracts of P. palmata showed a significant decrease in activity between the non-fermented and the fermented samples (p = 0.002).To confirm that the activity was not only limited to one parasite species, the crude extracts were also tested against first stage larvae (L1) of  systems . A simil of >87% . The remS. latissima, L. digitata) appeared to contain compounds with broad-spectrum anti-parasitic activity, against both clade III and V nematodes (latissima and L. digitata were chosen for further investigation and bio-guided fractionation via flash chromatography. DCM:MeOH fractions were prepared by firstly extracting with DCM:MeOH (2:1), followed by flash chromatography over a gradient and finally analysis by LC-MS/MS. Fractions from both S. latissima and L. digitata showed activity against A. suum L3 over several fractions after 24 h at a concentration of 500 μg/ml (S. latissima) or ten (L. digitata) subfractions , while subfractions F2-7 of fermented and subfraction F2-9 of non-fermented L. digitata were also highly active. The subfractions showed differences in activity between the fermented samples and the non-fermented samples. For S. latissima, mortality in the subfraction F2-6 was significantly higher in the fermented than non-fermented subfraction (P = <0.0001), but for fraction F2-3 and F2-7 activity was higher in the non-fermented fraction . For L. digitata the mortality was significantly higher in subfractions F2-2 to F2-7 for fermented samples as compared to corresponding non-fermented samples, but significantly lower in F2-9 (P = <0.0001).Based on the two parasite assays, two of the selected seaweed species (ematodes  with mor00 μg/ml . For botractions . For fer1–5) from subfraction S. latissima FER-F2-6 and four compounds (1–3+6) from subfraction L. digitata FER-F2-7. Using MS/MS data (1), Eicosapentaenoic acid (2), Alpha-Linolenic acid (3), Docosahexaenoic acid (4), Arachidonic acid (5), and MG 20:5 (6) since they achieved the highest scores in MS-FINDER  µg/ml for 1, 53.9 (47.6–61.0) µg/ml for 2 and 41.1 (34.6–48.6) µg/ml for 3.All six compounds were tested individually against L. digitata non-FER-F2-9 contained compounds 1, 2 and 3, with 5 and 6 in lesser amounts. The S. latissima non-FER-F2-6 subfraction with low larval mortality (7.9 ± 2.1%) at 100 μg/ml was also found to contain compounds 1, 2, 5 and 6, while the most potent compound 3 was only present at trace levels. This may explain the low activity compared to the corresponding fermented sub-fraction (20) of the two other compounds. The predicted activity of the compounds was calculated based on the assumption of them having an additive effect, with activity higher than these predicted values defined as synergy  with in vitro effects against nematode larvae. Furthermore, our results confirm that even though these compounds individually only have moderate effects, the synergistic effects enable a potent anti-parasitic effect. Fermentation did change the molecular network of the seaweeds and activity of different subfractions, but the same active compounds were present in both fermented and non-fermented active samples. Although similar in their basic structures, not all isolated compounds displayed anti-parasitic activity. trans-dihydroxytetrahydrofuran and its derivatives ). It is possible that the PUFAs isolated from seaweed hold higher bioactive properties than commercially available acids given the more than 10-fold higher activity at a lower dose in our study, although it should be kept in mind that the experiments were performed on different parasites. Fatty acids constitute important building blocks of cell membranes and take part in regulation of membrane fluidity  in calves infected with Ostertagia ostertagi and Cooperia oncophora did not significantly affect the number of worms, although the worm burdens were 28% lower than in infected controls for intestinal worms  against parasites in livestock, and our study show fatty acids to be among the active compounds. Further studies will reveal if the in vitro properties of seaweed against helminths translate to in vivo effects in infected animals fed diets with a seaweed component and if so which mechanisms are involved.In conclusion, the seaweeds Palmaria palmata, Laminaria digitata, Saccharina latissima and Ascophyllum nodosum and were supplied by the companies Nordic Seaweed ApS (NS) and Fermentation experts A/S (FEX). The seaweeds originated from Denmark, Faroe Islands and Norway, place of origin and time of harvest are listed in S. latissima from Faroe Islands and A. nodosum from Bergen, Norway were cultured. The samples were supplied as dried and milled material. Both fermented and non-fermented material (N = 12) was supplied for each seaweed species. Material from Nordic Seaweed ApS was fermented aerobically by "Lactobacillus plantarum NT" for 20–24 h and dried indoor by wind turbines at 30–35°C. Material from Fermentationexperts A/S was fermented using a two-step anaerobic solid state fermentation process with an in-house bacterial mix inoculum (Lactobacillus spp.), and then dried in a spin flash dryer as described elsewhere : lsewhere .2O (1:1) was added and sonicated for 10 min, followed by addition of 150 ml of a mixture of DCM:MeOH (2:1) to the mixture and sonicated for additional 10 min. After filtration on filter paper, the two phases  were separated and the solvents evaporated under reduced pressure, resulting in two extracts. The hydroalcoholic extracts (partition MeOH:H2O) were desalted on SPE cartridges  with 10 ml H2O to remove salts, followed by elution with 15 ml of MeOH to recover the desalted extracts. An overview of all samples and resulting extracts can be seen in Seaweed samples were extracted with two protocols leading to three extracts, using a similar approach as previously published . For theBased on the biological activities against parasites, four samples were chosen for further extraction and fractionation . Each sa2O/MeOH solvent system and a 25 ml/min flow. The dry deposit was realized by mixing 1.8 g C18 silica with the extract. The following gradient was employed: 1) MeOH 30% held for 2 min, 2) MeOH 30–100% over 33 min, 3) MeOH 100% held for 5 min (Equilibration time: 3 min). A total of 36 tubes (30 ml) were collected and grouped into 8 fractions (Sl (FER)-F2-1 to 2–8) for fermented S. latissima, and 10 fractions (Ld (FER)-F2-1 to 2–10) for fermented L. digitata. Fraction Sl (FER)-F2-6 was then fractionated again on flash chromatography using the same cartridge, H2O/MeOH solvent system and a 25 ml/min flow with the gradient: 1) MeOH 70% held for 2 min, 2) MeOH 70–100% over 20 min, 3) MeOH 100% held for 5 min (Equilibration time: 3 min). Nine fractions were collected (Sl (FER)-F2-6-1 to Sl (FER)-F2-6–9). Fraction Sl (FER)-F2-6-6, Sl-F2-6-7 and Sl (FER)-F2-6-8 were grouped into a single fraction which was subjected to reverse-phase HPLC purification  100 Å 250 × 10 mm, 5 µm) using an isocratic elution with 79% H2O-CH3CN (+0.1% formic acid) at a flow rate of 5 ml/min. Fraction Sl (FER)-F2-6-6 to -8 gave compound 1 , 2 , 3 , 4  and 5 . Fraction Ld (FER)-F2-7 was subjected to the same reverse-phase HPLC. The Ld (FER)-F2-7 fraction gave compound 1 , 2 , 3  and 6 .Fraction 1–2  was sele3CN in water over 10 min followed by 3 min at 100% CH3CN  with a flow rate of 0.5 ml/min. Analyses were performed in positive mode.All extracts were solubilized at concentration of 1 mg/ml, in isopropanol for hexane extracts or MeOH for the others extract. Pure compounds were prepared at a concentration of 0.2 mg/ml in MeOH. Ultra-high Performance Liquid Chromatography-High Resolution Mass Spectrometry (UHPLC-HRMS) was performed on a Dionex Ultimate 3000 RS equipped with a diode array detector and coupled to Bruker QTOF Maxis with an electrospray ionization source. The analyses (injection volume: 2 µL) were performed on a reversed-phase column  employing a gradient of 10–100% CHS. latissima and L. digitata (fermented and non-fermented samples) was performed using the online platform of the Global Natural Product Social Molecular Networking (GNPS) web platform (https://gnps.ucsd.edu). First, LC-MS chromatograms of the three crude extracts from each seaweed species  were converted to mzXML files and pre-processed (by the same seaweed species and fermentation status) with Mzmine (Molecular networking of the compounds present in the crude extracts of h Mzmine  using thhttps://ccms-ucsd.github.io/GNPSDocumentation/) on the GNPS website (http://gnps.ucsd.edu), as previously described (S. latissima extracts (non-fermented samples): https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=47f091c230c440a5b909bafde6456160; S. latissima extracts (fermented samples): http://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=610990c014c9403c97dba218e59f8b35; L. digitata extracts (non-fermented samples): http://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=a3ab8245b68a42a29dbf2b98f8949b69; L. digitata extracts  and then imported to MS-DIAL (version 4.24) . The detA. suum were collected from pig intestines at the local slaughterhouse. Eggs were isolated from dissected uteri and embryonated for two months at room temperature (22°C)  and larvae hatched by stirring with 2 mm glass beads for 40 min at 37°C in a 5% CO2 incubator. The L3 were allowed to migrate overnight at 37°C (5% CO2) from a 20 µm sieve into sterile HBSS  in a conical sedimentation glass. The larvae were collected from the bottom of the glass and suspended in heated larval culture media .Gravid e (22°C) . Once a T. circumcincta assay was performed essentially as described previously (T. circumcincta (susceptible to anthelmintics) maintained in the laboratory  dissolved in DMSO were added to 150 µL of larvae mixture (∼100 larvae) in triplicates and incubated at 37°C. DMSO (1%) was used as a negative control. Moving or coiled-up larvae were counted as alive, and immobile and straight larvae as dead. The survival percentage of each replicate was calculated  assay the plate was set up as described for the A. suum (L3) assay. Extracts were screened at a concentration of 1 mg/ml and 1% DMSO in triplicates in 96 well-plates on 2 adjacent days with different larval batches. The anthelmintic Ivermectin  at 100 µM was used as a positive control and DMSO 1% was used as a negative control. The assay was counted after 48 h incubation at 23°C, and the mortality rate was calculated as for A. suum.The 50 of isolated compounds by non-linear regression. Depending on the distribution and variance of the replicas, t-test, Welsh's t-test or Mann-Whitney test was used to compare the activity of the fermented sample to the corresponding non-fermented sample of the crude extracts as well as F2 corresponding fractions.Prism 7.04 was used for statistical analysis of mean and standard deviation and calculation of EC