Geobarrettin D, a Rare Herbipoline-Containing 6-Bromoindole Alkaloid from Geodia barretti.

Geobarrettin D (1), a new bromoindole alkaloid, was isolated from the marine sponge Geodia barretti collected from Icelandic waters. Its structure was elucidated by 1D, and 2D NMR (including 1H-15N HSQC, 1H-15N HMBC spectra), as well as HRESIMS data. Geobarrettin D (1) is a new 6-bromoindole featuring an unusual purinium herbipoline moiety. Geobarrettin D (1) decreased secretion of the pro-inflammatory cytokine IL-12p40 by human monocyte derived dendritic cells, without affecting secretion of the anti-inflammatory cytokine IL-10. Thus, compound 1 shows anti-inflammatory activity.

Bromotryptamine and Imidazole Alkaloids with Anti-inflammatory Activity from the Bryozoan Flustra foliacea.

Chemical investigation of the marine bryozoan Flustra foliacea collected in Iceland resulted in isolation of 13 new bromotryptamine alkaloids, flustramines Q-W (1-7) and flustraminols C-H (8-13), and two new imidazole alkaloids, flustrimidazoles A and B (14 and 15), together with 12 previously described compounds (16-27). Their structures were established by detailed spectroscopic analysis using 1D and 2D NMR and HRESIMS. Structure 2 was verified by calculations of the 13C and 1H NMR chemical shifts using density functional theory. The relative and absolute configurations of the new compounds were elucidated on the basis of coupling constant analysis, NOESY, [α]D, and ECD spectroscopic data, in addition to chemical derivatization. The compounds were tested for in vitro anti-inflammatory activity using a dendritic cell model. Eight compounds (1, 3, 5, 13, 16, 18, 26, and 27) decreased dendritic cell secretion of the pro-inflammatory cytokine IL-12p40, and two compounds (4 and 14) increased secretion of the anti-inflammatory cytokine IL-10. Deformylflustrabromine B (27) showed the most potent anti-inflammatory effect (IC50 2.9 µM). These results demonstrate that F. foliacea from Iceland expresses a broad range of brominated alkaloids, many without structural precedents. The potent anti-inflammatory activity in vitro of metabolite 27 warrants further investigations into its potential as a lead for inflammation-related diseases.

Minimizing Taxonomic and Natural Product Redundancy in Microbial Libraries Using MALDI-TOF MS and the Bioinformatics Pipeline IDBac.

Libraries of microorganisms have been a cornerstone of drug discovery efforts since the mid-1950s, but strain duplication in some libraries has resulted in unwanted natural product redundancy. In the current study, we implemented a workflow that minimizes both the natural product overlap and the total number of bacterial isolates in a library. Using a collection expedition to Iceland as an example, we purified every distinct bacterial colony off isolation plates derived from 86 environmental samples. We employed our mass spectrometry (MS)-based IDBac workflow on these isolates to form groups of taxa based on protein MS fingerprints (3-15 kDa) and further distinguished taxa subgroups based on their degree of overlap within corresponding natural product spectra (0.2-2 kDa). This informed the decision to create a library of 301 isolates spanning 54 genera. This process required only 25 h of data acquisition and 2 h of analysis. In a separate experiment, we reduced the size of an existing library based on the degree of metabolic overlap observed in natural product MS spectra of bacterial colonies (from 833 to 233 isolates, a 72.0% size reduction). Overall, our pipeline allows for a significant reduction in costs associated with library generation and minimizes natural product redundancy entering into downstream biological screening efforts.

Infraspecific Variation of Huperzine A and B in Icelandic Huperzia selago Complex.

The alkaloids huperzine A and huperzine B were originally isolated from the Chinese club moss Huperzia serrata. They are known inhibitors of acetylcholinesterase, and especially huperzine A shows pharmaceutical potential for the treatment of Alzheimer's disease. Its supply heavily relies on natural plant sources belonging to the genus Huperzia, which shows considerable interspecific huperzine A variations. Furthermore, taxonomic controversy remains in this genus, particularly in the Huperzia selago group. With focus on Icelandic H. selago taxa, we aimed to explore the relatedness of Huperzia species using multi-locus phylogenetic analysis, and to investigate correlations between huperzine A contents, morphotypes, and genotypes. Phylogenetic analysis was performed with five chloroplastic loci (the intergenic spacer between the photosystem II protein D1 gene and the tRNA-His gene, maturase K, ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit, tRNA-Leu, and the intergenic spacer region between tRNA-Leu and tRNA-Phe). Huperzine A and huperzine B contents were determined using an HPLC-UV method. The phylogenetic analysis suggests that previously proposed Huperzia appressa and Huperzia arctica should not be considered species, but rather subspecies of H. selago. Three genotypes of Icelandic H. selago were identified and presented in a haplotype networking diagram. A significantly (p < 0.05) higher amount of huperzine A was found in H. selago genotype 3 (264 - 679 µg/g) than genotype 1 (20 - 180 µg/g), where the former shows a typical green and reflexed "selago" morphotype. The huperzine A content in genotype 3 is comparable to Chinese H. serrata and a good alternative huperzine A source. Genotype 2 contains multiple morphotypes with a broad huperzine A content (113 - 599 µg/g). The content of huperzine B in Icelandic taxa (6 - 13 µg/g) is much lower than that in Chinese H. serrata (79 - 207 µg/g).

Lepadins I-K, 3- O-(3'-Methylthio)acryloyloxy-decahydroquinoline Esters from a Bahamian Ascidian Didemnum sp. Assignment of Absolute Stereostructures.

Three decahydroisoquinoline alkaloids, lepadins I-K, were isolated from a specimen of Didemnum sp. collected in the Bahamas. The structures of the new compounds were assigned by an integrated analysis of MS, IR, and 1H, 13C, and 2D NMR spectra. Like previously reported lepadins, the structures of the new compounds contain a decahydroquinoline heterocyclic core in lepadin I, and a new variation, an octahydroquinoline in lepadin J, but differ from earlier reported compounds by acylation of the 3-hydroxyl group by a rare 3'-methylthioacrylate. The absolute configuration of lepadin I was solved by interpretation of NOE measurements, and exciton coupled circular dichroism (ECCD) of the corresponding N- p-bromobenzoyl derivative. The latter constitutes a general method for determination of absolute configuration of the entire lepadin family. The configuration of the remote side-chain secondary carbinol was solved by the modified Mosher's esters method. Lepadin I inhibited butyrylcholineesterase (BuChE, IC50 3.1 µM), but only weakly inhibited acetylcholineesterase (AChE) (10% at 100 µM).

6-Bromoindole Derivatives from the Icelandic Marine Sponge Geodia barretti: Isolation and Anti-Inflammatory Activity.

An UPLC-qTOF-MS-based dereplication study led to the targeted isolation of seven bromoindole alkaloids from the sub-Arctic sponge Geodia barretti. This includes three new metabolites, namely geobarrettin A⁻C (1⁻3) and four known compounds, barettin (4), 8,9-dihydrobarettin (5), 6-bromoconicamin (6), and l-6-bromohypaphorine (7). The chemical structures of compounds 1⁻7 were elucidated by extensive analysis of the NMR and HRESIMS data. The absolute stereochemistry of geobarrettin A (1) was assigned by ECD analysis and Marfey's method employing the new reagent l-Nα-(1-fluoro-2,4-dinitrophenyl)tryptophanamide (l-FDTA). The isolated compounds were screened for anti-inflammatory activity using human dendritic cells (DCs). Both 2 and 3 reduced DC secretion of IL-12p40, but 3 concomitantly increased IL-10 production. Maturing DCs treated with 2 or 3 before co-culturing with allogeneic CD4⁺ T cells decreased T cell secretion of IFN-γ, indicating a reduction in Th1 differentiation. Although barettin (4) reduced DC secretion of IL-12p40 and IL-10 (IC50 values 11.8 and 21.0 µM for IL-10 and IL-12p40, respectively), maturing DCs in the presence of 4 did not affect the ability of T cells to secrete IFN-γ or IL-17, but reduced their secretion of IL-10. These results indicate that 2 and 3 may be useful for the treatment of inflammation, mainly of the Th1 type.

Metabolic Profiling as a Screening Tool for Cytotoxic Compounds: Identification of 3-Alkyl Pyridine Alkaloids from Sponges Collected at a Shallow Water Hydrothermal Vent Site North of Iceland.

Twenty-eight sponge specimens were collected at a shallow water hydrothermal vent site north of Iceland. Extracts were prepared and tested in vitro for cytotoxic activity, and eight of them were shown to be cytotoxic. A mass spectrometry (MS)-based metabolomics approach was used to determine the chemical composition of the extracts. This analysis highlighted clear differences in the metabolomes of three sponge specimens, and all of them were identified as Haliclona (Rhizoniera) rosea (Bowerbank, 1866). Therefore, these specimens were selected for further investigation. Haliclona rosea metabolomes contained a class of potential key compounds, the 3-alkyl pyridine alkaloids (3-APA) responsible for the cytotoxic activity of the fractions. Several 3-APA compounds were tentatively identified including haliclamines, cyclostellettamines, viscosalines and viscosamines. Among these compounds, cyclostellettamine P was tentatively identified for the first time by using ion mobility MS in time-aligned parallel (TAP) fragmentation mode. In this work, we show the potential of applying metabolomics strategies and in particular the utility of coupling ion mobility with MS for the molecular characterization of sponge specimens.

Lipophilic fractions from the marine sponge Halichondria sitiens decrease secretion of pro-inflammatory cytokines by dendritic cells and decrease their ability to induce a Th1 type response by allogeneic CD4+ T cells.

CONTEXT: Halichondria (Halichondriidae) marine sponges contain components possessing various biological activities, but immunomodulation is not among the ones reported. OBJECTIVE: This study evaluated the immunomodulatory effects of fractions/compounds from Halichondria sitiens Schmidt. MATERIALS AND METHODS: Crude dichloromethane/methanol extracts of H. sitiens were subjected to various chromatographic techniques to obtain fractions/compounds with immunomodulatory activity, using bioassay-guided isolation. The effects of the fractions/compounds were determined by measuring secretion of cytokines and expression of surface molecules by dendritic cells (DCs) and their ability to stimulate and modify cytokine secretion by allogeneic CD4+ T cells. The bioactive fractions were chemically analyzed to identify the immunomodulatory constituents by 1D, 2D NMR, and HRMS data. RESULTS: Several lipophilic fractions from H. sitiens at 10 µg/mL decreased secretion of the pro-inflammatory cytokines IL-12p40 and IL-6 by the DCs, with maximum inhibition being 64% and 25%, respectively. In addition, fractions B3b3F and B3b3J decreased the ability of DCs to induce T cell secretion of IFN-γ. Fraction B3b3 induced morphological changes in DCs, characterized by extreme elongation of dendrites and cell clustering. Chemical screening revealed the presence of glycerides and some minor unknown constituents in the biologically active fractions. One new glyceride, 2,3-dihydroxypropyl 2-methylhexadecanoate (1), was isolated from one fraction and two known compounds, 3-[(1-methoxyhexadecyl)oxy]propane-1,2-diol (2) and monoheptadecanoin (3), were identified in another, but none of them had immunomodulatory activity. DISCUSSION AND CONCLUSIONS: These results demonstrate that several lipophilic fractions from H. sitiens have anti-inflammatory effects on DCs and decrease their ability to induce a Th1 type immune response.

Comparative analysis of the antioxidant properties of Icelandic and Hawaiian lichens.

Antioxidant activity of symbiotic organisms known as lichens is an intriguing field of research because of its strong contribution to their ability to withstand extremes of physical and biological stress (e.g. desiccation, temperature, UV radiation and microbial infection). We present a comparative study on the antioxidant activities of 76 Icelandic and 41 Hawaiian lichen samples assessed employing the DPPH- and FRAP-based antioxidant assays. Utilizing this unprecedented sample size, we show that while highest individual sample activity is present in the Icelandic dataset, the overall antioxidant activity is higher for lichens found in Hawaii. Furthermore, we report that lichens from the genus Peltigera that have been described as strong antioxidant producers in studies on Chinese, Russian and Turkish lichens also show high antioxidant activities in both Icelandic and Hawaiian lichen samples. Finally, we show that opportunistic sampling of lichens in both Iceland and Hawaii will yield high numbers of lichen species that exclusively include green algae as photobiont.

Immunomodulatory N-acyl Dopamine Glycosides from the Icelandic Marine Sponge Myxilla incrustans Collected at a Hydrothermal Vent Site.

A chemical investigation of the sponge (Porifera) Myxilla incrustans collected from the unique submarine hydrothermal vent site Strytan, North of Iceland, revealed a novel family of closely related N-acyl dopamine glycosides. Three new compounds, myxillin A (1), B (2) and C (3), were isolated and structurally elucidated using several analytical techniques, such as HR-MS, 1D and 2D NMR spectroscopy. Myxillin A (1) and B (2)were shown to be structurally similar, composed of a dopamine moiety, but differ in the acyl chain length and saturation. The myxillin C (3) has a dehydrotyrosine moiety composing the same acyl chain and glycosylation as myxillin B (2). Myxillins A (1) and C (3) were tested for immunomodulating activity in an in vitro dendritic cell model. Dendritic cells matured and stimulated in the presence of myxillin A (1) secreted lower levels of IL-12p40, whilst dendritic cells matured and stimulated in the presence of myxillin C (3) secreted lower levels of IL-10 compared with dendritic cells matured and stimulated in the presence of the solvent alone. These opposing results indicate that the structural differences in the aromatic ring part of the molecules could have an impact on the immunological effects of dendritic cells. These molecules could, therefore, prove to be important in preventing inflammatory diseases on the one hand, and inducing a response to fight tumors and/or pathogens on the other hand. Further studies will be needed to confirm these potential uses.

Phylum-specific regulation of resistomycin production in a Streptomyces sp. via microbial coculture.

Actinomycete genomes are encoded with immense potential to produce secondary metabolites, however standard laboratory culture experiments rarely provide the conditions under which associated biosynthetic pathways are expressed. Despite years of research attempting to access these pathways and aside from a few well-studied bacterial quorum sensing systems, little is known about the specificity of secondary metabolite regulation in bacteria, such as the conditions under which a bacterium produces an antibiotic and the extent to which it does so in recognition of a particular species in the immediate environment. In the current study, we observed that the cocultivation of a Streptomyces sp. (strain B033) with four pathogenic strains of the phylum Proteobacteria resulted in the production of the antibiotic resistomycin. After further coculture experiments, we determined that Proteobacteria induced the production of resistomycin in B033 at significantly higher rates (65%) than strains from the phyla Firmicutes (5.9%) and Actinobacteria (9.1%), supporting that the regulation of secondary metabolism in bacteria can be dependent on the species present in the immediate environment. These results suggest a lack of promiscuity of antibiotic biosynthetic pathway regulation and indicate that it is feasible to mine existing microbial strain libraries for antibiotics in a phylum-specific manner.

The totality of evidence approach in the development of AVT02 (adalimumab), a biosimilar to Humira.

The development of a biosimilar is based on comparative structural, physicochemical, functional and clinical assessments. The sum of these analyses encompasses the 'totality of evidence', which demonstrates no clinically meaningful differences between the biosimilar and the reference product (RP). Once biosimilarity has been established, provided there is suitable scientific justification, clinical data may be extrapolated to other indications of the RP. AVT02 has been developed as a biosimilar to high-concentration, low-volume Humira (adalimumab), an anti-tumour necrosis factor-alpha monoclonal antibody approved for various chronic inflammatory indications. The totality of evidence for AVT02 is described, supporting its approval as an adalimumab biosimilar for all approved indications globally. Analytical similarity assessments using mass spectrometry methods demonstrated identical amino acid sequences for AVT02 and the RP, with high similarity in terms of primary structure, post-translational modifications and higher-order structural attributes. The mechanism of action was assessed by various cell-based potency assays and binding assays, and the results demonstrated that AVT02 is highly similar to the RP. No clinically meaningful differences in terms of purity, potency and safety were observed, and minor differences in a few physiochemical attributes did not impact the in vitro biologic activity and were not considered clinically relevant. Clinical similarity was demonstrated by comparing the pharmacokinetic, efficacy, safety and immunogenicity profiles of AVT02 with those of the RP. Clinical studies supported similar pharmacokinetic and comparable immunogenicity profiles between AVT02 and the RP in healthy participants and participants with moderate-to-severe chronic plaque psoriasis, with no new safety signals detected. The totality of evidence described demonstrates the biosimilarity of AVT02 to the RP, thereby fulfilling the scientific and regulatory requirements for AVT02 as a high-concentration biosimilar for the treatment of chronic plaque psoriasis and all approved indications of the RP.

Demonstrating the high similarity between the biosimilar AVT02 (adalimumab) and Humira, supporting AVT02 to be used to treat all conditions currently treated with Humira Biosimilars are drugs that have similar quality, effectiveness, and safety profiles to an already approved biological drug, which is referred to as the 'reference product (RP)'. Although biosimilars have identical amino acids (the building blocks that make up proteins) to the RPs, they are manufactured in living cells which leads to a small amount of natural variability. Therefore, extensive testing is required to confirm that a biosimilar is highly similar to the RP. The 'totality of evidence' is a set of tests to demonstrate that there are no meaningful differences between the biosimilar and the RP, in other words, that there is 'biosimilarity' between the biosimilar and RP. Once biosimilarity has been proven, the biosimilar may be used to treat all the diseases currently treated with the RP, without the need for separate clinical trials in each disease. AVT02 has been developed as a biosimilar to Humira, an antibody approved for various chronic inflammatory diseases such as chronic plaque psoriasis (PsO). A step-by-step approach was used to show biosimilarity of AVT02 to Humira. This included clinical studies (in healthy individuals and participants with moderate to severe chronic PsO) and non-clinical studies (comparisons of the chemistry of the drugs and how they work in the body). Clinical studies in healthy individuals and participants with PsO showed that AVT02 and Humira were taken up and degraded by the body in a similar way, peoples' immune response to the two drugs were similar, and both drugs had similar side effects. No clinically meaningful differences in the purity, effectiveness, and safety of AVT02 compared with Humira were seen. The evidence demonstrates the biosimilarity of AVT02 to Humira and supports the use of AVT02 to treat all conditions which are currently treated with Humira.

Litorilinea aerophila gen. nov., sp. nov., an aerobic member of the class Caldilineae, phylum Chloroflexi, isolated from an intertidal hot spring.

A thermophilic, aerobic, Gram-stain-negative, filamentous bacterium, strain PRI-4131(T), was isolated from an intertidal hot spring in Isafjardardjup, NW Iceland. The strain grew chemo-organotrophically on various carbohydrates. The temperature range for growth was 40-65 °C (optimum 55 °C), the pH range was pH 6.5-9.0 (optimum pH 7.0) and the NaCl range was 0-3 % (w/v) (optimum 0.5 %). Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain PRI-4131(T) represented a distinct lineage within the class Caldilineae of the phylum http://dx.doi.org/10.1601/nm.550Chloroflexi. The highest levels of sequence similarity, about 91 %, were with Caldilinea aerophila STL-6-O1(T) and Caldilinea tarbellica D1-25-10-4(T). Fermentative growth was not observed for strain PRI-4131(T), which, in addition to other characteristics, distinguished it from the two Caldilinea species. Owing to both phylogenetic and phenotypic differences from the described members of the class Caldilineae, we propose to accommodate strain PRI-4131(T) in a novel species in a new genus, Litorilinea aerophila gen. nov., sp. nov. The type strain of Litorilinea aerophila is PRI-4131(T) ( = DSM 25763(T)  = ATCC BAA-2444(T)).

Discovery of a New Antibiotic Demethoxytetronasin Using a Dual-Sided Agar Plate Assay (DAPA).

For over a century, researchers have cultured microorganisms together on solid support─typically agar─in order to observe growth inhibition via antibiotic production. These simple bioassays have been critical to both academic researchers that study antibiotic production in microorganisms and to the pharmaceutical industry's global effort to discover drugs. Despite the utility of agar assays to researchers around the globe, several limitations have prevented their widespread adoption in advanced high-throughput compound discovery and dereplication campaigns. To address a list of specific shortcomings, we developed the dual-sided agar plate assay (DAPA), which exists in a 96-well plate format, allows microorganisms to compete through opposing sides of a solid support in individual wells, is amenable to high-throughput screening and automation, is reusable, and is low-cost. Herein, we validate the use of DAPA as a tool for drug discovery and show its utility to discover new antibiotic natural products. From the screening of 217 bacterial isolates on multiple nutrient media against 3 pathogens, 55 hits were observed, 9 known antibiotics were dereplicated directly from agar plugs, and a new antibiotic, demethoxytetronasin (1), was isolated from a Streptomyces sp. These results demonstrate that DAPA is an effective, accessible, and low-cost tool to screen, dereplicate, and prioritize bacteria directly from solid support in the front end of antibiotic discovery pipelines.

Synergistic cytotoxic effect of the microtubule inhibitor marchantin A from Marchantia polymorpha and the Aurora kinase inhibitor MLN8237 on breast cancer cells in vitro.

Macrocyclic bisbibenzyls are a class of characteristic compounds, exclusively produced by liverworts. They are attracting increasing attention due to their wide range of biological activities, including antibacterial, antifungal, and antioxidative properties as well as cytotoxicity. Marchantin A is a cyclic bisbibenzyl that has previously been isolated from Marchantia polymorpha and other liverwort species and has been shown to exert cytotoxic effects. In the present study we found that the Icelandic M. polymorpha species produces marchantin A and through an in vitro cell growth inhibition assay, marchantin A was shown to induce a reduction in cell viability of breast cancer cell lines A256 (IC50 = 5.5 µM), MCF7 (IC50 = 11.5 µM), and T47D (IC50 = 15.3 µM). The effect was considerably increased in all cell lines in a synergistic manner when the Aurora-A kinase inhibitor MLN8237 was added simultaneously. Fluorescence microscopy confirmed the antimicrotubular effect of marchantin A, and cell cycle analysis indicated enhanced cell division failure when combining this mitotic-spindle inhibitor with the checkpoint modulator.

Novel methods to characterise spatial distribution and enantiomeric composition of usnic acids in four Icelandic lichens.

Usnic acid is an antibiotic metabolite produced by a wide variety of lichenized fungal lineages. The enantiomers of usnic acid have been shown to display contrasting bioactivities, and hence it is important to determine their spatial distribution, amounts and enantiomeric ratios in lichens to understand their roles in nature and grasp their pharmaceutical potential. The overall aim of the study was to characterise the spatial distribution of the predominant usnic acid enantiomer in lichens by combining spatial imaging and chiral chromatography. Specifically, separation and quantification of usnic acid enantiomers in four common lichens in Iceland was performed using a validated chiral chromatographic method. Molecular dynamics simulation was carried out to rationalize the chiral separation mechanism. Spatial distribution of usnic acid in the lichen thallus cross-sections were analysed using Desorption Electrospray Ionization-Imaging Mass Spectrometry (DESI-IMS) and fluorescence microscopy. DESI-IMS confirmed usnic acid as a cortical compound, and revealed that usnic acid can be more concentrated around the algal vicinity. Fluorescence microscopy complemented DESI-IMS by providing more detailed distribution information. By combining results from spatial imaging and chiral separation, we were able to visualize the distribution of the predominant usnic acid enantiomer in lichen cross-sections: (+)-usnic acid in Cladonia arbuscula and Ramalina siliquosa, and (-)-usnic acid in Alectoria ochroleuca and Flavocetraria nivalis. This study provides an analytical foundation for future environmental and functional studies of usnic acid enantiomers in lichens.

Cellular mechanisms of the anticancer effects of the lichen compound usnic acid.

The lichen compound usnic acid is used for its antimicrobial activities in cosmetic products and is also a component of slimming agents. Its effect against cancer cells was first noted over 30 years ago. In this study possible mechanisms of this effect were investigated using two human cell lines, the breast cancer cell line T-47D and the pancreatic cancer cell line Capan-2. Pure (+)-usnic acid from CLADONIA ARBUSCULA and (-)-usnic acid from ALECTORIA OCHROLEUCA were shown to be equally effective inhibitors of DNA synthesis, with IC (50) 4.2 microg/mL and 4.0 microg/mL for (+) and (-)-usnic acid against T-47D, and 5.3 microg/mL and 5.0 microg/mL against Capan-2, respectively. Flow cytometric analysis confirmed the inhibited entry into the S-phase and showed reduction in cell size. Classical apoptosis, as assessed by TUNEL staining, was not observed. Necrosis, measured by LDH release, was seen only in Capan-2 after exposure for 48 hours. Staining with the mitochondrial dye JC-1 demonstrated dose-dependent loss of mitochondrial membrane potential following treatment with usnic acid in both cell lines. In conclusion, usnic acid had a marked inhibitory effect on growth and proliferation of two different human cancer cell lines and led to loss of mitochondrial membrane potential. Cell survival was little affected; late necrosis was seen in one of the cell lines. No difference was noted between the two enantiomers.

Authentication of Iceland Moss (Cetraria islandica) by UPLC-QToF-MS chemical profiling and DNA barcoding.

The lichen Cetraria islandica or Iceland Moss is commonly consumed as tea, food ingredients (e.g. in soup or bread) and herbal medicines. C. islandica, which has two chemotypes, can be difficult to distinguish from the sister species Cetraria ericetorum. They are collectively referred to as the Cetraria islandica species complex. This study aimed to use an UPLC-QToF-MS chemical profiling together with DNA barcoding to distinguish species and chemotypes of the C. islandica species complex. Our results show that the two chemotypes of C. islandica are clearly distinguishable from each other and from C. ericetorum by the chemometric approach. The RPB2 barcode was able to differentiate C. islandica from C. ericetorum with a barcode gap, but the widely used nrITS barcode failed. Neither of them could discriminate chemotypes of C. islandica. In conclusion, this integrative approach involving chemical profiling and DNA barcoding could be applied for authentication of Iceland Moss materials.

DNA barcoding and LC-MS metabolite profiling of the lichen-forming genus Melanelia: Specimen identification and discrimination focusing on Icelandic taxa.

Taxa in the genus Melanelia (Parmeliaceae, Ascomycota) belong to a group of saxicolous lichens with brown to black foliose thalli, which have recently undergone extensive changes in circumscription. Taxa belonging to Parmeliaceae are prolific producers of bioactive compounds, which have also been traditionally used for chemotaxonomic purposes. However, the chemical diversity of the genus Melanelia and the use of chemical data for species discrimination in this genus are largely unexplored. In addition, identification based on morphological characters is challenging due to few taxonomically informative characters. Molecular identification methods, such as DNA barcoding, have rarely been applied to this genus. This study aimed to identify the Melanelia species from Iceland using DNA barcoding approach, and to explore their chemical diversity using chemical profiling. Chemometric tools were used to see if lichen metabolite profiles determined by LC-MS could be used for the identification of Icelandic Melanelia species. Barcoding using the fungal nuclear ribosomal internal transcribed spacer region (nrITS) successfully identified three Melalenlia species occurring in Iceland, together with Montanelia disjuncta (Basionym: Melanelia disjuncta). All species formed monophyletic clades in the neighbor-joining nrITS gene tree. However, high intraspecific genetic distance of M. stygia suggests the potential of unrecognized species lineages. Principal component analysis (PCA) of metabolite data gave a holistic overview showing that M. hepatizon and M. disjuncta were distinct from the rest, without the power to separate M. agnata and M. stygia due to their chemical similarity. Orthogonal partial least-squares to latent structures-discriminate analysis (OPLS-DA), however, successfully distinguished M. agnata and M. stygia by identifying statistically significant metabolites, which lead to class differentiation. This work has demonstrated the potential of DNA barcoding, chemical profiling and chemometrics in identification of Melanelia species.

Immunomodulating effects of lichen-derived polysaccharides on monocyte-derived dendritic cells.

Many naturally occurring polysaccharides from fungi and lichens have been found to have immunomodulating activity. However, the majority of these studies have focused on their effects on the innate arm of the immune system. Although dendritic cells (DCs) belong to the innate immune system, they play an important role as a bridge between the innate and the adaptive immune response. In this study, the effects of 11 chromatographically purified and well-characterised lichen polysaccharides (of different structural types) on the maturation of DCs were tested by analysing the secretion of IL-12p40 and IL-10 by human monocyte-derived dendritic cells in vitro. Four of the polysaccharides upregulated IL-10 secretion by the dendritic cells, as compared with unstimulated cells, the beta-glucans lichenan and Ths-2 and the heteroglycans Pc-4 and thamnolan. IL-12p40 secretion was significantly upregulated by the beta-glucan lichenan and the heteroglycans Pc-2, Pc-4, thamnolan and Ths-4, while the mature dendritic cells stimulated with the heteroglycan Pc-1 secreted significantly less IL-12p40 than the unstimulated cells. Proportional index (PI) was used to determine the relationship between the IL-12p40 and IL-10 secretion. The PI of all the beta-glucans, i.e. lichenan, pustulan and Ths-2, and the heteroglycan thamnolan was significantly lower than the PI observed for the unstimulated cells, which was mainly due to increased IL-10 secretion. Therefore, these polysaccharides could be considered suitable candidates in tolerance and anti-inflammatory studies, as IL-10 is one of the major cytokines involved in tolerance and anti-inflammatory responses.