New branched Porolithon species (Corallinales, Rhodophyta) from the Great Barrier Reef, Coral Sea, and Lord Howe Island.

Porolithon is one of the most ecologically important genera of tropical and subtropical crustose (non-geniculate) coralline algae growing abundantly along the shallow margins of coral reefs and functioning to cement reef frameworks. Thalli of branched, fruticose Porolithon specimens from the Indo-Pacific Ocean traditionally have been called P. gardineri, while massive, columnar forms have been called P. craspedium. Sequence comparisons of the rbcL gene both from type specimens of P. gardineri and P. craspedium and from field-collected specimens demonstrate that neither species is present in east Australia and instead resolve into four unique genetic lineages. Porolithon howensis sp. nov. forms columnar protuberances and loosely attached margins and occurs predominantly at Lord Howe Island; P. lobulatum sp. nov. has fruticose to clavate forms and free margins that are lobed and occurs in the Coral Sea and on the Great Barrier Reef (GBR); P. parvulum sp. nov. has short (<2 cm), unbranched protuberances and attached margins and is restricted to the central and southern GBR; and P. pinnaculum sp. nov. has a mountain-like, columnar morphology and occurs on oceanic Coral Sea reefs. A rbcL gene sequence of the isotype of P. castellum demonstrates it is a different species from other columnar species. In addition to the diagnostic rbcL and psbA marker sequences, the four new species may be distinguished by a combination of features including thallus growth form, margin shape (attached or unattached), and medullary system (coaxial or plumose). Porolithon species, because of their ecological importance and sensitivity to ocean acidification, need urgent documentation of their taxonomic diversity.

Phymatolithopsis gen. nov. (Hapalidiales, Corallinophycidae, Rhodophyta) based on molecular and morpho-anatomical evidence.

A multigene (psbA, rbcL, 18S rDNA) molecular phylogeny of the genus Phymatolithon showed a polyphyletic grouping of two monophyletic clades within the Hapalidiales. DNA sequence data integrated with morpho-anatomical comparisons of type material and of recently collected specimens were used to establish Phymatolithopsis gen. nov. with three species, P. prolixa comb. nov., the generitype, P. repanda comb. nov. and P. donghaensis sp. nov. Phymatolithopsis is sister to Mesophyllum and occurs in a clade distinct from Phymatolithon and boreal species currently assigned to Lithothamnion. Morpho-anatomically, Phymatolithopsis is comprised of species that are non-geniculate and encrusting, bear epithallial cells with rounded walls (not flared), subepithallial initials that are usually as short as or shorter than their immediate inward derivatives, conceptacle primordia from all stages forming superficially directly from subepithallial initials, mature carposporangial conceptacles with a discontinuous fusion cell, gonimoblast filaments that develop at the margins of the fusion cell around the periphery of the carposporangial conceptacle chambers, and multiporate tetra/bisporangial conceptacles. Phymatolithopsis can be distinguished from Phymatolithon by the origin of its conceptacle primordia, which are initiated superficially, directly from the layer of subepithallial initials below the epithallial cells and the distribution of gonimoblast filaments in carposporangial conceptacles, that are at the margins of the fusion cells.

Molecular and morphological analyses reveal new taxa additions to the tribe Streblocladieae (Rhodomelaceae, Rhodophyta).

The recent segregation of 12 genera in the tribe Streblocladieae suggests that the taxonomy of some species belonging to Polysiphonia sensu lato is updated with the transfer and the proposal of new combinations. Accordingly, six new additions to the tribe Streblocladieae on the basis of morphological and molecular analyses are presented as a consequence of this new segregation. These additions include the description of the new species Carradoriella platensis sp. nov., the proposal of the following new combinations Eutrichosiphonia paniculata comb. nov., E. tapinocarpa comb. nov., and the reinstatement of Vertebrata curta, V. decipiens, and V. patersonis. Additionally, our morphological observations identified additional diagnostic features for two genera of the Streblocladieae. Carradoriella has branches with sexual reproductive structures arranged adaxially on branchlets, and the recently described Eutrichosiphonia has rhizoids with multicellular digitate haptera. Our study gives insights in regards to the distribution, the diagnostic features for delimiting genera morphologically, and the molecular evolutionary relationships in the Streblocladieae.

Corallinapetrales and Corallinapetraceae: A new order and family of coralline red algae including Corallinapetra gabrielii comb. nov.

The coralline algal genus Corallinapetra is currently monospecific and was established on the species Corallinapetra novaezelandiae, known from a single collection from north-eastern New Zealand. On the basis of multi-gene phylogenetic analyses, Corallinapetra has been resolved apart from all currently recognized families and orders within the Corallinophycidae. We analyzed DNA sequence data from the holotype of Lithothamnion gabrielii, which has been considered a heterotypic synonym of L. muelleri, and an unidentified sample collected from Stewart Island in New Zealand, using psbA, rbcL, and COI-5P genes. We also observed detailed morpho-anatomical characters with light and scanning electron microscopy. Our phylogenetic analyses showed that L. gabrielii and the sample from New Zealand belonged to the same clade as Corallinapetra, distinct from other families and orders in the Corallinophycidae. Members of this clade are distinguishable from other families and orders in the Corallinophycidae by possessing sporangia that are surrounded by remnant sterile filaments that are weakly calcified in mature multiporate sporangial conceptacles that produce zonately divided tetrasporangia. Therefore, we propose that Corallinapetra be placed in its own family, Corallinapetraceae and order, Corallinapetrales, and that L. gabrielii should be assigned to Corallinapetra, as C. gabrielii, to reflect their phylogenetic relationships. We also obtained a partial rbcL sequence data from the lectotype of L. muelleri, the generitype of Lithothamnion. Comparison of the L. muelleri type sequence with L. gabrielii unambiguously demonstrated that these two species are not conspecific, and confirm the placement of L. muelleri within the Hapalidiales.

Sargassum miyabei Yendo Brown Algae Exert Anti-Oxidative and Anti-AdipogenicEffects on 3T3-L1 Adipocytes by Downregulating PPARγ.

Background and objectives: Sargassum miyabei Yendo, belonging to the family Sargassaceae, has been reported to have various biological effects such as anti-tyrosinase activity and anti-inflammation. However, the anti-obesity effect of Sargassum miyabei Yendo has not yet been reported. Materials and Methods: The effects of Sargassum miyabei Yendo extract (SME) on 3T3-L1 adipocytes were screened by3-(4,5)-dimethylthiazo-2-yl-2,5-diphenyltetrazolium bromide (MTT), Oil red O staining, western blot, and Real-time reverse transcription polymerase chain reaction analyses. Results: Here, we show that SME had potent 2,2'-azinobis-3-ehtlbezothiazoline-6-sulfonic acid radical decolorization (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) antioxidant activity with half maximal inhibitory concentration (IC50) value of 0.2868 ± 0.011 mg/mL and 0.2941 ± 0.014 mg/mL, respectively. In addition, SME significantly suppressed lipid accumulation and differentiation of 3T3-L1 preadipocytes, as shown by Oil Red O staining results. SME attenuated the expression of adipogenic- and lipogenic-related genes such as peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT-enhancer-binding protein alpha (C/EBPα), CCAAT-enhancer-binding protein delta (C/EBPδ), adiponectin, adipose triglyceride lipase (ATGL), fatty acid synthase (FAS), hormone-sensitive lipase (HSL), and lipoprotein lipase (LPL). Conclusions: These findings suggest that SME may have therapeutic implications for developing a new anti-obesity agent.

Dereplication by High-Performance Liquid Chromatography (HPLC) with Quadrupole-Time-of-Flight Mass Spectroscopy (qTOF-MS) and Antiviral Activities of Phlorotannins from Ecklonia cava.

Ecklonia cava is edible seaweed that is found in Asian countries, such as Japan and Korea; and, its major components include fucoidan and phlorotannins. Phlorotannins that are isolated from E. cava are well-known to have an antioxidant effect and strong antiviral activity against porcine epidemic diarrhea virus (PEDV), which has a high mortality rate in piglets. In this study, the bioactive components were determined based on two different approaches: (i) bio-guided isolation using the antiviral activity against the H1N1 viral strain, which is a representative influenza virus that originates from swine and (ii) high-resolution mass spectrometry-based dereplication, including relative mass defects (RMDs) and HPLC-qTOFMS fragmentation analysis. The EC70 fraction showed the strongest antiviral activity and contained thirteen phlorotannins, which were predicted by dereplication. Ten compounds were directly isolated from E. cava extract and then identified. Moreover, the dereplication method allowed for the discovery of two new phlorotannins. The structures of these two isolated compounds were elucidated using NMR techniques and HPLC-qTOFMS fragmentation analysis. In addition, molecular modelling was applied to determine the absolute configurations of the two new compounds. The antiviral activities of seven major phlorotannins in active fraction were evaluated against two influenza A viral strains (H1N1 and H9N2). Six of the compounds showed moderate to strong effects on both of the viruses and phlorofucofuroeckol A (12), which showed an EC50 value of 13.48 ± 1.93 µM, is a potential active antiviral component of E. cava.

Wilsonosiphonia gen. nov. (Rhodomelaceae, Rhodophyta) based on molecular and morpho-anatomical characters.

Morphological, anatomical, and molecular sequence data were used to assess the establishment and phylogenetic position of the genus Wilsonosiphonia gen. nov. Phylogenies based on rbcL and concatenated rbcL and cox1 loci support recognition of Wilsonosiphonia gen. nov., sister to Herposiphonia. Diagnostic features for Wilsonosiphonia are rhizoids located at distal ends of pericentral cells and taproot-shaped multicellular tips of rhizoids. Wilsonosiphonia includes three species with diagnostic rbcL and cox1 sequences, Wilsonosiphonia fujiae sp. nov. (the generitype), W. howei comb. nov., and W. indica sp. nov. These three species resemble each other in external morphology, but W. fujiae is distinguished by having two tetrasporangia per segment rather than one, W. indica by having abundant and persistent trichoblasts, and W. howei by having few and deciduous trichoblasts.

Protein Tyrosine Phosphatase 1B Inhibitors from the Stems of Akebia quinata.

PTP1B deficiency in mouse mammary tumor virus (MMTV)-NeuNT transgenic mice inhibited the onset of MMTV-NeuNT-evoked breast cancer, while its overexpression was observed in breast cancer. Thus, PTP1B inhibitors are considered chemopreventative agents for breast cancer. As part of our program to find PTP1B inhibitors, one new diterpene glycoside (1) and 13 known compounds (2-14) were isolated from the methanol extract of the stems of Akebia quinata. All isolates were identified based on extensive spectroscopic data analysis, including UV, IR, NMR and MS. Compounds 2, 3, 6, 8 and 11 showed significant inhibitory effects on the PTP1B enzyme, with IC50 values ranging from 4.08 ± 1.09 to 21.80 ± 4.74 µM. PTP1B inhibitors also had concentration-dependent cytotoxic effects on breast cancer cell lines, such as MCF7, MDA-MB-231 and tamoxifen-resistant MCF7 (MCF7/TAMR) (IC50 values ranging from 0.84 ± 0.04 to 7.91 ± 0.39 µM). These results indicate that compounds 6 and 8 from Akebia quinata may be lead compounds acting as anti-breast cancer agents.

Protoplast isolation from Dictyopteris pacifica and Scytosiphon lomentaria, using a simple commercial enzyme preparation.

BACKGROUND: Protoplasts (i.e., naked plant cells) can be used for in vitro manipulations and genetic improvement in cultivars with economic value. During the last decade, protoplast research in economic brown algae has been scarce, and it is usually hampered by the use of non-commercial enzymes or crude extracts for isolating protoplasts. Dictyopteris pacifica is part of a brown algal genus well known by its wide chemical diversity and biological properties. Scytosiphon lomentaria is an edible brown seaweed with antioxidant, antitumor, and antiviral properties. So far, there are no protoplast isolation protocols using commercial enzymes for these two economic brown algae. In this study, we obtained protoplasts from cultured samples of D. pacifica and S. lomentaria using commercially available enzymes. Additionally, we investigated the effects of Driselase inclusion and Ca-chelation pre-treatment on protoplast yields in order to optimize the conditions for protoplast preparations. RESULTS: Protoplasts were isolated from Dictyopteris pacifica and Scytosiphon lomentaria using the commercially available Cellulase Onozuka RS (1%) and Alginate lyase (4 U mL-1), and short incubation time (4 h). Driselase did not show significant effects on protoplast production in both species. Ca-chelation pre-treatment only increased the number of protoplasts in D. pacifica. Under optimal conditions, the protoplast yields from D. pacifica and S. lomentaria were 4.83 ± 2.08 and 74.64 ± 32.49 × 106 protoplasts g-1 fresh weight, respectively. The values obtained for S. lomentaria were 2-3 orders of magnitude higher than previously reported. CONCLUSIONS: Our results show that high protoplast yields can be obtained from D. pacifica and S. lomentaria using a simple mixture of commercial enzymes (Cellulase RS and Alginate lyase) and short incubation time (4 h). This work also represents the first report of protoplast isolation in D. pacifica. The method proposed here can help to expand protoplast technology in more brown algal species.

Mitochondrial and Plastid Genomes from Coralline Red Algae Provide Insights into the Incongruent Evolutionary Histories of Organelles.

Mitochondria and plastids are generally uniparentally inherited and have a conserved gene content over hundreds of millions of years, which makes them potentially useful phylogenetic markers. Organelle single gene-based trees have long been the basis for elucidating interspecies relationships that inform taxonomy. More recently, high-throughput genome sequencing has enabled the construction of massive organelle genome databases from diverse eukaryotes, and these have been used to infer species relationships in deep evolutionary time. Here, we test the idea that despite their expected utility, conflicting phylogenetic signal may exist in mitochondrial and plastid genomes from the anciently diverged coralline red algae (Rhodophyta). We generated complete organelle genome data from five coralline red algae (Lithothamnion sp., Neogoniolithon spectabile, Renouxia sp., Rhodogorgon sp., and Synarthrophyton chejuensis) for comparative analysis with existing organelle genome data from two other species (Calliarthron tuberculosum and Sporolithon durum). We find strong evidence for incongruent phylogenetic signal from both organelle genomes that may be explained by incomplete lineage sorting that has maintained anciently derived gene copies or other molecular evolutionary processes such as hybridization or gene flow during the evolutionary history of coralline red algae.

Functional restoration of replicative senescent mesenchymal stem cells by the brown alga Undaria pinnatifida.

The brown alga Undaria pinnatifida, which is called Mi-Yoek in Korea, has been traditionally consumed as a health food in East Asian countries. Recent studies have reported that U. pinnatifida has beneficial effects on arteriosclerosis, inflammation, fat metabolism, and tumors. In this study, we examined the anti-senescence effects of ethanol extracts of U. pinnatifida (UP-Ex) in human bone marrow mesenchymal stem cells (hBM-MSCs). UP-Ex protected hBM-MSCs against oxidative injury, as determined by MTT assays. This effect was confirmed by immunoblot analysis of the oxidation-sensitive protein p53 and the apoptotic protein cleaved caspase-3. Excessive intracellular reactive oxygen species (ROS) accumulation induced by oxidative stress was moderated in UP-Ex-treated hBM-MSCs (UP-Ex-MSCs). Similarly, expression of the ROS-scavenging enzymes superoxide dismutase 1 (SOD1), SOD2, and catalase was recovered in UP-Ex-MSCs. Excessive ROS induced by long-term cell expansion (passage 17) was significantly decreased along with restoration of the senescence proteins p53, p21, and p16 in UP-Ex-MSCs. UP-Ex treatment also improved the ability of these replicative, senescent hBM-MSCs (passage 17) to differentiate into osteocytes or adipocytes, suggesting that UP-Ex ameliorates the functional decline of senescent stem cells and may provide better therapeutic efficacy in stem cell therapy. Abbreviations: hBM-MSCs: human bone marrow mesenchymal stem cells; DCF: 2',7'-dichlorodihydrofluorescein; DCFH-DA: 2',7'-dichlorofluorescein diacetate; MTT: 3-(4,5-dimethylthiazol-2-yl-)2,5-diphenyltetrazolium bromide; PBS: phosphate-buffered saline; PFA: paraformaldehyde; RIPA: radioimmunoprecipitation assay; ROS: reactive oxygen species; SOD1: superoxide dismutase 1; SOD2: superoxide dismutase 2.

Antiviral phenolics from the leaves of Cleistocalyx operculatus.

During the screening program for anti-influenza agents from medicinal plants, the ethanolic extract of Cleistocalyx operculatus leaves was found to exhibit potential neuraminidase (NA) inhibitory activity. Bioassay-directed fractionation led to the isolation of two new acetophenones (1 and 2) and one new flavanone (3), along with six known compounds (4-9). The structures of all isolated compounds were elucidated using various spectroscopic methods and through comparison with the previous literature. Compounds 6 and 8 exhibited strong enzymatic inhibition on various neuraminidases from different influenza viruses, including H1N1, H9N2, novel H1N1, and oseltamivir-resistant novel H1N1 (H274Y mutation) expressed in HEK293 cells (IC50 values ranging from 5.07 ± 0.94 µM to 9.34 ± 2.52 µM, respectively). Kinetic experiments revealed the non-competitive inhibitory mode of both compounds 6 and 8. Furthermore, these flavonoids reduced the cytopathic effect of the H1N1 virus in MDCK cells. The present study suggests the potential of two flavonoids (6 and 8) as new lead compounds for the development of novel NA inhibitors in the future.

MORPHOLOGICAL AND MOLECULAR CHARACTERIZATION OF SPECIES OF THE GENUS CENTROCERAS (CERAMIACEAE, CERAMIALES), INCLUDING TWO NEW SPECIES(1).

Centroceras clavulatum (C. Agardh) Montagne is widely reported as being a prime example of a cosmopolitan red algal species. Instead, C. clavulatum is here determined as restricted to northern Chile, Peru, southern California, southern Australia, and New Zealand. Specimens identified using the current species concept for "C. clavulatum" fall into nine morphological groups that correspond to highly supported clades in phylogenetic analyses. Three of these clades correspond to the resurrected species Centroceras gasparrinii (Meneghini) Kützing, C. hyalacanthum Kützing, and C. micracanthum Kützing. Two others are recognized as new species: Centroceras rodmanii sp. nov. from southern Chile, which is characterized by hooked spines arranged in a whorl at the node, a spine or flattened gland cell cut off from the first cortical initials, and a single acropetal cortical cell issued from the second cortical initials; and C. tetrachotomum sp. nov. from South Africa, which has a tetrachotomous branching pattern, straight spines in a whorl, an acropetal cortical cell and a spine or a flattened gland cell cut off from the first cortical initials, and a two-celled acropetal filament cut off from the second cortical initials. Three additional species from South Africa are also recognized as distinct species. All phylogenetic analyses of the rbcL gene, LSU rDNA, and SSU rDNA were consistent with the vegetative and tetrasporangial morphological distinctions, thus supporting the resurrection of three species and the description of two new species.

GAYLIELLA GEN. NOV. IN THE TRIBE CERAMIEAE (CERAMIACEAE, RHODOPHYTA) BASED ON MOLECULAR AND MORPHOLOGICAL EVIDENCE(1).

On the basis of comparative morphology and phylogenetic analyses of rbcL and LSU rDNA sequence data, a new genus, Gayliella gen. nov., is proposed to accommodate the Ceramium flaccidum complex (C. flaccidum, C. byssoideum, C. gracillimum var. byssoideum, and C. taylorii), C. fimbriatum, and a previously undescribed species from Australia. C. transversale is reinstated and recognized as a distinct species. Through this study, G. flaccida (Kützing) comb. nov., G. transversalis (Collins et Hervey) comb. nov., G. fimbriata (Setchell et N. L. Gardner) comb. nov., G. taylorii comb. nov., G. mazoyerae sp. nov., and G. womersleyi sp. nov. are based on detailed comparative morphology. The species referred to as C. flaccidum and C. dawsonii from Brazil also belong to the new genus. Comparison of Gayliella with Ceramium shows that it differs from the latter by having an alternate branching pattern; three cortical initials per periaxial cell, of which the third is directed basipetally and divides horizontally; and unicellular rhizoids produced from periaxial cells. Our phylogenetic analyses of rbcL and LSU rDNA gene sequence data confirm that Gayliella gen. nov. represents a monophyletic clade distinct from most Ceramium species including the type species, C. virgatum. We also transfer C. recticorticum to the new genus Gayliella.