Microbiome in Cladonia squamosa Is Vertically Stratified According to Microclimatic Conditions.

Lichens are miniature ecosystems that contain fungi, microalgae, and bacteria. It is generally accepted that symbiosis between mycobiont and photobiont and microbial contribution to the ecosystem support the wide distribution of lichens in terrestrial ecosystems, including polar areas. The composition of symbiotic components can be affected by subtle microenvironmental differences within a thallus, as well as large-scale climate differences. In this study, we investigated fine-scale profiles of algal, fungal, and bacterial compositions through horizontal and vertical positions of the Antarctic lichen Cladonia squamosa colonies by next-generation sequencing of the nuclear large subunit rRNA gene (nucLSU) of eukaryotes and the 16S rRNA gene of bacteria. Apical parts of thalli were exposed to strong light, low moisture, and high variability of temperature compared with basal parts. Microbial diversity increased from apical parts to basal parts of thalli. Asterochloris erici was the major photobiont in apical positions of thalli, but other microalgal operational taxonomic units (OTUs) of Trebouxiophyceae and Ulvophyceae were major microalgal components in basal positions. Photochemical responses of algal components from apical and basal parts of thalli were quite different under variable temperature and humidity conditions. Several fungal OTUs that belonged to Arthoniomycetes and Lecanoromycetes, and diverse bacterial OTUs that belonged to Alphaproteobacteria, Acidobacteria_Gp1, and candidate division WPS-2 showed a clear distribution pattern according to their vertical positions within thalli. The overall lichen microbiome was significantly differentiated by the vertical position within a thallus. These results imply that different microclimate are formed at different lichen thallus parts, which can affect microbial compositions and physiological responses according to positions within the thalli.

Anti-Inflammatory Activity of Lobaric Acid via Suppressing NF-κB/MAPK Pathways or NLRP3 Inflammasome Activation.

Lobaric acid (LA) is a constituent of the lichen Stereocaulon alpinum. LA has multiple biological activities, including antibacterial and antioxidant ones. The purpose of this study was to investigate the effect of LA and its mechanism on lipopolysaccharide (LPS)-induced inflammatory responses in macrophages. Macrophages were pretreated with different concentrations of LA (0.2 - 20 µM), followed by LPS stimulation. LA treatment of LPS stimulated macrophages decreased their nitric oxide production and the expression of cyclooxygenase-2 and prostaglandin E2. LA also significantly reduced the production of tumor necrosis factor-α and interleukin (IL)-6 by inhibiting the activation of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB). Additionally, LA inhibited the production of IL-1ß and IL-18, as well as caspase-1 maturation, by inhibition of NLRP3 inflammasome activation in LPS/ATP-stimulated cells. These results strongly suggest that LA could inhibit inflammation by downregulating NF-κB/MAPK pathways and NLRP3 inflammasome activation in activated macrophages. These results reveal a new therapeutic approach to modulate inflammatory diseases linked to deregulated inflammasome activities.

Genomic Insight Into the Predominance of Candidate Phylum Atribacteria JS1 Lineage in Marine Sediments.

Candidate phylum Atribacteria JS1 lineage is one of the predominant bacterial groups in anoxic subseafloor sediments, especially in organic-rich or gas hydrate-containing sediments. However, due to the lack of axenic culture representatives, metabolic potential and biogeochemical roles of this phylum have remained elusive. Here, we examined the microbial communities of marine sediments of the Ross Sea, Antarctica, and found candidate phylum Atribacteria JS1 lineage was the most abundant candidate phylum accounting for 9.8-40.8% of the bacterial communities with a single dominant operational taxonomic unit (OTU). To elucidate the metabolic potential and ecological function of this species, we applied a single-cell genomic approach and obtained 18 single-cell amplified genomes presumably from a single species that was consistent with the dominant OTU throughout the sediments. The composite genome constructed by co-assembly showed the highest genome completeness among available Atribacteria JS1 genomes. Metabolic reconstruction suggested fermentative potential using various substrates and syntrophic acetate oxidation coupled with hydrogen or formate scavenging methanogens. This metabolic potential supports the predominance of Atribacteria JS1 in anoxic environments expanding our knowledge of the ecological function of this uncultivated group.

Leucothrix arctica sp. nov., isolated from Arctic seawater.

A Gram-stain-negative, non-motile, oxidase- and catalase-positive, rod-shaped bacterium was isolated from a coastal seawater sample from the Arctic Circle and designated strain IMCC9719T. On the basis of 16S rRNA gene sequence analysis, it was shown that strain IMCC9719T belonged to the genus Leucothrix and was closely related to the type strains of Leucothrix pacifica (97.6 % similarity) and Leucothrix mucor (95.1 %), while the strain shared <90.6 % sequence similarity with other bacterial species. The average nucleotide identity and genome-to-genome distance values between strain IMCC9719T and L. pacifica JCM 18388T were 71.7 and 16.9 %, respectively. The DNA G+C content of strain IMCC9719T was 43.5 mol%. Optimum growth of strain IMCC9719T was observed at 15 °C, at pH 7.5-8.5 and in the presence of 2.0-2.5 % (w/v) NaCl. The major fatty acids were summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), summed feature 8 (C18 : 1ω6c and/or C18 : 1ω7c) and C16 : 0. Cells of strain IMCC9719T contained diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, an unidentified polar lipid, an unidentified aminophospholipid and two unidentified phospholipids. The major respiratory quinone was ubiquinone-8 (Q-8). Based on the taxonomic data collected in this study, strain IMCC9719T represents a novel species of the genus Leucothrix, for which the name Leucothrix arctica sp. nov. is proposed. The type strain is IMCC9719T (=KACC 18010T=NBRC 110382T).

CspB of an arctic bacterium, Polaribacter irgensii KOPRI 22228, confers extraordinary freeze-tolerance

ABSTRACT Freezing temperatures are a major challenge for life at the poles. Decreased membrane fluidity, uninvited secondary structure formation in nucleic acids, and protein cold-denaturation all occur at cold temperatures. Organisms adapted to polar regions possess distinct mechanisms that enable them to survive in extremely cold environments. Among the cold-induced proteins, cold shock protein (Csp) family proteins are the most prominent. A gene coding for a Csp-family protein, cspB, was cloned from an arctic bacterium, Polaribacter irgensii KOPRI 22228, and overexpression of cspB greatly increased the freeze-survival rates of Escherichia coli hosts, to a greater level than any previously reported Csp. It also suppressed the cold-sensitivity of an E. coli csp-quadruple deletion strain, BX04. Sequence analysis showed that this protein consists of a unique domain at its N-terminal end and a well conserved cold shock domain at its C-terminal end. The most common mechanism of Csp function in cold adaption is melting of the secondary structures in RNA and DNA molecules, thus facilitating transcription and translation at low temperatures. P. irgensii CspB bound to oligo(dT)-cellulose resins, suggesting single-stranded nucleic acid-binding activity. The unprecedented level of freeze-tolerance conferred by P. irgensii CspB suggests a crucial role for this protein in survival in polar environments.

CspB of an arctic bacterium, Polaribacter irgensii KOPRI 22228, confers extraordinary freeze-tolerance.

Freezing temperatures are a major challenge for life at the poles. Decreased membrane fluidity, uninvited secondary structure formation in nucleic acids, and protein cold-denaturation all occur at cold temperatures. Organisms adapted to polar regions possess distinct mechanisms that enable them to survive in extremely cold environments. Among the cold-induced proteins, cold shock protein (Csp) family proteins are the most prominent. A gene coding for a Csp-family protein, cspB, was cloned from an arctic bacterium, Polaribacter irgensii KOPRI 22228, and overexpression of cspB greatly increased the freeze-survival rates of Escherichia coli hosts, to a greater level than any previously reported Csp. It also suppressed the cold-sensitivity of an E. coli csp-quadruple deletion strain, BX04. Sequence analysis showed that this protein consists of a unique domain at its N-terminal end and a well conserved cold shock domain at its C-terminal end. The most common mechanism of Csp function in cold adaption is melting of the secondary structures in RNA and DNA molecules, thus facilitating transcription and translation at low temperatures. P. irgensii CspB bound to oligo(dT)-cellulose resins, suggesting single-stranded nucleic acid-binding activity. The unprecedented level of freeze-tolerance conferred by P. irgensii CspB suggests a crucial role for this protein in survival in polar environments.

Planktotalea arctica sp. nov., isolated from Arctic seawater.

A Gram-staining-negative, non-motile, non-pigmented, rod-shaped bacterium was isolated from an Arctic coastal seawater sample and was designated strain IMCC9565T. Analysis of the 16S rRNA gene sequence of strain IMCC9565T revealed that the closest phylogenetic neighbours of the strain were members of the genus Planktotalea. Furthermore, the strain formed a robust clade with Planktotalea frisia SH6-1T, with which it shared 97.9 % 16S rRNA gene sequence similarity. Determination of genomic relatedness based on average nucleotide identity and genome-to-genome distance showed that strain IMCC9565T was distantly related to P. frisia, meaning the Arctic strain represents a novel species. Optimum growth of strain IMCC9565T was observed at 20 °C, pH 7.0 and in the presence of 2 % (w/v) NaCl. The major respiratory isoprenoid quinone was ubiquinone-10 (Q-10) and the major polar lipids consisted of phosphatidylglycerol, phosphatidylcholine, one unidentified aminolipid and two unidentified lipids. The principal fatty acids were C18 : 1ω7c and/or C18 : 1ω6c, C18 : 1ω7c 11-methyl and C16 : 0, and the DNA G+C content was 57.1 mol%. Based on these data, Planktotalea arctica sp. nov. is proposed to accommodate the bacterial isolate and the type strain is IMCC9565T (=KACC 18009T=NBRC 110393T).

Complete Genome Sequence of Cryobacterium arcticum Strain PAMC 27867, Isolated from a Sedimentary Rock Sample in Northern Victoria Land, Antarctica.

Cryobacterium arcticum PAMC 27867, a psychrotolerant, Gram-positive bacterium, was isolated from a sedimentary rock sample collected at Eureka Spurs in northern Victoria Land, Antarctica. Here, we report the genome sequence of C. arcticum PAMC 27867.

Ramalin inhibits differentiation of 3T3-L1 preadipocytes and suppresses adiposity and body weight in a high-fat diet-fed C57BL/6J mice.

Obesity is a serious global health problem. Natural substances that could be effective remedies for treatment of obesity, and which are relatively safe, are desired. The aim of this study was to examine the anti-obesity effect and the mechanism of ramalin in 3T3-L1 preadipocytes and high fat diet (HFD)-induced obese mice. In this study, 3T3-L1 cells were treated with various concentrations of ramalin (1, 5, and 10 µg/ml). Ramalin reduced the accumulation of intracellular lipid droplets in 3T3-L1 cells. In addition, ramalin inhibited 3T3-L1 adipocyte differentiation by blocking adipogenic gene expression including CCAAT enhancer binding proteins (C/EBPs), peroxisome proliferator-activated receptors γ (PPARγ), adipocyte fatty acid-binding protein (aP2), and leptin. The suppression of adipogenesis by ramalin was mediated through the inhibition of MAPK pathways. Ramalin also reduced the secretion of TNF-α and IL-6 in 3T3-L1 adipocytes. Oral administration of ramalin (50 and 100 mg/kg) to HFD-fed mice reduced body weight gain and abdominal fat accumulation without changes in food intake. Ramalin also attenuated organ weight and basal serum level by inhibiting Iiver X receptors (LXRs), sterol regulatory element-binding protein-1c (SREBP-1c), and lipoprotein lipase (LPL) mRNA expression in HFD-fed mice. Taken together, these results indicate that ramalin inhibits adipogenesis in 3T3-L1 preadipocytes and prevents HFD-induced obesity. The present study also provides insight into the mechanisms underlying the anti-obesity activity of ramalin and suggests that ramalin has the potential to prevent obesity.

Ramalin Isolated from Ramalina Terebrata Attenuates Atopic Dermatitis-like Skin Lesions in Balb/c Mice and Cutaneous Immune Responses in Keratinocytes and Mast Cells.

Atopic dermatitis (AD) is a chronic inflammatory skin disease that involves eczematous skin lesions with pruritic erythematous papules. In this study, we investigated the mitigating effects of ramalin, a component of the Antarctic lichen Ramalina terebrata against AD in vivo and in vitro. Oral administration of ramalin lessened scratching behaviors and significantly reduced both serum immunoglobulin E and IL-4 levels, and mRNA levels of IL-4 and IL-10 in AD-induced Balb/c mice. In vitro, treatment with ramalin produced significantly less inflammatory chemokines and cytokines, including TARC, MCP-1, RANTES, and IL-8 in TNF-α-stimulated HaCaT cells. In addition, ramalin inhibited the activation of nuclear factor-kappa B as well as the phosphorylation of mitogen-activated protein kinases (MAPK). Furthermore, ramalin treatment resulted in decreased production of ß-hexosaminidase and proinflammatory cytokines IL-4, IL-6, and TNF-α in 2,4 dinitrophenyl-human serum albumin-stimulated RBL-2H3 cells through blocking MAPK signaling pathways. The results suggest that ramalin modulates the production of immune mediators by inhibiting the nuclear factor-kappa B and MAPK signaling pathways. Taken together, ramalin effectively attenuated the development of AD and promoted the mitigating effects on Th2 cell-mediated immune responses and the production of inflammatory mediators in mast cells and keratinocytes. Thus, ramalin may be a potential therapeutic agent for AD. Copyright © 2016 John Wiley & Sons, Ltd.

Complete Genome Sequence of Psychrobacter alimentarius PAMC 27889, a Psychrophile Isolated from an Antarctic Rock Sample.

Psychrobacter alimentarius PAMC 27889, a Gram-negative, psychrophilic bacterium, was isolated from an Antarctic rock sample. Here, we report the complete genome of P. alimentarius PAMC 27889, which has the nonmevalonate methylerythritol phosphate pathway of terpenoid biosynthesis and a complete gene cluster for benzoate degradation.

Reclassification of Halomonas caseinilytica Wu et al. 2008 as a later synonym of Halomonas sinaiensis Romano et al. 2007, and emendation of the species description.

The taxonomic relationship between Halomonas sinaiensis DSM 18067(T) and Halomonas caseinilytica JCM 14802(T) has not been established, despite the high similarity (99.6 %) of their 16S rRNA gene sequences. To clarify their taxonomic positions, a polyphasic approach was applied to both type strains. Genomic relatedness analyses between H. sinaiensis DSM 18067(T) and H. caseinilytica JCM 14802(T) resulted in an average nucleotide identity of 99.5 % and an estimated DNA-DNA hybridization of 96.1 % by the genome-to-genome distance calculator, indicating that they belong to a single species. Phenotypic and chemotaxonomic characteristics showed no pronounced differences between the two type strains. Based on the results of this polyphasic study, it is proposed that H. caseinilytica JCM 14802(T) is a later heterotypic synonym of H. sinaiensis DSM 18067(T). An emended description for the species H. sinaiensis is given.

Complete genome sequence of Halocynthiibacter arcticus PAMC 20958(T) from an Arctic marine sediment sample.

Here, we present the first complete genome sequence of the strain PAMC 20958(T) from the genus Halocynthiibacter. Halocynthiibacter arcticus PAMC 20958(T), isolated from a marine sediment of the Arctic, is a gram-negative, aerobic, and rod-shaped bacterium. The complete genome contains 4,329,554 base pairs with 53.21% GC content and a 44,566 base pair plasmid with 48.72% GC content. This genome contained genes encoding alkaline phosphatase and lipase, and genes that confer resistance to arsenic, cadmium, tellurite, and acriflavin.

Complete genome sequence of Pedobacter cryoconitis PAMC 27485, a CRISPR-Cas system-containing psychrophile isolated from Antarctica.

Pedobacter cryoconitis PAMC 27485, an aerobic, Gram-negative, facultatively psychrophilic bacterium, was isolated from Antarctic soil. Here we report the complete genome of P. cryoconitis PAMC 27485, which contains a type II CRISPR-Cas system and genes encoding useful enzymes (e.g. proteases). The genome sequence of P. cryoconitis PAMC 27485 could provide insights into its adaptive immune system against foreign genetic elements and biotechnological potential.

Lobaric Acid Inhibits VCAM-1 Expression in TNF-α-Stimulated Vascular Smooth Muscle Cells via Modulation of NF-κB and MAPK Signaling Pathways.

Lichens have been known to possess multiple biological activities, including anti-proliferative and anti-inflammatory activities. Vascular cell adhesion molecule-1 (VCAM-1) may play a role in the development of atherosclerosis. Hence, VCAM-1 is a possible therapeutic target in the treatment of the inflammatory disease. However, the effect of lobaric acid on VCAM-1 has not yet been investigated and characterized. For this study, we examined the effect of lobaric acid on the inhibition of VCAM-1 in tumor necrosis factor-alpha (TNF-α)-stimulated mouse vascular smooth muscle cells. Western blot and ELISA showed that the increased expression of VCAM-1 by TNF-α was significantly suppressed by the pre-treatment of lobaric acid (0.1-10 µg/ml) for 2 h. Lobaric acid abrogated TNF-α-induced NF-κB activity through preventing the degradation of IκB and phosphorylation of extracellular signal-regulated kinases (ERK), c-Jun N-terminal kinases (JNK), and p38 mitogen activated protein (MAP) kinase. Lobaric acid also inhibited the expression of TNF-α receptor 1 (TNF-R1). Overall, our results suggest that lobaric acid inhibited VCAM-1 expression through the inhibition of p38, ERK, JNK and NF-κB signaling pathways, and downregulation of TNF-R1 expression. Therefore, it is implicated that lobaric acid may suppress inflammation by altering the physiology of the atherosclerotic lesion.

Inhibition of VCAM-1 expression on mouse vascular smooth muscle cells by lobastin via downregulation of p38, ERK 1/2 and NF-κB signaling pathways.

Atherosclerosis is a chronic inflammatory disease, the progression of which is associated with the increased expression of cell adhesion molecules on vascular smooth muscle cells (VSMCs). Lobastin is a new pseudodepsidone isolated from Stereocaulon alpinum, Antarctic lichen, which is known to have antioxidant and antibacterial activities. However, the nature of the biological effects of lobastin still remains unclear. In the present study, we examine the effect of lobastin on the expression of vascular cell adhesion molecules (VCAM-1) induced by TNF-α in the cultured mouse VSMC cell line, MOVAS-1. Pretreatment of VSMCs for 2 h with lobastin (0.1-10 µg/ml) concentration-dependently inhibited TNF-α-induced protein expression of VCAM-1. Lobastin also inhibited TNF-α-induced production of intracellular reactive oxygen species (ROS). Lobastin abrogated TNF-α-induced phosphorylation of p38 and ERK 1/2, but not JNK, and also inhibited TNF-α-induced NK-κB activation. In addition, lobastin suppressed TNF-α-induced IκB kinase activation, subsequent degradation of IκBα and nuclear translocation of p65 NF-κB. Our results indicate that lobastin downregulates the TNF-α-mediated induction of VCAM-1 in VSMC by inhibiting the p38, ERK 1/2 and NF-κB signaling pathways and intracellular ROS generation. Thus, lobastin may be an important regulator of inflammation in the atherosclerotic lesion and a novel therapeutic drug for the treatment of atherosclerosis.

Ramalin-Mediated Apoptosis Is Enhanced by Autophagy Inhibition in Human Breast Cancer Cells.

Breast cancer, the most commonly diagnosed cancer in women worldwide, is treated in various ways. Ramalin is a chemical compound derived from the Antarctic lichen Ramalina terebrata and is known to exhibit antioxidant and antiinflammatory activities. However, its effect on breast cancer cells remains unknown. We examined the ability of ramalin to induce apoptosis and its mechanisms in MCF-7 and MDA-MB-231 human breast cancer cell lines. Ramalin inhibited cell growth and induced apoptosis in both cell lines in a concentration-dependent manner. By upregulating Bax and downregulating Bcl-2, ramalin caused cytochrome c and apoptosis-inducing factor to be released from the mitochondria into the cytosol, thus activating the mitochondrial apoptotic pathway. In addition, activated caspase-8 and caspase-9 were detected in both types of cells exposed to ramalin, whereas ramalin activated caspase-3 only in the MDA-MB-231 cells. Ramalin treatment also increased the levels of LC3-II and p62. Moreover, the inhibition of autophagy by 3-methyladenine or Atg5 siRNA significantly enhanced ramalin-induced apoptosis, which was accompanied by a decrease in Bcl-2 levels and an increase in Bax levels. Therefore, autophagy appears to be activated as a protective mechanism against apoptosis in cancer cells exposed to ramalin. These findings suggest that ramalin is a potential anticancer agent for the treatment of patients with non-invasive or invasive breast cancer.