The new seriniquinone glycoside by biological transformation using the deep sea-derived bacterium Bacillus licheniformis KDM612.

Seriniquinone was isolated as a melanoma-selective anti-cancer agent from a culture broth of the marine-derived bacterium Serinicoccus marinus CNJ927 in 2014. It targets the unique small protein, dermcidin, which affects the drug resistance of cancer cells. Due to its significant activity against cancer cells, particularly melanoma, and its unique target, seriniquinone has been developed as a new pharmacophore. However, it has the disadvantage of poor solubility in drug discovery research, which needs to be resolved. A new seriniquinone glycoside (1) was synthesized by the biological transformation of seriniquinone using the deep sea-derived bacterium Bacillus licheniformis KDM612. Compound 1 exhibited selective anti-cancer activity against melanoma, similar to seriniquinone, and was 50-fold more soluble in DMSO than seriniquinone.

Community Structure and Predicted Functions of Actively Growing Bacteria Responsive to Released Coral Mucus in Surrounding Seawater.

A direct relationship exists between diverse corals and fish farming in Keten Bay, Amami-Oshima, Japan. The release of coral mucus has a significant impact on the microbial activity of surrounding seawater. To obtain a more detailed understanding of biogeochemical cycles in this environment, the effects of coral mucus on the community structure and function of bacteria in surrounding seawater need to be elucidated. We herein used a bromodeoxyuridine approach to investigate the structures and functions of bacterial communities growing close to mucus derived from two different Acropora corals, AC1 and AC2. The alpha diversities of actively growing bacteria (AGB) were lower in mucus-containing seawater than in control seawater and their community structures significantly differed, suggesting that the growth of specific bacteria was modulated by coral mucus. Rhodobacteraceae and Cryomorphaceae species were the most dominant AGB in response to the mucus of Acropora AC1 and AC2, respectively. In contrast, the growth of Actinomarinaceae, Alteromonadaceae, Flavobacteriaceae, and SAR86 clade bacteria was inhibited by coral mucus. The results of a Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt2) ana-lysis suggested that the predicted functions of AGB in mucus-containing seawater differed from those in seawater. These functions were related to the biosynthesis and degradation of the constituents of coral mucus, such as polysaccharides, sugar acids, and aromatic compounds. The present study demonstrated that complex bacterial community structures and functions may be shaped by coral mucus, suggesting that corals foster diverse bacterial communities that enhance the ecological resilience of this fish farming area.

Succession of the intestinal bacterial community in Pacific bluefin tuna (Thunnus orientalis) larvae.

We investigated the succession process of intestinal bacteria during seed production in full-cycle aquaculture of Pacific bluefin tuna (Thunnus orientalis). During the seed production, eggs, healthy fish, rearing water, and feeds from three experimental rounds in 2012 and 2013 were collected before transferring to offshore net cages and subjected to a fragment analysis of the bacterial community structure. We identified a clear succession of intestinal bacteria in bluefin tuna during seed production. While community structures of intestinal bacteria in the early stage of larvae were relatively similar to those of rearing water and feed, the bacterial community structures seen 17 days after hatching were different. Moreover, although intestinal bacteria in the late stage of larvae were less diverse than those in the early stage of larvae, the specific bacteria were predominant, suggesting that the developed intestinal environment of the host puts selection pressure on the bacteria in the late stage. The specific bacteria in the late stage of larvae, which likely composed 'core microbiota', were also found on the egg surface. The present study highlights that proper management of the seed production process, including the preparation of rearing water, feeds, and fish eggs, is important for the aquaculture of healthy fish.

Intra-arterial chemoradiotherapy for locally advanced buccal mucosal cancer in an elderly patient with multiple comorbidities: A case report.

BACKGROUND: The management of locally advanced oral cavity squamous cell carcinoma (LA-OCScc) in elderly patients with multiple comorbidities is difficult. CASE: We report the case of an elderly patient with buccal mucosal squamous cell carcinoma as well as chronic renal dysfunction, hepatic cirrhosis, rheumatoid arthritis, and metachronous diffuse large B-cell lymphoma. We performed radiation therapy (a total dose of 70 Gy in 35 fractions) and six cycles of intra-arterial chemotherapy with 40 mg/m2 cisplatin per week. After treatment, the tumor completely disappeared, and there was no recurrence or deterioration of comorbidities during the 12-month follow-up period. CONCLUSION: Intra-arterial chemoradiotherapy may be a good treatment option for LA-OCScc in elderly patients with multiple comorbidities.

Community structure of actively growing bacteria in a coastal fish-farming area.

In fish-farming areas, copious amounts of organic matter are released into the surrounding environment. Although it is well-known that bacterial community structures and activities are tightly coupled with organic conditions in the environment, actively growing bacteria (AGB) species that are responsible are still largely unknown. Here, we determined seasonal variations in the community structures of free-living and particle-attached AGB in surface and bottom seawater, and also in the easily resuspendable sediment boundary layer. Accordingly, we used bromodeoxyuridine (BrdU) magnetic bead immunocapture and PCR-denaturing gradient gel electrophoresis (BUMP-DGGE) analysis. Whereas overall bacterial communities in the resuspendable sediment were quite different from those of the free-living and particle-attached bacteria, the AGB community structures were similar among them. This result suggests that sediment resuspension in aquaculture environments functions as an organic source for bacteria in the water column, and that bacterial species contributing to the environmental capacity and carbon cycle are limited. We identified 25 AGB phylotypes, belonging to Alphaproteobacteria (Roseobacter clade, nine phylotypes), Gammaproteobacteria (five phylotypes), Deltaproteobacteria (one phylotype), Bacteroidetes (seven phylotypes), and Actinobacteria (three phylotypes). Among them, some AGB phylotypes appeared throughout the year with high frequency and were also identified in other coastal environments. This result suggests that these species are responsible for the environmental capacity and carbon cycle, and are key species in this fish-farming area, as well as other coastal environments.

Active populations of rare microbes in oceanic environments as revealed by bromodeoxyuridine incorporation and 454 tag sequencing.

The "rare biosphere" consisting of thousands of low-abundance microbial taxa is important as a seed bank or a gene pool to maintain microbial functional redundancy and robustness of the ecosystem. Here we investigated contemporaneous growth of diverse microbial taxa including rare taxa and determined their variability in environmentally distinctive locations along a north-south transect in the Pacific Ocean in order to assess which taxa were actively growing and how environmental factors influenced bacterial community structures. A bromodeoxyuridine-labeling technique in combination with PCR amplicon pyrosequencing of 16S rRNA genes gave 215-793 OTUs from 1200 to 3500 unique sequences in the total communities and 175-299 OTUs nearly 860 to 1800 sequences in the active communities. Unexpectedly, many of the active OTUs were not detected in the total fractions. Among these active but rare OTUs, some taxa (2-4% of rare OTUs) showed much higher abundance (>0.10% of total reads) in the active fraction than in the total fraction, suggesting that their contribution to bacterial community productivity or growth was much larger than that expected from their standing stocks at each location. An ordination plot by the principal component analysis presented that bacterial community compositions among 4 sampling locations and between total and active fractions were distinctive with each other. A redundancy analysis revealed that the variability of community compositions significantly correlated to seawater temperature and dissolved oxygen concentration. Also, a variation partitioning analysis showed that the environmental factors explained 49% of the variability of community compositions and the distance only explained 4.0% of its variability. These results implied very dynamic change of community structures due to environmental filtering. The active bacterial populations are more diverse and spread further in rare biosphere than we have ever seen. This study implied that rare microbes are important as an active part of microbial communities functioning ecosystems.

Seasonal variations in the community structure of actively growing bacteria in neritic waters of Hiroshima Bay, western Japan.

Using bromodeoxyuridine (BrdU) magnetic beads immunocapture and a PCR-denaturing gradient gel electrophoresis (DGGE) technique (BUMP-DGGE), we determined seasonal variations in the community structures of actively growing bacteria in the neritic waters of Hiroshima Bay, western Japan. The community structures of actively growing bacteria were separated into two clusters, corresponding to the timing of phytoplankton blooms in the autumn-winter and spring-summer seasons. The trigger for changes in bacterial community structure was related to organic matter supply from phytoplankton blooms. We identified 23 phylotypes of actively growing bacteria, belonging to Alphaproteobacteria (Roseobacter group, 9 phylotypes), Gammaproteobacteria (2 phylotypes), Bacteroidetes (8 phylotypes), and Actinobacteria (4 phylotypes). The Roseobacter group and Bacteroidetes were dominant in actively growing bacterial communities every month, and together accounted for more than 70% of the total DGGE bands. We revealed that community structures of actively growing bacteria shifted markedly in the wake of phytoplankton blooms in the neritic waters of Hiroshima Bay.

Differing growth responses of major phylogenetic groups of marine bacteria to natural phytoplankton blooms in the western North Pacific Ocean.

Growth and productivity of phytoplankton substantially change organic matter characteristics, which affect bacterial abundance, productivity, and community structure in aquatic ecosystems. We analyzed bacterial community structures and measured activities inside and outside phytoplankton blooms in the western North Pacific Ocean by using bromodeoxyuridine immunocytochemistry and fluorescence in situ hybridization (BIC-FISH). Roseobacter/Rhodobacter, SAR11, Betaproteobacteria, Alteromonas, SAR86, and Bacteroidetes responded differently to changes in organic matter supply. Roseobacter/Rhodobacter bacteria remained widespread, active, and proliferating despite large fluctuations in organic matter and chlorophyll a (Chl-a) concentrations. The relative contribution of Bacteroidetes to total bacterial production was consistently high. Furthermore, we documented the unexpectedly large contribution of Alteromonas to total bacterial production in the bloom. Bacterial abundance, productivity, and growth potential (the proportion of growing cells in a population) were significantly correlated with Chl-a and particulate organic carbon concentrations. Canonical correspondence analysis showed that organic matter supply was critical for determining bacterial community structures. The growth potential of each bacterial group as a function of Chl-a concentration showed a bell-shaped distribution, indicating an optimal organic matter concentration to promote growth. The growth of Alteromonas and Betaproteobacteria was especially strongly correlated with organic matter supply. These data elucidate the distinctive ecological role of major bacterial taxa in organic matter cycling during open ocean phytoplankton blooms.

Analysis of nanoplankton community structure using flow sorting and molecular techniques.

We developed a method for the separate and simultaneous analysis of the community structure of heterotrophic nanopkankton (HNP) and autotrophic nanoplankton (ANP). This method consists of three steps. First, nanoplankton cells were concentrated using a cross-flow filtration system because cell densities in natural seawater are usually too low for genetic studies. Second, HNP and ANP were separated by flow cytometric sorting ("flow sorting") on the basis of the presence or absence of chlorophyll. Finally, the community structure was analyzed using denaturing gradient gel electrophoresis targeting 18S rRNA gene. The newly developed method was applied to the coastal surface water of Aburatsubo Inlet, Japan, in July 2008. The separation of nanoplankton into HNP and ANP was validated by phylogenetic analysis, and the trophic mode of uncultured nanoplankton was confirmed (e.g. Marine Alveolata group II [MALV II] and Marine Stramenopile clade-2 [MAST-2]). This new method involving cell concentration, flow sorting and phylogenetic analysis is a potentially powerful tool for evaluating the population dynamics and ecology of marine protozoa.

Phylotype-Specific Productivity of Marine Bacterial Populations in Eutrophic Seawater, as Revealed by Bromodeoxyuridine Immunocytochemistry Combined with Fluorescence in situ Hybridization.

Among the fundamental questions in marine microbial ecology are which taxa or phylogenetic groups account for total bacterial productivity and what is the relative contribution of each. We combined bromodeoxyuridine (BrdU) immunocytochemistry and fluorescence in situ hybridization (BIC-FISH) to examine phylotype-specific contributions to total bacterial productivity in eutrophic seawater. We also examined year-round changes in phylotype-specific contributions and explored the factors controlling these changes. Monitoring by BIC-FISH throughout the year revealed the importance of the Roseobacter/Rhodobacter group as a constantly proliferating basic population (27% of all BrdU-positive cells), although their contribution was not significantly correlated with water temperature or with chlorophyll a or organic matter concentration. The Bacteroidetes were another important group, as they greatly increased in abundance after the end of phytoplankton blooms. Two other phylotypes tested, the SAR86 and Vibrio groups, changed their contributions to bacterial productivity with changes in water temperature. To our knowledge, this study was the first to estimate the yearly contribution of major subgroups of marine bacteria to total bacterial productivity in a seawater environment.

Community structures of actively growing bacteria shift along a north-south transect in the western North Pacific.

Bacterial community structures and their activities in the ocean are tightly coupled with organic matter fluxes and thus control ocean biogeochemical cycles. Bromodeoxyuridine (BrdU), halogenated nucleoside and thymidine analogue, has been recently used to monitor actively growing bacteria (AGB) in natural environments. We labelled DNA of proliferating cells in seawater bacterial assemblages with BrdU and determined community structures of the bacteria that were possible key species in mediating biochemical reactions in the ocean. Surface seawater samples were collected along a north-south transect in the North Pacific in October 2003 and subjected to BrdU magnetic beads immunocapture and PCR-DGGE (BUMP-DGGE) analysis. Change of BrdU-incorporated community structures reflected the change of water masses along a north-south transect from subarctic to subtropical gyres in the North Pacific. We identified 25 bands referred to AGB as BrdU-incorporated phylotypes, belonging to Alphaproteobacteria (5 bands), Betaproteobacteria (1 band), Gammaproteobacteria (4 bands), Cytophaga-Flavobacterium-Bacteroides (CFB) group bacteria (5 bands), Gram-positive bacteria (6 bands), and Cyanobacteria (4 bands). BrdU-incorporated phylotypes belonging to Vibrionales, Alteromonadales and Gram-positive bacteria appeared only at sampling stations in a subtropical gyre, while those belonging to Roseobacter-related bacteria and CFB group bacteria appeared at the stations in both subarctic and subtropical gyres. Our result revealed phylogenetic affiliation of AGB and their dynamic change along with north-south environmental gradients in open oceans. Different species of AGB utilize different amount and kinds of substrates, which can affect the change of organic matter fluxes along transect.

Actively growing bacteria in the inland sea of Japan, identified by combined bromodeoxyuridine immunocapture and denaturing gradient gel electrophoresis.

A fundamental question in microbial oceanography concerns the relationship between prokaryote diversity and biogeochemical function in an ecosystem context. We combined bromodeoxyuridine (BrdU) magnetic bead immunocapture and PCR-denaturing gradient gel electrophoresis (BUMP-DGGE) to examine phylotype-specific growth in natural marine assemblages. We also examined a broad range of marine bacterial isolates to determine their abilities to incorporate BrdU in order to test the validity of the method for application to diverse marine assemblages. We found that 27 of 29 isolates belonging to different taxa could incorporate BrdU. BUMP-DGGE analysis revealed phylogenetic affiliations of DNA-synthesizing, presumably actively growing bacteria across a eutrophic to mesotrophic transect in the Inland Sea of Japan. We found that the BrdU-incorporating (growing) communities were substantially different from the total communities. The majority (34/56) of phylotypes incorporated BrdU and were presumably growing, and these phylotypes comprised 10 alphaproteobacteria, 1 betaproteobacterium, 11 gammaproteobacteria, 11 Cytophaga-Flavobacterium-Bacteroides group bacteria, and 1 unclassified bacterium. All BrdU-responsive alphaproteobacteria were members of the Rhodobacterales, suggesting that such bacteria were dominant in the growing alphaproteobacterial populations in our samples. The BrdU-responsive gammaproteobacteria belonged to the Oceanospirillales, the SAR86 cluster, the Pseudomonadales, the Alteromonadales, and the Vibrionales. Thus, contemporaneous cooccurrence of diverse actively growing bacterial taxa was a consistent pattern in our biogeochemically varied study area.