Abstract

Endozoicomonas bacteria symbiose with various marine organisms and are known to be beneficial for coral health. However, genome analysis of coral-associated Endozoicomonas has been limited owing to the difficulty in cultivation and metagenomic approach by contamination of host-derived sequences. In this study, we applied a novel single-cell genomics technique using droplet microfluidics to obtain single-cell amplified genome (SAGs) for coral-associated Endozoicomonas spp. genome. We obtained seven novel Endozoicomonas genomes from Acropora tenuis coral. These genomes revealed that Endozoicomonas bacteria played host-associated functions in host corals and had undergone independent host-adaptive evolution in different clades. These adaptive evolutions were mediated by host-derived eukaryotic-like genes, some of which were speculated to influence host immune mechanisms. These genes are speculated to enhance coral tolerance to environmental stresses. This study suggests the possibility of host adaptation of Endozoicomonas spp. in symbiosis with corals and their contribution to coral bleaching tolerance.

Abstract

Spatial transcriptome analysis of formalin-fixed paraffin-embedded (FFPE) tissues using RNA-sequencing (RNA-seq) provides interactive information on morphology and gene expression, which is useful for clinical applications. However, despite the advantages of long-term storage at room temperature, FFPE tissues may be severely damaged by methylene crosslinking and provide less gene information than fresh-frozen tissues. In this study, we proposed a sensitive FFPE micro-tissue RNA-seq method that combines the punching of tissue sections (diameter: 100 μm) and the direct construction of RNA-seq libraries. We evaluated a method using mouse liver tissues at 2 years after fixation and embedding and detected approximately 7,000 genes in micro-punched tissue-spots (thickness: 10 μm), similar to that detected with purified total RNA (2.5 ng) equivalent to the several dozen cells in the spot. We applied this method to clinical FFPE specimens of lung cancer that had been fixed and embedded 6 years prior, and found that it was possible to determine characteristic gene expression in the microenvironment containing tumor and non-tumor cells of different morphologies. This result indicates that spatial gene expression analysis of the tumor microenvironment is feasible using FFPE tissue sections stored for extensive periods in medical facilities.

Abstract

Bacterial genome structure changes dynamically, and structural variants can change bacterial phenotype; However, obtaining the complete genome and analyzing genome structure of uncultured bacteria has been challenging. We aimed to develop a single-cell amplified genome long-read assembly (scALA) workflow to construct circular single-cell amplified genomes (cSAGs) from long-read single-cell sequencing data of targeted uncultured bacteria. In particular, scALA generated cSAGs from nanopore long-read sequencing data of SAGs by producing contiguous sequences with repeated bias reduction and assembly processes. From 12 human fecal samples, scALA generated 16 cSAGs of three specifically targeted bacterial species, Anaerostipes hadrus, Agathobacter rectalis, and Ruminococcus gnavus. A. hadrus cSAGs exhibited large, ten kbp-long, phage insertions, saccharide metabolic capacity, and frequent genomic recombination with related strains from cohabitant hosts. Noteworthy, cSAGs constructed using this method could expand bacterial genome databases and our understanding of within-species diversities in uncultured bacteria.

Abstract

The production of bioactive metabolites is increasingly recognized as an important function of host-associated bacteria. An example is defensive symbiosis that might account for much of the chemical richness of marine invertebrates including sponges (Porifera), 1 of the oldest metazoans. However, most bacterial members of sponge microbiomes have not been cultivated or sequenced, and therefore, remain unrecognized. Unequivocally linking metabolic functions to a cellular source in sponge microbiomes is, therefore, a challenge. Here, we report an analysis pipeline of microfluidic encapsulation, Raman microscopy, and integrated digital genomics (MERMAID) for an efficient identification of uncultivated producers. We applied this method to the chemically rich bacteriosponge (sponge that hosts a rich bacterial community) Theonella swinhoei, previously shown to contain ‘Entotheonella’ symbionts that produce most of the bioactive substances isolated from the sponge. As an exception, the antifungal aurantosides had remained unassigned to a source. Raman-guided single-bacterial analysis and sequencing revealed a cryptic, distinct multiproducer, ‘Candidatus Poriflexus aureus’ from a new Chloroflexi lineage as the aurantoside producer. Its exceptionally large genome contains numerous biosynthetic loci and suggested an even higher chemical richness of this sponge than previously appreciated. This study highlights the importance of complementary technologies to uncover microbiome functions, reveals remarkable parallels between distantly related symbionts of the same host, and adds functional support for diverse chemically prolific lineages being present in microbial dark matter.

Abstract

Partial mtDNA cytochrome oxidase subunit I (COI) fragments and near entire stretch of 12S rDNA (12S) and control region (Dloop) of the Japanese spiny lobster (Panulirus japonicus) (n = 3) were amplified by PCR and used for direct nucleotide sequencing and for clone library-based nucleotide sequence analysis. Nucleotide sequences of a total of 75 clones in COI, 77 in 12S and 92 in Dloop were determined. Haplotypes of the clones matched with those obtained by direct sequencing were determined to be genuine mtDNA sequence of the individual. Phylogenetic analysis revealed several distinct groups of haplotypes in all three regions. Genuine mtDNA sequences were observed to form a group with their closely related variables, and most of these variables may be due to amplification error but a few to be heteroplasmy. Haplotypes determined as nuclear mitochondrial pseudogenes (NUMTs) formed distinct groups. Nucleotide sequence divergence (K2P distance) between genuine haplotypes and NUMTs were substantial (7.169–23.880% for COI, 1.336–23.434% for 12S, and 7.897–71.862% for Dloop). These values were comparable to or smaller than those between species of the genus Panulirus, indicating that integration of mtDNA into the nuclear genome is a continuous and dynamic process throughout pre- and post-speciation events. Double peaks in electropherograms obtained by direct nucleotide sequencing were attributed to common nucleotides shared by multiple NUMTs. Information on the heteroplasmy and NUMTs would be very important for addressing their impact on direct nucleotide sequencing and for quality control of nucleotide sequences obtained.

<title>Abstract</title><sec>
<title>Background</title>
Obtaining high-quality (HQ) reference genomes from microbial communities is crucial for understanding the phylogeny and function of uncultured microbes in complex microbial ecosystems. Despite improvements in bioinformatic approaches to generate curated metagenome-assembled genomes (MAGs), existing metagenome binners obtain population consensus genomes but they are nowhere comparable to genomes sequenced from isolates in terms of strain level resolution. Here, we present a framework for the integration of single-cell genomics and metagenomics, referred to as single-cell (sc) metagenomics, to reconstruct strain-resolved genomes from microbial communities at once.


</sec><sec>
<title>Results</title>
Our sc-metagenomics integration framework, termed SMAGLinker, uses single-cell amplified genomes (SAGs) generated using microfluidic technology as binning guides and integrates them with metagenome-assembled genomes (MAGs) to recover improved draft genomes. We compared sc-metagenomics with the metagenomics-alone approach using conventional metagenome binners. The sc-metagenomics approach showed precise contig binning and higher recovery rates (&gt;97%) of rRNA and plasmids than conventional metagenomics in genome reconstruction from the cell mock community. In human microbiota samples, sc-metagenomics recovered the largest number of genomes with a total of 103 gut microbial genomes (21 HQ, with 65 showing &gt;90% completeness) and 45 skin microbial genomes (10 HQ, with 40 showing &gt;90% completeness), respectively. Conventional metagenomics recovered one <italic>Staphylococcus hominis</italic> genome, whereas sc-metagenomics recovered two <italic>S. hominis</italic> genomes from identical skin microbiota sample. Single-cell sequencing revealed that these <italic>S. hominis</italic> genomes were derived from two distinct strains harboring specifically different plasmids. We found that all conventional <italic>S. hominis</italic> MAGs had a substantial lack or excess of genome sequences and contamination from other <italic>Staphylococcus</italic> species (<italic>S. epidermidis).</italic>


</sec><sec>
<title>Conclusions</title>
SMAGLinker enabled us to obtain strain-resolved genomes in the mock community and human microbiota samples by assigning metagenomic sequences correctly and covering both highly conserved genes such as rRNA genes and unique extrachromosomal elements, including plasmids. SMAGLinker will provide HQ genomes that are difficult to obtain using metagenomics alone and will facilitate the understanding of microbial ecosystems by elucidating detailed metabolic pathways and horizontal gene transfer networks. SMAGLinker is available at <ext-link xmlns:xlink="http://www.w3.org/1999/xlink" ext-link-type="uri" xlink:href="https://github.com/kojiari/smaglinker">https://github.com/kojiari/smaglinker</ext-link>.



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Mechanism of production of extracellular vesicles (EVs) and their molecular contents are of great interest owing to their diverse roles in biological systems and are far from being completely understood. Even though, cellular cargo release mediated by EVs have been demonstrated in several cases, their role in secondary metabolite production and release remains elusive. In this study we investigate this aspect in detail using Raman micro-spectroscopic imaging. We provide considerable evidence to suggest that the release of antibiotic penicillin by filamentous fungus Penicillium chrysogenuminvolves EVs. Morphological modifications of the fungal body during biogenesis, changes in cell composition at the locus of biogenesis, and major molecular contents of the released EVs are also revealed in this study.

Abstract

Gene expression analysis at the single-cell level by next generation sequencing has revealed the existence of clonal dissemination and microheterogeneity in cancer metastasis. The current spatial analysis technologies can elucidate the heterogeneity of cell–cell interactions in situ. To reveal the regional and expressional heterogeneity in primary tumors and metastases, we performed transcriptomic analysis of microtissues dissected from a triple-negative breast cancer (TNBC) cell line MDA-MB-231 xenograft model with our automated tissue microdissection punching technology. This multiple-microtissue transcriptome analysis revealed three cancer cell-type clusters in the primary tumor and axillary lymph node metastasis, two of which were cancer stem cell (CSC)-like clusters (CD44/MYC-high, HMGA1-high). Reanalysis of public single-cell RNA-seq (scRNA-seq) datasets confirmed that the two CSC-like populations existed both in TNBC xenograft models and TNBC patients. In addition, the gene signature of the HMGA1-high CSC-like cluster has the potential to serve as a novel biomarker for diagnosis. The diversity of these multiple CSC-like populations may cause differential anticancer drug resistance, increasing the difficulty of curing this cancer.

Zika virus (ZIKV) is a mosquito-borne flavivirus that causes febrile illness. The recent spread of ZIKV from Asia to the Americas via the Pacific region has revealed unprecedented features of ZIKV, including transplacental congenital infection causing microcephaly. Amino acid changes have been hypothesized to underlie the spread and novel features of American ZIKV strains; however, the relationship between genetic changes and the epidemic remains controversial. A comparison of the characteristics of a Southeast Asian strain (NIID123) and an American strain (PRVABC59) revealed that the latter had a higher replication ability in cultured cells and higher virulence in mice. In this study, we aimed to identify the genetic region of ZIKV responsible for these different characteristics using reverse genetics. A chimeric NIID123 strain in which the E protein was replaced with that of PRVABC59 showed a lower growth ability than the recombinant wild-type strain. Adaptation of the chimeric NIID123 to Vero cells induced a Phe-to-Leu amino acid substitution at position 146 of the prM protein; PRVABC59 also has Leu at this position. Leu at this position was found to be responsible for the viral replication ability and partially, for the pathogenicity in mouse testes.

Species of infraorder Gryllidea, or crickets, are useful invertebrate models for studying developmental biology and neuroscience. They have also attracted attention as alternative protein sources for human food and animal feed. Mitochondrial genomic information on related invertebrates, such as katydids, and locusts, has recently become available in attempt to clarify the controversial classification schemes, although robust phylogenetic relationships with emphasis on crickets remain elusive. Here, we report newly sequenced complete mitochondrial genomes of crickets to study their phylogeny, genomic rearrangements, and adaptive evolution. First, we conducted de novo assembly of mitochondrial genomes from eight cricket species and annotated protein-coding genes (PCGs) and transfer and ribosomal RNAs using automatic annotations and manual curation. Next, by combining newly described PCGs with public data of the complete Gryllidea genomes and gene annotations, we performed phylogenetic analysis and found gene order rearrangements in several branches. We further analyzed genetic signatures of selection in ant-loving crickets (Myrmecophilidae), which are small wingless crickets that inhabit ant nests. Three distinct approaches revealed two positively selected sites in the cox1 gene in these crickets. Protein 3D structural analyses suggested that these selected sites could influence the interaction of respiratory complex proteins, conferring benefits to ant-loving crickets with a unique ecological niche and morphology. These findings enhance our understanding of the genetic basis of cricket evolution without relying on estimates based on a limited number of molecular markers.

Recent studies have shown that mutation at Ser522 causes inhibition of collapsin response mediator protein 2 (CRMP2) phosphorylation and induces axon elongation and partial recovery of the lost sensorimotor function after spinal cord injury (SCI). We aimed to reveal the intracellular mechanism in axotomized neurons in the CRMP2 knock-in (CRMP2KI) mouse model by performing transcriptome analysis in mouse sensorimotor cortex using micro-dissection punching system. Prior to that, we analyzed the structural pathophysiology in axotomized or neighboring neurons after SCI and found that somatic atrophy and dendritic spine reduction in sensorimotor cortex were suppressed in CRMP2KI mice. Further analysis of the transcriptome has aided in the identification of four hemoglobin genes Hba-a1, Hba-a2, Hbb-bs, and Hbb-bt that are significantly upregulated in wild-type mice with concomitant upregulation of genes involved in the oxidative phosphorylation and ribosomal pathways after SCI. However, we observed substantial upregulation in channel activity genes and downregulation of genes regulating vesicles, synaptic function, glial cell differentiation in CRMP2KI mice. Moreover, the transcriptome profile of CRMP2KI mice has been discussed wherein energy metabolism and neuronal pathways were found to be differentially regulated. Our results showed that CRMP2KI mice displayed improved SCI pathophysiology not only via microtubule stabilization in neurons, but also possibly via the whole metabolic system in the central nervous system, response changes in glial cells, and synapses. Taken together, we reveal new insights on SCI pathophysiology and the regenerative mechanism of central nervous system by the inhibition of CRMP2 phosphorylation at Ser522. All these experiments were performed in accordance with the guidelines of the Institutional Animal Care and Use Committee at Waseda University, Japan (2017-A027 approved on March 21, 2017; 2018-A003 approved on March 25, 2018; 2019-A026 approved on March 25, 2019).

Raman spectra are molecular structure-specific and hence are employed in applications requiring chemical identification. The advent of efficient handheld and smartphone-based Raman instruments is promoting widespread applications of the technique, which often involve less trained end users. Software modules that enable spectral library searches based on spectral pattern matching is an essential part of such applications. The Raman spectrum recorded by end users will naturally have varying levels of signal to noise (SN), baseline fluctuations, etc., depending on the sample environment. Further, in biological, forensic, food, pharmaceuticals, etc., fields where a vast amount of Raman spectral data is generated, careful removal of background is often impossible. In other words, a 100% match between the library spectrum and user input cannot be often guaranteed or expected. Often, such influences are discounted upon developing mathematical methods for general applications. In this manuscript, we carefully examine how such effects would determine the results of spectral similarity-based library search. We show that several popular mathematical spectral matching approaches give incorrect results under the influence of small changes in the baseline and/or the noise. We also discuss the points to be carefully considered while generating a spectral library. We believe our results will be a guiding note for developing applications of Raman spectroscopy that uses a standard spectral library and mathematical spectral matching.

Humanized mouse models are attractive experimental models for analyzing the development and functions of human dendritic cells (DCs) in vivo. Although various types of DC subsets, including DC type 3 (DC3s), have been identified in humans, it remains unclear whether humanized mice can reproduce heterogeneous DC subsets. CD14, classically known as a monocyte/macrophage marker, is reported as an indicator of DC3s. We previously observed that some CD14+ myeloid cells expressed CD1c, a pan marker for bona fide conventional DC2 (cDC2s), in humanized mouse models in which human FLT3L and GM-CSF genes were transiently expressed using in vivo transfection (IVT). Here, we aimed to elucidate the identity of CD14+CD1c+ DC-like cells in humanized mouse models. We found that CD14+CD1c+ cells were phenotypically different from cDC2s; CD14+CD1c+ cells expressed CD163 but not CD5, whereas cDC2s expressed CD5 but not CD163. Furthermore, CD14+CD1c+ cells primed and polarized naïve CD4+ T cells toward IFN-γ+ Th1 cells more profoundly than cDC2s. Transcriptional analysis revealed that CD14+CD1c+ cells expressed several DC3-specific transcripts, such as CD163, S100A8, and S100A9, and were clearly segregated from cDC2s and monocytes. When lipopolysaccharide was administered to the humanized mice, the frequency of CD14+CD1c+ cells producing IL-6 and TNF-α was elevated, indicating a pro-inflammatory signature. Thus, humanized mice are able to sustain development of functional CD14+CD1c+ DCs, which are equivalent to DC3 subset observed in humans, and they could be useful for analyzing the development and function of DC3s in vivo.

Raman microspectroscopy is a minimally invasive technique that can identify molecules without labeling. In this study, we demonstrate the detection of penicillin G inside Penicillium chrysogenum KF425 fungal cells. Raman spectra acquired from the fungal cells had highly overlapped spectroscopic signatures and hence were analyzed with multivariate curve resolution by alternating least-squares (MCR-ALS) to extract the spectra of individual molecular constituents. In addition to detecting spatial distribution of multiple constituents such as proteins and lipids inside the fungal body, we could also observe the subcellular localization of penicillin G. This methodology has the potential to be employed in screening the production of bioactive compounds by microorganisms.

Rickettsiales-like organisms are important for the survival and functioning of corals, prompting an investigation of their complete genomes. Earlier reports of the genomes of these organisms remain incomplete. Here, we report a novel draft genome of Rickettsiales bacterial strain SESOKO1, found in Acropora tenuis coral, using single-cell genome technology.

The genus Okeania is a globally distributed group of microorganisms that live in shallow seabed regions. These organisms play several environmentally important roles and are also known producers of several active secondary metabolites with potential human applications. Here, we present a draft genome of Okeania sp. strain KiyG1 (92.7% completeness) that was assembled from four single-amplified genomes.

Cancer chemoresistance is often attributed to the presence of cancer stem cell (CSC)-like cells, but whether they are homogeneously chemoresistant remains unclear. We previously showed that in colon tumors, a subpopulation of LGR5+ CSC-like cells driven by TCF1 (TCF7), a Wnt-responsive transcription factor, were responsible for tumorigenicity. Here we demonstrate that the tumorigenic subpopulation of mouse LGR5+ cells exists in a slow-cycling state and identify a unique 22-gene signature that characterizes these slow-cycling CSC. Seven of the signature genes are specifically expressed in slow-cycling LGR5+ cells from xenografted human colon tumors and are upregulated in colon cancer clinical specimens. Among these seven, four genes (APCDD1, NOTUM, PROX1, and SP5) are known to be direct Wnt target genes, and PROX1 was expressed in the invasive fronts of colon tumors. PROX1 was activated by TCF1 to induce CDKN1C and maintain a slow-cycling state in colon cancer organoids. Strikingly, PROX1 was required for recurrent growth after chemotherapeutic treatment, suggesting that inhibition of slow-cycling CSC by targeting the TCF1-PROX1-CDKN1C pathway is an effective strategy to combat refractory colon cancer in combination with conventional chemotherapy. SIGNIFICANCE: These findings illustrate the importance of a slow-cycling CSC subpopulation in colon cancer development and chemoresistance, with potential implications for the identified slow-cycling CSC signatures and the TCF1-PROX1-CDKN1C pathway as therapeutic targets.

Influenza A subtypes are categorized into group 1 and group 2 based on the hemagglutinin (HA) sequence. Owing to the phylogenetic distance of HAs in different groups, antibodies that bind multiple HA subtypes across different groups are extremely rare. In this study, we demonstrated that an immunization with acid-treated HA antigen elicits germinal center (GC) B cells that bind multiple HA subtypes in both group 1 and group 2. The cross-group GC B cells utilized mostly one VH gene (1S56) and exhibited a sign of clonal evolution within GCs. The 1S56-lineage IgGs derived from GC B cells were able to bind to HA protein on the infected cell surface but not to the native form of HA protein, suggesting the cryptic nature of the 1S56 epitope and its exposure in infected cells. Finally, the 1S56-lineage IgGs provided protection against lethal infection in an Fc-dependent manner, independent of the virus-neutralizing activity. Thus, we identified 1S56-lineage antibodies as a unique stereotype for achieving cross-group influenza specificity. The antigens exposing the 1S56 epitope may be good candidates for broadly protective immunogens.

Herpesviruses exist in nature within each host animal. Ten herpesviruses have been isolated from bats and their biological properties reported. A novel bat alphaherpesvirus, which we propose to name "Pteropus lylei-associated alphaherpesvirus (PLAHV)," was isolated from urine of the fruit bat Pteropus lylei in Vietnam and characterized. The entire genome sequence was determined to be 144,008 bp in length and predicted to include 72 genes. PLAHV was assigned to genus Simplexvirus with other bat alphaherpesviruses isolated from pteropodid bats in Southeast Asia and Africa. The replication capacity of PLAHV in several cells was evaluated in comparison with that of herpes simplex virus 1 (HSV-1). PLAHV replicated better in the bat-originated cell line and less in human embryonic lung fibroblasts than HSV-1 did. PLAHV was serologically related to another bat alphaherpesvirus, Pteropodid alphaherpesvirus 1 (PtAHV1), isolated from a Pteropus hypomelanus-related bat captured in Indonesia, but not with HSV-1. PLAHV caused lethal infection in mice. PLAHV was as susceptible to acyclovir as HSV-1 was. Characterization of this new member of bat alphaherpesviruses, PLAHV, expands the knowledge on bat-associated alphaherpesvirology.IMPORTANCE A novel bat alphaherpesvirus, Pteropus lylei-associated alphaherpesvirus (PLAHV), was isolated from urine of the fruit bat Pteropus lylei in Vietnam. The whole-genome sequence was determined and was predicted to include 72 open reading frames in the 144,008-bp genome. PLAHV is circulating in a species of fruit bats, Pteropus lylei, in Asia. This study expands the knowledge on bat-associated alphaherpesvirology.

Raman imaging has become an attractive technology in molecular biology because of its ability to detect multiple molecular components simultaneously without labeling. Two major limitations in accurately accounting for spectral features, viz., background removal and spectral unmixing, have been overcome by employing a modified and effective routine in multivariate curve resolution (MCR). With our improved strategy, we have spectrally isolated seven structurally specific biomolecules without any post-acquisition spectral treatments. Consequently, the isolated intensity profiles reflected concentrations of corresponding biomolecules with high statistical accuracy. Our study reveals the changes in the molecular composition of lipid droplets (LDs) inside HuH7 cells and its relation to the physiological state of the cell. Further, we show that the accurate separation of spectral components permits analysis of structural modification of molecules after cellular uptake. A detailed discussion is presented to highlight the potential of Raman spectroscopy with MCR in semi-quantitative molecular profiling of living cells.

In Japan, the imports of meat products have been increasing every year. Heat processing of meat is the current standard method for ensuring domestic animal health, particularly in case of meat products from areas where infectious diseases are known to have occurred in domestic animals. The Animal Quarantine Service needs to establish a method that detects the temperature at which the meat has been heat-processed (endpoint temperature) to ensure that the standard protocol is followed at the production location. Here, we developed a Raman spectroscopy and multivariate statistics (viz. multivariate curve resolution (MCR))-based simple and rapid method for accurately estimating the end point temperature. We showed that the temperature-dependent secondary structure modification of proteins can serve as an accurate indicator of the temperature of heat processing. This methodology can be easily automated for effective utilization by someone who is not an expert in spectroscopy. We envisage a wider application of this method in food analysis, although the present research investigated the application of this method in chicken meat heat processing analysis.

A diet rich in vegetables and fruit is generally considered healthy because of a high content of phytochemicals, vitamins, and fiber. The phytochemical indole-3-carbinol (I3C), a derivative of glucobrassicin, is sold as a dietary supplement promising diverse health benefits. I3C metabolites act as ligands of the aryl hydrocarbon receptor (AhR), an important sensor for environmental polyaromatic chemicals. Here, we investigated how dietary AhR ligand supplementation influences AhR target gene expression and intestinal microbiota composition. For this, we used AhR repressor (AhRR)-reporter mice as a tool to study AhR activation in the intestine following dietary I3C-supplementation in comparison with AhR ligand-deprived diets, including a high fat diet. AhRR expression in intestinal immune cells was mainly driven by dietary AhR ligands and was independent of microbial metabolites. A lack of dietary AhR ligands caused enhanced susceptibility to dextran sodium sulfate (DSS)-induced colitis and correlated with the expansion of Enterobacteriaceae, whereas Clostridiales, Muribaculaceae, and Rikenellaceae were strongly reduced. I3C supplementation largely reverted this effect. Comparison of I3C-induced changes in microbiota composition using wild-type (WT), AhRR-deficient, and AhR-deficient mice revealed both AhR-dependent and -independent alterations in the microbiome. Overall, our study demonstrates that dietary AhR ligand supplementation has a profound influence on Ahrr expression in intestinal immune cells as well as microbiota composition.

Bioprinting technology is expected to be applied in the fields of regenerative medicine and drug discovery. There are several types of bioprinters, especially inkjet-based bioprinter, which can be used not only as a printer for arranging cells but also as a precision cell-dispensing device with controlled cell numbers similar to a fluorescence activated cell sorter (FACS). Precise cell dispensers are expected to be useful in the fields of drug discovery and single-cell analysis. However, there are enduring concerns about the impacts of cell dispensers on cell integrity, particularly on sensitive cells, such as stem cells. In response to the concerns stated above, we developed a stress-free and media-direct-dispensing inkjet bioprinter. In the present study, in addition to conventional viability assessments, we evaluated the gene expression using RNA-seq to investigate whether the developed bioprinter influenced cell integrity in mouse embryonic stem cells. We evaluated the developed bioprinter based on three dispensing methods: manual operation using a micropipette, FACS and the developed inkjet bioprinter. According to the results, the developed inkjet bioprinter exhibited cell-friendly dispensing performance, which was similar to the manual dispensing operation, based not only on cell viability but also on gene expression levels.

Spatial transcriptomics is useful for understanding the molecular organization of a tissue and providing insights into cellular function in a morphological context. In order to obtain reproducible results in spatial transcriptomics, we have to maintain tissue morphology and RNA molecule stability during the image acquisition and biomolecule collection processes. Here, we developed a tissue processing method for robust and reproducible RNA-seq from tissue microdissection samples. In this method, we suppressed RNA degradation in fresh-frozen tissue specimens by dehydration fixation and effectively collected a small amount of RNA molecules from microdissection samples by magnetic beads. We demonstrated the spatial transcriptome analysis of the mouse liver and brain in serial microdissection samples (100 μm in a diameter and 10 μm in thickness) produced by a microdissection punching system. Using our method, we could prevent RNA degradation at room temperature and effectively produce a sequencing library with Smart-seq2. This resulted in reproducible sequence read mapping in exon regions and the detection of more than 2000 genes compared to non-fixed samples in the RNA-seq analysis. Our method would be applied to various transcriptome analyses, providing the information for region specific gene expression in tissue specimens.

How quiescent cells break dormancy is a key issue in eukaryotic cells including cancer. Fungal spores, for example, remain quiescent for long periods until nourished, although the mechanisms by which dormancy is broken remain enigmatic. Transcriptome analysis could provide a clue, but methods to synchronously germinate large numbers of spores are lacking, and thus it remains a challenge to analyse gene expression upon germination. Hence, we develop methods to assemble transcriptomes from individual, asynchronous spore cells of fission yeast undergoing germination to assess transcriptomic changes over time. The virtual time-lapse analyses highlights one of three copies of histone H3 genes whose transcription fluctuates during the initial stage of germination. Disruption of this temporal fluctuation causes defects in spore germination despite no visible defects in other stages of the life cycle. We conclude that modulation of histone H3 expression is a crucial 'wake-up' trigger at dormancy breaking.

<title>Abstract</title>To improve our understanding of the environmental microbiome, we developed a single-cell genome sequencing platform, named SAG-gel, which utilizes gel beads for single-cell isolation, cell lysis, and whole genome amplification (WGA) for sequencing. SAG-gel enables serial, parallel and independent reactions of &gt; 100,000 single cells in a single tube, delivering high-quality genome recovery with storable randomized single-cell genome libraries. From soil and marine environmental sources, we acquired 734 partial genomes that are recapitulated in 231 species, 35% of which were assigned as high-to-medium qualities. We found that each genome to be almost unique and 98.7% of them were newly identified, implying the complex genetic diversities across 44 phyla. The various metabolic capabilities including virulence factors and biosynthetic gene clusters were found across the lineages at single-cell resolution. This technology will accelerate the accumulation of reference genomes of uncharacterized environmental microbes and provide us new insights for their roles.

BACKGROUND: The gut microbiota can have dramatic effects on host metabolism; however, current genomic strategies for uncultured bacteria have several limitations that hinder their ability to identify responders to metabolic changes in the microbiota. In this study, we describe a novel single-cell genomic sequencing technique that can identify metabolic responders at the species level without the need for reference genomes, and apply this method to identify bacterial responders to an inulin-based diet in the mouse gut microbiota. RESULTS: Inulin-feeding changed the mouse fecal microbiome composition to increase Bacteroides spp., resulting in the production of abundant succinate in the mouse intestine. Using our massively parallel single-cell genome sequencing technique, named SAG-gel platform, we obtained 346 single-amplified genomes (SAGs) from mouse gut microbes before and after dietary inulin supplementation. After quality control, the SAGs were classified as 267 bacteria, spanning 2 phyla, 4 classes, 7 orders, and 14 families, and 31 different strains of SAGs were graded as high- and medium-quality draft genomes. From these, we have successfully obtained the genomes of the dominant inulin-responders, Bacteroides spp., and identified their polysaccharide utilization loci and their specific metabolic pathways for succinate production. CONCLUSIONS: Our single-cell genomics approach generated a massive amount of SAGs, enabling a functional analysis of uncultured bacteria in the intestinal microbiome. This enabled us to estimate metabolic lineages involved in the bacterial fermentation of dietary fiber and metabolic outcomes such as short-chain fatty acid production in the intestinal environment based on the fibers ingested. The technique allows the in-depth isolation and characterization of uncultured bacteria with specific functions in the microbiota and could be exploited to improve human and animal health. Video abstract.

In innate immunity, multiple autophagic processes eliminate intracellular pathogens, but it remains unclear whether noncanonical autophagy and xenophagy are coordinated, and whether they occur concomitantly or sequentially. Here, we show that Streptococcus pneumoniae, a causative of invasive pneumococcal disease, can trigger FIP200-, PI3P-, and ROS-independent pneumococcus-containing LC3-associated phagosome (LAPosome)-like vacuoles (PcLVs) in an early stage of infection, and that PcLVs are indispensable for subsequent formation of bactericidal pneumococcus-containing autophagic vacuoles (PcAVs). Specifically, we identified LC3- and NDP52-delocalized PcLV, which are intermediates between PcLV and PcAV. Atg14L, Beclin1, and FIP200 were responsible for delocalizing LC3 and NDP52 from PcLVs. Thus, multiple noncanonical and canonical autophagic processes are deployed sequentially against intracellular S. pneumoniae. The Atg16L1 WD domain, p62, NDP52, and poly-Ub contributed to PcLV formation. These findings reveal a previously unidentified hierarchical autophagy mechanism during bactericidal xenophagy against intracellular bacterial pathogens, and should improve our ability to control life-threating pneumococcal diseases.

Lymphoid-tissue-resident commensal bacteria (LRCs), including Alcaligenes faecalis, are present in intestinal lymphoid tissue including the Peyer's patches (PPs) of mammals and modulate the host immune system. Although LRCs can colonize within dendritic cells (DCs), the mechanisms through which LRCs persist in DCs and the symbiotic relationships between LRCs and DCs remain to be investigated. Here, we show an intracellular symbiotic system in which the LRC Alcaligenes creates a unique energy shift in DCs. Whereas DCs showed low mitochondrial respiration when they were co-cultured with Escherichia coli, DCs carrying A. faecalis maintained increased mitochondrial respiration. Furthermore, E. coli induced apoptosis of DCs but A. faecalis did not. Regarding an underlying mechanism, A. faecalis-unlike E. coli-did not induce intracellular nitric oxide (NO) production in DCs due to the low activity of its lipopolysaccharide (LPS). Therefore, A. faecalis, an example of LRCs, may persist within intestinal lymphoid tissue because they elicit little NO production in DCs. In addition, the symbiotic DCs exhibit characteristic physiologic changes, including a low rate of apoptosis and increased mitochondrial respiration.

In mammals, interleukin (IL)-17A and F are hallmark inflammatory cytokines that play key roles in protection against infection and intestinal mucosal immunity. In the gastrointestinal tract (GI), the induction of antimicrobial peptide (AMP) production via Paneth cells is a fundamental role of IL-17A and F in maintaining homeostasis of the GI microbiome and health. Although mammalian IL-17A and F homologs (referred to as IL-17A/F1-3) have been identified in several fish species, their function in the intestine is poorly understood. Additionally, the fish intestine lacks Paneth cells, and its GI structure is very different from that of mammals. Therefore, the GI microbiome modulatory mechanism via IL-17A/F genes has not been fully elucidated. In this study, Japanese medaka (Oryzias latipes) were used as a teleost model, and IL-17A/F1-knockout (IL-17A/F1-KO) medaka were established using the CRISPR/Cas9 genome editing technique. Furthermore, two IL-17A/F1-deficient medaka strains were generated, including one strain containing a 7-bp deletion (-7) and another with an 11-bp addition (+11). After establishing F2 homozygous KO medaka, transcriptome analysis (RNA-seq) was conducted to elucidate IL-17A/F1-dependent gene induction in the intestine. Results of RNA-seq and real-time PCR (qPCR) demonstrated down-regulation of immune-related genes, including interleukin-1β (IL-1β), complement 1q subunit C (C1qc), transferrin a (Tfa), and G-type lysozyme (LyzG), in IL-17A/F1-KO medaka. Interestingly, protein and lipid digestive enzyme genes, including phospholipase A2, group IB (pla2g1b), and elastase-1-like (CELA1), were also downregulated in the intestines of IL-17A/F1-KO medaka. Furthermore, to reveal the influence of these downregulated genes on the gut microbiome in IL-17A/F1-KO, 16S rRNA-based metagenomic sequencing analysis was conducted to analyze the microbiome constitution. Under a non-exposed state, the intestinal microbiome of IL-17A/F1-KO medaka differed at the phylum level from wild-type, with significantly higher levels of Verrucomicrobia and Planctomycetes. Additionally, at the operational taxonomic unit (OTU) level of the human and fish pathogens, the Enterobacteriaceae Plesiomonas shigelloides was the dominant species in IL-17A/F1-KO medaka. These findings suggest that IL-17A/F1 is involved in the maintenance of a healthy gut microbiome.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Germinal center (GC) B cells at viral replication sites acquire specificity to poorly immunogenic but conserved influenza hemagglutinin (HA) epitopes. Here, high-throughput epitope mapping of local GC B cells is used to identify conserved HA epitope selecting cross-reactive antibodies that mediate heterosubtypic protection. A distinct feature of this epitope is an occlusion in the naive trimeric HA structure that is exposed in the post-fusion HA structure to occur under low pH conditions during viral replication. Importantly, systemic immunization by the post-fusion HA antigen results in GC B cells targeting the occluded epitope, and induces a class of protective antibodies that have cross-group specificity and afford protection independent of virus neutralization activity. Furthermore, this class of broadly protective antibodies develops at late time points and persists. Our results identify a class of cross-protective antibodies that are selected at the viral replication site, and provide insights into vaccine strategies using the occluded epitope.

Since G-protein coupled receptors (GPCRs) are linked to various diseases, screening of functional ligands against GPCRs is vital for drug discovery. In the present study, we developed a high-throughput functional cell-based assay by combining human culture cells producing a GPCR, yeast cells secreting randomized peptide ligands, and a droplet microfluidic device. We constructed a reporter human cell line that emits fluorescence in response to the activation of human glucagon-like peptide-1 receptor (hGLP1R). We then constructed a yeast library secreting an agonist of hGLP1R or randomized peptide ligands. We demonstrated that high-throughput identification of functional ligands against hGLP1R could be performed by co-culturing the reporter cells and the yeast cells in droplets. We identified functional ligands, one of which had higher activity than that of an original sequence. The result suggests that our system could facilitate the discovery of functional peptide ligands of GPCRs.

After publication of the original article [1], the authors became aware of a miscalculation in the original Fig. 2d.

OBJECTIVE: Resting CD4+ T cells are major reservoirs of latent HIV-1 infection, and may be formed during the early phase of the infection. Although CCR5-tropic (R5) HIV-1 is highly transmissible during the early phase, newly infected individuals have usually been exposed to a mixture of R5 and CXCR4-tropic (X4) viruses, and X4 viral DNA is also detectable in the host. Our aim was to identify which subsets of resting CD4+ T cells contribute to forming the latent reservoir in the presence of both X4 and R5 viruses. RESULTS: Primary resting CD4+ naïve T (TN) cells, CCR5- memory T (TM) cells, and CCR5+ TM cells isolated by flow cytometry were infected simultaneously with X4 and R5 HIV-1, which harbored different reporter genes, and were cultured in the resting condition. Flow cytometry at 3 days post-infection demonstrated that X4 HIV-1+ cells were present in all three subsets of cells, whereas R5 HIV-1+ cells were present preferentially in CCR5+ TM cells, but not in TN cells. Following CD3/CD28-mediated activation at 3 days post-infection, numbers of R5 HIV-1+ cells and X4 HIV-1+ cells increased significantly only in the CCR5+ TM subset, suggesting that it provides a major reservoir of replication-competent, latently infected viruses.

Severe invasive group A Streptococcus (GAS) infection evades anti-bacterial immunity by attenuating the cellular components of innate immune responses. However, this loss of protection is compensated for by interferon (IFN)-γ-producing immature myeloid cells (γIMCs), which are selectively recruited upon severe invasive GAS infection in mice. Here, we demonstrate that γIMCs provide this IFN-γ-mediated protection by sequentially sensing GAS through two distinct pattern recognition receptors. In a mouse model, GAS is initially recognized by Toll-like receptor 2 (TLR2), which promptly induces interleukin (IL)-6 production in γIMCs. γIMC-derived IL-6 promotes the upregulation of a recently identified GAS-sensing receptor, macrophage-inducible C-type lectin (Mincle), in an autocrine or paracrine manner. Notably, blockade of γIMC-derived IL-6 abrogates Mincle expression, downstream IFN-γ production, and γIMC-mediated protection against severe invasive GAS infection. Thus, γIMCs regulate host protective immunity against severe invasive GAS infection via a TLR2-IL-6-Mincle axis.

Gut bacteria of phytophagous and omnivorous marine invertebrates often possess alginate lyases (ALGs), which are key enzymes for utilizing macroalgae as carbon neutral biomass. We hypothesized that the exclusive feeding of a target alga to marine invertebrates would shift the gut bacterial diversity suitable for degrading the algal components. To test this hypothesis, we reared sea hare (Dolabella auricularia) and sea snail (Batillus cornutus) for two to four weeks with exclusive feeding of a brown alga (Ecklonia cava). Pyrosequencing analysis of the gut bacterial 16S rRNA genes revealed shifts in the gut microbiota after rearing, mainly due to a decrease in the variety of bacterial members. Significant increases in six and four 16S rRNA gene phylotypes were observed in the reared sea hares and sea snails, respectively, and some of them were phylogenetically close to known alginate-degrading bacteria. Clone library analysis of PL7 family ALG genes using newly designed degenerate primer sets detected a total of 50 ALG gene phylotypes based on 90% amino acid identity. The number of ALG gene phylotypes increased in the reared sea hare but decreased in reared sea snail samples, and no phylotype was shared between them. Out of the 50 phylotypes, 15 were detected only after the feeding procedure. Thus, controlled feeding strategy may be valid and useful for the efficient screening of genes suitable for target alga fermentation.

Mucosal immunoglobulins comprise mainly secretory IgA antibodies (SIgAs), which are the major contributor to pathogen-specific immune responses in mucosal tissues. These SIgAs are highly heterogeneous in terms of their quaternary structure. A recent report shows that the polymerization status of SIgA defines their functionality in the human upper respiratory mucosa. Higher order polymerization of SIgA (i.e., tetramers) leads to a marked increase in neutralizing activity against influenza viruses. However, the precise molecular mechanisms underlying the effects of SIgA polymerization remain elusive. Here, we developed a method for generating recombinant tetrameric monoclonal SIgAs. We then compared the anti-viral activities of these tetrameric SIgAs, which possessed variable regions identical to that of a broadly neutralizing anti-influenza antibody F045-092 against influenza A viruses, with that of monomeric IgG or IgA. The tetrameric SIgA showed anti-viral inhibitory activity superior to that of other forms only when the antibody exhibits low-affinity binding to the target. By contrast, SIgA tetramerization did not substantially modify anti-viral activity against targets with high-affinity binding. Taken together, the data suggest that tetramerization of SIgA improved target breadth, but not peak potency of antiviral functions of the broadly neutralizing anti-influenza antibody. This phenomenon presumably represents one of the mechanisms by which SIgAs present in human respiratory mucosa prevent infection by antigen-drifted influenza viruses. Understanding the mechanisms involved in cross neutralization of viruses by SIgAs might facilitate the development of vaccine strategies against viral infection of mucosal tissues.

Dengue virus (DENV) infections are a major cause of morbidity and mortality in tropical and subtropical areas. Several compounds that act against DENV have been studied in clinical trials to date; however, there have been no compounds identified that are effective in reducing the severity of the clinical manifestations. To explore anti-DENV drugs, we examined small molecules that interact with DENV NS1 and inhibit DENV replication. Cyclofenil, which is a selective estrogen receptor modulator (SERM) and has been used clinically as an ovulation-inducing drug, showed an inhibitory effect on DENV replication in mammalian cells but not in mosquito cells. Other SERMs also inhibited DENV replication in mammalian cells, but cyclofenil showed the weakest cytotoxicity among these SERMs. Cyclofenil also inhibited the replication of Zika virus. A time-of-addition assay suggested that cyclofenil may interfere with two stages of the DENV life cycle: the translation-RNA synthesis and assembly-maturation stages. However, the level of intracellular infectious particles decreased more drastically after treatment with cyclofenil than the viral RNA level did, indicating that the assembly-maturation stage might be the main target of cyclofenil. In electron microscopy analysis, many aggregated particles were detected in DENV-infected cells in the presence of cyclofenil, supporting the possibility that cyclofenil impedes the process of assembly and maturation of DENV.

We would like to correct the information on the antibody used in this study. In Fig. 5 of the article, cellular β-actin was detected as an internal control using anti-β-actin antibody (Fujifilm Wako Pure Chemicals, #017-24573).

Regular exercise reduces the risks for cardiovascular diseases. Although the gut microbiota has been associated with fitness level and cardiometabolic risk factors, the effects of exercise-induced gut microbiota changes in elderly individuals are unclear. This study evaluated whether endurance exercise modulates the gut microbiota in elderly subjects, and whether these changes are associated with host cardiometabolic phenotypes. In a randomized crossover trial, 33 elderly Japanese men participated in a 5-week endurance exercise program. 16S rRNA gene-based metagenomic analyses revealed that the effect of endurance exercise on gut microbiota diversity was not greater than interindividual differences, whereas changes in α-diversity indices during intervention were negatively correlated with changes in systolic and diastolic blood pressure, especially during exercise. Microbial composition analyses showed that the relative abundance of Clostridium difficile significantly decreased, whereas that of Oscillospira significantly increased during exercise as compared to the control period. The changes in these taxa were correlated with the changes in several cardiometabolic risk factors. The findings indicate that short-term endurance exercise has little effect on gut microbiota in elderly individuals, and that the changes in gut microbiota were associated with cardiometabolic risk factors, such as systolic and diastolic blood pressure, providing preliminary insight into the associations between the gut microbiota and cardiometabolic phenotypes.

Several cases of herpes simplex encephalitis (HSE) caused by acyclovir (ACV)-resistant herpes simplex virus type 1 (HSV-1) have been reported. Amino acid substitutions of R41H, Q125H, and A156V in the viral thymidine kinase (vTK) gene have been reported to confer ACV resistance. Recombinant HSV-1 clones, containing each amino acid substitution in the vTK gene, were generated using the bacterial artificial chromosome system. A recombinant HSV-1 with the Q125H substitution showed ACV resistance while the R41H or A156V substitutions were ACV-sensitive. Furthermore, the Q125H recombinant HSV-1 was less virulent than the repaired virus, but it maintained neurovirulence in mice at relatively high levels. Substitution of Q125H, which was detected in the neonatal HSE patient, conferred ACV resistance, but the substitutions of R41H and A156V, which were detected in immunocompetent adult HSE patients, did not. This suggests that HSE caused by ACV-resistant HSV-1 might be a very rare event to occur during the course of ACV treatment in immunocompetent patients. Showing resistance to ACV treatment does not always indicate emergence of ACV-resistant HSV-1 in HSE patients.

Streptococcus pneumoniae is the most common causative agent of community-acquired pneumonia and can penetrate epithelial barriers to enter the bloodstream and brain. We investigated intracellular fates of S. pneumoniae and found that the pathogen is entrapped by selective autophagy in pneumolysin- and ubiquitin-p62-LC3 cargo-dependent manners. Importantly, following induction of autophagy, Rab41 was relocated from the Golgi apparatus to S. pneumoniae-containing autophagic vesicles (PcAV), which were only formed in the presence of Rab41-positive intact Golgi apparatuses. Moreover, subsequent localization and regulation of K48- and K63-linked polyubiquitin chains in and on PcAV were clearly distinguishable from each other. Finally, we found that E3 ligase Nedd4-1 was recruited to PcAV and played a pivotal role in K63-linked polyubiquitin chain (K63Ub) generation on PcAV, promotion of PcAV formation, and elimination of intracellular S. pneumoniae. These findings suggest that Nedd4-1-mediated K63Ub deposition on PcAV acts as a scaffold for PcAV biogenesis and efficient elimination of host cell-invaded pneumococci.

Coral reefs are one of the most biologically diverse and economically important ecosystems on earth. However, the destruction of coral reefs has been reported worldwide owing to rising seawater temperature associated with global warming. In this study, we investigated the potential of a redox nanoparticle (RNPO) to scavenge reactive oxygen species (ROS), which are overproduced under heat stress and play a crucial role in causing coral mortality. When reef-building coral (Acropora tenuis) larvae, without algal symbionts, were exposed to thermal stress at 33 °C, RNPO treatment significantly increased the survival rate. Proteome analysis of coral larvae was performed using nano-liquid chromatography-tandem mass spectrometry for the first time. The results revealed that several proteins related to ROS-induced oxidative stress were specifically identified in A. tenuis larvae without RNPO treatment, whereas these proteins were absent in RNPO-treated larvae, which suggested that RNPO effectively scavenged ROS from A. tenuis larvae. Results from this study indicate that RNPO treatment can reduce ROS in aposymbiotic coral larvae and would be a promising approach for protecting corals from thermal stress.

A novel system was developed for generating highly attenuated vaccinia virus LC16m8 (m8, third-generation smallpox vaccine) that expresses foreign genes. The innovations in this system are its excisable selection marker, specificity of the integration site of a gene of interest, and easy identification of clones with a fluorescent signal. Using this system, recombinant m8s, which expressed herpes simplex virus 2 (HSV-2) glycoprotein B (gB)-, gD-, or both gB and gD (gB + gD), were generated, and their efficacy was evaluated. First, the induction of a specific IgG against these HSV-2 glycoproteins in mice infected with one of these recombinant m8s was confirmed by an immunofluorescent assay. Next, mice preinfected with one of the recombinant m8s were infected with HSV-2 at a lethal dose to examine the vaccine efficacy. The fatality rate among the mice preinfected with either the recombinant gB + gD- or gD-expressing m8 significantly decreased in comparison with the control. The survival rate in male and female mice preinfected with either the recombinant gB + gD- or gD-expressing m8 increased to 100% and 60%, respectively, while most of the control mice died. In summary, this new system may be applicable to creation of a novel m8-based vaccine.

INTRODUCTION: Genetic associations for endophenotypes of Alzheimer's disease (AD) in cognitive stages preceding AD have not been thoroughly evaluated. METHODS: We conducted genome-wide association studies for AD-related endophenotypes including hippocampal volume, logical memory scores, and cerebrospinal fluid Aβ42 and total/phosphorylated tau in cognitively normal (CN), mild cognitive impairment, and AD dementia subjects from the Alzheimer's Disease Neuroimaging Initiative study. RESULTS: In CN subjects, study-wide significant (P < 8.3 × 10-9) loci were identified for total tau near SRRM4 and C14orf79 and for hippocampal volume near MTUS1. In mild cognitive impairment subjects, study-wide significant association was found with single nucleotide polymorphisms (SNPs) near ZNF804B for logical memory test of delayed recall scores. We found consistent expression patterns of C14orf40 and MTUS1 in carriers with risk alleles of expression SNPs and in brains of AD patients, compared with in the noncarriers and in brains of controls. DISCUSSION: Our findings for AD-related brain changes before AD provide insight about early AD-related biological processes.

Coral reefs are one of the most biologically diverse and economically important ecosystems on earth. However, the destruction of coral reefs has been reported worldwide owing to rising seawater temperature associated with global warming. In this study, we investigated the potential of a redox nanoparticle (RNP) to scavenge reactive oxygen species (ROS), which are overproduced under heat stress and play a crucial role in causing coral mortality. When reef-building coral (Acropora tenuis) larvae, without algal symbionts, were exposed to thermal stress at 33 °C, RNP treatment significantly increased the survival rate. Proteome analysis of coral larvae was performed using nano-liquid chromatography-tandem mass spectrometry for the first time. The results revealed that several proteins related to ROS-induced oxidative stress were specifically identified in A. tenuis larvae without RNP treatment, whereas these proteins were absent in RNP-treated larvae, which suggested that RNP effectively scavenged ROS from A. tenuis larvae. Results from this study indicate that RNP treatment can reduce ROS in aposymbiotic coral larvae and would be a promising approach for protecting corals from therm

Streptococcus pneumoniae is the most common causative agent of community-acquired pneumonia and can penetrate epithelial barriers to enter the bloodstream and brain. We investigated intracellular fates of S. pneumoniae and found that the pathogen is entrapped by selective autophagy in pneumolysin- and ubiquitin-p62-LC3 cargo-dependent manners. Importantly, following induction of autophagy, Rab41 was relocated from the Golgi apparatus to S. pneumoniae-containing autophagic vesicles (PcAV), which were only formed in the presence of Rab41-positive intact Golgi apparatuses. Moreover, subsequent localization and regulation of K48- and K63-linked polyubiquitin chains in and on PcAV were clearly distinguishable from each other. Finally, we found that E3 ligase Nedd4-1 was recruited to PcAV and played a pivotal role in K63-linked polyubiquitin chain (K63Ub) generation on PcAV, promotion of PcAV formation, and elimination of intracellular S. pneumoniae. These findings suggest that Nedd4-1-mediated K63Ub deposition on PcAV acts as a scaffold for PcAV biogenesis and efficient elimination of host cell-invaded pneumococci.

Single-cell RNA-seq is a powerful tool for revealing heterogeneity in cancer cells. However, each of the current single-cell RNA-seq platforms has inherent advantages and disadvantages. Here, we show that combining the different single-cell RNA-seq platforms can be an effective approach to obtaining complete information about expression differences and a sufficient cellular population to understand transcriptional heterogeneity in cancers. We demonstrate that it is possible to estimate missing expression information. We further demonstrate that even in the cases where precise information for an individual gene cannot be inferred, the activity of given transcriptional modules can be analyzed. Interestingly, we found that two distinct transcriptional modules, one associated with the Aurora kinase gene and the other with the DUSP gene, are aberrantly regulated in a minor population of cells and may thus contribute to the possible emergence of dormancy or eventual drug resistance within the population.

Marine sponges are prolific sources of unique bioactive natural products. The sponge Theonella swinhoei is represented by several distinct variants with largely nonoverlapping chemistry. For the Japanese chemotype Y harboring diverse complex polyketides and peptides, we previously provided genomic and functional evidence that a single symbiont, the filamentous, multicellular organism "Candidatus Entotheonella factor," produces almost all of these compounds. To obtain further insights into the chemistry of "Entotheonella," we investigated another phylotype, "Candidatus Entotheonella serta," present in the T. swinhoei WA sponge chemotype, a source of theonellamide- and misakinolide-type compounds. Unexpectedly, considering the lower chemical diversity, sequencing of individual bacterial filaments revealed an even larger number of biosynthetic gene regions than for Ca E. factor, with virtually no overlap. These included genes for misakinolide and theonellamide biosynthesis, the latter assigned by comparative genomic and metabolic analysis of a T. swinhoei chemotype from Israel, and by biochemical studies. The data suggest that both compound families, which were among the earliest model substances to study bacterial producers in sponges, originate from the same bacterium in T. swinhoei WA. They also add evidence that metabolic richness and variability could be a more general feature of Entotheonella symbionts.

Single-cell genomics is a straightforward approach to obtain genomes from uncultured microbes. However, sequence reads from a single-cell amplified genome (SAG) contain significant bias and chimeric sequences. Here, we describe Cleaning and Co-assembly of a Single-Cell Amplified Genome (ccSAG), a novel analytical workflow to obtain composite single-cell genomes with elimination of sequence errors. By the integration of ccSAG with a massively parallel single-cell genome amplification platform based on droplet microfluidics, we can generate multiple SAGs and effectively integrate them into the composite genomes quality equivalent to the data obtained from bulk DNA. We obtained two novel draft genomes from single gut microbial cells with high completeness (>96.6%) and extremely low contamination (<1.25%). Moreover, we revealed the presence of single nucleotide polymorphisms in the specific gene by sequence comparison at the single-cell level. Thus, the workflow yields near-complete genomes from uncultured microbes, and enables analyses of genetic heterogeneity within identical strains.

Two cytokines, fms-related tyrosine kinase 3 ligand (Flt3-L) and granulocyte-macrophage colony-stimulating factor (GM-CSF) are considered to be the essential regulators of dendritic cell (DC) development in vivo. However, the combined effect of Flt3-L and GM-CSF on human DCs has not been evaluated in vivo. In this study, we, therefore, aimed at evaluating this using a humanized mouse model. Humanized non-obese diabetic/SCID/Jak3null (hNOJ) mice were constructed by transplanting hematopoietic stem cells from human umbilical cord blood into newborn NOJ mice, and in vivo transfection (IVT) was performed by hydrodynamic injection-mediated gene delivery using plasmids encoding human Flt3-L and GM-CSF. Following IVT, Flt3-L and GM-CSF were successfully induced in hNOJ mice. At 10 days post-IVT, we found, in the spleen, that treatment with both Flt3-L and GM-CSF enhanced the reconstitution of two myeloid DC subsets, CD14-CD1c+ conventional DCs (cDCs) and CD14-CD141+ cDCs, in addition to CD14+ monocyte-like cells expressing CD1c and/or CD141. GM-CSF alone had less effect on the reconstitution of these myeloid cell populations. By contrast, none of the cytokine treatments enhanced CD123+ plasmacytoid DC (pDC) reconstitution. Regardless of the reconstitution levels, three cell populations (CD1c+ myeloid cells, CD141+ myeloid cells, and pDCs) could be matured by treatment with cytokines, in terms of upregulation of CD40, CD80, CD86, and CD184/CXCR4 and downregulation of CD195/CCR5. In particular, GM-CSF contributed to upregulation of CD80 in all these cell populations. Interestingly, we further observed that Foxp3+ cells within splenic CD4+ T cells were significantly increased in the presence of GM-CSF. Foxp3+ T cells could be subdivided into two subpopulations, CD45RA-Foxp3hi and CD45RA-Foxp3lo T cells. Whereas CD45RA-Foxp3hi T cells were increased only after treatment with GM-CSF alone, CD45RA-Foxp3lo T cells were increased only after treatment with both Flt3-L and GM-CSF. Treatment with Flt3-L alone had no effect on the number of Foxp3+ T cells. The correlation analysis demonstrated that the development of these Foxp3+ subpopulations was associated with the maturation status of DC(-like) cells. Taken together, this study provides a platform for studying the in vivo effect of Flt3-L and GM-CSF on human DCs and regulatory T cells.

The cytotoxic T lymphocyte (CTL) response plays a key role in host recovery from influenza virus infection and in subsequent immunity. Compared to natural infection with influenza virus, however, intranasal vaccination with adjuvant-combined inactivated vaccine elicits only moderate CTL responses. Here we demonstrate that 5 days of consecutive, intranasal vaccination with a combination of inactivated influenza vaccine and poly(I:C) elicits a strong CTL response in the lung. Antigen-captured respiratory DCs did efficiently migrate from the lung to the mediastinal lymph node (mLN) after the 5 day series of inoculations with vaccine and poly(I:C). Importantly, formalin-inactivated whole virus vaccine and poly(I:C) adjuvant have synergic effects on consecutive vaccinations to elicit a strong CTL response in the lung. Although the CTL response was less effective against heterologous influenza virus, we show for the first time that intranasal administration of inactivated influenza virus vaccine and poly(I:C) for 5 consecutive days can elicit high levels of influenza virus-specific CD8(+) T cells in the lung. (C) 2017 Elsevier Ltd. All rights reserved.

Vibrio harveyi is a gram-negative bacterium reported as found in many aquaculture species. To increase knowledge of the immune response against V. harveyi, in this study we performed transcriptome analysis of head kidney and spleen in orange-spotted grouper (Epinephelus coioides) at 1 and 2 days post-infection (dpi), using the Illumina sequencing platform. After de novo assembly, a total of 79,128 unigenes was detected with an N50 of 2511 bp. After alignments with sequences recorded in the major databases (NT, NR, Swiss-Prot COG, KEGG, Interpro and GO), based on sequence similarity, 61,208 (77.4%) of the unigene total could be annotated using at least one database. Comparison of gene expression levels between V. harveyi and a control group at each time point revealed differentially expressed genes (DEGs) (P &lt; 0.05): a total of 7918 (5536 upregulated and 2282 downregulated genes) from head kidney at 1 day post infection (dpi), 4260 (1444 upregulated and 2816 downregulated genes) from head kidney at 2 dpi, 7887 (4892 upregulated and 2995 downregulated genes) from spleen at 1 dpi, and 8952 (7388 upregulated and 1564 downregulated genes) from spleen at 2 dpi. The DEGs were mainly annotated into signal transduction and immune system categories, based on the KEGG database. The DEGs were enriched in immune-related pathway functions, NOD-like receptor signaling pathways, Toll-like receptor signaling pathways, NF-kappa B signaling pathways, and jak-STAT signaling pathways. Additionally, we selected several DEGs and validated their expression level by RT-qPCR. The data generated in this study may provide a valuable resource for further immune response research and offer improved strategies against V. harveyi infection in teleost fishes. (C) 2017 Elsevier Ltd. All rights reserved.

Single-cell genomics has enabled the exploration of cellular diversity in environmental microbes. However, current genome sequencing techniques, which utilizes next-generation sequencing (NGS), typically require nanogram to microgram levels of input DNA sample. Since single bacterial cells contain only a few femtograms of DNA, we have to amplify their genomes to adequate amount for sequencing. We aimed to develop a novel system for precise and high throughput single-cell genomics, to elucidate environmental microbial diversity. In this study, we have developed droplet-based microfluidic system to produce the compartmentalized reaction vessels for single-cell genome sequencing.

Massively parallel single-cell genome sequencing is required to further understand genetic diversities in complex biological systems. Whole genome amplification (WGA) is the first step for single-cell sequencing, but its throughput and accuracy are insufficient in conventional reaction platforms. Here, we introduce single droplet multiple displacement amplification (sd-MDA), a method that enables massively parallel amplification of single cell genomes while maintaining sequence accuracy and specificity. Tens of thousands of single cells are compartmentalized in millions of picoliter droplets and then subjected to lysis and WGA by passive droplet fusion in microfluidic channels. Because single cells are isolated in compartments, their genomes are amplified to saturation without contamination. This enables the high-throughput acquisition of contamination-free and cell specific sequence reads from single cells (21,000 single-cells/h), resulting in enhancement of the sequence data quality compared to conventional methods. This method allowed WGA of both single bacterial cells and human cancer cells. The obtained sequencing coverage rivals those of conventional techniques with superior sequence quality. In addition, we also demonstrate de novo assembly of uncultured soil bacteria and obtain draft genomes from single cell sequencing. This sd-MDA is promising for flexible and scalable use in single-cell sequencing.

Photobacterium damselae subsp. piscicida is a causative bacterium of fish pasteurellosis, which has caused serious economic damage to aquaculture farms worldwide. Here, the whole-genome sequence of P. damselae subsp. piscicida 91-197, isolated in the United States, suggests that this genome consists of two chromosomes and two plasmids.

Site-specific gene expression analyses are important for understanding tissue functions. Despite rapid developments in DNA-related technologies, the site-specific analysis of whole genome expression for a tissue remains challenging. Thus, a new tool is required for capturing multiple tissue micro-dissections or single cells while retaining the positional information. Here, we describe the development of such a system, which can pick up micro-dissections by punching a tissue repeatedly in a very short period, e.g., 5 s/sampling cycle. A photo of the punched tissue provides information on the dissected positions, allowing site-specific gene expression analysis. We demonstrate the site-specific analysis of a frozen tissue slice of mouse brain by analyzing many micro-dissections produced from the tissue at a 300-mu m pitch. The site-specific analysis provided new insights into the gene expression profiles in a tissue and on tissue functions. The analysis of site-specific whole genome expression may therefore, open new avenues in life science.

Antimicrobial peptides (AMPs) are important defense molecules of the innate immune system. High levels of AMPs are induced in response to infections to fight pathogens, whereas moderate levels induced by metabolic stress are thought to shape commensal microbial communities at barrier tissues. We expressed single AMPs in adult flies either ubiquitously or in the gut by using the inducible GeneSwitch system to tightly regulate AMP expression. We found that activation of single AMPs, including Drosocin, resulted in a significant extension of Drosophila lifespan. These animals showed reduced activity of immune pathways over lifetime, less intestinal regenerative processes, reduced stress response and a delayed loss of gut barrier integrity. Furthermore, intestinal Drosocin induction protected the animals against infections with the natural Drosophila pathogen Pseudomonas entomophila, whereas a germ-reduced environment prevented the lifespan extending effect of Drosocin. Our study provides new insights into the crosstalk of innate immunity, intestinal homeostasis and ageing.

A new technology employing Raman spectroscopy is attracting attention as a powerful biochemical technique for the detection of beneficial and functional food nutrients, such as carotenoids and unsaturated fatty acids. This technique allows for the dynamic characterization of food nutrient substances for the rapid determination of food quality. In this study, we attempt to detect and measure astaxanthin from salmon fillets using this technology. The Raman spectra showed specific bands corresponding to the astaxanthin present in salmon and the value of astaxanthin (Raman band, 1518 cm(-1)) relative to those of protein/lipid (Raman band, 1446 cm(-1)) in the spectra increased in a dose-dependent manner. A standard curve was constructed by the standard addition method using astaxanthin as the reference standard for its quantification by Raman spectroscopy. The calculation formula was established using the Raman bands typically observed for astaxanthin (i.e., 1518 cm(-1)). In addition, we examined salmon fillets of different species (Atlantic salmon, coho salmon, and sockeye salmon) and five fillets obtained from the locations (from the head to tail) of an entire Atlantic salmon. Moreover, the sockeye salmon fillet exhibited the highest astaxanthin concentration (14.2 mg/kg), while coho salmon exhibited an intermediate concentration of 7.0 mg/kg. The Raman-based astaxanthin concentration in the five locations of Atlantic salmon was more strongly detected from the fillet closer to the tail. From the results, a rapid, convenient Raman spectroscopic method was developed for the detection of astaxanthin in salmon fillets.

Influenza vaccines of H7N9 subtype are consistently less immunogenic in humans than vaccines developed for other subtypes. Although prior immunoinformatic analysis identified T-cell epitopes in H7 hemagglutinin (HA) which potentially enhance regulatory T cell response due to conservation with the human genome, the links between the T-cell epitopes and low immunogenicity of H7 HA remains unknown due to the lack of animal models reproducing the response observed in humans. Here, we utilized a humanized mouse model to recapitulate the low immunogenicity of H7 HA. Our analysis demonstrated that modification of a single H7 epitope by changing 3 amino acids so that it is homologous with a known H3 immunogenic epitope sequence significantly improved the immunogenicity of the H7 HA in the humanized mouse model, leading to a greater than 4-fold increase in HA-binding IgG responses. Thus, we provide experimental evidence for the important contribution of this H7-specific T cell epitope in determining the immunogenicity of an influenza vaccine. Furthermore, this study delineates strategies that can be used for screening and selecting vaccine strains using immunoinformatics tools and a humanized mouse model.

Background: Whole genome amplification techniques have enabled the analysis of unexplored genomic information by sequencing of single-amplified genomes (SAGs). Whole genome amplification of single bacteria is currently challenging because contamination often occurs in experimental processes. Thus, to increase the confidence in the analyses of sequenced SAGs, bioinformatics approaches that identify and exclude non-target sequences from SAGs are required. Since currently reported approaches utilize sequence information in public databases, they have limitations when new strains are the targets of interest. Here, we developed a software SAG-QC that identify and exclude non-target sequences independent of database.
Results: In our method, "no template control" sequences acquired during WGA were used. We calculated the probability that a sequence was derived from contaminants by comparing k-mer compositions with the no template control sequences. Based on the results of tests using simulated SAG datasets, the accuracy of our method for predicting non-target sequences was higher than that of currently reported techniques. Subsequently, we applied our tool to actual SAG datasets and evaluated the accuracy of the predictions.
Conclusions: Our method works independently of public sequence information for distinguishing SAGs from non-target sequences. This method will be effective when employed against SAG sequences of unexplored strains and we anticipate that it will contribute to the correct interpretation of SAGs.

Family B DNA polymerases comprise polymerase and 3' - &gt; 5' exonuclease domains, and detect a mismatch in a newly synthesized strand to remove it in cooperation with Proliferating cell nuclear antigen (PCNA), which encircles the DNA to provide a molecular platform for efficient protein-protein and protein-DNA interactions during DNA replication and repair. Once the repair is completed, the enzyme must stop the exonucleolytic process and switch to the polymerase mode. However, the cue to stop the degradation is unclear. We constructed several PCNA mutants and found that the exonuclease reaction was enhanced in the mutants lacking the conserved basic patch, located on the inside surface of PCNA. These mutants may mimic the Pol/PCNA complex processing the mismatched DNA, in which PCNA cannot interact rigidly with the irregularly distributed phosphate groups outside the dsDNA. Indeed, the exonuclease reaction with the wild type PCNA was facilitated by mismatched DNA substrates. PCNA may suppress the exonuclease reaction after the removal of the mismatched nucleotide. PCNA seems to act as a "brake" that stops the exonuclease mode of the DNA polymerase after the removal of a mismatched nucleotide from the substrate DNA, for the prompt switch to the DNA polymerase mode.

Background: Acyclovir (ACV)-resistant (ACVr) herpes simplex virus type 1 (HSV-1) infections are concern in immunocompromised patients. Most clinical ACVr HSV-1 isolates have mutations in the viral thymidine kinase (vTK) genes. The vTK-associated ACVr HSV-1 shows reduced virulence, but the association between the level of resistance and the virulence of the vTK-associated ACVr HSV-1 is still unclear.
Methods: The virulence in mice of 5 vTK-associated ACVr HSV-1 clones with a variety of ACV sensitivities, when inoculated through intracerebral and corneal routes, was evaluated in comparison with ACV-sensitive (ACVs) parent HSV-1 TAS.
Results: Although all the 5 ACVr HSV-1 clones and ACVs HSV-1 TAS showed a similar single-step growth capacity in vitro, the virulence of ACVr HSV-1 clones significantly decreased. A 50% lethal dose (LD50) of each clone was closely correlated with 50% inhibitory concentrations (IC50), demonstrating that the higher the ACV-sensitvity, the the higher the virulence among the ACVr clones. One of the ACVr HSV-1 clones with a relatively low IC50 value maintained similar virulence to that of the parent TAS. The infection in mice with ACVr HSV-1 due to a single amino acid substitution in vTK induced local diseases, keratitis and dermatitis, while vTK-deficient clone did not.
Conclusions: A statistically significant correlation between the virulence and susceptibility to ACV among ACVr HSV-1 clones was demonstrated.

Pseudotuberculosis caused by infection of Photobacterium damselae subsp. piscicida has caused serious economic damages to aquaculture farms worldwide. Here, the whole-genome sequence of P. damselae subsp. piscicida strain OT- 51443, isolated in Japan, was determined and suggests that this genome consists of two chromosomes and five plasmids.

Homeostatic proliferation (HSP) is a major mechanism by which long-lived naive and memory CD4(+) T cells are maintained in vivo and suggested to contribute to the persistence of the latent HIV-1 reservoir. However, while many in vitro latency models rely on CD4(+) T cells that were initially differentiated via T-cell receptor (TCR) stimulation into memory/effector cells, latent infection of naive resting CD4(+) T cells maintained under HSP conditions has not been fully addressed. Here, we describe an in vitro HSP culture system utilizing the cytokines IL-7 and IL-15 that allows studying latency in naive resting CD4(+) T cells. CD4(+) T cells isolated from several healthy donors were infected with HIV pseudotypes expressing GFP and cultured under HSP conditions or TCR conditions as control. Cell proliferation, phenotype, and GFP expression were analyzed by flow cytometry. RNA expression was quantified by qRT-PCR. Under HSP culture conditions, latently HIV-1 infected naive cells are in part maintained in the non-dividing (= resting) state. Although a few HIV-1 provirus(+) cells were present in these resting GFP negative cells, the estimated level of GFP transcripts per infected cell seems to indicate a block at the post-transcriptional level. Interestingly, neither TCR nor the prototypic HDAC inhibitor SAHA were able to reactivate HIV-1 provirus from these cells. This lack of reactivation was not due to methylation of the HIV LTR. These results point to a mechanism of HIV control in HSP-cultured resting naive CD4(+) T cells that may be distinct from that in TCR-stimulated memory/effector T cells.

Directly acting antivirals recently have become available for the treatment of hepatitis C virus (HCV) infection, but there is no prophylactic vaccine for HCV. In the present study, we took advantage of the properties of Japanese encephalitis virus (JEV) to develop antigens for use in a HCV vaccine. Notably, the surface-exposed JEV envelope protein is tolerant of inserted foreign epitopes, permitting display of novel antigens. We identified 3 positions that permitted insertion of the HCV E2 neutralization epitope recognized by HCV1 antibody. JEV subviral particles (SVP) containing HCV-neutralization epitope (SVP-E2) were purified from culture supernatant by gel chromatography. Sera from mice immunized with SVP-E2 inhibited infection by JEV and by trans-complemented HCV particles (HCVtcp) derived from multi-genotypic viruses, whereas sera from mice immunized with synthetic E2 peptides did not show any neutralizing activity. Furthermore, sera from mice immunized with SVP-E2 neutralized HCVtcp with N415K escape mutation in E2. As with the SVP-E2 epitope-displaying particles, JEV SVPs with HCV E1 epitope also elicited neutralizing antibodies against HCV. Thus, this novel platform harboring foreign epitopes on the surface of the particle may facilitate the development of a bivalent vaccine against JEV and other pathogens.

Alginate-degrading bacteria play an important role in alginate degradation by harboring highly efficient and unique alginolytic genes. Although the general mechanism for alginate degradation by these bacteria is fairly understood, much is still required to fully exploit them. Here, we report the isolation of a novel strain, Falsirhodobacter sp. alg1, the first report for an alginate-degrading bacterium from the family Rhodobacteraceae. Genome sequencing reveals that strain alg1 harbors a primary alginate degradation pathway with only single homologs of an endo-and exo-type alginate lyase, AlyFRA and AlyFRB, which is uncommon among such bacteria. Subsequent functional analysis showed that both enzymes were extremely efficient to depolymerize alginate suggesting evolutionary interests in the acquirement of these enzymes. The exo-type alginate lyase, AlyFRB in particular could depolymerize alginate without producing intermediate products making it a highly efficient enzyme for the production of 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH). Based on our findings, we believe that the discovery of Falsirhodobacter sp. alg1 and its alginolytic genes hints at the potentiality of a more diverse and unique population of alginate-degrading bacteria.

As a sensor of polyaromatic chemicals the aryl hydrocarbon receptor (AhR) exerts an important role in immune regulation besides its requirement for xenobiotic metabolism. Transcriptional activation of AhR target genes is counterregulated by the AhR repressor (AhRR) but the exact function of the AhRR in vivo is currently unknown. We here show that the AhRR is predominantly expressed in immune cells of the skin and intestine, different from other AhR target genes. Whereas AhRR antagonizes the anti-inflammatory function of the AhR in the context of systemic endotoxin shock, AhR and AhRR act in concert to dampen intestinal inflammation. Specifically, AhRR contributes to the maintenance of colonic intraepithelial lymphocytes and prevents excessive IL-1 beta production and Th17/Tc17 differentiation. In contrast, the AhRR enhances IFN-gamma-production by effector T cells in the inflamed gut. Our findings highlight the physiologic importance of cell-type specific balancing of AhR/AhRR expression in response to microbial, nutritional and other environmental stimuli.

Metagenome research has brought much attention for the identification of important and novel genes of industrial and pharmaceutical value. Here, using a metagenome library constructed from bacteria associated with the marine sponge, Styllisa massa, a high-throughput screening technique using radioisotope was implemented to screen for cadmium (Cd) binding or accumulation genes. From a total of 3301 randomly selected clones, a clone 247-11C was identified as harboring an open reading frame (ORF) showing Cd accumulation characteristics. The ORF, termed as ORF5, was further analyzed by protein functional studies to reveal the presence of a protein, Cdae-1. Cdae-1, composed of a signal peptide and domain harboring an E(G/A)KCG pentapeptide motif, enhanced Cd accumulation when expressed in Escherichia colt. Although showing no direct binding to Cd in vitro, the presence of important amino acid residues related to Cd detoxification suggests that Cdae-1 may possess a different mechanism from known Cd binding proteins such as metallothioneins (MTs) and phytochelatins (PCs). In summary, using the advantage of bacterial metagenomes, our findings in this work suggest the first report on the identification of a unique protein involved in Cd accumulation from bacteria associated with a marine sponge. (C) 2015 Elsevier B.V. All rights reserved.

Introduction: Recently, we reported that intranasal vaccination of humans with whole inactivated influenza vaccine in the absence of mucosal adjuvant induced neutralizing antibody responses in the serum and nasal mucus. The mucoadhesive excipient carboxy-vinyl polymer (CVP) increases the viscosity and therefore mucoadhesiveness of intranasal medicaments and is an authorized excipient in Japan. In the present study, we analyzed the effect of adding CVP on intranasal whole inactivated influenza vaccine antigen dynamics and antibody responses.
Methods: Mice and nonhuman primates (NHPs) were intranasally administered the [F-18]-radiolabeled vaccine and subjected to positron emission tomography analysis for 6 h. Dendritic cells were stimulated in vitro with the vaccine mixed with or without a mucosal adjuvant (Ampligen) and/or CVP, after which the tumor necrosis factor (TNF)-alpha and interferon (IFN)-beta levels in the supernatants were measured. Cynomolgus monkeys were immunized intranasally with the vaccine mixed with Ampligen and/or CVP and their vaccine-specific serum IgG and IgA titers were measured on days 0 and 33.
Results: The vaccine was retained significantly longer in the nasal cavity of both mice and NHPs when it was delivered with CVP rather than PBS. Accumulation of the radiolabeled vaccine in the central nervous system was not detected in either model regardless of whether CVP was used. CVP only very weakly increased the TNF-alpha production of vaccine-stimulated dendritic cells. IFN-beta production was not observed regardless of the presence or absence of CVP. CVP increased the vaccine-specific IgA antibody responses of the intranasally vaccinated cynomolgus macaques.
Conclusion: CVP increased intranasal retention of whole inactivated influenza vaccine, did not promote antigen redirection to the central nervous system, and improved mucosal antibody responses. The mechanism probably relates to its mucoadhesive properties rather than its ability to directly stimulate the immune system. Intranasal vaccines with CVP may be a promising candidate vaccine formulation for humans. (C) 2016 Elsevier Ltd. All rights reserved.

Acute hepatopancreatic necrosis disease (AHPND), also called early mortality syndrome (EMS), is a recently emergent shrimp bacterial disease that has resulted in substantial economic losses since 2009. AHPND is known to be caused by strains of Vibrio parahaemolyticus that contain a unique virulence plasmid, but the pathology of the disease is still unclear. In this study, we show that AHPND-causing strains of V. parahaemolyticus secrete the plasmid encoded binary toxin PirAB(vp) into the culture medium. We further determined that, after shrimp were challenged with AHPND-causing bacteria, the bacteria initially colonized the stomach, where they started to produce PirAB(vp) toxin. At the same early time point (6 hpi), PirB(vp) toxin, but not PirA(vp) toxin, was detected in the hepatopancreas, and the characteristic histopathological signs of AHPND, including sloughing of the epithelial cells of the hepatopancreatic tubules, were also seen. Although some previous studies have found that both components of the binary PirAB(vp) toxin are necessary to induce a toxic effect, our present results are consistent with other studies which have suggested that PirB(vp) alone may be sufficient to cause cellular damage. At later time points, the bacteria and PirA(vp) and PirB(vp) toxins were all detected in the hepatopancreas. We also show that Raman spectroscopy "Whole organism fingerprints" were unable to distinguish between AHPND-causing and non-AHPND causing strains. Lastly, by using minimum inhibitory concentrations, we found that both virulent and non-virulent V. parahaemolyticus strains were resistant to several antibiotics, suggesting that the use of antibiotics in shrimp culture should be more strictly regulated. (C) 2015 Elsevier Ltd. All rights reserved.

Actin-targeting macrolides comprise a large, structurally diverse group of cytotoxins isolated from remarkably dissimilar micro- and macroorganisms. In spite of their disparate origins and structures, many of these compounds bind actin at the same site and exhibit structural relationships reminiscent of modular, combinatorial drug libraries. Here we investigate biosynthesis and evolution of three compound groups: misakinolides, scytophycin-type compounds and luminaolides. For misakinolides from the sponge Theonella swinhoei WA, our data suggest production by an uncultivated 'Entotheonella' symbiont, further supporting the relevance of these bacteria as sources of bioactive polyketides and peptides in sponges. Insights into misakinolide biosynthesis permitted targeted genome mining for other members, providing a cyanobacterial luminaolide producer as the first cultivated source for this dimeric compound family. The data indicate that this polyketide family is bacteria-derived and that the unusual macrolide diversity is the result of combinatorial pathway modularity for some compounds and of convergent evolution for others.

Whole genome amplification (WGA) is essential for obtaining genome sequences from single bacterial cells because the quantity of template DNA contained in a single cell is very low. Multiple displacement amplification (MDA), using Phi29 DNA polymerase and random primers, is the most widely used method for single-cell WGA. However, single-cell MDA usually results in uneven genome coverage because of amplification bias, background amplification of contaminating DNA, and formation of chimeras by linking of non-contiguous chromosomal regions. Here, we present a novel MDA method, termed droplet MDA, that minimizes amplification bias and amplification of contaminants by using picoliter-sized droplets for compartmentalized WGA reactions. Extracted DNA fragments from a lysed cell in MDA mixture are divided into 10(5) droplets (67 pL) within minutes via flow through simple microfluidic channels. Compartmentalized genome fragments can be individually amplified in these droplets without the risk of encounter with reagent-borne or environmental contaminants. Following quality assessment of WGA products from single Escherichia coli cells, we showed that droplet MDA minimized unexpected amplification and improved the percentage of genome recovery from 59% to 89%. Our results demonstrate that microfluidic-generated droplets show potential as an efficient tool for effective amplification of low-input DNA for single-cell genomics and greatly reduce the cost and labor investment required for determination of nearly complete genome sequences of uncultured bacteria from environmental samples.

Bacterial xylose isomerases (XI) are promising resources for efficient biofuel production from xylose in lignocellulosic biomass. Here, we investigated xylose isomerase gene (xylA) diversity in three soil metagenomes differing in plant vegetation and geographical location, using an amplicon pyrosequencing approach and two newly-designed primer sets. A total of 158,555 reads from three metagenomic DNA replicates for each soil sample were classified into 1127 phylotypes, detected in triplicate and defined by 90% amino acid identity. The phylotype coverage was estimated to be within the range of 84.0-92.7%. The xylA gene phylotypes obtained were phylogenetically distributed across the two known xylA groups. They shared 49-100% identities with their closest-related XI sequences in GenBank. Phylotypes demonstrating &lt;90% identity with known Xis in the database accounted for 89% of the total xylA phylotypes. The differences among xylA members and compositions within each soil sample were significantly smaller than they were between different soils based on a UniFrac distance analysis, suggesting soil-specific xylA genotypes and taxonomic compositions. The differences among xylA members and their compositions in the soil were strongly correlated with 16S rRNA variation between soil samples, also assessed by amplicon pyrosequencing. This is the first report of xylA diversity in environmental samples assessed by amplicon pyrosequencing. Our data provide information regarding xylA diversity in nature, and can be a basis for the screening of novel xylA genotypes for practical applications. 2015, The Society for Biotechnology, Japan. All rights reserved.

This paper proposes a high-throughput, function-based screening approach of a metagenomic library for isolating novel microbial enzymes by droplet-based microfluidics. We used gel microdroplets (GMDs) dispersed in oil as picoliter-volume reaction vessels for lipolytic enzyme by encapsulating cells in individual GMDs. Using this approach, we monitored the growth of individual cells encapsulated in GMDs and assessed the enzyme reaction activities at the level of an individual GMD. We then applied this method to screen lipolytic enzyme genes from the metagenomic library constructed from soil collected from a quercus serrate forest of Mount Tsukuba, Ibaraki, Japan. In the workflow presented in this study, metagenomic library clones were encapsulated in 100-pL GMDs with a fluorogenic reporter substrate. A total of 67,000 metagenomic library clones can be screened in only 24 h with reduced consumption of reagents (i.e., &lt; 10 mu L). As a result, we identified a novel lipolytic enzyme, EstT1, belonging to the EstD2 family of esterases and containing a putative signal peptide, which facilitates enzyme export and catalyzation of substrates in the periplasm. Our study demonstrates the potential of microfluidic GMDs as an efficient tool for metagenomic library screening of industrially relevant enzymes with the potential of significantly reducing the cost and time factors involved in successful practical application of microbial enzymes. (C) 2014 Elsevier B.V. All rights reserved.

Bacteria are known to be highly diverse and unique to the various environments they reside in. Covering more than 70% of the earth’s surface, marine bacterial ecosystems in particular have long been regarded as reservoirs for novel and unique genes important to industry and pharmaceutics. In the first part of the chapter, we reviewed the importance and potential of bacteria from marine environments as an important genetic resource and some of the recent efforts in the implementation of marine metagenomic research to screen for genes applicable in bioprocesses, bioremediation and bioethanol production. Nevertheless, metagenomic research has also provided new challenges that will need to be addressed in order to use these resources efficiently. Here, in the second part of the chapter, we introduced several supporting technologies that show great potential in assisting metagenomic research to overcome such challenges including high-throughput screening using microfluidics, single-cell analysis and in sil-ico data mining of metagenomic data. The introduction of such technologies, with metagenomic research, does not only allowed us to exploit these genetic resources to the fullest but may also provide new perspectives and insights towards living organisms and natural ecosystems.

We developed a titanium-binding-peptide-1 (TBP-1)-tagged DNA polymerase, for self-oriented immobilization onto a titanium oxide (TiO2) substrate. The enzymatic function of a polymerase immobilized on a solid state device is strongly dependent on the orientation of the enzyme. The TBP-tagged DNA polymerase, which was derived from a hyperthermophilic archaeon, was designed to incorporate the RKLPDA peptide at the N-terminus, and synthesized by translation processes in Escherichia coli (E. coli). The specific binding of the TBP-tagged DNA polymerase onto a TiO2 substrate was clearly monitored by surface plasmon resonance spectroscopy (SPR) and by surface potential detection with an extended-gate field effect transistor (FET). In the SPR analyses, constant quantities of the DNA polymerase were stably immobilized on the titanium substrate under flow conditions, regardless of the concentration of the DNA polymerase, and could be completely removed by a 4 M MgCl2 wash after measurement. The FET signal showed the contribution of the molecular charge in the TBP motif to the binding with TiO2. In addition, the TBP-tagged DNA polymerase-tethered TiO2 gate electrode enabled the effective detection of the positive charges of hydrogen ions produced by the DNA extension reaction, according to the FET principle. Therefore, the self-oriented immobilization platform based on the motif-inserted enzyme is suitable for the quick and stable immobilization of functional enzymes on biosensing devices.

Mycobacterium is a genus within the order Actinomycetales that comprises of a large number of well-characterized species, several of which includes pathogens known to cause serious disease in human and animal. Here, we report the whole genome sequence of Mycobacterium sp. strain 012931 isolated from the marine fish, yellowtail (Seriola quinqueradiata). Mycobacterium sp. 012931 is a fish pathogen causing serious damage to aquaculture farms in Japan. DNA dot plot analysis showed that Mycobacterium sp. 012931 was more closely related to Mycobacterium marinum when compared across several Mycobacterium species. However, little conservation of the gene order was observed between Mycobacterium sp. 012931 and M. marinum genome. The annotated 5,464 genes of Mycobacterium sp. 012931 was classified into 26 subsystems. The insertion/deletion gene analysis shows Mycobacterium sp. 012931 had 643 unique genes that were not found in the M. marinum strains. In the virulence, disease, and defense subsystem, both insertion and deletion genes of Mycobacterium sp. 012931 were associated with the PPE gene cluster of Mycobacteria. Of seven plcB genes in Mycobacterium sp. 012931, plcB_2 and plcB_3 showed low identities with those of M. marinum strains. Therefore, Mycobacterium sp. 012931 has differences on genetic and virulence from M. marinum and may induce different interaction mechanisms between host and pathogen.

The study of spatial distribution of secondary metabolites within microbial cells facilitates the screening of candidate strains from marine environments for functional metabolites and allows for the subsequent assessment of the production of metabolites, such as antibiotics. This paper demonstrates the first application of Raman microspectroscopy for in situ detection of the antifungal antibiotic amphotericin B (AmB) produced by actinomycetes-Streptomyces nodosus. Raman spectra measured from hyphae of S. nodosus show the specific Raman bands, caused by resonance enhancement, corresponding to the polyene chain of AmB. In addition, Raman microspectroscopy enabled us to monitor the time-dependent change of AmB production corresponding to the growth of mycelia. The Raman images of S. nodosus reveal the heterogeneous distribution of AmB within the mycelia and individual hyphae. Moreover, the molecular association state of AmB in the mycelia was directly identified by observed Raman spectral shifts. These findings suggest that Raman microspectroscopy could be used for in situ monitoring of antibiotic production directly in marine microorganisms with a method that is non-destructive and does not require labeling.

Cultivated bacteria such as actinomycetes are a highly useful source of biomedically important natural products. However, such 'talented' producers represent only a minute fraction of the entire, mostly uncultivated, prokaryotic diversity. The uncultured majority is generally perceived as a large, untapped resource of new drug candidates, but so far it is unknown whether taxa containing talented bacteria indeed exist. Here we report the single-cell- and metagenomics-based discovery of such producers. Two phylotypes of the candidate genus 'Entotheonella' with genomes of greater than 9 megabases and multiple, distinct biosynthetic gene clusters co-inhabit the chemically and microbially rich marine sponge Theonella swinhoei. Almost all bioactive polyketides and peptides known from this animal were attributed to a single phylotype. 'Entotheonella' spp. are widely distributed in sponges and belong to an environmental taxon proposed here as candidate phylum 'Tectomicrobia'. The pronounced bioactivities and chemical uniqueness of 'Entotheonella' compounds provide significant opportunities for ecological studies and drug discovery.

Falsirhodobacter sp. alg1 is an alginate-degrading bacterium, the second species from the nonphototrophic bacterial genus Falsirhodobacter. We report the first draft genome of a bacterium from this genus and point out possible important features related to alginate assimilation and its evolutionary aspects.

Streptococcus parauberis, which is the main causative agent of streptococcosis among olive flounder (Paralichthys olivaceus) in northeast Asia, can be distinctly divided into two groups (type I and type II) by an agglutination test. Here, the whole genome sequences of two Japanese strains (KRS-02083 and KRS-02109) were determined and compared with the previously determined genome of a Korean strain (KCTC 11537). The genomes of S. parauberis are intermediate in size and have lower GC contents than those of other streptococci. We annotated 2,236 and 2,048 genes in KRS-02083 and KRS-02109, respectively. Our results revealed that the three S. parauberis strains contain different genomic insertions and deletions. In particular, the genomes of Korean and Japanese strains encode different factors for sugar utilization; the former encodes the phosphotransferase system (PTS) for sorbose, whereas the latter encodes proteins for lactose hydrolysis, respectively. And the KRS-02109 strain, specifically, was the type II strain found to be able to resist phage infection through the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system and which might contribute valuably to serologically distribution. Thus, our genome-wide association study shows that polymorphisms can affect pathogen responses, providing insight into biological/biochemical pathways and phylogenetic diversity.

Live attenuated measles virus (MV) has long been recognized as a safe and effective vaccine, and it has served as the basis for development of various MV-based vaccines. However, because MV is a human-tropic virus, the evaluation of MV-based vaccines has been hampered by the lack of a small-animal model. The humanized mouse, a recently developed system in which an immunodeficient mouse is transplanted with human fetal tissues or hematopoietic stem cells, may represent a suitable model. Here, we developed a sensitive one-step quantitative reverse transcription (qRT)-PCR that simultaneously measures nucleocapsid (N) and human RNase P mRNA levels. The results can be used to monitor MV infection in a humanized mouse model. Using this method, we elucidated the replication kinetics of MV expressing enhanced green fluorescent protein both in vitro and in humanized mice in parallel with flow-cytometric analysis. Because our qRT-PCR system was sensitive enough to detect MV expression using RNA extracted from a small number of cells, it can be used to monitor MV infection in humanized mice by sequential blood sampling.

Metabolic homeostasis is fundamental for the development and the survival of animals. It requires the proper functioning of pathways that control the sensing and processing of nutrients, the storage and mobilization of energy. Recent data demonstrate that energy homeostasis and immune responses are tightly connected and that inaccurate metabolic regulation can adversely influence immune functions. Dysfunctions of the immune system have been demonstrated to underlie many chronic metabolic diseases, including diabetes, metabolic syndrome and atherosclerosis. The molecular mechanisms behind the cross-regulation of metabolism and immunity in health and disease are only beginning to emerge. We use the model organism Drosophila melanogaster to gain insights into evolutionary conserved mechanisms by which energy homeostasis and innate immunity interact.

Mycobacterium marinum is difficult to distinguish from other species of Mycobacterium isolated from fish using biochemical methods. Here, we used genetic and proteomic analyses to distinguish three Mycobacterium strains: M. marinum strains MB2 and Europe were isolated from tropical and marine fish in Thailand and Europe, and Mycobacterium sp. 012931 strain was isolated from yellowtail in Japan. In phylogenetic trees based on gyrB, rpoB, and Ag85B genes, Mycobacterium sp. 012931 clustered with M. marinum strains MB2 and Europe, but in trees based on 16S rRNA, hsp65, and Ag85A genes Mycobacterium sp. 012931 did not cluster with the other strains. In proteomic analyses using a Bruker matrix-assisted laser desorption ionization Biotyper, the mass profile of Mycobacterium sp. 012931 differed from the mass profiles of the other two fish M. marinum strains. Therefore, Mycobacterium sp. 012931 is similar to M. marinum but is not the same, suggesting that it could be a subspecies of M. marinum.

Phage display libraries are used to screen for nucleotide sequences that encode immunoglobulin variable (V) regions that are specific for a target antigen. We previously constructed an immunoglobulin new antigen receptor (IgNAR) phage display library. Here we used this library to obtain an IgNAR V region that is specific for viral hemorrhagic septicemia virus (VHSV). A phage clone (clone 653) was found to be specific for VHSV by the biopanning method. The V region of clone 653 was used to construct a 6 × His tagged recombinant IgNAR-653 V protein (rIgNAR-653) using the Escherichia coli pET system. The rIgNAR-653 protein bound specifically to VHSV, confirming its activity. © 2012 Elsevier Ltd.

遺伝子 (mRNA) 発現量がタンパク質合成の量的指標となって久しいが，実際には両者の相関関係は低いことが多い．近年，翻訳活性化状態にある mRNA をリボソームプロファイリングによって詳細に解析することで定量する技術が開発され注目を浴びつつある．そこで本総説では，マイクロアレイなどの遺伝子発現の網羅的解析技術が抱えていた問題点，特に翻訳制御機構を含む転写後調節を考慮していなかった点に言及しつつ，これらの問題がリボソームプロファイリングによって大きく改善されることを概説する．さらに，同技術の特性を生かした新たなバイオマーカーの発見の可能性を探る．<br>

Aeromonas hydrophila is a pathogenic bacterium that has been implicated in fish, animal, and human disease. Recently, a multidrug resistance (MDR) plasmid, pR148, was isolated from A. hydrophila obtained from a tilapia (Oreochromis niloticus) farm in Thailand. pR148 is a 165,906-bp circular plasmid containing 147 coding regions showing highest similarity to pNDM-1-Dok1, an MDR plasmid isolated from a human pathogen. pR148 was also very similar to other IncA/C plasmids isolated from humans, animals, food, and fish. pR148 contains a mercuric resistance operon and encodes the complete set of genes for the type 4 secretion system. pR148 encodes a Tn21 type transposon. This transposon contains the drug resistance genes qacH, blaOXA-10, aadA1, and sul1 in a class 1 integron
tetA and tetR in transposon Tn1721
and catA2 and a duplicate sul1 in a locus showing 100% similarity to IncU plasmids isolated from fish. The blaOXA-10 and aadA1 genes showed 100% similarity to those from the Acinetobacter baumannii AYE genome. The similarity of pR148 to a human pathogen-derived plasmid indicates that the plasmids were either transferred between different genera or that they are derived from a common origin. Previous studies have shown that IncA/C plasmids retain a conserved backbone, while the accessory region points to lateral gene transfer. These observations point out the dangers of indiscriminate use of antibiotics in humans and in animals and the necessity of understanding how drug resistance determinants are disseminated and transferred. Copyright © 2013, American Society for Microbiology. All Rights Reserved.

The genus Mycobacterium comprises a large number of well-characterized species, several of which are human and animal pathogens. Here, we report the whole-genome sequence of Mycobacterium sp. strain 012931, a fish pathogen responsible for huge losses in aquaculture farms in Japan. The strain was isolated from a marine fish, yellowtail (Seriola quinqueradiata).

In innate immunity, LGP2 (laboratory of genetics and physiology 2) plays a very important role in the production of type I interferon (IFN) through recognition of cytosolic viral RNA. Although viral infection or stimulation with double-strand RNA dramatically induces expression of the LGP2 gene, the underlying transcriptional mechanism has never been studied. Here, we cloned and characterized the 5'-upstream region (-1,337 bp) of the LGP2 gene in olive flounder (Paralichthys olivaceus). Numerous canonical motifs for IFN-regulatory factors (IRFs) were found in this region, and reporter assays identified a poly I: C-responsive promoter region (-506 to -398) that regulated LGP2 transcription. Transcriptional activity of the LGP2 promoter was strongly enhanced by IRF3, which bound to IRF3 motif #3 (-480). The LGP2 promoter was also responsive to viral infection in vitro. These results suggest that LGP2 transcriptional control is crucially involved to regulated by IRF3 function after viral infection or stimulation with poly I:C.

Oysters are one of foodstuffs containing a relatively high amount of cadmium. Here we report on establishment of an immunochromatographic assay (ICA) method of cadmium levels in oysters. Cadmium was extracted with 0.1 mol L-1 HCl from oysters and cleaned up from other metals by the use of an anion-exchange column. The behavior of five metals Mn, Fe, Cu, Zn, and Cd was monitored at each step of extraction and clean-up procedure for the ICA method in an inductively coupled plasma-mass spectrometry (ICP-MS) analysis. The results revealed that a simple extraction method with the HCl solution was efficient enough to extract almost all of cadmium from oysters. Clean-up with an anion-exchange column presented almost no loss of cadmium adsorbed on the column and an efficient removal of metals other than cadmium. When a spiked recovery test was performed in the ICA method, the recovery ranged from 98% to 112% with relative standard deviations between 5.9% and 9.2%. The measured values of cadmium in various oyster samples in the ICA method were favorably correlated with those in ICP-MS analysis (r(2)=0.97). Overall results indicate that the ICA method established in the present study is an adequate and reliable detection method for cadmium levels in oysters. (C) 2012 Elsevier B.V. All rights reserved.

The Western Australian rock lobster fishery has been both a highly productive and sustainable fishery. However, a recent dramatic and unexplained decline in post-larval recruitment threatens this sustainability. Our lack of knowledge of key processes in lobster larval ecology, such as their position in the food web, limits our ability to determine what underpins this decline. The present study uses a high-throughput amplicon sequencing approach on DNA obtained from the hepatopancreas of larvae to discover significant prey items. Two short regions of the 18S rRNA gene were amplified under the presence of lobster specific PNA to prevent lobster amplification and to improve prey amplification. In the resulting sequences either little prey was recovered, indicating that the larval gut was empty, or there was a high number of reads originating from multiple zooplankton taxa. The most abundant reads included colonial Radiolaria, Thaliacea, Actinopterygii, Hydrozoa and Sagittoidea, which supports the hypothesis that the larvae feed on multiple groups of mostly transparent gelatinous zooplankton. This hypothesis has prevailed as it has been tentatively inferred from the physiology of larvae, captive feeding trials and co-occurrence in situ. However, these prey have not been observed in the larval gut as traditional microscopic techniques cannot discern between transparent and gelatinous prey items in the gut. High-throughput amplicon sequencing of gut DNA has enabled us to classify these otherwise undetectable prey. The dominance of the colonial radiolarians among the gut contents is intriguing in that this group has been historically difficult to quantify in the water column, which may explain why they have not been connected to larval diet previously. Our results indicate that a PCR based technique is a very successful approach to identify the most abundant taxa in the natural diet of lobster larvae.

Polymerase chain reaction (PCR)-clamping using blocking primer and DNA-analogs, such as peptide nucleotide acid (PNA), may be used to selectively amplify target DNA for molecular diet analysis. We investigated PCR-clamping efficiency by studying PNA position and mismatch with complementary DNA by designing PNAs at five different positions on the nuclear rDNA internal transcribed spacer 1 of the Japanese eel Anguilla japonica in association with intra-specific nucleotide substitutions. All five PNAs were observed to efficiently inhibit amplification of a fully complementary DNA template. One mismatch between PNA and template DNA inhibited amplification of the template DNA, while two or more mismatches did not. DNA samples extracted from dorsal muscle and intestine of eight wild-caught leptochephalus larvae were subjected to this analysis, followed by cloning, nucleotide sequence analysis, and database homology search. Among 12 sequence types obtained from the intestine sample, six were identified as fungi. No sequence similarities were found in the database for the remaining six types, which were not related to one another. These results, in conjunction with our laboratory observations on larval feeding, suggest that eel leptocephali may not be dependent upon living plankton for their food source.

The stomach contents of the larvae of marine animals are usually very small in quantity and amorphous, especially in invertebrates, making morphological methods of identification very difficult. Nucleotide sequence analysis using polymerase chain reaction (PCR) is a likely approach, but the large quantity of larval (host) DNA present may mask subtle signals from the prey genome. We have adopted peptide nucleic acid (PNA)-directed PCR clamping to selectively inhibit amplification of host DNA for this purpose. The Japanese spiny lobster (Panulirus japonicus) and eel (Anguilla japonica) were used as model host and prey organisms, respectively. A lobster-specific PNA oligomer (20 bases) was designed to anneal to the sequence at the junction of the 18 S rDNA gene and the internal transcribed spacer 1 (ITS1) of the lobster. PCR using eukaryote universal primers for amplifying the ITS1 region used in conjunction with the lobster-specific PNA on a mixed DNA template of lobster and eel demonstrated successful inhibition of lobster ITS1 amplification while allowing efficient amplification of eel ITS1. This method was then applied to wild-caught lobster larvae of P. japonicus and P. longipes bispinosus collected around Ryukyu Archipelago, Japan. ITS1 sequences of a wide variety of animals (Ctenophora, Cnidaria, Crustacea, Teleostei, Mollusca, and Chaetognatha) were detected.

Using a metagenome library constructed from a bacterial associated with a marine sponge Hyrtios erecta, we identified a novel esterase that belongs to the SGNH hydrolase superfamily of esterases. The substrate specificity of EstHE1 was determined using p-nitrophenyl (pNP) ester (C2: acetate, C4: butylate, C6: caproate, C12: laurate, C16: palmitate). EstHE1 exhibited activity against C2 (5.6 U/mg), C4 (5.1 U/mg), and C6 (2.8 U/mg) substrates. The optimal temperature for EstHE1 esterase activity of the pNP acetate substrate was 40A degrees C, and EstHE1 retained 60% of its enzymatic activity in the 30-50A degrees C range. This esterase showed moderate thermostability, retaining 58% of its activity even after preincubation for 12 h at 40A degrees C. EstHE1 also maintained activity in high concentrations of NaCl, indicating that this esterase is salt-tolerant. Thus, EstHE1 has the thermal stability and salt tolerance necessary for use as an industrial enzyme.

Magnetotactic bacteria are ubiquitous microorganisms that synthesize intracellular magnetite particles (magnetosomes) by accumulating Fe ions from aquatic environments. Recent molecular studies, including comprehensive proteomic, transcriptomic, and genomic analyses, have considerably improved our hypotheses of the magnetosome-formation mechanism. However, most of these studies have been conducted using pure-cultured bacterial strains of alpha-proteobacteria. Here, we report the whole-genome sequence of Desulfovibrio magneticus strain RS-1, the only isolate of magnetotactic microorganisms classified under delta-proteobacteria. Comparative genomics of the RS-1 and four alpha-proteobacterial strains revealed the presence of three separate gene regions (nuo and mamAB-like gene clusters, and gene region of a cryptic plasmid) conserved in all magnetotactic bacteria. The nuo gene cluster, encoding NADH dehydrogenase (complex I), was also common to the genomes of three iron-reducing bacteria exhibiting uncontrolled extracellular and/or intracellular magnetite synthesis. A cryptic plasmid, pDMC1, encodes three homologous genes that exhibit high similarities with those of other magnetotactic bacterial strains. In addition, the mamAB-like gene cluster, encoding the key components for magnetosome formation such as iron transport and magnetosome alignment, was conserved only in the genomes of magnetotactic bacteria as a similar genomic island-like structure. Our findings suggest the presence of core genetic components for magnetosome biosynthesis; these genes may have been acquired into the magnetotactic bacterial genomes by multiple gene-transfer events during proteobacterial evolution.

Detection and isolation of specific cell types from limited biological samples have become a major challenge in clinical diagnosis and cell biology research. Here, we report a high-density microcavity array for target cell detection in which thousands of single cells were neatly arrayed onto 10000 microcavities with high efficiency at approximately 90% of the loaded cells. Cell-specific immunophenotypes were exclusively identified at the single-cell level by measuring fluorescence intensities of cells labeled with antibodies targeting cell surface markers, and the purity of hematopoietic stem cells (HSCs) within human peripheral blood analyzed by this system was correlated with those obtained by conventional flow cytometry. Furthermore, gene expression of the stem cell marker, CD34, was determined from HSCs by isolating single cells using a micromanipulator. This technology has proven to be an effective tool for target cell detection and subsequent cellular analytical research at the single-cell level.

Length and guanine-cytosine (GC) content of the ribosomal first internal transcribed spacer (ITS1) were compared across a wide variety of marine animal species, and its phylogenetic utility was investigated. From a total of 773 individuals representing 599 species, we only failed to amplify the ITS1 sequence from 87 individuals by polymerase chain reaction with universal ITS1 primers. No species was found to have an ITS1 region shorter than 100 bp. In general, the ITS1 sequences of vertebrates were longer (318 to 2,318 bp) and richer in GC content (56.8% to 78%) than those of invertebrates (117 to 1,613 bp and 35.8% to 71.3%, respectively). Specifically, gelatinous animals (Cnidaria and Ctenophora) were observed to have short ITS1 sequences (118 to 422 bp) with lower GC content (35.8% to 61.7%) than the other animal taxa. Mollusca and Crustacea were diverse groups with respect to ITS1 length, ranging from 108 to 1,118 and 182 to 1,613 bp, respectively. No universal relationship between length and GC content was observed. Our data indicated that ITS1 has a limited utility for phylogenetic analysis as obtaining confident sequence alignment was often impossible between different genera of the same family and even between congeneric species. © 2008 Springer Science+Business Media, LLC.

There is a high demand for inexpensive and high-throughput DNA sequencing technologies in molecular biology and applied biosciences. In this study, novel nano-sized magnetic particles displaying enzymes for pyrosequencing, a rather novel bioluminometric DNA sequencing method based on the sequencing-by-synthesis principle by employing a cascade of several enzymatic reactions, was developed. A highly thermostable enzyme, pyruvate phosphate dikinase (PPDK) which converts PPi to ATP was successfully expressed onto bacterial magnetic particles (BacMPs) using a novel protein display system of Magnetospirillum magneticum AMB-1. The enzymatic stability of BacMPs displaying PPDK (PPDK-BacMPs) to pH and temperature was evaluated and its broad range of properties was shown. Subsequently, PPDK-BacMPs were applied in pyrosequencing and a target oligonucleotide was successfully sequenced. The PPDK enzyme displayed on BacMPs was shown to be recyclable in each sequence reaction as they can be manipulated by magnetic force. It was concluded that nano-sized PPDK-BacMPs are useful for the scale down of pyrosequencing reaction volumes, thus, permitting high-throughput. The recycling of enzymes was also shown to be promising and applicable for the development of an inexpensive DNA sequencing at a low running cost.

Specific molecular events, characteristic of each cell-cycle phase may have direct effect to the functionality of nuclear receptors. Based on this understanding, the evaluation of lipophilic chemicals at the different cell-cycle phases is significant and should be considered. In order to achieve the aim of performing large-scale dose-response analysis on the effects of lipophilic chemicals at the different cell-cycle phases, a stable, sensitive and highly selective human progesterone receptor (hPR) expressing HeLa reporter cell line, hPRLuc-20, was established. Upon the establishment of the hPRLuc-20 cells, they were synchronized to the G(1), S and G(2) phases and treated with progesterone (PROG) and promegestone (R5020). The cells successfully showed that at the different cell-cycle phase, both agonists resulted in different cellular responses. The differences in response supports that hPR expressed within the hPRLuc-20 cells do respond in a cell-cycle dependent manner, thus showing the cells&apos; compatibility in large-scale dose-response analyses of chemicals. It is hopeful that the advanced application of the hPRLuc-20 cells could contribute to provide fundamental hints to further understand the function of hPR, and provide key observations to elucidate the nature of these chemicals with hPR, its corresponding co-regulators and transcription factors. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Direct separation of target cells from mixed population, such as peripheral blood, umbilical cord blood, and bone marrow, is an essential technique for various therapeutic or diagnosis applications. In this study, novel particles were fabricated, and direct magnetic separation of immune cells from whole blood using such particles was performed. The magnetotactic bacterium Magnetospirillum magneticum AMB-1 synthesizes intracellular bacterial magnetic particles (BacMPs), and protein G was expressed on the surface of the BacMPs by gene fusion techniques with anchor proteins isolated from BacMP membrane. The BacMPs displaying protein G (protein G-BacMPs) had high binding capabilities to a wide range of antibody types, and various versions of protein G-BacMPs binding with different anti-CD monoclonal antibodies were constructed. Consequently, direct magnetic separation of immune cells from whole blood using protein G-BacMPs binding with anti-CD monoclonal antibodies was demonstrated. B lymphocytes (CD19(+) cells) or T lymphocytes (CD3(+) cells), which represent less than 0.05% in whole blood cells, were successfully separated at a purity level of more than 96%. This level was superior to that from previous reports using other magnetic separation approaches. The results of this study demonstrate the utility of protein G-BacMP and this particle may become a powerful tool for various therapeutic or diagnosis applications. 0 2009 American Institute of Chemical Engineers Biotechnol. Prog., 25: 219-226, 2009

Hemangioblasts are common progenitors of hematopoietic and angiogenic cells, which have been demonstrated in the mouse to possess a unique cell surface marker, podocalyxin-like protein 1 (PCLP1) (Hara, T. et al., Immunity, 11: 567-578. 1999). In this study, we prepared a novel monoclonal antibody against human PCLP1 (hPCLP1) and attempted to isolate human hematopoietic progenitor cells from umbilical cord blood and peripheral blood using nano-sized bacterial magnetic particles (BacMPs) coupled with the anti-hPCLP1 antibody. Flow cytometric analysis demonstrated that the purity of separated hPCLP1-positive cells from peripheral blood was approximately 95% whereas peripheral blood mononuclear cells contained only 0.1% PCLP1(+) cells. Umbilical cord blood was demonstrated to be a better source for PCLP1(+) cells than peripheral blood. These results suggest that the separation of human PCLP1(+) cells using BacMPs with anti-hPCLP1 were extremely effective and may be useful as a means to prepare human hematopoietic progenitor cells.

Magnetic nanoparticles produced by magnetotactic bacterium, bacterial magnetic particles (BacMPs), covered with it lipid bilayer membrane (magnetosome membrane) can be used to separate specific target cells from heterogeneous mixtures because they are easily manipulated by magnets and it is easy to display functional proteins on their surface via genetic engineering. Despite possessing unique and valuable characteristics, the potential toxicity of BacMPs to the separated cells has not been characterized in detail. Here, a novel technique was developed for the reconstruction of magnetosome membrane of BacMPs expressing protein A (protein A-BacMPs) to reduce cytotoxicity and the newly developed nanomaterial was then used for magnetic cell separation. The development of the magnetosome membrane-reconstructed protein A-BacMP was based on the characteristics of the Mms13 anchor protein, which strongly binds to the magnetite surface of BacMPs. Treatment of protein A-BacMPs with detergents removed contaminating protein but did not affect retention of Mms13-protein A fusion proteins. The particle surfaces were then reconstructed with phosphatidylcholine. The protein A-BacMPs containing reconstructed magnetosome membranes remained dispersible and retained the ability to immobilize antibody. In addition, they contained few membrane surface proteins and endotoxins, which were observed on non-treated protein A-BacMPs. Magnetic separation of monocytes and B-lymphocytes from the peripheral blood was achieved with high purity using magnetosome membrane-reconstructed protein A-BacMPs.

We established a novel method to evaluate endocrine disrupting chemicals (EDCs) by assembling the estrogen receptor-ligand binding domain (ERLBD) and GFP labeled coactivator on magnetic nanoparticles. EDC can promote or inhibit coactivator recruitment to the ligand-ERLBD complex. ERLBD was displayed on the surface of nano-sized bacterial magnetic particles (BacMPs) produced by the magnetic bacterium, Magnetospirillum magneticum AMB-1. Our method resulted in 38 molecules of ERLBD molecules on a BacMPs with diameter of 75 nm. Furthermore, ligand-dependent recruitment assays of GFP labeled coactivator to ERLBD-BacMPs was performed by measuring the fluorescence intensity. 17 beta-estradiol (E2), estriol, diethylstilbestrol, zeralenone (full agonist), octylphenol (partial agonist) and ICI 182,780 (antagonist) were evaluated by this method. Full agonists tested showed increased fluorescence with increasing agonist concentration. Octylphenol had lower fluorescence intensity than E2. ICT 182,780 did not produce any fluorescence. The method developed in this study can evaluate the estrogenic potential of chemicals by discriminating whether they are an ER full agonist, partial agonist, or antagonist, Finally, this method is amenable adaptation into a high throughput format by using automated magnetic separation. (C) 2008 Elsevier B.V. All rights reserved.

Here, we report a high-efficiency single-cell entrapment system with a poly(dimethylsiloxane) (PDMS) microfluidic device integrated with a micromesh, and its application to single-cell fluorescence in situ hybridization (FISH) analysis. A micromesh comprising of 10 x 10 microcavities was fabricated on a black poly(ethylene terephthalate) (PET) substrate by laser ablation. The cavity was approximately 2 mu m in diameter. Mammalian cells were driven and trapped onto the microcavities by applying negative pressure. Trapped cells were uniformly arrayed on the micromesh, enabling high-throughput microscopic analysis. Furthermore, we developed a method of PDMS surface modification by using air plasma and the copolymer Pluronic F-127 to prevent nonspecific adsorption on the PDMS microchannel. This method decreased the nonspecific adsorption of cells onto the microchannel to less than 1%. When cells were introduced into the microfluidic device integrated with the black PET micromesh, approximately 70-80% of the introduced cells were successfully trapped. Moreover, for mRNA expression analysis, on-chip fluorescence in situ hybridization (e.g., membrane permeabilization, hybridization, washing) can be performed in a microfluidic assay on an integrated device. Ibis microfluidic device has been employed for the detection of beta-actin mRNA expression in individual Raji cells. Differences in the levels of beta-actin mRNA expression were observed in serum-supplied or serum-starved cell populations.

Mammalian cell models for gene knock-out/knock-in experiments are important for functional analysis of genes and have a potential of useful tool for toxicological studies. However, uncontrolled insertion of transgenes has raised significant concerns over unwanted side effects. To address this issue, we established a stable HeLa55 cell line capable of site-specific transgenesis by means of Cre-mediated cassette exchange at a site on the long arm of human chromosome 9 containing no constitutive transcripts. We applied HeLa55 to transgenesis of the green fluorescent protein (GFP) gene based on recombinase-mediated cassette exchange. The transformants stably expressed GFP transgenes, even after cryopreservation, without compromising physiological properties. We produced an RNA interference (RNAi)-inducible knockdown stable cell line against human tumor necrosis factor (TNF) receptor 1, and one cloned stable cell line (TNFRIKD cells) exhibited long-term gene silencing with significant reduction (ca. 85%) and markedly resisted cytotoxicity induced by TNF alpha. Furthermore, xenobiotics were exposed to stable TNFRIKD cells and different cytotoxicity was exhibited based on various toxicological properties. Thus, we showed the feasibility of RNAi-based stable knockdown cells for xenobiotics-induced cytotoxicity, and HeLa55 has wide application for the generation of stable knock-in and knock-down cells mediated by RNAi. (c) 2009 Elsevier Ltd. All rights reserved.

Genomic recombination was performed in a genetically modified stable HeLa cell line, HeLa55, using a uniquely designed donor vector harboring an exchange cassette comprised of the human glucocorticoid receptor (hGR) gene, its response element, and a luciferase reporter gene, to generate stable hGRLuc clones. After screening for cassette insertion, the selected stable clone, hGRLuc-7, showed high integration stability of the exchange cassette over 20 passages with significantly high luciferase activity and fold inductions of up to 40- to 50-fold. In addition, the cells were evaluated with synthetic glucocorticoid, dexamethasone, and a reasonable EC50 value of approximately 2.3 x 10(-9) M was obtained. Strong and weak agonists, non-responsive chemicals, and hGR antagonists were also evaluated in which the stable hGRLuc-7 clone showed both high sensitivity and selectivity. The technology presented in this work is simple and reproducible, and shows great potential for the future development of genetically modified stable cell systems which are applicable in both fundamental and application researches of nuclear receptors.

To clarify the natural diet of phyllosoma larvae of the Japanese spiny lobster Panulirus japonicus, the sources of 18S rDNA clones obtained from the hepatopancreas were investigated. Of a total of 1537 clones examined, 160 had different restriction profiles from the host larvae, in which 21 restriction types were observed. Nucleotide sequences of 16 of 21 restriction types were successfully determined and their assignments were investigated by homology search and phylogenetic analysis. From seven late-stage larvae collected in spring to early summer, eukaryote DNA molecules of Teleostei, Oomycetes, Mycetozoa, and Fungi were identified. Exogenous DNA from four younger phyllosoma larvae collected in late autumn could not be recovered. A previous study identified DNAs of cnidarians and urochordates in late-stage phyllosoma larvae of a closely related species collected in winter. This indicates that the phyllosoma larvae are opportunistic carnivores, whose diets correlate with the relative abundance of prey organisms in the ambient water.

Magnetic beads have served as a conventional bioassay platform in biotechnology. In this study, a fully automated immunoassay was performed using novel nano- and microbead-composites constructed by assembling nano-magnetic beads onto polystyrene microbeads, designated 'Beads on Beads'. Nano-sized bacterial magnetic particles (BacMPs) displaying the immunoglobulin G (IgG)-binding domain of protein A (ZZ domain) were used for the construction of 'Beads on Beads' via the interaction of biotin-streptavidin. The efficient assembly of 'Beads on Beads' was performed by gradual addition of biotin-labeled BacMPs onto streptavidin-coated polystyrene microbeads. Approximately 2000 BacMPs were uniformly assembled on a single microbead without aggregation. The constructed 'Beads on Beads' were magnetized and separated from the suspension by using an automated magnetic separation system with a higher efficiency than BacMPs alone. Furthermore, fully automated detection of prostate-specific antigens was performed with the detection limit of 1.48 ng mL(-1). From this preliminary assay, it can be seen that 'Beads on Beads' could be a powerful tool in the development of high-throughput, fully automated multiplexed bioassays. (C) 2007 Elsevier B.V. All riahts reserved.

A non-magnetic mutant of Magnetospirilluin magneticum AMB-1 (NMA61), harboring a defective gene located in ORF4 (gene ID: amb4111) was generated by transposon mutagenesis. Biochemical characterization of the gene product of ORF4 revealed that it was localized in the cytoplasm and displayed ATPase activity. The ability of NMA61 to take up iron was severely compromised. Ferrous ion concentration in the medium decreased more with the wild-type than with NMA61, while the iron content in the cytoplasmic fraction of NMA61 was much lower than the wild-type strain. This cytoplasmic ATPase is essential for iron trafficking within M. magneticum AMB-1. (c) 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

A fully automated system using nano-scale engineered biomagnetite was developed to detect mutations in the epidermal growth factor receptor (EGFR) gene in non-small cell lung cancer (NSCLC). Bacterial magnetic particles (BacMPs) were isolated from the magnetic bacterium Magnetospirillum magneticum AMB-1 and conjugated to streptavidin. Biotin-labeled target PCR products were then captured with the BacMPs, hybridized with the detection probe and detected by fluorescence signaling. The process was performed using a newly designed automated processor equipped with an XYZ mobile arm containing a 96-way automated pipetter, reagent dispenser and fluorescence detector. Two types of somatic mutations (in-frame deletions and point substitutions) in the EGFR gene were successfully identified within 3.5 h using this system, suggesting that this system could be used in clinical tests of EGFR gene mutations in lung cancer, and potentially other cancer, patients. Additionally, a very low mutation rate could be detected in these samples. (c) 2006 Elsevier B.V. All rights reserved.

The number of repeat in the microsatellite region (AATG)(5-14) of the human thyroid peroxidase gene (TOPX) was determined using an automated DNA analysis system with nano-scale engineered biomagnetite. Thermal melting curve analysis of DNA duplexes on biomagnetite indicated that shorter repeat sequences (less than 9 repeats) were easily discriminated. However, it was difficult to determine the number of repeats at more than nine. In order to improve the selectivity of this method for the longer repeats, a "double probe hybridization assay" was performed in which an intermediate probe was used to replace a target repeat sequence having more than 9 repeats with a shorter sequence possessing less than 9 repeats. Thermal probe melting curve analyses and T,, determination confirmed that the target with 10 repeats was converted to 5 repeats, 11 repeats converted to 4 and 12 to 3, respectively. Furthermore, rapid determination of repeat numbers was possible by measuring fluorescence intensities obtained by probe dissociation at 56 and 66 degrees C, and 40, 60 and 80 degrees C for signal normalization. (c) 2006 Elsevier B.V. All rights reserved.

A semi-automated system for the large-scale detection of single nucleotide polymorphisms (SNPs) has been developed based on allele-specific oligonucleotide, hybridization and thermal dissociation curve analysis using nano-scale engineered biomagnetite (bacterial magnetic particles; BacMPs). For reliable detection in large numbers of samples, several conditions for the capture of target DNA on nano-sized BacMPs and the denaturation of double-stranded DNA were optimized. The most efficient target DNA capture was observed using short PCR amplicons (69 bp). Captured DNAs were denatured using 50 mM NaOH. With these optimizations, large-scale SNP detection was performed on 822 samples of the transforming growth factor (TGF)-beta 1 gene, which is rich in both GC content and repetitive sequences. High reliability for the semi-automated BacMP-based SNP detection system was confirmed following comparison to traditional sequencing-based methods. (c) 2007 Elsevier B.V. All rights reserved.

Development of a microfluidic device equipped with micromesh for detection of Cryptosporidium parvum oocyst was reported. A micromesh consisting of 10 x 10 cavities was microfabricated on the stainless steel plate by laser ablation. Each cavity size, approximately 2.7 mu m in diameter, was adopted to capture a single C. parvum oocyst. Under negative pressure operation, suspensions containing microbeads or C. parvum oocysts flowed into the micro- channel. Due to strong non-specific adsorption of microbe; ads onto the PDMS microchannel surface during sample injection, the surface was treated with air plasma, followed by treatment with 1% sodium dodecyl sulfate (SDS) solution. This process reduced the non-specific adsorption of microbeads on the microchannel to 10% or less in comparison to a non-treated microchannel. This microfluidic device equipped with the SUS micromesh was further applied for the capture of C parvum oocysts. Trapped C. parvum oocysts were visualized by staining with FITC-labeled anti-C. parvum oocyst antibody on a micromesh and counted under fluoroscopic observation. The result obtained by our method was consistent with that obtained by direct immunofluorescence assay coupled with immunomagnetic separation (DFA-IMS) method, indicating that the SUS micromesh is useful for counting of C. parvum oocysts. The newly designed microfluidic device exploits a geometry that allowed for the entrapment of oocysts on the micromesh while providing the rapid introduction of a j series of reagents and washes through the microfluidic structure. Our data indicate that this microfluidic device is useful for high-throughput counting of C. parvum oocysts from tap water sample.

RNA interference (RNAi) has become a popular tool for downregulating in many species including mammalian cells. Therefore, suppression of target genes in mammalian cultured cells using RNAi may represent an ideal alternative to knockout studies for understanding the molecular mechanisms of chemical toxicity. Here, we assessed the potential of RNAi mediated gene knockdown in HeLa and HepG2 cells to cytotoxicity studies. Tumor necrosis factor receptor I (TNFRI) was chosen as a target gene because its signaling has been implicated in xenobiotic-induced toxicity. We optimized the design and performance of a vector-based RNAi experiment and then investigated viability of both HeLa and HepG2 cells exposed to TNF alpha. In addition, we examined gene expression profile of TNFRI knockdown HeLa cells after TNF alpha treatment, and then protein expression levels for several apoptosis-related genes of the cells. In both HeLa and HepG2 cells, TNF alpha exposure resulted in significantly reduced susceptibility of the knockdown cells to the cytotoxicity as compared with those of mock-transfected cells. Furthermore, the gene expression profiling and western blotting revealed that several genes including apoptosis and/or NF-kappa B pathway were downregulated in the knockdown HeLa cells. These results suggest that downregulation of the TNFRI gene in both HeLa and HepG2 cells by RNAi participates in resistance to TNF alpha-induced cytotoxicity. Therefore, this study raises the possibility that RNAi-based gene silencing in mammalian cells may be a valuable tool for elucidating the relationships between phenotypic changes and target gene functions in response to xenobiotic-induced cytotoxicity. Further exposure study using xenobiotics needs to be done to validate the potential utility of RNAi technology. (c) 2006 Elsevier Ltd. All rights reserved.

A molecular method for detecting the epiphyte community on marine macroalgae was developed by using PCR-denaturing gradient gel electrophoresis. Selective amplification of 16S rRNA gene fragments from either cyanobacteria or algal plastids improved the detection of minor epiphytes. Two phylotypes of Acaryochloris, a chlorophyll d-containing cyanobacterium, were found not only on red macroalgae but also on green and brown macroalgae.

Herein the potential of a highly efficient cell separation system using bacterial magnetic particles expressing protein A (protein A-Bac-MPs) was demonstrated. Protein A was expressed on BacMPs using the transmembrane proteins Mms13 and MagA as anchor molecules. The evaluations of the numbers of bound antibody molecules and binding capabilities of the protein A-BacMPs using Mms13 indicated that the antibodies were efficiently introduced into protein A-BacMPs using Mms13 in comparison to MagA. In addition, the recovery ratio of the target cells on the magnetic cell separation system was enhanced by using protein A-BacMPs with Mms13. Using positive selection against peripheral blood mononuclear cells, the CD14(+) cells were separated at a purity of more than 99% by protein A-BacNlPs using Mms13. Furthermore, in the evaluation of the influence of protein A-BacMPs on the separated cells, the CD14(+) cells separated using protein A-BacMPs and were successfully differentiated into dendritic cells. (c) 2006 Elsevier Inc. All rights reserved.

This study reports the production of astaxanthin in the photosynthetic bacterium Rhodovulum sulfidophilum, which has adequate precursor pools and storage capabilities for heterologous carotenoid production. Chemical mutagenesis was carried out using ethylmethane sulfonate to produce mutants with a modified carotenoid biosynthesis pathway downstream of phytoene. Stable green- and gray-colored mutants were selected. Green mutants contained neurosporene or chloroxanthin as their major carotenoid (&gt; 90%), while the gray mutants accumulated phytoene. We previously demonstrated the production of beta-carotene in Rhodovulum sulfidophilum by cloning the Erythrobacter longus crtI (phytoene dehydrogenase) and crtY (lycopene cyclase) genes. In the present study, an expression vector for astaxanthin production was constructed that contained the Paracoccus crtW (beta-carotene oxygenase) and crtZ (beta-carotene hydroxylase) genes in addition to the E. longus crtI and crtY genes. A transconjugant, which can synthesize astaxanthin, was successfully generated (2.0 mu g g(-1) DCW).

This report describes the development of a direct and rapid detection method for the pathogenic protozoan, Cryptosporidium parvum, from environmental water samples using fluorescence in situ hybridization (FISH) on a membrane filter. The hydrophilic polytetrafluoroethylene (PTFE) membrane filter with FISH-stained oocysts yielded the highest signal to noise (S/N) ratio of the different membrane filters tested. PTFE membranes retained 98.8 +/- 10.4% of the concentrated oocysts after washing, simultaneous permeabilization and fixation with a hot ethanol solution, and hybridization with a fluorescently labeled oligonucleotide probe. This procedure eliminates subsequent time-consuming recovery steps that often result in a loss of the actual oocysts in a given environmental water sample. Furthermore, C. parvum was successfully distinguished from Cryptosporidium muris and other species in environmental water samples with the addition of formamide into the hybridization solution. In tap water samples, the S/N ratio was heightened by washing the membrane filter prior to FISH with a 1 M HCl solution in order to reduce the large amounts of impurities and background fluorescence from the non-specific adsorption of the fluorescently labeled oligonucleotide probe. (c) 2006 Elsevier B.V. All rights reserved.

A thin film transistor (TFT) photosensor fabricated by semiconductor integrated circuit (IC) technology was applied to DNA chip technology. The surface of the TFT photosensor was coated with TiO2 using a vapor deposition technique for the fabrication of optical filters. The immobilization of thiolated oligonucleotide probes onto a TiO2-Coated TFT photosensor using gamma-aminopropyltriethoxysilane (APTES) and N-(gamma-maleimidobutyloxy) sulfosuccinimide ester (GMBS) was optimized. The coverage value of immobilized oligonucleotides reached a plateau at 33.7 pmol/cm(2), which was similar to a previous analysis using radioisotope-labeled oligonucleotides. The lowest detection limits were 0.05 pmol/cm(2) for quantum dot and 2.1 pmol/cm(2) for Alexa Fluor 350. Furthermore, single nucleotide polymorphism (SNP) detection was examined using the oligonucleotide-arrayed TFT photosensor. A SNP present in the alclehyde dehydrogenase 2 (ALDH2) gene was used as a target. The SNPs in ALDH2*1 and ALDH2*2 target DNA were detected successfully using the TFT photosensor. DNA hybridization in the presence of both ALDH2*1 and ALDH2*2 target DNA was observed using both ALDH2*1 and ALDH2*2 detection oligonucleotides-arrayed TFT photosensor. Use of the TFT photosensor will allow the development of a disposable photodetecting device for DNA chip systems. (c) 2006 Wiley Periodicals, Inc.

Bacterial magnetic particles (BMPs) were modified with 3-[2-(2-aminoethylamino)-ethylaminol-propyltrimethoxysilane (AEEA) to produce a dense amine surface. Modification of BMPs in a toluene solution resulted in an increased amine yield, and approximately 11.3 x 10(4) surface amines were detected on a single particle. The modified BMPs were capable of efficient electrostatic capture of DNA. The maximum amount of DNA captured on 10 mu g of aminosilane-modified BMPs was 600 ng. A 10 mM phosphate buffer effectively released the captured DNA. This efficiency was dramatically enhanced by incubation at 80 degrees C and DNA recovery from aminosilane-modified BMPs approached 95%. DNA extraction from whole blood using these modified BMPs, followed by PCR, was successfully performed. Furthermore, automated single nucleotide polymorphism (SNP) detection of the aldehyde clehydrogenase 2 (ALDH2) was demonstrated. (c) 2006 Wiley Periodicals, Inc.

Limitations in obtaining sufficient specimens and difficulties in extracting high quality DNA from environmental samples have impeded understanding of the structure of microbial communities. In this study, multiple displacement amplification (MDA) using phi 29 polymerase was applied to overcome these hindrances. Optimization of the reaction conditions for amplification of the bacterial genome and evaluation of the MDA product were performed using cyanobacterium Synechocystis sp. strain PCC6803. An 8-h MDA reaction yielded a sufficient quantity of DNA from an initial amount of 0.4 ng, which is equivalent to approximately 10(5) cells. Uniform amplification of genes randomly selected from the cyanobacterial genome was confirmed by real-time polymerase chain reaction. The metagenome from bacteria associated with scleractinian corals was used for whole-genome amplification using phi 29 polymerase to analyse the microbial diversity. Unidentified bacteria with less than 93% identity to the closest 16S rDNA sequences deposited in DNA Bata Bank of Japan were predominantly detected from the coral-associated bacterial community before and after the MDA procedures. Sequencing analysis indicated that alpha-Proteobacteria was the dominant group in Pocillopora damicornis. This study demonstrates that MDA techniques are efficient for genome wide investigation to understand the actual microbial diversity in limited bacterial samples.

Siderophore activity was detected in the culture supernatant of the magnetotactic bacterium Magnetospirillum magneticum AMB-1. Here we report the first structural elucidation of a siderophore produced by a magnetotactic bacterium. The structure of the purified compound was 3,4-dihydroxybenzoic acid as determined by nuclear magnetic resonance (NMR) and electro-spray ionization mass spectroscopy (ESI-MS).

A molecular approach was adopted to investigate the natural diets of palinurid and scyllarid lobster phyllosoma larvae. The central domain of the 18S rDNA gene was amplified using nested polymerase chain reaction (PCR) and genomic DNA extracted from the larval hepatopancreas. The resulting 18S rDNA clones were screened using restriction fragment length polymorphism (RFLP) analysis, and then FASTA homology search and phylogenetic analysis were performed on the nucleotide sequences to identify the source of the eukaryotic organisms. The feasibility of this method was confirmed using the laboratory-reared phyllosoma larvae of the Japanese spiny lobster Panulirus japonicus that were fed either with common mussel Mytilus edulis gonads or with Artemia nauplii exclusively. Among the 804 clones isolated from five wild-caught mid- to late-stage phyllosoma larvae (three palinurids and two scyllarids), 0-132 clones per sample possessed restriction profiles distinct from those of the hosts. The Cnidaria and Urochordata DNA were identified from both the palinurid and the scyllarid larvae, which were thought to be prey animals for the mid- to late-stage phyllosoma larvae.

Iron uptake systems were identified by global expression profiling of Magnetospirillum magneticum AMB-1. feo, tpd, and ftr, which encode ferrous iron transporters, were up-regulated under iron-rich conditions. The concomitant rapid iron uptake and magnetite formation suggest that these uptake systems serve as iron supply lines for magnetosome synthesis.

The entire structure of a 98 kb genomic region that abounds in genes related to magnetosome synthesis was first described in the Magnetospirillum sp. strain AMB-1. The deletion of this 98 kb genomic region and the circular form after excision from the chromosome was detected by PCR amplification. This strongly suggests that the region has undergone a lateral gene transfer. The region has the characteristics of a genomic island: low GC content, location between two repetitive sequences, and the presence of an integrase in the flanking region of the first repetitive sequence. This 98 kb genomic region has the potential for transfer by the integrase activity. Comparative genome analysis revealed other regions with a high concentration of orthologs in magnetic bacteria besides the 98 kb region, and magnetosome synthesis seemed to need not only the exogenous 98 kb region, but also other orthologs and individually originating genes. (c) 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Hermatypic (or reef-building) corals live in obligatory Mutualistic symbiosis with the symbiotic dinoflagellates Symbiodinium spp. (generally known as zooxanthellae). In an attempt to establish a model symbiosis system consisting of a coral host and a monoclonal population of zooxanthellae, infectivity of five cultured Symbiodinium cell lines was tested on naturally aposymbiotic juveniles of Acropora tenuis. A clade A3 strain (PL-TS-1) infected the juveniles at high density and promoted growth of the host. To identify host genes involved in the establishment or maintenance of symbiosis, mRNA expression patterns were compared between aposymbiotic and PL-TS-1-infected juvenile polyps using the suppression subtractive hybridization technique. Two mRNAs, the expression levels of which were augmented more than twofold by the presence of the symbionts, were thereby identified. One of the mRNAs, AtSym-02, encodes a novel protein of 322 amino acids which is predicted to be a glycosylated trans-membrane protein. (c) 2005 Elsevier Inc. All rights reserved.

Magnetospirillum sp. strain AMB-1 is a Gram-negative alpha-proteobacterium that synthesizes nano-sized magnetites, referred to as magnetosomes, aligned intracellularly in a chain. The potential of this nano-sized material is growing and will be applicable to broad research areas. It has been expected that genome analysis would elucidate the mechanism of magnetosome formation by magnetic bacteria. Here we describe the genome of Magnetospirillum sp. AMB-1 wild type, which consists of a single circular chromosome of 4 967 148 bp. For identification of genes required for magnetosome formation, transposon mutagenesis and determination of magnetosome membrane proteins were performed. Analysis of a non-magnetic transposon mutant library focused on three unknown genes from 2752 unknown genes and three genes from 205 signal transduction genes. Partial proteome analysis of the magnetosome membrane revealed that the membrane contains numerous oxidation/reduction proteins and a signal response regulator that may function in magnetotaxis. Thus, oxidation/reduction proteins and elaborate multidomain signaling proteins were analyzed. This comprehensive genome analysis will enable resolution of the mechanisms of magnetosome formation and provide a template to determine how magnetic bacteria maintain a species-specific, nanosized, magnetic single domain and paramagnetic morphology.

Alignment of magnetic nanoparticles in nanotubes (see Figure) is produced using magnetic bacteria. The protein- and lipid-coated nanocrystals are extracted from the bacteria, and then introduced into peptide nanotubes where they are anchored by hydrogen bonding. The aligned nanoparticles behave its magnetic nanowires, with enhanced properties over unaligned magnetic nanocrystals.

A glass slide and micro-well array chip on which anti-Cryptosporidium parvum antibody was immobilized were used for the rapid capture and detection of C. parvum. Biotinylated anti-C. parvum antibodies were spotted onto the streptavidin-coated glass slides. C. parvum oocysts were captured specifically on the spot when more than 73 ng of anti-C. parvum antibody was applied onto the glass slide. However, C. parvum oocysts captured on the glass slide were detached by repeating washing steps. To improve the capture efficiency of oocysts, capture was performed in a micro-well format consisting of 1024 wells/2.5 mm(2) (32 x 32 wells) fabricated as a chip by photolithography. Instead of a flat surface on a glass slide, each well was 30 mu m diameter and 10 mu m in depth. Streptavidin was also immobilized onto the micro-well array. The biotinylated anti-C. parvum antibodies were immobilized efficiently onto the chip using a buffer containing 20% methanol. Using this technique C. parvum oocysts were stably captured onto the chip after repeated washing procedures. These data show that the newly designed micro-well array technique described here is useful for anti body-mediated C. parvum capture. (c) 2004 Elsevier B.V. All rights reserved.

A simple microchip device for DNA extraction was constructed based on electrostatic interactions between surface amine groups and DNA. Microchannel was fabricated on silicon wafer by photolithography and coated with 3-aminopropyltrietboxysilane (APTES) or 3-[2-(2-aminoethylamino)-ethylamino]-propyluimethoxysilane (AEEA) to introduce amine groups on the surface. Determination of the number of surface amine groups and optimization of DNA capture condition were demonstrated to characterize the microchip. Capacities of capturing DNA were approximately 97 ng/cm(2) in APTES and 194 ng/cm(2) in AEEA modified microchips, respectively. The amount of DNA captured in the microchip increased depending on surface amine density. Furthermore, DNA extraction using amine-coated microchip from whole blood was examined. Quantification of DNA and proteins in washing or eluting fraction indicates that proteins were removed at washing steps and only DNA was effectively eluted by changing alkalinity of buffer from pH 7.5 to 10.6. The amount of DNA extracted from whole blood was approximately 10 ng and its recovery ratio was 27-40%. Performance of PCR for the eluted fraction indicates that DNA extracted from whole blood was well purified using amine-coated microchip. (C) 2004 Elsevier B.V. All rights reserved.

Marine microalgae, the largest primary biomass, have been attracting attention as resources for new metabolites and biotechnologically useful genes. The diversified marine environment harbors a large variety of microalgae. In this paper, the biotechnological aspects and fundamental characteristics of marine microalgae are reviewed.

Bacterial magnetic particles (BacMPs) are efficient platforms of proteins for surface display systems. In this study, mononuclear cells from peripheral blood were separated using BacMPs expressing protein A on the BacMP membrane surface (protein A-BacMPs), which were complexed with the Fc fragment of anti-mouse IgG antibody. The procedure of positive selection involves incubation of mononuclear cells and mouse monoclonal antibodies against different cell surface antigens (CD8, CD14, CD19, CD20) prior to treatment with protein A-BacMP binding with rabbit anti-mouse IgG secondary antibodies. Flow cytometric analysis showed that similar to97.5 +/- 1.7% of CD19(+) and CD20(+) cells were involved in the positive fraction after magnetic separation. The ratio of the negative cells in the negative fraction was -97.6 +/- 1.4%. This indicates that CD19(+) and CD20(+) cells can be efficiently separated from mononuclear cells. Stem cell marker (CD34) positive cells were also separated using protein A-BacMP binding with antibody. May-Grunwald Giemsa stain showed a high nuclear/cytoplasm ratio, which indicates a typical staining pattern of stem cells. The separated cells had the capability of colony formation as hematopoietic stem cells. Furthermore, the inhibitory effect of magnetic cell separation on CD14(+) cells was evaluated by measurement of cytokine in the culture supernatant by ELISA when the cells were cultured with or without lipopolysaccharide (LPS). The induction of IL1-beta, TNFalpha, and IL6 was observed in the presence of 1 ng/mL LPS in all fractions. On the other hand, in the absence of LPS, BacMPs had little immunopotentiation to CD14(+) cells as well as that of artificial magnetic particles, although TNFa and IL6 were slightly induced in the absence of LPS in the positive fraction.

A non-magnetic mutant, NMA61, of the magnetic bacterium Magnetospirillum magneticum AMB-1 was generated by transposon mutagenesis to identify genes involved in magnetosome synthesis. The genomic region of NMA61 interrupted by a Mini-Tn5 transposon was analyzed. The transposon was inserted in an open reading frame (ORF)coding for a periplasmic transport binding protein kinase gene homologue. Three adjacent ORFs and a promoter were identified upstream, indicating that the sequences comprised an operon. Phenotype characterizations showed that the growth inhibition imposed by the exogenous non-assimilable iron chelator nitrilotri acetate was relieved in wild type but not in NMA61, by the addition of the isolated wild type siderophore. Higher concentration of siderophores accumulated in the culture medium of NMA61 than in wild type. These data suggest that the interrupted periplasmic transport binding protein kinase gene homologue is required for siderophore transport into M. magneticum AMB-1. (C) 2004 Elsevier Inc. All rights reserved.

Single nucleotide polymorphism (SNP) detection for aldehyde dehydrogenase 2 (ALDH2) gene based on DNA thermal dissociation curve analysis was successfully demonstrated using an automated system with bacterial magnetic particles (BMPs) by developing a new method for avoiding light scattering caused by nanometer-size particles when using commercially available fluorescent dyes such as FITC, Cy3, and Cy5 as labeling chromophores. Biotin-labeled PCR products in ALDH2, two allele-specific probes (Cy3-labeled detection probe for ALDH2*1 and Cy5-labeled detection probe for ALDH2*2), streptavidinimmobilized BMPs (SA-BMPs) were simultaneously mixed. The mixture was denatured at 70degreesC for 3 min, cooled slowly to 25degreesC, and incubated for 10 min, allowing the DNA duplex to form between Cy3- or Cy5-labeled detection probes and biotin-labeled PCR products on SA-BMPs. Then duplex DNA-BMP complex was heated to 58degreesC, a temperature determined by dissociation curve analysis and a dissociated single-base mismatched detection probe was removed at the same temperature under precise control. Furthermore, fluorescence signal from the detection probe was liberated into the supernatant from completely matched duplex DNA-BMP complex by heating to 80degreesC and measured. In the homozygote target DNA (ALDH2*1/*1 and ALDH2*2/*2), the fluorescence signals from single-base mismatched were decreased to background level, indicating that mismatched hybridization was efficiently removed by the washing process. In the heterozygote target DNA (ALDH2*1/*2), each fluorescence signals was at a similar level. Therefore, three genotypes of SNP in ALDH2 gene were detected using the automated detection system with BMPs. (C) 2004 Wiley Periodicals, Inc.

G protein-coupled receptors (GPCRs) play a central role in a wide range of biological processes and are prime targets for drug discovery. GPCRs have large hydrophobic domains, and therefore purification of GPCRs from cells is frequently time-consuming and typically results in loss of native conformation. In this work, GPCRs have been successfully assembled into the lipid membrane of nanosized bacterial magnetic particles (BMPs) produced by the magnetic bacterium Magnetospirillum magneticum AMB-1. A BMP-specific protein, Mms16, was used as an anchor molecule, and localization of heterologous Mms16 on BMPs was confirmed by luciferase fusion studies. Stable luminescence was obtained from BMPs bearing Mms16 fused with luciferase at the C-terminal region. D1 dopamine receptor (D1R), a GPCR, was also efficiently assembled onto BMPs by using Mms16 as an anchor molecule. D1R-BMP complexes were simply extracted by magnetic separation from ruptured AMB-1 transformants. After washing, the complexes were ready to use for analysis. This system conveniently refines the native conformation of GPCRs without the need for detergent solubilization, purification, and reconstitution after cell disruption.

Halomonas variabilis and phylogenetically related organisms were isolated from various habitats such as Antarctic terrain and saline ponds, deep-sea sediment, deep-sea waters affected by hydrothermal plumes, and hydrothermal vent fluids. Ten strains were selected for physiological and phylogenetic characterization in detail. All of those strains were found to be piezotolerant and psychrotolerant, as well as euryhaline halophilic or halotolerant. Their stress tolerance may facilitate their wide occurrence, even in so-called extreme environments. The 16S rDNA-based phylogenetic relationship was complemented by analyses of the DNA gyrase subunit B gene (gyrB) and genes involved in the synthesis of the major compatible solute, ectoine: diaminobutyric acid aminotransferase gene (ectB) and ectoine synthase gene (ectC). The phylogenetic relationships of H. variabilis and related organisms were very similar in terms of 16S rDNA, gyrB, and ectB. The ectC-based tree was inconsistent with the other phylogenetic trees. For that reason, ectC was inferred to derive from horizontal transfer.

A fully automated immunoassay of 17beta-estradiol (E2) was performed using anti-E2 monoclonal antibody immobilized on bacterial magnetic particles (AntiE2-BMPs) and alkaline phosphatase-conjugated E2 (ALP-E2). E2 concentration in environmental water samples was evaluated by decrease in luminescence based on competitive reaction. A linear correlation between the luminescence intensity and E2 concentration was obtained between 0.5 and 5 ppb. The minimum detectable concentration of E2 was 20 ppt. All measurement steps were done within 0.5 h. The analysis of environmental water samples by a commercially available ELISA kit and the BMP-based immunoassay gave good correlation plots with a correlation efficient of 0.992. These results suggest that the fully automated system using the BMP-based immunoassay has some advantages in the high rapidity and sensitivity of the measurement. This system will enable us to determine low E2 concentrations without sample condensation. (C) 2003 Elsevier B.V. All rights reserved.

We designed an automated workstation for magnetic particle-based single nucleotide polymorphism (SNP) discrimination of ALDH genotypes. Bacterial magnetic particles (BMPs) extracted from Magnetospirillum magneticum AMB-1 were used as DNA carriers. The principle for SNP discrimination in this study was based on fluorescence resonance energy transfer (FRET) between FITC (donor) and POPO-3 (acceptor) bound to double-stranded DNA. The workstation is equipped with a 96-way automated pipetter which collects and dispenses fluids as it moves in x- and z-directions. The platform contains a disposable tip rack station, a reagent vessel serving as a stock for POPO-3 and FITC-labeled probes and a reaction station for a 96-well microtiter plate. BMPs were collected by attaching a neodymium iron boron sintered (Nd-Fe-B) magnet on the bottom of the microtiter plate. This system permits the simultaneous heating and magnetic separation of 96 samples per assay. The genotypes ALDH2*1 and ALDH2*2 were discriminated by calculating the relative fluorescence intensities on BMPs. (C) 2003 Elsevier B.V. All rights reserved.

To develop an analytical system for single-nucleotide polymorphisms (SNPs), the fluorescence resonance energy transfer (FRET) technique was employed on a bacterial magnetic particle (BMP) surface. A combination of fluorescein isothiocyanate (FITC; excitation 490 nm/emission 520 nm) labeled at the 5' end of DNA and an intercalating compound (POPO-3, excitation 534 nm/emission 570 nm) was used to avoid the interference from light scattering caused by nanoparticles. After hybridization between target DNA immobilized onto BMPs and FITC-labeled probes, fluorescence from POPO-3, which was excited by the energy from the FITC, was detected. The major homozygous (ALDH2*1), heterozygous (ALDH2*11*2), and minor homozygous (ALDH2*2) genotypes in the blood samples were discriminated by this method. The assay described herein allows for a simple and rapid SNP analysis using a fully automated system. (C) 2003 Wiley Periodicals, Inc.

A 3.7-kb cryptic plasmid designated pMGT was found in Magnetospirillum magneticum MGT-1. It was characterized and used for the development of an improved expression system in strain AMB-1 through the construction of a shuttle vector, pUMG. An electroporation method for magnetic bacteria that uses the cryptic plasmid was also developed.

Fluorescence in situ hybridization has been used for the identification and analysis of populations of the dinoflagellate Symbiodinium that lives symbiotically in marine invertebrates. Conditions for in situ hybridization of Symbiodinium were optimized and used to identify the clade to which the isolate belongs using specific probes. The optimized in situ hybridization procedure used a combination of chlorophyll removal and permeabilization with hot ethanol. Incubation of the cells in 50% ethanol at 80 degreesC for 20 min rendered the cell wall permeable to Cy3-labeled probes. Symbiodinium clade-specific probes were designed based on 18S rRNA sequences. Symbiodinium A, B and C were distinguished by in situ hybridization with the specific probes SymA, SymB and SymC, respectively. The hybridization results using clade-specific probes corresponded with results obtained using restriction fragment length polymorphism (RFLP) analysis. Symbiodinium isolated from jellyfish Cassiopea sp. and sea anemone Aiptasia sp. were classified as belonging to clades A and B using the FISH procedure established in this study. (C) 2003 Elsevier Science B.V. All rights reserved.

A single nucleotide polymorphism (SNP) genotyping for aldehyde dehydrogenase 2 gene (ALDH2) has been developed by using a nano-sized magnetic particle, which was synthesized intracellularly by magnetic bacteria. Streptavidin-immobilized on bacterial magnetic particles (BMPs) were prepared using biotin labeled cross-linkers reacting with the amine group on BMPs. ALDH2 fragments from genomic DNA were amplified using a TRITC labeled primer and biotin labeled primer pair, and conjugated onto BMP surface by biotin-streptavidin interaction. PCR product-BMP complex was observed at a single particle level by fluorescence microscopy. These complexes were treated with restriction enzyme, specifically digesting the wild-type sequence of ALDH2 (normal allele of ALDH2). The homozygous (ALDH2*11*1), heterozygous (ALDH2*11*2), and mutant (ALDH2*21*2) genotypes were discriminated by three fluorescence patterns of each particle. SNP genotyping of ALDH2 has been successfully achieved at a single particle level using BMP. (C) 2003 Elsevier Science B.V. All rights reserved.

A single nucleotide polymorphism (SNP) within the transforming growth factor-beta1 (TGF-beta1) gene was detected by hybridization-based method using bacterial magnetic particles (BMPs). TGF-beta1 is commonly associated with a single base change resulting in a Leu(10) --&gt; Pro (T-869 --&gt; C) polymorphism and is a genetic marker for susceptibility to osteoporosis. Short (9 bases) and specific probes were designed to detect SNP in TGF-beta1. Detection probes were immobilized on BMPs using cross-linking reagents. TGF-beta1 PCR products (139 bp) were labeled with the fluorescent dye coumarin and hybridized with detection probes on BMPs. Complementary hybridized targets gave over four times higher fluorescent intensities, compared with one base mismatched hybridizations. The SNP genotype was successfully discriminated using this technique. (C) 2003 Elsevier Science B.V. All rights reserved.

A non-magnetic mutant of Magnetospirillum magneticum AMB-1, designated as NMA2 1, was generated by mini-Tn5 transposon mutagenesis to identify genes involved in bacterial magnetic particle (BMP) synthesis. Alignment of the DNA sequences flanking the transposon allowed the isolation of an open reading frame (ORF2) within an operon consisting of five genes. The amino acid sequence of ORF2 showed homology with tungsten-containing aldehyde ferredoxin oxidoreductase (AOR) from Pyrococcus furiosus (48% identity and 64% similarity), which functions for aldehyde oxidation. AOR was found to be expressed under microaerobic conditions and localized in the cytoplasm of AMB-1. Iron uptake and growth of NMA21 were lower than wild type. Transmission electron microscopy (TEM) of NMA21 revealed that no BMPs were completely synthesized, but polyhydroxybutyrate (PHB)-like granules were persistently produced. These results indicate that AOR may contribute to ferric iron reduction during BMP synthesis in M. magneticum AMB-1 under microacrobic respiration. (C) 2003 Elsevier Science (USA). All rights reserved.

An approach to analyze mismatches using short and specific oligonucleotide probes directly synthesized on bacterial magnetic particles (BMPs) by phosphoramidite methods was exploited. Approximately 126 molecules of 4-mer oligonucleotides/particle were synthesized on BMPs with high reaction efficiencies. Hybridization between FITC-labeled oligonucleotides and chemically synthesized oligonucleotides on BMPs was performed. Perfect matched and mismatched hybridizations were successfully discriminated by using the oligonucleotide probes on BMPs. © 2003 Elsevier Science B.V. All rights reserved.

Bacterial and artificial magnetic particles were modified using a polyamidoamine (PAMAM) dendrimer and outer shell amines determined. Bacterial magnetic particles were the most consistently modified. Transmission electron microscopic (TEM) analysis showed that the artificial magnetic particles were structurally damaged by the modification process including sonication. Furthermore, laser particle analysis of the magnetite also revealed damage. Small quantities of dendrimer-modified bacterial magnetic particles were used to extract DNA from blood. The efficiency of DNA recovery was consistently about 30 ng of DNA using 2-10 mug of dendrimer-modifled bacterial magnetite. This technique was fully automated using newly developed liquid handling robots and bacterial magnetic particles.

Soil bacteria were studied for the production of biodegradable cleaning agents. Among 86 bacterial strains resistant to liquid paraffin, 58 showed hemolytic activity. These strains were cultured, and the supernatant of culture broths was evaluated for cleaning activity against a dirty porcelain tile. Potent activity was exhibited in 18 strains. The lowest value of surface tension was obtained from Bacillus sp. NKB03 suggesting the presence of a biosurfactant. Aeromonas sp. NKB26c and Bacillus cereus NKB46b exhibited enzymatic cleaning activity. A cleaning efficiency of 82% was achieved when using a mixture of supernatants from culture broths of Bacillus sp. NKB03 and Aeromonas sp. NKB26c in synthetic minimal media. The cleaning efficiency using this mixture was higher than that of sodium dodecyl sulfate. These results suggest that a mixture of supernatants from culture broths of Bacillus sp. NKB03 and Aeromonas sp. NKB26c has potential for commercial use as a biocleaner.

Cadmium recovery by a sulfate-reducing magnetotactic bacterium, Desulfovibrio magneticus strain RS-1, was investigated. D. magneticus precipitated &gt
95% of cadmium at an initial concentration of 1.3 ppm in the growth medium. Electron microscopic analysis revealed that D. magneticus formed electron-dense particles on its surface when cultivated in the presence of cadmium ions (Cd2+). Sulfide was also found in the precipitate, and the composition ratio of sulfide/cadmium was 0.7. Sixty percent of viable RS-1 cells was recovered by a simple magnetic separation revealing the removal of 58% cadmium from the culture medium.

Variants in the calmodulin gene intron 4 (CaM) and the mtDNA D-loop region (D-loop) detected by RFLP and nucleotide sequence analyses were used to investigate the global stock structure of the swordfish Xiphias gladius. Two alleles (A and B) were observed at the CaM locus among 8 samples (Mediterranean, Tarifa, four Atlantic areas, Indian and Pacific oceans) comprising 567 individuals. Genotype distributions at this locus within samples were in accordance with Hardy-Weinberg equilibrium. Significant differences in allele frequencies were observed between Mediterranean-Northwest Atlantic samples (A=0.347-0.493) and those from tropical Atlantic and Indo-Pacific (A=0.81-1). In the D-loop region, Alu I and Rsa I digestions detected 4 (A-D) and 8 (A-H) genotypes, respectively. The frequency of type C for Alu I was lower in Indo-Pacific samples (0.2) than Atlantic and Mediterranean samples (0.365-0.643). Frequency of type C for Rsa I increased from the Pacific (0.398) to the Northwest Atlantic (0.698) and Tarifa (0.949) samples, and the Mediterranean sample was fixed for type C. These samples were classified into three groups (Mediterranean-Tarifa, Atlantic and Indo-Pacific) using a heterogeneity test of mtDNA genotype distributions. Combined analysis of the CaM and D-loop loci indicated that there are at least four breeding units: Mediterranean, northwestern Atlantic, tropical to South Atlantic, and Indo-Pacific. (C) 2000 The Fisheries Society of the British Isles.

Twenty four strains out of 191 marine microalgal strains exhibited cadmium (Cd) resistance. They were tested for their Cd removal ability in growth media containing 50 μM Cd. Six strains out of 19 green algae and one out of five cyanobacteria removed more than 10% of total Cd from the medium. The marine green alga Chlorella sp. NKG16014 showed the highest removal of Cd 48.7% of total. Cd removal by NKG 16014 was further quantitatively evaluated by measuring the amount of cell adsorption and intracellular accumulation. After 12 days incubation, 67% of the removed Cd was accumulated intracellularly and 25% of the Cd removed was adsorbed on the algal cell surface. The maximum Cd adsorption (qmax) was estimated to be 37.0 mg Cd (g dry cells)- 1 using the Langmuir sorption model. The Cd removal by freeze-dried NKG16014 cells was also determined. Cd was more quickly adsorbed by dried cells than that by living cells, with a qmax of 91.0 mg Cd (g dry cells)- 1. © 1999 Elsevier B.V. All rights reserved.

Introns may accumulate much higher genetic variation than exons. Universal primers were designed from the conservative nucleotide sequences of exons to amplify the flanking intron. Length variations in the S7 ribosomal protein (RP) gene intron 1 and mitochondrial creatine kinase (CK) gene intron 6 of the swordfish Xiphias gladius were found. Single or two banded fragment patterns in each individual were observed by agarose gel electrophoresis. Nucleotide sequence analysis revealed that highly polymorphic fragment patterns observed in the RP gene intron 1 were due to different numbers of a TG repeat (microsatellite). The length of the CK gene intron 6 was dimorphic, in which presence or absence of a 24 bp block was responsible for longer or shorter introns. Additional minor nucleotide insertion/deletions were observed independent of the RP microsatellite and the CK 24 bp block regions. The results of this investigation indicate that introns may be good sources of intraspecific genetic variation for population genetic studies and that the same set of primers can be used to amplify homologous intron regions even among distant species. Further, the conserved exon primed PCR strategy may be useful to prevent appearance of priming site polymorphism (null allele).

Restriction fragment length polymorphism (RFLP) analysis was performed on PCR amplified DNA fragments containing the control region of the swordfish (Xiphias gladius Linnaeus, 1758) mitochondrial DNA. A total of 456 individuals comprising 13 local samples (six Pacific, three Atlantic, the Mediterranean Sea, two Indian Ocean and the Cape of Good Hope) were surveyed with four endonucleases (Alu I, Dde I, Hha I and Rsa I), yielding a total of 52 composite genotypes. Within-sample genotypic diversity (H) was high ranging from 0.702 to 0.962 with a value of 0.922 for the pooled sample. Significant geographic variation in the frequencies of genotypes and restriction patterns was revealed. The Mediterranean sample was highly distinct from all other samples. Further, Rsa I digestion revealed high levels of polymorphism in all but the Mediterranean samples, indicating that exogenous swordfishes rarely enter that body of water. Heterogeneity between the North and South Atlantic samples was significant, both of which differed from those of the Pacific. In contrast, the Indian Ocean samples were not significantly different from the samples of South Atlantic and Pacific. Genetic differentiation among the Pacific samples was low. The results indicate that the worldwide swordfish population is genetically structured not only among, but also within ocean basins and suggest that gene flow is restricted despite the absence of geographic barriers.

There are many kinds of microalgae who have adapted to extremely diversified marine environments. Screening of new useful materials from marine microalgae has been carried out. High density culture system using photo-bioreactor and genetic manipulation technology have been employed in order to enhance productivity of those materials from marine microalgae.
We have found many useful materials, such as W-A absorbing substance, plant growth regulating factor, polysaccharides, unsaturated fatty acids and antibiotics, from marine microalgae. Here, we describe production of those useful materials and their application. Marine coccolithophorid algae convert CO2 to CaCO3 and they have been expected to contribute to CO2 recycling system. Metabolic engineering in marine microalgae, especially cyanobacteria, has been established. Eicosapentaenoic acid is successfully synthesized in marine cyanobacterium which can not synthesize it.

Bacterial magnetite particles (BMPs) of 50 to 100nm diam were used as DNA carriers for the ballistic transformation of the marine cyanobacterium Synechococcus. BMPs were bombarded into the cyanobacterial cells at several bombardment velocities using a particle gun. Successful transformation and gene expression were confirmed by Southern hybridization and CAT assay, respectively. The BMPs were also observed in the cyanobacterial cells by transmission electron microscopy. These results suggested that BMPs can be used as carriers for introducing DNA into bacterial cells. © 1995 Chapman &amp
Hall.

Versatility of gene transfer by transconjugation in marine cyanobacteria was demonstrated. In this study, seven different marine cyanobacteria were used as recipient cells. First, transconjugation was carried out using the mobilizable transposon (Tn5) carrying plasmid pSUP1021. Transconjugants were observed in all marine cyanobacteria tested. Second, the broad-host-range vector pKT230 (IncQ) was tested for transconjugation. pKT230 has been successfully transferred in a marine cyanobacterium Synechococcus sp. NKBG15041C, and replicated as an autonomous replicon without alteration in the restriction enzyme pattern. A maximum transfer efficiency of 5.2 x 10(-4) transconjugants/recipient cell was observed, when mating was performed on agar plates containing low salinity (0.015 M NaCl) medium. This is the first study to demonstrate gene transfer in marine cyanobacteria via transconjugation.

We have investigated the application of bacterial magnetite particles as DNA carriers for microprojectile based gene transfer. The magnetic bacterium, Aquaspirillum sp. AMB-1, can grow aerobically to high cell densities, allowing high yields of bacterial magnetite particles to be sonicated. The optimum sonication conditions were determined and found to be dependent on culture age. Excessive sonication damaged the magnetosome membrane surrounding magnetite particles. This decreased dispersion resulted in a reduced surface area available for DNA adsorption. When compared with gold, tungsten and non membrane coated magnetite, membrane coated magnetite was a better material for DNA binding (5-mu-g DNA/mg magnetite). The greatest amount of DNA was bound following treatment of membrane coated magnetite with glutaraldehyde-triamine prior to incubation with DNA.

The marine environment is remarkably diverse and harbors a wide variety of organisms that have acquired novel metabolic functions and corresponding genes through evolutionary adaptation. Marine organisms are potentially a treasure of gene resources. Especially, much attention has been paid to photosynthetic organisms because they can grow with inorganic carbon fixation, produce useful materials and convert photo-energy into bio-fuel. On the other hand, it is known that standard culturing techniques account for only 1 % or less of the bacterial diversity in the environments. Therefore, recently, marine metagenomic approach with construction of genomic libraries by direct extraction and cloning of DNA from environmental samples has been applied to analyze and utilize the unculturable microbes as gene resources. Furthermore, it has also become important tool for screening useful enzyme genes in biofuel production.

Magnetic separation of target cells from mixtures, such as peripheral blood and bone marrow, has considerable practical potential in research and medical applications. Among the current cell separation techniques, magnetic cell separation using immunomagnetic particles has been routinely applied and has proven rapidness and simplicity. Magnetospirillum magneiicum AMB-1 synthesizes intracellular nano-sized bacterial magnetic particles (BacMPs) that are individually enveloped by a stable lipid bilayer membrane. BacMPs, which exhibit strong ferrimagnetism, can be collected easily with commercially available permanent magnets. In this study, a novel magnetic nanoparticle displaying protein G (protein G-BacMPs) was fabricated, and one-step cell separation for direct cell separation from whole blood was performed using the protein G-BacMPs. B lymphocytes (CD20+ cells), which cover less than 0.3 × 10-2 % of whole blood cells, were separated with 93% purity using protein G-BacMPs binding with anti-CD20 monoclonal antibodies. The results of this study demonstrate the utility of protein G-BacMPs and the magnetic cell separation approach based on protein G-BacMPs in numerous applications. © 2008 Materials Research Society.

To elucidate the mechanism of the electrochemical killing, the global gene expression of synchronously cultured Saccharomyces cerevisiae exposed to electrochemical stimulus was analyzed using DNA microarray. By applying a potential of 1.0 V vs. saturated calomel electrode (SCE), oxidative stress related genes were expressed after only 5 minutes. The gene for cta1 exhibited the highest expression level, while ROS gradually accumulated inside the cells. Genes for signal transduction which may be related to cellular apoptosis were also seen expressed.

Streptomycin-resistantMycobacterium tuberculosis has been attributed to two distinct classes of mutations, including point mutations within therpsL gene (three mutation sites) and therrs gene (seven mutation sites). We have developed an automated simultaneous detection system of multiple mutations based on thermal dissociation curve analysis for streptomycin resistance inM. tuberculosis using streptavidin-labeled bacterial magnetic particles (SA-BacMPs). With consideration for time and cost effectiveness, we used fewer PCR reactions, with a long PCR target (rpsL, 182 bp
rrs, 467 bp) including multiple mutation sites. In order to improve the amount of target DNA captured on BacMPs through streptavidin-biotin binding, several reaction conditions, such as salt species and concentration in the buffer, and reaction temperature were examined. Compared to the commonly used 1M NaCl solution, the amount of DNA captured on SA-BacMPs was about six times greater (approx 5 pmoles/50 μg BacMPs) in the 2M LiCl solution. Under these conditions, automated nucleotide discriminations of 10 targets inrpsL andrrs genes of streptomycin-resistant and wild-type strains were successfully performed at the same time. Copyright © 2006 Humana Press Inc. All rights of any nature whatsoever are reserved.

The interaction between oligonucleotides and long DNA fragments was analyzed by force curve measurements using atomic force microscopy (AFM). DNA fragments (150-base or 406-base) from the mitochondrial ATPase and cytochrome oxidase subunit III genes that contained a mismatch of one to three bases among Tunnus species were immobilized on glass slides. The statistical distribution of disruption forces between oligonucleotide probes (21-mer or 29-mer) and single stranded DNA fragments (150-base or 406-base) were analyzed by 40 or 180 force curve measurements. Histograms plotting the frequencies of disruption forces showed a wide distribution with a highest peak. The highest mean values in disruption force were obtained when DNA fragments with perfectly match sequences were employed. These results demonstrated that the specific sequence differences between long DNA fragments can be measured using force-based detection. A single base mismatch yielded a statistically significant 10% decrease in disruption force, furthermore, 2-base and 3-base mismatches provided approximately 20 and 25-30% decreases, respectively. Our results indicated that force-based detection potentially can be applied toward many other mismatched DNA detection techniques besides species-specific identification of tuna. © 2006 Elsevier B.V. All rights reserved.

Intra and interspecific nucleotide sequence variation of rDNA first internal transcribed spacer (ITS1) was analysed using all eight species of the genus Thunnus plus two out-group species within the same family, skipjack tuna Katsuwonus pelamis and striped bonito Sarda orientalis. Intraspecific nucleotide sequence variation in ITS1, including intra-genomic variation, was low, ranging from 0.003 to 0.014 [Kimura's two parameter distance (K2P)], whereas variation between species within the genus Thunnus ranged from 0.009 to 0.05. The Atlantic and Pacific northern bluefin tunas Thunnus thynnus thynnus and Thunnus thynnus orientalis, recently proposed to be distinct species, were found to share nearly identical ITS1 sequences (mean K2P = 0.006) well within the range of intraspecific variation. The northern bluefin tuna appeared to be a sister group to albacore Thunnus alalunga, with all other Thunnus species in a distinct clade. The ITS1 phylogeny was consistent with mtDNA phylogeny in clustering the three tropical Thunnus species (T. albacares, T. atlanticus and T. tonggol). Southern bluefin Thunnus maccoyii and bigeye Thunnus obesus tunas showed a closer affinity to this tropical tuna group than to the northern bluefin tuna and albacore. The molecular data supported mitochondrial introgression between species and contradicted morphological subdivision of the genus into two subgenera Neothunnus and Thunnus. © 2006 The Fisheries Society of the British Isles.

A novel bionanotechnological method for fabricating magnetic nanotubes with the aid of magnetic bacteria was investigated. Magnetospirillum magneticum strain AMB-1 was selected as a model to immobilize the extracted bacterial magnetic nanocrystals on the peptide nanotubes. Then, the magnetic nanocrystals were separated by using a magnet placed at the bottom of the glass tube containing the disrupted cells. The main advantage in using bacterial magnetic nanocrystals is that the mineral type, crystal size and morphology of the magnetic nanocrystals can be controlled by the selection of the bacterial species or strains.

In this study, nano-sized bacterial magnetic particles (BMPs) displaying human estrogen receptor ligand binding domain (ERLBD) on the surface was successfully produced by the magnetic bacterium, Magnetospirillum magneticum AMB-1. Furthermore, a non-isotopic binding assay for estrogenic compounds using the BMPs displaying ERLBD was developed. A BMP membrane-specific protein, Mms16, was used as an anchor molecule to localize ERLBD on the surface of BMPs. ERLBD-BMP complexes were simply extracted by magnetic separation from ruptured AMB-1 transformants and used for the assay based on the competitive binding of alkaline phosphatase conjugated 17β-estradiol (ALP-E2) as a tracer. Dissociation constant of the receptor was 2.3 nM. Inhibition curves were evaluated by the decrease in luminescence intensity resulting from the enzymatic reaction of alkaline phosphatase. The overall simplicity of this receptor binding assay results in a method that can be easily adapted to a high throughput format. Moreover, this method can be integrated into a fully-automated ligand screening system using magnetic separation. © 2004 Elsevier B.V. All rights reserved.

The marine environment is remarkably diverse and harbors a wide variety of organisms that have acquired novel metabolic functions and corresponding genes through evolutionary adaptation. We have screened many useful material such as UV-A resistance molecule, antibiotics, polysaccharide, from our culture collection containing cyanobacteria and photosynthetic bacteria isolated from marine environment. We introduce our research for screening of useful material from marine photosynthetic microorganisms. Furthermore, genetic manipulation for the purpose of metabolic engineering in marine microorganisms is described as the application of marine genomes. The uses of molecular approaches for analyzing microbial diversity have enhanced our knowledge of their population and lead the fact of abundant unculturable microbes. The isolation of novel bioactive compounds from environmental sources has been carried out by molecular approaches where the metagenomic libraries are constructed with directly extracted DNA from environmental samples.

1974年に&lt;I&gt;Anabaena cylindrica&lt;/I&gt;を用いた光水素生産が報告されて以降、シアノバクテリアを用いた水素生産研究には、&lt;I&gt;Anabaena&lt;/I&gt;、&lt;I&gt;Calothrix&lt;/I&gt;、&lt;I&gt;Nostoc&lt;/I&gt;、&lt;I&gt;Scytonema&lt;/I&gt;、&lt;I&gt;Synechococcus&lt;/I&gt; (&lt;I&gt;Cyanothece&lt;/I&gt;)など様々な窒素固定株が用いられている。これら窒素固定株は細胞形態の上、単細胞、ヘテロシストをもたない糸状細胞体、ヘテロシストをもつ糸状細胞体に大別可能で、それぞれの細胞形態ごとに異なった水素生産方式をとっているものと考えられている。一方、近年シアノバクテリアのSSUrDNA配列情報が加速度的に蓄積され、各株の分子系統位置を解析することが容易になっている。そこで我々は、保存株の水素生産能を測定し、同時にSSUrDNAを用いた分子系統解析をおこなって、水素生産能の高低と分子系統上の位置との関係について検討した。その結果、シアノバクテリアの水素生産能の高低が、ある程度系統位置を反映しており遺伝的に決定されている可能性が示唆された。&lt;br&gt; シアノバクテリアを用いた光水素生産システムの今後の開発には、高い水素生産能力をもつ株の更なる探索と、バイオテクノロジーを駆使して遺伝的に高いポテンシャルをもった株をさらに改良・育種してゆくことが重要であると考えられる。これら探索や遺伝子改変のモデル生物の選定にあたって、分子系統位置情報がどのように活用できるのか議論してみたい。

The development of a rapid and high-throughput detection system for endocrine disrupting chemicals (EDCs) has been required in the recent years. A fully automated immunoassay system was described for the detection of EDCs, such as alkylphenol ethoxylates (APEs), bisphenol A (BPA) and linear alkylbenzene sulfonates (LASs), using monoclonal antibodies chemically conjugated to bacterial magnetic particles (BMPs) and alkaline phosphatase (ALP)-conjugated EDCs. EDC concentrations were evaluated by the decrease in luminescence based on the competitive reaction of EDCs and ALP-conjugated EDCs. Full automation of the BMP-based immunoassay was achieved by using an automated eight-way pipet moving at X-, Y- and Z-direction and a B/F separation unit. B/F separation was performed on the tip surface of eight-way pipet with a retractable magnet mounted close to the pipet tip. Immunoreactions were saturated after 10min, and the assay was completed within 15min. The detection ranges for APE, BPA and LAS were 6.6ppb-66ppm, 2.3ppt-2.3ppm, and 35ppt-35ppm, respectively. This BMP-based immunoassay system has advantages due to its high sensitivity and rapid measurement of samples. © 2002 Elsevier Science B.V. All rights reserved.

A centrifugation method using a sucrose density gradient was established to distinguish buoyant cyanobacterial cells based on their cell density. The marine strain Nostoc NKBG 500017 was selected, because it showed the lowest density among 71 strains examined from our culture collection and also exhibited stable buoyancy. The cell suspension was homogeneous in a 100 mL graduated cylinder in the dark for at least 12 h. Stability was also confirmed using an artificial water column consisting of polyethylene pipe 2 m in height and 11 cm in diameter with a halogen light source at the top. Cells were suspended well throughout the water column and no precipitation was observed at the bottom of the column after 24 h incubation. Most cells were retained in the upper part of the water column from 10 cm to 30 cm depth. Growth was inhibited by the addition of tributyltin (TBT), an endocrine disruptor. The autofluorescence intensity of the cells decreased with increasing TBT concentration. The stable buoyancy and TBT sensitivity of Nostoc NKBG 500017 indicate that the strain is a possible candidate for monitoring contamination by toxic chemicals in the marine environment.

Bacterial magnetic particles (BMPs) were used for the identification of cyanobacterial DNA. Genus-specific oligonucleotide probes for the detection of Anabaena spp., Microcystis spp., Nostoc spp., Oscillatoria spp., and Synechococcus spp. were designed from the variable region of the cyanobacterial 16S rDNA of 148 strains. These oligonucleotide probes were immobilized on BMPs via streptavidin-biotin conjugation and employed for magnetic-capture hybridization against digoxigenin-labeled cyanobacterial 16S rDNA. Bacterial magnetic particles were magnetically concentrated, spotted in 100-μm-size microwell on MAG-microarray, and the fluorescent detection was performed. This work details the development of an automated technique for the magnetic isolation, the concentration of hybridized DNA, and the detection of specific target DNA on MAG-microarray. The entire process of hybridization and detection was automatically performed using a magnetic-separation robot and all five cyanobacterial genera were successfully discriminated. © 2001 John Wiley &amp
Sons, Inc.

Nonmagnetic mutants of Magnetospirillum magneticum AMB-1 were recovered following mini-Tn5 transposon mutagenesis. Transconjugants with kanamycin resistance were obtained at a frequency of 2.7 × 10-7 per recipient. Of 3327 transconjugants, 62 were defective for bacterial magnetic particle (BMP) synthesis. The frequency of independent transposition events for nonmagnetic mutants was about 1.4% in transconjugants. Further analysis of DNA sequences flanking transposon by inverted polymerase chain reaction allowed isolation of at least 10 genes or DNA sequences involved in BMP synthesis in M. magneticum AMB-1.

16S rRNA-targeted identification of cyanobacterial genera, Anabaena, Microcystis, Nostoc, Oscillatoria, Synechococcus was developed using bacterial magnetic particles (BMPs). 16S rRNA-targeted capture probes designed from the genus specific region of the 16S rRNA sequence were immobilized on BMPs. Identification of cyanobacteria was performed by a sandwich hybridization using the capture probes - BMP conjugates and a digoxigenin (DIG)-labeled detector probe complementary to the highly conserved 16S rRNA sequence for cyanobacteria. The luminescence intensity of the probe/target-BMP hybrids was measured after reaction with alkaline phosphatase conjugated anti-DIG antibody. Five species of cyanobacteria from five different genera were successfully discriminated using this magnetic capture system.

Restriction analysis detected two types of bigeye tuna (α and β)
the α type was in the majority in the Atlantic but nearly absent in the Indo-Pacific. The α type shared a larger number of restriction sites with other species than the conspecific β type, but bigeye-specific nucleotide substitutions with a novel diagnostic restriction profile were found. Although the nucleotide sequence difference between Atlantic and Pacific sub-species of the northern bluefin tuna was nearly the largest among species, individuals possessing the Atlantic type of mtDNA were found at very low frequency in the Pacific and vice versa. Previous RFLP markers were found to be diagnostic for the other five species (albacore, blackfin, longtail, southern bluefin and yellowfin tunas). Genetic information is provided to discriminate all Thunnus species regardless of their origin and to identify the ocean of capture in the northern bluefin and bigeye tunas. © 2001 The Fisheries Society of the British Isles.

We investigated the relationship between the synthesis of bacterial magnetic particles (BMPs) and the transcription of magA gene-encoding iron transport protein using synchronous culture of Magnetospirillum magneticum AMB-1. Synchronously cultured cells were subjected to transmission electron microscopic observation and fluorescence in situ hybridization. The average number of BMPs slowly increased in the cell with increasing cell size. A sharp increase in BMPs occurred just before cell division and resulted in maximum BMP production of 30 particles/cell. The transcription of magA was regulated immediately before and after cell division.

The production of bacterial magnetic particles (BMPs) by recombinant Magnetospirillum magneticum AMB-1 harboring the plasmid pKML was enhanced in pH-regulated fed-batch culture. The addition of fresh nutrients was feedback-controlled as a function of the pH of the culture. The yield of BMPs was optimized by adjusting the rate of ferric iron addition. Feeding ferric quinate at 15.4 μg/min resulted in a BMP yield of 7.5 mg/l, which is the highest yield so far reported. Expression of a plasmid-encoded fusion protein and segregation of the plasmid during bacterial growth were both stable during fed-batch culture. More than 75% of the cells retained the plasmid for 130 h under antibiotic-free conditions. In addition, the fusion protein permitting the display of a specific protein on the BMP surface was also stably expressed.

Proteins located within the lipid bilayer, surrounding the intracellular bacterial magnetic particles (BMP) from Magnetospirillum sp. AMB-1, were separated using SDS-PAGE. Several major proteins of approximate molecular weight 66.2, 35.6, and 24.8 kDa were identified. The N-terminal amino acid sequence of one of these proteins, designated MpsA, was determined and used to design a pair of PCR primers which amplified a 105 bp DNA fragment from AMB-1 genomic DNA. Gene-walking, using anchored PCR, was used to determine the complete nucleotide sequence (954 bp) of the mpsA gene. The mpsA encodes a 317 amino acid protein which does not have an N-terminal cytoplasmic transport signal sequence. Intracellular localization studies were carried out using an mpsA-luc gene fusion expressed in AMB-1 following gene transfer by conjugation. The gene fusion was constructed by cloning a 1.6 kb mpsA fragment upstream of luc in the conjugal plasmid pKLC. The MpsA-Luc fusion protein was preferentially located on the magnetic particle membrane. Although the function of MpsA remains unknown, homology searches suggest similarity with the α subunit of acetyl-CoA carboxylase and the CoA-binding motif. (C) 2000 Academic Press.

The endogenous plasmid pSY10 in the marine cyanobacterium Synechococcus sp. NKBG042902 is maintained at a high copy number when cells are grown in seawater and at a low copy number when cultured in freshwater. The mechanism of salinity-regulated replication of this plasmid was investigated. Transcription of repA was depressed under freshwater, which was accompanied by a low copy number of pSY10 and the appearance of a new protein that was expressed only in cells cultured in freshwater. This protein was observed to bind to putative repA promoters (Prep1 and Prep2) on pSY10. Moreover, this protein was observed only in Synechococcus sp. NKBG042902. The data suggest that this protein(s) regulates repA transcription in pSY10, stress responsive and encoded by the host chromosome.

The eicosapentaenoic acid (EPA) synthesis gene cluster from an EPA-producing bacterium, Shewanella sp. SCRC-2738, was cloned into a broad-host range vector, pJRD215, and then introduced into a marine cyanobacterium, Synechococcus sp. NKBG15041c, by conjugation. The transconjugant cyanobacteria produced 3.7 ± 0.2% (2.24 ± 0.13 mg/L) EPA (n-3) and 2.5 ± 0.2% (1.49 ± 0.06 mg/L) eicosatetraenoic acid (n-3) of the total fatty acids when the cells were cultured at 23°C at a light intensity of 1,000-1,500 Lux. The EPA and eico-satetraenoic acid contents of the cells were increased to 4.6 ± 0.6% (3.86 ± 1.11 mg/L) and 4.7 ± 0.3% (3.86 ± 0.82 mg/L), and 7.5 ± 0.3% (1.76 ± 0.10 mg/L) and 5.1 ± 0.2% (1.19 ± 0.06 mg/L) when they were cultured at low temperature (18°C) and at lower light intensity (40 Lux), respectively.

We report the identification of five proteins expressed specifically on the bacterial magnetic particle (BMP) membrane of Magnetospirillum sp. AMB-1. These proteins are major components of the BMP membrane. The molecular weights were determined to be 12.0, 16.0, 24.8, 35.6, and 66.2 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Of these five, the 16.0- kDa protein was the most abundant in the BMP membrane. Furthermore, the 16.0- kDa protein consisted of two components each of differing pI. The 35.6-kDa protein was the second most abundant protein of the five detected.

The aim of this study was to develop improved methodologies for bulk culturing of biotechnologically useful marine cyanobacteria in the open ocean. We have investigated the viability of using coal fly ash (CFA) blocks as the support medium in a novel floating culture system for marine microalgae. The marine cyanobacterium Synechococcus sp. NKBG 040607 was found to adhere to floating CFA blocks in liquid culture medium. Maximum density of attached cells of 2.0 x 108 cells/cm2 was achieved using seawater. The marine cyanobacterium Synechococcus sp. NKBG 042902 weakly adhered to floating CFA blocks in BG-11 medium. Increasing the concentration of calcium ion in the culture medium enhanced adherence to CFA blocks.

Twenty four strains out of 191 marine microalgal strains exhibited cadmium (Cd) resistance. They were tested for their Cd removal ability in growth media containing 50 μM Cd. Six strains out of 19 green algae and one out of five cyanobacteria removed more than 10% of total Cd from the medium. The marine green alga Chlorella sp. NKG16014 showed the highest removal of Cd 48.7% of total. Cd removal by NKG16014 was further quantitatively evaluated by measuring the amount of cell adsorption and intracellular accumulation. After 12 days incubation, 67% of the removed Cd was accumulated intracellularly and 25% of the Cd removed was adsorbed on the algal cell surface. The maximum Cd adsorption (q(max)) was estimated to be 37.0 mg Cd (g dry cells)-1 using the Langmuir sorption model. The Cd removal by freeze-dried NKG16014 cells was also determined. Cd was more quickly adsorbed by dried cells than that by living cells, with a q(max) of 91.0 mg Cd (g dry cells)-1. Copyright (C) 1999 Elsevier Science B.V.

The authors have examined the response to UV-A irradiation of the UV-A-resistant marine cyanobacterium Oscillatoria sp. NKBG 091600, which produces the UV-A-absorbing compound biopterin glucoside. The expression of a 60 kDa protein was markedly induced at 500 min after UV-A irradiation. This protein was identified by N-terminal amino acid sequence analysis as GroEL. Northern blot analysis demonstrated that GroEL synthesis was controlled by UV-A at the transcriptional level. A CIRCE element and a putative SOS consensus sequence were found upstream of the groESL operon, overlapping two putative promoter sequences. Primer extension analysis revealed that groESL transcription in UV-A-induced cells starts from the proximal promoter overlapped by the SOS consensus sequence. This indicates that an SOS response regulation is instrumental in UV-A-induced GroEL expression of Oscillatoria sp. NKBG 091600. Furthermore, this UV-A-inducible GroEL may function to upregulate biopterin glucoside biosynthesis, thereby allowing growth under UV-A irradiation.

&nbsp;&nbsp;Magnetospirillum sp. AMB-1 is a magnetic bacterium which synthesizes intracellular particles of magnetite (Fe₃O₄). We isolated magA gene required for the synthesis of bacterial magnetic particles (BMPs) and it encodes a polypeptide which is an iron transport protein. Intracellular localization of the MagA protein was studied using magA - luc fusion proteins. The fusion protein was also detected on the surface of the lipid bilayer covering the BMPs. One particular BMP-associated protein designated &ldquo;Mps&rdquo; was also isolated and characterized. The localization analysis using mps-luc fusion protein showed that there is a significant preference for the magnetosome membrane during partitioning of the Mps and the Mps protein preferentially associates with the magnetosome membrane.

The eicosapentaenoic acid (EPA) synthesis gene cluster isolated from a marine bacterium, Shewanella putrefaciens strain SCRC-2738, was cloned and expressed in the marine cyanobacterium Synechococcus sp. A broad-host-range cosmid vector, pJRD215 (10.2 kb, Sm-r Km(r)), was used to clone a 38 kb insert, pEPA, containing the EPA synthesis gene cluster, creating plasmid pJRDEPA (approx. 48 kb). This plasmid was transferred to the cyanobacterial host at a frequency of 2.2 x 10(-7). Cyanobacterial transconjugants grown at 29 degrees C produced 0.12 mg EPA (g dry weight)(-1), whereas those grown at 23 degrees C produced 0.56 mg EPA (g dry weight)(-1). The yield was further improved to 0.64 mg (g dry weight)(-1) by incubation for 1 d at 17 degrees C. This is believed to be the first successful cloning and expression of such a large heterologous gene cluster in a marine cyanobacterium.

Two genes which encode the enzymes lycopene cyclase and phytoene desaturase in the aerobic photosynthetic bacterium Erythrobacter longus sp. strain Och101 have been cloned and sequenced. The gene for lycopene cyclase, designated crtY, was expressed in a strain of Escherichia coli which contained the crtE, B, I and Z genes encoding geranylgeranyl pyrophosphate synthase, phytoene synthase, phytoene desaturase, and β-carotene hydroxylase, respectively. As a result, zeaxanthin production was observed in E. coli transformants. In addition, expression of the E. longus gene crtI for phytoene desaturase in E. coli containing crtE and B resulted in the accumulation of lycopene in transformants. Zeaxanthin and lycopene were also determined by mass spectrum. Nucleotide sequence similarities between E. longus crtY gene and other microbial lycopene cyclase genes are 40.2% (Erwinia herbicola), 37.4% (Erwinia uredovora) and 22.9% (Synechococcus sp.), and those between phytoene desaturase genes are 50.3% (E. herbicola), 54.7% (E. uredovora) and 39.6% (Rhodobacter capsulatus).

Euglena gracilis Z is one of the few microorganisms which simultaneously produces antioxidant vitamins such as β-carotene and vitamins C and E. Photoheterotrophically cultured E. gracilis Z produced larger levels of biomass but with a lower content of antioxidant vitamins than photoautotrophically grown cultures. For efficient production of these vitamins, a two-step culture was performed. Cells were grown photoheterotrophically and then transferred to photoautotrophic conditions. When E. gracilis Z cells were grown in fed-batch culture under photoheterotrophic conditions, their density reached 19 g/L after 145 h. Subsequent transfer of these cells to photoautotrophic conditions increased vitamin content, enhancing the total vitamin yields, which were 71.0 mg/L of β-carotene, 30.1 mg/L of vitamin E, and 86.5 mg/L of vitamin C.

Screening of fatty acid composition in 150 strains of marine microalgae, cyanobacteria and green algae was carried out, and 20 strains showed relatively high contents of palmitoleic acid. Among them, two cyanobacteria, Phormidium sp. NKBG 041105 and Oscillatoria sp. NKBG 091600, showed an unusually high cis-palmitoleic acid content (54.5% and 54.4% of total fatty acid, respectively). Phormidium sp. NKBG 041105 had the highest cis-palmitoleic acid content per biomass (46.3 mg (g dry cell weight)-1), and cis-palrnitoleic acid composition was found to be constant with varying temperature. These results indicate that this cyanobacterium could be considered as a new source for palmitoleic acid. © 1995.

Inhibitors of melanin biosynthesis from marine microalgae were screened against a melanin-producing microorganism, Streptomyces bikiniensis. From 28 marine microalgal strains, 5 were found showing inhibitory activity. Of these, the extracts (50μl, 2μg total organic carbon/μl) from two marine green algae showed strongly inhibited melanin biosynthesis, but showed less than 30 % inhibitory activity against mushroom tyrosinase. © 1995 Chapman &amp
Hall.

Most of the 16S ribosomal RNA gene of a sulfate-reducing magnetic bacterium, RS-1, was sequenced, and phylogenetic analysis was carried out. The results suggest that RS-1 is a member of the δ-Proteobacteria, and it appears to represent a new genus. RS-1 is the first bacterium reported outside the α-Proteobacteria that contains magnetite inclusions. RS-1 therefore disrupts the correlation between the α-Proteobacteria and possession of magnetite inclusions, and that between the δ-Proteobacteria and possession of greigite inclusions. The existence of RS-1 also suggests that intracellular magnetite biomineralization is of multiple evolutionary origins. © 1995.

Many species of microalgae producing useful materials have been isolated from marine environments. For their industrial application, widely applicable and stable gene expression is required. It is necessary to establish gene transfer methods as an essential first step in genetic manipulation. Although gene transfer techniques for cyanobacteria have been developed, only naturally transformable strains have been used. Here, we describe recent progress made in developing gene transfer methods for marine cyanobacteria. The following are covered: (1) transformation, (2) electroporation, (3) conjugation, (4) particle gun. A plasmid from the marine cyanobacterium, Synechococcus sp., whose copy number is dependent on salinity, was characterized. This plasmid is being used to develop a stable and controllable gene expression system. © 1995 Kluwer Academic Publishers.

We have shown previously that the copy number of plasmid pSY10 from the marine cyanobacterium Synechococcus sp. NKBG 042902 is dependent on the salinity of the growth medium. We report here the complete nucleotide sequence (2561 bp) of this plasmid. The longest open reading frame, ORF-B (1.08 kb), occurs on a 1.6-kb EcoRI fragment. This ORF encodes a putative protein which is 360 aa residues in length and is 37.8% homologous to the replication protein of plasmid pCA2.4 from Synechocystis sp. strain PCC 6803, 35.8% homologous to an ORF from the Nostoc plasmid pGL2, and 33.2% homologous to the ORF of a plasmid from Lactobacillus plantarum, pC30il. Highly conserved regions of amino acid sequence were also found between ORF-B and other bacterial plasmids. © 1994 Academic Press, Inc.

A light diffusing optical fiber (LDOF) photobioreactor with an improved gas input system has been used for the high-density culture of a marine cyanobacterium Synechococcus sp. Optimum conditions for CO2 removal and biomass production were investigated. Maximum CO2 removal of 4.44 g/L/d was achieved using an initial cell concentration of 6.8 g/L. The biomass yield was 0.97 g/L for a 12-culture time. Continuous cultures, in which medium was filtered using a ceramic membrane module, showed enhanced growth, with a final cell concentration of 11.2 g/L. These results demonstrate the potential of LDOF photobioreactor units for CO2 removal and biomass production using marine cyanobacteria. © 1992 Humana Press Inc.

A marine cyanobacterium Synechococcus sp. NKBG 042902 grown in the presence of various NaCl concentrations showed different plasmid profiles. This strain possesses a 2.7-kb endogenous plasmid pSY10, whose copy number increased five-fold when the salinity of the growth medium was increased from 0 to 3% NaCl. Cells were grown initially in a fresh water medium and then transferred to a NaCl supplemented medium. The copy number of pSY10 began to increase after 7 h of incubation in 3% NaCl BG11 medium. This phenomenon was specific to NaCl and did not occur when other osmotica such as KCl and sorbitol were used. This is the first report of a cyanobacterial plasmid with a salinity-dependent copy number. © 1991.

A photobioreactor was constructed in the form of a Perspex column 900 mm tall with an internal diameter of 70 mm. The reactor volume was 1.8 L and the light source consisted of a metal-halide lamp to reproduce sunlight. Light was distributed through the culture using a new type of optical fiber that diffuses light out through its surface, perpendicular to the fiber axis. A cluster of 661 light-diffusing optical fibers (LDOFs) pass from the light source through the reactor column (60-cm culture depth) and are connected to a mirror at the top of the reactor. This biosolar reactor has been used for the production of glutamate from CO2 by the marine cyanobacterium Synechococcus sp. NKBG040607. We present here details of the construction of the biosolar reactor and characterization of its properties. The effect of light intensity on glutamate production was measured. Carbon dioxide-to-glutamate conversion ratios were determined at different cell densities: the maximum conversion ratio (28%) was achieved at a cell density of 3x108 cells/mL. A comparison of glutamate production using the LDOF biosolar reactor described here with production by batch culture using free or immobilized cells showed that use of an optical-fiber biosolar reactor increased glutamate-production efficiency 6.75-fold. We conclude that as a result of its high surface-to-volume ratio (692/m) increased photoproduction of useful compounds may be achieved. Such a system is generally applicable to all aspects of photobiotechnology. © 1991 Humana Press Inc.

A novel on-line fluorescence monitoring system for marine cyanobacterial cultivation was developed. This method is based on the measurement of intracellular phycocyanin content, which is the major light harvesting protein. A fluorescence spectrophotometer, equipped with a flow cell connected with a culture liquid recycling tube was used. Experiments were carried out using a marine unicellular cyanobacteria Synechococcus sp. NKBG 042902 isolated from Japanese coastal sea water. We have optimized excitation wavelength to avoid the light scattering, using non-pigmented old cells which no longer contained phycocyanin. At an excitation wavelength of 590 nm, light scattering was minimized. Viable cell concentration could be measured in the range of 2×106 to 2×108 cells per ml, without pronounced light scattering. Continuous monitoring of marine cyanobacteria cultivation was performed. Cell concentrations were determined by both culture fluorescence and by using a hemacytometer. A good linear correlation was obtained. We conclude that on-line monitoring of cyanobacterial culture fluorescence based on phycocyanin is a rapid, efficient and also versatile method for determining viable cell concentration. © 1991.

Among forty strains of marine cyanobacteria isolated in our laboratory, five strains had 1-3 different plasmids. The unicellular marine cyanobacterium, Synechococcus sp. NKBG 042902, contains at least three plasmids (pSY09, pSY10, and pSY11). However, these plasmids are cryptic. Therefore, a hybrid plasmid pUSY02 containing the 1.4kb HindIII fragment of pSY11 and Escherichia coli plasmid pUC18 was constructed. The plasmid pUSY02 transformed both marine Synechococcus sp. NKBG042902-YG1116, which is a cured strain, and fresh water Anacystis nidulans R2 by dark incubation or Ca2+ treatment. However, the plasmid pSG111 constructed from the plasmid DNA of A. nidulans R2 failed to transform marine Synechococcus sp. Electroporation was also applicable to transformation of marine Synechococcus sp. and fresh water A. nidulans R2. The plasmid pUSY02 was rapidly introduced into marine Synechococcus sp. © 1990 Humana Press Inc.

水産増養殖の発展を妨げている大きな要因は魚病の問題である。一方、トラフグは他の多くの海産養殖魚種と異なり、多くの疾病に強いことが知られている。申請者は、これまでに、トラフグを中心とした魚類の免疫系の解明を行い、その研究成果を利用して多くの免疫賦活剤の開発を行い魚病の制御に貢献してきた。本研究は、トラフグの細菌感染におけるサイトカインを中心とした自然免疫機構の役割をヒラメなどの他魚種と比較することによって、トラフグの耐病性のメカニズムを明らかにし、その知見を利用してサイトカインを用いた新しい免疫賦活剤を開発し、魚病を制御することを目的としている。H31/R1年度は、新規サイトカイン遺伝子の機能解明の一環として、作製したIL-17A/F1の役割を明らかにするために、IL-17A/F1-KO（knockout）メダカと野生メダカの腸管についてメタゲノム解析を行ったところ、IL-17A/F1-KOメダカの腸内細菌叢は、野生メダカと比べて明らかに異なる細菌叢の組成をしており、日和見感染する病原菌が増加していた。この現象は、Edwardsiella piscicida（旧名: tarda）が感染したKOおよび野生メダカの腸管においても観察できた。さらに、IL-17A/Fのより詳細な役割を理解するために、IL-17受容体A（IL-17RA）遺伝子についてもクローン化を行い、ゲノム編集による変異メダカの作製を試みた。一方、ヒラメとトラフグのE. piscicida感染症に対する抵抗性を調べるために、E. piscicida感染時のヒラメおよびトラフグの血清におけるリゾチーム活性（溶菌活性）を調べたところ、トラフグ血清のリゾチーム活性の方がヒラメに比べて有意に高かった。

”Frugality and fl uorescence”

”メタゲノム解析で海洋資源を生かす”

特集’微細藻類を用いた炭化水素燃料生産技術’の中で
「海洋微細藻類の工学的応用とエネルギー資源の可能性」

「知の市場」コンソーシアム進展

科学の話、出前します「研究の理解者増やしたい」