Melting temperature mapping method using imperfect-match linear long probes.

Identifying pathogenic microorganisms as early as possible is critical for selecting the appropriate antimicrobial therapy in infected patients. We previously reported the development of the Tm mapping method for identifying a broad range of pathogenic bacteria within 3 h of blood collection. However, the Tm mapping identification requires an analytical instrument with a tube-to-tube variation of no more than 0.1 °C, so we can only use a few instruments that have such high thermal accuracy. To address the problem, we developed the improved Tm mapping method using imperfect-match linear long quenching probes (IMLL Q-probes). Using IMLL Q-probes, almost all commercially available analytical instruments can be used for the Tm mapping method. Some bacterial species cannot be narrowed down to one species, but they can at least be narrowed down to the genus level. The Tm mapping method using IMLL Q-probes is useful for deciding on antimicrobial therapy in infected patients.

Novel rapid method for identifying and quantifying pathogenic bacteria within four hours of blood collection.

Sepsis is life-threatening organ dysfunction and is considered a major cause of health loss. However, since the current biomarkers of sepsis reflect the host's immune response to microorganisms, they would inevitably cause a time-lag. This means that there is still no truly reliable biomarker of sepsis. In the present study, we developed a novel method for identifying and quantifying unknown pathogenic bacteria within four hours of sample collection. The most important point of this study is that the novel method can be used to determine the number of bacteria in a sample as a novel biomarker of infectious diseases. Indeed, based on the number of bacteria, we were able to accurately estimate the severity of microbial infection. Furthermore, using the time-dependent changes in the number of bacteria, we were able to monitor the therapeutic effect accurately. The rapid identification and quantification of bacteria may change our approach to medical care.

Evolutionary Developments in Plant Specialized Metabolism, Exemplified by Two Transferase Families.

Plant specialized metabolism emerged from the land colonization by ancient plants, becoming diversified along with plant evolution. To date, more than 1 million metabolites have been predicted to exist in the plant kingdom, and their metabolic processes have been revealed on the molecular level. Previous studies have reported that rates of evolution are greater for genes involved in plant specialized metabolism than in primary metabolism. This perspective introduces topics on the enigmatic molecular evolution of some plant specialized metabolic processes. Two transferase families, BAHD acyltransferases and aromatic prenyltransferases, which are involved in the biosynthesis of paclitaxel and meroterpenes, respectively, have shown apparent expansion. The latter family has been shown to beinvolved in the biosynthesis of a variety of aromatic substances, including prenylated coumarins in citrus plants and shikonin in Lithospermum erythrorhizon. These genes have evolved in the development of each special subfamily within the plant lineage. The broadness of substrate specificity and the exon-intron structure of their genes may provide hints to explain the evolutionary process underlying chemodiversity in plants.

Self-organization of hepatocyte morphogenesis depending on the size of collagen microbeads relative to hepatocytes.

Recent advances in microfabrication technologies have enabled us to construct collagen gel microbeads, which can be cultured with hepatocytes. However, little is known about the hepatocyte-collagen gel microbead interactions. Here, we aimed to clarify the effects of the balance between cell-cell and cell-collagen gel microbead interactions on hepatocyte morphogenesis and functions. The magnitude of cell-microbead interactions was controlled by changing the size of the microbeads, which were smaller than, comparable to, and larger than hepatocytes. These small, medium, and large microbeads were cultured separately with primary hepatocytes. Phase-contrast and time-lapse imaging revealed that the medium microbeads significantly induced the construction of 3D structures composed of the microbeads and hepatocytes in a self-organizing manner, whereas hepatocytes formed 2D monolayers with the small or large microbeads. These results suggest that only the medium microbeads induced the 3D tissue formation of hepatocytes. Furthermore, liver-specific functions, such as albumin secretion and ammonia clearance, were significantly upregulated in the 3D structures. These findings are critical to understand how to control the construction of 3D hepatocyte tissues with hydrogel microbeads in the context of biofabrication.

Structural basis of autoinhibition and activation of the DNA-targeting ADP-ribosyltransferase pierisin-1.

ADP-ribosyltransferases transfer the ADP-ribose moiety of ßNAD+ to an acceptor molecule, usually a protein that modulates the function of the acceptor. Pierisin-1 is an ADP-ribosyltransferase from the cabbage butterfly Pieris rapae and is composed of N-terminal catalytic and C-terminal ricin B-like domains. Curiously, it ADP-ribosylates the DNA duplex, resulting in apoptosis of various cancer cells, which has raised interest in pierisin-1 as an anti-cancer agent. However, both the structure and the mechanism of DNA ADP-ribosylation are unclear. Here, we report the crystal structures of the N-terminal catalytic domain of pierisin-1, its complex with ßNAD+, and the catalytic domain with the linker connecting it to the ricin B-like domains. We found that the catalytic domain possesses a defined, positively charged region on the molecular surface but that its overall structure is otherwise similar to those of protein-targeting ADP-ribosyltransferases. Electrophoretic mobility shift assays and site-directed mutagenesis indicated that pierisin-1 binds double-stranded but not single-stranded DNA and that Lys122, Lys123, and Lys124, which are found in a loop, and Arg181 and Arg187, located in a basic cleft near the loop, are required for DNA binding. Furthermore, the structure of the catalytic domain with the linker revealed an autoinhibitory mechanism in which the linker occupies and blocks both the ßNAD+- and DNA-binding sites, suggesting that proteolytic cleavage to remove the linker is necessary for enzyme catalysis. Our study provides a structural basis for the DNA-acceptor specificity of pierisin-1 and reveals that a self-regulatory mechanism is required for its activity.

Macrocyclization Using RCM Reactions. Synthesis of Simple Metacyclophanes and Synthetic Efforts Toward Platycarynol, a 15-Membered Aromatic Ether.

- Preparation of 13- to 19-membered carbocycles (metacyclophanes) from 1,3-disubstituted benzene derivatives has been successfully carried out using the RCM reaction, but similar starting materials having diphenyl ether did not cyclize to 15-membered compounds, whose ground state conformations were calculated and discussed. Several attempts to cyclize to form platycarynol are described.

Melting Temperature Mapping Method: A Novel Method for Rapid Identification of Unknown Pathogenic Microorganisms within Three Hours of Sample Collection.

Acquiring the earliest possible identification of pathogenic microorganisms is critical for selecting the appropriate antimicrobial therapy in infected patients. We herein report the novel "melting temperature (Tm) mapping method" for rapidly identifying the dominant bacteria in a clinical sample from sterile sites. Employing only seven primer sets, more than 100 bacterial species can be identified. In particular, using the Difference Value, it is possible to identify samples suitable for Tm mapping identification. Moreover, this method can be used to rapidly diagnose the absence of bacteria in clinical samples. We tested the Tm mapping method using 200 whole blood samples obtained from patients with suspected sepsis, 85% (171/200) of which matched the culture results based on the detection level. A total of 130 samples were negative according to the Tm mapping method, 98% (128/130) of which were also negative based on the culture method. Meanwhile, 70 samples were positive according to the Tm mapping method, and of the 59 suitable for identification, 100% (59/59) exhibited a "match" or "broad match" with the culture or sequencing results. These findings were obtained within three hours of whole blood collection. The Tm mapping method is therefore useful for identifying infectious diseases requiring prompt treatment.

Eukaryote-Made Thermostable DNA Polymerase Enables Rapid PCR-Based Detection of Mycoplasma, Ureaplasma and Other Bacteria in the Amniotic Fluid of Preterm Labor Cases.

BACKGROUND: Intra-amniotic infection has long been recognized as the leading cause of preterm delivery. Microbial culture is the gold standard for the detection of intra-amniotic infection, but several days are required, and many bacterial species in the amniotic fluid are difficult to cultivate. METHODS: We developed a novel nested-PCR-based assay for detecting Mycoplasma, Ureaplasma, other bacteria and fungi in amniotic fluid samples within three hours of sample collection. To detect prokaryotes, eukaryote-made thermostable DNA polymerase, which is free from bacterial DNA contamination, is used in combination with bacterial universal primers. In contrast, to detect eukaryotes, conventional bacterially-made thermostable DNA polymerase is used in combination with fungal universal primers. To assess the validity of the PCR assay, we compared the PCR and conventional culture results using 300 amniotic fluid samples. RESULTS: Based on the detection level (positive and negative), 93.3% (280/300) of Mycoplasma, 94.3% (283/300) of Ureaplasma, 89.3% (268/300) of other bacteria and 99.7% (299/300) of fungi matched the culture results. Meanwhile, concerning the detection of bacteria other than Mycoplasma and Ureaplasma, 228 samples were negative according to the PCR method, 98.2% (224/228) of which were also negative based on the culture method. Employing the devised primer sets, mixed amniotic fluid infections of Mycoplasma, Ureaplasma and/or other bacteria could be clearly distinguished. In addition, we also attempted to compare the relative abundance in 28 amniotic fluid samples with mixed infection, and judged dominance by comparing the Ct values of quantitative real-time PCR. CONCLUSIONS: We developed a novel PCR assay for the rapid detection of Mycoplasma, Ureaplasma, other bacteria and fungi in amniotic fluid samples. This assay can also be applied to accurately diagnose the absence of bacteria in samples. We believe that this assay will positively contribute to the treatment of intra-amniotic infection and the prevention of preterm delivery.

Diversity in the complexity of phosphate starvation transcriptomes among rice cultivars based on RNA-Seq profiles.

Rice has developed several morphological and physiological strategies to adapt to phosphate starvation in the soil. In order to elucidate the molecular basis of response to phosphate starvation, we performed mRNA sequencing of 4 rice cultivars with variation in growth response to Pi starvation as indicated by the shoot/root dry weight ratio. Approximately 254 million sequence reads were mapped onto the IRGSP-1.0 reference rice genome sequence and an average of about 5,000 transcripts from each cultivar were found to be responsive under phosphate starvation. Comparative analysis of the RNA-Seq profiles of the 4 cultivars revealed similarities as well as distinct differences in expression of these responsive transcripts. We elucidated a set of core responsive transcripts including annotated and unannotated transcripts commonly expressed in the 4 cultivars but with different levels of expression. De novo assembly of unmapped reads to the Nipponbare genome generated a set of sequence contigs representing potential new transcripts that may be involved in tolerance to phosphate starvation. This study can be used for identification of genes and gene networks associated with environmental stress and the development of novel strategies for improving tolerance to phosphate starvation in rice and other cereal crops.

Genomic inversion caused by gamma irradiation contributes to downregulation of a WBC11 homolog in bloomless sorghum.

Epicuticular wax (bloom) plays important roles in protecting the tissues of sorghum (Sorghum bicolor (L.) Moench) plants from abiotic stresses. However, reducing wax content provides resistance to greenbug and sheath blight-a useful trait in agricultural crops. We generated a sorghum bloomless (bm) mutant by gamma irradiation. One bm population segregated for individuals with and without epicuticular wax at a frequency of 72:22, suggesting that the bm mutation was under the control of a single recessive nuclear gene. Genes differentially expressed in the wild-type and the bm mutant were identified by RNA-seq technology. Of the 31 downregulated genes, Sb06g023280 was the most differentially expressed and was similar to WBC11, which encodes an ABC transporter responsible for wax secretion in Arabidopsis. An inversion of about 1.4 Mb was present in the region upstream of the Sb06g023280 gene in the bm mutant; it is likely that this inversion changed the promoter sequence of the Sb06g023280 gene. Using genomic PCR, we confirmed that six independent F2 bm mutant-phenotype plants carried the same inversion. Therefore, we concluded that the inversion involving the Sb06g023280 gene inhibited wax secretion in the bloomless sorghum.

RiceXPro version 3.0: expanding the informatics resource for rice transcriptome.

A wide range of resources on gene expression profiling enhance various strategies in plant molecular biology particularly in characterization of gene function. We have updated our gene expression profile database, RiceXPro (http://ricexpro.dna.affrc.go.jp/), to provide more comprehensive information on the transcriptome of rice encompassing the entire growth cycle and various experimental conditions. The gene expression profiles are currently grouped into three categories, namely, 'field/development' with 572 data corresponding to 12 data sets, 'plant hormone' with 143 data corresponding to 13 data sets and 'cell- and tissue-type' comprising of 38 microarray data. In addition to the interface for retrieving expression information of a gene/genes in each data set, we have incorporated an interface for a global approach in searching an overall view of the gene expression profiles from multiple data sets within each category. Furthermore, we have also added a BLAST search function that enables users to explore expression profile of a gene/genes with similarity to a query sequence. Therefore, the updated version of RiceXPro can be used more efficiently to survey the gene expression signature of rice in sufficient depth and may also provide clues on gene function of other cereal crops.

RiceFREND: a platform for retrieving coexpressed gene networks in rice.

Similarity of gene expression across a wide range of biological conditions can be efficiently used in characterization of gene function. We have constructed a rice gene coexpression database, RiceFREND (http://ricefrend.dna.affrc.go.jp/), to identify gene modules with similar expression profiles and provide a platform for more accurate prediction of gene functions. Coexpression analysis of 27 201 genes was performed against 815 microarray data derived from expression profiling of various organs and tissues at different developmental stages, mature organs throughout the growth from transplanting until harvesting in the field and plant hormone treatment conditions, using a single microarray platform. The database is provided with two search options, namely, 'single guide gene search' and 'multiple guide gene search' to efficiently retrieve information on coexpressed genes. A user-friendly web interface facilitates visualization and interpretation of gene coexpression networks in HyperTree, Cytoscape Web and Graphviz formats. In addition, analysis tools for identification of enriched Gene Ontology terms and cis-elements provide clue for better prediction of biological functions associated with the coexpressed genes. These features allow users to clarify gene functions and gene regulatory networks that could lead to a more thorough understanding of many complex agronomic traits.

DaizuBase, an integrated soybean genome database including BAC-based physical maps.

Soybean [Glycine max (L) Merrill] is one of the most important leguminous crops and ranks fourth after to rice, wheat and maize in terms of world crop production. Soybean contains abundant protein and oil, which makes it a major source of nutritious food, livestock feed and industrial products. In Japan, soybean is also an important source of traditional staples such as tofu, natto, miso and soy sauce. The soybean genome was determined in 2010. With its enormous size, physical mapping and genome sequencing are the most effective approaches towards understanding the structure and function of the soybean genome. We constructed bacterial artificial chromosome (BAC) libraries from the Japanese soybean cultivar, Enrei. The end-sequences of approximately 100,000 BAC clones were analyzed and used for construction of a BAC-based physical map of the genome. BLAST analysis between Enrei BAC-end sequences and the Williams82 genome was carried out to increase the saturation of the map. This physical map will be used to characterize the genome structure of Japanese soybean cultivars, to develop methods for the isolation of agronomically important genes and to facilitate comparative soybean genome research. The current status of physical mapping of the soybean genome and construction of database are presented.

Global transcriptome analysis reveals distinct expression among duplicated genes during sorghum-interaction.

BACKGROUND: Sorghum (Sorghum bicolor L. Moench) is a rich source of natural phytochemicals. We performed massive parallel sequencing of mRNA to identify differentially expressed genes after sorghum BTx623 had been infected with Bipolaris sorghicola, a necrotrophic fungus causing a sorghum disease called target leaf spot. RESULT: Seventy-six-base-pair reads from mRNAs of mock- or pathogen-infected leaves were sequenced. Unannotated transcripts were predicted on the basis of the piling-up of mapped short reads. Differentially expressed genes were identified statistically; particular genes in tandemly duplicated putative paralogs were highly upregulated. Pathogen infection activated the glyoxylate shunt in the TCA cycle; this changes the role of the TCA cycle from energy production to synthesis of cell components. The secondary metabolic pathways of phytoalexin synthesis and of sulfur-dependent detoxification were activated by upregulation of the genes encoding amino acid metabolizing enzymes located at the branch point between primary and secondary metabolism. Coordinated gene expression could guide the metabolic pathway for accumulation of the sorghum-specific phytochemicals 3-deoxyanthocyanidin and dhurrin. Key enzymes for synthesizing these sorghum-specific phytochemicals were not found in the corresponding region of the rice genome. CONCLUSION: Pathogen infection dramatically changed the expression of particular paralogs that putatively encode enzymes involved in the sorghum-specific metabolic network.

Evaluation of the effectiveness of light streamer tori-lines and characteristics of bait attacks by seabirds in the western North Pacific.

To improve the effectiveness of tori-lines it is necessary to evaluate the ability of tori-lines to mitigate seabird bycatch and determine what kind of seabird species gather during line settings, attack the bait and are incidentally caught. We conducted two experiments in the western North Pacific and examined the effectiveness for seabird mitigation of light streamer tori-lines which have no long streamers but many light (short) streamers and are mainly used in the North Pacific area. Firstly, the effectiveness of two different types of tori-line (light streamer (1 m) and long streamer (up to 7 m) tori-line) and of two different colors (yellow and red) of light streamers for seabird bycatch avoidance was evaluated using 567 sets based on data from 20 offshore surface commercial longliners. No significant difference in the bycatch number between the different tori-line types and streamer colors was found. Secondly, we investigated the characteristics of the seabird bycatch in the North Pacific and the effectiveness of three different types of streamers (light, hybrid and modified light types) by detailed observations of seabird attacks using a chartered longline vessel. Although the appearance rate of albatrosses and shearwaters were 40.9% and 27.7%, Laysan albatross was the main seabird species that followed the vessel but shearwaters seldom followed the vessel and did not aggregate during line setting. In all attacks on bait observed during line settings, 81% and 7% were by albatrosses and shearwaters, respectively. In the number of primary attacks by Laysan albatrosses which attacked most aggressively of all seabirds, there were no significant differences among the tori-line types. No individuals of shearwater were caught. The results of both experiments indicated that light streamer tori-lines were as effective as tori-lines with long streamers for mitigating seabird bycatch in the North Pacific.

Genome-wide transcriptome dissection of the rice root system: implications for developmental and physiological functions.

The root system is a crucial determinant of plant growth potential because of its important functions, e.g. uptake of water and nutrients, structural support and interaction with symbiotic organisms. Elucidating the molecular mechanism of root development and functions is therefore necessary for improving plant productivity, particularly for crop plants, including rice (Oryza sativa). As an initial step towards developing a comprehensive understanding of the root system, we performed a large-scale transcriptome analysis of the rice root via a combined laser microdissection and microarray approach. The crown root was divided into eight developmental stages along the longitudinal axis and three radial tissue types at two different developmental stages, namely: epidermis, exodermis and sclerenchyma; cortex; and endodermis, pericycle and stele. We analyzed a total of 38 microarray data and identified 22,297 genes corresponding to 17,010 loci that showed sufficient signal intensity as well as developmental- and tissue type-specific transcriptome signatures. Moreover, we clarified gene networks associated with root cap function and lateral root formation, and further revealed antagonistic and synergistic interactions of phytohormones such as auxin, cytokinin, brassinosteroids and ethylene, based on the expression pattern of genes related to phytohormone biosynthesis and signaling. Expression profiling of transporter genes defined not only major sites for uptake and transport of water and nutrients, but also distinct signatures of the radial transport system from the rhizosphere to the xylem vessel for each nutrient. All data can be accessed from our gene expression profile database, RiceXPro (http://ricexpro.dna.affrc.go.jp), thereby providing useful information for understanding the molecular mechanisms involved in root system development of crop plants.

A novel eukaryote-made thermostable DNA polymerase which is free from bacterial DNA contamination.

To achieve the production of a thermostable DNA polymerase free from bacterial DNA contamination, we developed eukaryote-made thermostable DNA (Taq) polymerase. The novel eukaryote-made thermostable DNA polymerase resolves the problem of contaminating bacterial DNA in conventional bacterially made thermostable DNA polymerase as a result of its manufacture and incomplete purification. Using eukaryote-made thermostable DNA polymerase, the sensitive and reliable detection of bacteria becomes feasible for large fields, thereby making the development of a wide range of powerful applications possible.

Distinct evolutionary patterns of Oryza glaberrima deciphered by genome sequencing and comparative analysis.

Here we present the genomic sequence of the African cultivated rice, Oryza glaberrima, and compare these data with the genome sequence of Asian cultivated rice, Oryza sativa. We obtained gene-enriched sequences of O. glaberrima that correspond to about 25% of the gene regions of the O. sativa (japonica) genome by methylation filtration and subtractive hybridization of repetitive sequences. While patterns of amino acid changes did not differ between the two species in terms of the biochemical properties, genes of O. glaberrima generally showed a larger synonymous-nonsynonymous substitution ratio, suggesting that O. glaberrima has undergone a genome-wide relaxation of purifying selection. We further investigated nucleotide substitutions around splice sites and found that eight genes of O. sativa experienced changes at splice sites after the divergence from O. glaberrima. These changes produced novel introns that partially truncated functional domains, suggesting that these newly emerged introns affect gene function. We also identified 2451 simple sequence repeats (SSRs) from the genomes of O. glaberrima and O. sativa. Although tri-nucleotide repeats were most common among the SSRs and were overrepresented in the protein-coding sequences, we found that selection against indels of tri-nucleotide repeats was relatively weak in both African and Asian rice. Our genome-wide sequencing of O. glaberrima and in-depth analyses provide rice researchers not only with useful genomic resources for future breeding but also with new insights into the genomic evolution of the African and Asian rice species.

Rice TOGO Browser: A platform to retrieve integrated information on rice functional and applied genomics.

The Rice TOGO Browser is an online public resource designed to facilitate integration and visualization of mapping data of bacterial artificial chromosome (BAC)/P1-derived artificial chromosome (PAC) clones, genes, restriction fragment length polymorphism (RFLP)/simple sequence repeat (SSR) markers and phenotype data represented as quantitative trait loci (QTLs) onto the genome sequence, and to provide a platform for more efficient utilization of genome information from the point of view of applied genomics as well as functional genomics. Three search options, namely keyword search, region search and trait search, generate various types of data in a user-friendly interface with three distinct viewers, a chromosome viewer, an integrated map viewer and a sequence viewer, thereby providing the opportunity to view the position of genes and/or QTLs at the chromosomal level and to retrieve any sequence information in a user-defined genome region. Furthermore, the gene list, marker list and genome sequence in a specified region delineated by RFLP/SSR markers and any sequences designed as primers can be viewed and downloaded to support forward genetics approaches. An additional feature of this database is the graphical viewer for BLAST search to reveal information not only for regions with significant sequence similarity but also for regions adjacent to those with similarity but with no hits between sequences. An easy to use and intuitive user interface can help a wide range of users in retrieving integrated mapping information including agronomically important traits on the rice genome sequence. The database can be accessed at http://agri-trait.dna.affrc.go.jp/.

Field transcriptome revealed critical developmental and physiological transitions involved in the expression of growth potential in japonica rice.

BACKGROUND: Plant growth depends on synergistic interactions between internal and external signals, and yield potential of crops is a manifestation of how these complex factors interact, particularly at critical stages of development. As an initial step towards developing a systems-level understanding of the biological processes underlying the expression of overall agronomic potential in cereal crops, a high-resolution transcriptome analysis of rice was conducted throughout life cycle of rice grown under natural field conditions. RESULTS: A wide range of gene expression profiles based on 48 organs and tissues at various developmental stages identified 731 organ/tissue specific genes as well as 215 growth stage-specific expressed genes universally in leaf blade, leaf sheath, and root. Continuous transcriptome profiling of leaf from transplanting until harvesting further elucidated the growth-stage specificity of gene expression and uncovered two major drastic changes in the leaf transcriptional program. The first major change occurred before the panicle differentiation, accompanied by the expression of RFT1, a putative florigen gene in long day conditions, and the downregulation of the precursors of two microRNAs. This transcriptome change was also associated with physiological alterations including phosphate-homeostasis state as evident from the behavior of several key regulators such as miR399. The second major transcriptome change occurred just after flowering, and based on analysis of sterile mutant lines, we further revealed that the formation of strong sink, i.e., a developing grain, is not the major cause but is rather a promoter of this change. CONCLUSIONS: Our study provides not only the genetic basis for functional genomics in rice but also new insight into understanding the critical physiological processes involved in flowering and seed development, that could lead to novel strategies for optimizing crop productivity.