Dietary habits and plasma lipid concentrations in a general Japanese population.

INTRODUCTION: Accumulating data on the associations between food consumption and lipid composition in the body is essential for understanding the effects of dietary habits on health. OBJECTIVES: As part of omics research in the Tohoku Medical Megabank Community-Based Cohort Study, this study sought to reveal the dietary impact on plasma lipid concentration in a Japanese population. METHODS: We conducted a correlation analysis of food consumption and plasma lipid concentrations measured using mass spectrometry, for 4032 participants in Miyagi Prefecture, Japan. RESULTS: Our analysis revealed 83 marked correlations between six food categories and the concentrations of plasma lipids in nine subclasses. Previously reported associations, including those between seafood consumption and omega-3 fatty acids, were validated, while those between dairy product consumption and odd-carbon-number fatty acids (odd-FAs) were validated for the first time in an Asian population. Further analysis suggested that dairy product consumption is associated with odd-FAs via sphingomyelin (SM), which suggests that SM is a carrier of odd-FAs. These results are important for understanding odd-FA metabolism with regards to dairy product consumption. CONCLUSION: This study provides insight into the dietary impact on plasma lipid concentration in a Japanese population.

Uncovering Ecdysozoa-specific Sphingomyelin Synthase by Phylogenetic Analysis of Metazoan Sequences.

Sphingomyelin (SM) is a membrane phospholipid that is widely distributed in Metazoa; it is the major constituent of myelin sheaths in vertebrates. In mammals, two genes (SMS1 and SMS2) are responsible for its synthesis. No SM-producing genes have been clearly identified in insects and crustaceans (Ecdysozoa) despite the presence of a myelin sheath-like structure in shrimps. Since the rapid transmission of electrical signals requires the use of an insulating material in the nerve, it is possible that the convergent evolution of enzymes to synthesize the insulating compounds for the nervous system also occurred in animals other than vertebrates. Our exhaustive phylogenetic search for metazoan SM synthase identified an Ecdysozoa-specific SM synthase candidate, SMSe, which is absent in Drosophila and Lophotrochozoa. All Ecdysozoa lack the homolog of myelin basic- and proteolipid proteins present in mammals. We propose an evolutionary path of SM synthase and discuss the origin of the myelin structure in Metazoa.

The Validation of the VereBeef™ Detection Kit.

VereBeef™ Detection Kit, incorporating both multiplex PCR and microarray technologies on a lab-on-chip platform, is intended for qualitative detection and differentiation of Escherichia coli O157:H7, E. coli O26, E. coli O45, E. coli O103, E. coli O111, E. coli O121, E. coli O145, Shiga toxin-producing E. coli (STEC) virulence factors (stx1A, stx2A, eae), and Salmonella species in one test using raw beef trim samples. This product underwent extensive evaluations, including inclusivity-exclusivity, method comparison, robustness, lot-to-lot variability, and stability studies. The inclusivity/exclusivity study demonstrated that VereBeef Detection Kit specifically detects and identifies target analytes without occurrence of false-positive and false-negative detection. In the method comparison study, the performance of the VereBeef Detection Kit was compared with U.S. Department of Agriculture Food Safety and Inspection Service Microbiology Laboratory Guidebook's methods for target organism detection in raw beef trim using E. coli O157:H7 single inoculation and Salmonella and non-O157 STEC dual inoculation. Data demonstrated equivalence in both methods. The robustness study showed that changes in the test parameters do not impact assay performance. Collectively, VereBeef Detection Kit is able to detect target pathogens in raw beef trim with a minimum enrichment time of 8 h for E. coli O157:H7 detection and 10 h for Salmonella and non-O157 STEC detection.

Phylogenetic Network Analysis Revealed the Occurrence of Horizontal Gene Transfer of 16S rRNA in the Genus Enterobacter.

Horizontal gene transfer (HGT) is a ubiquitous genetic event in bacterial evolution, but it seldom occurs for genes involved in highly complex supramolecules (or biosystems), which consist of many gene products. The ribosome is one such supramolecule, but several bacteria harbor dissimilar and/or chimeric 16S rRNAs in their genomes, suggesting the occurrence of HGT of this gene. However, we know little about whether the genes actually experience HGT and, if so, the frequency of such a transfer. This is primarily because the methods currently employed for phylogenetic analysis (e.g., neighbor-joining, maximum likelihood, and maximum parsimony) of 16S rRNA genes assume point mutation-driven tree-shape evolution as an evolutionary model, which is intrinsically inappropriate to decipher the evolutionary history for genes driven by recombination. To address this issue, we applied a phylogenetic network analysis, which has been used previously for detection of genetic recombination in homologous alleles, to the 16S rRNA gene. We focused on the genus Enterobacter, whose phylogenetic relationships inferred by multi-locus sequence alignment analysis and 16S rRNA sequences are incompatible. All 10 complete genomic sequences were retrieved from the NCBI database, in which 71 16S rRNA genes were included. Neighbor-joining analysis demonstrated that the genes residing in the same genomes clustered, indicating the occurrence of intragenomic recombination. However, as suggested by the low bootstrap values, evolutionary relationships between the clusters were uncertain. We then applied phylogenetic network analysis to representative sequences from each cluster. We found three ancestral 16S rRNA groups; the others were likely created through recursive recombination between the ancestors and chimeric descendants. Despite the large sequence changes caused by the recombination events, the RNA secondary structures were conserved. Successive intergenomic and intragenomic recombination thus shaped the evolution of 16S rRNA genes in the genus Enterobacter.

PCR Primer Design for 16S rRNAs for Experimental Horizontal Gene Transfer Test in Escherichia coli.

We recently demonstrated that the Escherichia coli ribosome is robust enough to accommodate foreign 16S rRNAs from diverse gamma- and betaproteobacteria bacteria (Kitahara et al., 2012). Therein, we used the common universal primers Bac8f and UN1541r to obtain a nearly full-length gene. However, we noticed that these primers overlap variable sites at 19[A/C] and 1527[U/C] in Bac8f and UN1541r, respectively, and thus, the amplicon could contain mutations. This is problematic, particularly for the former site, because the 19th nucleotide pairs with the 916th nucleotide, which is a part of the "central pseudoknot" and is critical for function. Therefore, we mutationally investigated the role of the base pair using several 16S rRNAs from gamma- and betaproteobacteria. We found that both the native base pairs (gammaproteobacterial 19A-916U and betaproteobacterial 19C-916G) and the non-native 19A-916G pair retained function, whereas the non-native 19C-916U was defective 16S rRNAs. We next designed a new primer set, Bac1f and UN1542r, so that they do not overlap the potential mismatch sites. 16S rRNA amplicons obtained from the environmental metagenome using the new primer set were dominated by proteobacterial species (~85%). Subsequent functional screening identified various 16S rRNAs from proteobacteria, all of which contained native 19A-916U or 19C-916G base pairs. The primers developed in this study are thus advantageous for functional characterization of foreign 16S rRNA in E. coli with no artifacts.

An integrated lab-on-chip for rapid identification and simultaneous differentiation of tropical pathogens.

Tropical pathogens often cause febrile illnesses in humans and are responsible for considerable morbidity and mortality. The similarities in clinical symptoms provoked by these pathogens make diagnosis difficult. Thus, early, rapid and accurate diagnosis will be crucial in patient management and in the control of these diseases. In this study, a microfluidic lab-on-chip integrating multiplex molecular amplification and DNA microarray hybridization was developed for simultaneous detection and species differentiation of 26 globally important tropical pathogens. The analytical performance of the lab-on-chip for each pathogen ranged from 102 to 103 DNA or RNA copies. Assay performance was further verified with human whole blood spiked with Plasmodium falciparum and Chikungunya virus that yielded a range of detection from 200 to 4×105 parasites, and from 250 to 4×107 PFU respectively. This lab-on-chip was subsequently assessed and evaluated using 170 retrospective patient specimens in Singapore and Thailand. The lab-on-chip had a detection sensitivity of 83.1% and a specificity of 100% for P. falciparum; a sensitivity of 91.3% and a specificity of 99.3% for P. vivax; a positive 90.0% agreement and a specificity of 100% for Chikungunya virus; and a positive 85.0% agreement and a specificity of 100% for Dengue virus serotype 3 with reference methods conducted on the samples. Results suggested the practicality of an amplification microarray-based approach in a field setting for high-throughput detection and identification of tropical pathogens.

Analysis of a Larger SNP Dataset from the HapMap Project Confirmed That the Modern Human A Allele of the ABO Blood Group Genes Is a Descendant of a Recombinant between B and O Alleles.

The human ABO blood group gene consists of three main alleles (A, B, and O) that encode a glycosyltransferase. The A and B alleles differ by two critical amino acids in exon 7, and the major O allele has a single nucleotide deletion (Δ261) in exon 6. Previous evolutionary studies have revealed that the A allele is the most ancient, B allele diverged from the A allele with two critical amino acid substitutions in exon 7, and the major O allele diverged from the A allele with Δ261 in exon 6. However, a recent phylogenetic network analysis study showed that the A allele of humans emerged through a recombination between the B and O alleles. In the previous study, a restricted dataset from only two populations was used. In this study, therefore, we used a large single nucleotide polymorphism (SNP) dataset from the HapMap Project. The results indicated that the A101-A201-O09 haplogroup was a recombinant lineage between the B and O haplotypes, containing the intact exon 6 from the B allele and the two critical A type sites in exon 7 from the major O allele. Its recombination point was assumed to be located just behind Δ261 in exon 6.

Polypyrimidine tract-binding protein regulates the cell cycle through IRES-dependent translation of CDK11(p58) in mouse embryonic stem cells.

Polypyrimidine tract-binding protein (PTB/PTBP1/hnRNP I) is a member of the heterogeneous nuclear ribonucleoprotein family that binds specifically to pyrimidine-rich sequences of RNAs. Although PTB is a multifunctional protein involved in RNA processing and internal ribosome entry site (IRES)-dependent translation, the role of PTB in early mouse development is unclear. Ptb knockout mice exhibit embryonic lethality shortly after implantation and Ptb-/- embryonic stem (ES) cells have a severe proliferation defect that includes a prolonged G2/M phase. The present study shows that PTB promotes M phase progression by the direct repression of CDK11(p58) IRES activity in ES cells. The protein expression and IRES activity of CDK11(p58) in Ptb-/- ES cells is higher than that of wild-type ES cells, indicating that PTB is involved in the repression of CDK11(p58) expression through IRES-dependent translation in ES cells. Interestingly, CDK11(p58) IRES activity is activated by upstream of N-Ras (UNR) in 293T and NIH3T3 cells, whereas UNR is not present in the Cdk11 mRNA-protein complex in ES cells. In addition, PTB interacts directly with the IRES region of CDK11(p58) in ES cells. These results suggest that PTB regulates the precise expression of CDK11(p58) through direct interaction with CDK11(p58) IRES and promotes M phase progression in ES cells.

Mastermind-like 1 (MamL1) and mastermind-like 3 (MamL3) are essential for Notch signaling in vivo.

Mastermind (Mam) is one of the elements of Notch signaling, a system that plays a pivotal role in metazoan development. Mam proteins form transcriptionally activating complexes with the intracellular domains of Notch, which are generated in response to the ligand-receptor interaction, and CSL DNA-binding proteins. In mammals, three structurally divergent Mam isoforms (MamL1, MamL2 and MamL3) have been identified. There have also been indications that Mam interacts functionally with various other transcription factors, including the p53 tumor suppressor, ß-catenin and NF-κB. We have demonstrated previously that disruption of MamL1 causes partial deficiency of Notch signaling in vivo. However, MamL1-deficient mice did not recapitulate total loss of Notch signaling, suggesting that other members could compensate for the loss or that Notch signaling could proceed in the absence of Mam in certain contexts. Here, we report the generation of lines of mice null for MamL3. Although MamL3-null mice showed no apparent abnormalities, mice null for both MamL1 and MamL3 died during the early organogenic period with classic pan-Notch defects. Furthermore, expression of the lunatic fringe gene, which is strictly controlled by Notch signaling in the posterior presomitic mesoderm, was undetectable in this tissue of the double-null embryos. Neither of the single-null embryos exhibited any of these phenotypes. These various roles of the three Mam proteins could be due to their differential physical characteristics and/or their spatiotemporal distributions. These results indicate that engagement of Mam is essential for Notch signaling, and that the three Mam isoforms have distinct roles in vivo.

VereFlu™: an integrated multiplex RT-PCR and microarray assay for rapid detection and identification of human influenza A and B viruses using lab-on-chip technology.

Threatening sporadic outbreaks of avian influenza and the H1N1 pandemic of 2009 highlight the need for rapid and accurate detection and typing of influenza viruses. In this paper, we describe the validation of the VereFlu™ Lab-on-Chip Influenza Assay, which is based on the integration of two technologies: multiplex reverse transcription (RT)-PCR followed by microarray amplicon detection. This assay simultaneously detects five influenza virus subtypes, including the 2009 pandemic influenza A (H1N1), seasonal H1N1, H3N2, H5N1 and influenza B virus. The VereFlu™ assay was clinically validated in Singapore and compared against reference methods of real-time PCR, virus detection by immunofluorescence of cell cultures and sequencing. A sensitivity and specificity of 96.8% and 92.8%, respectively, was demonstrated for pandemic H1N1; 95.7% and 100%, respectively, for seasonal H1N1; 91.2% and 97.6%, respectively, for seasonal H3N2; 95.2% and 100%, respectively, for influenza B. Additional evaluations carried out at the World Health Organization (WHO) Collaborating Centre, Melbourne, Australia, confirmed that the test was able to reliably detect H5N1. This portable, fast time-to-answer (3 hours) device is particularly suited for diagnostic applications of detection, differentiation and identification of human influenza virus subtypes.

Polypyrimidine tract-binding protein is essential for early mouse development and embryonic stem cell proliferation.

Polypyrimidine tract-binding protein (PTB) is a widely expressed RNA-binding protein with multiple roles in RNA processing, including the splicing of alternative exons, mRNA stability, mRNA localization, and internal ribosome entry site-dependent translation. Although it has been reported that increased expression of PTB is correlated with cancer cell growth, the role of PTB in mammalian development is still unclear. Here, we report that a homozygous mutation in the mouse Ptb gene causes embryonic lethality shortly after implantation. We also established Ptb(-/-) embryonic stem (ES) cell lines and found that these mutant cells exhibited severe defects in cell proliferation without aberrant differentiation in vitro or in vivo. Furthermore, cell cycle analysis and a cell synchronization assay revealed that Ptb(-/-) ES cells have a prolonged G(2)/M phase. Thus, our data indicate that PTB is essential for early mouse development and ES cell proliferation.

Mastermind-1 is required for Notch signal-dependent steps in lymphocyte development in vivo.

Mastermind (Mam) is one of the elements of Notch signaling, an ancient system that plays a pivotal role in metazoan development. Genetic analyses in Drosophila and Caenorhabditis elegans have shown Mam to be an essential positive regulator of this signaling pathway in these species. Mam proteins bind to and stabilize the DNA-binding complex of the intracellular domains of Notch and CBF-1, Su(H), Lag-1 (CSL) DNA-binding proteins in the nucleus. Mammals have three Mam proteins, which show remarkable similarities in their functions while having an unusual structural diversity. There have also been recent indications that Mam-1 functionally interacts with other transcription factors including p53 tumor suppressor. We herein describe that Mam-1 deficiency in mice abolishes the development of splenic marginal zone B cells, a subset strictly dependent on Notch2, a CSL protein and Delta1 ligand. Mam-1 deficiency also causes a partially impaired development of early thymocytes, while not affecting the generation of definitive hematopoiesis, processes that are dependent on Notch1. We also demonstrate the transcriptional activation of a target promoter by constitutively active forms of Notch to decrease severalfold in cultured Mam-1-deficient cells. These results indicate that Mam-1 is thus required to some extent for Notch-dependent stages in lymphopoiesis, thus supporting the notion that Mam is an essential component of the canonical Notch pathway in mammals.

Morphological change caused by loss of the taxon-specific polyalanine tract in Hoxd-13.

Sequence comparison of Hoxd-13 among vertebrates revealed the presence of taxon-specific polyalanine tracts in amniotes. To investigate their function at the organismal level, we replaced the wild-type Hoxd-13 gene with one lacking the 15-residue polyalanine tract by using homologous recombination. Sesamoid bone formation in knock-in mice was different from that in the wild type; this was observed not only in the homozygotes but also in the heterozygotes. The present study provides the first direct evidence that taxon-specific homopolymeric amino acid repeats are involved in phenotypic diversification at the organismal level.

Significance of thymidylate synthase gene expression level in patients with adenocarcinoma of the lung.

BACKGROUND: Thymidylate synthase (TS) catalyzes the methylation of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP) and is a key enzyme for DNA synthesis. High expression of TS is thought to be associated with poor prognosis in some kinds of cancers. However, this association has not been clarified for nonsmall cell lung carcinoma. In the current study, the authors investigated the clinicopathologic significance of TS mRNA levels and the correlation with cellular proliferation in patients with lung adenocarcinoma. METHODS: The expression levels of TS mRNA were measured in 47 lung adenocarcinoma tissues using the Taq-Man real-time reverse transcriptase-polymerase chain reaction (RT-PCR) method and examined the clinicopathologic significance of TS expression. To clarify the correlation between TS expression level and cell proliferation, the Ki-67 labeling index was also examined using immunohistochemical staining. RESULTS: A positive correlation was observed between the expression levels of TS mRNA and stage of disease, lymph node metastasis, or tumor differentiation (P = .015). The higher expression group of TS mRNA showed a significantly poorer prognosis than the group with lower expression (P = .042). Moreover, there was a strong correlation noted between the expression levels of TS mRNA and the Ki-67 labeling index (P = .009). CONCLUSIONS: The results of the current study demonstrated that TS may be associated with stage of disease, lymph node metastasis, tumor differentiation, prognosis, and tumor cell proliferation. These results suggest that the expression levels of TS mRNA may be useful for predicting the malignant potential in patients with lung adenocarcinoma.

IRF-7 is the master regulator of type-I interferon-dependent immune responses.

The type-I interferon (IFN-alpha/beta) response is critical to immunity against viruses and can be triggered in many cell types by cytosolic detection of viral infection, or in differentiated plasmacytoid dendritic cells by the Toll-like receptor 9 (TLR9) subfamily, which generates signals via the adaptor MyD88 to elicit robust IFN induction. Using mice deficient in the Irf7 gene (Irf7-/- mice), we show that the transcription factor IRF-7 is essential for the induction of IFN-alpha/beta genes via the virus-activated, MyD88-independent pathway and the TLR-activated, MyD88-dependent pathway. Viral induction of MyD88-independent IFN-alpha/beta genes is severely impaired in Irf7-/- fibroblasts. Consistently, Irf7-/- mice are more vulnerable than Myd88-/- mice to viral infection, and this correlates with a marked decrease in serum IFN levels, indicating the importance of the IRF-7-dependent induction of systemic IFN responses for innate antiviral immunity. Furthermore, robust induction of IFN production by activation of the TLR9 subfamily in plasmacytoid dendritic cells is entirely dependent on IRF-7, and this MyD88-IRF-7 pathway governs the induction of CD8+ T-cell responses. Thus, all elements of IFN responses, whether the systemic production of IFN in innate immunity or the local action of IFN from plasmacytoid dendritic cells in adaptive immunity, are under the control of IRF-7.

Identification of IFN regulatory factor-1 binding site in IL-12 p40 gene promoter.

IL-12 is a heterodimer composed of p40 and p35 and is a key cytokine that functions to protect the host from viral and microbial infections. IL-12 links the innate immune system with the acquired immune system during infection, and induces differentiation of type 1 T cells that play an important role in the eradication of microbes. The induction of the IL-12 p40 gene is regulated by NF-kappaB in the presence of IFN-gamma. IFN-gamma induces IFN regulatory factor-1 (IRF-1), which in turn induces the transcription of the IL-12 p40 gene. However, the IRF-1 binding site in the promoter region of the IL-12 p40 gene has not yet been formally determined. In the present study, we demonstrated that IRF-1 directly binds to the IL-12 p40 gene promoter and identified its binding site. The IRF-1 binding site in the promoter region of the IL-12 p40 gene is shown to be in the -72 to -58 area of the 5'-upstream region. The -63 to -61 position is the critical site within this region for the binding of IRF-1 to the IL-12 p40 gene promoter. While IFN-gamma must be present for IL-12 p40 gene induction, the p35 gene is strongly induced by LPS, even in the absence of IFN-gamma, and therefore the induction of the p35 gene is IRF-1 independent.