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1.  Transcriptome Analysis of Mouse Brain Infected with Toxoplasma gondii 
Infection and Immunity  2013;81(10):3609-3619.
Toxoplasma gondii is an obligate intracellular parasite that invades a wide range of vertebrate host cells. Chronic infections with T. gondii become established in the tissues of the central nervous system, where the parasites may directly or indirectly modulate neuronal function. However, the mechanisms underlying parasite-induced neuronal disorder in the brain remain unclear. This study evaluated host gene expression in mouse brain following infection with T. gondii. BALB/c mice were infected with the PLK strain, and after 32 days of infection, histopathological lesions in the frontal lobe were found to be more severe than in other areas of the brain. Total RNA extracted from infected and uninfected mouse brain samples was subjected to transcriptome analysis using RNA sequencing (RNA-seq). In the T. gondii-infected mice, 935 mouse brain genes were upregulated, whereas 12 genes were downregulated. GOstat analysis predicted that the upregulated genes were primarily involved in host immune responses and cell activation. Positive correlations were found between the numbers of parasites in the infected mouse brains and the expression levels of genes involved in host immune responses. In contrast, genes that had a negative correlation with parasite numbers were predicted to be involved in neurological functions, such as small-GTPase-mediated signal transduction and vesicle-mediated transport. Furthermore, differential gene expression was observed between mice exhibiting the clinical signs of toxoplasmosis and those that did not. Our findings may provide insights into the mechanisms underlying neurological changes during T. gondii infection.
doi:10.1128/IAI.00439-13
PMCID: PMC3811780  PMID: 23856619
2.  Dengue transmission model by means of viremic adult immuno-competent mouse 
Parasites & Vectors  2014;7:143.
Background
Dengue virus infection manifests in three distinct forms in humans: dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. Infection with the virus is a fatal disease; no vaccine is available and prevention depends on interruption of the chain of transmission. The study of dengue viral transmission by mosquitoes is hindered due to the lack of an affordable animal model. In general, immuno-competent mice are used as a simple and inexpensive animal model, but mice are not susceptible to dengue virus infection and therefore viremia will not occur following the inoculation of the virus in such mice. Here, we report a method for creating artificial viremia in immuno-competent mice, and further demonstrate the use of viremic mice to simultaneously infect a large number of Aedes aegypti.
Methods
We infected K562 cells with DENV-2 in the presence of an antibody against DENV-4. We then incubated the cells for 2 d before injecting the infected cells into C3H mice. After 5 h incubation, we allowed 100–150 female Aedes aegypti to feed on blood from the mice directly. We collected blood samples from the mice and from randomly selected Ae. aegypti at 2, 6, 12, and 24 h post-blood meal and screened the samples for DENV-2 genome as well as for virus concentration.
Results
Our procedure provided high virus concentrations in the mice for at least 7 h after viral inoculation. We found that 13 out of 14 randomly picked mosquitoes were infected with DENV-2. High concentrations of virus were detected in the mosquitoes until at least 12 h post-infection.
Conclusions
Using the viremic immuno-competent mouse, we show that mass infection of Ae. aegypti is achievable. Compared to other infection techniques using direct inoculation, membrane-feeding, or immuno-deficient/humanized mice, we are confident that this method will provide a simpler and more efficient infection technique.
doi:10.1186/1756-3305-7-143
PMCID: PMC3976050  PMID: 24685121
Aedes aegypti; Dengue virus type 2; Mass-infection; Viremia; Immuno-competent mouse
3.  Genome-Wide Analysis of the Chromatin Composition of Histone H2A and H3 Variants in Mouse Embryonic Stem Cells 
PLoS ONE  2014;9(3):e92689.
Genome-wide distribution of the majority of H2A and H3 variants (H2A, H2AX, H2AZ, macroH2A, H3.1, H3.2 and H3.3) was simultaneously investigated in mouse embryonic stem cells by chromatin immunoprecipitation sequencing. Around the transcription start site, histone variant distribution differed between genes possessing promoters of high and low CpG density, regardless of their expression levels. In the intergenic regions, regulatory elements were enriched in H2A.Z and H3.3, whereas repeat elements were abundant in H2A and macroH2A, and H3.1, respectively. Analysis of H2A and H3 variant combinations composing nucleosomes revealed that the H2A.Z and H3.3 combinations were present at a higher frequency throughout the genome than the other combinations, suggesting that H2A.Z and H3.3 associate preferentially with each other to comprise the nucleosomes independently of genome region. Finally, we found that chromatin was unstable only in regions where it was enriched in both H2A.Z and H3.3, but strongly quantified stable in regions in which only H3.3 was abundant. Therefore, histone variant composition is an important determinant of chromatin structure, which is associated with specific genomic functions.
doi:10.1371/journal.pone.0092689
PMCID: PMC3962432  PMID: 24658136
4.  A Single-Cell and Feeder-Free Culture System for Monkey Embryonic Stem Cells 
PLoS ONE  2014;9(2):e88346.
Primate pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), hold great potential for research and application in regenerative medicine and drug discovery. To maximize primate PSC potential, a practical system is required for generating desired functional cells and reproducible differentiation techniques. Much progress regarding their culture systems has been reported to date; however, better methods would still be required for their practical use, particularly in industrial and clinical fields. Here we report a new single-cell and feeder-free culture system for primate PSCs, the key feature of which is an originally formulated serum-free medium containing FGF and activin. In this culture system, cynomolgus monkey ESCs can be passaged many times by single-cell dissociation with traditional trypsin treatment and can be propagated with a high proliferation rate as a monolayer without any feeder cells; further, typical PSC properties and genomic stability can be retained. In addition, it has been demonstrated that monkey ESCs maintained in the culture system can be used for various experiments such as in vitro differentiation and gene manipulation. Thus, compared with the conventional culture system, monkey ESCs grown in the aforementioned culture system can serve as a cell source with the following practical advantages: simple, stable, and easy cell maintenance; gene manipulation; cryopreservation; and desired differentiation. We propose that this culture system can serve as a reliable platform to prepare primate PSCs useful for future research and application.
doi:10.1371/journal.pone.0088346
PMCID: PMC3915054  PMID: 24505480
5.  Innate Immunity Interactome Dynamics 
Innate immune response involves protein–protein interactions, deoxyribonucleic acid (DNA)–protein interactions and signaling cascades. So far, thousands of protein–protein interactions have been curated as a static interaction map. However, protein–protein interactions involved in innate immune response are dynamic. We recorded the dynamics in the interactome during innate immune response by combining gene expression data of lipopolysaccharide (LPS)-stimulated dendritic cells with protein–protein interactions data. We identified the differences in interactome during innate immune response by constructing differential networks and identifying protein modules, which were up-/down-regulated at each stage during the innate immune response. For each protein complex, we identified enriched biological processes and pathways. In addition, we identified core interactions that are conserved throughout the innate immune response and their enriched gene ontology terms and pathways. We defined two novel measures to assess the differences between network maps at different time points. We found that the protein interaction network at 1 hour after LPS stimulation has the highest interactions protein ratio, which indicates a role for proteins with large number of interactions in innate immune response. A pairwise differential matrix allows for the global visualization of the differences between different networks. We investigated the toll-like receptor subnetwork and found that S100A8 is down-regulated in dendritic cells after LPS stimulation. Identified protein complexes have a crucial role not only in innate immunity, but also in circadian rhythms, pathways involved in cancer, and p53 pathways. The study confirmed previous work that reported a strong correlation between cancer and immunity.
doi:10.4137/GRSB.S12850
PMCID: PMC3885269  PMID: 24453478
innate immunity; protein-protein interactions; gene expression; differential networks; interactome dynamics
6.  Distinct Effects of Alcohol Consumption and Smoking on Genetic Alterations in Head and Neck Carcinoma 
PLoS ONE  2013;8(11):e80828.
Background
Tobacco and alcohol consumption are risk factors for head and neck squamous cell carcinoma (HNSCC). Recently, whole-exome sequencing clarified that smoking increased TP53 and other mutations in HNSCC; however, the effects of alcohol consumption on these genetic alterations remain unknown. We explored the association between alcohol consumption and somatic copy-number alterations (SCNAs) across the whole genome in human papillomavirus (HPV)-negative HNSCCs, and compared with the effects of smoking on genetic alterations.
Methods
SCNA and TP53 mutations in tumor samples were examined by high-resolution comparative genomic hybridization microarray 180K and by direct sequencing, respectively, and statistically analyzed for associations with alcohol consumption and smoking during the 20 years preceding diagnosis of HNSCC. Probes with a corrected p-value (=q-value) less than 0.05 and fold change greater than 1.2 or less than -1.2 were considered statistically significant.
Results
A total of 248 patients with HNSCC were enrolled. In the HPV-negative patients (n=221), heavy alcohol consumption was significantly associated with SCNAs of oncogenes/oncosuppressors that were previously reported to occur frequently in HNSCCs: CDKN2A (q=0.005), FHIT (q=0.005), 11q13 region including CCND1, FADD and CTTN (q=0.005), ERBB2 (HER2) (q=0.009), 3q25-qter including CCNL1, TP63, DCUN1D1 and PIK3CA (q=0.014), and CSMD1 (q=0.019). But, TP53 mutations were not affected. In contrast, smoking was associated with increased risk of TP53 mutations, but did not induce any significant SCNAs of oncogenes/oncosuppressors.
Conclusion
These results suggest that both alcohol consumption and smoking had distinct effects on genetic alterations in HNSCCs. Heavy alcohol consumption may trigger previously known and unknown SCNAs, but may not induce TP53 mutation. In contrast, smoking may induce TP53 mutation, but may not trigger any SCNAs.
doi:10.1371/journal.pone.0080828
PMCID: PMC3835411  PMID: 24278325
7.  Molecular basis of wing coloration in a Batesian mimic butterfly, Papilio polytes 
Scientific Reports  2013;3:3184.
Batesian mimicry protects animals from predators through resemblance with distasteful models in shape, color pattern, or behavior. To elucidate the wing coloration mechanisms involved in the mimicry, we investigated chemical composition and gene expression of the pale yellow and red pigments of a swallowtail butterfly, Papilio polytes, whose females mimic the unpalatable butterfly Pachliopta aristolochiae. Using LC/MS, we showed that the pale yellow wing regions in non-mimetic females consist of kynurenine and N-β-alanyldopamine (NBAD). Moreover, qRT-PCR showed that kynurenine/NBAD biosynthetic genes were upregulated in these regions in non-mimetic females. However, these pigments were absent in mimetic females. RNA-sequencing showed that kynurenine/NBAD synthesis and Toll signaling genes were upregulated in the red spots specific to mimetic female wings. These results demonstrated that drastic changes in gene networks in the red and pale yellow regions can switch wing color patterns between non-mimetic and mimetic females of P. polytes.
doi:10.1038/srep03184
PMCID: PMC3822385  PMID: 24212474
8.  Linking Transcriptional Changes over Time in Stimulated Dendritic Cells to Identify Gene Networks Activated during the Innate Immune Response 
PLoS Computational Biology  2013;9(11):e1003323.
The innate immune response is primarily mediated by the Toll-like receptors functioning through the MyD88-dependent and TRIF-dependent pathways. Despite being widely studied, it is not yet completely understood and systems-level analyses have been lacking. In this study, we identified a high-probability network of genes activated during the innate immune response using a novel approach to analyze time-course gene expression profiles of activated immune cells in combination with a large gene regulatory and protein-protein interaction network. We classified the immune response into three consecutive time-dependent stages and identified the most probable paths between genes showing a significant change in expression at each stage. The resultant network contained several novel and known regulators of the innate immune response, many of which did not show any observable change in expression at the sampled time points. The response network shows the dominance of genes from specific functional classes during different stages of the immune response. It also suggests a role for the protein phosphatase 2a catalytic subunit α in the regulation of the immunoproteasome during the late phase of the response. In order to clarify the differences between the MyD88-dependent and TRIF-dependent pathways in the innate immune response, time-course gene expression profiles from MyD88-knockout and TRIF-knockout dendritic cells were analyzed. Their response networks suggest the dominance of the MyD88-dependent pathway in the innate immune response, and an association of the circadian regulators and immunoproteasomal degradation with the TRIF-dependent pathway. The response network presented here provides the most probable associations between genes expressed in the early and the late phases of the innate immune response, while taking into account the intermediate regulators. We propose that the method described here can also be used in the identification of time-dependent gene sub-networks in other biological systems.
Author Summary
The innate immune response is the first level of protection in organisms against invading pathogens. It consists of a large number of proteins functioning in signaling cascades triggered by the binding of fragments from microbes to specific cellular receptors. Disruptions in these pathways can lead to numerous diseases. As such, the innate immune system has been the subject of a large number of studies. However, due to its complexity and the interplay of a large number of pathways, it is not yet completely understood. In this study, we measured transcriptional changes in activated immune cells and used this information in the context of a large network of protein-protein and protein-DNA interactions to identify a smaller network of response genes. We did this by identifying the most probable network paths connecting genes showing large changes in their expression patterns at successive stages of the response. Analysis of this activated gene network revealed the associations between various temporal regulators of the innate immune response. We also identified response networks for immune cells lacking important mediators, MyD88 and TRIF, to clarify the distinct functional modules affected by their associated pathways in the innate immune response.
doi:10.1371/journal.pcbi.1003323
PMCID: PMC3820512  PMID: 24244133
9.  Identification of hundreds of novel UPF1 target transcripts by direct determination of whole transcriptome stability 
RNA Biology  2012;9(11):1370-1379.
UPF1 eliminates aberrant mRNAs harboring premature termination codons, and regulates the steady-state levels of normal physiological mRNAs. Although genome-wide studies of UPF1 targets performed, previous studies did not distinguish indirect UPF1 targets because they could not determine UPF1-dependent altered RNA stabilities. Here, we measured the decay rates of the whole transcriptome in UPF1-depleted HeLa cells using BRIC-seq, an inhibitor-free method for directly measuring RNA stability. We determined the half-lives and expression levels of 9,229 transcripts. An amount of 785 transcripts were stabilized in UPF1-depleted cells. Among these, the expression levels of 76 transcripts were increased, but those of the other 709 transcripts were not altered. RNA immunoprecipitation showed UPF1 bound to the stabilized transcripts, suggesting that UPF1 directly degrades the 709 transcripts. Many UPF1 targets in this study were newly identified. This study clearly demonstrates that direct determination of RNA stability is a powerful approach for identifying targets of RNA degradation factors.
doi:10.4161/rna.22360
PMCID: PMC3597577  PMID: 23064114
BRIC-seq; RNA decay; RNA-seq; UPF1; nonsense-mediated mRNA decay
10.  Giardia lamblia Transcriptome Analysis Using TSS-Seq and RNA-Seq 
PLoS ONE  2013;8(10):e76184.
Giardia lamblia is a protozoan parasite that is found worldwide and has both medical and veterinary importance. We applied the transcription start sequence (TSS-seq) and RNA sequence (RNA-seq) techniques to study the transcriptome of the assemblage A WB strain trophozoite. We identified 8000 transcription regions (TR) with significant transcription. Of these regions, 1881 TRs were more than 500 nucleotides upstream of an annotated ORF. Combining both techniques helped us to identify 24 ORFs that should be re-annotated and 60 new ORFs. From the 8000 TRs, we were able to identify an AT-rich consensus that includes the transcription initiation site. It is possible that transcription that was previously thought to be bidirectional is actually unidirectional.
doi:10.1371/journal.pone.0076184
PMCID: PMC3792122  PMID: 24116096
11.  Identification and Characterization of Cancer Mutations in Japanese Lung Adenocarcinoma without Sequencing of Normal Tissue Counterparts 
PLoS ONE  2013;8(9):e73484.
We analyzed whole-exome sequencing data from 97 Japanese lung adenocarcinoma patients and identified several putative cancer-related genes and pathways. Particularly, we observed that cancer-related mutation patterns were significantly different between different ethnic groups. As previously reported, mutations in the EGFR gene were characteristic to Japanese, while those in the KRAS gene were more frequent in Caucasians. Furthermore, during the course of this analysis, we found that cancer-specific somatic mutations can be detected without sequencing normal tissue counterparts. 64% of the germline variants could be excluded using a total of 217 external Japanese exome datasets. We also show that a similar approach may be used for other three ethnic groups, although the discriminative power depends on the ethnic group. We demonstrate that the ATM gene and the PAPPA2 gene could be identified as cancer prognosis related genes. By bypassing the sequencing of normal tissue counterparts, this approach provides a useful means of not only reducing the time and cost of sequencing but also analyzing archive samples, for which normal tissue counterparts are not available.
doi:10.1371/journal.pone.0073484
PMCID: PMC3772023  PMID: 24069199
12.  Large Scale Full-Length cDNA Sequencing Reveals a Unique Genomic Landscape in a Lepidopteran Model Insect, Bombyx mori 
G3: Genes|Genomes|Genetics  2013;3(9):1481-1492.
The establishment of a complete genomic sequence of silkworm, the model species of Lepidoptera, laid a foundation for its functional genomics. A more complete annotation of the genome will benefit functional and comparative studies and accelerate extensive industrial applications for this insect. To realize these goals, we embarked upon a large-scale full-length cDNA collection from 21 full-length cDNA libraries derived from 14 tissues of the domesticated silkworm and performed full sequencing by primer walking for 11,104 full-length cDNAs. The large average intron size was 1904 bp, resulting from a high accumulation of transposons. Using gene models predicted by GLEAN and published mRNAs, we identified 16,823 gene loci on the silkworm genome assembly. Orthology analysis of 153 species, including 11 insects, revealed that among three Lepidoptera including Monarch and Heliconius butterflies, the 403 largest silkworm-specific genes were composed mainly of protective immunity, hormone-related, and characteristic structural proteins. Analysis of testis-/ovary-specific genes revealed distinctive features of sexual dimorphism, including depletion of ovary-specific genes on the Z chromosome in contrast to an enrichment of testis-specific genes. More than 40% of genes expressed in specific tissues mapped in tissue-specific chromosomal clusters. The newly obtained FL-cDNA sequences enabled us to annotate the genome of this lepidopteran model insect more accurately, enhancing genomic and functional studies of Lepidoptera and comparative analyses with other insect orders, and yielding new insights into the evolution and organization of lepidopteran-specific genes.
doi:10.1534/g3.113.006239
PMCID: PMC3755909  PMID: 23821615
Bombyx mori; large-scale full-length cDNA collection; tissue-specific genes; sexual dimorphism; gene cluster; silkworm
13.  Detection of phase-dependent transcriptomic changes and Rubisco-mediated CO2 fixation into poly (3-hydroxybutyrate) under heterotrophic condition in Ralstonia eutropha H16 based on RNA-seq and gene deletion analyses 
BMC Microbiology  2013;13:169.
Background
Ralstonia eutropha H16 is well known to produce polyhydroxyalkanoates (PHAs), which are potential bio-based biodegradable plastics, in an efficient manner as an energy storage material under unbalanced growth conditions. To obtain further knowledge of PHA biosynthesis, this study performed a quantitative transcriptome analysis based on deep sequencing of the complementary DNA generated from the RNA (RNA-seq) of R. eutropha H16.
Results
Total RNAs were extracted from R. eutropha cells in growth, PHA production, and stationary phases on fructose. rRNAs in the preparation were removed by repeated treatments with magnetic beads specific to bacterial rRNAs, and then the 36 bp sequences were determined using an Illumina high-throughput sequencer. The RNA-seq results indicated the induction of gene expression for transcription, translation, cell division, peptidoglycan biosynthesis, pilus and flagella assembly, energy conservation, and fatty acid biosynthesis in the growth phase; and the repression trends of genes involved in central metabolisms in the PHA production phase. Interestingly, the transcription of genes for Calvin-Benson-Bassham (CBB) cycle and several genes for β-oxidation were significantly induced in the PHA production phase even when the cells were grown on fructose. Moreover, incorporation of 13C was observed in poly(3-hydroxybutyrate) synthesized by R. eutropha H16 from fructose in the presence of NaH13CO3, and further gene deletion analyses revealed that both of the two ribulose 1,5-bisphosphate carboxylase (Rubiscos) in CBB cycle were actually functional in CO2 fixation under the heterotrophic condition.
Conclusions
The results revealed the phase-dependent transcriptomic changes and a CO2 fixation capability under heterotrophic conditions by PHA-producing R. eutropha.
doi:10.1186/1471-2180-13-169
PMCID: PMC3734047  PMID: 23879744
RNA-seq; Ralstonia eutropha; Polyhydroxyalkanoates; CO2 fixation; Cavin-benson-bassham cycle; Rubisco
14.  Genome-Wide Gene Expression Profiling Revealed a Critical Role for GATA3 in the Maintenance of the Th2 Cell Identity 
PLoS ONE  2013;8(6):e66468.
Functionally polarized CD4+ T helper (Th) cells such as Th1, Th2 and Th17 cells are central to the regulation of acquired immunity. However, the molecular mechanisms governing the maintenance of the polarized functions of Th cells remain unclear. GATA3, a master regulator of Th2 cell differentiation, initiates the expressions of Th2 cytokine genes and other Th2-specific genes. GATA3 also plays important roles in maintaining Th2 cell function and in continuous chromatin remodeling of Th2 cytokine gene loci. However, it is unclear whether continuous expression of GATA3 is required to maintain the expression of various other Th2-specific genes. In this report, genome-wide DNA gene expression profiling revealed that GATA3 expression is critical for the expression of a certain set of Th2-specific genes. We demonstrated that GATA3 dependency is reduced for some Th2-specific genes in fully developed Th2 cells compared to that observed in effector Th2 cells, whereas it is unchanged for other genes. Moreover, effects of a loss of GATA3 expression in Th2 cells on the expression of cytokine and cytokine receptor genes were examined in detail. A critical role of GATA3 in the regulation of Th2-specific gene expression is confirmed in in vivo generated antigen-specific memory Th2 cells. Therefore, GATA3 is required for the continuous expression of the majority of Th2-specific genes involved in maintaining the Th2 cell identity.
doi:10.1371/journal.pone.0066468
PMCID: PMC3688927  PMID: 23824597
15.  Youngest case of an early gastric cancer after successful eradication therapy 
A 28-year-old woman visited our clinic with a chief complaint of epigastralgia. She had received successful Helicobacter pylori (H. pylori) eradication therapy 5 years before. We repeated esophagogastroduodenoscopy, and a discolored depressed area with reddish spots and converging folds, 20 mm in size, was detected. No atrophic change including intestinal metaplasia or nodular gastritis was seen endoscopically. Two endoscopic biopsies revealed undifferentiated adenocarcinoma. No H. pylori was found, and the 13C-urea breath test was also negative. Abdominal computed tomography demonstrated no nodal involvement, distant metastasis or fluid collection. She underwent a laparoscopy-assisted distal gastrectomy. Histologically, the resected specimen revealed an early undifferentiated gastric cancer that had invaded deeply into the submucosal layer. Nodal involvement was histologically confirmed. No atrophic change or H. pylori infection was evident histologically. This is the youngest patient ever reported to have developed a node-positive early gastric cancer after eradication of H. pylori.
doi:10.4253/wjge.v5.i6.300
PMCID: PMC3680621  PMID: 23772269
Early gastric cancer; Helicobacter pylori; Eradication therapy; Undifferentiated adenocarcinoma; Intestinal-type adenocarcinoma; Point of no return theory
17.  Coordinated Changes in DNA Methylation in Antigen-Specific Memory CD4 T Cells 
Memory CD4+ T cells are central regulators of both humoral and cellular immune responses. T cell differentiation results in specific changes in chromatin structure and DNA methylation of cytokine genes. Although the methylation status of a limited number of gene loci in T cells has been examined, the genome-wide DNA methylation status of memory CD4+ T cells remains unexplored. To further elucidate the molecular signature of memory T cells, we conducted methylome and transcriptome analyses of memory CD4+ T cells generated using T cells from TCR-transgenic mice. The resulting genome-wide DNA methylation profile revealed 1144 differentially methylated regions (DMRs) across the murine genome during the process of T cell differentiation, 552 of which were associated with gene loci. Interestingly, the majority of these DMRs were located in introns. These DMRs included genes such as CXCR6, Tbox21, Chsy1, and Cish, which are associated with cytokine production, homing to bone marrow, and immune responses. Methylation changes in memory T cells exposed to specific Ag appeared to regulate enhancer activity rather than promoter activity of immunologically relevant genes. In addition, methylation profiles differed between memory T cell subsets, demonstrating a link between T cell methylation status and T cell differentiation. By comparing DMRs between naive and Ag-specific memory T cells, this study provides new insights into the functional status of memory T cells.
doi:10.4049/jimmunol.1202267
PMCID: PMC3619526  PMID: 23509353
18.  HDAC8 mutations in Cornelia de Lange Syndrome affect the cohesin acetylation cycle 
Nature  2012;489(7415):313-317.
Cornelia de Lange syndrome (CdLS) is a dominantly inherited congenital malformation disorder caused by mutations in the cohesin-loading protein NIPBL1,2 for nearly 60% of individuals with classical CdLS3-5 and in the core cohesin components SMC1A (~5%) and SMC3 (<1%) for a smaller fraction of probands6,7. In humans, the multi-subunit complex cohesin is comprised of SMC1, SMC3, RAD21 and a STAG protein to form a ring structure proposed to encircle sister chromatids to mediate sister chromatid cohesion (SCC)8 as well as play key roles in gene regulation9. SMC3 is acetylated during S-phase to establish cohesiveness of chromatin-loaded cohesin10-13 and in yeast, HOS1, a class I histone deacetylase, deacetylates SMC3 during anaphase14-16. Here we report the identification of HDAC8 as the vertebrate SMC3 deacetylase as well as loss-of-function HDAC8 mutations in six CdLS probands. Loss of HDAC8 activity results in increased SMC3 acetylation (SMC3-ac) and inefficient dissolution of the “used” cohesin complex released from chromatin in both prophase and anaphase. While SMC3 with retained acetylation is loaded onto chromatin, ChIP-Seq analysis demonstrates decreased occupancy of cohesin localization sites that results in a consistent pattern of altered transcription seen in CdLS cell lines with either NIPBL or HDAC8 mutations.
doi:10.1038/nature11316
PMCID: PMC3443318  PMID: 22885700
19.  Position-dependent and neuron-specific splicing regulation by the CELF family RNA-binding protein UNC-75 in Caenorhabditis elegans 
Nucleic Acids Research  2013;41(7):4015-4025.
A large fraction of protein-coding genes in metazoans undergo alternative pre-mRNA splicing in tissue- or cell-type-specific manners. Recent genome-wide approaches have identified many putative-binding sites for some of tissue-specific trans-acting splicing regulators. However, the mechanisms of splicing regulation in vivo remain largely unknown. To elucidate the modes of splicing regulation by the neuron-specific CELF family RNA-binding protein UNC-75 in Caenorhabditis elegans, we performed deep sequencing of poly(A)+ RNAs from the unc-75(+)- and unc-75-mutant worms and identified more than 20 cassette and mutually exclusive exons repressed or activated by UNC-75. Motif searches revealed that (G/U)UGUUGUG stretches are enriched in the upstream and downstream introns of the UNC-75-repressed and -activated exons, respectively. Recombinant UNC-75 protein specifically binds to RNA fragments carrying the (G/U)UGUUGUG stretches in vitro. Bi-chromatic fluorescence alternative splicing reporters revealed that the UNC-75-target exons are regulated in tissue-specific and (G/U)UGUUGUG element-dependent manners in vivo. The unc-75 mutation affected the splicing reporter expression specifically in the nervous system. These results indicate that UNC-75 regulates alternative splicing of its target exons in neuron-specific and position-dependent manners through the (G/U)UGUUGUG elements in C. elegans. This study thus reveals the repertoire of target events for the CELF family in the living organism.
doi:10.1093/nar/gkt097
PMCID: PMC3627589  PMID: 23416545
20.  Whole-Genome Sequencing of Theileria parva Strains Provides Insight into Parasite Migration and Diversification in the African Continent 
The disease caused by the apicomplexan protozoan parasite Theileria parva, known as East Coast fever or Corridor disease, is one of the most serious cattle diseases in Eastern, Central, and Southern Africa. We performed whole-genome sequencing of nine T. parva strains, including one of the vaccine strains (Kiambu 5), field isolates from Zambia, Uganda, Tanzania, or Rwanda, and two buffalo-derived strains. Comparison with the reference Muguga genome sequence revealed 34 814–121 545 single nucleotide polymorphisms (SNPs) that were more abundant in buffalo-derived strains. High-resolution phylogenetic trees were constructed with selected informative SNPs that allowed the investigation of possible complex recombination events among ancestors of the extant strains. We further analysed the dN/dS ratio (non-synonymous substitutions per non-synonymous site divided by synonymous substitutions per synonymous site) for 4011 coding genes to estimate potential selective pressure. Genes under possible positive selection were identified that may, in turn, assist in the identification of immunogenic proteins or vaccine candidates. This study elucidated the phylogeny of T. parva strains based on genome-wide SNPs analysis with prediction of possible past recombination events, providing insight into the migration, diversification, and evolution of this parasite species in the African continent.
doi:10.1093/dnares/dst003
PMCID: PMC3686427  PMID: 23404454
Theileria parva; genome sequence; SNPs; recombination; dN/dS
21.  MAML1 Enhances the Transcriptional Activity of Runx2 and Plays a Role in Bone Development 
PLoS Genetics  2013;9(1):e1003132.
Mastermind-like 1 (MAML1) is a transcriptional co-activator in the Notch signaling pathway. Recently, however, several reports revealed novel and unique roles for MAML1 that are independent of the Notch signaling pathway. We found that MAML1 enhances the transcriptional activity of runt-related transcription factor 2 (Runx2), a transcription factor essential for osteoblastic differentiation and chondrocyte proliferation and maturation. MAML1 significantly enhanced the Runx2-mediated transcription of the p6OSE2-Luc reporter, in which luciferase expression was controlled by six copies of the osteoblast specific element 2 (OSE2) from the Runx2-regulated osteocalcin gene promoter. Interestingly, a deletion mutant of MAML1 lacking the N-terminal Notch-binding domain also enhanced Runx2-mediated transcription. Moreover, inhibition of Notch signaling did not affect the action of MAML1 on Runx2, suggesting that the activation of Runx2 by MAML1 may be caused in a Notch-independent manner. Overexpression of MAML1 transiently enhanced the Runx2-mediated expression of alkaline phosphatase, an early marker of osteoblast differentiation, in the murine pluripotent mesenchymal cell line C3H10T1/2. MAML1−/− embryos at embryonic day 16.5 (E16.5) had shorter bone lengths than wild-type embryos. The area of primary spongiosa of the femoral diaphysis was narrowed. At E14.5, extended zone of collagen type II alpha 1 (Col2a1) and Sox9 expression, markers of chondrocyte differentiation, and decreased zone of collagen type X alpha 1 (Col10a1) expression, a marker of hypertrophic chondrocyte, were observed. These observations suggest that chondrocyte maturation was impaired in MAML1−/− mice. MAML1 enhances the transcriptional activity of Runx2 and plays a role in bone development.
Author Summary
To identify new molecules involved in bone and cartilage development and/or homeostasis, we utilized approximately 10,000 arrayed and addressable cDNA clones, which allowed systematic, efficient, and unbiased screening of cDNAs encoding factors that could activate critical bone differentiation activity via activation of Runx2, master regulator of bone development. We analyzed MAML1−/− mice to investigate the role of MAML1 in bone development. MAML1−/− embryos at embryonic day 14.5 and 16.5 had shorter bone lengths than wild-type embryos. The area of primary spongiosa of the femoral diaphysis was narrowed, indicated that chondrocyte maturation was impaired. This revealed that MAML1 plays an important role in proper bone development and may provide us with a new basis for identifying potential therapeutic targets for bone diseases.
doi:10.1371/journal.pgen.1003132
PMCID: PMC3542067  PMID: 23326237
22.  The comprehensive epigenome map of piRNA clusters 
Nucleic Acids Research  2012;41(3):1581-1590.
PIWI-interacting RNA (piRNA) clusters act as anti-transposon/retrovirus centers. Integration of selfish jumping elements into piRNA clusters generates de novo piRNAs, which in turn exert trans-silencing activity against these elements in animal gonads. To date, neither genome-wide chromatin modification states of piRNA clusters nor a mode for piRNA precursor transcription have been well understood. Here, to understand the chromatin landscape of piRNA clusters and how piRNA precursors are generated, we analyzed the transcriptome, transcription start sites (TSSs) and the chromatin landscape of the BmN4 cell line, which harbors the germ-line piRNA pathway. Notably, our epigenomic map demonstrated the highly euchromatic nature of unique piRNA clusters. RNA polymerase II was enriched for TSSs that transcribe piRNA precursors. piRNA precursors possessed 5′-cap structures as well as 3′-poly A-tails. Collectively, we envision that the euchromatic, opened nature of unique piRNA clusters or piRNA cluster-associated TSSs allows piRNA clusters to capture new insertions efficiently.
doi:10.1093/nar/gks1275
PMCID: PMC3561999  PMID: 23258708
24.  Comparative Genome Analysis of Three Eukaryotic Parasites with Differing Abilities To Transform Leukocytes Reveals Key Mediators of Theileria-Induced Leukocyte Transformation 
mBio  2012;3(5):e00204-12.
ABSTRACT
We sequenced the genome of Theileria orientalis, a tick-borne apicomplexan protozoan parasite of cattle. The focus of this study was a comparative genome analysis of T. orientalis relative to other highly pathogenic Theileria species, T. parva and T. annulata. T. parva and T. annulata induce transformation of infected cells of lymphocyte or macrophage/monocyte lineages; in contrast, T. orientalis does not induce uncontrolled proliferation of infected leukocytes and multiplies predominantly within infected erythrocytes. While synteny across homologous chromosomes of the three Theileria species was found to be well conserved overall, subtelomeric structures were found to differ substantially, as T. orientalis lacks the large tandemly arrayed subtelomere-encoded variable secreted protein-encoding gene family. Moreover, expansion of particular gene families by gene duplication was found in the genomes of the two transforming Theileria species, most notably, the TashAT/TpHN and Tar/Tpr gene families. Gene families that are present only in T. parva and T. annulata and not in T. orientalis, Babesia bovis, or Plasmodium were also identified. Identification of differences between the genome sequences of Theileria species with different abilities to transform and immortalize bovine leukocytes will provide insight into proteins and mechanisms that have evolved to induce and regulate this process. The T. orientalis genome database is available at http://totdb.czc.hokudai.ac.jp/.
IMPORTANCE
Cancer-like growth of leukocytes infected with malignant Theileria parasites is a unique cellular event, as it involves the transformation and immortalization of one eukaryotic cell by another. In this study, we sequenced the whole genome of a nontransforming Theileria species, Theileria orientalis, and compared it to the published sequences representative of two malignant, transforming species, T. parva and T. annulata. The genome-wide comparison of these parasite species highlights significant genetic diversity that may be associated with evolution of the mechanism(s) deployed by an intracellular eukaryotic parasite to transform its host cell.
doi:10.1128/mBio.00204-12
PMCID: PMC3445966  PMID: 22951932
25.  Intestinal malrotation with suspected cow’s milk allergy: a case report 
BMC Research Notes  2012;5:481.
Background
Intestinal malrotation is an incomplete rotation of the intestine. Failure to rotate leads to abnormalities in intestinal positioning and attachment that leave obstructing bands across the duodenum and a narrow pedicle for the midgut loop, thus making it susceptible to volvulus. One of the important differential diagnoses for malrotation is an allergy to cow’s milk. Several studies have described infants with surgical gastrointestinal diseases and cow’s milk allergy. However, to our knowledge, no study has reported infants with intestinal malrotation who have been symptomatic before surgery was performed and have been examined by allergen-specific lymphocyte stimulation test and food challenge tests with long-term follow-up.
Case presentation
The patient was a Japanese male born at 39 weeks of gestation. He was breast-fed and received commercial cow’s milk supplementation starting the day of birth and was admitted to our hospital at 6 days of age due to bilious vomiting. Plain abdominal radiography showed a paucity of gas in the distal bowel. Because we demonstrated malpositioning of the intestine by barium enema, we repositioned the bowel in a normal position by laparotomy. The patient was re-started on only breast milk 2 days post surgery because we suspected the presence of a cow’s milk allergy, and the results of an allergen-specific lymphocyte stimulation test showed a marked increase in lymphocyte response to kappa-casein. At 5 months of age, the patient was subjected to a cow’s milk challenge test. After the patient began feeding on cow’s milk, he had no symptoms and his laboratory investigations showed no abnormality. In addition, because the patient showed good weight gain and no symptoms with increased cow’s milk intake after discharge, we concluded that the present case was not the result of a cow’s milk allergy. At 1 year, the patient showed favorable growth and development, and serum allergy investigations revealed no reaction to cow’s milk.
Conclusion
When physicians encounter infants with surgical gastrointestinal disease, including intestinal malrotation, they should consider cow’s milk allergy as a differential diagnosis or complication and should utilize food challenge tests for a definitive diagnosis.
doi:10.1186/1756-0500-5-481
PMCID: PMC3490812  PMID: 22943656
Allergen-specific lymphocyte stimulation test; Cow’s milk allergy; Food challenge test; Infant; Intestinal malrotation

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