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1.  Physiological Properties and Genome Structure of the Hyperthermophilic Filamentous Phage φOH3 Which Infects Thermus thermophilus HB8 
A filamentous bacteriophage, φOH3, was isolated from hot spring sediment in Obama hot spring in Japan with the hyperthermophilic bacterium Thermus thermophilus HB8 as its host. Phage φOH3, which was classified into the Inoviridae family, consists of a flexible filamentous particle 830 nm long and 8 nm wide. φOH3 was stable at temperatures ranging from 70 to 90°C and at pHs ranging from 6 to 9. A one-step growth curve of the phage showed a 60-min latent period beginning immediately postinfection, followed by intracellular virus particle production during the subsequent 40 min. The released virion number of φOH3 was 109. During the latent period, both single stranded DNA (ssDNA) and the replicative form (RF) of phage DNA were multiplied from min 40 onward. During the release period, the copy numbers of both ssDNA and RF DNA increased sharply. The size of the φOH3 genome is 5688 bp, and eight putative open reading frames (ORFs) were annotated. These ORFs were encoded on the plus strand of RF DNA and showed no significant homology with any known phage genes, except ORF 5, which showed 60% identity with the gene VIII product of the Thermus filamentous phage PH75. All the ORFs were similar to predicted genes annotated in the Thermus aquaticus Y51MC23 and Meiothermus timidus DSM 17022 genomes at the amino acid sequence level. This is the first report of the whole genome structure and DNA multiplication of a filamentous T. thermophilus phage within its host cell.
doi:10.3389/fmicb.2016.00050
PMCID: PMC4763002  PMID: 26941711
hyperthermophilic phage; Thermus thermophilus; filamentous phage; Inoviridae; replicative form
2.  Diabetes Caused by Elastase-Cre-Mediated Pdx1 Inactivation in Mice 
Scientific Reports  2016;6:21211.
Endocrine and exocrine pancreas tissues are both derived from the posterior foregut endoderm, however, the interdependence of these two cell types during their formation is not well understood. In this study, we generated mutant mice, in which the exocrine tissue is hypoplastic, in order to reveal a possible requirement for exocrine pancreas tissue in endocrine development and/or function. Since previous studies showed an indispensable role for Pdx1 in pancreas organogenesis, we used Elastase-Cre-mediated recombination to inactivate Pdx1 in the pancreatic exocrine lineage during embryonic stages. Along with exocrine defects, including impaired acinar cell maturation, the mutant mice exhibited substantial endocrine defects, including disturbed tip/trunk patterning of the developing ductal structure, a reduced number of Ngn3-expressing endocrine precursors, and ultimately fewer β cells. Notably, postnatal expansion of the endocrine cell content was extremely poor, and the mutant mice exhibited impaired glucose homeostasis. These findings suggest the existence of an unknown but essential factor(s) in the adjacent exocrine tissue that regulates proper formation of endocrine precursors and the expansion and function of endocrine tissues during embryonic and postnatal stages.
doi:10.1038/srep21211
PMCID: PMC4758062  PMID: 26887806
4.  The impact of a single-nucleotide mutation of bgl2 on cellulase induction in a Trichoderma reesei mutant 
Background
The filamentous fungus Trichoderma reesei (anamorph of Hypocrea jecorina) produces increased cellulase expression when grown on cellulose or its derivatives as a sole carbon source. It has been believed that β-glucosidases of T. reesei not only metabolize cellobiose but also contribute in the production of inducers of cellulase gene expression by their transglycosylation activity. The cellulase hyper-producing mutant PC-3-7 developed in Japan has enhanced cellulase production ability when cellobiose is used as the inducer. The comparative genomics analysis of PC-3-7 and its parent revealed a single-nucleotide mutation within the bgl2 gene encoding intracellular β-glucosidase II (BGLII/Cel1a), giving rise to an amino acid substitution in PC-3-7, which could potentially account for the enhanced cellulase expression when these strains are cultivated on cellulose and cellobiose.
Results
To analyze the effects of the BGLII mutation in cellulase induction, we constructed both a bgl2 revertant and a disruptant. Enzymatic analysis of the transformant lysates showed that the strain expressing mutant BGLII exhibited weakened cellobiose hydrolytic activity, but produced some transglycosylation products, suggesting that the SNP in bgl2 strongly diminished cellobiase activity, but did not result in complete loss of function of BGLII. The analysis of the recombinant BGLII revealed that transglycosylation products might be oligosaccharides, composed probably of glucose linked β-1,4, β-1,3, or a mixture of both. PC-3-7 revertants of bgl2 exhibited reduced expression and inducibility of cellulase during growth on cellulose and cellobiose substrates. Furthermore, the effect of this bgl2 mutation was reproduced in the common strain QM9414 in which the transformants showed cellulase production comparable to that of PC-3-7.
Conclusion
We conclude that BGLII plays an important role in cellulase induction in T. reesei and that the bgl2 mutation in PC-3-7 brought about enhanced cellulase expression on cellobiose. The results of the investigation using PC-3-7 suggested that other mutation(s) in PC-3-7 could also contribute to cellulase induction. Further investigation is essential to unravel the mechanism responsible for cellulase induction in T. reesei.
Electronic supplementary material
The online version of this article (doi:10.1186/s13068-015-0420-y) contains supplementary material, which is available to authorized users.
doi:10.1186/s13068-015-0420-y
PMCID: PMC4696228  PMID: 26719764
Trichoderma reesei; Hypocrea jecorina; β-Glucosidase; Cellobiose; Cellulase induction; Gene regulation
5.  Identification of miR-15b as a transformation-related factor in mantle cell lymphoma 
International Journal of Oncology  2015;48(2):485-492.
Mantle cell lymphoma (MCL) is an aggressive B cell lymphoma with a poor prognosis. It is characterized by the t(11;14)(q13;q32) translocation, resulting in overexpression of CCND1. Morphologically, MCL is categorised into two types: classical MCL (cMCL) and aggressive MCL (aMCL), with a proportion of cMCL progressing to develop into aMCL. miRNAs are currently considered to be important regulators for cell behavior and are deregulated in many malignancies. Although several genetic alterations have been implicated in the transformation of cMCL to aMCL, the involvement of miRNAs in transformation is not known. In an effort to identify the miRNAs related to the transformation of MCL, miRNA microarray analyses were used for cMCL and aMCL cases. These analyses demonstrated significant differences in the expression of seven microRNAs based on a t-test (p-value <0.05); miR-15b was greatly upregulated in aMCL. Locked nucleic acid in situ hybridization showed increased staining of miR-15b in formalin-fixed paraffin-embedded sections of aMCL. These results correlated well with the microRNA microarray analysis. Although the molecular functions of miR-15b are largely unknown, it has been found to be associated with the cell cycle and apoptosis. However, the physiological significance of increased miR-15b in MCL is still unknown. Our present findings suggest that the upregulated expression of miR-15b is likely to play an important role in the transformation of cMCL to aMCL.
doi:10.3892/ijo.2015.3295
PMCID: PMC4725451  PMID: 26676972
mantle cell lymphoma; laser microdissection; miRNA microarray; in situ hybridization; miR-15b
6.  α-Ketoglutaramate: An overlooked metabolite of glutamine and a biomarker for hepatic encephalopathy and inborn errors of the urea cycle 
Metabolic brain disease  2013;29(4):991-1006.
Glutamine metabolism is generally regarded as proceeding via glutaminase-catalyzed hydrolysis to glutamate and ammonia, followed by conversion of glutamate to α-ketoglutarate catalyzed by glutamate dehydrogenase or by a glutamate-linked aminotransferase (transaminase). However, another pathway exists for the conversion of glutamine to α-ketoglutarate that is often overlooked, but is widely distributed in nature. This pathway, referred to as the glutaminase II pathway, consists of a glutamine transaminase coupled to ω-amidase. Transamination of glutamine results in formation of the corresponding α-keto acid, namely, α-ketoglutaramate (KGM). KGM is hydrolyzed by ω-amidase to α-ketoglutarate and ammonia. The net glutaminase II reaction is: L-Glutamine + α-keto acid + H2O → α-ketoglutarate + L-amino acid + ammonia. In this mini-review the biochemical importance of the glutaminase II pathway is summarized, with emphasis on the key component KGM. Forty years ago it was noted that the concentration of KGM is increased in the cerebrospinal fluid (CSF) of patients with hepatic encephalopathy (HE) and that the level of KGM in the CSF correlates well with the degree of encephalopathy. In more recent work, we have shown that KGM is markedly elevated in the urine of patients with inborn errors of the urea cycle. It is suggested that KGM may be a useful biomarker for many hyperammonemic diseases including hepatic encephalopathy, inborn errors of the urea cycle, citrin deficiency and lysinuric protein intolerance.
doi:10.1007/s11011-013-9444-9
PMCID: PMC4020999  PMID: 24234505
ω-Amidase; ammonia; glutaminase II; hepatic encephalopathy; α-ketoglutaramate; urea cycle disorders
7.  Impaired ATP6V0A2 expression contributes to Golgi dispersion and glycosylation changes in senescent cells 
Scientific Reports  2015;5:17342.
Many genes and signaling pathways have been found to be involved in cellular senescence program. In the present study, we have identified 16 senescence-associated genes by differential proteomic analysis of the normal human diploid fibroblast cell line, TIG-1, and focused on ATP6V0A2. The aim of this study is to clarify the role of ATP6V0A2, the causal gene for ARCL2, a syndrome of abnormal glycosylation and impaired Golgi trafficking, in cellular senescence program. Here we showed that ATP6V0A2 is critical for cellular senescence; impaired expression of ATP6V0A2 disperses the Golgi structure and triggers senescence, suggesting that ATP6V0A2 mediates these processes. FITC-lectin staining and glycoblotting revealed significantly different glycosylation structures in presenescent (young) and senescent (old) TIG-1 cells; reducing ATP6V0A2 expression in young TIG-1 cells yielded structures similar to those in old TIG-1 cells. Our results suggest that senescence-associated impaired expression of ATP6V0A2 triggers changes in Golgi structure and glycosylation in old TIG-1 cells, which demonstrates a role of ATP6V0A2 in cellular senescence program.
doi:10.1038/srep17342
PMCID: PMC4661525  PMID: 26611489
8.  Identification and Characterization of a Novel Galactofuranose-Specific β-D-Galactofuranosidase from Streptomyces Species 
PLoS ONE  2015;10(9):e0137230.
β-D-galactofuranose (Galf) is a component of polysaccharides and glycoconjugates and its transferase has been well analyzed. However, no β-D-galactofuranosidase (Galf-ase) gene has been identified in any organism. To search for a Galf-ase gene we screened soil samples and discovered a strain, identified as a Streptomyces species by the 16S ribosomal RNA gene analysis, that exhibits Galf-ase activity for 4-nitrophenyl β-D-galactofuranoside (pNP-β-D-Galf) in culture supernatants. By draft genome sequencing of the strain, named JHA19, we found four candidate genes encoding Galf-ases. Using recombinant proteins expressed in Escherichia coli, we found that three out of four candidates displayed the activity of not only Galf-ase but also α-L-arabinofuranosidase (Araf-ase), whereas the other one showed only the Galf-ase activity. This novel Galf-specific hydrolase is encoded by ORF1110 and has an optimum pH of 5.5 and a Km of 4.4 mM for the substrate pNP-β-D-Galf. In addition, this enzyme was able to release galactose residue from galactomannan prepared from the filamentous fungus Aspergillus fumigatus, suggesting that natural polysaccharides could be also substrates. By the BLAST search using the amino acid sequence of ORF1110 Galf-ase, we found that there are homolog genes in both prokaryotes and eukaryotes, indicating that Galf-specific Galf-ases widely exist in microorganisms.
doi:10.1371/journal.pone.0137230
PMCID: PMC4560423  PMID: 26340350
9.  Transcriptomic Analysis of Temperature Responses of Aspergillus kawachii during Barley Koji Production 
The koji mold Aspergillus kawachii is used for making the Japanese distilled spirit shochu. During shochu production, A. kawachii is grown in solid-state culture (koji) on steamed grains, such as rice or barley, to convert the grain starch to glucose and produce citric acid. During this process, the cultivation temperature of A. kawachii is gradually increased to 40°C and is then lowered to 30°C. This temperature modulation is important for stimulating amylase activity and the accumulation of citric acid. However, the effects of temperature on A. kawachii at the gene expression level have not been elucidated. In this study, we investigated the effect of solid-state cultivation temperature on gene expression for A. kawachii grown on barley. The results of DNA microarray and gene ontology analyses showed that the expression of genes involved in the glycerol, trehalose, and pentose phosphate metabolic pathways, which function downstream of glycolysis, was downregulated by shifting the cultivation temperature from 40 to 30°C. In addition, significantly reduced expression of genes related to heat shock responses and increased expression of genes related with amino acid transport were also observed. These results suggest that solid-state cultivation at 40°C is stressful for A. kawachii and that heat adaptation leads to reduced citric acid accumulation through activation of pathways branching from glycolysis. The gene expression profile of A. kawachii elucidated in this study is expected to contribute to the understanding of gene regulation during koji production and optimization of the industrially desirable characteristics of A. kawachii.
doi:10.1128/AEM.03483-14
PMCID: PMC4309687  PMID: 25501485
10.  A Highly Conserved Region Between Amino Acids 221 and 266 of Dengue Virus Non-Structural Protein 1 Is a Major Epitope Region in Infected Patients 
The immune response to dengue virus (DENV) infection generates high levels of antibodies (Abs) against the DENV non-structural protein 1 (NS1), particularly in cases of secondary infection. Therefore, anti-NS1 Abs may play a role in severe dengue infections, possibly by interacting (directly or indirectly) with host factors or regulating virus production. If it does play a role, NS1 may contain epitopes that mimic those epitopes of host molecules. Previous attempts to map immunogenic regions within DENV-NS1 were undertaken using mouse monoclonal Abs (MAbs). The aim of this study was to characterize the epitope regions of nine anti-NS1 human monoclonal Abs (HuMAbs) derived from six patients secondarily infected with DENV-2. These anti-NS1 HuMAbs were cross-reactive with DENV-1, -2, and -3 but not DENV-4. All HuMAbs bound a common epitope region located between amino acids 221 and 266 of NS1. This study is the first report to map a DENV-NS1 epitope region using anti-DENV MAbs derived from patients.
doi:10.4269/ajtmh.13-0624
PMCID: PMC4080554  PMID: 24778195
11.  Late-occurring coil migration into the duodenum 
BMJ Case Reports  2013;2013:bcr2012007759.
doi:10.1136/bcr-2012-007759
PMCID: PMC3603895  PMID: 23345483
12.  Galanin plays an important role in cancer invasiveness and is associated with poor prognosis in stage II colorectal cancer 
Oncology Reports  2014;33(2):539-546.
Reliable predictors of tumor recurrence for patients with stage II colorectal cancer (CRC) are needed to select patients who should receive adjuvant chemotherapy. Although galanin (GAL) is expressed in several malignant tumors and is associated with cell proliferation and tumor growth, the prognostic value of GAL expression in CRC is poorly understood. We compared GAL expression between 56 patients with stage II and III CRC who developed tumor recurrences and 56 patients who did not. The clinical and prognostic significance of GAL expression was examined using our data and independent public datasets. We also analyzed the influence of GAL expression on the proliferation and invasive activity of CRC cells. Higher expression of GAL was associated with tumor recurrence among the CRC patients (P<0.001). Stage II CRC patients who presented with high expression levels of GAL had significantly poorer prognosis than those with low expression levels of GAL [5-year overall survival: hazard ratio (HR), 7.31; 95% confidence interval (CI), 2.38–24.04; P<0.001; 5-year recurrence-free survival: HR, 3.99; 95% CI, 1.61–9.44; P=0.004], but there was no association between GAL expression and survival in stage III CRC patients. These findings were supported by analysis of two public datasets. Functionally, siRNA-mediated silencing of GAL resulted in a significant decrease in the proliferative and invasive activities of CRC cells. In conclusion, high expression of GAL is associated with poor prognosis of stage II CRC patients and GAL expression may be related to the aggressive behavior of CRC.
doi:10.3892/or.2014.3660
PMCID: PMC4306273  PMID: 25504183
galanin; colorectal cancer; stage II; prognostic factor
13.  Light and pheromone-sensing neurons regulates cold habituation through insulin signalling in Caenorhabditis elegans 
Nature Communications  2014;5:4412.
Temperature is a critical environmental stimulus that has a strong impact on an organism’s biochemistry. Animals can respond to changes in ambient temperature through behaviour or altered physiology. However, how animals habituate to temperature is poorly understood. The nematode C. elegans stores temperature experiences and can induce temperature habituation-linked cold tolerance. Here we show that light and pheromone-sensing neurons (ASJ) regulate cold habituation through insulin signalling. Calcium imaging reveals that ASJ neurons respond to temperature. Cold habituation is abnormal in a mutant with impaired cGMP signalling in ASJ neurons. Insulin released from ASJ neurons is received by the intestine and neurons regulating gene expression for cold habituation. Thus, temperature sensation in a light and pheromone-sensing neuron produces a robust effect on insulin signalling that controls experience-dependent temperature habituation.
Cold tolerance in Caenorhabditis elegans is regulated by signalling pathways and neuronal circuits, but the exact mechanisms are unclear. Here the authors show that cold tolerance requires activity from specific light and pheromone-sensing neurons that release insulin to regulate gene expression in the intestine.
doi:10.1038/ncomms5412
PMCID: PMC4109018  PMID: 25048458
14.  Management of visceral artery embolization using 0.010-inch detachable microcoils 
Transcatheter coil embolization is used primarily to treat arterial hemorrhages, tumors, aneurysms, and vascular malformations. However, conventional microcatheter systems cannot always be employed in difficult cases. In this technical note, we describe how small-diameter primary coils and microcatheter tips that are thinner than normal can be used to increase the safety and reliability of coil embolization.
doi:10.5152/dir.2014.13382
PMCID: PMC4463280  PMID: 24509180
15.  A novel method for gathering and prioritizing disease candidate genes based on construction of a set of disease-related MeSH® terms 
BMC Bioinformatics  2014;15:179.
Background
Understanding the molecular mechanisms involved in disease is critical for the development of more effective and individualized strategies for prevention and treatment. The amount of disease-related literature, including new genetic information on the molecular mechanisms of disease, is rapidly increasing. Extracting beneficial information from literature can be facilitated by computational methods such as the knowledge-discovery approach. Several methods for mining gene-disease relationships using computational methods have been developed, however, there has been a lack of research evaluating specific disease candidate genes.
Results
We present a novel method for gathering and prioritizing specific disease candidate genes. Our approach involved the construction of a set of Medical Subject Headings (MeSH) terms for the effective retrieval of publications related to a disease candidate gene. Information regarding the relationships between genes and publications was obtained from the gene2pubmed database. The set of genes was prioritized using a “weighted literature score” based on the number of publications and weighted by the number of genes occurring in a publication. Using our method for the disease states of pain and Alzheimer’s disease, a total of 1101 pain candidate genes and 2810 Alzheimer’s disease candidate genes were gathered and prioritized. The precision was 0.30 and the recall was 0.89 in the case study of pain. The precision was 0.04 and the recall was 0.6 in the case study of Alzheimer’s disease. The precision-recall curve indicated that the performance of our method was superior to that of other publicly available tools.
Conclusions
Our method, which involved the use of a set of MeSH terms related to disease candidate genes and a novel weighted literature score, improved the accuracy of gathering and prioritizing candidate genes by focusing on a specific disease.
doi:10.1186/1471-2105-15-179
PMCID: PMC4068192  PMID: 24917541
16.  A Biomarker Found in Cadmium Exposed Residents of Thailand by Metabolome Analysis 
First, the urinary metabolic profiling by gas chromatography-mass spectrometry (GC-MS), was performed to compare ten cadmium (Cd) toxicosis cases from a Cd-polluted area in Mae Sot (Thailand) with gender-matched healthy controls. Orthogonal partial list square-discrimination analysis was used to identify new biomarker candidates in highly Cd exposed toxicosis cases with remarkable renal tubular dysfunction. The results of the first step of this study showed that urinary citrate was a negative marker and myo-inositol was a positive marker for Cd toxicosis in Thailand. In the second step, we measured urinary citrate in the residents (168 Cd-exposed subjects and 100 controls) and found significantly lower levels of urinary citrate and higher ratios of calcium/citrate and magnesium/citrate, which are risk factors for nephrolithiasis, in highly Cd-exposed residents. Additionally, this inverse association of urinary citrate with urinary Cd was observed after adjustment for age, smoking and renal tubular dysfunction, suggesting a direct effect of Cd on citrate metabolism. These results indicate that urinary citrate is a useful biomarker for the adverse health effects of Cd exposure in a Thai population with a high prevalence of nephrolithiasis.
doi:10.3390/ijerph110403661
PMCID: PMC4025033  PMID: 24699029
cadmium; metabolomics; orthogonal partial list square-discrimination analysis (OPLS-DA); urinary citrate; Thailand
17.  Follow-up of true visceral artery aneurysm after coil embolization by three-dimensional contrast-enhanced MR angiography 
PURPOSE
We aimed to evaluate the outcomes of coil embolization of true visceral artery aneurysms by three-dimensional contrast-enhanced magnetic resonance (MR) angiography.
MATERIALS AND METHODS
We used three-dimensional contrast-enhanced MR angiography, which included source images, to evaluate 23 patients (mean age, 60 years; range, 28–83 years) with true visceral artery aneurysms (splenic, n=15; hepatic, n=2; gastroduodenal, n=2; celiac, n=2; pancreaticoduodenal, n=1; gastroepiploic, n=1) who underwent coil embolization. Angiographic aneurysmal occlusion was revealed in all cases. Follow-up MR angiography was conducted with either a 1.5 or 3 Tesla system 3–25 months (mean, 18 months) after embolization. MR angiography was evaluated for aneurysmal occlusion, hemodynamic status, and complications.
RESULTS
Complete aneurysmal occlusion was determined in 22 patients (96%) on follow-up MR angiography (mean follow-up period, 18 months). Neck recanalization, which was observed at nine and 20 months after embolization, was confirmed in one of eight patients (13%) using a neck preservation technique. In this patient, a small neck recanalization covered by a coil mass was demonstrated. The complete hemodynamic status after embolization was determined in 21 patients (91%); the visualization of several collateral vessels, such as short gastric arteries, after parent artery occlusion was poor compared with that seen on digital subtraction angiography in the remaining two patients (9%). An asymptomatic localized splenic infarction was confirmed in one patient (4%).
CONCLUSION
Our study presents the follow-up results from three-dimensional contrast-enhanced MR angiography, which confirmed neck recanalization, the approximate hemodynamic status, and complications. This effective and less invasive method may be suitable for serial follow-up after coil embolization of true visceral aneurysms.
doi:10.5152/dir.2013.13236
PMCID: PMC4463299  PMID: 24356294
18.  Clinical, biochemical and molecular analysis of 13 Japanese patients with β-ureidopropionase deficiency demonstrates high prevalence of the c.977G > A (p.R326Q) mutation 
β-ureidopropionase (βUP) deficiency is an autosomal recessive disease characterized by N-carbamyl-β-amino aciduria. To date, only 16 genetically confirmed patients with βUP deficiency have been reported. Here, we report on the clinical, biochemical and molecular findings of 13 Japanese βUP deficient patients. In this group of patients, three novel missense mutations (p.G31S, p.E271K, and p.I286T) and a recently described mutation (p.R326Q) were identified. The p.R326Q mutation was detected in all 13 patients with eight patients being homozygous for this mutation. Screening for the p.R326Q mutation in 110 Japanese individuals showed an allele frequency of 0.9 %. Transient expression of mutant βUP enzymes in HEK293 cells showed that the p.E271K and p.R326Q mutations cause profound decreases in activity (≤ 1.3 %). Conversely, βUP enzymes containing the p.G31S and p.I286T mutations possess residual activities of 50 and 70 %, respectively, suggesting we cannot exclude the presence of additional mutations in the non-coding region of the UPB1 gene. Analysis of a human βUP homology model revealed that the effects of the mutations (p.G31S, p.E271K, and p.R326Q) on enzyme activity are most likely linked to improper oligomer assembly. Highly variable phenotypes ranging from neurological involvement (including convulsions and autism) to asymptomatic, were observed in diagnosed patients. High prevalence of p.R326Q in the normal Japanese population indicates that βUP deficiency is not as rare as generally considered and screening for βUP deficiency should be included in diagnosis of patients with unexplained neurological abnormalities.
Electronic supplementary material
The online version of this article (doi:10.1007/s10545-014-9682-y) contains supplementary material, which is available to authorized users.
doi:10.1007/s10545-014-9682-y
PMCID: PMC4158181  PMID: 24526388
21.  Unique Regulatory Mechanism of Sporulation and Enterotoxin Production in Clostridium perfringens 
Journal of Bacteriology  2013;195(12):2931-2936.
Clostridium perfringens causes gas gangrene and gastrointestinal (GI) diseases in humans. The most common cause of C. perfringens-associated food poisoning is the consumption of C. perfringens vegetative cells followed by sporulation and production of enterotoxin in the gut. Despite the importance of spore formation in C. perfringens pathogenesis, the details of the regulation of sporulation have not yet been defined fully. In this study, microarray and bioinformatic analyses identified a candidate gene (the RNA regulator virX) for the repression of genes encoding positive regulators (Spo0A and sigma factors) of C. perfringens sporulation. A virX mutant constructed in the food poisoning strain SM101 had a much higher sporulation efficiency than that of the wild type. The transcription of sigE, sigF, and sigK was strongly induced at 2.5 h of culture of the virX mutant. Moreover, the transcription of the enterotoxin gene was also strongly induced in the virX mutant. Western blotting confirmed that the levels of enterotoxin production were higher in the virX mutant than in the wild type. These observations indicated that the higher levels of sporulation and enterotoxin production in the virX mutant were specifically due to inactivation of the virX gene. Since virX homologues were not found in any Bacillus species but were present in other clostridial species, our findings identify further differences in the regulation of sporulation between Bacillus and certain Clostridium species. The virX RNA regulator plays a key role in the drastic shift in lifestyle of the anaerobic flesh eater C. perfringens between the vegetative state (for gas gangrene) and the sporulating state (for food poisoning).
doi:10.1128/JB.02152-12
PMCID: PMC3697249  PMID: 23585540
22.  Dissection of the Octoploid Strawberry Genome by Deep Sequencing of the Genomes of Fragaria Species 
Cultivated strawberry (Fragaria x ananassa) is octoploid and shows allogamous behaviour. The present study aims at dissecting this octoploid genome through comparison with its wild relatives, F. iinumae, F. nipponica, F. nubicola, and F. orientalis by de novo whole-genome sequencing on an Illumina and Roche 454 platforms. The total length of the assembled Illumina genome sequences obtained was 698 Mb for F. x ananassa, and ∼200 Mb each for the four wild species. Subsequently, a virtual reference genome termed FANhybrid_r1.2 was constructed by integrating the sequences of the four homoeologous subgenomes of F. x ananassa, from which heterozygous regions in the Roche 454 and Illumina genome sequences were eliminated. The total length of FANhybrid_r1.2 thus created was 173.2 Mb with the N50 length of 5137 bp. The Illumina-assembled genome sequences of F. x ananassa and the four wild species were then mapped onto the reference genome, along with the previously published F. vesca genome sequence to establish the subgenomic structure of F. x ananassa. The strategy adopted in this study has turned out to be successful in dissecting the genome of octoploid F. x ananassa and appears promising when applied to the analysis of other polyploid plant species.
doi:10.1093/dnares/dst049
PMCID: PMC3989489  PMID: 24282021
Fragariax ananassa; wild Fragaria species; genome sequence assembly; comparative analysis; polyploidy
24.  Emerging Antigenic Variants at the Antigenic Site Sb in Pandemic A(H1N1)2009 Influenza Virus in Japan Detected by a Human Monoclonal Antibody 
PLoS ONE  2013;8(10):e77892.
The swine-origin pandemic A(H1N1)2009 virus, A(H1N1)pdm09, is still circulating in parts of the human population. To monitor variants that may escape from vaccination specificity, antigenic characterization of circulating viruses is important. In this study, a hybridoma clone producing human monoclonal antibody against A(H1N1)pdm09, designated 5E4, was prepared using peripheral lymphocytes from a vaccinated volunteer. The 5E4 showed viral neutralization activity and inhibited hemagglutination. 5E4 escape mutants harbored amino acid substitutions (A189T and D190E) in the hemagglutinin (HA) protein, suggesting that 5E4 recognized the antigenic site Sb in the HA protein. To study the diversity of Sb in A(H1N1)pdm09, 58 viral isolates were obtained during the 2009/10 and 2010/11 winter seasons in Osaka, Japan. Hemagglutination-inhibition titers were significantly reduced against 5E4 in the 2010/11 compared with the 2009/10 samples. Viral neutralizing titers were also significantly decreased in the 2010/11 samples. By contrast, isolated samples reacted well to ferret anti-A(H1N1)pdm09 serum from both seasons. Nonsynonymous substitution rates revealed that the variant Sb and Ca2 sequences were being positively selected between 2009/10 and 2010/11. In 7,415 HA protein sequences derived from GenBank, variants in the antigenic sites Sa and Sb increased significantly worldwide from 2009 to 2013. These results indicate that the antigenic variants in Sb are likely to be in global circulation currently.
doi:10.1371/journal.pone.0077892
PMCID: PMC3797713  PMID: 24147093
25.  Statins Decrease Lung Inflammation in Mice by Upregulating Tetraspanin CD9 in Macrophages 
PLoS ONE  2013;8(9):e73706.
Tetraspanins organize protein complexes in tetraspanin-enriched membrane microdomains that are distinct from lipid rafts. Our previous studies suggested that reduction in the levels of tetraspanins CD9 and CD81 may be involved in the progression of inflammatory lung diseases, especially COPD. To search for agents that increase the levels of these tetraspanins, we screened 1,165 drugs in clinical use and found that statins upregulate CD9 and CD81 in RAW264.7 macrophages. The lipophilic statins, fluvastatin and simvastatin, reversed LPS-induced downregulation of CD9 and CD81, simultaneously preventing TNF-α and matrix metalloproteinase-9 production and spreading of RAW264.7 cells. These statins exerted anti-inflammatory effects in vitro in wild-type macrophages but not in CD9 knockout macrophages, and decreased lung inflammation in vivo in wild-type mice but not in CD9 knockout mice, suggesting that their effects are dependent on CD9. Mechanistically, the statins promoted reverse transfer of the LPS-signaling mediator CD14 from lipid rafts into CD9-enriched microdomains, thereby preventing LPS receptor formation. Finally, upregulation of CD9/CD81 by statins was related to blockade of GTPase geranylgeranylation in the mevalonate pathway. Our data underscore the importance of the negative regulator CD9 in lung inflammation, and suggest that statins exert anti-inflammatory effects by upregulating tetraspanin CD9 in macrophages.
doi:10.1371/journal.pone.0073706
PMCID: PMC3767596  PMID: 24040034

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