Ammoniibacillus agariperforans gen. nov., sp. nov., a thermophilic, agar-degrading bacterium isolated from compost.

A thermophilic, agar-degrading bacterium, strain FAB2(T), was isolated from sewage sludge compost. According to phylogenetic analysis based on 16S rRNA gene sequences, strain FAB2(T) belonged to the family Paenibacillaceae within the phylum Firmicutes. However, FAB2(T) was different enough at the genus level from closely related species. The percentages of 16S rRNA gene sequence similarity with related organisms were 90.4 % for Thermobacillus xylanilyticus, 91.8 % for Paenibacillus barengoltzii, 89.4 % for Cohnella lupini, 90.1 % for Fontibacillus aquaticus, and 89.0 % for Saccharibacillus sacchari. Morphological and physiological analyses revealed that the strain was motile, rod-shaped, Gram-stain-positive, aerobic and able to form oval endospores in swollen sporangia. Ammonium was required as a nitrogen source while nitrate, nitrite, urea and glutamate were not utilized. Catalase and oxidase activities were weakly positive and positive, respectively. The bacterium grew in the temperature range of 50-65 °C and in media with pH 7.5 to 9.0. Optimal growth occurred at 60 °C and pH 8.0-8.6. Growth was inhibited at pH≤7.0 and NaCl concentrations ≥2.5 % (w/v). In chemotaxonomic characterization, MK-7 was identified as the dominant menaquinone. Major fatty acids were iso-C16 : 0 and C16 : 0. Dominant polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. Phosphatidylcholine was present in a moderate amount. The diamino acid in the cell wall was meso-diaminopimelic acid. The G+C content of the genomic DNA was 49.5 mol% in a nucleic acid study. On the basis of genetic and phenotypic characteristics, strain FAB2(T) ( = NBRC 109510(T) = KCTC 33130(T)) showed characteristics suitable for classification as the type strain of a novel species of a new genus in the family Paenibacillaceae, for which the name Ammoniibacillus agariperforans gen. nov., sp. nov. is proposed.

Development of LNA oligonucleotide-PCR clamping technique in investigating the community structures of plant-associated bacteria.

Simultaneous extraction of plant organelle (mitochondria and plastid) genes during the DNA extraction step is major limitation in investigating the community structures of plant-associated bacteria. Although locked nucleic acid (LNA) oligonucleotides was designed to selectively amplify the bacterial small subunit rRNA genes by applying the PCR clamping technique, those for plastids were applicable only for particular plants, while those for mitochondria were available throughout most plants. To widen the applicable range, new LNA oligonucleotides specific for plastids were designed, and the efficacy was investigated. PCR without LNA oligonucleotides predominantly amplified the organelle genes, while bacterial genes were predominantly observed in having applied the LNA oligonucleotides. Denaturing gradient gel electrophoresis (DGGE) analysis displayed additional bacterial DGGE bands, the amplicons of which were prepared using the LNA oligonucleotides. Thus, new designed LNA oligonucleotides specific for plastids were effective and have widened the scope in investigating the community structures of plant-associated bacteria.

Cell-autonomous signal transduction in the Xenopus egg Wnt/ß-catenin pathway.

Wnt proteins are thought to bind to their receptors on the cell surfaces of neighboring cells. Wnt8 likely substitutes for the dorsal determinants in Xenopus embryos to dorsalize early embryos via the Wnt/ß-catenin pathway. Here, we show that Wnt8 can dorsalize Xenopus embryos working cell autonomously. Wnt8 mRNA was injected into a cleavage-stage blastomere, and the subcellular distribution of Wnt8 protein was analyzed. Wnt8 protein was predominantly found in the endoplasmic reticulum (ER) and resided at the periphery of the cells; however, this protein was restricted to the mRNA-injected cellular region as shown by lineage tracing. A mutant Wnt8 that contained an ER retention signal (Wnt8-KDEL) could dorsalize Xenopus embryos. Finally, Wnt8-induced dorsalization occurred only in cells injected with Wnt8 mRNA. These experiments suggest that the Wnt8 protein acts within the cell, likely in the ER or on the cell surface in an autocrine manner for dorsalization.

High cell-autonomy of the anterior endomesoderm viewed in blastomere fate shift during regulative development in the isolated right halves of four-cell stage Xenopus embryos.

The isolated right half (RH) or left half (LH) of Xenopus embryos can undergo regulation so as to form well-proportioned larvae. To assess how the combined actions of maternal determinants and cell-cell interactions contribute to form the well-proportioned larvae, we quantitatively compared four-cell stage blastomere fate between normal larvae and regulated larvae from RH embryos. In normal larvae, the clones of the right dorsal blastomere (RD) and right ventral blastomere (RV) were located unilaterally. In contrast, in regulated larvae: (i) the RD clone exclusively occupied the anterior endomesoderm (AE) derivatives, coinciding no RV progeny in those derivatives of normal larvae. The clone bilaterally populated tissues along the dorsal midline, which characteristically included the medial regions of both somites adjoining the notochord, with higher percentages on the right and anterior sides. (ii) The RV clone extensively compensated for the missing left side at the expense of its right side contribution, and bilaterally occupied the ventroposterior and also dorsal regions excluding the AE derivatives. This clone considerably populated, with altered orientations, the derivatives of the left half gastrocoel roof plate (GRP), the left half GRP being essential for laterality determination. These results show that the high cell-autonomy in the AE constitutes a mechanism common to both normal and regulative development. In regulated larvae, cell-cell interactions shifted the midlines on the dorsal side slightly and the ventral side to a greater extent. The cell lineage difference in the left half GRP could result in a different utilization of maternal determinants in that area.

Mobile and accurate detection system for infection by the 2009 pandemic influenza A (H1N1) virus with a pocket-warmer reverse-transcriptase loop-mediated isothermal amplification.

The 2009 pandemic H1N1 influenza A virus spread quickly worldwide in 2009. Since most of the fatal cases were reported in developing countries, rapid and accurate diagnosis methods that are usable in poorly equipped laboratories are necessary. In this study, a mobile detection system for the 2009 H1N1 influenza A virus was developed using a reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) kit with a disposable pocket-warmer as a heating device (designated as pwRT-LAMP). The pwRT-LAMP can detect as few as 100 copies of the virus--which is nearly as sensitive as real-time reverse-transcription polymerase chain reaction (RT-PCR)--and does not cross-react with RNA of seasonal influenza viruses. To evaluate the usefulness of the pwRT-LAMP system, nasal swab samples were collected from 56 patients with flu-like symptoms and were tested. Real-time RT-PCR confirmed that the 2009 H1N1 influenza A virus was present in 27 of the 56 samples. Of these 27 positive samples, QuickVue Influenza A+B immunochromatography detected the virus in only 11 samples (11/27; 40.7%), whereas the pwRT-LAMP system detected the virus in 26 of the 56 samples (26/27 of the positive samples; 96.3%). These findings indicate that the mobile pwRT-LAMP system is an accurate diagnostic system for the 2009 H1N1 influenza A virus, and has great potential utility in diagnosing future influenza pandemics.

Characterization and Distribution of Agar-degrading Steroidobacter agaridevorans sp. nov., Isolated from Rhizosphere Soils.

The environment of plant rhizosphere soil differs from that of non-rhizosphere soil due to the secretion of mucilage polysaccharides from the roots. This environment is regarded as one of the preferential habitats for agar-degrading bacteria. In a previous study, agar-degrading Steroidobacter agariperforans KA5-BT was isolated from agar-enriched agricultural soil using diffusible metabolites from Rhizobiales bacteria. Based on the hypothesis that similar characteristic bacteria still exist in the rhizosphere, isolation was performed using rhizosphere soils. Agar-degrading SA29-BT and YU21-B were isolated from onion and soybean rhizosphere soils. The 16S rRNA genes of these strains showed ≥98.7% identities with the most closely related strain KA5-BT. However, differences were noted in polysaccharide utilization, and average nucleotide identities were <95-96% against strain KA5-BT, indicating that they are different species from S. agariperforans KA5-BT. To investigate the distribution of bacterial sequences affiliated with novel strains, a primer set was designed and a meta-analysis of the 16S rRNA gene was performed. Sequences were widely distributed in rhizospheres throughout Japan, but varied in plant- and region-dependent manners. Regarding phenotypic characterization, distinguishable features were observed in growth temperatures, pH, and dominant fatty acids. SA29-BT and YU21-B grew at 15-40°C and pH 6.0-12 and contained C16:0 as the dominant cell fatty acid, whereas KA5-BT showed no growth at 40°C and pH 12 and contained a moderate amount of C16:0. Based on these characteristics, SA29-BT (JCM 333368T=KCTC 72223T) and YU21-B (JCM 333367=KCTC 72222) represent novel species in the genus Steroidobacter, for which the name Steroidobacter agaridevorans sp. nov. is proposed.

Ventricular fibrillation diagnosed during electrophysiological study for non-sustained tachycardia.

Ventricular fibrillation diagnoses such as Brugada syndrome pose a risk of sudden incapacitation or death in aircrew. This case report presents a 44-yr-old male fighter pilot who unexpectedly developed ventricular fibrillation (VF) during an electrophysiological study (EPS) prior to therapy for non-sustained ventricular tachycardia (nsVT). The initial aeromedical disposition for this case was "qualified for flying duties". with the restriction that he must fly with another pilot due to repeatedly observed nsVT. This pilot wanted to return to flight duty in single-seat aircraft without any restrictions. Therefore, this patient decided to undergo catheter therapy for nsVT. Unexpectedly, not VT but VF was induced by catheter manipulation during EPS. Pilsicainide-induced coved-type ST wave elevation consistent with Brugada syndrome was noted in this patient's electrocardiogram. He was ultimately disqualified due to the diagnosis of VF. This report suggests EPS on rare occasions may uncover another severe disease similar to this case report.

Survey of severe spatial disorientation episodes in Japan Air Self-Defense Force fighter pilots showing increased severity in night flight.

Spatial disorientation (SD) is one of the most severe causative factors in aviation accidents. We analyzed the reported SD episodes to evaluate the characteristics of severe SD in fighter pilots. Three hundred seventeen cases (95.5%) of 332 total valid cases experienced SD, and the ratio of night and day SD experiences (52.7% vs. 47.3%) (p < 0.05) shows a clear prevalence of night SD events. The severity of SD episodes at night (2.23 +/- 1.09) was higher than at day (1.89 +/- 1.04) (p < 0.01). In addition, the severity of visual illusions was significantly higher at night. A significant difference was found for meteorological conditions, such as visual meteorological conditions (VMC), instrument meteorological conditions (IMC) and VMC-IMC (VI) transition, among times of days. In conclusion, the severity of the SD episodes was higher at night. This may be due to an increase in visual severe SD episodes at night.

VegT, eFGF and Xbra cause overall posteriorization while Xwnt8 causes eye-level restricted posteriorization in synergy with chordin in early Xenopus development.

We examined several candidate posterior/mesodermal inducing molecules using permanent blastula-type embryos (PBEs) as an assay system. Candidate molecules were injected individually or in combination with the organizer factor chordin mRNA. Injection of chordin alone resulted in a white hemispherical neural tissue surrounded by a large circular cement gland, together with anterior neural gene expression and thus the development of the anterior-most parts of the embryo, without mesodermal tissues. When VegT, eFGF or Xbra mRNAs were injected into a different blastomere of the chordin-injected PBEs, the embryos elongated and formed eye, muscle and pigment cells, and expressed mesodermal and posterior neural genes. These embryos formed the full spectrum of the anteroposterior embryonic axis. In contrast, injection of CSKA-Xwnt8 DNA into PBEs injected with chordin resulted in eye formation and expression of En2, a midbrain/hindbrain marker, and Xnot, a notochord marker, but neither elongation, muscle formation nor more posterior gene expression. Injection of chordin and posteriorizing molecules into the same cell did not result in elongation of the embryo. Thus, by using PBEs as the host test system we show that (i) overall anteroposterior neural development, mesoderm (muscle) formation, together with embryo elongation can occur through the synergistic effect(s) of the organizer molecule chordin, and each of the 'verall posteriorizing molecules'eFGF, VegT and Xbra; (ii) Xwnt8-mediated posteriorization is restricted to the eye level and is independent of mesoderm formation; and (iii) proper anteroposterior patterning requires a separation of the dorsalizing and posteriorizing gene expression domains.

Improvements in Bacterial Primers to Enhance Selective SSU rRNA Gene Amplification of Plant-associated Bacteria by Applying the LNA Oligonucleotide-PCR Clamping Technique.

PCR clamping by locked nucleic acid (LNA) oligonucleotides is an effective technique for selectively amplifying the community SSU rRNA genes of plant-associated bacteria. However, the original primer set often shows low amplification efficiency. In order to improve this efficiency, new primers were designed at positions to compete with LNA oligonucleotides. Three new sets displayed higher amplification efficiencies than the original; however, efficiency varied among the primer sets. Two new sets appeared to be available in consideration of bacterial profiles by next-generation sequencing. One new set, KU63f and KU1494r, may be applicable to the selective gene amplification of plant-associated bacteria.

Effectiveness of CT for clinical stratification of occupational lung edema.

We treated two occupational lung diseases in different situations during military training. The purpose of this study is to investigate the availability of CT scanning for the evaluation of inhalation pulmonary edema. Two soldiers suffered severe lung edema after using a spray for the daily maintenance of their firearms. Four soldiers suffered severe dyspnea after undertaking drills in a narrow zone where numerous smoke bombs had been used. We evaluated these patients from several aspects. CT scans of the chest of spray-induced patients revealed bilateral infiltration predominantly in the upper lung fields. The patients received steroid pulse treatment and gradually recovered. CT scans of the chest of smoke-induced patients revealed bilateral ground-glass attenuation with peripheral lung sparing. The patients gradually recovered with steroid therapy. In accordance with previous studies, CT scans of the chest in our patients demonstrated that the periphery of the lungs remained normal, except in cases of serious injury. When differential diagnosis is required, we consider that CT scans of the chest are particularly useful; CT findings are useful in determining the severity of lung injury as well as the diagnosis of inhalation pulmonary edema.

Application of Locked Nucleic Acid (LNA) Primer and PCR Clamping by LNA Oligonucleotide to Enhance the Amplification of Internal Transcribed Spacer (ITS) Regions in Investigating the Community Structures of Plant-Associated Fungi.

The simultaneous extraction of host plant DNA severely limits investigations of the community structures of plant-associated fungi due to the similar homologies of sequences in primer-annealing positions between fungi and host plants. Although fungal-specific primers have been designed, plant DNA continues to be excessively amplified by PCR, resulting in the underestimation of community structures. In order to overcome this limitation, locked nucleic acid (LNA) primers and PCR clamping by LNA oligonucleotides have been applied to enhance the amplification of fungal internal transcribed spacer (ITS) regions. LNA primers were designed by converting DNA into LNA, which is specific to fungi, at the forward primer side. LNA oligonucleotides, the sequences of which are complementary to the host plants, were designed by overlapping a few bases with the annealing position of the reverse primer. Plant-specific DNA was then converted into LNA at the shifted position from the 3' end of the primer-binding position. PCR using the LNA technique enhanced the amplification of fungal ITS regions, whereas those of the host plants were more likely to be amplified without the LNA technique. A denaturing gradient gel electrophoresis (DGGE) analysis displayed patterns that reached an acceptable level for investigating the community structures of plant-associated fungi using the LNA technique. The sequences of the bands detected using the LNA technique were mostly affiliated with known isolates. However, some sequences showed low similarities, indicating the potential to identify novel fungi. Thus, the application of the LNA technique is considered effective for widening the scope of community analyses of plant-associated fungi.

Draft Genome Sequences of Streptomyces scabiei S58, Streptomyces turgidiscabies T45, and Streptomyces acidiscabies a10, the Pathogens of Potato Common Scab, Isolated in Japan.

The draft genome sequences of the three pathogens of potato common scab, Streptomyces scabiei S58, Streptomyces turgidiscabies T45, and Streptomyces acidiscabies a10, isolated in Japan, are presented here. The genome size of each strain is >10 Mb, and the three pathogenic strains share genes located in a pathogenicity island previously described in other pathogenic Streptomyces species.

[Effects of manure application on the diversity of corn root endophytic bacterial communities at seedling stage in eroded Mollisols.] / æ–½ç”¨æœ‰æœºè‚¥å¯¹ä¾µèš€é»‘åœŸçŽ‰ç±³è‹—æœŸæ ¹å† ç”Ÿç»†èŒå¤šæ ·æ€§çš„å½±å“.

In order to investigate the change of root endophytic bacterial communities under soil erosion condition, and to evaluate the response of root endophytic bacteria to manure fertilizer, we adopted the LNA-PCR clamping and 454 bar-coded pyrosequencing methods to study the corn root endophytic bacterial communities under 30 cm topsoil erosion and manure fertilization conditions. No topsoil removing (0 cm) and only chemical fertilizer treatment were used as control. A total of 37820 valid sequences of 16S rDNA were obtained, mainly distributed in 4 phyla, 35 classes, 214 genera and 782 OTUs. The dominant phyla were Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes, but their proportions varied in different samples. The diversity of corn root endophytic bacteria decreased in soil erosion condition. In the topsoil removing soil and no erosion soil, the diversity of corn root endophytic bacterial communities increased by manure application, and the effect was more obvious in the topsoil removing soil.

Heat-Induced Reversal of Dorsal-Ventral Polarity in Xenopus Eggs: (Xenopus eggs/heat-treatment/D-V axis).

Heat-treatment of fertilized Xenopus laevis eggs at 30°C induced; 1. conspicuous concentration of the pigment toward the sperm entry point (SEP), 2. eccentric first cleavage furrow formation, and 3. reversal of the dorsal-ventral polarity of the embryos. The optimal treatment was for 2.5 min applied at 20 min postfertilization (p.f.). The rotation movement of the Nile-blue stained spots in the vegetal hemisphere of the heated eggs accurately located the future dorsal midline as in untreated embryos (ref. 22). Exposure of eggs to D2 O also reversed the dorsal-ventral polarity of the embryo suggesting that stabilization of microtubules is involved in the dorsal-ventral axis reversal.

Subcortical Rotation and Specification of the Dorsoventral Axis in Newt Eggs: (newt eggs/subcortical rotation/dorsoventral axis).

The specification of the dorsoventral axis in naturally polyspermic eggs of the Japanese newt, Cynops pyrrhogaster, was first examined by studies on the spatial relationship between the dorsal midline of the future body plan and the sperm entrance points (SEPs1 ). On local insemination, the dorsal blastopore lip was usually found to be formed opposite the SEPs, as in anuran monospermic eggs. Next the movements of the subcortical layer and the cortex were analyzed. "Subcortical rotation" was observed, similar to that of Xenopus laevis eggs with respect to its timing and extent, and its direction was shown to predict the embryonic axis of the eggs. Thus, the dorsoventral axis was concluded to be determined by essentially the same mechanism in the newt as in Xenopus. Owing to their large size and long first cell cycle, newt eggs appear to be suitable material for study of subcortical rotation, but their behavior is unique in that subcortical rotation occurs in only the vegetal hemisphere so that the subcortical layer stretches in the future dorsal side. Studies on the movement of Nile blue spots suggested that the cytoplasm under the cortex in newt eggs consists of two layers.

CYCLIC SURFACE CHANGES IN THE NON-NUCLEATE EGG FRAGMENT OF XENOPUS LAEVIS.

Fertilized uncleaved eggs of Xenopus laevis were divided into nucleate and non-nucleate egg fragments. Both fragments, together with the whole egg of the same batch, were observed by time-lapse cinematography. Two kinds of cyclic surface changes, (1) rounding-up and relaxing movements and (2) surface contraction waves, accompanying each cleavage in the whole eggs and the nucleate fragments, were also observed even in the non-nucleate fragments although they do not cleave. Cleavage intervals of the whole egg and the nucleate fragment were nearly equal, but the rounding-up intervals of the non-nucleate fragment were slightly but definitely longer than the cleavage intervals of the nucleate fragment and the whole egg.

Application of a new PCR primer for terminal restriction fragment length polymorphism analysis of the bacterial communities in plant roots.

Contamination with plastid small subunit (SSU) rDNA is a major drawback when analyzing the bacterial communities of plant roots using culture-independent methods. In this study, a polymerase chain reaction (PCR) primer, 783r, was designed and tested to specifically amplify the SSU rDNA of various bacterial species without amplifying the SSU rDNA of plant plastids. To confirm how useful the community analysis of rhizobacteria is using 783r, the terminal restriction fragment length polymorphism (T-RFLP) method was performed with wheat (Triticum aestivum) and spinach (Spinacea oleracea) root samples. Using the standard T-RFLP method, a large T-RF peak of plant plastid SSU rDNA interfered with the bacterial community analysis. In contrast, the T-RFLP method using the 783r primer was able to detect the bacterial DNA while directly eliminating the influence of the plant-derived DNA extracted from the plant roots. Primer 783r might, therefore, be a useful PCR primer for the culture-independent analysis of bacterial communities in plant roots using SSU rDNA.

Application of Locked Nucleic Acid (LNA) oligonucleotide-PCR clamping technique to selectively PCR amplify the SSU rRNA genes of bacteria in investigating the plant-associated community structures.

The simultaneous extraction of plant organelle (mitochondria and plastid) genes during the DNA extraction step is a major limitation in investigating the community structures of bacteria associated with plants because organelle SSU rRNA genes are easily amplified by PCR using primer sets that are specific to bacteria. To inhibit the amplification of organelle genes, the locked nucleic acid (LNA) oligonucleotide-PCR clamping technique was applied to selectively amplify bacterial SSU rRNA genes by PCR. LNA oligonucleotides, the sequences of which were complementary to mitochondria and plastid genes, were designed by overlapping a few bases with the annealing position of the bacterial primer and converting DNA bases into LNA bases specific to mitochondria and plastids at the shifted region from the 3' end of the primer-binding position. PCR with LNA oligonucleotides selectively amplified the bacterial genes while inhibited that of organelle genes. Denaturing gradient gel electrophoresis (DGGE) analysis revealed that conventional amplification without LNA oligonucleotides predominantly generated DGGE bands from mitochondria and plastid genes with few bacterial bands. In contrast, additional bacterial bands were detected in DGGE patterns, the amplicons of which were prepared using LNA oligonucleotides. These results indicated that the detection of bacterial genes had been screened by the excessive amplification of the organelle genes. Sequencing of the bands newly detected by using LNA oligonucleotides revealed that their similarity to the known isolated bacteria was low, suggesting the potential to detect novel bacteria. Thus, application of the LNA oligonucleotide-PCR clamping technique was considered effective for the selective amplification of bacterial genes from extracted DNA containing plant organelle genes.