Assembly properties of bacterial actin MreB involved in Spiroplasma swimming motility.

Bacterial actin MreB forms filaments composed of antiparallel double-stranded units. The wall-less helical bacterium Spiroplasma has five MreB homologs (MreB1-5), some of which are involved in an intracellular ribbon for driving the bacterium's swimming motility. Although the interaction between MreB units is important for understanding Spiroplasma swimming, the interaction modes of each ribbon component are unclear. Here, we examined the assembly properties of Spiroplasma eriocheiris MreB5 (SpeMreB5), one of the ribbon component proteins that forms sheets. Electron microscopy revealed that sheet formation was inhibited under acidic conditions and bundle structures were formed under acidic and neutral conditions with low ionic strength. We also used solution assays and identified four properties of SpeMreB5 bundles as follows: (I) bundle formation followed sheet formation; (II) electrostatic interactions were required for bundle formation; (III) the positively charged and unstructured C-terminal region contributed to promoting lateral interactions for bundle formation; and (IV) bundle formation required Mg2+ at neutral pH but was inhibited by divalent cations under acidic pH conditions. During these studies, we also characterized two aggregation modes of SpeMreB5 with distinct responses to ATP. These properties will shed light on SpeMreB5 assembly dynamics at the molecular level.

Sequence analyses of a lipoprotein conserved with bacterial actins responsible for swimming motility of wall-less helical Spiroplasma.

Spiroplasma is a genus of pathogenic or commensal cell-wall-deficient helical bacterium. Spiroplasma -specific protein fibril and five classes of bacterial actins, MreB1-5, are involved in a helical ribbon structure responsible for helical-cell morphology and swimming motility. A gene for a hypothetical protein-SPE_1229, 7th protein-has been found in the locus coding mreB s. In this study, we characterized the 7th protein using in silico methods and found that it could be a lipoprotein whose gene is encoded downstream of mreB3 and conserved in a clade of Spiroplasma .

Wheat Ym2 originated from Aegilops sharonensis and confers resistance to soil-borne Wheat yellow mosaic virus infection to the roots.

Wheat yellow mosaic virus (WYMV) is a pathogen transmitted into its host's roots by the soil-borne vector Polymyxa graminis. Ym1 and Ym2 genes protect the host from the significant yield losses caused by the virus, but the mechanistic basis of these resistance genes remains poorly understood. Here, it has been shown that Ym1 and Ym2 act within the root either by hindering the initial movement of WYMV from the vector into the root and/or by suppressing viral multiplication. A mechanical inoculation experiment on the leaf revealed that the presence of Ym1 reduced viral infection incidence, rather than viral titer, while that of Ym2 was ineffective in the leaf. To understand the basis of the root specificity of the Ym2 product, the gene was isolated from bread wheat using a positional cloning approach. The candidate gene encodes a CC-NBS-LRR protein and it correlated allelic variation with respect to its sequence with the host's disease response. Ym2 (B37500) and its paralog (B35800) are found in the near-relatives, respectively, Aegilops sharonensis and Aegilops speltoides (a close relative of the donor of bread wheat's B genome), while both sequences, in a concatenated state, are present in several accessions of the latter species. Structural diversity in Ym2 has been generated via translocation and recombination between the two genes and enhanced by the formation of a chimeric gene resulting from an intralocus recombination event. The analysis has revealed how the Ym2 region has evolved during the polyploidization events leading to the creation of cultivated wheat.

Purification and ATPase Activity Measurement of Spiroplasma MreB.

Spiroplasma is a genus of wall-less helical bacteria with swimming motility unrelated to conventional types of bacterial motility machinery, such as flagella and pili. The swimming of Spiroplasma is suggested to be driven by five classes of MreB (MreB1-MreB5), which are members of the actin superfamily. In vitro studies of Spiroplasma MreBs have recently been conducted to evaluate their activities, such as ATPase, which is essential for the polymerization dynamics among classic actin superfamily proteins. In this chapter, we describe methods of purification and Pi release measurement of Spiroplasma MreBs using column chromatography and absorption spectroscopy with the molecular probe, 2-amino-6-mercapto-7-methylpurine riboside (MESG). Of note, the methods described here are applicable to other proteins that possess NTPase activity.

ATP-dependent polymerization dynamics of bacterial actin proteins involved in Spiroplasma swimming.

MreB is a bacterial protein belonging to the actin superfamily. This protein polymerizes into an antiparallel double-stranded filament that determines cell shape by maintaining cell wall synthesis. Spiroplasma eriocheiris, a helical wall-less bacterium, has five MreB homologous (SpeMreB1-5) that probably contribute to swimming motility. Here, we investigated the structure, ATPase activity and polymerization dynamics of SpeMreB3 and SpeMreB5. SpeMreB3 polymerized into a double-stranded filament with possible antiparallel polarity, while SpeMreB5 formed sheets which contained the antiparallel filament, upon nucleotide binding. SpeMreB3 showed slow Pi release owing to the lack of an amino acid motif conserved in the catalytic centre of MreB family proteins. Our SpeMreB3 crystal structures and analyses of SpeMreB3 and SpeMreB5 variants showed that the amino acid motif probably plays a role in eliminating a nucleophilic water proton during ATP hydrolysis. Sedimentation assays suggest that SpeMreB3 has a lower polymerization activity than SpeMreB5, though their polymerization dynamics are qualitatively similar to those of other actin superfamily proteins, in which pre-ATP hydrolysis and post-Pi release states are unfavourable for them to remain as filaments.

One-Pot γ-Lactonization of Homopropargyl Alcohols via Intramolecular Ketene Trapping.

A one-pot γ-lactonization of homopropargyl alcohols via an alkyne deprotonation/boronation/oxidation sequence has been developed. Oxidation of the generated alkynyl boronate affords the corresponding ketene intermediate, which is trapped by the adjacent hydroxy group to furnish the γ-lactone. We have optimized the conditions as well as examined the substrate scope and synthetic applications of this efficient one-pot lactonization.

Magnetorheological Response for Magnetic Elastomers Containing Carbonyl Iron Particles Coated with Poly(methyl methacrylate).

The magnetorheological response for magnetic elastomers containing carbonyl iron (CI) particles with a diameter of 6.7 µm coated with poly(methyl methacrylate) (PMMA) was investigated to estimate the diameter of secondary particles from the amplitude of magnetorheological response. Fourier-transformed infrared spectroscopy revealed that the CI particles were coated with PMMA, and the thickness of the PMMA layer was determined to be 71 nm by density measurement. The change in the storage modulus for magnetic elastomers decreased by coating and it was scaled by the number density of CI particles as ΔG~N2.8. The diameter of secondary particle of CI particles coated with PMMA was calculated to be 8.4 µm. SEM images revealed that the CI particles coated with PMMA aggregated in the polyurethane matrix.

Phylogenetic origin and sequence features of MreB from the wall-less swimming bacteria Spiroplasma.

Spiroplasma are wall-less bacteria which belong to the phylum Tenericutes that evolved from Firmicutes including Bacillus subtilis. Spiroplasma swim by a mechanism unrelated to widespread bacterial motilities, such as flagellar motility, and caused by helicity switching with kinks traveling along the helical cell body. The swimming force is likely generated by five classes of bacterial actin homolog MreBs (SMreBs 1-5) involved in the helical bone structure. We analyzed sequences of SMreBs to clarify their phylogeny and sequence features. The maximum likelihood method based on around 5000 MreB sequences showed that the phylogenetic tree was divided into several radiations. SMreBs formed a clade adjacent to the radiation of MreBH, an MreB isoform of Firmicutes. Sequence comparisons of SMreBs and Bacillus MreBs were also performed to clarify the features of SMreB. Catalytic glutamic acid and threonine were substituted to aspartic acid and lysine, respectively, in SMreB3. In SMreBs 2 and 4, amino acids involved in inter- and intra-protofilament interactions were significantly different from those in Bacillus MreBs. A membrane-binding region was not identified in most SMreBs 1 and 4 unlike many walled-bacterial MreBs. SMreB5 had a significantly longer C-terminal region than the other MreBs, which possibly forms protein-protein interactions. These features may support the functions responsible for the unique mechanism of Spiroplasma swimming.

Particle Size in Secondary Particle and Magnetic Response for Carrageenan Magnetic Hydrogels.

The relation between the number of magnetic particles and the change in storage modulus induced by a magnetic field was investigated for weak hydrogels containing carbonyl iron, iron oxide, and barium ferrite particles with different diameters in primary particles while maintaining the magnetization of magnetic particles. The change in storage modulus exhibited a power dependency against the number of magnetic particles, which was nearly independent of the magnetic particles. The change in storage modulus was successfully scaled by the reduced number of magnetic particles using the diameter of secondary particles. Microphotographs revealed that iron oxide and barium ferrite particles form aggregations while carbonyl iron particles are well dispersed in carrageenan gels. The diameter of secondary particles determined by a relation between the change in storage modulus and the reduced number of magnetic particles showed similar values with those observed in microphotographs.

Risk Factor for Diabetes Mellitus and High Blood Glucose With HMG-CoA Reductase Inhibitors Using a Postmarketing Surveillance Database in Japan.

To investigate whether 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor (statin) use is associated with an increased risk of diabetes mellitus and hyperglycemia, we performed a nested case-control study using a postmarketing surveillance database in Japan. The database cohort included 26,849 cases of statin use and 5308 cases of other lipid-lowering drug use in patients with hyperlipidemia. Participants received at least 1 type of statin, had a clear medication history of statin use, and had no complications of diabetes mellitus. Cases were defined as onset of diabetes mellitus or hyperglycemia during statin intake. For each case, 20 controls were randomly selected and matched by time point. The factors associated with an increased risk of diabetes mellitus and hyperglycemia during statin intake examined included sex, age, body mass index, statin use duration, complications, concomitant medication, and clinical laboratory tests. Statin-associated diabetes mellitus or hyperglycemia was identified based on abnormal elevation of blood glucose concentrations beyond the reference range. A total of 19,868 patients met the inclusion criteria, of whom 24 were patients in the case group. Two complicating factors, fatty liver (adjusted odds ratio 16.10) and hyperuricemia (adjusted odds ratio 28.96), were extracted for onset of diabetes mellitus or hyperglycemia. Nonalcoholic fatty liver was associated with diabetes mellitus, obesity, and insulin resistance, and hyperuricemia was associated with lifestyle. This study suggested that the onset of diabetes mellitus or hyperglycemia might be increased with statin use in patients with complications of fatty liver and hyperuricemia.

Village health volunteers' individual social capital and caretakers' health service utilization for febrile children in Malaria-endemic villages in Papua New Guinea.

Background: Little is known about the association between the social capital of village health volunteers (VHVs) and their performance in relation to malarial care. Methods: Data came from 337 children and 13 VHVs working in Dagua, Papua New Guinea. The outcome variable was whether caretakers brought their children to health care services on the incidence of a febrile episode. The social capital of VHVs was assessed by inquiring about relationships with people in 25 social positions/roles. Results: Caretakers were more likely to bring their febrile children to health care services when they lived in a village whose VHVs frequently discussed their activities with people in positions/roles outside their village (prevalence ratio [PR]=1.47 [95% confidence interval {CI} 1.22 to 1.78]). On the other hand, caretakers were less likely to do so when their VHVs had known people in informal positions/roles inside their village (PR=0.85 [95% CI 0.77 to 0.93]) and when they discussed their activities with people in formal positions/roles inside their village (PR=0.76 [95% CI 0.61 to 0.95]). Conclusions: Our results suggest that the social interactions of VHVs with people in positions/roles outside the village may benefit residents while those with people in positions/roles inside the village might not necessarily benefit them.

A New Contrast Enhancement Protocol for Subtraction Coronary Computed Tomography Requiring a Short Breath-Holding Time.

RATIONALE AND OBJECTIVES: We have developed a new contrast enhancement protocol for subtraction coronary computed tomography (SCCTA) requiring a short breath-holding time. In the protocol, test and main boluses were sequentially and automatically injected, and correct timings for pre-contrast and contrast-enhanced scans for main bolus were automatically determined only by the test bolus tracking. Combined with a fixed short main bolus injection for 7 seconds, the breath-holding time was shortened as possible. The purpose of this study was to evaluate whether use of this new protocol produced adequate quality images, taking into account calcified lesions and in-stent lumens. MATERIALS AND METHODS: Patients (n = 127) with calcium scores of >400 Agatston units or a history of stent placement were enrolled. Breath-holding times were recorded, and image quality was visually evaluated by two observers. RESULTS: The mean ± standard deviation breath-holding time was 13.2 ± 0.6 seconds. The mean ± SD computed tomography (CT) number of coronary arteries for the pre-contrast scan was sufficiently low [99.2 ± 32.2 Hounsfield units (HU)] and, simultaneously, that for SCCTA was 367.0 ± 77.2 HU. The rate of segments evaluated as unreadable was sufficiently low (3.8%). CONCLUSIONS: Use of the SCCTA protocol was efficient and allowed for a shorter breath-holding time and adequate diagnostic accuracy of SCCTA images, including images of calcified and stent implantation segments.

Sensitive and selective detection of prostate-specific antigen using a photonic crystal nanolaser.

The detection of low-concentration biomarkers is expected to facilitate the early diagnosis of severe diseases, including malignant tumors. Using photonic crystal nanolaser sensors, we detected prostate-specific antigen (PSA) from a concentration of 1 fM, which is difficult to detect by conventional enzyme-linked immunosorbent assay. The signal intensity and stability were improved by using a surfactant (i.e., ethanolamine). Even when a contaminant such as bovine serum albumin was mixed into the PSA sample, thereby increasing the concentration of the contaminant ten billion times, it was still possible to maintain a high level of detection.

The right central amygdala shows greater activation in response to an auditory conditioned stimulus in male rats.

Pavlovian fear conditioning is an experimental procedure in which a conditioned stimulus (CS) acquires an ability to elicit fear responses. This type of conditioning depends on the basolateral complex of the amygdala (BLA) and/or central amygdala (CeA). We previously found that rats showed reduced fear responses to an auditory CS when they were subjected to a pre-training chemical lesion of the entire right amygdala as compared with the left amygdala. Based on this finding, we hypothesize that the BLA and/or CeA in the right hemisphere will be more strongly activated by an auditory CS than those in the left hemisphere. To test this hypothesis, we re-exposed fear-conditioned and non-conditioned rats to an auditory CS 1 day after fear conditioning. We assessed Fos expression in the BLA and CeA in each hemisphere. We found that fear-conditioned subjects showed fear responses, such as increased freezing and decreased walking, as well as increased Fos expression in the BLA and CeA. When we compared Fos expression between hemispheres, Fos expression in the CeA, but not the BLA, was greater in the right hemisphere compared with the left hemisphere. These results suggest that the right CeA is more strongly activated by the auditory CS.

Experimental and Numerical Investigations of Effects of Flow Control Devices Upon Flat-Plate Film Cooling Performance.

This study deals with the experimental and numerical studies of the effect of flow control devices (FCDs) on the film cooling performance of a circular cooling hole on a flat plate. Two types of FCDs with different heights are examined in this study, where each of them is mounted to the flat plate upstream of the cooling hole by changing its lateral position with respect to the hole centerline. In order to measure the film effectiveness as well as heat transfer downstream of the cooling hole with upstream FCD, a transient method using a high-resolution infrared camera is adopted. The velocity field downstream of the cooling hole is captured by 3D laser Doppler velocimeter (LDV). Furthermore, the aerodynamic loss associated with the cooling hole with/without FCD is measured by a total pressure probe rake. The experiments are carried out at blowing ratios ranging from 0.5 to 1.0. In addition, numerical simulations are also made to have a better understanding of the flow field. LES approach is employed to solve the flow field and visualize the vortex structure around the cooling hole with FCD. When a taller FCD is mounted to the plate, the film effectiveness tends to increase due to the vortex structure generated by the FCD. As FCD is laterally shifted from the centerline, the film effectiveness increases, while the lift-off of cooling air is also promoted when FCD is put on the center line.

[Examination of the effectiveness of heart rate control using intravenous ß-blocker in 64-slice coronary computed tomography angiography].

The purpose of this study is to clarify the effectiveness of the use of ß-blocker in coronary computed tomography angiography (CCTA). In 1783 patients, heart rate was controlled by propranolol injection to patients with heart rates of 61 bpm or more. As a result, the scan heart rate (58.8±6.5 bpm) decreased significantly compared with the initial heart rate (72.7±9.4 bpm). Prospective gating method was used by 61.9% including 64.3% of the intravenous ß-blocker injection group. Moreover, daily use of oral ß-blocker had influence on reduction of the scan heart rate (daily use group: 60.1±6.5 bpm vs. unuse group: 58.5±6.3 bpm p<0.01). When we evaluated the image quality of CCTA by the score, the improvement of the score was obviously admitted by 65 bpm or less of the scan heart rate. The ratio of scan heart rate that was controlled by 65 bpm or less was decreased in the initial heart rate groups that were 81 bpm or more. The incidence of adverse reactions by the propranolol injection was few, and these instances only involved slight symptoms. Therefore, heart rate control with the use of ß-blocker is useful for the image quality improvement of CCTA. This form of treatment can be safely enforced.

[Development of test bolus tracking method and usefulness in coronary CT angiography].

The test bolus tracking (TBT) method is a new injection method of contrast medium that we developed. The TBT method is an injection technique that continuously performs the test bolus injection and the main bolus injection, such that the best acquisition of scan timing and the improvement of examination efficiency can be expected. We compared the TBT method and the test injection method by coronary CT angiography. The results demonstrated that the contrast enhancement of the coronary arteries was high and the variation of the CT value was also small in the TBT method. When the scan timing expected by the TI method and the TBT method were compared, it was different of two seconds or more by the case with 43%. However, the variation of CT value was small for the TBT method in these cases. Therefore, the TBT method is a very useful method for CCTA.