A Single Shot of Vesicles.

Bacteria communicate through signaling molecules that coordinate group behavior. Hydrophobic signals that do not diffuse in aqueous environments are used as signaling molecules by several bacteria. However, limited information is currently available on the mechanisms by which these molecules are transported between cells. Membrane vesicles (MVs) with diverse functions play important roles in the release and delivery of hydrophobic signaling molecules, leading to differences in the dynamics of signal transportation from those of free diffusion. Studies on Paracoccus denitrificans, which produces a hydrophobic long-chain N-acyl homoserine lactone (AHL), showed that signals were loaded into MVs at a concentration with the potential to trigger the quorum sensing (QS) response with a "single shot" to the cell. Furthermore, stimulating the formation of MVs increased the release of signals from the cell; therefore, a basic understanding of MV formation is important. Novel findings revealed the formation of MVs through different routes, resulting in the production of different types of MVs. Methods such as high-speed atomic force microscopy (AFM) phase imaging allow the physical properties of MVs to be analyzed at a nanometer resolution, revealing their heterogeneity. In this special minireview, we introduce the role of MVs in bacterial communication and highlight recent findings on MV formation and their physical heterogeneity by referring to our research. We hope that this minireview will provide basic information for understanding the functionality of MVs in ecological systems.

Physical Properties and Shifting of the Extracellular Membrane Vesicles Attached to Living Bacterial Cell Surfaces.

Bacterial cells release nanometer-sized extracellular membrane vesicles (MVs) to deliver cargo molecules for use in mediating various biological processes. However, the detailed processes of transporting these cargos from MVs to recipient cells remain unclear because of the lack of imaging techniques to image nanometer-sized fragile vesicles in a living bacterial cell surface. Herein, we quantitatively demonstrated that the direct binding of MV to the cell surface significantly promotes hydrophobic quorum-sensing signal (C16-HSL) transportation to the recipient cells. Moreover, we analyzed the MV-binding process in the Paracoccus denitrificans cell surface using high-speed atomic force microscopy phase imaging. Although MV shapes were unaltered after binding to the cell surface, the physical properties of a group of single MV particles were shifted. Additionally, the phase shift values of MVs were higher than that of the cell's surfaces upon binding, whereas the phase shift values of the group of MVs were decreased during observation. The shifting physical properties occurred irreversibly only once for each MV during the observations. The decreasing phase shift values indicated alterations of chemical components in the MVs as well, thereby suggesting the dynamic process in which single MV particles deliver their hydrophobic cargo into the recipient cell. IMPORTANCE Compared to the increasing knowledge about MV release mechanisms from donor cells, the mechanism by which recipient cells receive cargo from MVs remains unknown. Herein, we have successfully imaged single MV-binding processes in living bacterial cell surfaces. Accordingly, we confirmed the shift in the MV hydrophobic properties after landing on the cell surface. Our results showed the detailed states and the attaching process of a single MV into the cell surface and can aid the development of a new model for MV reception into Gram-negative bacterial cell surfaces. The insight provided by this study is significant for understanding MV-mediated cell-cell communication mechanisms. Moreover, the AFM technique presented for nanometer-scaled mapping of dynamic physical properties alteration on a living cell could be applied for the analyses of various biological phenomena occurring on the cell surface, and it gives us a new view into the understanding of the phenotypes of the bacterial cell surface.

Geometrical Characterization of Glass Nanopipettes with Sub-10 nm Pore Diameter by Transmission Electron Microscopy.

Glass nanopipettes are widely used for various applications in nanosciences. In most of the applications, it is important to characterize their geometrical parameters, such as the aperture size and the inner cone angle at the tip region. For nanopipettes with sub-10 nm aperture and thin wall thickness, transmission electron microscopy (TEM) must be most instrumental in their precise geometrical measurement. However, this measurement has remained a challenge because heat generated by electron beam irradiation would largely deform sub-10 nm nanopipettes. Here, we provide methods for preparing TEM specimens that do not cause deformation of such tiny nanopipettes.

Diversity of physical properties of bacterial extracellular membrane vesicles revealed through atomic force microscopy phase imaging.

Bacteria release nanometer-scale extracellular membrane vesicles (MVs) to mediate a variety of biological processes. We analyzed individual MVs under physiological conditions by phase imaging of high-speed atomic force microscopy to assess the physiological heterogeneity of MVs isolated from bacterial cultures. Phase imaging makes it possible to map the physical properties of an individual, fragile MV in an isolated MV population containing a broad variety of vesicle diameters, from 20 to 150 nm. We also developed a method for quantitatively comparing the physical properties of MVs among samples. This allowed for the comparison of the physical properties of MVs isolated from different bacterial species. We compared bacterial MVs isolated from four bacterial species and artificially synthesized liposomes. We demonstrate that each bacterial species generates physically heterogeneous types of MVs, unlike the physical homogeneity displayed by liposomes. These results indicate that the physical heterogeneity of bacterial MVs is mainly caused by compositional differences mediated through biological phenomena and could be unique to each species. We provide a new methodology using phase imaging that would pave the way for single-vesicle analysis of extracellular vesicles of a broad size range.

Dynamics of oligomer and amyloid fibril formation by yeast prion Sup35 observed by high-speed atomic force microscopy.

The yeast prion protein Sup35, which contains intrinsically disordered regions, forms amyloid fibrils responsible for a prion phenotype [PSI+]. Using high-speed atomic force microscopy (HS-AFM), we directly visualized the prion determinant domain (Sup35NM) and the formation of its oligomers and fibrils at subsecond and submolecular resolutions. Monomers with freely moving tail-like regions initially appeared in the images, and subsequently oligomers with distinct sizes of ∼1.7 and 3 to 4 nm progressively accumulated. Nevertheless, these oligomers did not form fibrils, even after an incubation for 2 h in the presence of monomers. Fibrils appeared after much longer monomer incubation. The fibril elongation occurred smoothly without discrete steps, suggesting gradual conversions of the incorporated monomers into cross-ß structures. The individual oligomers were separated from each other and also from the fibrils by respective, identical lengths on the mica surface, probably due to repulsion caused by the freely moving disordered regions. Based on these HS-AFM observations, we propose that the freely moving tails of the monomers are incorporated into the fibril ends, and then the structural conversions to cross-ß structures gradually occur.

Auditory gap detection: psychometric functions and insights into the underlying neural activity.

The detection of a silent interval or gap provides important insight into temporal processing by the auditory system. Previous research has uncovered a multitude of empirical findings leaving the mechanism of gap detection poorly understood and key issues unresolved. Here, we expand the findings by measuring psychometric functions for a number of conditions including both across-frequency and across-intensity gap detection as a first study of its kind. A model is presented which not only accounts for our findings in a quantitative manner, but also helps frame the body of work on auditory gap research. The model is based on the peripheral response and postulates that the identification of gap requires the detection of activity associated with silence.

Tethered Magnets Are the Key to Magnetotaxis: Direct Observations of Magnetospirillum magneticum AMB-1 Show that MamK Distributes Magnetosome Organelles Equally to Daughter Cells.

Magnetotactic bacteria are a unique group of bacteria that synthesize a magnetic organelle termed the magnetosome, which they use to assist with their magnetic navigation in a specific type of bacterial motility called magneto-aerotaxis. Cytoskeletal filaments consisting of the actin-like protein MamK are associated with the magnetosome chain. Previously, the function of MamK was thought to be in positioning magnetosome organelles; this was proposed based on observations via electron microscopy still images. Here, we conducted live-cell time-lapse fluorescence imaging analyses employing highly inclined and laminated optical sheet microscopy, and these methods enabled us to visualize detailed dynamic movement of magnetosomes in growing cells during the entire cell cycle with high-temporal resolution and a high signal/noise ratio. We found that the MamK cytoskeleton anchors magnetosomes through a mechanism that requires MamK-ATPase activity throughout the cell cycle to prevent simple diffusion of magnetosomes within the cell. We concluded that the static chain-like arrangement of the magnetosomes is required to precisely and consistently segregate the magnetosomes to daughter cells. Thus, the daughter cells inherit a functional magnetic sensor that mediates magneto-reception.IMPORTANCE Half a century ago, bacterial cells were considered a simple "bag of enzymes"; only recently have they been shown to comprise ordered complexes of macromolecular structures, such as bacterial organelles and cytoskeletons, similar to their eukaryotic counterparts. In eukaryotic cells, the positioning of organelles is regulated by cytoskeletal elements. However, the role of cytoskeletal elements in the positioning of bacterial organelles, such as magnetosomes, remains unclear. Magnetosomes are associated with cytoskeletal filaments that consist of the actin-like protein MamK. In this study, we focused on how the MamK cytoskeleton regulates the dynamic movement of magnetosome organelles in living magnetotactic bacterial cells. Here, we used fluorescence imaging to visualize the dynamics of magnetosomes throughout the cell cycle in living magnetotactic bacterial cells to understand how they use the actin-like cytoskeleton to maintain and to make functional their nano-sized magnetic organelles.

Between-Frequency and Between-Ear Gap Detections and Their Relation to Perception of Stop Consonants.

OBJECTIVES: The objective of this study was to examine the hypothesis that between-channel gap detection, which includes between-frequency and between-ear gap detection, and perception of stop consonants, which is mediated by the length of voice-onset time (VOT), share common mechanisms, namely relative-timing operation in monitoring separate perceptual channels. DESIGN: The authors measured gap detection thresholds and identification functions of /ba/ and /pa/ along VOT in 49 native young adult Japanese listeners. There were three gap detection tasks. In the between-frequency task, the leading and trailing markers differed in terms of center frequency (Fc). The leading marker was a broadband noise of 10 to 20,000 Hz. The trailing marker was a 0.5-octave band-passed noise of 1000-, 2000-, 4000-, or 8000-Hz Fc. In the between-ear task, the two markers were spectrally identical but presented to separate ears. In the within-frequency task, the two spectrally identical markers were presented to the same ear. The /ba/-/pa/ identification functions were obtained in a task in which the listeners were presented synthesized speech stimuli of varying VOTs from 10 to 46 msec and asked to identify them as /ba/ or /pa/. RESULTS: The between-ear gap thresholds were significantly positively correlated with the between-frequency gap thresholds (except those obtained with the trailing marker of 4000-Hz Fc). The between-ear gap thresholds were not significantly correlated with the within-frequency gap thresholds, which were significantly correlated with all the between-frequency gap thresholds. The VOT boundaries and slopes of /ba/-/pa/ identification functions were not significantly correlated with any of these gap thresholds. CONCLUSIONS: There was a close relation between the between-ear and between-frequency gap detection, supporting the view that these two types of gap detection share common mechanisms of between-channel gap detection. However, there was no evidence for a relation between the perception of stop consonants and the between-frequency/ear gap detection in native Japanese speakers.

Biochemical blood markers and sampling sites in forensic autopsy.

Forensic pathologists often hesitate to use biochemical blood markers due to the risk of large postmortem changes and deviations from healthy subjects. Biochemical analyses of postmortem blood, if possible, may help to evaluate pathological status and determining the cause of death in forensic diagnosis, for example, in sudden unexpected death without obvious cause, or young adults with no apparent cause of death or antemortem information. Even commercially available biochemical markers were re-evaluated in the blood samples of 164 forensic autopsy cases. Biochemical markers examined were HbA1c, fructosamine, blood nitrogen urea (BUN), creatinine, total protein, total bilirubin, gamma-glutamyl transpeptidase (gamma-GTP), triglyceride, total cholesterol, C-reactive protein (CRP) and pseudocholine esterase (pChE). We collected cardiac blood (left cardiac blood and right cardiac blood) and peripheral blood (femoral vein blood) to clarify the differences in measured values by sampling site. The measured values were analyzed in relation to postmortem interval, etiology of death and sampling sites. Of all eleven markers, HbA1c is the most useful and reliable because of its negligible postmortem changes and small deviation from healthy subjects. Total bilirubin, BUN, CRP and total cholesterol can be useful if we set appropriate limit ranges and pay attention to the interpretation. For the evaluation of changes due to postmortem intervals, none of the markers except for triglyceride showed significant changes up to three days postmortem. As for sampling sites, femoral vein blood is generally recommended considering postmortem changes, but left cardiac blood was suitable for creatinine, pChE, and total cholesterol. For clinical forensic diagnosis of biochemical blood markers, we must determine the "forensic abnormal value" after collecting more cases by known causes with more information about the population.

A fatal case of post-operative pulmonary thromboembolism with cosmetic liposuction.

Pulmonary thromboembolism (PTE) has been regarded as rare in Japan. However, PTE has been increasingly recognised because either of increased incidence, diagnostic progress, or social recognition. Recently, 10 Japanese Medical Associations have submitted preventive guidelines for PTE in post-operative patients and the government decided to fund this, as results of the increase in cases and concern regarding medical negligence. A fatal case of PTE after liposuction is reported. A female patient was in the home toilet after two days of immobilization following day-surgery liposuction. Clinicians must be aware of appropriate methods for the prevention of post operative PTE with cosmetic surgery.

Endothelial NO Synthase (eNOS) phosphorylation regulates coronary diameter during ischemia-reperfusion in association with oxidative stress.

The link between endothelial nitric oxide synthase (eNOS) activation and vascular diameter during ischemia-reperfusion was investigated in the rat heart. After short (<30 min) and long (>45 min) time of ischemia conferred by coronary artery occlusion of the rats, reperfusion caused dilatation and constriction of arterioles, respectively. Partial oxygen pressure (pO2) measurement of the heart by the electrode confirmed the hyper-perfusion and no-reflow phenomena during reperfusion, as well as myocardial ischemia. The vascular diameter was correlated with phosphorylation of Akt and serine 1177 residue of eNOS, and formation of NO-bound guanylate cyclase (GC) by immuoflorescence study. Western blotting confirmed the phosphorylation of eNOS-Ser1177 depending on ischemia time. The constriction during reperfusion after 45 min of ischemia is supposedly caused by the inhibition of Akt-mediated eNOS-Ser1177 phosphorylation, which was suppressed by a PKC inhibitor chelerythrine, or ROS scavengers N-2-mercaptopropionyl glycine (MPG) and 4,5-Dihydroxy-1, 3-benzenedisulfonic acid disodium salt (Tiron). However, an endothelin receptor antagonist BQ123 alleviated the vasoconstriction by increasing NO availability but not eNOS-Ser1177 phosphorylation. Thus, vascular patency is correlated with eNOS-Ser1177 phosphorylation in association with ROS, and PKC during reperfusion. Endothelin inhibits vasodilatation by reducing NO availability during reperfusion.