Ibaraki virus enters host cells by macropinocytosis.

Ibaraki virus (IBAV) is the pathogen associated with Ibaraki disease. In a previous study, we suggested that IBAV enters hamster lung (HmLu-1) cells via endocytosis and subsequently escapes into the cytoplasm upon endosomal acidification. However, it is unclear which of the endocytic pathways IBAV utilizes. In this study, we aimed to further elucidate the pathway of IBAV entry into host cells. We found that IBAV replication was not suppressed by inhibitors of clathrin-mediated or caveolin-mediated endocytosis but was markedly suppressed by 5-(N-ethyl-N-isopropyl) amiloride (EIPA) and cytochalasin D, both of which inhibit macropinocytosis. Monensin, which inhibits endosomal acidification, also suppressed IBAV replication. To assess the inhibitory effects of these reagents on endocytosis, dextran and transferrin were used as indicators of macropinocytosis and clathrin-mediated endocytic activity, respectively. Our data confirmed that EIPA and monensin inhibited dextran uptake, and cytochalasin D inhibited the uptake of both. Additionally, we confirmed that endosomal/lysosomal acidification was inhibited by monensin. These results suggest that the macropinocytosis pathway is the major route of IBAV entry and confirm that IBAV infection of HmLu-1 cells is dependent on endosomal acidification.

Amino acid starvation accelerates replication of Ibaraki virus.

Ibaraki virus (IBAV) is a strain of epizootic hemorrhagic disease virus 2 that belongs to the genus Orbivirus of the family Reoviridae. IBAV replication is suppressed by the inhibition of autophagy, and since mechanistic target of rapamycin complex 1 (mTORC1) is a key regulator of autophagy, we examined if mTORC1 inhibition by amino acid starvation or mTOR inhibitors (Torin 1 and rapamycin) affects IBAV replication. We found that IBAV replication is significantly enhanced after amino acid starvation of host cells, but not after treatment with mTOR inhibitors, during early stages of viral infection (0-1 hpi). Notably, inhibition of mTORC1 by amino acid starvation was reversible and thus restricted to 0-1 hpi, whereas mTOR inhibitors sustainably suppressed mTORC1 even after the 1-h treatment, suggesting that mTORC1 suppression itself does not affect IBAV replication. To investigate the mechanism of enhanced IBAV replication by amino acid starvation, we examined the endocytic pathway, since IBAV utilizes acidification of endosomes as a trigger for viral replication. Accordingly, we found that amino acid starvation, but not mTOR inhibitors, strongly induced acidification of endosomes/lysosomes and that inhibition of endosomal acidification by bafilomycin A1 effectively blocked enhancement of IBAV replication. Altogether, the inactivation of mTORC1 by amino acid starvation during early stages of infection enhances acidification of endosomes, which in turn enhances IBAV replication.

[Medroxyprogesterone Acetate as Part of Palliative Care for Terminal-Stage Breast Cancer Patients--A Report of Two Cases].

Various effective strategies have recently been described in the treatment of breast cancer, including endocrine therapy, chemotherapy, and molecular-targeted therapy, providing long-term survival benefits even after cancer recurrence. However, terminal-stage patients experience side effects and worse quality of life (QOL), in addition to deterioration of their general condition caused by the progression of the disease itself. When providing the best supportive care, use of anti-cancer drugs is not taboo and can represent a good option as long as physical, social, psychological, and spiritual supports are provided to both the patients and their families. Medroxyprogesterone acetate (MPA) is an endocrine therapeutic drug. In Japan, MPA is used only as a late-line endocrine therapy for breast cancer recurrence because many other endocrine therapy drugs are much more effective and MPA increases the risk of thrombosis and obesity. Here, we report 2 patients with breast cancer who reached terminal stage more than 10 years after the first diagnosis. MPA was administered as the final-line treatment. During that time, their appetite and QOL improved and the patients became more active than when they had been undergoing aggressive anticancer treatment. Both patients spent quality time with their families until their death. MPA may be a good option as part of palliative care of breast cancer patients in terminal stage.

High aspect ratio AFM Probe processing by helium-ion-beam induced deposition.

A Scanning Helium Ion Microscope (SHIM) is a high resolution surface observation instrument similar to a Scanning Electron Microscope (SEM) since both instruments employ finely focused particle beams of ions or electrons [1]. The apparent difference is that SHIMs can be used not only for a sub-nanometer scale resolution microscopic research, but also for the applications of very fine fabrication and direct lithography of surfaces at the nanoscale dimensions. On the other hand, atomic force microscope (AFM) is another type of high resolution microscopy which can measure a three-dimensional surface morphology by tracing a fine probe with a sharp tip apex on a specimen's surface.In order to measure highly uneven and concavo-convex surfaces by AFM, the probe of a high aspect ratio with a sharp tip is much more necessary than the probe of a general quadrangular pyramid shape. In this paper we report the manufacture of the probe tip of the high aspect ratio by ion-beam induced gas deposition using a nanoscale helium ion beam of SHIM.Gas of platinum organic compound was injected into the sample surface neighborhood in the vacuum chamber of SHIM. The decomposition of the gas and the precipitation of the involved metal brought up a platinum nano-object in a pillar shape on the normal commercial AFM probe tip. A SHIM system (Carl Zeiss, Orion Plus) equipped with the gas injection system (OmniProbe, OmniGIS) was used for the research. While the vacuum being kept to work, we injected platinum organic compound ((CH3)3(CH3C5H4)Pt) into the sample neighborhood and irradiated the helium ion beam with the shape of a point on the apex of the AFM probe tip. It is found that we can control the length of the Pt nano-pillar by irradiation time of the helium ion beam. The AFM probe which brought up a Pt nano-pillar is shown in Figure 1. It is revealed that a high-aspect-ratio Pt nano-pillar of ∼40nm diameter and up to ∼2000 nm length can be grown. In addition, for possible heating by the helium ion beam, it was observed that an original probe shape was transformed. AFM measurement of a reference sample (pitch 100-500 nm, depth 100 nm) of the lines and spaces was performed using the above probes. The conventional probes which did not bring up platinum was not able to get into the ditch enough. Therefore it was found that a salient was big and a reentrant was shallow. On the other hand, the probe which brought up platinum was able to enter enough to the depths of the ditch.jmicro;63/suppl_1/i30-a/DFU075F1F1DFU075F1Fig.1.SHIM image of the AFM probe with the Pt nano-pillar fabricated by ion-beam induced deposition.

Factors predicting adverse events associated with pregabalin administered for neuropathic pain relief.

BACKGROUND: Pregabalin administration is occasionally abandoned due to adverse events such as somnolence, dizziness, unsteadiness, weight gain and edema. However, the exact causes of these differences in adverse events associated with pregabalin have not been elucidated. OBJECTIVE: To identify factors predicting adverse events associated with pregabalin administered for neuropathic pain. METHODS: The present study was a retrospective analysis involving 208 patients with neuropathic pain who had been treated with pregabalin in the pain clinic at the authors' hospital between July 2010 and September 2011. Variables were extracted from the clinical records for regression analysis of factors related to the occurrence of adverse events associated with pregabalin administration. Multivariate logistic regression analysis was used to examine the relationship between various predictive factors and the adverse events. RESULTS: Predictive factors were: duration of therapy (OR 1.684 [95% CI 1.179 to 2.406]; P=0.0042) for somnolence; nonsteroidal anti-inflammatory drugs (OR 0.132 [95% CI 0.030 to 0.578]; P=0.0072), age (OR 3.137 [95% CI 1.220 to 8.066]; P=0.0177) and maintenance dose (OR 0.437 [95% CI 0.217 to 0.880]; P=0.0205) for unsteadiness; serum creatinine (OR 6.439 [95% CI 1.541 to 26.902]; P=0.0107) for body weight gain; and neurotropin (OR 8.538 [95% CI 1.159 to 62.901]; P=0.0353) and serum creatinine (OR 6.912 [95% CI 1.118 to 42.726]; P=0.0375) for edema. CONCLUSIONS: The results of the present study indicate that care is warranted regarding long durations of therapy for somnolence, advanced age rather than dose-dependent adverse events for unsteadiness, elevated serum creatinine level for weight gain, and elevated serum creatinine level and combination use of neurotropin for edema. The safety of the combined use of pregabalin and nonsteroidal anti-inflammatory drugs were also suggested.

The effect of magnesium ions on chromosome structure as observed by helium ion microscopy.

One of the few conclusions known about chromosome structure is that Mg2+ is required for the organization of chromosomes. Scanning electron microscopy is a powerful tool for studying chromosome morphology, but being nonconductive, chromosomes require metal/carbon coating that may conceal information about the detailed surface structure of the sample. Helium ion microscopy (HIM), which has recently been developed, does not require sample coating due to its charge compensation system. Here we investigated the structure of isolated human chromosomes under different Mg2+ concentrations by HIM. High-contrast and resolution images from uncoated samples obtained by HIM enabled investigation on the effects of Mg2+ on chromosome structure. Chromatin fiber information was obtained more clearly with uncoated than coated chromosomes. Our results suggest that both overall features and detailed structure of chromatin are significantly affected by different Mg2+ concentrations. Chromosomes were more condensed and a globular structure of chromatin with 30 nm diameter was visualized with 5 mM Mg2+ treatment, while 0 mM Mg2+ resulted in a less compact and more fibrous structure 11 nm in diameter. We conclude that HIM is a powerful tool for investigating chromosomes and other biological samples without requiring metal/carbon coating.

Predictive factors for postherpetic neuralgia using ordered logistic regression analysis.

OBJECTIVES: To identify predictive factors for the occurrence of postherpetic neuralgia (PHN). METHODS: The participants were 73 herpes zoster patients who had been treated at the pain clinic of our hospital between January 2008 and June 2010. Variables present at the initial visit were extracted from the clinical records for regression analysis of factors related to the occurrence of PHN. The following scores for response were used: 0=no PHN after 3 months; 1=PHN present after 3 months but absent after 6 months; and 2=PHN present after 6 months. Multivariate ordered logistic regression analysis was performed to identify the predictive factors for PHN. RESULTS: Advanced age [odds ratio (OR)=2.740, confidence interval (CI)=1.110-6.761; P=0.0288] and deep pain (OR=4.244, CI=1.114-16.163; P=0.0341) at the initial visit to our outpatient pain clinic were found to be significant predictive factors for the occurrence of PHN. Diabetes mellitus (OR=3.075) and pain reduced by bathing (OR=3.389) also had high OR, although they were not significant. DISCUSSION: Our study indicates that advanced age and deep pain at the initial visit are significant predictors for PHN. Our results are considered likely to contribute to the establishment of evidence-based medicine in the optimal treatment of PHN.

Novel tip shape reconstruction method for restoration of AFM topography images using nano-structures with given shapes.

The establishment of more accurate imaging of surface microstructures is needed. The most significant distortion in atomic force microscopy (AFM) imaging is induced by the probe tip shape, whenever the sample surface contains features whose dimensions are comparable to the probe tip size. The acquired AFM image is the dilation between the tip shape and the sample topography. To restore the original topographical profile, a numerical erosion procedure using a precise probe shape function is required. Here, a new technique for reconstruction of probe shape function using a well-defined nanostructure is proposed. First, AFM topography images of the given-shape nanostructure dispersed on flat substrates are taken. Then, a probe shape function is determined by a numerical calculation procedure. By using the experimentally determined probe shape function, the most probable surface morphologies from the observed AFM topography images of unknown samples can be extracted.

Unique synthesis of few-layer graphene films on carbon-doped Pt(83)Rh(17) surfaces.

We report a unique synthesis of single- and few-layer graphene films on carbon-doped Pt(83)Rh(17) surfaces by surface segregation and precipitation. The ultrathin graphene films were characterized by atomic force microscopy, Auger electron spectroscopy, and micro-Raman spectroscopy measurements, providing evidence of graphene film thickness and structural quality. The G and 2D band intensity images from micro-Raman spectroscopy measurements confirm that the graphene films with different coverage have very limited defects. Additionally, the 2D band peak can be well-fitted by a single Lozentian peak, indicating that graphene films are characteristic of single layer graphene. Graphene film thickness can be determined by analysis of Auger spectra, indicating that graphene films after 850 degrees C annealing mainly consist of monolayer graphene. By precise adjustment of annealing temperature, graphene film thickness and area size can be controlled and uniform large-area single-layer and double-layer graphene can be achieved.

Improving accuracy of sample surface topography by atomic force microscopy.

We describe method for improving the accuracy of sample surface topography by atomic force microscopy (AFM). It takes into account the effect of the AFM tip shape and image reconstruction on the acquired AFM images. The dilation effect due to the use of a finite-sized tip shape can be minimized by using a sharp AFM tip and scanning at the most symmetric direction of tip geometry. Reconstruction of AFM image could produce more accurate sample surface features. The method is useful to AFM measurement and is significant because AFM has become a fundamental tool in nanoscience and nanotechnology with multiple applications in a wide range of disciplines ranging from biology to physics and material science.

Reconstruction of atomic force microscopy image by using nanofabricated tip characterizer toward the actual sample surface topography.

The atomic force microscopy (AFM) image is a dilation of the sample surface topography due to the finite-sized AFM tip. We accurately estimated the tip apex shape with a nanofabricated Si tip characterizer and applied the estimated tip shape function to a dilation-erosion algorithm for image reconstruction. The reconstructed images from the original AFM images attained with different AFM tips show consistent surface features and closely match the high-resolution field-emission scanning electron microscope image. The results demonstrate the reliability of our method and suggest the importance of AFM image reconstruction for a variety of technologies requiring new strategies of measuring, interpreting, manipulating, and positioning in the submicrometer and nanometer range.

Light emission induced by tunneling electrons from surface nanostructures observed by novel conductive and transparent probes.

We have developed an ultrahigh-vacuum low-temperature scanning tunneling microscope (STM) equipped with a near-field optical detection system using novel conductive and optically transparent probes. Tunneling-electron induced photons generated in a nanometer-scale area just under the STM probe can be collected directly into the core of the optical fiber probe within the optical near-field region. Firstly, optical fiber probes coated with indium-tin-oxide thin film are applied to quantitative analysis of p-type GaAs(110) surface, where a decrease of light emission in photon mapping clearly extracts the existence of Zn accepter atoms located at the sub-surface layers. Secondly, in order to enhance the efficiency for inelastic tunneling excitation of a tip-induced plasmon mode, a STM probe coated with an Ag/ITO dual-layer film has been developed and applied to an Ag(111) surface, where photon mapping with a step resolution has been achieved by near-field detection.

Novel local density of state mapping technique for low-dimensional systems.

A novel local density of state (LDOS) probing method for low-dimensional electron systems is proposed. By applying a two-dimensional fast Fourier transform to a real-space image obtained by scanning tunneling microscopy (STM), visualization of a complementary image in k-space can be realized. Especially, low-dimensional Fermi contours can be extracted by applying the k-space imaging to real-space images containing sufficient LDOS information around the Fermi level. To realize a more enhanced LDOS visualization in both spaces, we have proposed the use of special materials for STM tips, which have relatively large LDOSs at the Fermi level. To demonstrate this idea, several kinds of STM tips (Ag, Au, W and Nb) with different types of LDOSs were developed. An Au(111)-(22 x square root(3)) reconstructed surface, where Shockley surface-state electrons form a nearly free electron gas, was selected as a test sample for the LDOS extraction. Visualization of standing waves in the surface LDOS modulated by herringbone reconstruction was attempted using the various types of STM tips. Significant effects of the LDOSs of the STM tips were clarified.