In vitro gene transfer to mammalian cells by the use of laser-induced stress waves: effects of stress wave parameters, ambient temperature, and cell type

Laser-mediated gene transfection has received much attention as a new method for targeted gene therapy because of the high spatial controllability of laser energy. We previously demonstrated both in vivo and in vitro that plasmid DNA can be transfected by applying nanosecond pulsed laser-induced stress waves (LISWs). In the present study, we investigated the dependence of transfection efficiency on the laser irradiation conditions and hence stress wave conditions in vitro. We measured characteristics of LISWs used for gene transfection. For NIH 3T3 cells, transfection efficiency was evaluated as functions of laser fluence and number of pulses. The effect of ambient temperature was also investigated, and it was found that change in ambient temperature in a specific range resulted in drastic change in transfection efficiency for NIH 3T3 cells. Gene transfection of different types of cell lines were also demonstrated, where cellular heating increased transfection efficiency for nonmalignant cells, while heating decreased transfection efficiency for malignant cells.     

The kinetics of proinflammatory cytokines in murine peritoneal macrophages infected with envelope protein-glycosylated or non-glycosylated West Nile virus

The envelope (E) protein glycosylation status of the New York strain of West Nile (WN) virus is an important determinant of virus neuroinvasiveness. To elucidate the determinant of the difference between E protein-glycosylated and non-glycosylated WN virus infections, the cytokine expression of murine peritoneal macrophages infected with each virus was examined. Tumor necrosis factor (TNF) alpha and interleukin (IL)-1beta were up-regulated with replication of the E protein-glycosylated virus. Interferon (IFN) beta and IL-6 were up-regulated with the clearance of both viruses. These results suggest that TNFalpha and IL-1beta expression are related to the virulence of E protein-glycosylated WN virus.     

Experimental determination of subjective similarity for pairs of clustered microcalcifications on mammograms: observer study results

Presentation of images of lesions similar to that of an unknown lesion might be useful to radiologists in distinguishing between benign and malignant clustered microcalcifications on mammograms. Investigators have been developing computerized schemes to select similar images from large databases. However, whether selected images are really similar in appearance is not examined for most of the schemes. In order to retrieve images that are useful to radiologists, the selected images must be similar from radiologists' diagnostic points of view. Therefore, in this study, the data of radiologists' subjective similarity for pairs of clustered microcalcification images were obtained from a number of observers, and the intra- and inter-observer variations and the intergroup correlations were determined to investigate whether reliable similarity ratings by human observers can be determined. Nineteen images of clustered microcalcifications, each of which was paired with six other images, were selected for the observer study. Thus, subjective similarity ratings for 114 pairs of clustered microcalcifications were determined by each observer. Thirteen breast, ten general, and ten nonradiologists participated in the observer study; some of them completed the study multiple times. Although the intraobserver variations for the individual readings and the interobserver variations for pairs of observers were not small, the interobserver agreements were improved by taking the average of readings by the same observers. When the similarity ratings by a number of observers were averaged among the groups of breast, general, and nonradiologists, the mean differences of the ratings between the groups decreased, and good concordance correlations (0.846, 0.817, and 0.785) between the groups were obtained. The result indicates that reliable similarity ratings can be determined by use of this method, and the average similarity ratings by breast radiologists can be considered meaningful and useful for the development and evaluation of a computerized scheme for selection of similar images.     

Computerized detection of vertebral compression fractures on lateral chest radiographs: preliminary results with a tool for early detection of osteoporosis

Vertebral fracture (or vertebral deformity) is a very common outcome of osteoporosis, which is one of the major public health concerns in the world. Early detection of vertebral fractures is important because timely pharmacologic intervention can reduce the risk of subsequent additional fractures. Chest radiographs are used routinely for detection of lung and heart diseases, and vertebral fractures can be visible on lateral chest radiographs. However, investigators noted that about 50% of vertebral fractures visible on lateral chest radiographs were underdiagnosed or under-reported, even when the fractures were severe. Therefore, our goal was to develop a computerized method for detection of vertebral fractures on lateral chest radiographs in order to assist radiologists' image interpretation and thus allow the early diagnosis of osteoporosis. The cases used in this study were 20 patients with severe vertebral fractures and 118 patients without fractures, as confirmed by the consensus of two radiologists. Radiologists identified the locations of fractured vertebrae, and they provided morphometric data on the vertebral shape for evaluation of the accuracy of detecting vertebral end plates by computer. In our computerized method, a curved search area, which included a number of vertebral end plates, was first extracted automatically, and was straightened so that vertebral end plates became oriented horizontally. Edge candidates were enhanced by use of a horizontal line-enhancement filter in the straightened image, and a multiple thresholding technique, followed by feature analysis, was used for identification of the vertebral end plates. The height of each vertebra was determined from locations of identified vertebral end plates, and fractured vertebrae were detected by comparison of the measured vertebral height with the expected height. The sensitivity of our computerized method for detection of fracture cases was 95% (19/20), with 1.03 (139/135) false-positive fractures per image. The accuracy of identifying vertebral end plates, marked by radiologists in a morphometric study, was 76.6% (400/522) and 70.9% (420/592) for cases used for training and those for testing, respectively. We prepared 32 additional fracture cases for a validation test, and we examined the detection accuracy of our computerized method. The sensitivity for these cases was 75% (24/32) at 1.03 (33/32) false-positive fractures per image. Our preliminary results show that the automated computerized scheme for detecting vertebral fractures on lateral chest radiographs has the potential to assist radiologists in detecting vertebral fractures.     

Practical application to time indicator of a novel white film formed by interaction of calcium salts with hydroxypropyl methylcellulose

We have found that a cast film forms a white film when an aqueous solution comprising hydroxypropyl methylcellulose (HPMC) and calcium salts such as calcium lactate pentahydrate (CLP) and calcium chloride (CaCl₂) is used. In contrast, the obtained white film was transformed into a transparent film by the addition of purified water. The transformation time for the change from the white film to the transparent film was dependent on film thickness. The relationship between the transformation time and the film thickness was significantly correlated, and it was found that the white film could be adaptable as time indicator. The formation of a white film comprising HPMC and calcium salts was strongly dependent on temperature conditions. The objective of the present study is to investigate the mechanism of the formation of this white film because of the interaction between HPMC and calcium salts. The DSC and XRPD results indicate that the calcium salts affect the HPMC polymer phase in the cast film comprising HPMC and calcium salts. By carrying out attenuated total reflection Fourier transform infrared (ATR FT-IR) analysis, we found that the white film could be formed by the calcium salts affecting the region associated with the C–O–C, C–O, and CH₃ stretching of the HPMC polymer phase.     

Impaired megabenthic community structure caused by summer hypoxia in a eutrophic coastal bay

Eutrophication and hypoxia are major problems affecting the health of coastal ecosystems throughout the world. Tokyo Bay, Japan, is a eutrophic coastal area where the abundance of the megabenthic community has been decreasing. To assess factors associated with the impaired biota, seasonal surveys of the megabenthic community and water and sediment quality were conducted in the bay. Cluster analysis showed a difference in the community structure between the northern and southern parts of the bay. The density of species and species diversity were high throughout the year in the southern part of the bay, whereas in the northern part of the bay species diversity was low and defaunation occurred in August. At this time, bottom hypoxia due to temperature and salinity stratification, and high concentrations of nutrients, chlorophyll a, and organic matter in the water column and/or sediment, dominated the northern part of the bay. In October, bottom hypoxia was less severe but was still present in the northern part of the bay, and recolonization by mobile fishes and sessile mussels occurred. Multivariate analyses of the megabenthic community and environmental parameters in August showed the spatial pattern of the community could be explained by concentrations of dissolved oxygen and particulate organic carbon in the bottom water, and total sulfide and total organic carbon in the sediments. In particular, impairment of the biota in the northern area could be explained by the threshold concentrations of dissolved oxygen <1.7 mL L⁻¹ and total organic carbon >20.3 mg g⁻¹.     

Medicinal history and ginsenosides composition of Panax ginseng rhizome, "Rozu"

Ginseng is prepared from Panax ginseng C.A. Meyer root. The root of wild P. ginseng has long tortuous rhizome called traditionally "Rozu" in Japanese. In the present historical studies on ginseng, it has been proven that ginseng has sometimes been used after removing "Rozu" due to its emetic effects. However, ginseng with "Rozu" is prescribed in almost all the present Kampo formulations used clinically in China and Japan. Possible reasons for this are (1) some formulations including "Rozu" have been used for vomiting resulting from the retention of fluid in the intestine and stomach, "tan-in" in Japanese, and (2) the present cultivated ginseng has shorter "Rozu" than wild ginseng. Furthermore, it is proved that "Rozu", rich in ginsenoside Ro with oleanane-type aglycone, is distinguished from ginseng roots rich in ginsenosides Rb1 and Rg1 with dammarane-type aglycone. This is the first report to declare the distribution of ginsenosides in underground parts of wild P. ginseng. Ginsenoside Ro is a minor ginsenoside in ginseng whereas it is the major ginsenoside in P. japonicus rhizome (chikusetsu-ninjin in Japanese). Ginsenoside Ro is characterized by antiinflammatory effects which differ from ginsenosides Rb1 and Rg1 responsible for adaptogenic effects of ginseng. These results suggest that "Rozu" containing both oleanane- and dammarane-type ginsenosides might be a promising raw material distinct from ginseng root or P. japonicus rhizome.     

Two cases of mycosis fungoides treated by reduced-intensity cord blood transplantation

Mycosis fungoides is a cutaneous T-cell lymphoma, which is clinically divided into three stages: patch, plaque and tumor. Despite a variety of treatments the prognosis is poor in advanced mycosis fungoides. Recently, allogeneic hematopoietic stem cell transplantation has been successfully applied for such cases. We performed reduced-intensity umbilical cord blood transplantation for two advanced mycosis fungoides patients. Case 1 was a 56-year-old man and case 2 was a 30-year-old woman. Tumors of each case were refractory to conventional chemotherapy. Although radiation therapy was considerably effective, tumors relapsed after several months. Reduced-intensity umbilical cord blood transplantation was performed because case 1 had no human leukocyte antigen-identical siblings and the sibling of case 2 did not agree to be the donor. The male patient died of pulmonary failure 23 days after reduced-intensity umbilical cord blood transplantation. The case 2 patient succeeded in reduced-intensity umbilical cord blood transplantation and remained in complete/partial remission for 13 months. However, chemotherapy-resistant tumors relapsed, and allogeneic hematopoietic stem cell transplantation was performed at 17 months. She died of cerebral hemorrhage 23 days after the procedure. Reduced-intensity umbilical cord blood transplantation may be included in the treatments for advanced mycosis fungoides, where graft-versus-lymphoma effect seems to be a significant factor for the success of the treatment.     

Murine typhus in two travelers returning from Bali, Indonesia: an underdiagnosed disease

Two Japanese travelers from Bali were diagnosed with murine typhus in Japan during the same period. Although one had only mild illness, the other experienced liver and kidney dysfunction. Murine typhus may be missed not only in endemic areas around the world, but also in travelers, especially those returning from marine resorts in these areas.