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Euglena gracilis is a microalga, which has been used as a model organism for decades. Recent technological advances have enabled mass cultivation of this species for industrial applications such as feedstock in nutritional foods and cosmetics. E. gracilis degrades its storage polysaccharide (paramylon) under hypoxic conditions for energy acquisition by an oxygen-independent process and accumulates high amount of wax-ester as a by-product. Using this sequence of reactions referred to as wax-ester fermentation, E. gracilis is studied for its application in biofuel production. Although the wax-ester production pathway is well characterized, little is known regarding the biochemical reactions underlying the main metabolic route, especially, the existence of an unknown sulfur-compound metabolism implied by the nasty odor generation accompanying the wax-ester fermentation. In this study, we show sulfur-metabolomics of E. gracilis in aerobic and hypoxic conditions, to reveal the biochemical reactions that occur during wax-ester synthesis. Our results helped us in identifying hydrogen sulfide (H2S) as the nasty odor-producing component in wax-ester fermentation. In addition, the results indicate that glutathione and protein degrades during hypoxia, whereas cysteine, methionine, and their metabolites increase in the cells. This indicates that this shift of abundance in sulfur compounds is the cause of H2S synthesis.

High-throughput large-droplet sorting is demonstrated by an array of sequentially addressable dielectrophoretic sources., Droplet microfluidics has become a powerful tool in precision medicine, green biotechnology, and cell therapy for single-cell analysis and selection by virtue of its ability to effectively confine cells. However, there remains a fundamental trade-off between droplet volume and sorting throughput, limiting the advantages of droplet microfluidics to small droplets (

Microalgae have been attracting increasing attention as a renewable energy source and feedstock because of their potential for use in the production of bio-based fuels and materials. In this chapter, we provide an overview of bioproduction based on microalgae species. Specifically, we describe the taxonomic distribution of major industrially exploited microalgae species and highlight their utilities and recent advances. We also introduce recent advances in breeding and engineering techniques to improve the productivity of microalgae to enhance their biomass use.

Single-cell transfection is a powerful technique for delivering chemicals, drugs, or probes into arbitrary, specific single cells. This technique is especially important when the analysis of molecular function and cellular behavior in individual microscopic organisms such as protists requires the precise identification of the target cell, as fluorescence labeling of bulk populations makes tracking of individual motile protists virtually impossible. Herein, we have modified current single-cell electroporation techniques for delivering fluorescent markers into single Euglena gracilis, a motile photosynthetic microalga. Single-cell electroporation introduced molecules into individual living E. gracilis cells after a negative pressure was applied through a syringe connected to the micropipette to the target cell. The new method achieves high transfection efficiency and viability after electroporation. With the new technique, we successfully introduced a variety of molecules such as GFP, Alexa Fluor 488, and exciton-controlled hybridization-sensitive fluorescent oligonucleotide (ECHO) RNA probes into individual motile E. gracilis cells. We demonstrate imaging of endogenous mRNA in living E. gracilis without interfering with their physiological functions, such as swimming or division, over an extended period of time. Thus the modified single-cell electroporation technique is suitable for delivering versatile functional molecules into individual motile protists.

Euglena gracilisis a green photosynthetic microalga that swims using its flagellum. This species has been used as a model organism for over half a century to study its metabolism and the mechanisms of its behavior. The development of mass-cultivation technology has led toE. gracilisapplication as a feedstock in various products such as foods. Therefore, breeding ofE. gracilishas been attempted to improve the productivity of this feedstock for potential industrial applications. For this purpose, a characteristic that preserves the microalgal energy e.g., reduces motility, should be added to the cultivars. The objective of this study was to verify our hypothesis thatE. gracilislocomotion-defective mutants are suitable for industrial applications because they save the energy required for locomotion. To test this hypothesis, we screened forE. gracilismutants from Fe-ion-irradiated cell suspensions and established a mutant strain, ${\mathrm{M}}_{3}^{-}$ZFeL, which shows defects in flagellum formation and locomotion. The mutant strain exhibits a growth rate comparable to that of the wild type when cultured under autotrophic conditions, but had a slightly slower growth under heterotrophic conditions. It also stores 1.6 times the amount of paramylon, a crystal ofβ-1,3-glucan, under autotrophic culture conditions, and shows a faster sedimentation compared with that of the wild type, because of the deficiency in mobility and probably the high amount of paramylon accumulation. Such characteristics makeE. gracilismutant cells suitable for cost-effective mass cultivation and harvesting.

Microalgae-based metabolic engineering has been proven effective for producing valuable substances such as food supplements, pharmaceutical drugs, biodegradable plastics, and biofuels in the past decade. The ability to accurately visualize and quantify intracellular metabolites in live microalgae is essential for efficient metabolic engineering, but remains a major challenge due to the lack of characterization methods. Here we demonstrate it by synthesizing fluorogenic peptide aptamers with specific binding affinity to a target metabolite and delivering them into live microalgae by femtosecond laser photoporation at single-cell resolution. As a proof-of-principle demonstration of our method, we use it to characterize Euglena gracilis, a photosynthetic unicellular motile microalgal species, which is capable of producing paramylon (a carbohydrate granule similar to starch). Specifically, we synthesize a peptide aptamer containing a paramylon-binding fluorescent probe, 7-nitrobenzofurazan, and introduce it into E. gracilis cells one-by-one by suppressing their mobility with mannitol and transiently perforating them with femtosecond laser pulses at 800 nm for photoporation. To demonstrate the method’s practical utility in metabolic engineering, we perform spatially and temporally resolved fluorescence microscopy of single live photoporated E. gracilis cells under different culture conditions. Our method holds great promise for highly efficient microalgae-based metabolic engineering.

Euglena gracilis Z (Euglena) is a unicellular, photosynthesizing, microscopic green alga. It contains several nutrients such as vitamins, minerals, and unsaturated fatty acids. In this study, to verify the potential role of Euglena consumption on human health and obesity, we evaluated the effect of Euglena on human adipose-derived stem cells. We prepared a Euglena extract and evaluated its effect on cell growth and lipid accumulation, and found that cell growth was promoted by the addition of the Euglena extract. Interestingly, intracellular lipid accumulation was inhibited in a concentration-dependent manner. Quantitative real-time PCR analysis and western blotting analysis indicated that the Euglena extract suppressed adipocyte differentiation by inhibiting the gene expression of the master regulators peroxisome proliferator-activated receptor-γ (PPARγ) and one of three CCAAT-enhancer-binding proteins (C/EBPα). Further Oil Red O staining experiments indicated that the Euglena extract inhibited the early stage of adipocyte-differentiation. Consistent with these results, we observed that down-regulation of gene expression was involved in the early stage of adipogenesis represented by the sterol regulatory element binding protein 1 c (SREBP1c), two of three CCAAT-enhancer-binding proteins (C/EBPβ, C/EBPδ), and the cAMP regulatory element-binding protein (CREB). Taken together, these data suggest that Euglena extract is a promising candidate for the development of a new therapeutic treatment for obesity.

The marine diatom Phaeodactylum tricornutum is attractive for basic and applied diatom research. We isolated putative endogenous gene promoters derived from genes that are highly expressed in P. tricornutum: the fucoxanthin chlorophyll a/c-binding protein (FCP) C gene, the vacuolar ATP synthase 16-kDa proteolipid subunit (V-ATPase C) gene, the clumping factor A gene and the solute carrier family 34 member 2 gene. Five putative promoter regions were isolated, linked to an antibiotic resistance gene (Sh ble) and transformed into P. tricornutum. Using quantitative RT-PCR, the promoter activities in the transformants were analyzed and compared to that of the diatom endogenous gene promoter, the FCP A gene promoter which has been used for the transformation of P. tricornutum. Among the five isolated potential promoters, the activity of the V-ATPase C gene promoter was approximately 2.73 times higher than that of the FCP A gene promoter. The V-ATPase C gene promoter drove the expression of Sh ble mRNA transcripts under both light and dark conditions at the stationary phase. These results suggest that the V-ATPase C gene promoter is a novel tool for the genetic engineering of P. tricornutum.

Carotenoids exert beneficial effects on human health through their excellent antioxidant activity. To increase carotenoid productivity in the marine Pennales Phaeodactylum tricornutum, we genetically engineered the phytoene synthase gene (psy) to improve expression because RNA-sequencing analysis has suggested that the expression level of psy is lower than other enzyme-encoding genes that are involved in the carotenoid biosynthetic pathway. We isolated psy from P. tricornutum, and this gene was fused with the enhanced green fluorescent protein gene to detect psy expression. After transformation using the microparticle bombardment technique, we obtained several P. tricornutum transformants and confirmed psy expression in their plastids. We investigated the amounts of PSY mRNA and carotenoids, such as fucoxanthin and β-carotene, at different growth phases. The introduction of psy increased the fucoxanthin content of a transformants by approximately 1.45-fold relative to the levels in the wild-type diatom. However, some transformants failed to show a significant increase in the carotenoid content relative to that of the wild-type diatom. We also found that the amount of PSY mRNA at log phase might contribute to the increase in carotenoids in the transformants at stationary phase.

Microalgae have been receiving great attention for their ability to produce biomaterials that are applicable for food supplements, drugs, biodegradable plastics, and biofuels. Among such microalgae, Euglena gracilis has become a popular species by virtue of its capability of accumulating useful metabolites including paramylon and lipids. In order to maximize the production of desired metabolites, it is essential to find ideal culturing conditions and to develop efficient methods for genetic transformation. To achieve this, understanding and controlling cell-to-cell variations in response to external stress is essential, with chemically specific analysis of microalgal cells including E. gracilis. However, conventional analytical tools such as fluorescence microscopy and spontaneous Raman scattering are not suitable for evaluation of diverse populations of motile microalgae, being restricted either by the requirement for fluorescent labels or a limited imaging speed, respectively. Here we demonstrate video-rate label-free metabolite imaging of live E. gracilis using stimulated Raman scattering (SRS) – an optical spectroscopic method for probing the vibrational signatures of molecules with orders of magnitude higher sensitivity than spontaneous Raman scattering. Our SRS’s highspeed image acquisition (27 metabolite images per second) allows for population analysis of live E. gracilis cells cultured under nitrogen-deficiency - a technique for promoting the accumulation of paramylon and lipids within the cell body. Thus, our SRS system’s fast imaging capability enables quantification and analysis of previously unresolvable cell-to-cell variations in the metabolite accumulation of large motile E. gracilis cell populations.

Euglena gracilis is a eukaryotic, unicellular phytoflagellate that has been widely studied in basic science and applied science. Under dark, anaerobic conditions, the cells of E. gracilis produce a wax ester that can be converted into biofuel. Here, we demonstrate that under dark, anaerobic conditions, E. gracilis excretes organic acids, such as succinate and lactate, which are bulk chemicals used in the production of bioplastics. The levels of succinate were altered by changes in the medium and temperature during dark, anaerobic incubation. Succinate production was enhanced when cells were incubated in CM medium in the presence of NaHCO3. Excretion of lactate was minimal in the absence of external carbon sources, but lactate was produced in the presence of glucose during dark, anaerobic incubation. E. gracilis predominantly produced L-lactate; however, the percentage of D-lactate increased to 28.4% in CM medium at 30°C. Finally, we used a commercial strain of E. gracilis for succinate production and found that nitrogen-starved cells, incubated under dark, anaerobic conditions, produced 869.6 mg/L succinate over a 3-day incubation period, which was 70-fold higher than the amount produced by nitrogen-replete cells. This is the first study to demonstrate organic acid excretion by E. gracilis cells and to reveal novel aspects of primary carbon metabolism in this organism.

A study was conducted 1) to develop a scale of the frequency of environmentally responsible behavior (ERB), 2) to investigate how ERB is related to voluntary simplicity lifestyle (VSL), control of needs, social responsibility and concern about global environment for future generations, and 3) to investigate how VSL is related to the control of needs, social responsibility and the concern about global environment for future generations. Complete data were obtained from 137 undergraduates (102 male and 35 female). Variables were measured by factor-analyzed multiple-item 5-or 7-point scales. Analysis of multiple regression shows 1) VSL predicts ERB; 2) the control of needs predicts VSL; and 3) the concern about global environment for future generations predicts both ERB and VSL.

Understanding metabolism in live microalgae is crucial for efficient biomaterial engineering, but conventional methods fail to evaluate heterogeneous populations of motile microalgae due to the labelling requirements and limited imaging speeds. Here, we demonstrate label-free video-rate metabolite imaging of live Euglena gracilis and statistical analysis of intracellular metabolite distributions under different culture conditions. Our approach provides further insights into understanding microalgal heterogeneity, optimizing culture methods and screening mutant microalgae. Label-free, video-rate metabolite imaging of live Euglena gracilis shows spatiotemporal intracellular metabolite distributions under different culture conditions.

Microbes, especially microalgae, have recently been of great interest for developing novel biofuels, drugs, and biomaterials. Imaging-based screening of live cells can provide high selectivity and is attractive for efficient bio-production from microalgae. Although conventional cellular screening techniques use cell labeling, labeling of microbes is still under development and can interfere with their cellular functions. Furthermore, since live microbes move and change their shapes rapidly, a high-speed imaging technique is required to suppress motion artifacts. Stimulated Raman scattering (SRS) microscopy allows for label-free and high-speed spectral imaging, which helps us visualize chemical components inside biological cells and tissues. Here we demonstrate high-speed SRS imaging, with temporal resolution of 0.14 seconds, of intracellular distributions of lipid, polysaccharide, and chlorophyll concentrations in rapidly moving Euglena gracilis, a unicellular phytoflagellate. Furthermore, we show that our method allows us to analyze the amount of chemical components inside each living cell. Our results indicate that SRS imaging may be applied to label-free screening of living microbes based on chemical information.

Euglena gracilis, a microalgal species of unicellular flagellate protists, has attracted much attention in both the industrial and academic sectors due to recent advances in the mass cultivation of E. gracilis that have enabled the cost-effective production of nutritional food and cosmetic commodities. In addition, it is known to produce paramylon (β-1,3-glucan in a crystalline form) as reserve polysaccharide and convert it to wax ester in hypoxic and anaerobic conditions–a promising feedstock for biodiesel and aviation biofuel. However, there remain a number of technical challenges to be solved before it can be deployed in the competitive fuel market. Here we present a method for efficient selective breeding of live oil-rich E. gracilis with fluorescence-activated cell sorting (FACS). Specifically, the selective breeding method is a repetitive procedure for one-week heterotrophic cultivation, staining intracellular lipids with BODIPY505/515 and FACS-based isolation of top 0.5% lipid-rich E. gracilis cells with high viability, after inducing mutation with Fe-ion irradiation to the wild type (WT). Consequently, we acquire a live, stable, lipid-rich E. gracilis mutant strain, named B1ZFeL, with 40% more lipid content on average than the WT. Our method paves the way for rapid, cost-effective, energy-efficient production of biofuel.

We report a patient with congenital Chagas disease in Japan. This report reemphasizes the role of neglected and emerging tropical diseases in the era of globalization. It also indicates the need for increased vigilance for detecting Chagas disease in non-disease-endemic countries.

Laser-scanning confocal fluorescence microscopy is an indispensable tool for biomedical research by virtue of its high spatial resolution. Its temporal resolution is equally important, but is still inadequate for many applications. Here we present a confocal fluorescence microscope that, for the first time to our knowledge, surpasses the highest possible frame rate constrained only by the fluorescence lifetime of fluorophores (typically a few to several nanoseconds). This microscope is enabled by integrating a broadband, spatially distributed, dual-frequency comb or spatial dual-comb and quadrature amplitude modulation for optimizing spectral efficiency into frequency-division multiplexing with single-pixel photodetection for signal integration. Specifically, we demonstrate confocal fluorescence microscopy at a record high frame rate of 16,000 frames/s. To show its broad biomedical utility, we use the microscope to demonstrate 3D volumetric confocal fluorescence microscopy of cellular dynamics at 104 volumes/s and confocal fluorescence imaging flow cytometry of hematological and microalgal cells at up to 2 m/s.

大学生を対象に高度達成者に対する態度に関する研究が行われた。この態度を測定する20項目,6種の架空の成功体験に対する4種の人物の認知された嫉妬/祝福反応に関する項目および6つの心理的変数に関する項目が使用された。高度達成者に対する「賞賛」と「嫉妬」の因子が抽出された。各場面について「きょうだい」「親友」および「いとこ」の嫉妬/祝福反応の得点を加算した。これらの他者の認知された嫉妬/祝福の得点と高度達成者に対する態度の相関から,嫉妬に関してのみ部分的に有意な負の相関が得られた。また,幸福感・自尊心と他者の認知された嫉妬/祝福との間で有意な正の相関が見出された。最後に,「賞賛」と心理的変数の相関は有意ではなかったが,「嫉妬」には幸福感・自尊心との間で有意な負の相関が認められた。, Questionnaires were administered to male and female undergraduates at a national university to investigate relationships among attitudes toward high achievers, judged jealousy/blessing to imaginary successful experiences of six scenarios and six psychological variables. A 20-item five-point scale of attitudes toward high achievers produced two factors, "praise" and "jealousy." Regarding the scenarios, subjects were imagined to have a successful experience in each scenario and was asked to judge four target persons' degree of jealousy/blessing to the success on a seven-point scale. Although jealousy was partially correlated with the perceived jealousy/blessing, praise was not significantly correlated with this perceived measure. A few of the six psychological variables were partially correlated with the percived jealousy/blessing. Regarding attitudes toward high achievers, praise did not correlate with the psychological variables. On the other hand, jealousy significantly correlated with these variables partially.

Euglena gracilis is a microalgae used as a model organism. Recently, mass cultivation of this species has been achieved for industrial applications. The genus Euglena includes more than 200 species that share common useful features, but the potential industrial applications of other Euglena species have not been evaluated. Thus, we conducted a pilot screening study to identify other species that proliferate at a sufficiently rapid rate to be used for mass cultivation; we found that Euglena anabaena var. minor had a rapid growth rate. In addition, its cells accumulated more than 40% weight of carbohydrate, most of which is considered to be a euglenoid specific type of beta-1-3-glucan, paramylon. Carbohydrate is stored in E. anabaena var. minor cells during normal culture, whereas E. gracilis requires nitrogen limitation to facilitate paramylon accumulation. These results suggest the potential industrial application of E. anabaena var. minor.

Data from a questionnaire were collected from 189 Japanese female undergraduates at a women's university. They were 18, 19, or 20 years of age. The measures included a 22-item 7-point voluntary simplicity lifestyle scale, a 9-item 5-point scale of the evaluation of a voluntarily simple life, an 11-item 4-point scale of environmentally responsible consumerism and a 28-item 5-point scale of a nonsimplicity lifestyle. Factor analysis was conducted for each scale and Cronbach's αcoefficients were calculated for selected items associated with each scale. Both the evaluation of a voluntarily simple life and environmentally responsible consumerism were positively associated with a voluntary simplicity lifestyle and its three factors, that is, cautious attitudes in shopping, acceptance of self-sufficiency and a desire for a voluntarily simple life. A voluntary simplicity lifestyle was significantly associated with more than half of the nonsimplicity lifestyle statements.

因子分析を実施した複数項目の5段階評定尺度を用いて、2つの研究で日本人大学生における自己報告による「環境に対する責任ある行動」の心理的相関要因について検討した。研究Iでは、「将来世代に対する責任」と「実行力」が「環境に対する責任ある行動」と有意に関連していたが、「環境の汚染と破壊の認知された深刻さ」と「都会-田舎志向」は有意に関連していなかった。研究IIでは、「長期的視野」と「人間の自然への服従」は「環境に対する責任ある行動」と有意に関連していたが、「環境の汚染と破壊の認知された深刻さ」と他の2つの自然観には有意な関連が認められなかった。, Two studies, using factor-analyzed multiple-item five-point scales, investigated some psychological correlates of self-reported environmentally responsible behavior in Japanese undergraduates. In Study I, "responsibility to future generations" and "power of execution" were significantly correlated with environmentally responsible behavior, but "perceived seriousness of environmental pollution and destruction" and "urban-rural orientation" were not. In Study II, "long-term perspective" and "human obedience to nature" were significantly correlated with environmentally responsible behavior, but "perceived seriousness of environmental pollution and destruction" and two other concepts of nature were not.

某国立大学学部学生139名を対象として、環境に対する責任ある行動と環境保全態度・地球環境保全への動機の関連について5段階評定尺度の質問紙を用いて検討した。被験者の性別は、男性が97名、女性が36名、記入漏れの者が6名であった。また、年齢は記入漏れの12名を除き、平均が18.99、標準偏差が1.29であった。重回帰分析の結果、全体としての環境保全態度と地球環境保全への動機がそれぞれ強いほど環境に対する責任ある行動がより頻繁であるという関係が認められた。また、環境保全態度の5因子を独立変数とする重回帰分析を行ったところ、環境問題に関する情報への接近、反「自動車利用」および反「環境汚染」の行動意図の得点が高いほど環境に対する責任ある行動が頻繁であるという関係が明らかになった。, Using five-point scale questionnaire data of 139 undergraduates from a national university, the relationship was investigated between environmentally responsible behavior on the one hand and proenvironmental attitudes and the motive for global environmental protection on the other. Among the undergraduates, ninety-seven were male, thirty-six were female and six did not give any response about sex. According to an analysis of multiple regression, higher frequency of environmentally responsible behavior was associated with stronger proenvironmental attitude and stronger motive for global environmental protection. Also, when five factors of proenvironmental attitudes were introduced as independent variables, higher frequency of environmentally responsible behavior was associated with more frequent "approach to information of environmental problems," stronger objection to car use or stronger anti-pollution behavioral intention.

This study investigated the relationship between six psychological variables and the environmentally responsible behavior of Japanese undergraduates; 106 males and 44 females. Their average age was 18.66 with a standard deviation of 1.38. The six variables were attitudes toward growth and technology, efficacy of personal proenvironmental behavior, sensitivity to noise, social awareness, emotional sensitivity, and behavioral independence. Multiple regression analysis was conducted for environmentally responsible behavior with the six variables entered simultaneously as predictors. Negative attitudes toward growth and technology, efficacy of personal proenvironmental behavior, and emotional sensitivity were significantly related to environmentally responsible behavior, while behavioral independence was marginally related, and sensitivity to noise and social awareness were not related.

本研究は某国立大学生132名(男性76名、女性52名)を対象に「子供を持つこと」の価値に関する実態を調べ、、「子供を持つこと」の価値と快楽希求、男女平等主義、親和欲求、自己中心主義、目標達成指向、男性性及び女性性との関係について検討した。これらすべての変数は5段階評定尺度によって測定し、因子分析により項目を選択し内的整合性について検討した。「子供を持つこと」の価値について7つの心理的変数を独立変数として一括投入し重回帰分析を行った。その結果、自己中心主義が強いほど「子供を持つこと」の価値が低いという関係が認められた。「子供を持つこと」の価値に関して、将来欲しい子供の人数の多少による差違は認められたが、性差は見られなかった。, This study investigated value of children among 132 undergraduates at a national university (76 males and 52 females) and examined the relationship between value of children on the one hand and pleasure seeking, sexual egalitarianism, need for affiliation, egocentric attitude, need for goal achievement, masculinity and femininity on the other. All variables were measured by five-point rating scales. Items were selected by factor analysis and internal consistency was examined for each variable. Analysis of multiple regression was carried out for value of children with seven predictors entered simultaneously. Results indicated that stronger egocentric attitude was associated with lower value of children. Desired number of children produced significant difference in value of children, but there was no sex difference in value of children.

Questionnaires containing five multi-item 5-point scales were administered to 153 Japanese male and female undergraduates. Data of each of the scales were factor analyzed and, as a rule, items with a factor loading of .40 or over were selected. The scale for coping style produced three factors: avoidance, self-deceptive optimism and problem solving. Each of the other four scales produced one factor. Using the total score for each scale or factor, multiple regression analysis was applied to environmentally responsible behavior with six predictors entered simultaneously. Self-deceptive optimism and willingness to accept sacrifices for global environmental protection proved to be significant predictors of environmentally responsible behavior, but the four other predictors did not.

A study was conducted to investigate how voluntary simplicity lifestyle (VSL), hedonism and anti-materialism affect environmentally responsible behavior (ERB). Questionnaires containing multi-item 5-point or 7-point scales of ERB, VSL, hedonism and anti-materialism were administered to 130 undergraduates (51 males and 79 females). Factor analysis was applied to each of the scales. The VSL scale yielded two factors: 1) Careful shopping attitudes intended for long-term use and 2) self-sufficiency oriented attitudes. A measure of positive attitudes toward ERB was also obtained from this VSL scale. Multiple regression analysis was carried out on ERB, with other indices entered simultaneously as predictors. Results indicate that careful shopping attitudes intended for long-term use and positive attitudes toward ERB make a substantial contribution to ERB.

A study was conducted to investigate correlations between voluntary simplicity lifestyles (VSLs.) and perceptual or behavioral characteristics regarding VSLs.. Questionnaires each of which contained a 20-item 5-point scale of VSLs., a 13-item 4-point scale of the frequency of proenvironmental behaviors, and 23 5-point scales of various perceptual or behavioral characteristics regarding VSLs were administered to 250 female adults. The scale of VSLs yielded four variables. Product-moment correlation coefficients were calculated 1) between these four variables and variables of the frequency of proenvironmental behaviors and 2) between the four variables and perceptual or behavioral characteristics associated with VSLs. Many correlations were significant, and were in assumed directions.

We demonstrate high-throughput label-free single-cell image cytometry and image-based classification of Euglena gracilis (a microalgal species) under different culture conditions. We perform it with our high-throughput optofluidic image cytometer composed of a time-stretch microscope with 780-nm resolution and 75-Hz line rate, and an inertial-focusing microfluidic device. By analyzing a large number of single-cell images from the image cytometer, we identify differences in morphological and intracellular phenotypes between E. gracilis cell groups and statistically classify them under various culture conditions including nitrogen deficiency for lipid induction. Our method holds promise for real-time evaluation of culture techniques for E. gracilis and possibly other microalgae in a non-invasive manner.

This paper deals with advances and problems related to anesthesia of the bronchoesophageal region, including various anesthetic agents and discussion of some of the peculiar difficulties entailed.Regarding inhalational agents, although halothane's effectiveness is recognized, the risks associated with halothane induced hepatitis and malignant hyperthermia have also been long-recognized. Various alternative inhalational agents, and their relative merits, are discussed. Among the various intravenous agents, of particular interest for its effectiveness, as well as unique attributes such as rapid induction and quick recovery, is propofol, developed over ten years ago and expected to come into use in Japan in 1995. These properties have made propofol a more well-controlled and safe method available for total intravenous anesthesia (TIVA). As to muscle relaxants, vecuronium is noteworthy for its relative lack of effect on the circulatory system, and for its short duration.Operations in the bronchoesophageal region necessarily find the surgeon and anesthesiologist placing competing demands on the area, and resolution of the various priorities continues to pose challenges. The use of vasoconstrictors (epinephrine) during surgery also poses risks, and the guidelines espoused by Katz are discussed in this paper. The incidence of intubation difficulties is addressed, as are minitracheostomies.The advantages in this regard of Brain's laryngeal airway mask (LAM) are stressed. In particular, the LAM provides for an unobstructed view during the use of optic fibroscopy in larygeal microsurgery. Finally, the importance of monitoring, especially with respect to the measurement of end-tidal CO2 (monitoring the capnometer) is emphasized.

First, the connotations in Japan of crowded situations were discussed. In the second section, some personal characteristics of those who might experience crowding, and of partners who might share a space with the former, were discussed as determinants of individual differences in the experience of crowding. In the third section, it was suggested that high collectivism, a non-contact culture, and high population density combined with a high crime rate might encourage the experience of crowding in public or semi-public spaces. In the last section, various structural or physical devices widely observed in Japan, which were assumed to be effective in inhibiting the experience of crowding or reducing crowding stress by maintaining isolation, were discussed.

Endothelial cells are indispensable components of the vascular system, and play pivotal roles during development and in health and disease. Their properties have been studied extensively by in vivo analysis of genetically modified mice. However, further analysis of the molecular and cellular phenotypes of endothelial cells and their heterogeneity at various developmental stages, in vascular beds and in various organs has often been hampered by difficulties in culturing mouse endothelial cells. In order to overcome these difficulties, we developed a new transgenic mouse line expressing the SV40 tsA58 large T antigen (tsA58T Ag) under the control of a binary expression system based on Cre/loxP recombination. tsA58T Ag-positive endothelial cells in primary cultures of a variety of organs proliferate continuously at 33 degrees C without undergoing cell senescence. The resulting cell population consists of blood vascular and lymphatic endothelial cells, which could be separated by immunosorting. Even when cultured for two months, the cells maintained endothelial cell properties, as assessed by expression of endothelium-specific markers and intracellular signaling through the vascular endothelial growth factor receptors VEGFR-2 and VEGFR-3, as well as their physiological characteristics. In addition, lymphatic vessel endothelial hyaluronan receptor-1 (Lyve-1) expression in liver sinusoidal endothelial cells in vivo was retained in vitro, suggesting that an organ-specific endothelial characteristic was maintained. These results show that our transgenic cell culture system is useful for culturing murine endothelial cells, and will provide an accessible method and applications for studying endothelial cell biology.

times difficult to repair, resulting in delayed union or nonunion. For intractable cases, we often perform surgical treatment using some internal fixation device with or without bone grafting. Hulkko and Orava reported 368 stress fractures of which 26 fractures (7.1%) required surgical treatment because of dislocation or delayed union/ nonunion. However, surgical treatment can sometimes lead to complications. Consequently, there is interest in developing an effective nonsurgical technique for treating stress fracture. Recently, extracorporeal shock wave therapy (ESWT) has been reported as an effective noninvasive technique for fracture healing and delayed union/nonunion. We performed ESWT for resistant stress fractures that were not healed conservatively or operatively, and we report efficacy and short-term results. The purpose of this study was to evaluate the effects of ESWT for stress fracture, especially in resistant cases.

Class I antiarrhythmic agents are not always effective in the treatment of life-threatening ventricular tachycardia/ventricular fibrillation (VT/VF) especially in patients with cardiopulmonary arrest. Nifekalant hydrochloride(NIF) is a novel class III antiarrhythmic agent for malignant VT/VF. This study prospectively evaluated NIF efficacy for life-threatening VT/VF observed after cardiopulmonary arrest.Thirty-two of 145 patients who were transferred to the emergency room in Tokai University Hospital showed VT/VF after resuscitation from cardiopulmonary arrest from June 2000 to March 2001. These 32 patients were treated with 12 mg (mean) epinephrine and 1.0-2.0 mg/kg lidocaine following direct current application(200 to 360J), and then classified into two groups. Eleven patients received intravenous 0.15 to 0.3 mg/kg NIF followed by intravenous infusion of 0.3 to 0.4 mg/kg/hr NIF(NIF group). The other 21 patients received 1.0 to 2.0 mg/kg of lidocaine(non-NIF group).Sinus rhythm was restored in the nine patients(82%) in the NIF group but only four patients (19%) in the non-NIF group. QTc was not prolonged(0.45 +/- 0.04 sec, n = 9) and no torsades de pointes was observed in the NIF group. Two patients survived but the remaining nine patients died in the NIF group. Five patients died of cardiac standstill following sinus bradycardia and repeated sinus arrest within 2 to 27 hr after admission, two patients died of sudden cardiac arrest from sinus rhythm, and two patients died of persistent VT/VF. In contrast, all 21 patients in the non-NIF group died. Seventeen patients died of persistent VT/VF before hospitalization, one patient died of recurrent VT/VF, and three patients died of cardiac standstill following sinus bradycardia.NIF effectively suppresses VT/VF which is refractory to direct current shock in patients with cardiopulmonary arrest. However, NIF may rather worsen electrophysiological function in the sinus node after administration of high doses of epinephrine, and may induce sinus bradycardia and/or sinus arrest. Careful observation, such as monitoring of electrocardiography and blood pressure and temporary cardiac pacemaker use, is needed to prevent death in patients surviving after cardiopulmonary arrest if NIF is administered following high dose epinephrine infusion.

The author investigated relationships between proenvironmental attitudes and concepts of nature. Proenvironmental attitudes were measured on a 5-point scale containing 27 items. The author developed a 5-point scale containing 20 items to assess concepts of nature. The author administered those 2 scales to 140 young adults and adolescents at 2 Japanese institutions of higher education. An investigation of 6 proenvironmental attitudes and 20 concepts of nature showed some significant relationships. Factor analysis of the 20 concepts of nature produced 2 factors: Positive Evaluation of Nature and Rejection of Manipulation of Human Life. Most relationships were significant between the scores for the 6 proenvironmental attitudes and those for the 2 factors of concepts of nature.

The aim of this study was to determine whether a quantitative relation exists between changes in regional myocardial blood flow (RMBF) and those in electrophysiologic determinants recorded via left ventricular endocardial and epicardial bipolar electrograms after administration of disopyramide (DP) and a class III antiarrhythmic drug, MS-551 (MS), during myocardial ischemia in the dog. Dogs were given DP ( 1 mg/kg, i.v., n = 14), MS (1 mg/kg, i.v., and 0.1 mg/kg/min, d.i.v., n = 13), or saline (n = 12). The effective refractory period (ERP) was determined by an S 1 -S 2 extrastimulus method, and RMBF by a nonradioactive microsphere technique. The duration of regional electrograms (DRE) was measured as an indicator of conduction time in the myocardium. DP blunted ischemia-induced shortening of ERPs and lengthened DREs at the endocardial and epicardial sites, with a greater effect seen epicardially (p < 0.01 each). DP reduced RMBF, especially at the endocardial surfaces of the ischemic zone (p < 0.05). MS prolonged ERPs at the endocardial and epicardial sites in the ischemic and normal zones (p < 0.05-0.01), but there were no significant differences between the two sites. MS prolonged DREs (p < 0.05), but the magnitude of the prolongation of the DREs was similar to the values in the control group. MS had no effects on RMBF. DP treatment prolonged DREs at both sites in the ischemic zone more markedly than MS or saline treatment (p < 0.01 each). DP reduced RMBF at the endocardial site of the ischemic zone more markedly than MS or saline (p < 0.05 in each). Accordingly, MS prolonged ERPs, but did not increase disparities between endocardial and epicardial sites in the ischemic myocardium, whereas DP had a greater ERP-prolonging effect at the epicardial site than at the endocardial site. DP reduced endocardial RMBF more markedly than epicardial RMBF. These observations suggest that differences in ERPs between endocardial and epicardial ischemic myocardium caused by DP treatment are not due to the difference in RMBF reduction between the two tissue layers, and that DP and MS do not affect the same population of ion channel(s) when ERPs are prolonged.

2 experiments were conducted to examine the degree of concentration on performance as a determinant in the effect of acoustic stimuli upon performance. In the Exp. I, 2 groups of female college students whose concentration on performance was high or low took part in a choice reaction time task in which 2 visual signals were presented in 2 conditions. One was a quiet condition and the other was an instrumental music condition. In the Exp. II, another group of medium concentration was added to the 2 groups and radio news and cross-talk were presented to S instead of music. The results showed that concentration on performance was a very important determinant in the effect of acoustic stimuli upon performance.