Label-free cell detection of acute leukemia using ghost cytometry.

Early diagnosis and prompt initiation of appropriate treatment are critical for improving the prognosis of acute leukemia. Acute leukemia is diagnosed by microscopic morphological examination of bone marrow smears and flow cytometric immunophenotyping of bone marrow cells stained with fluorophore-conjugated antibodies. However, these diagnostic processes require trained professionals and are time and resource-intensive. Here, we present a novel diagnostic approach using ghost cytometry, a recently developed high-content flow cytometric approach, which enables machine vision-based, stain-free, high-speed analysis of cells, leveraging their detailed morphological information. We demonstrate that ghost cytometry can detect leukemic cells from the bone marrow cells of patients diagnosed with acute lymphoblastic leukemia and acute myeloid leukemia without relying on biological staining. The approach presented here holds promise as a precise, simple, swift, and cost-effective diagnostic method for acute leukemia in clinical practice.

[Developing a Japanese version of the Digital Health Literacy Instrument].

Objectives　The Internet has made it possible to search for, obtain, transmit, and share information. Accordingly, the ability to use health information and skills related to interactivity taken from the Internet have become important in the medical and health fields. However, there is no scale to evaluate these abilities. Therefore, this study was conducted to assess the validity and reliability of the Digital Health Literacy Instrument (DHLI) and examine the association of digital health literacy (DHL) with the characteristics of the study participants.Methods　The Japanese version of the DHLI was developed using the basic guidelines for scale translation. The participants included 2,000 Japanese adults (men: 50.0%, mean age: 40.7±12.0 years) who responded to an Internet-based cross-sectional survey. The Japanese version of the DHLI, attributes, sources of health information, contents of health information taken from the Internet, and eHealth Literacy Scale (eHEALS) scores were obtained using a questionnaire. Confirmatory factor analysis and correlation with eHEALS scores were used to assess construct and criterion validities. Cronbach's alpha and correlation coefficients were computed for internal consistency and test-retest reliability. Differences in DHLI scores for each attribute and variables related to health information were examined using the analysis of variance and t-test.Results　Confirmatory factor analysis revealed a goodness-of-fit index of .946, a comparative fit index of .969, and a root mean square error of approximation of .054, confirming that the Japanese version has the same seven-factor structure as the original version does. A significant positive correlation was found between DHLI and eHEALS scores (r=.40, P<.001). Cronbach's alpha was .92, and test-retest reliability was r=.88 (P<.001). DHLI scores were mainly associated with household income, health status, frequency of information searches on the Internet, and devices used. The subscale scores found difficulties in evaluating reliability, determining relevance, and adding self-generated content. Differences in DHL were observed among some sources and contents of health information on the Internet.Conclusion　The Japanese version of the DHLI was a sufficiently reliable and valid instrument for assessing DHL among Japanese adults. Our results suggest that low DHL may lead to health information disparities. Therefore, it is necessary to consider support strategies for individuals who need to improve their DHL and for skills that need to be strengthened.

Patterns of alcohol and alcohol-flavoured non-alcoholic beverage advertisements over Japanese free-to-air television networks.

BACKGROUND: Alcohol use is a serious public health challenge worldwide. Japan has no government regulations or legal penalties against advertising alcoholic beverages on television (TV). Instead, advertisements depend on the Japanese alcohol industry's self-regulation on airtime (no advertisements from 5 am to 6 pm) and the content of alcoholic beverages, which must not tempt minors. However, many adolescents (10 to 19 years old) watch TV from 6 pm to 11 pm. The aim of this study was to describe the pattern in the advertising of alcoholic beverages and alcohol-flavoured non-alcoholic beverages (AFNAB) in Japan during the popular TV viewing time for adolescents. METHODS: A secondary analysis of advertising airtime data from five free-to-air Japanese TV networks in the Greater Tokyo area that aired between 12 August and 3 November 2019, was performed. RESULTS: During the study period, 5215 advertisements for alcoholic beverages and AFNABs aired (1451.75 min). In total, 2303 advertisements (44.2%) were beer, low-malt beer, or beer-taste beverages, 277 (5.3%) were whisky, 2334 (44.8%) were local alcoholic beverages (shochu and seishu), and 301 (5.8%) were AFNAB. On average, more advertisements aired on weekends (67.6 advertisements) than on weekdays (59.3 advertisements) per day. Approximately 30% of advertisements for AFNABs were aired during the time restricted for alcohol advertising, although AFNABs are considered alcohol according to industry guidelines. During the popular television viewing time for young adolescents, about two to three times more advertisements were aired per hour than during the rest of the day, on both weekdays and weekends (p < 0.001). CONCLUSION: The number of alcohol advertisements aired at times when adolescents often watch TV is 2 to 3.2 times higher than that at other times of the day. Furthermore, despite the industry's self-imposed regulations, some alcoholic beverages are still advertised. Therefore, other methods to protect children and adolescents from exposure to advertisements for alcoholic beverages should be investigated and implemented.

Use of Ghost Cytometry to Differentiate Cells with Similar Gross Morphologic Characteristics.

Imaging flow cytometry shows significant potential for increasing our understanding of heterogeneous and complex life systems and is useful for biomedical applications. Ghost cytometry is a recently proposed approach for directly analyzing compressively measured signals of cells, thereby relieving a computational bottleneck for real-time data analysis in high-throughput imaging cytometry. In our previous work, we demonstrated that this image-free approach could distinguish cells from two cell lines prepared with the same fluorescence staining method. However, the demonstration using different cell lines could not exclude the possibility that classification was based on non-morphological factors such as the speed of cells in flow, which could be encoded in the compressed signals. In this study, we show that GC can classify cells from the same cell line but with different fluorescence distributions in space, supporting the strength of our image-free approach for accurate morphological cell analysis. © 2020 International Society for Advancement of Cytometry.

Children's Knowledge of Cancer Prevention and Perceptions of Cancer Patients: Comparison Before and After Cancer Education with the Presence of Visiting Lecturer -Guided Class.

The purpose of this study was to investigate the effect of the presence of visiting lecturer-guided class on children's knowledge of cancer prevention and perceptions of cancer patients, conducting surveys before and after the cancer education classes at local elementary schools that are pioneering cancer education. We implemented self-administered questionnaire surveys with 571 sixth-grade children at nine elementary schools receiving cancer education in the Tokyo metropolitan area from September 2013 to February 2014. The surveys were conducted twice in each classroom: 1 week before the cancer education class and 1 week after. The questionnaire items included participants' gender, cancer prevention information, and perceptions of cancer patients with the following description: "looks pitiful," "always depressed," "bedridden and hospitalized," "excessive smoking and drinking," "looks thin and pale," "no visible change despite cancer," and "always bright and cheerful." Children who had not attended the cancer education day, or had not completed both the pre- and post-test, were excluded from the analysis. Regardless of whether there visiting lecturer-guided class was provided, the possibility of improving children's knowledge of cancer prevention was demonstrated. On the other hand, in the post-class surveys, compared to the group with visiting lecturer-guided class, the group without it had a significantly lower percentage of persons selecting "Looks pitiful" (76.2%/63.5%, p = 0.002) and significantly higher percentages for "Too much smoking and drinking," and "Looks thin and pale" (50.2%/63.5%, p = 0.002; 18.8%/31.1%, p = 0.001). Therefore, study is needed into cancer education coordinated with relevant institutions in order to more effectively utilize visiting lecturers and the like.

[Consideration of the Evaporation Residue Test for Plastics Intended to Contact with Oils and Fatty Foods].

The Japanese Food Sanitation Act designates the evaporation residue test as a specification for plastics that intended to contact with foods. The test conditions and migration limits for oils and fatty foods were considered on the basis of the results obtained from the evaporation residue test according to the Japanese Act and the overall migration test into olive oil according to EN1186-2. The evaporation residue test was conducted using heptane at 25℃ for 1 hour. The results of most samples were under 30 µg/mL although those of high impact polystyrene,polymethylpentene and polyvinyl chloride were found to be over 30 µg/mL. However, these results were within the acceptable range of the relaxed limits (240, 120, 150 µg/mL). Regarding the overall migration into olive oil, most plastics were under the determination limit at 60℃ for 30 min. But the results for polyethylene, polypropylene and polyvinyl chloride were over 30 µg/mL at 95 and 121℃, which were higher than their evaporation residues. In other words, the existing test conditions and limits of the evaporation residue test could be used for testing plastics that come into contact with oils and fatty foods at lower temperatures. However, they are not adequate for evaluating some plastics that come into contact with oils and fatty foods at higher temperatures.

[Establishment of Test Methods for Overall Migration Test into Vegetable Oil Applied to Food Contact Materials Intended for Contact with Oils and Fatty Foods].

The evaporation residue test designated in the Japanese Food Sanitation Act is used to determine the total migration amount of substances that food contact materials release into foods. Vegetable oil would be the most suitable food simulant for oils and fatty foods, but it is difficult to remove by heating due to its high boiling point, so heptane and 20％ ethanol are used as substitute food simulants in the test for plastics and rubbers. The EU has introduced an overall migration test into olive oil for plastics intended to come into contact with oils and fatty foods. This test method is described in European Standard EN1186-2. However, this method has several problems. Therefore, we improved the procedures for weighing samples in a desiccator containing 43％ sulfuric acid, extraction of the absorbed vegetable oil using the soaking method with an internal standard, methyl esterification of vegetable oil using sodium methoxide, and the GC conditions for the determination of vegetable oil. The improved method is simpler and much quicker than the original method, and the harmfulness of the reagents is reduced. It can be applied to both plastics and rubbers. Comparative trials showed that the results obtained with this method and EN1186-2 method are equivalent.

Label-free enrichment of human pluripotent stem cell-derived early retinal progenitor cells for cell-based regenerative therapies.

Pluripotent stem cell-based therapy for retinal degenerative diseases is a promising approach to restoring visual function. A clinical study using retinal organoid (RO) sheets was recently conducted in patients with retinitis pigmentosa. However, the graft preparation currently requires advanced skills to identify and excise suitable segments from the transplantable area of the limited number of suitable ROs. This remains a challenge for consistent clinical implementations. Herein, we enabled the enrichment of wild-type (non-reporter) retinal progenitor cells (RPCs) from dissociated ROs using a label-free ghost cytometry (LF-GC)-based sorting system, where a machine-based classifier was trained in advance with another RPC reporter line. The sorted cells reproducibly formed retinal spheroids large enough for transplantation and developed mature photoreceptors in the retinal degeneration rats. This method of enriching early RPCs with no specific surface antigens and without any reporters or chemical labeling is promising for robust preparation of graft tissues during cell-based therapy.

Pooled CRISPR screening of high-content cellular phenotypes using ghost cytometry.

Recent advancements in image-based pooled CRISPR screening have facilitated the mapping of diverse genotype-phenotype associations within mammalian cells. However, the rapid enrichment of cells based on morphological information continues to pose a challenge, constraining the capacity for large-scale gene perturbation screening across diverse high-content cellular phenotypes. In this study, we demonstrate the applicability of multimodal ghost cytometry-based cell sorting, including both fluorescent and label-free high-content phenotypes, for rapid pooled CRISPR screening within vast cell populations. Using the high-content cell sorter operating in fluorescence mode, we successfully executed kinase-specific CRISPR screening targeting genes influencing the nuclear translocation of RelA. Furthermore, using the multiparametric, label-free mode, we performed large-scale screening to identify genes involved in macrophage polarization. Notably, the label-free platform can enrich target phenotypes without requiring invasive staining, preserving untouched cells for downstream assays and expanding the potential for screening cellular phenotypes even when suitable markers are absent.

Absolute quantitation of stevioside and rebaudioside A in commercial standards by quantitative NMR.

The extract prepared from the leaves of Stevia rebaudiana BERTONI (Asteraceae) contains sweet steviol glycosides, mainly stevioside and rebaudioside A. Highly purified stevia extracts have become popular worldwide as a natural, low-calorie sweetener. They contain various types of steviol glycosides, and their main components are stevioside and rebaudioside A. The content of each steviol glycoside is quantified by comparing the ratios of the molecular weights and the chromatographic peak areas of the samples to those of stevioside or rebaudioside A standards of the Food and Agriculture Organization of the United Nations (FAO)/World Health Organization (WHO) Joint Expert Committee on Food Additives (JECFA) and other specifications. However, various commercial standard reagents of stevioside and rebaudioside A are available. Their purities are different and their exact purities are not indicated. Therefore, the measured values of stevioside and rebaudioside A contained in a sample vary according to the standard used for the quantification. In this study, we utilized an accurate method, quantitative NMR (qNMR), for determining the contents of stevioside and rebaudioside A in standards, with traceability to the International System of Units (SI units). The purities of several commercial standards were determined to confirm their actual values.

Thermal stability and surface passivation of Ge nanowires coated by epitaxial SiGe shells.

Epitaxial growth of a highly strained, coherent SiGe alloy shell around a Ge nanowire core is investigated as a method to achieve surface passivation and carrier confinement, important in realizing nanowire devices. The high photoluminescence intensity observed from the core-shell nanowires with spectral features similar to that of bulk Ge indicates effective surface passivation. Thermal stability of these core-shell heterostructures has been systematically investigated, with a method demonstrated to avoid misfit strain relaxation during postgrowth annealing.

Cultural Tightness-Looseness: How Was It Related to Vaccination Behavior among University Students in Japan, the United States, and India?

As a next step to better understand the role of cultural tightness-looseness (CTL), this study aimed to examine whether CTL is associated with COVID-19 vaccination behavior among university students, taking into consideration sociocultural perceptions of vaccination across countries. A global online survey was conducted. University students from Japan, the US, and India participated. The average CTL score, three sociocultural perceptions related to COVID-19 vaccination, side effects, infection experience of themselves and family members, and other demographic variables were used to identify the model and to explain the second vaccination status using stepwise logistic regression methods with Akaike Information Criterion (AIC) scores which was for both the total, with the country as a variable, and for each country. Analyses of data from 1289 respondents who received the first vaccine revealed the essential role of CTL in individuals getting the second vaccine, while also revealing differences between countries. Regardless of the limitations, this study adds knowledge about CTL's roles in the COVID-19 vaccination behavior among young generations and provides insights into public health communication practices for issues like COVID-19.

Sex-related perceptions associated with sexual activity status among Japanese adolescents who heavily use text messaging.

This study examines the relationship between sex-related perceptions and engagement in sexual intercourse among adolescents in Japan who were heavy users of text massaging. Using the data from the 6th National Survey on Youth Sexual Behavior of 548 high school students who heavily use text messaging, multinomial logistic regression analyses on variables constructing sexual norms and gender-role attitudes were conducted to assess the relationship with sexual activity status as the first step. A backward stepwise elimination method of multinomial logistic regression was used as the second step at which variables for each set of two factors were tested, and as the third step at which variables of two factors were simultaneously tested. The study results showed that perceptions were related to engagement in sexual intercourse among adolescents who heavily used text messaging. In particular, those who perceived that sex is an act to be engaged in at an earlier stage of a relationship and that men have a stronger sex drive tended to be sexually active or have experienced sexual intercourse. These findings could be utilized to design more effective sexual health education messages for Japanese adolescents who are at an elevated risk.

[Improved methodology for quantitative determination of thiabendazole].

The method prescribed in the 8th edition of Japan's Specifications and Standards for Food Additives (JSSFA) for the quantitative analysis of thiabendazole was improved by eliminating the use of toxic reagents such as mercuric acetate and chromium trioxide. For exclusion of mercuric acetate, a nonaqueous titration was performed using four types of solvent systems, including acetic acid:acetic anhydride (1:5), acetic acid:acetic anhydride (3:7), acetic acid alone, and formic acid:acetic acid (1:10), that did not contain mercuric compounds. Because precipitates were formed in titrations using acetic acid alone and formic acid:acetic acid (1:10), we considered that it was difficult to determine the purity using these solvent systems. However, it was confirmed that the purity of thiabendazole dissolved in the two acetic acid:acetic anhydride solvent systems can be determined using either a visual indicator or potentiometry. Specifically, the purity of thiabendazole was determined to be 99.9% (relative standard deviation (RSD) = 0.07%) for acetic acid:acetic anhydride (1:5) and 99.7% (RSD = 0.13%) for acetic acid:acetic anhydride (3:7) With respect to chromium trioxide, it was determined that chromium trioxide can be excluded using acetic acid, which conforms to the JIS K8001 standard for nonaqueous titrations. Therefore, in this study, an improved method for the quantitative determination of thiabendazole was developed without the use of toxic reagents.

[Study of purity tests for silicone resins].

In the 8th edition of Japan's Specifications and Standards for Food Additives, the purity test for silicone resins requires the determination of the refractive index and kinetic viscosity of the extracted silicone oil, and allows for only a limited amount of silicon dioxide. In the purity test, carbon tetrachloride is used to separate the silicone oil and silicon dioxide. To exclude carbon tetrachloride, methods were developed for separating the silicone oil and silicon dioxide from silicone resin, which use hexane and 10% n-dodecylbenzenesulfonic acid in hexane. For silicone oil, the measured refractive index and kinetic viscosity of the silicone oil obtained from the hexane extract were shown to be equivalent to those of the intact silicone oil. In regard to silicon dioxide, it was confirmed that, following the separation with 10% n-dodecylbenzenesulfonic acid in hexane, the level of silicon dioxide in silicone resin can be accurately determined. Therefore, in this study, we developed a method for testing the purity of silicone resins without the use of carbon tetrachloride, which is a harmful reagent.

Tigloylshikonin, a new minor Shikonin derivative, from the roots and the commercial root extract of lithospermum erythrorhizon.

Tigloylshikonin, a new shikonin derivative esterified with tiglic acid ((E)-2-methylbut-2-enoic acid), was isolated as a minor pigment from a food colorant "Shikon color," a commercial root extract from Lithospermum erythrorhizon SIEBOLD et ZUCCARINI. The structure of tigloylshikonin was elucidated using (1)H, (13)C, the difference nuclear Overhauser effect (NOE), and 2D NMR techniques. Its stereochemistry was determined by chiral-phase HPLC analysis. Tigloylshikonin was also found in the roots of L. erythrorhizon, which indicated that this new shikonin derivative is a typical component of naphthoquinone pigments in the roots of L. erythrorhizon.

[Analytical method for natamycin in wine using high-performance liquid chromatography].

An analytical method was developed for determining amount of natamycin in wine using a C18 minicartridge column and high-performance liquid chromatography (HPLC) with photodiode array (PDA) detection. Natamycin purified from wine was identified in accordance with the retention time and UV spectrum obtained from PDA detection. The limit of quantification of natamycin in wine was estimated as 0.05 microg/ml. Recovery of natamycin in wine was acceptable at 91.0% with low relative standard deviation (2.3%).

In silico-labeled ghost cytometry.

Characterization and isolation of a large population of cells are indispensable procedures in biological sciences. Flow cytometry is one of the standards that offers a method to characterize and isolate cells at high throughput. When performing flow cytometry, cells are molecularly stained with fluorescent labels to adopt biomolecular specificity which is essential for characterizing cells. However, molecular staining is costly and its chemical toxicity can cause side effects to the cells which becomes a critical issue when the cells are used downstream as medical products or for further analysis. Here, we introduce a high-throughput stain-free flow cytometry called in silico-labeled ghost cytometry which characterizes and sorts cells using machine-predicted labels. Instead of detecting molecular stains, we use machine learning to derive the molecular labels from compressive data obtained with diffractive and scattering imaging methods. By directly using the compressive 'imaging' data, our system can accurately assign the designated label to each cell in real time and perform sorting based on this judgment. With this method, we were able to distinguish different cell states, cell types derived from human induced pluripotent stem (iPS) cells, and subtypes of peripheral white blood cells using only stain-free modalities. Our method will find applications in cell manufacturing for regenerative medicine as well as in cell-based medical diagnostic assays in which fluorescence labeling of the cells is undesirable.

Analysis of acrylonitrile, 1,3-butadiene, and related compounds in acrylonitrile-butadiene-styrene copolymers for kitchen utensils and children's toys by headspace gas chromatography/mass spectrometry.

A headspace gas chromatography/mass spectrometry method was developed for the simultaneous determination of the residual levels of acrylonitrile (AN), 1,3-butadiene (1,3-BD), and their related compounds containing propionitrile (PN) and 4-vinyl-1-cyclohexene (4-VC) in acrylonitrile-butadiene-styrene (ABS) copolymers for kitchen utensils and children's toys. A sample was cut into small pieces, then N,N-dimethylacetamide and an internal standard were added in a sealed headspace vial. The vial was incubated for 1 h at 90 degrees C and the headspace gas was analyzed by gas chromatography/mass spectrometry. The recovery rates of the analytes were 93.3-101.8% and the coefficients of variation were 0.3-6.5%. In ABS copolymers, the levels were 0.3-50.4 microg/g for AN, ND-4.5 microg/g for PN, 0.06-1.58 microg/g for 1,3-BD, and 1.1-295 microg/g for 4-VC. The highest level was found for 4-VC, which is a dimer of 1,3-BD, and the next highest was for AN, which is one of the monomers of the ABS copolymer. Furthermore, the method was also applied to acrylonitrile-styrene (AS) copolymers and polystyrenes (PS) for kitchen utensils, and nitrile-butadiene rubber (NBR) gloves. In AS copolymers, AN and PN were detected at 16.8-54.5 and 0.8-6.9 microg/g, respectively. On the other hand, the levels in PS and NBR samples were all low.

[Endocrine disruptors in food contact articles and baby toys with their transition].

A number of endocrine disruptors have been reported in food contact articles and baby toys mainly during the second half of the 1990s. Bisphenol A, nonylphenol, phthalates, styrene dimers and trimers, and their transision are described in this article. Bisphenol A was found in polycarbonate tableware, nursing bottles and the epoxy resin coating of cans, therefore, it was also found in the canned foods and drinks. Polycarbonate is now only slightly used for tableware or nursing bottles in Japan because consumers refused them. The can manufacturers changed their coating material to the low bisphenol A resin or PET films and voluntarily regulate its migration limit to under 5 or 10 ng/ml. Nonylphenol was found in most PVC wrapping films and gloves. It was generated from an oxidant, tris (nonylphenyl) phosphite. PVC wrapping film was extensively used in markets, thus many kinds of foods were contaminated. Among them, fillet or minced fish and meat contained it at high levels. In 2000, manufacturers voluntarily changed their composition and all wrapping films in Japan no longer contain nonylphenol. Di(2-ethylhexyl) phthalate (DEHP) was found in PVC gloves, which contaminated packed lunches and hospital meals at high levels. The government prohibited these gloves for all food contact use in 2000, moreover, other PVC food contact articles containing DEHP were prohibited for contact use with fatty foods in 2002. DEHP was also found in PVC toys which was prohibited in 2002. Styrene dimers and trimers were found in PS products, which migrated into cupped noodles after cooking. No changes have been made in them. In Japan, the exposure to bisphenol A, nonylphenol and DEHP have been significantly reduced and people also have more concerns with the safety of food contact articles.