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.

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.

[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.

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.

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.

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.

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.

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.

[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.

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.

[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.

Ghost cytometry.

Ghost imaging is a technique used to produce an object's image without using a spatially resolving detector. Here we develop a technique we term "ghost cytometry," an image-free ultrafast fluorescence "imaging" cytometry based on a single-pixel detector. Spatial information obtained from the motion of cells relative to a static randomly patterned optical structure is compressively converted into signals that arrive sequentially at a single-pixel detector. Combinatorial use of the temporal waveform with the intensity distribution of the random pattern allows us to computationally reconstruct cell morphology. More importantly, we show that applying machine-learning methods directly on the compressed waveforms without image reconstruction enables efficient image-free morphology-based cytometry. Despite a compact and inexpensive instrumentation, image-free ghost cytometry achieves accurate and high-throughput cell classification and selective sorting on the basis of cell morphology without a specific biomarker, both of which have been challenging to accomplish using conventional flow cytometers.

Migration of nonylphenol and plasticizers from polyvinyl chloride stretch film into food simulants, rapeseed oil, and foods.

Nonylphenol (NP) has been suspected as an endocrine-disrupting chemical. Japanese polyvinyl chloride (PVC) stretch films contained 0.5-3.3 mg/g of NP and 100-400 mg/g of plasticizers such as diisononyl adipate (DINA), di-n-alkyl adipate (DAA), and diacetyllauroyl glycerol (DALG). Migration of NP and plasticizers from PVC stretch films into food simulants (water; 4% acetic acid; 20%, 50%, and 95% ethanol; and heptane), rapeseed oil, and foods was investigated. Plasticizers migrated only in small amounts into aqueous simulants and foods, although they migrated at much higher levels into 50% and 95% ethanol, heptane, rapeseed oil, and fatty foods, whereas NP more easily migrated into aqueous simulants and foods. At 5°C for 24 hr, migration of NP into vegetable and fruit was 2.9%-6.4% of their contents, and that of DINA and DAA was 0.1%-0.3%. The migration ratios of NP into aqueous foods were much higher than those of DINA and DAA. The migration ratio of NP into fatty foods, such as minced tuna and pork, was 33% and 24%, which was almost similar to that of DINA and DAA. The estimated daily intakes of NP and DINA for Japanese individuals of those days were 35 and 1,050 µg, respectively, and should not be associated with any safety concerns.

Contents of Ag and other metals in food-contact plastics with nanosilver or Ag ion and their migration into food simulants.

Six nanosilver-labelled products and five silver ion (Ag(+))-labelled products were investigated to measure the migration of Ag from food-contact plastics, including nanosilver into various food simulants. The products were obtained in Japanese markets in 2012. Zinc (Zn), another major antimicrobial agent, and three harmful metals, cadmium (Cd), lead (Pb) and arsenic (As), were also examined. Ag and Zn were detected in all six nanosilver products at concentrations of 21-200 and 8.4-140 mg kg(-1), respectively. These metals were also detected in all five Ag(+) products at the same level as nanosilver products. Cd, Pb and As were not detected in any sample. Migrations of Ag and Zn were highest in 4% acetic acid, but also observed in water and 20% ethanol. Big differences were not observed in the migration ratio between nanosilver products and Ag(+) products. The ultrafiltration experiments suggested that the Ag that migrated from nanosilver products into 4% acetic acid was in its ionic form, while that into water and 20% ethanol was in its nanoparticle form.

Isolation and Quantification of Polyamide Cyclic Oligomers in Kitchen Utensils and Their Migration into Various Food Simulants.

Small amounts of cyclic monomers and oligomers are present in polyamide (PA)-based kitchen utensils. In this study, we isolated eight PA-based cyclic monomers and oligomers from kitchen utensils made from PA6 (a polymer of ε-caprolactam) and PA66 (a polymer of 1,6-diaminohexane and adipic acid). Their structures were identified using high-resolution mass spectrometry and 1H- and 13C-nuclear magnetic resonance spectroscopy, and their residual levels in PA-based kitchen utensils and degree of migration into food simulants were quantified by high-performance liquid chromatography/mass spectrometry using purchased PA6 monomer and isolated PA66 monomers, and isolated PA6 and PA66 oligomers as calibration standards. Their total residual levels among 23 PA-based kitchen utensils made from PA6, PA66, and copolymers of PA6 and PA66 (PA6/66) ranged from 7.8 to 20 mg/g. Using water, 20% ethanol, and olive oil as food simulants, the total migration levels of the PA monomers and oligomers ranged from 0.66 to 100 µg/cm2 under most examined conditions. However, the total migration levels of the PA66 monomer and oligomers from PA66 and PA6/66 kitchen utensils into 20% ethanol at 95°C were very high (1,700 and 2,200 µg/cm2, respectively) due to swelling by high-temperature ethanol.

Survey of Primary Aromatic Amines and Colorants in Polyurethane, Nylon and Textile Toys.

The residual and migration levels of 28 primary aromatic amines (PAAs) in polyurethane and nylon toys were determined using LC-MS/MS, and the migration and residual amounts of PAAs and 15 colorants in textile toys were determined using LC-MS/MS and LC-TOF-MS according to the European Standard EN71. Among 34 polyurethane toy samples, 2,6-diaminotoluene and 2,4-diaminotoluene were detected in the same 12 samples at residual levels ranging from 2.1 to 19.7 and from 7.6 to 39.6 µg/g, respectively. Furthermore, 4,4'-diaminodiphenylmethane (4,4'-MDA) and aniline were detected in 9 samples (from 0.2 to 8.7 µg/g), and one sample (0.4 µg/g), respectively. PAAs were not detected in the 8 samples of nylon toys. As for the migration test into water, only 4,4'-MDA migrated from 3 polyurethane toys at levels ranging from 0.4 to 2.5 µg/g. PAAs did not migrate from the 43 textile toys, but colorants such as Solvent Yellow 1 and Basic Red 9 were detected at the residual level of 0.02 µg/g. The residual levels of PAAs and colorants detected in this study were significantly lower than the limit values established by the European Union regulation.

[Validation Study on Headspace-GC Analytical Method for Residual Volatile Substances in Food Contact Polystyrene and Its Application for Surveillance (1998-2014)].

A headspace-GC analysis method for the determination of residual volatile substances (styrene, toluene, ethylbenzene, isopropylbenzene and propylbenzene) in food contact polystyrene (PS) was evaluated. Ten PS products were analyzed by this headspace-GC method and the Japanese official method, and the values obtained were almost equal. The performance of the method was evaluated, and the trueness, repeatability and reproducibility were 100.4-102.8%, 3.7-6.3% and 6.0-11.1%, respectively. The values of the performance parameters of the headspace-GC method fulfilled the requirements, and this method was confirmed to be extremely precise. Moreover, contamination of the GC equipment was minimized. The residual volatile substances in 58 PS products were surveyed with this method. All products met the specifications defined in the Japanese Food Sanitation Law, and no relationship was found between volatile substances and the sampling year or country of origin.

[Interlaboratory study on migration test of cadmium and lead for food contact articles].

An interlaboratory study was performed to evaluate a migration test method of cadmium (Cd) and lead (Pb), based on the Japanese Food Sanitation Law for glassware, ceramicware, enamelware and metal cans. Seventeen laboratories participated, and quantified Cd and Pb in eight test solutions as blind duplicates using flame atomic absorption spectrometry (AAS), graphite furnace atomic absorption spectrometry (GF-AAS), inductively coupled plasma-optical emission spectrometry (ICP-OES) or induced coupled plasma-mass spectrometry (ICP-MS). Statistical analysis revealed that the trueness, repeatability (RSDr) and reproducibility (RSDr) were 93-105%, 0.7-8.4% and 2.6-19.3% by using AAS, ICP-OES and ICP-MS (internal standard method). The performance of these methods is sufficient for testing specifications. However, some of the RSDr values exceeded 10% in GF-AAS, and careful control of accuracy is required.

Survey of volatile substances in kitchen utensils made from acrylonitrile-butadiene-styrene and acrylonitrile-styrene resin in Japan.

Residual levels of 14 volatile substances, including 1,3-butadiene, acrylonitrile, benzene, ethylbenzene, and styrene, in 30 kitchen utensils made from acrylonitrile-butadiene-styrene resin (ABS) and acrylonitrile-styrene resin (AS) such as slicers, picks, cups, and lunch boxes in Japan were simultaneously determined using headspace gas chromatography/mass spectroscopy (HS-GC/MS). The maximum residual levels in the ABS and AS samples were found to be 2000 and 2800 µg/g of styrene, respectively. The residual levels of 1,3-butadiene ranged from 0.06 to 1.7 µg/g in ABS, and three of 15 ABS samples exceeded the regulatory limit for this compound as established by the European Union (EU). The residual levels of acrylonitrile ranged from 0.15 to 20 µg/g in ABS and from 19 to 180 µg/g in AS. The levels of this substance in seven ABS and six AS samples exceeded the limit set by the U.S. Food and Drug Administration (FDA). Furthermore, the levels of acrylonitrile in three AS samples exceeded the voluntary standard established by Japanese industries. These results clearly indicate that the residual levels of some volatile compounds are still high in ABS and AS kitchen utensils and further observations are needed.

Quantification of isocyanates and amines in polyurethane foams and coated products by liquid chromatography-tandem mass spectrometry.

An analytical method for the identification and quantification of 10 different isocyanates and 11 different amines in polyurethane (PUR) foam and PUR-coated products was developed and optimized. Isocyanates were extracted and derivatized with di-n-butylamine, while amines were extracted with methanol. Quantification was subsequently performed by liquid chromatography-tandem mass spectrometry. Using this methodology, residual levels of isocyanates and amines in commercial PUR products were quantified. Although the recoveries of certain isocyanates and amines were low, the main compounds used as monomers in the production of PUR products, and their decomposition species, were clearly identified at quantifiable levels. 2,4-and 2,6-toluenediisocyanate were detected in most PUR foam samples and a pastry bag in the range of 0.02-0.92 mg/kg, with their decomposition compounds, 2,4-and 2,6-toluenediamine, detected in all PUR foam samples in the range of 9.5-59 mg/kg. PUR-coated gloves are manufactured using 4,4'-methylenebisphenyl diisocyanate as the main raw material, and a large amount of this compound, in addition to 4,4'-methylenedianiline and dicyclohexylmethane-4,4'-diamine were found in these samples.