Parental experience of initiation of alcohol drinking has been identified as one of the early causes of alcohol drinking in preadolescents in many countries, including Japan. This study identified the association between parental alcohol-related knowledge and the initiation of alcohol use among preadolescent students in an urban area in Japan. Self-administrated questionnaires were distributed to 420 parents of kindergarteners and elementary school students, of which 339 were filled and returned (response rate: 81%). The parents’ experience in initiating alcohol drinking in their children and their knowledge about the effects of alcohol on youth were explored. The requirements for drinking prevention programs for youth were also investigated. The result showed that a significantly higher proportion of parents of elementary school students had experiences of initiating alcohol use in their children compared to parents of kindergarten children. The parents’ knowledge regarding the effects of alcohol on youth showed no significant difference between the two parent groups. These data indicate that the age of children is the only factor as opposed to parents’ knowledge. We also found significant differences in the requirements of prevention programs between the two parent groups. The results of this study can contribute to the design of alcohol prevention programs for these parents, which could reduce the onset of children’s drinking. Underage alcohol use is a serious problem associated with various health issues ,2 and prAlcohol misuse negatively affects young people and has become one of the leading causes of premature death . The preGiven the negative health effects of alcohol drinking at younger ages, a large number of studies have been conducted on alcohol-related drinking among adolescents. For instance, several studies have sought to find out behavioral problems associated with the early onset of drinking such as whether students who began drinking alcohol at early adolescence frequently engaged in delinquency in later adolescence . In addiParental factors have repeatedly been identified as one of the risk factors for students’ alcohol use (reviewed in ). The poAlthough both preadolescent prevention efforts and parental education seem important for reducing adolescent drinking, there is no available study for such interventions in Japan. Based on the frequently reported association between parental behavior and children’s alcohol drinking, we hypothesized that alcohol supply by parents would initiate alcohol drinking in children. We also analyzed the relationship between parents’ knowledge of alcohol-related problems on youth and their alcohol supply. n = 172 parents of kindergarteners and 167 parents of elementary students) returned the filled and completed questionnaires (response rate: 81%). The completed questionnaires were placed in an envelope to ensure anonymity along with parents’ written informed consent, and then parents were required to post them to boxes at these schools. The questionnaire sheet and written informed consent were anonymized. The data manager separated them and the data analyst analyzed the anonymized data. The study was approved by the Institutional Review Board of Yokohama Soei University (No. 27-010).A total of 420 parents of children in one elementary school (6–12 years old) and three kindergartens (5–6 years old) in Yokohama, Japan, an urban area neighboring the Tokyo metropolitan area, were recruited to participate in the study. The parents of the students in the four schools were notified about the study through the teachers. To ensure anonymity and security, no online tools were used in this study. Thereafter, a paper-based self-administered questionnaire was delivered to the 420 parents of the students. Of this figure, 339 parents  and whether their children are in elementary school or kindergarten. The parents were asked a binary variable (yes/no) question concerning their provision of alcoholic beverages to their children. Since underage alcohol drinking is commonly known as illegal in Japan, to prevent parents’ concerns about disclosing this information, we did not provide the details regarding this question; that is, the information concerning the frequency and volume of alcohol beverages supplied to children was not included. The answers that enable us to identify the responder were also eliminated, as well as any other type of identifying information. Parents’ knowledge of the health effects of alcohol drinking on youth was determined using nine items. These items were selected from previous studies ,32. PartParents’ perceived requirements for alcohol prevention programs for elementary students were assessed using six items selected from a previous survey conducted in Japan . Again, p-values were adjusted by false discovery rate  to yield Q-values, considering the multiple independent tests. P-values were adjusted among the items of parents’ knowledge of the health effects of alcohol drinking and the items of requirements for the alcohol drinking prevention programs, respectively. All analyses were conducted using GraphPad Prism .We examined differences between the parents of elementary school students and kindergarten students on their knowledge of the effects of alcohol on youths and their perceived requirements for the alcohol prevention program. The chi-square test was used to assess age differences, and Fisher’s exact test was used for all other items, including responses for the main variables. Logistic regression analysis was used to calculate odds ratios and 95% confidential intervals (CIs) of odds ratios (OR) for all variables for parents of elementary school students compared to the parents of kindergarteners. p = 0.93) between the groups. There was a significant age difference  between the groups: mode—the largest proportion of parents of kindergarteners and elementary school students was in their 30s  and 40s , respectively; thus, the latter group was generally older, as expected.n = 161, 96.4%) had such an experience. This difference was significant (p < 0.0001).n = 164, 94.2%) and parents of elementary school students : 0.26–1.9). More than 80% of both groups reported knowledge of “(7) Excessive drinking sometimes induces death”  and “(8) Drinking is addictive,” , while less than 50% of these parents knew that “(4) Drinking induces deterioration of the stomach lining”  and “(6) Drinking induces pancreatic disease such as pancreatitis and diabetes” .We observed no significant group differences in parents’ responses to the nine items evaluating their knowledge of the health effects of alcohol drinking on youth. The odds ratios (ORs) ranged from 0.80 to 1.6. Among the nine items, “(2) Large quantity of drinking induces acute alcoholism” garnered the highest responses among both parents of kindergarteners , “(3) Lectures on laws regarding youth drinking” , “(4) Presentations by alcoholics on their alcoholism experiences” , and “(6) Establishment of a consultation center for drinking” . All these items showed higher demand by the parents of kindergarten children .As for the six items concerning parents’ requirements for drinking prevention programs, we observed significant differences in four items between the parent groups: “(1) Lectures on effects of drinking on youth health” (p = 0.86), but there was a significant age difference . Among parents who did not have the initiating experience, those in their 30s were the dominant age group , while among those with such experience, those in their 40s were the dominant group. We observed no significant difference among the nine knowledge items. As for the requirements of a drinking prevention program, “(3) Prognosis of students with alcohol addiction” (p < 0.0001) showed the greatest difference; items (1), (4), and (6) also showed significant differences (p < 0.05 and Q < 0.05).Here, we analyzed the drinking initiation induced by parents for elementary school children and kindergarten and the association of this initiation with parental knowledge regarding youth alcohol drinking. One of the difficulties in this line of research is assuring the validity of data collected from such young students. Furthermore, research on adolescents has shown that parents are major alcohol suppliers . Given tThe most profound difference between the parents of elementary school students and kindergarteners was their experience of initiating their children’s drinking; that is, very few parents of kindergarteners had such initiation experiences (1.16%) while nearly all parents of elementary school students had such experiences (96.41%). Meanwhile, no significant differences were observed in alcohol-related knowledge. Therefore, initiation experiences seem to be more dependent on children’s age than on parents’ knowledge. These initiation experiences were pervasive regardless of elementary school students’ age, so it seems that proceeding to elementary school (5 or 6 years old) is a clear borderline for initiation experiences.A survey among Japanese students showed that students who initiated alcohol drinking and cigarette smoking at 12 years of age or less showed greater rates of dependency or addiction to these substances at 15 years of age or older . As noteIt is increasingly difficult to obtain accurate data from elementary school students and kindergarteners using self-reported questionnaires because of their limited ability to respond to the questions. Therefore, we analyzed parents’ data instead. We found that parents’ knowledge did not differ according to their experience of initiating alcohol drinking in children. One possible reason is that the data were collected in an urban area close to Tokyo, where most of the residents have similar and high levels of education and economic backgrounds. The current results might not be generalizable to the provincial area, including families with more heterogeneous backgrounds. In addition, based on the extremely high occurrence of parents of elementary school students providing alcoholic beverages to them, the parents without this experience might not be concerned with legal limitations but instead may consider the impacts of drinking on their children’s health; in other words, these parents might regard their children who grew up as elementary school students to be strong enough to resist the drinking of alcohol.Participants’ requirements for drinking prevention programs differed according to their children’s school. We might consider their required contents as parents’ demands for specific knowledge. Thus, the selected items could contribute to the design of parental education programs, which could help to reduce the initiation of children’s alcohol usage. While school programs are known to be more effective for managing problematic behaviors among students in Japan  when compared to neighborhood programs, the parents were not included in the comparisons . HoweverThis study has several limitations. First, we collected data only in urban areas of Japan, and the sample size was small. In Japan, the constitutive members of a family and cultural events, such as festivals and ceremonial occasions, differ widely between urban and rural areas, and these factors might affect the initiation of alcohol drinking. A larger sample collected from various regions of Japan  is necessary to evaluate the generalizability of our findings. Second, we hypothesized that parental knowledge would influence the initiation of alcohol drinking and administered the questionnaire only to parents of elementary school students and kindergarteners. Therefore, other factors that affect the initiation of alcohol drinking were not explored. Thirdly, all questionnaires were designed as binary questions, with no dummy or test questions. One of the major problems of this study is that parents are the source of the information even though the questions include inducing their children to illegal alcohol drinking. The quantitative value  was not determined. The different effect of parental and maternal alcohol use on children was reported; for example, paternal drinking showed a higher association of male adolescents rather than maternal drinking . That diIn conclusion, this study investigated the differences in parents’ experience of initiating alcohol use in their children. Parents of elementary school children, in general, had experience in initiating alcohol drinking in their children, whereas parents of kindergartners rarely had such an experience. We also investigated young people’s knowledge of alcohol drinking and found no significant difference between the two parent groups, suggesting that the age of the children is the only factor. The elementary school students reported a significantly greater number of requirements for drinking prevention programs. Taken together, effective education, especially for parents of elementary school children, is necessary.


Acquired glucocorticoid (GC) resistance remains the main obstacle in acute lymphoblastic leukemia (ALL) therapy. The aim of the present study was to establish a novel GC-resistant B-ALL cell line and investigate its biological characteristics.A cell culture technique was used to establish the GC-resistant cell line from the parental cell, NALM-6. Molecular and cellular biological techniques including flow cytometry, MTT assay, western blotting, DNA fingerprinting analysis and whole transcriptome sequencing (WTS) were used to characterize the GC-resistant cell lines. Nude mice were used for xenograft studies.The GC-resistant cell line, NALM-6/HDR, was established by culturing NALM-6 cells under hypoxia for 5 weeks with a single dexamethasone (Dex) treatment. We subcloned the NALM-6/HDR cell lines, and got 6 monoclone Dex-resistant cell lines, NALM-6/HDR-C1, C3, C4, C5, C6 and C9 with resistance index (RI) ranging from 20,000–50,000. NALM-6/HDR and its monoclone cell line, NALM-6/HDR-C5, exhibited moderate (RI 5–15) to high resistance (RI > 20) to Ara-c; low or no cross-resistance to L-Asp, VCR, DNR, and MTX (RI < 5). STR analysis confirmed that NALM-6/HDR and NALM-6/H were all derived from NALM-6. All these cells derived from NALM-6 showed similar morphology, growth curves, immunophenotype, chromosomal karyotype and tumorigenicity. WTS analysis revealed that the main metabolic differences between NALM-6 or NALM-6/H (GC-sensitive) and NALM-6/HDR (GC-resistant) were lipid and carbohydrates metabolism. Western blotting analysis showed that NALM-6/HDR cells had a low expression of GR and p-GR. Moreover, AMPK, mTORC1, glycolysis and de novo fatty acid synthesis (FAS) pathway were inhibited in NALM-6/HDR when compared with NALM-6.NALM-6/HDR cell line may represent a subtype of B-ALL cells in patients who acquired GC and Ara-c resistance during the treatment. These patients may get little benefit from the available therapy target of AMPK, mTORC1, glycolysis and FAS pathway.The online version contains supplementary material available at 10.1186/s12935-021-02335-7. Although contemporary chemotherapy regimens have improved the long-term survival rate of childhood acute lymphoblastic leukemia (ALL) to nearly 90% , the outDrug-resistant cell lines that are established from drug-sensitive cell lines are the most useful models in tumor research, which have been used to investigate the mechanisms of drug resistance for nearly 50 years . TraditiHypoxia plays a central role in ALL progression and resistance to therapy . Glycoly2) and 21% oxygen .The GC-sensitive B-ALL cell line, NALM-6 was purchased from Shanghai Institute Cell Resources Bank. NALM-6/HDR and NALM-6/H were established from NALM-6 as described previously [Dexamethasone , trypan blue, methylcellulose (MethoCult GFH4434) and 3--2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma-Aldrich . The Annexin V-PI Kit was purchased from Roche . Antibodies to ATP-citrate lyase (ACLY), p-ACLY (Ser455), acetyl-CoA carboxylase (ACC), p-ACC (Ser79), fatty acid synthase (FASN), long-chain acyl-CoA synthetase 1 (ACSL1), acetyl-CoA synthetase (AceCS1), AMPK, p-AMPK (Thr172), Glut-1, HKII, LDHA, p-LDHA (Tyr10), 4E-BP1, p-4E-BP1 (Thr37/46), p70S6K, p-p70S6K (Thr389), glucocorticoid receptor (GR), and p-GR (Ser211) were obtained from Cell Signaling Technology . Horseradish peroxidase (HRP)–conjugated donkey anti-rabbit antibody and HRP-conjugated sheep anti-mouse antibodies were obtained from Santa Cruz Biotech . The β-Actin and GAPDH antibody was purchased from Kangchen Bio-Tech .2/ml in methylcellulose RPMI-1640 medium containing 0.9% methylcellulose and 10% FBS at 37 °C under a humidified atmosphere with 5% CO2 and 21% O2. Monoclonal cells were picked randomly on day 8 of the culture, and cultured in RPMI-1640 complete medium.As described previously , exponent/N0), where t is the time of continuous culture, Nt is the final number of cells, and N0 is the initial number of cells.Exponentially growing cells were cultured in a 6-well sterile plastic culture plates for seven days. The number of viable cells was counted using heamocytometer and trypan blue staining every day. Doubling time (Td) of cells was calculated according to the following formula: Td (h) = t × lg2/lg were added to each well and incubated overnight at 37 °C. Finally, the optical density was measured at 570 nm using a multiplate reader . Values were obtained by comparing the experimental cells with their respective controls. Mean values were calculated from triplicate cultures.50 was calculated by linear interpolation.The chemotherapeutic drugs, Dex, asparaginase (L-Asp), daunorubicin (DNR), vincristine (VCR), arabinoside (Ara-c), and methotrexate (MTX), were purchased from Sigma. All cells were treated with increasing concentrations of different drugs for 48 h, followed by assessment of cell viability by MTT assay. The ICCells were harvested 48 h after treatment and fixed overnight in 70% ethanol at 4 °C. The cells were then washed twice with PBS and stained with 5 μg/ml PI in the presence of DNAse-free RNAse (Sigma) for 30 min at room temperature. Then, the cell cycle distribution was analyzed by flow cytometry .Antibodies against the following targets: HLA-DR, cCD3, CD3, CD79α, CD10, CD19, CD20, CD22, CD38, and CD45  were used. Positivity for the antigens was determined using a FACSCalibur flow cytometer (Becton Dickinson Inc.).Amelogenin, CSF1PO, D13S317, D16S539, D5S818, D7S820, TH01, TPOX, vWA, Penta E, Penta D, D2S441, D2S1338, D3S1358, D6S1043, D8S1179, D10S1248, D12S391, D18S51, D19S433, D21S11 and FGA.The identity of the NALM-6, NALM-6/HDR and NALM-6/H cell lines was checked using DNA fingerprinting. DNA was prepared from these cells using the Qiagen DNeasy Blood Kit (Qiagen), according to the instructions provided by the manufacturer. The following 22 highly polymorphic short tandem repeat (STR) loci were tested by a multiplex PCR: Total RNA was extracted by Trizol reagent  according to the manufacturer's instructions. RNA quality and purity were assessed by RNA 6000 Nano assay on Agilent 2100 Bioanalyzer . RNA-sequencing libraries were acquired on the BGISEQ-500 Platform in total and generated approximately 10.98 Gb per sample. After reads filtering, clean reads were mapped to the reference genome using HISAT. Then, gene expression levels for each sample were calculated with RSEM. Based on the gene expression level, the differentially expression genes (DEGs) between samples can be identified. PossionDis algorithms were used to detect the DEGs. With DEGs, we perform Gene Ontology (GO) classification, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway classification and functional enrichment.6) were washed twice in cold PBS and then lysed by RIPA lysis buffer (Bio-Rad). Equivalent amounts of proteins were separated by 8–15% SDS–polyacrylamide gel electrophoresis and transferred to PVDF membranes . Immunoblots were assayed by using primary antibodies. HRP conjugated secondary antibodies were used for detection. The level of the β-Actin protein was used as a control for the amount of protein loaded into each lane.Proteins were visualized by incubation with ECL plus (Millipore). All experiments were conducted independently at least 3 times.Briefly, cells  nude mice , and 0.1 ml of PBS was injected into the left flanks as the control. Tumor size was measured by calipers every 2 days. The approximate tumor volume was calculated using the equation V = (length × width × width)/2. All animals were ear-tagged and monitored individually throughout the experiment. All animal care was in compliance with the guidelines established by the internal Institutional Animal Care and Use Committee and Ethics Committee of Sichuan University. Mice were euthanized by cervical dislocation after anesthetizing with a mixture of 100 mg/kg ketamine and 10 mg/kg xylazine via intraperitoneal injection. Then, the tumor mass was excised, fixed in 10% formalin, and routinely processed for paraffin embedding. Five-millimeter-thick sections were obtained and prepared for standard histopathological examination.Cultured 5 × 10p-value < 0.05 was considered to be significant.All assays were performed in triplicate, and data are expressed as mean values ± SD. One-way ANOVA was used to compare two groups. A 50 and RI of Dex in the NALM-6/HDR cells were 55–70 µM and 25,000–30,000, respectively [50 and RI ranging from 50 to 125 µM and 25,000–50,000, NALM-6/HDR-C5 cells were the most resistant cells to Dex among the monoclonal cell lines  , are recognized as one of the key hallmarks in several cancer cells , 12. BesHypoxia may induce a metabolic switch in energy production, which is characterized by an increase in glycolysis phenotype in cancer cells . We geneLoss of GR expression plays a key role in GC resistance and is associated with poor prognosis in leukemia patients , 16. As 3 (n = 4) and 346.9 ± 102.7 mm3 (n = 3), respectively . After 40 days, the mean volume of the tumors generated by subcutaneous injection with NALM-6 and NALM-6/HDR were 413.2 ± 140.0 mmOver the last century, studies on patient-derived cancer cell lines have provided crucial insights into the biological processes in tumorigenesis and high-throughput screening for drug development . MoleculGCs specifically induce apoptosis in malignant lymphoblasts and are thus pivotal in the treatment of lymphoid malignancies, especially in ALL . However2+ signaling, which cause the metabolic reprogramming, with an enhanced level of glycolysis and oxidative phosphorylation, apoptosis resistance, and multidrug resistance [However, the mechanism of GC resistance is much more complicated than a GR alternation. The level of the GRs expression among ALL patients and derived cell lines appears to be highly heterogeneous . There asistance . Accordisistance , 29, 30.sistance . Leukemisistance . SimilarCancer cells reprogram lipid metabolism during their malignant progression . Enzymes or in vivo. NALM-6/H showed an increased Glut-1, LDHA and p-LDHA. These results suggest that AMPK and glycolysis pathway may not be a therapeutic target in GC resistant NALM-6/HDR. A recent paper reported that cancer cells preferred to uptake glutamine while myeloid cells in tumor microenvironment (TME) took up the most glucose [Reprogramming of carbohydrate metabolism, particularly increased glycolysis, has been recognized as a hallmark of cancer  and play glucose . Therefo glucose . A progr glucose . We obse glucose . Initial glucose , 23, 54. glucose . It is i glucose ; both AM glucose . TherefoHere, we constructed a new GC-resistant B-ALL cell line by mimicking the clinical setting in vivo. Compared with its parental cell line, NALM-6, the novel cell line, NALM-6/HDR, has a low expression of GR, a common feature of GC-resistant ALL cells. Moreover, NALM-6/HDR has some distinct characters, including depression in AMPK, mTORC1, glycolysis and FAS pathway, which may hint that the previous therapeutic strategies, such as targeting AMPK, mTORC1, glycolysis and FAS pathway, could not reverse the GC resistant, or even induce some subtype of ALL aggravation. In addition, NALM-6/HDR got cross-resistance to Ara-C. Therefore, NALM-6/HDR may represent a novel cell type in B-ALL patients who acquired GC and Ara-c resistance during the treatment. NALM-6/HDR may serve as valuable in vitro and in vivo tools for further investigation on the potential mechanisms and therapeutic targets of relapsed/refractory B-ALL, especially the roles of the hypoxic TME in GC resistance.Additional file 1: Fig. S1. Immunophenotypic characteristics of NALM-6/HDR cell line. The immunophenotype was analyzed using a FACSCalibur flow cytometer. NALM-6/HDR and NALM-6 displayed identical immunophenotype with CD10, CD19, CD22, cCD79α and HLA-DR positive.Additional file 2: Fig. S2. Venn diagram and DEGs analysis on NALM-6, NALM-6/H, and NALM-6/HDR. a Venn diagram was used to display expressed gene between samples. b Based on the gene expression level, PossionDIS algorithms was used to detect the DEGs. X axis represented the sample. Y axis represented the DEGs.Additional file 3: Fig. S3. Heatmap of DEGs and the DEGs in metabolism pathway between NALM-6 and NALM-6/HDR cells. a X axis represented the sample, NALM-6 and NALM-6/HDR. Y axis represented the DEGs. The color represents the log10 transformed gene expression level. The dark color means the high expression level while the light color means the low expression level. b The metabolic functional enrichment results of NALM-6 compared with NALM-6/HDR after hierarchical clustering were shown in the table.


YouTube (Alphabet Incorporated) has become the most popular video-sharing platform in the world. The Fukushima Daiichi Nuclear Power Plant (FDNPP) disaster resulted in public anxiety toward nuclear power and radiation worldwide. YouTube is an important source of information about the FDNPP disaster for the world.This study's objectives were to examine the characteristics of YouTube videos related to the FDNPP disaster, analyze the content and comments of videos with a quantitative method, and determine which features contribute to making a video popular with audiences. This study is the first to examine FDNPP disaster–related videos on YouTube.We searched for the term “Fukushima nuclear disaster” on YouTube on November 2, 2019. The first 60 eligible videos in the relevance, upload date, view count, and rating categories were recorded. Videos that were irrelevant, were non-English, had inappropriate words, were machine synthesized, and were <3 minutes long were excluded. In total, 111 videos met the inclusion criteria. Parameters of the videos, including the number of subscribers, length, the number of days since the video was uploaded, region, video popularity , the tone of the videos, the top ten comments, affiliation, whether Japanese people participated in the video, whether the video recorder visited Fukushima, whether the video contained theoretical knowledge, and whether the video contained information about the recent situation in Fukushima, were recorded. By using criteria for content and technical design, two evaluators scored videos and grouped them into the useful (score: 11-14), slightly useful (score: 6-10), and useless (score: 0-5) video categories.P<.001), likes per day (P<.001), and comments per day (P=.02) than useless and slightly useful videos. The popularity of videos had a significant correlation with clear sounds , vivid information , understanding content . There was no significant difference in likes per day (P=.72) and comments per day (P=.11) between negative and neutral- and mixed-tone videos. Videos about the recent situation in Fukushima had more likes and comments per day. Video recorders who personally visited Fukushima Prefecture had more subscribers and received more views and likes.Of the 111 videos, 43 (38.7%) videos were useful, 43 (38.7%) were slightly useful, and 25 (22.5%) were useless. Useful videos had good visual and aural effects, provided vivid information on the Fukushima disaster, and had a mean score of 12 (SD 0.9). Useful videos had more views per day (The possible features that made videos popular to the public included video quality, videos made in Fukushima, and information on the recent situation in Fukushima. During risk communication on new forms of media, health institutes should increase publicity and be more approachable to resonate with international audiences. The Great East Japan Earthquake (magnitude 9.0) and subsequent tsunami on March 11, 2011, caused an accident at the Fukushima Daiichi Nuclear Power Plant (FDNPP). The FDNPP  is located approximately 200 km northeast of Tokyo. This disaster released massive amounts of radioactive material into the environment .Social media can be defined as interactive communication media that have been fused into human lives worldwide ,8. YouTuAnxiety toward nuclear power is an important social issue. The public lacks knowledge about radiological issues and distrusts information provided by authorities . They neThis study aimed to examine the characteristics of YouTube videos related to the FDNPP disaster, analyze the content and comments of videos with a quantitative method, and determine which features contribute to making a video popular with audiences. On November 2, 2019, videos were identified by using the search term “Fukushima nuclear disaster” on YouTube via the Google Chrome browser. YouTube sorts videos into the following four filter categories: relevance, upload date, view count, and rating. The first 60 eligible videos in each category were recorded . Videos Characteristics of the videos, including the number of subscribers, length, the number of days since the video was uploaded, video popularity , region, and affiliation , were recorded .We divided videos into the following five types based on format parameters: news and interviews, formal presentations, informal presentations, educational videos, documentaries. The following parameters of videos were also recorded: whether Japanese people effectively participated in the video, whether the video recorder personally visited Fukushima, whether the video contained theoretical knowledge of radiation, and whether the video contained information about the recent situation in Fukushima (after January 2017).The tone of the videos was coded as neutral and mixed or negative by two independent evaluators. No videos were coded as having a positive tone. Neutral and mixed videos were those that reported news or recorded the situation in Fukushima and interviews without personal comments and judgment. Negative videos presented conspiracy theories about Japan and described serious health risks to people in the world. The top 10 comments of each video were collected and divided into the following six categories by two independent evaluators: (1) conspiracy theories ; (2) serious health risks ; (3) positive comments on the video ; (4) negative comments on the video ; (5) negative comments on official agencies ; and (6) other .Based on previous studies on educational and medical videos ,15,20-22Each item in the major criteria has two scores and in the minor criteria has one score . Videos P<.05.Data were expressed as medians, minimums, and maximums. Normality was checked by using the Kolmogorov-Smirnov test. Since the variables were not normally distributed, nonparametric statistical tests were used. A Spearman correlation analysis was conducted to determine if video popularity correlated with the key parameters and scores. The Kruskal-Wallis test and Mann-Whitney U test were used to compare continuous variables. Statistical analysis was performed by using SPSS 21.0 (IBM Corporation). The significance level was set to The descriptive information of 111 videos is summarized in P=.17; comments/day: P=.38).The format of the videos, authorship type, and the nationality of video uploaders are shown in P=.01) and laypersons (P=.03). Videos uploaded by private individuals and laypersons (P=.02), for-profit companies or organizations (P=.004), and news sources (P=.02) received more likes/day than nonprofit organizations or universities.Private individuals and laypersons posted 40/111 (36%) of the videos, news sources posted 32/111 (29%) of the videos, nonprofit organizations or universities posted 19/111 (17%) of the videos, and for-profit companies or organizations posted 17/111 (15%) of the videos. News sources and for-profit companies or organizations had more subscribers than nonprofit organizations or universities and private individuals and laypersons. In addition to news sources, organizations and individuals uploaded different types of videos, including on-the-spot interviews, press conferences, news about the accident, wastewater treatment, explanations of the principles and processes of the nuclear accident, and personal opinions. However, only videos made by for-profit companies or organizations received more views per day than those made by nonprofit organizations or universities (P=.01) and likes per day (P=.04) than those from Japan. In total, 60% (24/40), 21% (9/43), 18% (3/17), and 16% (3/19) of videos with a negative tone were uploaded by private individuals and laypersons, news sources, for-profit companies or organizations, and nonprofit organizations or universities, respectively.Of the 111 video uploaders, 45 (40.5%) uploaders were from the United States and only 14 (13%) uploaders were from Japan. Video posters from the United States received more subscribers, views per day, likes per day, and comments per day than those from Japan. Videos from other countries received more views per day (P=.99). Useful videos had good visual and aural effects, provided vivid information on the Fukushima disaster, and had a mean score of 12 (SD 0.9). The correlations between the total video scores and the number of subscribers, views per day, and likes per day were significant (P<.001). Useful videos had more views per day (P<.001), likes per day (P<.001), and comments per day (P=.02) than useless and slightly useful videos.Of the 111 videos, 43 (38.7%) videos were useful, 43 (38.7%) were slightly useful, and 25 (22.5%) were useless . The meaP=.001; comments/day: P=.02), vivid information , and understanding content .The popularity of videos had a significant correlation with clear sounds (likes/day: P=.01) and comments per day (P=.04). Video posters who made videos in Fukushima had more subscribers (P<.001), views per day (P=.003), and likes per day (P=.01). Videos with theoretical knowledge of radiation had more subscribers (P=.04) and dislikes per day (P=.02). There was no significant difference in likes per day (P=.72) and comments per day (P=.11) between negative and neutral- and mixed-tone videos.We defined 4 key parameters after reviewing the videos . Videos Of the 588 comments extracted from videos, 292 (49.7%) comments were negative and included conspiracy theories and criticisms of official agencies and the Japanese government. For example:Their government said they would use rice from Fukushima for Olympic.711 likesJapan is lying to its people and neighbors. They lie that their radioactive food is safe.659 likesPositive comments on videos comprised 73 (12.4%) of comments. For example:The older employees should be considered a hero. I believe nuclear plant workers who saved or slowed a devastating reaction should be internationally recognized as a hero.5 likesIn this study, videos had a high number of views, likes, dislikes, and comments, indicating that viewers seek FDNPP-related information on YouTube. Private individuals and laypersons uploaded the greatest number of videos that provided information about personal experiences in Fukushima Prefecture, summaries of news, personal opinions, and their perspectives on the nuclear accident. Japanese health-related institutions’ videos comprised 12.6% (14/111) of FDNPP accident videos; this shows that these institutions did not pay sufficient attention to the YouTube platform in terms of the distribution of information overseas. It should be noted that no private individual and layperson uploaders were from Japan in this study. One of the reasons for this may be that only English videos were collected. Experts and the government should encourage people living in Fukushima Prefecture to engage with YouTube and show their real lives to the world.Useful videos that had good quality and provided reliable radiation knowledge received more views, likes, and comments. The uploaders of these videos had more subscribers. The evaluation tool in this study was scientifically correct, balanced, and unbiased, but for the public, scientifically correct information and video tone were not statistically important elements of video popularity. This indicated the possibility of a large amount of misleading information being accessible to the public. A previous study also reported that governmental organizations' health information videos had limited impact . The pubIn our sample of videos, educational and documentary videos that held a neutral position accounted for 15.3% (17/111) of the videos. Compared to the number of medical videos, there were fewer educational videos about nuclear accidents on YouTube . One reaThe most commonly watched videos were uploaded by for-profit companies or organizations, while the lowest number of likes per day was found on videos from nonprofit organizations or universities. The primary purpose of some videos was to attract viewers; thus, it is not surprising that the majority of video content was misinformation and conspiracy theories. Some studies have also reported on misleading health information on social media ,25. AfteOur results show that half of the comments  were negative while the negative-tone videos comprised only 35.1% (39/111) of the total number of examined videos. For example, following a documentary uploaded by National Geographic, 1 comment said:Fukushima was dangerous, is dangerous and will be dangerous.We believe that videos that portrayed personal experiences elicited more engagement, as evidenced by the high number of views and likes and by the fact that videos describing the recent situation in Fukushima had more likes and comments.This study has several limitations. First, only 1 search term—“Fukushima nuclear disaster”—was used, which might have resulted in videos on the study topic being missed. Second, we only analyzed videos in English and recorded publicly available metrics. In a follow-up study, it will be necessary to analyze Japanese videos of the Fukushima nuclear accident. Second, ours is a cross-sectional study that was conducted in November 2019; more videos have been made available since then. Last, although we used an evaluation tool, the subjectivity of judgment existed in this study. Conducting further research on content analysis of YouTube and improving the evaluation tools of popular science videos are necessary.This study is the first to examine the Fukushima nuclear disaster–related information specifically found on YouTube. A large amount of information on the Fukushima nuclear disaster is available on YouTube. In this cross-sectional study, 38.7% (43/111) of YouTube videos provided useful information about the Fukushima nuclear disaster. However, for the public, scientifically correct information and video tone were not statistically essential elements in video popularity. The possible features that made videos popular to the public included good technical design, understandable content, personal experiences, and publishers’ popularity. During risk communication on new forms of media, health institutes should increase publicity and try to be more approachable to resonate with international audiences.


Laser-induced incandescence (LII) is a widely used combustion diagnostic for in situ measurements of soot primary particle sizes and volume fractions in flames, exhaust gases, and the atmosphere. Increasingly, however, it is applied to characterize engineered nanomaterials, driven by the increasing industrial relevance of these materials and the fundamental scientific insights that may be obtained from these measurements. This review describes the state of the art as well as open research challenges and new opportunities that arise from LII measurements on non-soot nanoparticles. An overview of the basic LII model, along with statistical techniques for inferring quantities-of-interest and associated uncertainties is provided, with a review of the application of LII to various classes of materials, including elemental particles, oxide and nitride materials, and non-soot carbonaceous materials, and core–shell particles. The paper concludes with a discussion of combined and complementary diagnostics, and an outlook of future research. Gas-phase synthesis of nanoparticles offers the possibility of generating high-purity materials via a continuous flow process. Controlled variation of size and morphology at the nanoscale brings exciting new possibilities for the design of materials having unique size-dependent properties that enhance the performance of current devices and processes, and lays a foundation for emerging technology . ApplicaWhile ex situ techniques are often considered the “gold standard” for characterizing the product properties, they do not possess the spatial resolution needed to map out nanoparticle attributes within the reactor flow field, nor do they have the temporal resolution needed for online control. Moreover, extracting particles from within a reactor is complicated by limited physical access , and theOptical diagnostics have long been used to characterize soot particle sizes, morphologies, and volume fractions within flames and engines, as well as in exhaust gases of combustion processes and in atmospheric research. Optical measurement approaches may be based on light scattering , pyromet2 for soot, with variations depending on the experimental setup 1/2 and the number density of evaporated species iskB is the Boltzmann constant and pv is the vapor pressure.Evaporation heat transfer is calculated by assuming that the condensed-phase and gas-phase material on either side of the nanoparticle interface is in equilibrium. Under these conditions, the evaporative heat transfer rate isFrom a thermodynamics perspective, TiRe-LII amounts to perturbing a system initially at thermal equilibrium with a laser pulse, and then measuring how quickly the system returns to equilibrium through heat transfer between the nanoparticles and the surrounding gas. While the microsecond time-scales typical of the nanoparticle cooling rate are much longer than the nanosecond-scale laser pulse, the sub-femtosecond timescales important to the phase equilibrium across the solid–liquid and vapor interface are much shorter , and thedp < 10 nm), it may also be necessary to account for the increased energy of the curved interface via the Kelvin 1/2 from the equilibrium bath gas at pg and Tg, and a molecular mass mg, and <Eg,o–Eg,i> is the average energy transferred when a gas molecule scatters from the nanoparticle surface. This last term is written in terms of the thermal accommodation coefficient, α, which specifies the average surface energy transferred to a gas molecule relative to the maximum value allowed by the 2nd Law of Thermodynamicsint is the number of internal energy modes of the gas molecule. Given the brevity of the interaction between the gas molecules and the particle surface, the vibrational modes of the gas molecule can be usually be neglected, so ζint only accounts for the rotational degrees-of-freedom . Some studies have instead phrased Eq.  and semiconductors, leads to large evaporation rates. In these scenarios, it may be necessary to model the evolving mass of the nanoparticle according toTp,i = Tg, and mp,i = ρπdp,i3/6. These equations are coupled by the evaporation term in Eq. (dp = [6mp/(πρ)]1/3, which affects the heat transfer terms in Eq. 18) are cs in Eq. . The chas in Eq. , but thiWhile this basic model provides a general basis for analysis, it also presents several key limitations. For instance, Eq.  assumes Evaporation heat transfer introduces further complexities into the model, particularly for low boiling point materials. In contrast to heat conduction, which can be reasonably modeled assuming a stationary gas, evaporated species leaving the nanoparticle are highly nonstationary, and can produce a “snowplow effect”, wherein the evaporated species push the bath gas molecules away from the particle . To date2, N2) can be emitted upon thermal decomposition. In addition to its impact on evaporative heat transfer (due to the thermochemistry of the related reactions), this phenomenon could also fundamentally change the radiative properties of the nanoparticles through phase changes and defect formation, while the evaporating species may also contribute to non-incandescent laser-induced emission ) Most TiRe-LII studies target aerosol attributes such as particle size and volume fraction, including some very early studies , 101. FoExperimental conditions are sometimes sufficiently well-controlled to permit inference of more fundamental parameters that are otherwise difficult to measure through alternate means, particularly at the high temperatures typical of LII measurements. For example, during the development of the TiRe-LII technique, the thermal accommodation coefficient was usually treated as a “tuning parameter” that could be adjusted to “force” modeled cooling curves or TEM-derived particle sizes to match LII signal traces. TACs inferred in this way were confounded by model errors, and could not be interpreted in a strict physical sense. This situation has changed as the LII community has developed an increasingly detailed knowledge of the nanoparticle cooling mechanisms. In particular, a comparative analysis of TACs obtained from various elemental nanoparticle-gas combinations has provided key insights into the gas–surface scattering process underlying this parameter, including the influence of the mass ratio of the involved gas-phase molecules and surface atoms Fig. , the gasKock et al.  and EremLikewise, both the latent heat of vaporization and vapor pressure have been inferred for metal nanoparticles , 74, 92.−C2/λT) with C2 = hc/kB, so this difference in temperatures translates into a significantly lower intensity, and consequently a lower signal-to-noise ratio.While LII studies on metal nanoparticles offer advantages over experiments on carbonaceous nanoparticles, there are also a number of key drawbacks. The signal-to-noise ratio of LII measurements largely depends on the maximum temperature to which the nanoparticles can be heated, which is governed by a balance between laser heating and cooling by evaporation or sublimation during the pulse . In the The situation is further confounded by the fact that the absorption cross-sections of metal nanoparticles, particularly those of high electrical conductivity, are much lower than carbonaceous nanoparticles. Combined, the lower evaporation temperatures and low absorption cross-sections of metal and solid metalloid nanoparticles result in a much weaker spectral intensity compared to what is typical for LII measurements on carbonaceous materials. Accordingly, significant experimental effort must be invested to provide highly efficient signal detection in order to achieve an acceptable signal-to-noise ratio. Of particular note, the weak absorption of metalloids at room temperature may limit studies at room temperature, with measurements easier when the particles start at an elevated temperature, e.g., in gas-phase synthesis reactors , 72. Othm· xp||< 1, is not generally satisfied for metals. Consequently, the spectral absorption cross-section must be calculated using Mie theory 140]. Sin2, TiO2, and Al2O3, only weakly absorb in the visible and near infrared, making them inherently more challenging as LII targets. Their small absorption cross-sections make them difficult to heat, and also contribute to a weak incandescence signal that is susceptible to interference from other laser-induced emission sources. Due to these challenges, LII is used less frequently to characterize oxide (and nitride) nanoparticles compared to metal and carbonaceous nanoparticles, with some notable exceptions: in what is widely considered the first deployment of TiRe-LII, Weeks and Duley [2 laser.According to Drude–Lorentz theory, oxides of light elements such as silicon, titanium, or aluminum couple with EM fields via bound charges that oscillate within the lattice. Therefore, in contrast to metals, oxide materials, such as SiOnd Duley  targetedIn the case of transparent oxides like titania and silica (that appear white due to light scattering), laser heating and emission is expected to arise mainly through defects , which c2 nanoparticles indicate that LII quickly transitions into a LIBS regime with increasing laser fluence. Zizak, De Iuliis, and co-workers have reported LII measurements on TiO2 nanoparticles in a series of publications [2), which makes particle sizing, derived from signal decay curves, difficult.Experiments on TiOications , 146–149ications . In manyications , in orde2 nanoparticles at 266 nm causes emission dominated by photoluminescence in the 400–600 nm range at low fluence (< 1 mJ/cm2), which rapidly decays and becomes undetectable within ~ 300 ns. Broadband emission was observed with fluences reported as 17 mJ/cm2, while higher fluence (56 mJ/cm2) caused strong overlap from narrowband emission through the transition to laser-induced breakdown  [Many other oxides, especially brown- and black-colored transition-metal oxides, have strong interaction within the visible and near infrared light, such that particle heating is more easily accomplished. As an additional complication, in these cases, a variety of oxide phases exist and, from a thermodynamic point of view, less-oxidized phases are favored with increasing temperature (FeeO → Fe) . These peO → Fe)  -based particle sizing in the particle formation zone as an independent method. Additional reports have been published in brief conference proceedings on manganese oxide [2 nanoparticles, show LII signals that decline very quickly in comparison to what is typical for carbonaceous nanoparticles.Dörr  adopted se oxide  and ironse oxide , which, While most LII studies on non-elemental nanoparticles have focused on oxides Table , in an e2 versus sp3 hybridization and degree of long-range order. Figure Engineered carbon nanomaterials are distinguished from soot based on their relatively low PAH and hydrogen content, as well as by how they are generated. Carbonaceous nanomaterials can be further classified by their internal structure, such as the relative prominence of spCarbon nanotubes (CNTs) are among the most frequently studied carbon allotropes due to their unique properties and industrial potency. Aerosol synthesis of CNTs is often done using metal catalyst nanoparticles. Puretzky et al. , 173 and3 hybridization; carbon dots; and graphene platelets) trapped using a single nanoparticle mass spectrometer. The nanoparticles were heated with 532 or 1064 nm pulses. The recovered spectra were compared to elucidate the connection between carbon structure and the spectral emission cross-section . Measurements on pure nanodiamonds were not possible due to their low absorption cross-section at the laser wavelength.Other allotropes feature less commonly in the literature. Carbon arc reactors have been used to generate a range of nanomaterials, including CNTs, fullerenes, carbon nanowires, and nanosheets; many of these structures have been probed with TiRe-LII , 184–186In terms of emission spectra, laser-heated carbonaceous nanoparticles are largely consistent across the alloptropes; this is likely due to the fact that most of these particles emit in the Rayleigh limit of Mie theory, so the constituent dipoles oscillate in phase  and the absorption cross-section only depends on dipole number density and not particle morphology. Osswald et al.  measuredThe variation of peak temperature or peak incandescence with laser fluence  also provides important insight into absorption efficiency and evaporation. These measurements have been carried out for carbon black , 188, CN2 hybridization, the heat transfer models used for these other materials are the same as those applied to soot. However, there are likely differences in some of the properties, most notably with respect to the evaporation submodel [The morphology of CNTs and FLG particles differ substantially from the particle aggregates typical of soot and carbon black. Nevertheless, TiRe-LII shows promise for interrogating this morphology, since signal decay rate is roughly proportional to the heat transfer surface area. Cau et al.  interpresubmodel , that reApplications of LII to more complex or composite nanomaterials are relatively rare. Candidate nanomaterials include homogeneous mixtures of two or more compounds, core–shell arrangements, or special morphologies, such as prisms or hollow spheres. To date, LII studies have only considered carbon-coated iron in a series of papers by Eremin and coworkers –51 and c5/Ar mixtures before either methane or acetylene were added to the mixture as a means to coat the iron nanoparticles with a thin carbon layer. Particle growth was tracked via time-resolved optical extinction. The acetylene-containing mixture produced carbon-coated nanoparticles, attributed to the presence of catalytically active surface sites, while the mixture containing methane did not.Eremin et al.  generate5 with benzene, toluene, or butanol [E(m) ratio of unity. This may be questionable given that the refractive index of iron varies over the detection wavelength and the Rayleigh model is inapplicable, cf. Sec. 2.1. Peak pyrometric temperatures of laser-heated, carbon-encapsulated iron particles increased from 1900 to 2850 K when the laser fluence increased from 150 to 900 mJ/cm2  butanol . The meacm2 Fig. a, which cm2 Fig. b. The au5, Ar, and either C2H2 or C6H6. TiRe-LII was triggered after the reflected shock wave passed the probe volume and allowed for in situ determination of particle size, with laser fluences kept between 70 and 150 mJ/cm2 to avoid particle evaporation and emission from evaporating atomic/molecular species, while providing a reasonable signal-to-noise ratio. The generated nanoparticles typically feature an iron core of 3–10 nm covered by a carbon shell containing between 2 and 20 atomic layers, with benzene producing the smaller particles. In the LII model refined from their previous work, the mass ratio between the iron core and carbon shell was estimated from TEM images and used to calculate a mean density. The authors found that when utilizing thermal accommodation coefficients between 0.35 and 0.46 reported in the literature for similar systems, model-retrieved particle sizes deviated between 21 and 25% from the values with α = 0.44 in experiments in mixtures with benzene as soot precursor. A thorough error analysis led the authors to claim uncertainty ranges in LII particle sizing for an iron:carbon mass ratio of 5:1 between – 31 and + 17%, and that density, heat capacity, and TAC are the most sensitive parameters affecting deduced particle sizes. Unfortunately, predicted mean primary particle sizes deviated from TEM results 4, loading rate typically 100 μL/min) as the prevaporized precursor  mixed in the oxygen flow from below; both flows were diluted with N2. In such a configuration, the metal oxide nanoparticles quickly grow on the oxidizer side of the flame sheet without interaction with the carbon-containing fuel, whereas their carbonaceous shell is formed as the nanoparticles travel through the fuel side of the flame. Specific crystalline structures and coating thicknesses may be realized by controlling particle temperature–time histories as they transition through flame regions of varying temperature and gaseous composition.Ren et al.  investig2* Swan emission (at 516.1 nm); for high enough laser fluences, Ti* atomic emission  particles  composition of the CMO nanocomposite. This data, along with the intensity profiles along the burner axial direction, was used to derive a CMO formation mechanism, with particle size  increasing with precursor seeding rate keeping an almost constant carbonaceous shell thickness. Contrary to the laser photolysis [From the laser fluence dependence of the LII, PS-LIBS, and the Cotolysis  or shockotolysis  initiateThese largely empirical results highlight the promise of TiRe-LII as a tool for characterizing composite and otherwise complex nanoparticles. As it stands, qualitative results remain useful in assessing changes in nanoparticle characteristics and determining the location where the changes happen in a reaction system and thus provide a route to further study these unique nanomaterials. However, considerable further research is needed to develop the sophisticated spectroscopic and heat transfer models needed to support quantitative analysis of these materials.The laser pulse used to induce an incandescence signal may also elicit other types of signals from the nanoparticles, collectively referred to as non-incandescent laser-induced emission (LIE). While these signals are often a nuisance, particularly when they are difficult to distinguish from incandescence, they may also provide additional insight about the nanoparticle quantities-of-interest that complement the information contained in the LII signal.Most non-incandescent LIE arises from species evaporated from the nanoparticle by the LII excitation pulse. For example, as noted in Sect. 2 swan bands for carbonaceous materials, are another common form of laser-induced emission. The underlying mechanism depends on the fluence regime, shown schematically in Fig. 2 3p4 s 1P° 1 → 3s2 3p2 1D 2 at 288 nm for Si [Atomic emission lines, such as those observed in Fig. m for Si ) using aFigure xOy) nanoparticles formed in a spray flame, where strong line emissions between 404 and 407 nm originate from atomic iron (Fe I). Guo et al. [At still higher fluences, Zhang et al.  showed to et al.  applied While the most obvious application of PS-LIBS is to determine the chemical composition of the nanoparticle phase from the relative strengths of atomic emission lines, the fluence level of PS-LIBS saturation can also be used to estimate the total nanoparticle mass/volume concentration in the observation volume, which can complement LII measurements. Zhang et al. , 198 andIf the particles have suitable chromophores, several authors have shown that conclusions about the particle size can also be drawn from the photoluminescence emitted after laser excitation , 204.Several techniques have been used in conjunction with TiRe-LII, as summarized in Table 2 nanoparticles in a flame reactor for in situ identification of the crystal structure [At laser fluences below the threshold that produces detectable incandescence, spontaneous Raman scattering has been successfully applied to gas-borne TiOtructure . The deptructure  in orderxp << 1, ||m · xp|| << 1), the scattering cross-section, Csca,λ, scales with dp6 and, thus, strongly depends on particle size and wavelength. Santra et al. [1 = 1064 nm and λ2 = 532 nm in horizontal and vertical polarization, respectively) to measure the aspect ratio length over diameter of carbon nanotubes.Elastic light scattering has been carried out using either pulsed or continuous-wave lasers, sometimes in parallel with LII studies. In the case of spherical particles that satisfy the Rayleigh criterion , where θ is the angle between the direction of laser propagation and the detection view angle [The information content of the light scattering data can be enhanced by measuring the scattered light at multiple wavelengths or multiple angles; these two dimensions influence the scattering measurement model via the modulus of the scattering wavevector, ew angle , 210. Anew angle ). Martinew angle  present ew angle , silica ew angle  and ethaew angle .Often LII and elastic light scattering may be combined to improve the robustness of the recovered variables. Combined elastic scattering and TiRe-LII measurements are particularly appealing in the case of aggregated nanoparticles, since the scattering measurement is more sensitive to the aggregate structure, while the TiRe-LII data indicates the primary particle size. As such, using light scattering in combination with laser-induced incandescence enables the detection of the radius of gyration of soot particle aggregates . While tWhile it lies outside the scope of this review, it is worth noting that the SP2 instrument , which u2/g through surface desorption of a gas (most often nitrogen). The BET-inferred specific surface area is influenced by agglomerate structures of the product powder and the value can be strongly affected in case a second (large) particle phase is present. Pabisch et al. [In many works reported in literature, TiRe-LII measurements are calibrated against ex situ techniques like transmission electron microscopy (TEM), particle mass spectrometry (PMS), scanning mobility particle sizing (SMPS), and Brunauer–Emmett–Teller (BET) analysis. Each of these measurement methods can determine specific nanoparticle properties, but they have different interpretations in the context of TiRe-LII-inferred sizes. In principle, both TEM and TiRe-LII may be used to infer primary particle size, although, in the case of TiRe-LII, the particle diameter corresponds to the specific heat transfer area in the same way that SMPS measurements correspond to a “mobility diameter”. While TEM measurements are widely considered the “gold standard” for particle sizing, especially in the context of the many LII measurement model uncertainties addressed in this paper, it is important to acknowledge that TEM-derived particle sizes may differ to those found in the gas phase. Reasons for this include: inadequate sample sizes due to the laborious nature of TEM analysis (even with automation); artifacts introduced by the extraction process ; and surface forces  that dish et al.  showed th et al. . OverallWhile laser-induced incandescence is a standard combustion diagnostic for measuring soot primary particle size and volume fraction, it is increasingly applied to characterize other types of aerosols, including metal, metal oxide, and manufactured carbonaceous particles. This trend is driven, in large part, by the increasing industrial relevance of nanoparticles, which are often produced in the gas phase.Although the same basic LII measurement principle applies to both soot and synthetic nanoparticles, there are notable differences between these two scenarios. In the case of aerosols of elemental nanoparticles, the thermophysical properties needed for the LII measurement model are often much better known compared to those of soot, which vary with fuel composition and local combustion conditions, and evolve as soot “ages”. Moreover, many synthetic aerosols consist of “pure” spherical nanoparticles that obey a narrow size distribution, while soot particles usually have a more complicated and heterogeneous aggregate structure, and both primary particle and aggregate sizes often have wide size distributions. For these reasons, LII measurements on well-characterized nanoparticle systems may be used to better understand the fundamental aspects of electromagnetic theory and transport phenomena that underlie this diagnostic. These lessons can then be applied to more complex measurement scenarios, including soot. LII may also be deployed to carry out more fundamental scientific inquiries, e.g., to determine the thermophysical properties of materials at extreme temperatures and thermal accommodation coefficients between various gas/surface systems, both of which are difficult to infer from experiments on larger-scale materials.On the other hand, in the case of more complex nanoparticles , LII model parameters may be difficult or impossible to quantify. In these scenarios the measurement model may need to be purely empirical, derived by fitting simulated LII traces with measurements in the context of nanoparticle sizes obtained through non-LII means . A particular challenge concerns the spectroscopic models that connect the measured spectral intensity to the nanoparticle temperature. While the spectral absorption cross-sections of soot particles can almost always be modeled using the Rayleigh approximation, this is not the case for synthetic nanoparticles, particularly those made of metal. Modeling the radiative properties of spherical metal nanoparticles requires the full Mie equations, which depend on the particle diameter. For non-spherical nanoparticles, even more sophisticated techniques, like the discrete dipole approximation, are needed to quantify the spectral absorption cross-section; otherwise, an empirical “fitting” approach would again need to be employed.A further complication concerns non-incandescent laser-induced emission (LIE), which, under some experimental conditions, may contaminate LII signals and complicate data analysis. On the other hand, non-incandescent LIE may provide additional information about the aerosol. For example, at higher fluences, atoms and molecules evaporated from the nanoparticle produce atomic emission lines that can be used to determine nanoparticle composition, and, in some cases size, volume fraction, and other properties.The capabilities of LII will continue to evolve in lockstep with the number and range of applications for synthetic nanoparticles. This diagnostic will prove particularly important for designing and controlling gas phase synthesis routes used to produce complex nanostructures, including few-layer graphene particles “decorated” with silicon nanoparticles, aggregates of metal and semiconductor nanoparticles, metal alloys, and core–shell structures. Analyzing these nanoparticle types will require a more detailed understanding of the spectroscopic and transport processes that connect the LII signal to the aerosol quantities-of-interest than is presently available. However, higher-fidelity LII measurement models are being developed using sophisticated electromagnetic and molecular dynamics simulations, enabled by high performance computing and machine learning. Parallel advancements in optoelectronics hardware  are presently being exploited to develop highly sensitive LII systems that are far more informative compared to traditional single-pulse, two-wavelength detection setups. In this context, particularly potent diagnostics may be realized by combining LII with complementary optical techniques, e.g., elastic and inelastic scattering and plasma signals related to laser-induced breakdown spectroscopy. Statistical analysis techniques must also be developed in order to fully exploit advancements in theoretical modeling and hardware. Bayesian-based approaches are particularly useful for quantifying the uncertainty in LII-derived quantities, synthesizing data from multiple diagnostics, and even developing LII measurement models.The diagnostic tools enabled through these advancements will allow engineers to develop new types of synthetic nanoparticles and applications, allow combustion researchers to understand soot formation and its impact on human health and the environment, and equip scientists who wish to advance the frontiers of knowledge of thermodynamics, electromagnetics, and transport phenomena at the nanoscale.


Escherichia coli.The human Bri2 BRICHOS domain inhibits amyloid formation and toxicity and could be used as a therapeutic agent against amyloid diseases. For translation into clinical use, large quantities of correctly folded recombinant human (rh) Bri2 BRICHOS are required. To increase the expression and solubility levels of rh Bri2 BRICHOS it was fused to NT*, a solubility tag derived from the N-terminal domain of a spider silk protein, which significantly increases expression levels and solubility of target proteins. To increase the expression levels even further and reach the g/L range, which is a prerequisite for an economical production on an industrial scale, we developed a fed-batch expression protocol for A fed-batch production method for NT*-Bri2 BRICHOS was set up and systematically optimized. This gradual improvement resulted in expression levels of up to 18.8 g/L. Following expression, NT*-Bri2 BRICHOS was purified by chromatographic methods to a final yield of up to 6.5 g/L. After removal of the NT*-tag and separation into different oligomeric species, activity assays verified that different assembly states of the fed-batch produced rh Bri2 BRICHOS have the same ability to inhibit fibrillar and non-fibrillar protein aggregation as the reference protein isolated from shake flask cultures.The protocol developed in this work allows the production of large quantities of rh Bri2 BRICHOS using the solubility enhancing NT*-tag as a fusion partner, which is required to effectively conduct pre-clinical research.The online version contains supplementary material available at 10.1186/s12934-021-01638-8. Alzheimer’s disease (AD) is the most common cause of dementia and belongs to a group of diseases that are characterized by the formation of deposits of specific proteins in amyloid fibrillar conformations . DespiteBri2, chondromodulin-1, and prosurfactant protein C) has emerged as the first example of an endogenous molecular chaperone-like domain that protects amyloidogenic protein segments from forming amyloid :1\d:1\d2 lev22 g g−1 . A summag for 20 min. Then, the cell pellet was re-suspended by gentle agitation at 4 °C in Buffer A , using 20 mL buffer per 10 g wet cell weight. Cell resuspensions were stored at −20 °C.After cultivation, the cells were harvested in a Sorvall LYNX 6000 (Thermo Scientific) centrifuge, using an F9-6 × 1000 LEX rotor at 4000×g in a Sorvall RC 6 + (Thermo Fisher Scientific) using an F21-8 × 50y rotor (4 °C for 30 min). After centrifugation, the supernatant was filtered through a Filtropur S 0.45 . All subsequent purification steps were carried out on an Äkta Explorer liquid chromatographic system  at 6 °C. The lysate was loaded onto 4 × 5 mL HiTrap Chelating HP columns  equilibrated with buffer A using a flow rate of 2.5 mL/min. Next, the proteins bound to the column were washed with buffer A containing 40 mM imidazole, before NT*-Bri2 BRICHOS was eluted with buffer A containing 200 mM imidazole. After elution, the flow-through was re-applied onto the columns, and NT*-Bri2 BRICHOS was washed and eluted in the same manner one more time. The combined eluate was subsequently dialyzed against 5 L buffer A overnight using a Spectra Por membrane  with a molecular weight cut-off between 6 and 8 kDa. All samples were stored at −20 °C.A 50 mL cell resuspension was thawed and lysed using a cell disruptor  at 1.35 kbar. Then DNAse I  was added to the lysed cells to a final concentration of 10 µg/mL, followed by incubation on ice for 30 min, and by centrifugation at 38,000×To estimate the expression levels of NT*-Bri2 BRICHOS in the different cultures before harvest, a 100 µL sample  was centrifuged to pellet the cells. The supernatant was discarded, and the pellet was re-suspended into 100 µl 8 M urea. 10 µL of each sample was loaded onto a 4–20% Mini-protean TGX stain-free precast SDS-PAGE gel . To estimate the amount of protein in the samples, already purified NT*-Bri2 BRICHOS was used as standard: 0.24, 0.48, 0.95, and 1.9 mg/ml unless noted otherwise. To create a standard curve, the standard bands were integrated with ImageLab software , which correlated the band intensity to protein concentration.The concentration of purified NT*-Bri2 BRICHOS was calculated using the protein specific extinction coefficient, Abs 0.1% (= 1 g/L) = 0.492, after measuring the absorbance at 280 nm in triplicates. This method was also used to determine the concentration of the NT*-Bri2 BRICHOS standards used to estimate the expression level with SDS-PAGE.The dry cell mass of culture #9–11 was determined by transferring 1 mL cell resuspensions (in triplicates), to already dried and weighed Eppendorf tubes. The cells were pelleted by centrifugation, the supernatant was removed, and the cell paste was dried at 65 °C for at least 24 h until a stable weight was obtained. This method enabled an alternative way to estimate the expression level of culture #9-11, by determining the % of NT*-Bri2 BRICHOS relative to the total cellular proteins, using GelAnalyzer 19.1, and considering that the protein fraction of the total dry cell weight is 55% .g and 4 °C. Then, the different rh NT*-Bri2 BRICHOS oligomeric species were isolated by size exclusion chromatography (SEC) using a Superdex 200 26/600 column , which was equilibrated with Buffer B (20 mM NaPi buffer pH 8.0 containing 0.2 mM EDTA), and an ÄKTA Explorer liquid chromatographic system . The individual peaks corresponding to the differently sized oligomeric species were narrowly collected. Then, the rh NT*-Bri2 BRICHOS species were cleaved separately with 1: 600 thrombin  in a cold room overnight. After cleavage, the NT* tag was removed by reapplying the sample onto an IMAC column with buffer B (reverse IMAC), where rh Bri2 BRICHOS was found in the flow-through, and the NT*-tag bound to the column was eluted with buffer B containing 200 mM imidazol. To polish the Bri2 BRICHOS species, they were further purified with SEC one more time, using a Superdex 200 26/600 , or a Superdex 75 26/600 (for monomer) column . The purified Bri2-BRICHOS species were stored at −20 °C.IMAC  purified NT*-Bri2 BRICHOS was concentrated to 3.4 mg/mL with a Vivaspin protein concentrator  at 4000×The ability of the different rh Bri2 BRICHOS species to prevent non-fibrillar protein aggregation was assessed by suppressing thermo-denaturation of citrate synthase (CS). CS from porcine heart  was diluted in 40 mM HEPES/KOH pH 7.5 to 600 nM and then incubated at 45 °C with and without different concentrations of rh Bri2 BRICHOS oligomer or tetramer. The aggregation kinetics were monitored in triplicate using a microplate reader  by reading the apparent absorbance at 360 nm, as a measure of turbidity, under quiescent conditions.E. coli (B strain) cells (Novagen) and purified as described previously [−1 cm−1 for (A280-A300).Aβ42 fused with a solubility tag (NT*) was expressed in BL21*(DE3) pLysS eviously . BrieflyA is the amplitude and F0 the base value.For testing the activities of rh Bri2 BRICHOS species against Aβ42 fibril formation, 80 µL reaction solution containing 3 µM Aβ42 monomers, 10 µM Thioflavin T (ThT) and different concentrations of Bri2 BRICHOS species at molar ratios 0, 10, 50, and 100% relative to Aβ42 were added to each well of half-area 96-well microplates . The fluorescence was recorded using a 440 nm excitation filter and a 480 nm emission filter  under quiescent conditions at 37 °C. Aggregation traces are averages of four replicates for all the experiments. For comparing the activities of each Bri2 BRICHOS species, the aggregation half time Rh Bri2 BRICHOS oligomers (10 μM) and monomers (5 μM) expressed in the bioreactor were incubated in 20 mM sodium phosphate pH 8.0 with 0.2 mM EDTA overnight at 37℃, and samples were analyzed by SDS-PAGE under native, reducing, and non-reducing conditions.600 is below 1. This is comparable to the yield of purified NT*-Bri2 BRICHOS produced by bioreactor culture #1, which essentially follows this simple shake flask expression protocol . After a total culture time of 39 h, the da Silva-medium yielded an OD600 of 120, Wyre-medium an OD600 of 61, and Korz-medium an OD600 of 50. Even though the medium according to da Silva yielded the best results in terms of cell density, after purification the yield was 0.6 g/L soluble NT*-Bri2 BRICHOS for Wyre and 0.5 g/L for da Silva medium . According to the OD600 measured before culture harvest and the wet cell mass after harvest, there is a possible negative correlation between the amount of IPTG used to induce the culture and the final cell mass, however, the expression levels of NT*-Bri2 BRICHOS were comparable  by reverse IMAC. Finally, the rh Bri2 BRICHOS species were concentrated before isolation with SEC to obtain pure oligomeric, tetrameric, dimeric, and monomeric species , the aggregation was completely inhibited  Fig. . The effe flasks , in termE. coli is a favorite workhorse for the production of biopharmaceuticals and is used for the production of about 30% of all clinically approved proteins [E. coli, hence the next logical step was to employ the NT*-domain for protein expression in a high-cell density bioreactor cultivation. In this study, we attempted to express and purify the rh Bri2 BRICHOS domain on a g/L scale employing the NT*-tag. Rh Bri2 BRICHOS has emerged as an interesting biopharmaceutical candidate for treating AD, as the domain is a potent inhibitor of Aβ42 amyloid fibril formation and in particular its associated neurotoxicity in vitro, in hippocampal slice preparations ex vivo, and in Drosophila models in vivo [. In addition, rh Bri2 BRICHOS passes the blood-brain barrier [To use recombinant proteins for medical or biotechnological applications, the production needs to be scalable and the typical expression levels need to be in the high g/L scale to be economical , 35. E. proteins , but a fproteins , 38. To proteins , 23. The in vivo . In addi barrier .E. coli SHuffle cells in a bioreactor using a fed-batch setup [600 above 100 was only obtained for the medium formulation described by da Silva et al., why we chose this medium for all subsequent cultivations.To increase the expression yield of rh Bri2 BRICHOS compared to standard shake flask cultivations, we reasoned that the medium composition and feeding with a suitable carbon source are key elements to obtain a high cell density culture, in addition to proper oxygen supply and pH control. Accordingly, three previously suggested medium formulations that are supposed to support cell densities higher than 70 g/L (dry cell weight) were tested for culturing ch setup –28. Inte600 of 30 resulted in an NT*-Bri2 BRICHOS expression level of 7.9 g/L. A minimal adjustment of this protocol by inducing protein expression at OD600 of 60 instead, elevated the expression level to 18.8 g/L (15.2 g/L if estimated from the dry cell weight), which competes with the highest reported recombinant protein expression levels for E. coli (Table Furthermore, the influence of different IPTG concentrations for induction on the cell density at harvest and the expression level was tested (Table Purification of Bri2-BRICHOS was executed in the same manner as previously described , and inve.g. metals, could have potential effects on the equilibrium of the different species, and future work is necessary to elucidate the exact mechanisms behind this behavior. Nevertheless, in accordance with earlier results, CS aggregation was only inhibited by the presence of Bri2 BRICHOS oligomers. Likewise, the inhibition profile of Bri2 BRICHOS against Aβ42 amyloid formation is comparable, suggesting that the chaperoning capacities against fibrillar amyloid aggregation and non-fibrillar amorphous aggregation of Bri2 BRICHOS are independent of the expression and purification protocol.Another relevant question that we wanted to investigate was whether the protein aggregation inhibition profiles of bioreactor produced rh Bri2 BRICHOS are comparable to previously published data for the shake flask-produced protein. To answer this, after removal of the NT*-tag with thrombin, rh Bri2 BRICHOS was separated into the individual oligomeric species using SEC. Interestingly, we were not only able to isolate monomers, dimers, and higher order oligomers but also tetramers, a species that we were not able to purify from our previous shake-flask preparations. The tetramer was predominantly formed after the removal of NT* from the NT*-Bri2 BRICHOS dimer. So far, we do not know what can trigger the transformation of the tetramer from the dimer, but the high concentration is probably an explanation as the Bri2 BRICHOS oligomerization process is sensitive to increased concentration . Also, wE. coli. The high yield is likely attributed to the use of the NT*-tag, which drastically increases the solubility and expression levels of certain peptides and proteins. Since rh Bri2 BRICHOS is a promising candidate for treating AD, the protocol described in this paper is important to enable future research and successful translation into clinical trials.In this study, we establish an expression protocol of the human small heat shock protein chaperone domain Bri2 BRICHOS. Optimization of the process resulted in an expression level of up to 18.8 g/L, which is among the highest protein expression levels reported for Additional file 1.Table S1. Summary of key parameters for the expression and purification of  NT*-Bri2 BRICHOS. Figure S1. SDS PAGE of NT*-Bri2 BRICHOS IMAC eluates. Figure S2-3. Estimation of the NT*-Bri2 BRICHOS expression level using SDS-PAGE. Figure S4. Native PAGE and SDS-PAGE of rh Bri2 BRICHOS.