Association and dose-response relationship between exposure to alcohol advertising media and current drinking: a nationwide cross-sectional study of Japanese adolescents.

BACKGROUND: Underage drinking is a public health concern. However, few studies have examined the association between alcoholic beverage advertising and underage drinking, particularly in countries with low underage drinking rates, such as Japan. Therefore, we aimed to investigate the relationship between exposure to advertising in various media and alcohol drinking among Japanese adolescents. METHODS: We conducted a cross-sectional study involving 15,683 adolescents (51% girls) using data from a nationwide lifestyle survey in 2021 among junior and senior high schools across Japan. Media types were websites, stores, and public transportation. We defined current drinking as alcohol consumption of ≥1 day in the 30 days preceding the survey. Multivariable logistic regression was used to examine the association between exposure to alcohol advertisements and current drinking, adjusting for sex, grades, school area, lifestyle (bedtime and having fun at school), and addictive behaviors (smoking status and parents' alcohol consumption). RESULTS: The prevalence of current drinking was 2.2% (2.3% of boys and 2.0% of girls). Students who were exposed to any alcohol advertising media had higher odds of current drinking compared with those who were not (odds ratio, 1.48; 95% confidence interval [CI], 1.18-1.87). Students who were exposed to web, in-store, and public transportation advertisements had odds ratios of 1.44 (95% CI, 1.14-1.81), 1.62 (1.28-2.05), and 1.45 (1.06-1.98) of current drinking, respectively, compared with those who were not. The association of exposure to alcohol advertising media with the prevalence of current drinking was similar among boys and girls (all p for sex interaction >0.1), except for that of exposure to web advertisements; its association with current drinking was more pronounced in girls (p for sex interaction = 0.046). Exposure to a larger cumulative number of different alcohol advertising media was independently associated with a higher prevalence of current drinking among all students, boys, and girls (p-values for trend <0.001, 0.031, and <0.001, respectively; p for sex interaction = 0.085). CONCLUSIONS: We found an association with a dose-response relationship between exposure to alcohol advertisements and current drinking among adolescents in junior and senior high schools across Japan. Our findings highlight the need for further advertising regulations to prevent underage drinking.

Oligodeoxynucleotides Modified with 2'-O-(Cysteinylaminobutyl)carbamoylethylribothymidine Residues for Native Chemical Ligation with Peptide at Internal Positions.

We used native chemical ligation (NCL) to synthesize a 2'-O-{N-[N-(S-tert-butylthiocysteinyl)aminobutyl]carbamoylethyl} (CysBCE) ribothymidine-derived oligonucleotide to expand the variety of peptide conjugation sites, allowing the incorporation of peptides at the 2'-hydroxy group when the oligonucleotide forms a duplex with the complementary strand. The NCL reaction with a peptide thioester and the modified oligonucleotide proceeded smoothly even when the CysBCE modification was in the middle of the oligonucleotide sequence. In addition, we incorporated two CysBCEs into an oligonucleotide to conjugate two peptides to one oligonucleotide. The results indicated that the tandem NCL reactions proceeded efficiently when the oligonucleotide hybridized to the complementary strand to avoid intramolecular disulfide formation between the two CysBCE groups. This method could be useful for peptide conjugation on the 2'-position.

Expression of T-cell immunoreceptor with immunoglobulin and tyrosine-based inhibitory motif domains (TIGIT) in anaplastic thyroid carcinoma.

BACKGROUND: Immune checkpoint proteins have not been fully examined in follicular cell-derived thyroid carcinoma and medullary thyroid carcinoma (MTC). Anaplastic thyroid carcinoma (ATC) is one of the most aggressive carcinomas. Even multimodal treatment does not result in favorable clinical outcomes for patients with ATC. Anti-tumor immunity has therefore been highlighted as having therapeutic promise for ATC. METHODS: We examined a novel immune checkpoint receptor, T-cell immunoreceptor with immunoglobulin and tyrosine-based inhibitory motif domains (TIGIT), in variable thyroid lesions: adenomatous goiter, follicular adenoma, and thyroid carcinoma (TC) using immunohistochemistry (IHC). RESULTS: Our IHC results showed that TIGIT expression was detected in cancer cells of MTC and high-grade TC: poorly differentiated thyroid carcinoma (PDTC) and ATC. Neoplastic cells were positive for TIGIT in four of five MTCs (80.0%), 17 of 31 ATCs (54.8%) and in 3 of 12 PDTCs (25.0%). TIGIT was not detected in any adenomatous goiters, thyroid benign tumors, or differentiated thyroid carcinoma (DTCs). Intriguingly, ATC cells showing pleomorphic/giant cell features were positive for TIGIT, while ATC cells with other cell morphologies lacked the immunoreactivity. Intra-tumoral immune cell was inclined to be enriched in TIGI-positive ATC. Although coexisting papillary thyroid carcinoma (PTC) components demonstrated high-grade microscopic features, neither the PTC nor follicular thyroid carcinoma (FTC) components expressed TIGT in any composite ATCs. CONCLUSION: TIGIT was immunohistochemically found in MTC with high frequency and partially in high-grade TC. TIGIT expression in cancer cells may be beneficial for a potential utility in MTC and a subset of high-grade TC, especially ATC therapy.

Hox13 is essential for formation of a sensory organ at the terminal end of the sperm duct in Ciona.

Species-specific traits are thought to have been acquired by natural selection. Transcription factors play central roles in the evolution of species-specific traits. Hox genes encode a set of conserved transcription factors essential for establishing the anterior-posterior body axis of animals. Changes in the expression or function of Hox genes can lead to the diversification of animal-body plans. The tunicate ascidian Ciona intestinalis Type A has an orange-colored structure at the sperm duct terminus. This orange-pigmented organ (OPO) is the characteristic that can distinguish this ascidian from other closely related species. The OPO is formed by the accumulation of orange-pigmented cells (OPCs) that are present throughout the adult body. We show that Hox13 is essential for formation of the OPO. Hox13 is expressed in the epithelium of the sperm duct and neurons surrounding the terminal openings for sperm ejection, while OPCs themselves do not express this gene. OPCs are mobile cells that can move through the body vasculature by pseudopodia, suggesting that the OPO is formed by the accumulation of OPCs guided by Hox13-positive cells. Another ascidian species, Ciona savignyi, does not have an OPO. Like Hox13 of C. intestinalis, Hox13 of C. savignyi is expressed at the terminus of its sperm duct; however, its expression domain is limited to the circular area around the openings. The genetic changes responsible for the acquisition or loss of OPO are likely to occur in the expression pattern of Hox13.

Hox-mediated endodermal identity patterns pharyngeal muscle formation in the chordate pharynx.

The chordate pharynx, possessing gill slits and the endostyle, is a complex of multiple tissues that are highly organized along the anterior-posterior (AP) axis. Although Hox genes show AP coordinated expression in the pharyngeal endoderm, tissue-specific roles of these factors for establishing the regional identities within this tissue have not been demonstrated. Here, we show that Hox1 is essential for the establishment of AP axial identity of the endostyle, a major structure of the pharyngeal endoderm, in the ascidian Ciona intestinalis We found that knockout of Hox1 causes posterior-to-anterior transformation of the endostyle identity, and that Hox1 represses Otx expression and anterior identity, and vice versa. Furthermore, alteration of the regional identity of the endostyle disrupts the formation of body wall muscles, suggesting that the endodermal axial identity is essential for coordinated pharyngeal development. Our results demonstrate an essential role of Hox genes in establishment of the AP regional identity in the pharyngeal endoderm and reveal crosstalk between endoderm and mesoderm during development of chordate pharynx.

Synthesis of 2'-O-(N-methylcarbamoylethyl) 5-methyl-2-thiouridine and its application to splice-switching oligonucleotides.

The effect of 2'-O-(N-methylcarbamoyl)ethyl (MCE) modification on splice-switching oligonucleotides (SSO) was systematically evaluated. The incorporation of five MCE nucleotides at the 5'-termini of SSOs effectively improved the splice switching effect. In addition, the incorporation of 2'-O-(N-methylcarbamoylethyl)-5-methyl-2-thiouridine (s2TMCE), a duplex-stabilizing nucleotide with an MCE modification, into SSOs further improved splice switching. These SSOs may be useful for the treatment of genetic diseases associated with splicing errors.

Modification of oligonucleotides with weak basic residues via the 2'-O-carbamoylethyl linker for improving nuclease resistance without loss of duplex stability and antisense activity.

For the improvement of nuclease resistance, four kinds of new modifications through a carbamoylethyl linker were designed. Among them, the 2'-O-[2-N-{2-(benzimidazol-1-yl)ethyl}carbamoylethyl] modification showed 20-fold longer half-life when treated with a 3' to 5' exonuclease compared to the 2'-O-methoxyethyl (MOE) modification, which is used in approved drugs. In addition, this large modification did not disturb the binding affinity or RNase H-dependent antisense activity. From these findings, it could be concluded that an adequate linker, such as carbamoylethyl in this study, could extend the utility of 2'-O-modification without loss of the properties of nucleic acids. This strategy would be useful for the development of nucleic acid therapeutics.

Germ cell regeneration-mediated, enhanced mutagenesis in the ascidian Ciona intestinalis reveals flexible germ cell formation from different somatic cells.

The ascidian Ciona intestinalis has a high regeneration capacity that enables the regeneration of artificially removed primordial germ cells (PGCs) from somatic cells. We utilized PGC regeneration to establish efficient methods of germ line mutagenesis with transcription activator-like effector nucleases (TALENs). When PGCs were artificially removed from animals in which a TALEN pair was expressed, somatic cells harboring mutations in the target gene were converted into germ cells, this germ cell population exhibited higher mutation rates than animals not subjected to PGC removal. PGC regeneration enables us to use TALEN expression vectors of specific somatic tissues for germ cell mutagenesis. Unexpectedly, cis elements for epidermis, neural tissue and muscle could be used for germ cell mutagenesis, indicating there are multiple sources of regenerated PGCs, suggesting a flexibility of differentiated Ciona somatic cells to regain totipotency. Sperm and eggs of a single hermaphroditic, PGC regenerated animal typically have different mutations, suggesting they arise from different cells. PGCs can be generated from somatic cells even though the maternal PGCs are not removed, suggesting that the PGC regeneration is not solely an artificial event but could have an endogenous function in Ciona. This study provides a technical innovation in the genome-editing methods, including easy establishment of mutant lines. Moreover, this study suggests cellular mechanisms and the potential evolutionary significance of PGC regeneration in Ciona.

TALEN-Based Knockout System.

Targeted mutagenesis of genes-of-interest is a powerful method of addressing the functions of genes. Genome editing techniques, such as transcriptional activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 systems, have enabled this approach in various organisms because of their ease of use. In the ascidian, Ciona intestinalis, recent studies show that TALEN-based knockout can be applied to establishing both mutant lines and tissue-specific knockout for addressing gene functions. Here, we introduce recent updates to the TALEN toolkit that facilitate detailed functional analysis of genes in ascidians.

Effective Postoperative Analgesia Using Intravenous Flurbiprofen and Acetaminophen.

PURPOSE: Management of postoperative pain is one of the most important components in postoperative care, because most patients have pain after dental surgery. The aim of this study was to evaluate whether acetaminophen could be an alternative to fentanyl in combination with a nonsteroidal anti-inflammatory drug (NSAID) as an analgesic after dental surgery in cases in which narcotic drugs were contraindicated. MATERIALS AND METHODS: Patients were 24- to 54-year-old men who underwent enucleation of a mandibular cyst under general anesthesia. The authors measured time from discontinuation of anesthetic administration until discharge from the operating room and postoperative pain during 4 hours after discharge. They compared these parameters between patients who were intravenously administered an NSAID such as flurbiprofen with fentanyl (NSAID/fentanyl group) and those administered an NSAID with acetaminophen (NSAID/acetaminophen group). Parametric data of time were analyzed using Student t test. Nonparametric data of the analgesic effect were analyzed using Mann-Whitney U test. RESULTS: Time until discharge from the operating room after discontinuation of anesthetics in the NSAID/fentanyl group was significantly longer than that in the NSAID/acetaminophen group (P < .05). In contrast, there was no significant difference in analgesic effect between the NSAID/acetaminophen and NSAID/fentanyl groups (P > .05). CONCLUSION: Although recovery time in the operating room of the flurbiprofen and acetaminophen group was markedly shorter than that of the flurbiprofen and fentanyl group, the postoperative analgesic effects of the 2 drugs were equipotent. Therefore, acetaminophen can be an alternative to fentanyl in cases in which narcotic drugs are contraindicated.

Tissue-specific and ubiquitous gene knockouts by TALEN electroporation provide new approaches to investigating gene function in Ciona.

Custom designed nucleases can simplify gene targeting experiments and have the potential to allow these techniques to be performed in a wide range of organisms. Transcriptional activator-like effector nucleases (TALENs) are starting to fulfill this potential with the advantages of low cost and fast construction times. Here, we report that TALENs are highly effective at inducing mutations in specific genomic loci in the ascidian chordate Ciona intestinalis. In Ciona there are well-established methods to introduce exogenous DNA by electroporation, and we show that this method can be used to introduce constructs that can express TALENs ubiquitously or in specific tissues. Our current protocols enable the rapid analysis of hundreds of TALEN-induced mutants. TALEN electroporations result in a high rate of mutations. These mutations can result in gene knockouts that recapitulate previously described functions of Fgf3 and Hox12. We show that TALENs can work efficiently to cause tissue-specific knockouts and demonstrate this by knocking out Hox12 in the epidermis and Fgf3 in neural tissues. We also use tissue-specific knockouts to reveal a new function of Fgf3 during ascidian larval metamorphosis.

A Case With Deteriorating Palmoplantar Pustulosis and Hyperthyroidism After Simultaneous Bimaxillary Orthognathic Surgery.

A case of palmoplantar pustulosis and hyperthyroidism following orthognathic surgery is presented. Both diseases may have been related to allergic phenomena.

Transcriptional regulation of a horizontally transferred gene from bacterium to chordate.

The horizontal transfer of genes between distantly related organisms is undoubtedly a major factor in the evolution of novel traits. Because genes are functionless without expression, horizontally transferred genes must acquire appropriate transcriptional regulations in their recipient organisms, although the evolutionary mechanism is not known well. The defining characteristic of tunicates is the presence of a cellulose containing tunic covering the adult and larval body surface. Cellulose synthase was acquired by horizontal gene transfer from Actinobacteria. We found that acquisition of the binding site of AP-2 transcription factor was essential for tunicate cellulose synthase to gain epidermal-specific expression. Actinobacteria have very GC-rich genomes, regions of which are capable of inducing specific expression in the tunicate epidermis as the AP-2 binds to a GC-rich region. Therefore, the actinobacterial cellulose synthase could have been potentiated to evolve its new function in the ancestor of tunicates with a higher probability than the evolution depending solely on a spontaneous event.

Chondroitin 6-O-sulfotransferases are required for morphogenesis of the notochord in the ascidian embryo.

BACKGROUND: Chondroitin sulfate (CS) is a sulfated polysaccharide chain that binds to various core proteins to form proteoglycans. The amount and position of sulfate groups in CS are variable among different tissues, and are determined by specific sulfotransferases. Although the ascidians are the closest relatives of vertebrates, the functions of their sulfotransferases have not been studied. RESULTS: The genome of the ascidian Ciona intestinalis contains eight genes encoding proteins similar to chondroitin 6-O-sulfotransferases (C6STs), which appear to have independently diverged in the ascidian lineage during evolution. Among them, Ci-C6ST-like1 and Ci-C6ST-like7 were predominantly expressed in the developing notochord. In addition, they were weakly expressed in the neural tube. The disruption of either one of them affected the convergent extension movement of notochordal cells. Presumptive notochord cells coming from both sides of the embryo did not intercalate. The results suggest that both of them are necessary. In some cases, the anterior neural tube failed to close. Forced expression of Ci-C6ST-like1 or Ci-C6ST-like7 in the notochord restored the normal intercalation of notochordal cells, indicating that the effects of morpholino oligos are specific. CONCLUSIONS: Ci-C6ST-like1 and Ci-C6ST-like7 are required for the morphogenesis of the notochord in the ascidian embryo.

Nonreproductive role of gonadotropin-releasing hormone in the control of ascidian metamorphosis.

BACKGROUND: Gonadotropin-releasing hormones (GnRHs) are neuropeptides that play central roles in the reproduction of vertebrates. In the ascidian Ciona intestinalis, GnRHs and their receptors are expressed in the nervous systems at the larval stage, when animals are not yet capable of reproduction, suggesting that the hormones have non-reproductive roles. RESULTS: We showed that GnRHs in Ciona are involved in the animal's metamorphosis by regulating tail absorption and adult organ growth. Absorption of the larval tail and growth of the adult organs are two major events in the metamorphosis of ascidians. When larvae were treated with GnRHs, they completed tail absorption more frequently than control larvae. cAMP was suggested to be a second messenger for the induction of tail absorption by GnRHs. tGnRH-3 and tGnRH-5 (the "t" indicates "tunicate") inhibited the growth of adult organs by arresting cell cycle progression in parallel with the promotion of tail absorption. CONCLUSIONS: This study provides new insights into the molecular mechanisms of ascidian metamorphosis conducted by non-reproductive GnRHs.

Germ cell mutations of the ascidian Ciona intestinalis with TALE nucleases.

Targeted mutagenesis of genes-of-interest, or gene-knockout, is a powerful method to address the functions of genes. Engineered nucleases have enabled this approach in various organisms because of their ease of use. The ascidian Ciona intestinalis is an excellent organism to analyze gene functions by means of genetic technologies. In our previous study, we reported mutagenesis of Ciona somatic cells with TALE nucleases (TALENs) by electroporating expression constructs. In this study, we report germ cell mutagenesis of Ciona by microinjecting mRNAs encoding TALENs. TALEN mRNAs introduced mutations to target genes in both somatic and germ cells. TALEN-mediated mutations in the germ cell genome were inherited by the next generation. We conclude that knockout lines of Ciona that have disrupted target genes can be established through TALEN-mediated germ cell mutagenesis.

CRISPR/Cas9-mediated gene knockout in the ascidian Ciona intestinalis.

Knockout of genes with CRISPR/Cas9 is a newly emerged approach to investigate functions of genes in various organisms. We demonstrate that CRISPR/Cas9 can mutate endogenous genes of the ascidian Ciona intestinalis, a splendid model for elucidating molecular mechanisms for constructing the chordate body plan. Short guide RNA (sgRNA) and Cas9 mRNA, when they are expressed in Ciona embryos by means of microinjection or electroporation of their expression vectors, introduced mutations in the target genes. The specificity of target choice by sgRNA is relatively high compared to the reports from some other organisms, and a single nucleotide mutation at the sgRNA dramatically reduced mutation efficiency at the on-target site. CRISPR/Cas9-mediated mutagenesis will be a powerful method to study gene functions in Ciona along with another genome editing approach using TALE nucleases.

Protocol for forward genetic screening to identify novel factors involved in a biological process in Caenorhabditis elegans.

Forward genetic screens have been a powerful tool for discovering genes involved in various biological processes in Caenorhabditis elegans. Here, we present a protocol for forward genetic screening to identify novel factors involved in a biological process in C. elegans. We describe steps for mutagenesis, screening, and backcrossing. To save time and effort, we also detail procedures for utilizing whole-genome sequencing to exclude mutants of previously characterized genes from crosses for mapping mutations. For complete details on the use and execution of this protocol, please refer to Yoshida et al.1.

Gastric metastasis of renal cell carcinoma treated with endoscopic resection: A case report.

Gastric metastasis of renal cell carcinoma (RCC) is rarely encountered. The time interval between the primary diagnosis of RCC and the occurrence of gastric metastasis tends to occur after more than 10 years. Clinicians should be diligent in checking the general symptoms of patients for more than 10 years.

Prognostic Evaluation of HER2-positive Early Breast Cancers Using Clinico-pathological Criteria.

Background/Aim: HER2-positive breast carcinomas (BCs) generally behave more aggressively and show higher cytological and histological grade than HER2-negative BCs. However, the clinical properties of HER2-positive early BCs have not been studied extensively. Hence, the therapeutic significance of neoadjuvant chemotherapy (NAC) for this BC remains debatable. Patients and Methods: We retrospectively examined the clinicopathological features of 94 HER2-positive early BCs who perioperatively received anti-HER2 drugs, without undergoing NAC prior to surgery. Results: The patients' five year-disease free survival (DFS) and overall survival (OS) rates were 95.6% and 100%, respectively. Univariate analysis demonstrated significant differences in distant metastasis-free survival (DMFS) between clinical and pathological tumor stages (T stages). Pathological T1 stage and clinical T1 stage tumors showed significantly higher DMSF than pT2-3 and cT2-3 (p=0.0002 and 0.0294). Multivariate analysis disclosed no significant differences in DFS, OS, and DMFS with respect to preoperative clinical tumor stage, patient age, type of surgery, postoperative therapy, and pathological factors. Recurrences occurred in nine patients: four (4.3%) and five (5.3%) patients showed local and distant recurrences, respectively. One patient with cT2 BC died of disease. Interestingly, four of the five BCs with distant recurrence pathologically demonstrated lymph vessel invasion. The prognoses of patients with HER2-positive stage cT1/2N0M0 BC were highly favorable. Conclusion: The indications for NAC in small, localized, and node-negative HER2-positive BC should be carefully assessed based on the presence of a larger tumor size, postoperative pathological evaluation of tumor size, and lymph vessel invasion.