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Abstract Background Previously, Japanese Environmental Mutagen and Genome Society/Mammalian Mutagenicity Study Group/Toxicogenomics Study Group (JEMS/MMS toxicogenomic study group) proposed 12 genotoxic marker genes (Aen, Bax, Btg2, Ccnf, Ccng1, Cdkn1a, Gdf15, Lrp1, Mbd1, Phlda3, Plk2, and Tubb4b) to discriminate genotoxic hepatocarcinogens (GTHCs) from non-genotoxic hepatocarcinogens (NGTHCs) and non-genotoxic non-hepatocarcinogens (NGTNHCs) in mouse and rat liver using qPCR and RNA-Seq and confirmed in public rat toxicogenomics data, Open TG-GATEs, by principal component analysis (PCA). On the other hand, the U.S. Environmental Protection Agency (US EPA) suggested seven genotoxic marker genes (Bax, Btg2, Ccng1, Cgrrf1, Cdkn1a, Mgmt, and Tmem47) with Open TG-GATEs data. Four genes (Bax, Btg2, Ccng1, and Cdkn1a) were common in these two studies. In the present study, we examined the performance of these four genes in Open TG-GATEs data using PCA. Results The study's findings are of paramount significance, as these four genes proved to be highly effective in distinguishing five typical GTHCs (2-acetylaminofluorene, aflatoxin B1, 2-nitrofluorene, N-nitrosodiethylamine and N-nitrosomorpholine) from seven typical NGTHCs (clofibrate, ethanol, fenofibrate, gemfibrozil, hexachlorobenzene, phenobarbital, and WY-14643) and 11 NGTNHCs (allyl alcohol, aspirin, caffeine, chlorpheniramine, chlorpropamide, dexamethasone, diazepam, indomethacin, phenylbutazone, theophylline, and tolbutamide) by PCA at 24 h after a single administration with 100% accuracy. These four genes also effectively distinguished two typical GTHCs (2-acetylaminofluorene and N-nitrosodiethylamine) from seven NGTHCs and ten NGTNHCs by PCA on 29 days after 28 days-repeated administrations, with a similar or even better performance compared to the previous 12 genes. Furthermore, the study's analysis revealed that the three intermediate GTHC/NGTHCs (methapyrilene, monocrotaline, and thioacetamide, which were negative in the Salmonella test but positive in the in vivo rat liver test) were located in the intermediate region between typical GTHCs and typical NGTHCs by PCA. Conclusions The present results unequivocally demonstrate the availability of four genotoxic marker genes ((Bax, Btg2, Ccng1, and Cdkn1a) and PCA in discriminating GTHCs from NGTHCs and NGTNHCs in Open TG-GATEs. These findings strongly support our recommendation that future rat liver in vivo toxicogenomics tests prioritize these four genotoxic marker genes, as they have proven to be highly effective in discriminating between different types of hepatocarcinogens.

Abstract Background Error-corrected next-generation sequencing (ecNGS) technologies have enabled the direct evaluation of genome-wide mutations after exposure to mutagens. Previously, we reported an ecNGS methodology, Hawk-Seq™, and demonstrated its utility in evaluating mutagenicity. The evaluation of technical transferability is essential to further evaluate the reliability of ecNGS-based assays. However, cutting-edge sequencing platforms are continually evolving, which can affect the sensitivity of ecNGS. Therefore, the effect of differences in sequencing instruments on mutation data quality should be evaluated. Results We assessed the performance of four sequencing platforms (HiSeq2500, NovaSeq6000, NextSeq2000, and DNBSEQ-G400) with the Hawk-Seq™ protocol for mutagenicity evaluation using DNA samples from mouse bone marrow exposed to benzo[a]pyrene (BP). The overall mutation (OM) frequencies per 106 bp in vehicle-treated samples were 0.22, 0.36, 0.46, and 0.26 for HiSeq2500, NovaSeq6000, NextSeq2000, and DNBSEQ-G400, respectively. The OM frequency of NextSeq2000 was significantly higher than that of HiSeq2500, suggesting the difference to be based on the platform. The relatively higher value in NextSeq2000 was a consequence of the G:C to C:G mutations in NextSeq2000 data (0.67 per 106 G:C bp), which was higher than the mean of the four platforms by a ca. of 0.25 per 106 G:C bp. A clear dose-dependent increase in G:C to T:A mutation frequencies was observed in all four sequencing platforms after BP exposure. The cosine similarity values of the 96-dimensional trinucleotide mutation patterns between HiSeq and the three other platforms were 0.93, 0.95, and 0.92 for NovaSeq, NextSeq, and DNBSeq, respectively. These results suggest that all platforms can provide equivalent data that reflect the characteristics of the mutagens. Conclusions All platforms sensitively detected mutagen-induced mutations using the Hawk-Seq™ analysis. The substitution types and frequencies of the background errors differed depending on the platform. The effects of sequencing platforms on mutagenicity evaluation should be assessed before experimentation.

Abstract Background To prospectively evaluate binocular visual outcomes after implantation of trifocal intraocular lenses (IOLs) with high-water-content hydrophobic acrylic material in Japanese patients. Methods In 59 patients (mean age 65.1 ± 7.9 years), Clareon PanOptix (CNWTT0: Alcon) with a high-water-content hydrophobic acrylic material was implanted bilaterally. Three months postoperatively, binocular uncorrected visual acuity (BUCVA) and distance-corrected visual acuity (BDCVA) at distances of 5 m, 80, 60, and 40 cm, binocular defocus curves, and binocular photopic contrast sensitivity were examined. Subjective symptoms (night vision disturbance, glare, halos, haze, or blurry vision) were also assessed. Results The mean postoperative BUCVA/BDCVA at 5 m, 80 cm, 60 cm, and 40 cm were − 0.115/-0.163, -0.052/-0.047, -0.054/-0.075, and − 0.043/-0.067 logMAR, respectively. A smooth defocus curve, contrast sensitivity within the normal range, and acceptable subjective symptom rates were obtained. Conclusions The trifocal IOL, composed of a high-water-content hydrophobic acrylic material, provides good continuous binocular vision from distance to near. Trial registration This investigator-initiated study was registered in the Japan Registry for Clinical Trials (identifier: jRCTs032220042) on April 26, 2022.

IntroductionTraditionally formal assessment of surgical skills has not been part of a surgeon’s accreditation process. The widely adopted apprentice model of “on-the-job training” does create additional risk for the patients. In the past surgical training has used cadavers, but these are expensive, require dedicated wet-lab facilities and are in increasingly short supply. In many countries religious and cultural practices also preclude cadaveric use. Recent 3D-printed technology allows mass reproduction of high-fidelity 3D models. In this study, we examined the utility of 3D sinus models compared to cadaver dissection for surgical skill assessment for endoscopic sinus surgery (ESS).Materials and methodsA total of 17 otolaryngologists performed Endoscopic Sinus Surgery (ESS) on 3D printed sinus models and then repeated these procedures on cadavers. Their surgical performance was assessed with the Objective Structured Assessment of Technical Skills (OSATS) score for ESS and time was taken to complete an ESS procedure. Their performance on the 3D models and cadavers was compared.ResultsThere were no significant differences in the OSATS score between 3D models and cadavers (50.41 ± 13.31 vs. 48.29 ± 16.01, p = 0.36). There was a strong positive correlation between the score in 3D models and those in cadavers (r = 0.84, p

Background: We developed a technique to preserve the entire nasolacrimal duct and inferior turbinate, by swinging the nasolacrimal duct upward and the inferior turbinate backward; that is, a nasolacrimal duct and inferior turbinate swing technique for sinonasal inverted papilloma of the maxillary sinus. In this study, we evaluated the long-term results of this technique. Methods: A retrospective analysis was performed on the data for 53 consecutive pathologically confirmed inverted papilloma patients who were treated using nasolacrimal duct and inferior turbinate swing technique. The frequency of recurrence, the degree of nasolacrimal duct and inferior turbinate preservation, and the frequency of adverse events were assessed. Results: The median follow-up period was 51 months (mean 62.5 months; range 10–187 months). No cases of recurrence within the maxillary sinus were observed among the cases treated using this technique, whereas three (5.7%) of the 53 cases experienced recurrence in areas other than the maxillary sinus. We could preserve the nasolacrimal duct in all cases. On the other hand, the inferior turbinate was resected during surgery in one case. No epiphora, dacryocystiits, numbness of the cheek, or collapse of the ala of the nose was observed. Conclusion: This nasolacrimal duct and inferior turbinate swing technique method is a safe and effective method for the excision of primary or recurrent inverted papilloma and demonstrates low rates of recurrence and complications.

Plant homeodomain finger protein 8 (PHF8) is a histone demethylase that regulates the expression of various genes. PHF8 targets repressor histone markers and activates gene expression. Although PHF8 has been involved in X-linked mental retardation and certain types of cancers, the role of PHF8 remains largely unknown, and its relevance to the pathogenesis of these diseases is also uncertain. In the present study, we aimed to clarify the cellular function of PHF8 in P19 cells using Phf8 knockout (KO) cells generated via the CRISPR-Cas9 system and by performing PHF8 specific inhibitor experiments, instead of using PHF8 small interfering RNA transfection. After establishing Phf8 KO cells, we analyzed the effects of PHF8 on neuronal differentiation and cell proliferation. Both PHF8 deficiency and inhibition of its activity did not considerably affect neuronal differentiation, however, they showed an increased trend of promoted neurite outgrowth. Moreover, we found that PHF8 regulated cell proliferation via the MEK/ERK pathway. PHF8 deficiency and activity inhibition reduced the phosphorylation of ERK and MEK. The MEK expression level was associated with PHF8 expression, as revealed by chromatin immunoprecipitation analysis. These results suggested that PHF8 regulates cell proliferation via the MEK/ERK pathway in P19 embryonic carcinoma cells.

Abstract Background and Aim The use of a hood at the tip of a colonoscope enables aspiration, inversion of the diverticulum, and observation of the inside of the diverticulum. In most previous studies, a short hood was used; however, observation of the diverticulum is often inadequate. Long food is promising by previous research, but it was a retrospective study using propensity matching and has some limitations. We compared the identification rate of stigmata of recent hemorrhage (SRH) between the long and standard hoods in cases of suspected colonic diverticular hemorrhage (CDH) to confirm the usefulness of long hood by prospective randomized controlled trial. Methods Eighty patients (42 in the long hood group [L group] and 38 in the short hood group [S group]) who visited the Saitama Medical University Hospital and Tokai University Hachioji Hospital between December 2018 and July 2021 with a chief complaint of bloody stool and suspected CDH, based on the clinical course and imaging studies, were included. Patients were randomly assigned to the L or S group. Results Regarding patient background, age was significantly higher in the L group; however, no significant differences were found in medical history or history of antithrombotic medication or nonsteroidal anti‐inflammatory drug use. Identification rate of SRH was significantly higher in the L group (58.5%, 24/42 patients) than in the S group (26.3%, 10/38 patients) (P

Introduction: Hemobilia, which refers to bleeding from the bile duct, is rare and difficult to treat. We report a case of successful hemostasis of a pancreatic tumor complicated by hemobilia. Case Presentation: A 76-year-old man was referred to our hospital with a pancreatic head tumor. Endoscopic retrograde cholangiopancreatography (ERCP) and endoscopic ultrasonography-FNA were performed, and the patient was diagnosed with pancreatic metastasis of renal cell carcinoma. After discharge, the patient noted worsening jaundice and progressive anemia and was readmitted. ERCP reveals active bleeding from the duodenal papillae. The patient was placed on a fully covered metallic stent and discharged after confirming hemostasis. Conclusion: Renal cell carcinoma is a tumor with abundant blood flow. If hemobilia occurs, bleeding from pancreatic metastatic tumors should be considered. Additionally, hemostasis using a fully covered metallic stent is useful for treating hemobilia in tumors.

Abstract Background Histone acetylation, which is regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs), plays a crucial role in the control of gene expression. HDAC inhibitors (HDACi) have shown potential in cancer therapy; however, the specific roles of HDACs in early embryos remain unclear. Moreover, although some pan-HDACi have been used to maintain cellular undifferentiated states in early embryos, the specific mechanisms underlying their effects remain unknown. Thus, there remains a significant knowledge gap regarding the application of selective HDACi in early embryos. Results To address this gap, we treated early embryos with two selective HDACi (MGCD0103 and T247). Subsequently, we collected and analyzed their transcriptome data at different developmental stages. Our findings unveiled a significant effect of HDACi treatment during the crucial 2-cell stage of zygotes, leading to a delay in embryonic development after T247 and an arrest at 2-cell stage after MGCD0103 administration. Furthermore, we elucidated the regulatory targets underlying this arrested embryonic development, which pinpointed the G2/M phase as the potential period of embryonic development arrest caused by MGCD0103. Moreover, our investigation provided a comprehensive profile of the biological processes that are affected by HDACi, with their main effects being predominantly localized in four aspects of zygotic gene activation (ZGA): RNA splicing, cell cycle regulation, autophagy, and transcription factor regulation. By exploring the transcriptional regulation and epigenetic features of the genes affected by HDACi, we made inferences regarding the potential main pathways via which HDACs affect gene expression in early embryos. Notably, Hdac7 exhibited a distinct response, highlighting its potential as a key player in early embryonic development. Conclusions Our study conducted a comprehensive analysis of the effects of HDACi on early embryonic development at the transcriptional level. The results demonstrated that HDACi significantly affected ZGA in embryos, elucidated the distinct actions of various selective HDACi, and identified specific biological pathways and mechanisms via which these inhibitors modulated early embryonic development.

Abstract Background Carcinogenic risk assessment studies have been repeatedly improved and are still being debated to find a goal. Evaluation might be changed if new approaches would be applied to some chemicals which means that new approaches may change the final assessment. In this paper, the risk assessment of a chemical, in particular the proper carcinogenicity, is examined using the long-banned food additive, 2-(2-furyl)-3-(5-nitro-2-furyl)-acrylamide, AF-2, as a case study. Results First, Ames tests were carried out using strains TA1535, TA100, TA1538, and TA98 and their nitroreductase-deficient strains YG7127, YG7128, YG7129, and YG7130. The results showed that mutagenic activity was reduced by about 50% in the nitroreductase-deficient strains, indicating that part of the mutagenic activity shown in Ames test was due to bacterial metabolism. Second, in vivo genotoxicity tests were conducted, including the one that had not been developed in 1970’s. Both a micronucleus test and a gene mutation assay using transgenic mice were negative. Third, assuming it is a genotoxic carcinogen, the virtual safety dose of 550 μg/day was calculated from the TD50 in rats with a probability of 10−5. Conclusion AF-2 has been shown to be carcinogenic to rodents and has previously been indicated to be genotoxic in vitro. However, the present in vivo genotoxicity study, it was negative in the forestomach, a target organ for cancer, particularly in the gene mutation assay in transgenic mice. Considering the daily intake of AF-2 in the 1970s and its virtually safety dose, the carcinogenic risk of AF-2 could be considered acceptable.

Abstract Background Tri-weekly cisplatin and radiotherapy (CDDP + RT) is a standard of care for locally advanced head and neck squamous cell carcinoma (LA-HNSCC) but is sometimes challenging to complete in older patients. Weekly CDDP + RT has shown mild toxicity compared to tri-weekly CDDP + RT for LA-HNSCC and is a promising option for older adults. We aimed to report the treatment outcomes and prognostic factors in patients with LA-HNSCC treated with weekly CDDP + RT. Methods We analyzed patients aged ≥ 70 years who started weekly CDDP + RT for LA-HNSCC between July 2006 and October 2022. LA-HNSCC includes cancer in the oropharynx, hypopharynx, or larynx with a clinical stage of 3 or 4 without distant metastases based on the Union for International Cancer Control staging system 8th edition. The radiation dose of 70 Gy was delivered in 35 fractions by 3-dimensional conformal radiotherapy, intensity-modulated radiotherapy, or proton beam therapy. The primary endpoint was the 3-year overall survival (OS), and the secondary endpoints were the 3-year progression-free survival (PFS) and 3-year cause-specific survival (CSS). The Kaplan–Meier method was used to calculate survival rates, and the log-rank test was used to evaluate statistical significance. A Cox proportional hazards model was used for the multivariate analysis of prognostic factors. Results The median age of the 49 patients was 72 (range: 70–78) years. The median CDDP dose was 200 (40–280) mg/ m2, and 47 patients completed scheduled radiotherapy. Forty-eight patients (98.0%) had a performance status of ≥ 1 at the initial visit. The 3-year OS, PFS, and CSS were 80.9% (95% confidence interval [CI]: 64.8–90.7), 58.9% (95%CI: 42.7–73.3), and 85.0% (95%CI: 68.7–93.4), respectively. In the multivariate analysis, the cumulative CDDP dose (

Abstract Background Mutagenicity, the ability of chemical agents to cause mutations and potentially lead to cancer, is a critical aspect of substance safety assessment for protecting human health and the environment. Metabolic enzymes activate multiple mutagens in living organisms, thus in vivo animal models provide highly important information for evaluating mutagenicity in human. Rats are considered suitable models as they share a similar metabolic pathway with humans for processing toxic chemical and exhibit higher responsiveness to chemical carcinogens than mice. To assess mutagenicity in rats, transgenic rodents (TGRs) are widely used for in vivo gene mutation assays. However, such assays are labor-intensive and could only detect transgene mutations inserted into the genome. Therefore, introducing a technology to directly detect in vivo mutagenicity in rats would be necessary. The next-generation sequencing (NGS) based error-corrected sequencing technique is a promising approach for such purposes. Results We investigated the applicability of paired-end and complementary consensus sequencing (PECC-Seq), an error-corrected sequencing technique, for detecting in vivo mutagenicity in the rat liver samples. PECC-Seq allows for the direct detection of ultra-rare somatic mutations in the genomic DNA without being constrained by the genomic locus, tissue, or organism. We tested PECC-Seq feasibility in rats treated with diethylnitrosamine (DEN), a mutagenic compound. Interestingly, the mutation and mutant frequencies between PECC-Seq and the TGR assay displayed a promising correlation. Our results also demonstrated that PECC-Seq could successfully detect the A:T > T:A mutation in rat liver samples, consistent with the TGR assay. Furthermore, we calculated the trinucleotide mutation frequency and proved that PECC-Seq accurately identified the DEN treatment-induced mutational signatures. Conclusions Our study provides the first evidence of using PECC-Seq for in vivo mutagenicity detection in rat liver samples. This approach could provide a valuable alternative to conventional TGR assays as it is labor- and time-efficient and eliminates the need for transgenic rodents. Error-corrected sequencing techniques, such as PECC-Seq, represent promising approaches for enhancing mutagenicity assessment and advancing regulatory science.

BackgroundOlfactory neuroblastoma (ONB) is a rare malignant tumor arising from the olfactory neuroepithelium. The standard of care for ONB is surgical resection; however, detailed treatment protocols vary by institution. Our treatment protocol consists of endoscopic skull base surgery (ESBS) for endoscopically resectable cases and induction chemotherapy followed by craniotomy combined with ESBS for locally advanced cases, with postoperative radiotherapy performed for all cases. Chemoradiotherapy (CRT) is performed in unresectable cases. In this study, we evaluate our treatment protocol and outcomes for ONB.MethodsA retrospective review of patients with ONB was conducted. Outcomes included survival outcomes and perioperative data.ResultsFifteen patients (53.6%) underwent ESBS, 12 (42.9%) underwent craniotomy combined with ESBS, and 1 (3.6%) received CRT. The 5- and 10-year overall survival rates for all patients were 92.9% and 82.5%, respectively, with a median follow-up period of 81 months. The 5- and 10-year disease-free survival rates were 77.3% and 70.3%, respectively, and the 5- and 10-year local control rates were 88.2% and 80.2%, respectively. Patients undergoing ESBS demonstrated a significantly shorter operating time, period from operation to ambulation, hospitalization period, and less blood loss than those undergoing craniotomy combined with ESBS.ConclusionOur treatment protocol was found to afford favorable outcomes. Patients who underwent endoscopic resection showed lower complication rates and better perioperative data than those who underwent craniotomy combined with ESBS. With appropriate case selection, ESBS is considered a useful approach for ONB.

IntroductionThe rise in antimicrobial resistance (AMR) that is affecting humans, animals, and the environment, compromises the human immune system and represents a significant threat to public health. Regarding the impact on water sanitation, the risk that antimicrobial-resistant genes (ARGs) and antimicrobial-resistant bacteria in surface water in cities pose to human health remains unclear. To determine the prevalence of AMR in environmental surface water in Japan, we used DNA sequencing techniques on environmental water DNA (eDNA) and the DNA of multidrug-resistant bacteria (mrDNA).MethodsThe eDNA was extracted from four surface water samples obtained from the Tokyo area and subjected to high- throughput next-generation DNA sequencing using Illumina-derived shotgun metagenome analysis. The sequence data were analyzed using the AmrPlusPlus pipeline and the MEGARes database. Multidrug-resistant bacteria were isolated using a culture-based method from water samples and were screened by antimicrobial susceptibility testing (for tetracycline, ampicillin-sulbactam, amikacin, levofloxacin, imipenem, and clarithromycin). Of the 284 isolates, 22 were identified as multidrug-resistant bacteria. The mrDNA was sequenced using the Oxford nanopore MinION system and analyzed by NanoARG, a web service for detecting and contextualizing ARGs.Results and discussionThe results from eDNA and mrDNA revealed that ARGs encoding beta-lactams and multidrug resistance, including multidrug efflux pump genes, were frequently detected in surface water samples. However, mrDNA also revealed many sequence reads from multidrug-resistant bacteria, as well as nonspecific ARGs, whereas eDNA revealed specific ARGs such as pathogenic OXA-type and New Delhi metallo (NDM)-beta-lactamase ARGs.ConclusionTo estimate potential AMR pollution, our findings suggested that eDNA is preferable for detecting pathogen ARGs.

Abstract Next-generation RNA sequencing (RNA-Seq) has identified more differentially expressed protein-coding genes (DEGs) and provided a wider quantitative range of expression level changes than conventional DNA microarrays. JEMS·MMS·Toxicogenomics group studied DEGs with targeted RNA-Seq on freshly frozen rat liver tissues and on formalin-fixed paraffin-embedded (FFPE) rat liver tissues after 28 days of treatment with chemicals and quantitative real-time PCR (qPCR) on rat and mouse liver tissues after 4 to 48 h treatment with chemicals and analyzed by principal component analysis (PCA) as statics. Analysis of rat public DNA microarray data (Open TG-GATEs) was also performed. In total, 35 chemicals were analyzed [15 genotoxic hepatocarcinogens (GTHCs), 9 non-genotoxic hepatocarcinogens (NGTHCs), and 11 non-genotoxic non-hepatocarcinogens (NGTNHCs)]. As a result, 12 marker genes (Aen, Bax, Btg2, Ccnf, Ccng1, Cdkn1a, Gdf15, Lrp1, Mbd1, Phlda3, Plk2, and Tubb4b) were proposed to discriminate GTHCs from NGTHCs and NGTNHCs. U.S. Environmental Protection Agency studied DEGs induced by 4 known GTHCs in rat liver using DNA microarray and proposed 7 biomarker genes, Bax, Bcmp1, Btg2, Ccng1, Cdkn1a, Cgr19, and Mgmt for GTHCs. Studies involving the use of whole-transcriptome RNA-Seq upon exposure to chemical carcinogens in vivo have also been performed in rodent liver, kidney, lung, colon, and other organs, although discrimination of GTHCs from NGTHCs was not examined. Candidate genes published using RNA-Seq, qPCR, and DNA microarray will be useful for the future development of short-term in vivo studies of environmental carcinogens using RNA-Seq.

Background and Aims: Japan has experienced 8 waves of the coronavirus disease 2019 (COVID-19) outbreak over the past 3 years, resulting in an increasing number of deaths and incidence of severe infections. This study aimed to analyze the data from the Japanese inflammatory bowel disease (IBD) patients with COVID-19 registry (J-COSMOS) up to the eighth wave to investigate the clinical course of IBD patients with COVID-19 and factors contributing to disease severity. Methods: In this multicenter, observational, cohort study, we analyzed a cohort of 1308 IBD patients diagnosed with COVID-19, enrolled across 77 participating facilities in the J-COSMOS registry from June 2020 to December 2022. Data on age, sex, IBD (classification, treatment, and activity), and COVID-19 (symptoms, severity, and treatment) were analyzed. Results: The majority of patients (76%) were in clinical remission. According to the World Health Organization classification of COVID-19 severity, 98.4% of IBD patients had nonsevere disease, while 1.6% of patients had severe or critical disease. COVID-19 did not affect disease activity in most IBD patients. Stepwise logistic regression analysis revealed that high body mass index, and cerebrovascular disease were risk factors for severe COVID-19. Corticosteroids could affect COVID-19 severity, whereas anti-tumor necrosis factor α antibodies and thiopurines were associated with a reduced risk of severe COVID-19. No deaths were observed among IBD patients with COVID-19 registered in this cohort. Conclusion: The impact of COVID-19 on IBD disease activity and factors associated with COVID-19 severity were consistent with findings of previous reports. No deaths in Japanese patients with IBD were observed.

This real-world study examined the prevalence of programmed death ligand-1 (PD-L1) expression and assessed the frequency of microsatellite instability-high (MSI-H) status and Epstein-Barr virus (EBV) positivity in Japanese patients with advanced gastric and gastroesophageal junction (GEJ) adenocarcinoma. This multicenter (5 sites), retrospective, observational study (November 2018–March 2019) evaluated Japanese patients with advanced gastric and GEJ adenocarcinoma after surgical resection (Stage II/III at initial diagnosis) or unresectable advanced cancer (Stage IV). The primary objectives were prevalence of PD-L1 expression (combined positive score [CPS] ≥1), MSI status, and EBV positivity. Tumor specimens of 389/391 patients were analyzed (male, 67.1%; mean age, 67.6 ± 12.2 years); 241/389 (62%) were PD-L1 positive, 24/379 (6.3%) had MSI-H tumors, and 13/389 (3.3%) were EBV positive. PD-L1 expression was higher in tumor-infiltrating immune cells than in tumor cells for lower CPS cutoffs. Among patients with MSI-H tumors and EBV-positive tumors, 19/24 (79.2%) and 9/13 (69.2%), respectively, were PD-L1 positive. A greater proportion of patients with MSI-H tumors (83.3% [20/24]) were PD-L1 positive than those with MSI-low/stable tumors (60.8% [216/355]; p = .0297); similarly, an association was observed between history of H pylori infection and PD-L1 expression. A higher proportion of patients with MSI-H tumors demonstrated PD-L1 expression with a CPS ≥10 (66.7% [16/24]) vs those with MSI-low/stable tumors (24.8% [88/355]; p

Abstract Molecular targets and predictive biomarkers for prognosis in salivary duct carcinoma (SDC) have not been fully identified. We conducted comprehensive molecular profiling to discover novel biomarkers for SDC. A total of 67 SDC samples were examined with DNA sequencing of 464 genes and transcriptome analysis in combination with the clinicopathological characteristics of the individuals. Prognostic biomarkers associated with response to combined androgen blockade (CAB) treatment were explored using mRNA expression data from 27 cases. Oncogenic mutations in receptor tyrosine kinase (RTK) genes or genes in the MAPK pathway were identified in 55 cases (82.1%). Alterations in the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway were identified in 38 cases (56.7%). Interestingly, patient prognosis could be predicted using mRNA expression profiles, but not genetic mutation profiles. The risk score generated from the expression data of a four-gene set that includes the ADAMTS1, DSC1, RNF39, and IGLL5 genes was a significant prognostic marker for overall survival in the cohort (HR = 5.99, 95% confidence interval (CI) = 2.73–13.1, p = 7.8 × 10−6). Another risk score constructed from the expression of CD3E and LDB3 was a strong prognostic marker for progression-free survival for CAB treatment (p = 0.03). Mutations in RTK genes, MAPK pathway genes, and PI3K/AKT pathway genes likely represent key mutations in SDC tumorigenesis. The gene expression profiles identified in this study may be useful for stratifying patients who are good candidates for CAB treatment and may benefit from additional systemic therapies.

Abstract Background The purpose of this study was to find a utility of a newly developed 3D‐printed sinus model and to evaluate the educational benefit of simulation training with the models for functional endoscopic sinus surgery (FESS). Material and methods Forty‐seven otolaryngologists were categorized as experts (board‐certified physicians with ≥200 experiences of FESS, n = 9), intermediates (board‐certified physicians with

Abstract Background Pneumatosis intestinalis (PI) is a rare condition characterized by gas collection in the intestinal wall. We aimed to determine the etiology and affected segments associated with complications, treatment, and outcome. Methods We conducted a multicenter epidemiological survey using a standardized data collection sheet in Japan. Complicating PI was defined as strangulation or bowel necrosis, bowel obstruction, adynamic ileus, sepsis, shock, and massive gastrointestinal bleeding requiring blood transfusion. Results We enrolled 167 patients from 48 facilities. Multivariate analysis revealed that older age (adjusted OR, 1.05 and 95% confidence intervals [CI], 1.02–1.09, P = 0.0053) and chronic kidney disease (adjusted OR, 13.19 and 95% CI 1.04–167.62, P = 0.0468) were independent predictors of the small-bowel-involved type. Complicating PI was associated with the small-bowel-involved combined type (adjusted OR, 27.02 and 95% CI 4.80–152.01, P = 0.0002), the small-bowel-only type (adjusted OR, 3.94 and 95% CI 1.02–15.27, P = 0.0472), and symptomatic PI (adjusted OR, 16.24 and 95% CI 1.82–145.24, P = 0.0126). Oxygen therapy was performed in patients with a past history of bowel obstruction (adjusted OR, 13.77 and 95% CI 1.31–144.56, P = 0.0288) and surgery was performed in patients with complicating PI (adjusted OR, 8.93 and 95% CI 1.10–72.78, P = 0.0408). Antihypertensives (adjusted OR, 12.28 and 95% CI 1.07–140.79, P = 0.0439) and complicating PI (adjusted OR, 11.77 and 95% CI 1.053–131.526; P = 0.0453) were associated with exacerbation of PI. The complicating PI was the only indicator of death (adjusted OR, 14.40 and 95% CI 1.09–189.48, P = 0.0425). Discussion Small-bowel-involved type and symptomatic PI were associated with complications which were indicators of poor prognosis.

IntroductionSurgeons’ mental workload during endoscopic sinus surgery (ESS) has not been fully evaluated. The assessment was challenging due to the great diversity of each patient’s anatomy and the consequence variety of surgical difficulties. In this study, we examined the mental workload of surgeons with various surgical skill levels during ESS under the standardized condition provided by novel-designed 3D sinus models.Materials and methodsForty-seven participants performed a high-fidelity ESS simulation with 3D-printed sinus models. Surgeons’ mental workload was assessed with the national aeronautics and space administration-task load index (NASA-TLX). Associations between the total and subscales score of NASA-TLX and surgical skill index, including the board certification status, the number of experienced ESS cases, and the objective structured assessment of technical skills (OSATS), were analyzed. In addition, 10 registrars repeated the simulation surgery, and their NASA-TLX score was compared before and after the repetitive training.ResultsThe total NASA-TLX score was significantly associated with OSATS score (p = 0.0001). Primary component analysis classified the surgeons’ mental burden into three different categories: (1) the skill-level-dependent factors (temporal demand, effort, and performance), (2) the skill-level-independent factors (mental and physical demand), and (3) frustration. After the repetitive training, the skill-level-dependent factors were alleviated (temporal demand; z = −2.3664, p = 0.0091, effort; z = −2.1704, p = 0.0346, and performance; z = −2.5992, p = 0.0017), the independent factors were increased (mental demand; z = −2.5992, p = 0.0023 and physical demand; z = −2.2509, p = 0.0213), and frustration did not change (p = 0.3625).ConclusionSome of the mental workload during ESS is associated with surgical skill level and alleviated with repetitive training. However, other aspects remain a burden or could worsen even when surgeons have gained surgical experience. Routine assessment of registrars’ mental burdens would be necessary during surgical training to sustain their mental health.

Abstract Breast cancer is one of the most common malignancies in women worldwide. Although most breast cancers are curable, in cases of metastasis, many are often found in the lungs, bones, liver, and central nervous system; however, metastasis to the gastrointestinal tract is rare. Invasive lobular carcinoma, which represents only 5%–10% of breast cancers, has a higher risk of metastasis to the gastrointestinal tract than invasive ductal carcinoma. Here, we report a rare case of gastrointestinal metastasis of invasive lobular carcinoma that spread extensively to the colonic mucosa. Given the improved survival rates of breast cancer patients with current treatments, many rarer metastatic diseases, including gastrointestinal metastases, are likely to be increased in the future.

Background and aimAdherent-invasive E. coli (AIEC) has been identified as a pathobiont associated with Crohn’s disease (CD), that prefers to grow in inflammatory conditions. Although the colonization by AIEC is implicated in the progression of the disease and exacerbates inflammation in murine colitis models, the recognition and response of host immunity to AIEC remains elusive.MethodsAntibiotic treated female C57BL/6 mice were inoculated by commensal E. coli and LF82 AIEC strains. Luminal-IgA fractions were prepared from feces and their binding to AIEC and other strains was assessed to confirm specificity. IgA binding to isogenic mutant strains was performed to identify the functional molecules that are recognized by AIEC specific IgA. The effect of IgA on epithelial invasion of LF82 strain was confirmed using in vitro invasion assay and in vivo colonization of the colonic epithelium.ResultsPersistent colonization by AIEC LF82 induced secretion of luminal IgA, while commensal E. coli strain did not. Induced anti-LF82 IgA showed specific binding to other AIEC strains but not to the commensal, non-AIEC E. coli strains. Induced IgA showed decreased binding to LF82 strains with mutated adhesin and outer membrane proteins which are involved in AIEC – epithelial cell interaction. Consistently, LF82-specific IgA limited the adhesion and invasion of LF82 in cultured epithelial cells, which seems to be required for the elimination in the colonic epithelium in mice.ConclusionThese results demonstrate that host immunity selectively recognizes pathobiont E. coli, such as AIEC, and develop specific IgA. The induced IgA specific to pathobiont E. coli, in turn, contributes to preventing the pathobionts from accessing the epithelium.

Recently, Schlafen family member 11 (SLFN11) has been reported to increase the sensitivity of cancer cells to DNA-damaging agents, including platinum derivatives; thus, SLFN11 may be a predictive biomarker for platinum-based chemoradiotherapy (CRT). In this study, we examined whether SLFN11 expression was associated with the therapeutic outcome of platinum-based CRT in head and neck squamous cell carcinoma (HNSCC). We performed immunohistochemical analyses for SLFN11 expression in 161 HNSCC tissues from patients who had been administered cisplatin-based CRT and examined the correlation between SLFN11 expression and progression-free survival (PFS). Additionally, SLFN11 expression was examined in 10 paired samples obtained before and after CRT in patients with local failure. Furthermore, in vitro experiments were performed using several HNSCC cell lines and isogenic SLFN11-knockout cells to assess the association between SLFN11 expression and drug sensitivity. PFS was found to be significantly better in the SLFN11-positive group than in the SLFN11-negative group among the 161 patients (5-year PFS: 78.8% vs. 52.8%, respectively, p < 0.001). Similar results were observed for the PFS at each primary site. The percentage of SLFN11 positivity was lower in tumor samples from patients with local failure after CRT than that in the corresponding primary tumors before CRT in 8 of 10 cases. Results of the in vitro assay demonstrated that SLFN11-knockout cells exhibited reduced sensitivity to DNA-damaging agents but not to the non-DNA-damaging agent docetaxel. Our findings suggest that SLFN11 may serve as a potential biomarker for predicting the response of HNSCC patients to platinum-based CRT.

The crystal structures and spectroscopic properties of both E- and Z-isomers of N'-(1H-imidazol-4-ylmethylene)-picolinohydrazide (H2Lim) were investigated. In addition, an aerobic reaction of H2Lim with Co(BF4)2 in methanol afforded a mononuclear cobalt(III) complex, [Co(Z-HLim)2]BF4 (1) bearing tridentate hydrazonate ligands, and a dinuclear complex of [Co2(μ-E-HLim)3](BF4)3 (2), where the hydrazonate bridged two CoIII centers in the μ-κ2N,N': κ2N'',N''' mode to form an C3-symmetry triple helicate complex.

Abstract Background Gene mutation assays in transgenic rodents are useful tools to investigate in vivo mutagenicity in a target tissue. Using a lambda EG10 transgene containing reporter genes, gpt delta transgenic mice and rats have been developed to detect point mutations and deletions. The transgene is integrated in the genome and can be rescued through an in vitro packaging reaction. However, the packaging efficiency is lower in gpt delta rats than in mice, because of the transgene in gpt delta rats being heterozygous and in low copy number. To improve the packaging efficiency, we herein describe a newly developed homozygous gpt delta rat strain. Results The new gpt delta rat has a Wistar Hannover background and has been successfully maintained as homozygous for the transgene. The packaging efficiency in the liver was 4 to 8 times higher than that of existing heterozygous F344 gpt delta rats. The frequency of gpt point mutations significantly increased in the liver and bone marrow of N-nitroso-N-ethylurea (ENU)- and benzo[a]pyrene (BaP)-treated rats. Spi− deletion frequencies significantly increased in the liver and bone marrow of BaP-treated rats but not in ENU-treated rats. Whole genome sequencing analysis identified ≥ 30 copies of lambda EG10 transgenes integrated in rat chromosome 1. Conclusions The new homozygous gpt delta rat strain showed a higher packaging efficiency, and could be useful for in vivo gene mutation assays in rats.

Zinc is an essential micronutrient in human body and a vital cofactor for the function of numerous proteins encoded by the human genome. Zinc has a critical role in maintaining many biochemical and physiological processes at the molecular, cellular, and multiple organ and systemic levels. The alteration of zinc homeostasis causes dysfunction of many organs and systems.In the immune system, zinc regulates the differentiation, proliferation and function of inflammatory cells, including T cells, eosinophils, and B cells, by modifying several signaling pathways such as NFκB signaling pathways and TCR signals. An adequate zinc level is essential for proper immune responses and decreased zinc levels were reported in many allergic inflammatory diseases, including atopic dermatitis, bronchial asthma, and chronic rhinosinusitis. Decreased zinc levels often enhance inflammatory activation. On the other hand, the inflammatory conditions alter the intracellular homeostasis of zinc, often decreasing zinc levels. These findings implied that there could be a vicious cycle between zinc deficiency and inflammatory conditions.In this review, we present recent evidence on the involvement of zinc in atopic dermatitis, bronchial asthma, and chronic rhinosinusitis, with insights into the involvement of zinc in the underlying molecular and cellular mechanisms related to these allergic inflammatory diseases.

We report a case of eosinophilic cholecystitis associated with eosinophilic granulomatosis with polyangiitis (EGPA) complicated by cerebral hemorrhage. A 60-year-old man presented to a local hospital with a diagnosis of acute cholecystitis, with persistent fever and epigastric pain for 2 weeks. His symptoms persisted despite 3-week hospitalization; therefore, he was transferred to our hospital for further evaluation. Laboratory investigations upon admission showed white blood cells 26,300/µL and significant eosinophilia (eosinophils 61%). Abdominal computed tomography revealed no gallbladder enlargement but a circumferentially edematous gallbladder wall. Additional blood test results were negative for antineutrophil cytoplasmic and perinuclear antineutrophil cytoplasmic antibodies; however, immunoglobulin (Ig)G and IgE levels were high at 1,953 mg/dL and 3,040/IU/mL, respectively. He improved following endoscopic transnasal gallbladder drainage for cholecystitis and was diagnosed with EGPA and received corticosteroid and immunosuppressant combination therapy. The eosinophil count decreased immediately after treatment, and abdominal pain and numbness resolved. He returned with left-sided suboccipital hemorrhage likely attributed to EGPA 6 months after discharge. EGPA is characterized by inflammation of small blood vessels and clinically manifests with an allergic presentation of bronchial asthma, as well as renal dysfunction, interstitial pneumonia, enteritis, and cerebral hemorrhage. Few reports have described cholecystitis as a presenting symptom of EGPA. We report a rare case of such a presentation with added considerations.

Background/Aims Intestinal fibrosis is a major complication of Crohn’s disease (CD). The profibrotic protein transforming growth factor-β (TGF-β) has been considered to be critical for the induction of the fibrotic program. TGF-β has the ability to induce not only the expression of extracellular matrix (ECM) including collagen, but also the production of plasminogen activator inhibitor-1 (PAI-1) that prevents enzymatic degradation of the ECM during the onset of fibrotic diseases. However, the significance of PAI-1 in the developing intestinal fibrosis has not been fully understood. In the present study, we examined the actual expression of PAI-1 in fibrotic legion of intestinal inflammation and its correlation with the abnormal ECM deposition. Methods Chronic intestinal inflammation was induced in BALB/c mice using 8 repeated intrarectal injections of 2,4,6-trinitrobenzene sulfonic acid (TNBS). TM5275, a PAI-1 inhibitor, was orally administered as a carboxymethyl cellulose suspension each day for 2 weeks after the sixth TNBS injection. Results Using a publicly available dataset (accession number, GSE75214) and TNBS-treated mice, we observed increases in PAI-1 transcripts at active fibrotic lesions in both patients with CD and mice with chronic intestinal inflammation. Oral administration of TM5275 immediately after the onset of intestinal fibrosis upregulated MMP-9 (matrix metalloproteinase 9) and decreased collagen accumulation, resulting in attenuation of the fibrogenesis in TNBS-treated mice. Conclusions PAI-1-mediated fibrinolytic system facilitates collagen degradation suppression. Hence, PAI-1 inhibitor could be applied as an anti-fibrotic drug in CD treatment.

Abstract Introduction Various challenges have been overcome with regard to applying ‘omics technologies for chemical risk assessments. Previously we published results detailing targeted mRNA sequencing (RNA-Seq) on a next generation sequencer using intact RNA derived from freshly frozen rat liver tissues. We successfully discriminated genotoxic hepatocarcinogens (GTHCs) from non-genotoxic hepatocarcinogens (NGTHCs) using 11 selected marker genes. Based on this, we next attempted to use formalin-fixed paraffin-embedded (FFPE) pathology specimens for RNA-Seq analyses. Findings In this study we performed FFPE RNA-Seq to compare a typical GTHC, 2-acetylaminofluorene (AAF) to genotoxicity equivocal p-cresidine (CRE). CRE is used as a synthetic chemical intermediate, and this compound is classified as an IARC 2B carcinogen and is mutagenic in S. typhimurium, which is non-genotoxic to rat livers as assessed by single strand DNA damage analysis. RNA-Seq was used to examine liver FFPE samples obtained from groups of five 10-week-old male F344 rats that were fed with chemicals (AAF: 0.025% and CRE: 1% in food) for 4 weeks or from controls that were fed a basal diet. We extracted RNAs from FFPE samples and RNA-Seq was performed on a MiniSeq (Illumina) using the TruSeq custom RNA panel. AAF induced remarkable differences in the expression of eight genes (Aen, Bax, Btg2, Ccng1, Gdf15, Mbd1, Phlda3 and Tubb4b) from that in the control group, while CRE only induced expression changes in Gdf15, as shown using Tukey’s test. Gene expression profiles for nine genes (Aen, Bax, Btg2, Ccng1, Cdkn1a, Gdf15, Mbd1, Phlda3, and Plk2) differed. between samples treated with AAF and CRE. Finally, principal component analysis (PCA) of 12 genes (Aen, Bax, Btg2, Ccnf, Ccng1, Cdkn1a, Gdf15, Lrp1, Mbd1, Phlda3, Plk2, and Tubb4b) using our previous Open TG-GATE data plus FFPE-AAF and FFPE-CRE successfully differentiated FFPE-AAF, as GTHC, from FFPE-CRE, as NGHTC. Conclusion Our results suggest that FFPE RNA-Seq and PCA are useful for evaluating typical rat GTHCs and NGTHCs.

ObjectiveSalivary duct carcinoma (SDC) is a highly aggressive and uncommon tumor arising not only de novo but also in pleomorphic adenoma. Androgen receptor (AR)- and HER2-targeted therapy have recently been introduced for SDC as promising treatment options; however, no predictive biomarkers have yet been established. EZH2 and H3K27me3 are closely linked to the development and progression of various cancers, and EZH2 is also expected to be a desirable therapeutic target. We therefore explored the clinicopathological and prognostic implications of EZH2 and H3K27me3 in a large cohort of SDC patients, focusing on their impact on the therapeutic efficacy of AR- or HER2-targeted therapy.Materials and MethodsThe EZH2 and H3K27me3 immunohistochemical expression and EZH2 Y646 gain-of-function mutation status were examined in 226 SDCs, and the relationship with the clinicopathological factors as well as clinical outcomes were evaluated within the three groups depending on the treatment: AR-targeted (combined androgen blockade with leuprorelin acetate and bicalutamide; 89 cases), HER2-targeted (trastuzumab and docetaxel; 42 cases), and conventional therapy (112 cases).ResultsEZH2 and H3K27me3 were variably immunoreactive in most SDCs. A positive correlation was found between the expression of EZH2 and H3K27me3. The EZH2 expression in the SDC component was significantly higher than that in the pre-existing pleomorphic adenoma component. EZH2 Y646 was not identified in any cases. EZH2-high cases more frequently had an advanced clinical stage and aggressive histological features than EZH2-low cases. An EZH2-high status in patients treated with AR-targeted therapy was associated with a significantly shorter progression-free and overall survival as well as a lower objective response rate and clinical benefit rate. In addition, a H3K27me3-high status in patients treated with AR-targeted therapy was related to a shorter overall survival. Conversely, there was no association between the EZH2 and H3K27me3 expression and the clinical outcomes in the conventional or HER2-targeted therapy groups.ConclusionsA high expression of EZH2 and H3K27me3 in SDC might be a predictor of a poor efficacy of AR-targeted therapy. Our data provide new insights into the role of EZH2 and H3K27me3 in therapeutic strategies for SDC.

Oral conditions are relatively common in patients with inflammatory bowel disease (IBD). However, the contribution of oral maladies to gut inflammation remains unexplored. Here, we investigated the effect of periodontitis on disease phenotypes of patients with IBD. In all, 60 patients with IBD (42 with ulcerative colitis [UC] and 18 with Crohn’s disease [CD]) and 45 healthy controls (HCs) without IBD were recruited for this clinical investigation. The effects of incipient periodontitis on the oral and gut microbiome as well as IBD characteristics were examined. In addition, patients were prospectively monitored for up to 12 months after enrollment. We found that, in both patients with UC and those with CD, the gut microbiome was significantly more similar to the oral microbiome than in HCs, suggesting that ectopic gut colonization by oral bacteria is increased in patients with IBD. Incipient periodontitis did not further enhance gut colonization by oral bacteria. The presence of incipient periodontitis did not significantly affect the clinical outcomes of patients with UC and CD. However, the short CD activity index increased in patients with CD with incipient periodontitis but declined or was unchanged during the study period in patients without periodontitis. Thus, early periodontitis may associate with worse clinically symptoms in some patients with CD.

The respiratory tract is constantly at risk of invasion by microorganisms such as bacteria, viruses, and fungi. In particular, the mucosal epithelium of the nasal cavity and paranasal sinuses is at the very forefront of the battles between the host and the invading pathogens. Recent studies have revealed that the epithelium not only constitutes a physical barrier but also takes an essential role in the activation of the immune system. One of the mechanisms equipped in the epithelium to fight against microorganisms is the Toll-like receptor (TLR) response. TLRs recognize common structural components of microorganisms and activate the innate immune system, resulting in the production of a plethora of cytokines and chemokines in the response against microbes. As the epithelia-derived cytokines are deeply involved in the pathogenesis of inflammatory conditions in the nasal cavity and paranasal sinuses, such as chronic rhinosinusitis (CRS) and allergic rhinitis (AR), the molecules involved in the TLR response may be utilized as therapeutic targets for these diseases. There are several differences in the TLR response between nasal and bronchial epithelial cells, and knowledge of the TLR signals in the upper airway is sparse compared to that in the lower airway. In this review, we provide recent evidence on TLR signaling in the upper airway, focusing on the expression, regulation, and responsiveness of TLRs in human nasal epithelial cells (HNECs). We also discuss how TLRs in the epithelium are involved in the pathogenesis of, and possible therapeutic targeting, for CRS and AR.

Abstract Background Next Generation Sequencer (NGS) is a powerful tool for a high-throughput sequencing of human genome. It is important to ensure reliability and sensitivity of the sequence data for a clinical use of the NGS. Various cancer-related gene panels such as Oncomine™ or NCC OncoPanel have been developed and used for clinical studies. Because these panels contain multiple genes, it is difficult to ensure the performance of mutation detection for every gene. In addition, various platforms of NGS are developed and their cross-platform validation has become necessity. In order to create mutant standards in a defined background, we have used CRISPR/Cas9 genome-editing system in HEK 293 T/17 cells. Results Cancer-related genes that are frequently used in NGS-based cancer panels were selected as the target genes. Target mutations were selected based on their frequency reported in database, and clinical significance and on the applicability of CRISPR/Cas9 by considering distance from PAM site, and off-targets. We have successfully generated 88 hetero- and homozygous mutant cell lines at the targeted sites of 36 genes representing a total of 125 mutations. Conclusions These knock-in HEK293T/17 cells can be used as the reference mutant standards with a steady and continuous supply for NGS-based cancer panel tests from the JCRB cell bank. In addition, these cell lines can provide a tool for the functional analysis of targeted mutations in cancer-related genes in the isogenic background.

Librarians at a university had planned to check the collection prior to the library renovations that began in 2015. They had previous knowledge of the presence of a light greyish-white powder with an unpleasant odour (hereinafter referred to as 'powder') sprinkled between the pages of antiquarian books in the library archive. The purpose of this study was to identify this powder with the help of experts from both inside and outside the university. The powder was qualitatively analysed using gas chromatography with mass spectrometry after hexane extraction. The powder was examined under a polarised light microscope and a field-emission scanning electron microscope equipped with an energy-dispersive X-ray spectrometer. Benzene hexachloride (BHC) was detected in the powder. Talc was the most abundant particle in the powder. The powder also contained 0.52 wt% asbestos, which belonged to the tremolite-actinolite series. No other types of asbestos were detected. The powder was presumed to be a bulking agent for BHC, and its major constituent was talc. This is the first report on asbestos-containing insecticides. [ABSTRACT FROM AUTHOR]

A mixture of 2-pyridine carboxaldehyde, 4-formylimidazole (or 2-methyl-4-formylimidazole), and NiCl2·6H2O in a molar ratio of 2:2:1 was reacted with two equivalents of hydrazine monohydrate in methanol, followed by the addition of aqueous NH4PF6 solution, afforded a NiII complex with two unsymmetric azine-based ligands, [Ni(HLH)2](PF6)2 (1) or [Ni(HLMe)2](PF6)2 (2), in a high yield, where HLH denotes 2-pyridylmethylidenehydrazono-(4-imidazolyl)methane and HLMe is its 2-methyl-4-imidazolyl derivative. The spectroscopic measurements and elemental analysis confirmed the phase purity of the bulk products, and the single-crystal X-ray analysis revealed the molecular and crystal structures of the NiII complexes bearing an unsymmetric HLH or HLMe azines in a tridentate κ3N, N’, N” coordination mode. The HLH complex with a methanol solvent, 1·MeOH, crystallizes in the orthorhombic non-centrosymmetric space group P212121 with Z = 4, affording conglomerate crystals, while the HLMe complex, 2·H2O·Et2O, crystallizes in the monoclinic and centrosymmetric space group P21/n with Z = 4. In the crystal of 2·H2O·Et2O, there is intermolecular hydrogen-bonding interaction between the imidazole N–H and the neighboring uncoordinated azine-N atom, forming a one-dimensional polymeric structure, but there is no obvious magnetic interaction among the intra- and interchain paramagnetic NiII ions.

Salivary duct carcinoma (SDC) is morphologically similar to breast cancer, with HER2-overexpression reported. With regard to the pattern of disease onset, SDC can arise from de novo or carcinoma ex-pleomorphic adenoma (Ca-ex-PA). Recently, multiple molecular profiles of SDC as well as breast cancer have been reported, with significant differences in HER2 expression between Ca-ex-PA and de novo. We assessed the differences in gene expression between onset classifications. We conducted immunohistochemical analysis and HER2-DISH for 23 patients and classified SDCs into three subtypes as follows: “HER2-positive” (HER2+/any AR), “Luminal-AR” (HER2-/AR+), and “Basal-like” (HER2-/AR-). We assessed the expression levels of 84 functional genes for 19 patients by using a qRT-PCR array. Ten cases were classified as HER2-positive, seven cases as Luminal-AR, and six cases as Basal-like. The gene expression pattern was generally consistent with the corresponding immunostaining classification. The expression levels of VEGFA, ERBB2(HER2), IGF1R, RB1, and XBP1 were higher, while those of SLIT2 and PTEN were lower in Ca-ex-PA than in de novo. The functions of those genes were concentrated in angiogenesis and AKT/PI3K signaling pathway (Fisher’s test: p-value = 0.025 and 0.004, respectively). Multiple machine learning methods, OPLS-DA, LASSO, and RandomForest, also show that VEGFA can be a candidate for the characteristic differences between Ca-ex-PA and de novo. In conclusion, the AKT/PI3K signaling pathway leading to angiogenesis was hyper-activated in all SDCs, particularly in those classified into the Ca-ex-PAs. VEGFA was over-expressed significantly in the Ca-ex-PA, which can be a crucial factor in the malignant conversion to SDC.

Three palladium(II) complexes with amino-amidato-phenolato-type tridentate ligands were synthesized and characterized by 1H NMR spectroscopy and X-ray crystallography. The strategic arrangement of a hydrogen-bond donor and acceptor adjacent to the substitution site of the PdII complex allowed the selective coordination of nucleosides. Among two pyrimidine-nucleosides, cytidine and 5-methyluridine, cytidine was successfully coordinated to the PdII complex while 5-methyluridne was not. On the other hand, both purine-nucleosides, adenosine and guanosine, were coordinated to the PdII complex. As purines have several coordination sites, adenosine afforded three kinds of coordination isomers expected from the three different donors. However, guanosine afforded a sole product according to the ligand design such that the formation of double intramolecular hydrogen-bond strongly induced the specific coordination by N1-position of guanine moiety. Furthermore, the preference of the nucleosides was evaluated by scrambling reactions. It was found that the preference of guanosine is nearly twice as high as adenosine and cytidine, owing to the three-point interaction of a coordination bond and two hydrogen bonds. These results show that the combination of a coordination and hydrogen bonds, which is reminiscent of the Watson–Crick base pairing, is an effective tool for the precise recognition of nucleosides.

Abstract Background Sirtuin 2 (SIRT2) is a member of the sirtuin family, nicotinamide adenine dinucleotide+-dependent deacylases, which participates in modulation of cell cycle control, neurodegeneration, and tumorigenesis. SIRT2 expression increases in acute myeloid leukemia blasts. Downregulation of SIRT2 using siRNA causes apoptosis of HeLa cells. Therefore, selective inhibitors of SIRT2 are candidate therapeutic agents for cancer. Adult T-cell leukemia/lymphoma (ATL) is a T-cell malignancy that has a poor prognosis and develops after long-term infection with human T-cell leukemia virus (HTLV)-1. Sirtuin 1 inhibition has been shown to induce apoptosis and autophagy in HTLV-1-infected cell lines, whereas the effects of SIRT2 inhibition alone have not been elucidated. Methods We assessed the efficacy of our small molecule selective SIRT2 inhibitors NCO-90/141 to induce leukemic cell death. Cell viability was examined using the cell proliferation reagent Cell Count Reagent SF. Apoptotic cells were detected by annexin V-FITC and terminal deoxynucleotidyl transferase dUTP nick end labeling assays by flow cytometry. Caspase activity was detected using an APOPCYTO Intracellular Caspase Activity Detection Kit. The presence of autophagic vacuoles was assessed using a Cyto-ID Autophagy Detection Kit. Results Our novel small molecule SIRT2-specific inhibitors NCO-90/141 inhibited cell growth of leukemic cell lines including HTLV-1-transformed T-cells. NCO-90/141 induced apoptosis via caspase activation and mitochondrial superoxide generation in leukemic cell lines. However, a caspase inhibitor did not prevent this caspase-associated cell death. Interestingly, NCO-90/141 increased the LC3-II level together with autophagosome accumulation, indicating autophagic cell death. Thus, NCO-90/141 simultaneously caused apoptosis and autophagy. Conclusions These results suggest that NCO-90/141 are highly effective against leukemic cells in caspase-dependent or -independent manners via autophagy, and they may have a novel therapeutic potential for treatment of leukemias including ATL.

Abstract Background The carcinogenic potential of dimethylaniline (DMA) isomers in rodents and humans has been previously reported, and there is sufficient evidence for the carcinogenicity of 2,6-DMA in experimental animals. The target organ of carcinogenesis of 2,6-DMA is the nasal cavity. In the current study, six DMA isomers, 2,3-, 2,4-, 2,5-, 2,6-, 3,4- and 3,5-DMA, were evaluated for mutagenic properties. Results Male ddY mice (3/group) were treated intragastrically (i.g.) with 200 mg/kg of one of the six DMAs, and a comet assay was performed on samples of bone marrow, kidney, liver and lung at 3 and 24 h after the treatment. Positive responses were observed in the kidney, liver and lungs of mice from all of the DMA treatment groups after 3 h and in the bone marrow of mice treated with either 3,4- or 3,5-DMA after 3 h; however, these effects were diminished at the 24 h time point. The micronucleus induction in the bone marrow was analysed in the same mouse at 24 h after the treatment. No induction of micronucleated polychromatic erythrocytes was observed after treatment with any of the DMAs. Male transgenic Muta™ mice (five/group) were treated i.g. with 2,5-, 2,6- or 3,5-DMA at 100 mg/kg bw weekly for 4 weeks, and the lacZ and the cII mutation frequencies were examined in the nasal cavity, liver and bone marrow at 7 days after the last treatment. Statistically significant increases in the mutation frequencies of the lacZ and/or cII genes were observed in the nasal cavity of 2,5-DMA or 2,6-DMA treated mice. Sequence analysis showed increased incidences of AT to GC and GC to TA mutations in the nasal tissues. Conclusions These findings suggest that the carcinogenic activities of DMAs are associated with mutagenic events.

In non-small cell lung cancer (NSCLC), oncogenic driver mutations including those in KRAS and EGFR are typically mutually exclusive. However, recent reports indicate that multiple driver mutations are found in a certain percentage of cancers, and that the therapeutic responses of such cases with co-mutations of driver genes are largely unclear. Here, using CRISPR-Cas9-mediated genome editing, we generated isogenic cell lines harboring one or two copies of an EGFR-activating mutation from the human NSCLC cell line A549, which is known to harbor a homozygous KRAS gene mutation. In comparison with parent cells with KRAS mutation alone, cells with concomitant EGFR mutation exhibited higher sensitivity to EGFR-tyrosine kinase inhibitors (TKIs) but not to conventional anti-cancer drugs. In particular, cells with two copies of EGFR mutation were markedly more sensitive to EGFR-TKIs compared with parent cells. Thus, the presence of concomitant EGFR mutation can affect the TKI response of KRAS-mutated cells, implying that EGFR-TKI may represent an effective treatment option against NSCLC with EGFR/KRAS co-mutation.

Background IgG4-related disease is a new clinical entity frequently associated with swelling of the submandibular glands (SMGs). The long-term outcome of SMG swelling without steroid therapy remains unknown. Objective To examine whether swollen SMGs spontaneously regress without steroid therapy in the context of IgG4-related disease and to identify biomarkers that can predict the spontaneous regression of SMG swelling. Methods The SMG volume of 49 patients diagnosed with IgG4-related disease was calculated by measuring the axial and coronal planes of computed tomography scans. The change in SMG volume over time was measured and examined by treatment regimen, clinical data, and serum complement level. Results We found 28 of 49 (57%) IgG4-related disease patients to have swollen SMGs, with 15 of 20 (75%) of the swollen SMGs regressing without steroid therapy. The time required for the SMGs swelling to regress was significantly shorter in the steroid therapy group than in the no-steroid therapy group. Serum complement components at the initial visit were significantly lower in the regressed SMG group than in the nonregressed SMG group. Conclusion We observed 75% of swollen SMGs spontaneously regressed in patients with IgG4-related disease. The time required for the swollen SMGs to regress was longer in patients without steroid therapy than in those with steroid therapy. Serum complement level could be used as a predictor for the spontaneous regression of swollen SMGs in patients with IgG4-related disease.

Summary: Memory is formed by synapse-to-nucleus communication that leads to regulation of gene transcription, but the identity and organizational logic of signaling pathways involved in this communication remain unclear. Here we find that the transcription cofactor CRTC1 is a critical determinant of sustained gene transcription and memory strength in the hippocampus. Following associative learning, synaptically localized CRTC1 is translocated to the nucleus and regulates Fgf1b transcription in an activity-dependent manner. After both weak and strong training, the HDAC3-N-CoR corepressor complex leaves the Fgf1b promoter and a complex involving the translocated CRTC1, phosphorylated CREB, and histone acetyltransferase CBP induces transient transcription. Strong training later substitutes KAT5 for CBP, a process that is dependent on CRTC1, but not on CREB phosphorylation. This in turn leads to long-lasting Fgf1b transcription and memory enhancement. Thus, memory strength relies on activity-dependent changes in chromatin and temporal regulation of gene transcription on specific CREB/CRTC1 gene targets. : Uchida et al. link CRTC1 synapse-to-nucleus shuttling in memory. Weak and strong training induce CRTC1 nuclear transport and transient Fgf1b transcription by a complex including CRTC1, CREB, and histone acetyltransferase CBP, whereas strong training alone maintains Fgf1b transcription through CRTC1-dependent substitution of KAT5 for CBP, leading to memory enhancement. Keywords: memory enhancement, long-term potentiation, hippocampus, nuclear transport, epigenetics, FGF1, CRTC1, KAT5/Tip60, HDAC3, CREB

Introduction: The rise in antimicrobial resistance (AMR) that is affecting humans, animals, and the environment, compromises the human immune system and represents a significant threat to public health. Regarding the impact on water sanitation, the risk that antimicrobial-resistant genes (ARGs) and antimicrobialresistant bacteria in surface water in cities pose to human health remains unclear. To determine the prevalence of AMR in environmental surface water in Japan, we used DNA sequencing techniques on environmental water DNA (eDNA) and the DNA of multidrug-resistant bacteria (mrDNA). Methods: The eDNA was extracted from four surface water samples obtained from the Tokyo area and subjected to high-throughput next-generation DNA sequencing using Illumina-derived shotgun metagenome analysis. The sequence data were analyzed using the AmrPlusPlus pipeline and the MEGARes database. Multidrug-resistant bacteria were isolated using a culture-based method from water samples and were screened by antimicrobial susceptibility testing (for tetracycline, ampicillin-sulbactam, amikacin, levofloxacin, imipenem, and clarithromycin). Of the 284 isolates, 22 were identified as multidrug-resistant bacteria. The mrDNA was sequenced using the Oxford nanopore MinION system and analyzed by NanoARG, a web service for detecting and contextualizing ARGs. Results and discussion: The results from eDNA and mrDNA revealed that ARGs encoding beta-lactams and multidrug resistance, including multidrug efflux pump genes, were frequently detected in surface water samples. However, mrDNA also revealed many sequence reads from multidrug-resistant bacteria, as well as nonspecific ARGs, whereas eDNA revealed specific ARGs such as pathogenic OXAtype and New Delhi metallo (NDM)-beta-lactamase ARGs. Conclusion: To estimate potential AMR pollution, our findings suggested that eDNA is preferable for detecting pathogen ARGs. [ABSTRACT FROM AUTHOR]

Background/Aim: Our recent studies have indicated that trace copper co-existed with iron in hemosiderin particles of human genetic iron overload. To understand this phenomenon, we analyzed hemosiderin particles in iron-overloaded rat liver by using scanning transmission electron microscopy - energy-dispersive X-ray (STEM-EDX) spectroscopy. Materials and Methods: Samples for STEM-EDX spectroscopy were prepared from the liver of rats administered an intraperitoneal injection of dextran iron. Results: The micro-domain analysis with STEM-EDX spectroscopy showed that dense bodies contained high levels of iron and trace copper. Quantitative analysis of copper levels in the liver specimen using atomic spectrophotometry showed that copper concentration in the liver was not increased by iron overload. These findings suggest that the overload of iron induced distribution of trace copper to hemosiderin particles without changing cellular copper levels. Conclusion: Co-existence of copper with iron was observed in hemosiderin particles of the liver of an experimental model of iron overload, suggesting that iron overload induced distribution of trace copper into hemosiderin particles. [ABSTRACT FROM AUTHOR]