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Background and Aims Ulcerative colitis [UC] is a chronic inflammatory disease of the colon with frequent relapses. Telomere shortening in intestinal epithelial cells has been reported in severe or longstanding cases. However, its influence on UC pathogenesis remains unelucidated. To this end, we evaluated telomere shortening using a long-term organoid inflammation model that we had originally established. Methods A UC model using human colon organoids was established to assess telomere changes chronologically. MST-312 was used for the telomerase inhibition assay. The potential of telomerase activators as a novel UC treatment was evaluated with an in vitro model, including microarray analysis, and histological changes were assessed using xenotransplantation into mouse colonic mucosa. Results Our UC model reproduced telomere shortening in vitro, which was induced by the continuous suppression of telomerase activity via P53. MST-312-based analysis revealed that telomere shortening was involved in the pathogenesis of UC. Madecassoside [MD] improved the telomere length of the UC model and UC patient-derived organoids, which further promoted cell proliferation in vitro and improved the graft take-rate of xenotransplantation. Moreover, histological analysis revealed that MD induced normal crypt structure with abundant goblet cells. Conclusions This study is the first to reveal the mechanism and importance of telomere shortening in the pathogenesis of UC. MD could be a novel candidate for UC treatment beyond endoscopic mucosal healing.

ObjectiveEarly integration of palliative and cancer care improves the quality of life and is facilitated by discussions about the end of life after cessation of active cancer treatment between patients with advanced cancer and their physicians. However, both patients and physicians find end-of-life discussions challenging. The aim of this study was to assess the need for a question prompt list (QPL) that encourages end-of-life discussions between patients with advanced cancer and their physicians.MethodsFocus group interviews (FGIs) were conducted with 18 participants comprising 5 pancreatic cancer patients, 3 family caregivers, 4 bereaved family members, and 6 physicians. Three themes were discussed: question items that should be included in the QPL that encourages end-of-life discussions with patients, family caregivers, and physicians after cessation of active cancer treatment; when the QPL should be provided; and who should provide the QPL. Each interview was audio-recorded, and content analysis was performed.ResultsThe following 9 categories, with 57 question items, emerged from the FGIs: (1) preparing for the end of life, (2) treatment decision-making, (3) current and future quality of life, (4) current and future symptom management, (5) information on the transition to palliative care services, (6) coping with cancer, (7) caregivers’ role, (8) psychological care, and (9) continuity of cancer care. Participants felt that the physician in charge of the patient's care and other medical staff should provide the QPL early during active cancer treatment.Significance of resultsData were collected to develop a QPL that encourages end-of-life discussions between patients with advanced cancer and their physicians.

BACKGROUND: The COVID-19 pandemic has had wide-ranging impacts across international healthcare systems and direct impacts on rehabilitation professionals. Few outcome data for cardiac patients undergoing rehabilitation programs during the COVID-19 pandemic are available. AIM: We conducted a study to compare the effect of modified rehabilitation therapies mainly performed in wards versus conventional therapies mainly performed in rehabilitation units in which exercise on a treadmill and cardiopulmonary exercise testing were available. DESIGN: Observational study. SETTING: University hospital. POPULATION: Fifty-five consecutive inpatients admitted to a university hospital and underwent a cardiac rehabilitation program from August 2019 to June 2020. METHODS: The patients were divided into two groups: those admitted during the COVID-19 outbreak (Group A, N.=28) and those admitted before the COVID-19 outbreak (Group B, N.=27). The evaluation included age, sex, duration of the rehabilitation intervention program, days before initiation of the rehabilitation program, functional status, and Functional Independence Measure (FIM) Score. RESULTS: A higher proportion of patients in Group A than B underwent a cardiac rehabilitation program provided in wards (88.5% vs. 48.8%, respectively). Group A showed a significantly lower 6-minute walking distance and walking speed than Group B at discharge (P=0.031 and 0.014, respectively). Group A showed a significantly shorter exercise time using an ergometer than Group B (P=0.028). CONCLUSIONS: The difference in the cardiac rehabilitation location during the COVID-19 pandemic may affect the rehabilitation contents and lead to less improvement in physical function. CLINICAL REHABILITATION IMPACT: A cardiac rehabilitation program was performed mainly in wards instead of in rehabilitation units during the COVID-19 pandemic. Walking abilities were adversely affected by the modified cardiac rehabilitation program.

The preoperative diagnosis of thyroid follicular carcinomas (FCs) by fine-needle aspiration cytology is almost impossible. It was previously demonstrated that p53-binding protein 1 (53BP1) expression, based on immunofluorescence (IF),can serve as a valuable biomarker to estimate the malignant potential of various cancers. 53BP1 belongs to a class of DNA damage response molecules that rapidly localize to the site of DNA double-strand breaks,forming nuclear foci (NF). This study aimed to elucidate the utility of 53BP1 NF expression as a biomarker to differentiate follicular tumors (FTs). Methods: Associations between 53BP1 expression based on IF and histological types of FTs were analyzed using 27 follicular adenomas (FAs), 28 minimally invasive FCs, and 14 widely invasive FCs. Furthermore, the study clarified the relationship between 53BP1 NF and copy number aberrations (CNAs) based on array comparative genomic hybridization, a hallmark of genomic instability (GIN). Results: This study demonstrates differences in 53BP1 NF expression between FA and FC. The incidence of 53BP1 at NF significantly increased with FT progression in the following order: normal follicle < FA < minimally invasive FCs< widely invasive FCs. In contrast, no significant differences were observed in CNAs among the FT samples. Furthermore, there was no significant correlation between CNAs and 53BP1 at NF in FTs. Thus, based on a comparison of these two indicators of GIN, 53BP1 NF (by IF) was better able to estimate the malignancy of FTs compared to CNA (by array comparative genomic hybridization). Interestingly, IF revealed a heterogenous distribution of 53BP1 NF,which occurred more frequently in the invasive or subcapsular area than in the center of the tumor, suggesting intratumoral heterogeneity of GIN in FTs. Conclusions: It is proposed that IF analysis of 53BP1 expression could be a novel diagnostic method to estimate the malignant potential of FTs. Because 53BP1 NF reflect DNA double-strand breaks, it is hypothesized that the incidence of 53BP1 at NF can represent the level of GIN in tumor cells. IF analysis of 53BP1 expression will not only be an auxiliary histologic technique to diagnose FTs accurately, but also a novel technique for preoperative diagnosis using fine-needle aspiration cytology., Thyroid, 29(5), pp.657-665; 2019

Resistance-associated substitutions (RASs) of hepatitis C virus (HCV) in the NS5A region impair the efficacy of NS5A inhibitors. In this study, we evaluated the characteristics of the novel RASs observed in treatment-failure patients, A92K and a deletion at P32 (P32del), and the susceptibility of viruses with these RASs to various anti-HCV reagents by using JFH-1 based recombinant HCV with NS5A from a genotype 1b Con1 strain (JFH1/5ACon1). We introduced A92K or P32del solely or in combination with Q24K, L28M, R30Q or L31F into the NS5A of JFH1/5ACon1. Viruses harboring R30Q/A92K showed high extracellular core antigens and infectivity titers, whereas the other viruses with RASs showed low replication levels and infectivity titers. All the viruses with A92K or P32del were markedly resistant to ledipasvir, velpatasvir and elbasvir. Interestingly, viruses with R30Q/A92K were more susceptible to grazoprevir than viruses without RAS. All the viruses had a similar susceptibility to ribavirin and sofosbuvir. In conclusion, combination RASs R30Q/A92K enhanced virus production whereas other RASs impaired virus replication. Both A92K and P32del conferred severe resistance even to second generation NS5A inhibitors. However, these viruses were susceptible to grazoprevir, ribavirin and sofosbuvir. Thus, combination regimens with these reagents may eradicate viruses harboring A92K or P32del.

Senna laxatives are commonly used for bowel preparation before colonoscopies in Japan. However, this laxative frequently causes complications such as abdominal pain. This study aimed to establish a novel method of bowel preparation, which involved the pre-administration of super-low volume polyethylene glycol (PEG) for three days followed by the same-day administration of low volume PEG. This study was a prospective, multicenter, investigator-blinded, phase 2, randomized control trial. The intake of 13.9 g (120 mL) of PEG or 1 g of a senna laxative for 3 days before the examination was indicated for each group, and 2 L of PEG solution was used for preparation on the examination day. The primary endpoint was the efficacy of bowel cleansing, as assessed by the Boston bowel preparation scale. The secondary endpoints were the adenoma detection rate and occurrence of complications. A total of 250 patients were initially enrolled. A total of 122 patients from each group were included in the intention-to-treat analysis. In the intention-to-treat analysis, the responder rates were the same for the two groups (56.6% vs 50.8%). Additionally, the adenoma detection rate did not differ between the two groups (34.9% vs 41.8%, P = 0.3795). In contrast, adherence was higher in the PEG group (93.4% vs 82.8%, P = 0.0101), and the occurrence of complications was lower in the PEG group (1.7% vs 16.4%, P = 0.0001). The novel super-low volume PEG method for bowel preparation was as effective as the conventional method with senna laxatives.

Ribosomal RNA (rRNA), the most abundant non-coding RNA species, is a major component of the ribosome. Impaired ribosome biogenesis causes the dysfunction of protein synthesis and diseases called &ldquo, ribosomopathies,&rdquo, including genetic disorders with cancer risk. However, the potential role of rRNA gene (rDNA) alterations in cancer is unknown. We investigated germline and somatic single-nucleotide variants (SNVs) in the rDNA promoter region (positions &minus, 248 to +100, relative to the transcription start site) in 82 lung adenocarcinomas (LUAC). Twenty-nine tumors (35.4%) carried germline SNVs, and eight tumors (9.8%) harbored somatic SNVs. Interestingly, the presence of germline SNVs between positions +1 and +100 (n = 12, 14.6%) was associated with significantly shorter recurrence-free survival (RFS) and overall survival (OS) by univariate analysis (p &lt, 0.05, respectively), and was an independent prognostic factor for RFS and OS by multivariate analysis. LUAC cell line PC9, carrying rDNA promoter SNV at position +49, showed significantly higher ribosome biogenesis than H1650 cells without SNV. Upon nucleolar stress induced by actinomycin D, PC9 retained significantly higher ribosome biogenesis than H1650. These results highlight the possible functional role of SNVs at specific sites of the rDNA promoter region in ribosome biogenesis, the progression of LUAC, and their potential prognostic value.

Background: The risk factors associated with the development of hepatocellular carcinoma (HCC) in nonalcoholic fatty liver disease (NAFLD) are still unclear. The aim of the present study was to identify such risk factors in NAFLD patients who developed HCC. Methods: Between April 2000 and ­December 2016, a total of 182 patients with NAFLD were enrolled in this study; of these, only 22 patients had HCC. To identify risk factors, univariate and multivariate analyses were performed. To identify risk factors other than the degree of fibrosis, propensity matched analysis adjusted by the NAFLD fibrosis score (NFS) was carried out on 44 patients. Multivariate and survival analyses were also performed in HCC patients. Results: In 182 patients, multivariate analysis highlighted the NFS (OR 2.275; p < 0.001) and hypertension (OR 5.868; p = 0.037) as independent factors that were significantly associated with the development of HCC. After adjustment for the NFS, multivariate analysis identified diabetic retinopathy (OR 8.654; p = 0.017) as an independent factor that was significantly associated with the development of HCC. For predicting the development of HCC, the area under the receiver operating characteristic curve of diabetic retinopathy was significantly higher than that of diabetes (0.731 vs. 0.615; p < 0.001). In patients with HCC, multivariate analysis indicated that the NFS were significantly associated with diabetic retinopathy. Conclusions: Diabetic retinopathy as well as liver fibrosis is a risk factor that associates with the development of HCC in NAFLD patients. Therefore, NAFLD patients with diabetic retinopathy should undergo careful screening for HCC.

It is crucial to identify risk factors for life prognosis after hepatitis C virus (HCV) eradication among patients with or without a high risk of liver cancer or complications. This is a prospective, multicenter and observational study using the database of 1031 patients after HCV eradication by direct-acting antiviral agents (DAAs) to evaluate the development of hepatocellular carcinoma (HCC) and patients’ survival after a sustained virological response (SVR). The Cox proportional hazards regression model was used to estimate hazard ratios associated with HCC development and survival. AFP at SVR was significantly associated with HCC recurrence in the adjusted model. Liver fibrosis, Mac-2 binding protein glycosylation isomer (M2BPGi) at SVR and smoking status before treatment were positively associated with the development of HCC and M2BPGi was positively associated with HCC recurrence, although not reaching statistical significance. Among patients without a history of HCC, M2BPGi and estimated glomerular filtration rate (eGFR) at SVR were significantly associated with death after viral eradication [M2BPGi (HR 4.07, 95% CI 1.22, 13.57), eGFR (HR 0.97, 95% CI 0.94, 0.99)]. Strikingly, of 16 patients who died, among participants without a history of HCC, only two died of liver cancer associated with HCV, whereas 11 died of non-HCV- related cancer or cardiovascular diseases. M2BPGi at SVR is a potential predictor for patients’ survival and a candidate biomarker for detecting individuals who are at greater risk of death due to cancer-related and unrelated to HCV, as well as cardiovascular diseases, after viral eradication.

Background: There have been great concerns about pediatric thyroid cancers after the accident at the Fukushima Daiichi Nuclear Power Plant in 2011. Case presentation: We report a case of a 7-year-old Japanese girl with sporadic papillary thyroid carcinoma (PTC) harboring an ETV6/NTRK3 rearrangement. The patient presented with tumors in both lobes and underwent thyroidectomy followed by radioactive iodine (RAI) ablation. Histopathology showed a classic type of PTC with cervical lymph node metastasis. Conclusions: Genetic evaluation showed ETV6/NTRK3 fusion but no BRAF mutations or RET/PTC rearrangements. RET/PTC rearrangement and BRAF mutations often contribute to the pathogenesis of PTC; however, rearrangements of NTRK genes are relatively rare in pediatric PTC. Although NTRK rearrangement has been shown to often present unique pathological types and infiltrative architectures in the western population, such findings were not observed in this patient. Thus, the present case of classic PTC with ETV6/NTRK3 rearrangement highlights the disparate collection of clinic-pathological features compared to the trend in the western population. We therefore emphasize the need to further accumulate clinical as well as genetic data in pediatric PTCs.

Previous reports have suggested that a longer withdrawal time (WT) during colonoscopy led to an improved adenoma detection rate (ADR); however, there are few controlled studies that substantiated monitoring WT as an educational method. We aimed to validate a feedback and monitoring system to improve the ADR in screening colonoscopy in a prospective case–control setting. After collecting data in the pre-feedback period (3.5 months), the individual performance and the average ADR and WT values of the facility were provided to 6 endoscopists in the intervention group, while 3 endoscopists were isolated as the control group during the feedback period (2 weeks). The intervention group consisted of two subgroups, the Fast and Slow WT groups, according to the results from the pre-feedback period. The endoscopists in the intervention group were instructed to be aware of their own WT in each examination during the post-feedback period (4 months). The performances of all endoscopists in the post-feedback period were analyzed and compared with those in the pre-feedback period. Among the initial analyses, the correlation analysis and multivariate analysis revealed that WT was an independent predictor for the ADR (P = 0.0101). After providing individual performance feedback and instruction regarding real-time WT monitoring, the WT was significantly prolonged in the Fast WT group (P = 0.0346) but did not change in the Slow WT and control groups. In addition, the ADR of the Fast WT group significantly improved after the intervention (P = 0.024), whereas the ADR of the Slow WT and control groups did not change. Providing individual feedback on ADR and WT and monitoring WT helped improve the endoscopists’ ADRs.

Amino acid derivative reactivity assay (ADRA) has previously been developed as an alternative method to direct peptide reactivity assay (DPRA) to evaluate key event 1 in skin sensitization mechanisms. However, when using alternative methods for skin sensitization, integrated approaches to testing and assessment (IATA) that combine the results of multiple tests evaluating different key events are generally required. To verify whether ADRA can be used in IATA, we replaced DPRA with ADRA in five IATA methods combining DPRA, KeratinoSens, and h-CLAT: (i) the "2 out of 3" approach, (ii) the "3 out of 3" approach, (iii) sequential testing strategy (STS), (iv) integrated testing strategy by scoring approach (ITS-SA), and (v) the "ITS by two methods approach" (ITS-2MA). The prediction accuracy of the "2 out of 3" approach using ADRA (1 mM) and ADRA (0.5 mg/mL) was 90.0% and 91.1%, respectively, for human data, and was very similar to that obtained using DPRA (91.1%). The "3 out of 3" approach also showed good predictability (83.2%) using either ADRA (1 mM) or ADRA (0.5 mg/mL) compared to DPRA. Regarding the accuracy of the prediction of sensitization intensity for the human data by the third classification, prediction accuracy using ADRA was almost the same as STS, ITS-SA, or ITS-2MA using DPRA. As a result, this study showed that ADRA can be used as a test method for key event 1 in the evaluation of skin sensitization by combining multiple alternative methods.

Human induced pluripotent stem (iPS) cells can differentiate into hepatocyte lineages, although the phenotype of the differentiated cells is immature compared to adult hepatocytes. Improvement of cell-cell interactions between epithelium and mesenchyme is a potential approach to address this phenotype issue. In this study, we developed a model system for improving interactions between human iPS-derived hepatic progenitor cells (iPS-HPCs) and human iPS-derived hepatic stellate cell-like cells (iPS-HSCs). The phenotype of iPS-HSCs, including gene and protein expression profiles and vitamin A storage, resembled that of hepatic stellate cells. Direct co-culture of iPS-HSCs with iPS-HPCs significantly improved hepatocytic maturation in iPS-HPCs, such as their capacity for albumin production. Next, we generated iPS cell lines overexpressing LIM homeobox 2 (LHX2), which suppresses myofibroblastic changes in HSCs in mice. Hepatocytic maturation in iPS-HPCs was significantly increased in direct co-culture with iPS-HSCs overexpressing LHX2, but not in co-culture with a human hepatic stellate cell line (LX-2) overexpressing LHX2. LHX2 regulated the expression of extracellular matrices, such as laminin and collagen, in iPS-HSCs. In conclusion, this study provides an evidence that LHX2 upregulation in iPS-HSCs promotes hepatocytic maturation of iPS-HPCs, and indicates that genetically modified iPS-HSCs will be of value for research into cell-cell interactions.

Background: Recent studies have shown that genetic alterations are associated with the effect of patient geographic location on gallbladder cancer development. Peru has a high incidence of gallbladder cancer, but causative factors have not yet been identified. We examined the frequency of mutations in TP53 and K -ras genes in Peruvian patients with gallbladder cancer, and compared this with data from Bolivia, Hungary, Chile, and Japan, which have a high gallbladder cancer incidence. Methods: DNA was extracted from formalin-fixed paraffin-embedded gallbladder tissue sections of 30 gallbladder cancer patients (9 men and 21 women) obtained using microdissection. Mutations in exons 5 to 8 of TP53 and codons 12, 13, and 61 of K -ras were examined using direct sequencing. Results: TP53 mutations were observed in 10 (33.3%) of patients, but K-ras mutations were absent. Nine (90%) TP53 mutations were point mutations (7 missense and 2 silent mutations), and the most frequent substitution was a G:C to A:T transition. G:C to A:T transitions at the CpG site or G:C to T:A transversions were found in one patient each. No significant differences were found in the frequency of TP53 and K-ras mutations among patients in the 5 countries. Conclusions: Our findings suggest that endogenous mechanisms and exogenous carcinogens may affect the carcinogenic process in Peruvian gallbladder cancer patients, similar to that in Bolivian patients. Further studies with a larger sample size are needed to clarify these findings.

Background & Aims Congenital hepatic fibrosis (CHF) is a genetic liver disease resulting in abnormal proliferation of cholangiocytes and progressive hepatic fibrosis. CHF is caused by mutations in the PKHD1 gene and the subsequent dysfunction of the protein it encodes, fibrocystin. However, the underlying molecular mechanism of CHF, which is quite different from liver cirrhosis, remains unclear. This study investigated the molecular mechanism of CHF pathophysiology using a genetically engineered human induced pluripotent stem (iPS) cell model to aid the discovery of novel therapeutic agents for CHF. Methods PKHD1-knockout (PKHD1-KO) and heterozygously mutated PKHD1 iPS clones were established by RNA-guided genome editing using the CRISPR/Cas9 system. The iPS clones were differentiated into cholangiocyte-like cells in cysts (cholangiocytic cysts [CCs]) in a 3D-culture system. Results The CCs were composed of a monolayer of cholangiocyte-like cells. The proliferation of PKHD1-KO CCs was significantly increased by interleukin-8 (IL-8) secreted in an autocrine manner. IL-8 production was significantly elevated in PKHD1-KO CCs due to mitogen-activated protein kinase pathway activation caused by fibrocystin deficiency. The production of connective tissue growth factor (CTGF) was also increased in PKHD1-KO CCs in an IL-8-dependent manner. Furthermore, validation analysis demonstrated that both the serum IL-8 level and the expression of IL-8 and CTGF in the liver samples were significantly increased in patients with CHF, consistent with our in vitro human iPS-disease model of CHF. Conclusions Loss of fibrocystin function promotes IL-8-dependent proliferation of, and CTGF production by, human cholangiocytes, suggesting that IL-8 and CTGF are essential for the pathogenesis of CHF. IL-8 and CTGF are candidate molecular targets for the treatment of CHF. Lay summary Congenital hepatic fibrosis (CHF) is a genetic liver disease caused by mutations of the PKHD1 gene. Dysfunction of the protein it encodes, fibrocystin, is closely associated with CHF pathogenesis. Using an in vitro human induced pluripotent stem cell model and patient samples, we showed that the loss of fibrocystin function promotes proliferation of cholangiocytes and the production of connective tissue growth factor (CTGF) in an interleukin 8 (IL-8)-dependent manner. These results suggest that IL-8 and CTGF are essential for the pathogenesis of CHF.

GNAQ and GNA11 mutations are thought to be important for the tumorigenesis of uveal melanoma. Although previous studies have reported on mutation rates in cases of uveal melanoma, presently, no such report for the Japanese population exists. In this study, we examined the frequency of GNAQ and GNA11 somatic mutations in cases of uveal melanoma in Japan and their relationship with clinicopathologic features or Ki-67-positive cell rates (Ki-67 labeling index: Ki-67 LI) using immunofluorescence methods. The study involved 19 cases of uveal melanoma. We extracted the template DNA from formalin-fixed, paraffin-embedded specimens using a DNA extraction kit. We amplified the DNA sequences of GNAQ and GNA11 using polymerase chain reaction and analyzed mutations by direct sequencing. We evaluated Ki-67 LI using immunofluorescence methods. The frequencies of GNAQ and GNA11 somatic mutations were 26.3% (5/19) and 31.6% (6/19), respectively. The GNAQ and GNA11 mutations were mutually exclusive, as indicated in previous reports. The frequency of GNA11 mutations was significantly higher in epithelioid cells; however, no significant association between GNAQ mutations and cell type was evident, and there was no significant difference in Ki-67 LI between the mutation-positive and mutation-negative tumors. GNAQ and GNA11 mutations were identified in cases of uveal melanoma in Japan, although at lower frequencies than in white counterparts. The mutation frequency of GNA11 was significantly higher in epithelioid cells.

Myeloid cell differentiation is the process by which stem cells develop into mature monocytes or granulocytes. This process is achieved by the sequential activation of variety of genes. Disruption of this process can result in immunodeficiency, bone marrow failure syndrome, or leukemia. Acute promyelocytic leukemia (APL) is characterized by the t(15;17) translocation and can be treated by a combination of all-trans retinoic acid (ATRA) and anthracycline. This treatment can induce leukemic cell differentiation, leading to extremely high remission rates. XAB2, a molecule involved in nucleotide excision repair (NER), is downregulated during granulocyte differentiation and shows reduced expression in NB4 APL-derived cells in vitro. Differentiation of APL by ATRA treatment reduced XAB2 expression levels in vivo. These observations suggest that cellular differentiation is associated with reduced NER activity and provides new insights into combined differentiation induction. NB4 cells were more susceptible than the immature myeloid leukemic cell lines, Kasumi-3 and Kasumi-1, to the DNA interstrand crosslinking agent cisplatin.

The initial step of bone regeneration requires the migration of osteogenic cells to defective sites. Our previous studies suggest that a salmon DNA-based scaffold can promote the bone regeneration of calvarial defects in rats. We speculate that the salmon DNA may possess osteoinductive properties, including the homing of migrating osteogenic cells. In the present study, we investigated the influence of the salmon DNA on osteoblastic differentiation and induction of osteoblast migration using MG63 cells (human preosteoblasts) in vitro. Moreover, we analyzed the bone regeneration of a critical-sized in vivo calvarial bone defect (CSD) model in rats. The salmon DNA enhanced both mRNA and protein expression of the osteogenesis-related factors, runt-related transcription factor 2 (Runx2), alkaline phosphatase, and osterix (OSX) in the MG63 cells, compared with the cultivation using osteogenic induction medium alone. From the histochemical and immunohistochemical assays using frozen sections of the bone defects from animals that were implanted with DNA disks, many cells were found to express aldehyde dehydrogenase 1, one of the markers for mesenchymal stem cells. In addition, OSX was observed in the replaced connective tissue of the bone defects. These findings indicate that the DNA induced the migration and accumulation of osteogenic cells to the regenerative tissue. Furthermore, an in vitro transwell migration assay showed that the addition of DNA enhanced an induction of osteoblast migration, compared with the medium alone. The implantation of the DNA disks promoted bone regeneration in the CSD of rats, compared with that of collagen disks. These results indicate that the salmon DNA enhanced osteoblastic differentiation and induction of migration, resulting in the facilitation of bone regeneration.

Currently used elution methods for strong cation exchange (SCX) chromatography are based on two principles: salt and pH gradient. In this paper, we report the first observation of peptide elution by acid gradient. The degree of peptide separation using C18-SCX StageTip was greatly improved by our acid and salt-based elution method compared with a salt-based elution method. This development enabled us to identify over 22 000 phosphopeptides from 2 mg of protein without labor-intensive sample preparation. Our method is simple, robust, scalable, and low-cost and can be easily implemented without any special equipment or techniques.

As a response to the health effects associated with the Fukushima Daiichi Nuclear Power Plant accident that occurred after the Great East Japan Earthquake, the Thyroid Ultrasound Examination program as a part of the Fukushima Health Management Survey was started on Oct. 9th, 2011. Since this project required a large-scale cohort comprising all residents aged ≤ 18 years living in Fukushima when the earthquake happened, the nurturing of many ultrasound examiners was a matter of great urgency. Moreover, the standardization of examination procedures and skills of examiners were also important issues. Therefore, educational projects were established to develop ultrasound skills for medical doctors and technicians in Fukushima Prefecture. Marked efforts for this project resulted in increases in ultrasound examiners and institutions taking part in the Thyroid Ultrasound Examination project. Medical technicians specialized in ultrasound examinations are actively involved in these educational projects. We report the details of human resource development projects from the point of view of such medical technicians.

Interleukin (IL)-27, composed of p28 and EBV-induced gene 3 subunits, has diverse functions in enhancing cell-mediated immunity and silencing the immunity. We examined whether forced expression of the p28-linked EBI3 gene in human oesophageal carcinoma cells (Eca109) produced antitumour effects in a T cell-defective condition. Tumour growth of Eca109 cells expressing IL-27 (Eca109 ⁄ IL-27) was retarded in nude mice compared with parental and vector DNA-transduced tumours and survival of the mice inoculated with Eca109 ⁄ IL-27 cells was prolonged. Expression of the tumour necrosis factor-a, IL-1b and IL-6 genes was up-regulated in Eca109 ⁄ IL-27 tumour specimens while the tumours remained small in size but the increased transcription was subsequently down-regulated in enlarged tumours. Spleen cells from micebearing Eca109 ⁄ IL-27 tumours produced interferon-c and showed YAC-1-targeted cytotoxic activities greater than those of mice inoculated with parental or vector DNA-transducer tumours. Numbers of DX5 + CD69 + natural killer cells in spleen of mice-bearing Eca109 ⁄ IL-27 tumours and those of CD31 + cells within Eca109 ⁄ IL-27 tumours remained the same as found in micebearing parental or vector DNA-transduced tumours. These data collectively suggest that the IL-27-mediated antitumour effects produced in a mature T cell-defective condition were attributable to enhanced interferon-c production and natural killer activities.

The Gallyas method is a silver impregnation technique that is essential in the field of neuropathology because of its high sensitivity for the detection of argentophilic inclusion bodies in the central nervous system. In Japan, the Gallyas method has improved and is widely used as the “modified Gallyas method”. However, this method is not popularly used in general pathology laboratories because of the need for special reagents, several staining processes, and skilled techniques. The objective of the current study was to provide a simplified Gallyas method. We omitted the lanthanum nitrate step from the staining process and verified the adequacy in comparison with the original method as well as immunohistochemistry, using specimens from patients of Alzheimer's disease, argyrophilic grain disease, multiple system atrophy, Pick's disease, and Lewy body disease. The simplified method provided good staining to all the structures in archival tissues, compared with the modified Gallyas method in a significantly shorter staining time. The lanthanum nitrate step can be omitted from the modified Gallyas method, resulting in reduction in the number of reagents required and shortening of the staining time.

S-1 is a novel oral anticancer agent consisting of tegafur, a prodrug of 5-fluorouracil, and 2 modulators. A phase I study of sequential S-1 and cyclophosphamide(CPA)therapy was conducted to determine the dose-limiting toxicities(DLTs)and recommended doses(RDs)in patients with metastatic or recurrent breast cancer(MBC). Patients with MBC received sequential S-1 and CPA. Chemotherapy consisted of administration of S-1 twice daily on days 1-14 at escalating doses of 40, 50, 65, and 80mg/m2/day and CPA at 100 mg/body/day on days 15-28. The schedule was repeated twice at a 4-week interval. The purposes of this study were to determine the RDs, safety, and efficacy of the regimen. A total of 12 patients were registered. No patients experienced DLTs, and the RDs of S-1 and CPA were 80mg/m2/day and 100 mg/body/day, respectively. The response rate was 50. 0%. In conclusion, sequential therapy with S-1 and CPA could be safely and effectively used for the treatment of MBC, and the RDs for this regimen were determined to be 80mg/m2/day for S-1 and 100 mg/m2/day for CPA.

First-generation H1-antihistamines are known to cause fatigue and drowsiness, due to their poor receptor selectivity and their high penetration rate of the blood–brain barrier. However, little is known about the effects of first-generation H1-antihistamines on postural stability. The purpose of this study was to evaluate the effects of d-chlorpheniramine on postural stability using posturography with and without foam rubber. A double-blind study with three parallel groups was conducted. Twenty-seven healthy young volunteers (mean age 21.9 years) were recruited and orally administered d-chlorpheniramine, 2 or 4 mg, or placebo. Postural sway was measured every hour up to 8 h after administration. Two-legged stance tasks were performed by each subject in four conditions: eyes open or eyes closed and with or without foam rubber. Inter-group comparisons showed that the group receiving 4-mg d-chlorpheniramine showed significantly larger sway in the eyes open with foam rubber condition (visual and vestibular information available, somatosensory information reduced). Inter-subject analysis in the 4-mg d-chlorpheniramine group showed that the effect of d-chlorpheniramine on postural control was variable. Our results suggest that among the three main sensory systems responsible for postural control (visual, vestibular, and somatosensory), d-chlorpheniramine may have a larger effect on the visual and/or vestibular systems in susceptible individuals.

A method for rapid identification of antiseptic- and methicillin-resistant Staphylococcus aureus (MRSA) based on 3 loop-mediated isothermal amplification (LAMP) assays was developed. LAMP targeting the femB gene identified S. aureus with 100% specificity, and LAMP targeting the mecA gene associated with methicillin resistance identified methicillin-resistant staphylococci with 100% specificity. LAMP targeting the qacA/B gene encoding an efflux pump responsible for antiseptic resistance identified high-acriflavine-resistant (MIC≥100 mg/L) MRSA (92.5% positive) and acriflavine-susceptible (MIC

A phase II clinical trial was conducted to examine the clinical and pathologic efficacy and safety of neoadjuvant paclitaxel with or without trastuzumab in women with advanced or metastatic breast cancer. A total of 49 patients with advanced or metastatic breast cancer (clinical stage IIB -IV) were included. Patients with HER2-negative tumors received weekly paclitaxel 80 mg/m2 (days 1, 8, 15) followed by a 1-week break for 4 cycles. Patients with HER2-positive tumors received weekly paclitaxel 80 mg/m2 (days 1, 8, 15) followed by a 1-week break and a trastuzumab 4 mg/kg loading dose, intravenously, followed by 2 mg/kg weekly for 4 cycles. The age of the patients was 51.6 +/- 1.6 years (mean +/- SE) and the size of their tumors was 5.8 +/- 0.4 cm (mean +/- SE). Thirty-two patients had HER2-negative tumors and 17 had HER2-positive tumors. Of 49 patients, 13 (26.5%) had a clinical complete response and 24 (49.0%) had a clinical partial response. Five (10.2%) patients had a pathological complete response (pCR) and three (6.1%) patients had a near pCR in the breast. A total of eight (16.3%) patients had a pCR or near pCR in the breast. The pCR or near pCR rate was 3.1% in the HER2-negative group and 41.2% in the HER2-positive group. With a median follow-up of 28 months (range, 1-45), the 3-year overall survival was 88%. Clinical responders showed a significantly better overall survival than non-responders (p0.01). Pathological responders showed a better overall survival than non-responders. There was no significant difference in overall survival between patients with HER2-positive and -negative tumors. In conclusion, combined neoadjuvant weekly paclitaxel and trastuzumab achieved high clinical and pathological response rates for HER2 -overexpressing breast cancers, despite the omission of an anthracycline.

A dose-escalation study was conducted for patients with inoperable or recurrent breast cancer in order to determine the recommended dose (RD) of capecitabine combined with a fixed dose of weekly paclitaxel. Capecitabine was administered twice daily from day 1 through day 14 combined with paclitaxel given on days 1 and 8, every 21 days. Dose-limiting toxicities(DLT)were evaluated during the first two cycles. Three patients were recruited at one of two dose levels (capecitabine 1,255 mg/m2 or 1,657 mg/m2, paclitaxel 80 mg/m2). In this study, no DLT was seen in each level, and the RD of capecitabine was determined to be 1,657 mg/m2.

Selective expression of a therapeutic gene in tumors contributes to the efficacy and the safety of cancer therapy. Regulatory regions of genes that are preferentially expressed in tumors have been examined. The regions of the survivin gene exhibited the greatest activity in human hepatocellular carcinoma cells. Deletion of the survivin regulatory region from the 5'-side demonstrated that the 0.5 kb and the 1.4 kb fragments possessed a strong promoter activity with relative tumor specificity. Human tumors transfected with the herpes simplex virus-thymidine kinase gene, that was powered by the survivin region, became susceptible to a prodrug, ganciclovir. Although survivin gene expression was up-regulated in the G2/M-phase of the cell cycle, taxol or vincristine treatment, which induce cell cycle arrest at the M-phase, did not enhance the transcriptional activity of the survivin promoter. These data collectively suggest that the survivin regulatory region induces the expression of an exogenous gene in tumors, but the transcriptional activity is not directly linked with M-phase induction.

We quantitatively studied factors influencing the environment cleanliness for intravenous hyperalimentation (IVH) admixing. The environment cleanliness was evaluated by measuring the counts of particles (> 0.5 micron) and bacteria floating in 1 ft3 of the air inside the clean room (23.6 m3) and in the clean bench built in the department of pharmacy, The University of Tokyo Hospital in 1998. The number of particles at the center of the clean room during IVH admixing by 4 pharmacists was higher than that at the medicine passing area (150 +/- 50/ft3 vs. 260 +/- 60/ft3; mean +/- S.D., n = 12). The cleanliness inside the clean room was improved as the measurement point became higher from the floor (600 +/- 180/ft3, 150 +/- 50/ft3, and 35 +/- 15/ft3 at 50, 100, and 150 cm height, respectively) and the number of persons working inside the room decreased. The changes in the counts of floating bacteria were similar to that of floating particles under the same conditions. In addition the effect of disinfection on the counts of bacteria was clearly observed. When the cleanliness of the room became lower by turning off the air conditioning, the particle counts inside the clean bench became lower along with the distance from the front glass becoming deeper (i.e., 1400 +/- 550/ft3, 140 +/- 70/ft3, and 40 +/- 30/ft3 at 0, 5, and 15 cm, respectively). From these lines of evidence, the following items were suggested in order to maintain the environment cleanliness for IVH admixing. First, the number of persons residing in the clean room should be kept to be minimum. Second, the clean bench should be set up in the center of the clean room. Finally IVH admixing operation should be performed at more than 15 cm depth inside the front glass surface of the clean bench. Moreover, the effect of mopping-up of the clean room with 0.1% benzethonium chloride clearly demonstrated the importance of disinfection on a routine basis.