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BackgroundLiver tumors are rare in children with histologic heterogeneity that makes diagnosis challenging. Systematic histopathological review, performed as part of collaborative therapeutic protocols, identified relevant histologic subtypes that are important to distinguish. The Children's Hepatic tumors International Collaboration (CHIC) was established to study pediatric liver tumors on a global scale and led to establishment of a provisional consensus classification for use in international clinical trials. The current study is the validation of this initial classification and first large-scale application by international expert reviewers.ProcedureThe CHIC initiative includes data from 1605 children treated on eight multicenter hepatoblastoma (HB) trials. Review of 605 available tumors was performed by seven expert pathologists from three consortia (US, EU, Japan). Cases with discordant diagnoses were collectively reviewed to reach a final consensus diagnosis.ResultsOf 599 cases with sufficient material for review, 570 (95.2%) were classified as HB by all consortia, and 29 (4.8%) as non-HB, which included "hepatocellular neoplasm, NOS" and malignant rhabdoid tumors. 453 of 570 HBs were classified as epithelial by final consensus. Some patterns (i.e., small cell undifferentiated, macrotrabecular, cholangioblastic) were selectively identified by reviewers from different consortia. All consortia identified a similar number of mixed epithelial-mesenchymal HB.ConclusionsThis study represents the first large-scale application and validation of the pediatric malignant hepatocellular tumors consensus classification. It is a valuable resource to train future generations of investigators on accurate diagnosis of these rare tumors and provides a framework for further international collaborative studies and refinement of the current classification of pediatric liver tumors.

Small cell undifferentiated (SCU) histology and alpha-fetoprotein (AFP) levels below 100 ng/mL have been reported as poor prognostic factors in hepatoblastoma (HB); subsequent studies reported SMARCB1 mutations in some SCU HBs confirming the diagnosis of rhabdoid tumor. The Children's Hepatic tumors International Collaboration (CHIC) database was queried for patients with HB who had AFP levels less than 100 ng/mL at diagnosis or were historically diagnosed as SCU HBs. Seventy-three of 1605 patients in the CHIC database were originally identified as SCU HB, HB with SCU component, or HB with low AFP levels. Upon retrospective review, they were re-classified as rhabdoid tumors (n = 11), HB with SCU component (n = 41), and HB with low AFP (n = 14). Seven were excluded for erroneously low AFP levels. Overall survival was 0% for patients with rhabdoid tumors, 76% for patients with HB with SCU component, and 64% for patients with HB with AFP less than 100 ng/mL. Patients with HB with SCU component or low AFP should be assessed for SMARCB1 mutations and, if confirmed, treated as rhabdoid tumors. When rhabdoid tumors are excluded, the presence of SCU component and low AFP at diagnosis were not associated with poor prognosis in patients diagnosed with HB.

type:text, Identifying the electrical properties of cancer relies on the understanding of the electric potential (EP) of cancer tissues. This study aimed to investigate the EP properties in 49 pancreatic head cancer tissues using a digital multimetre. The anode was placed at the central side of the tumour, and the electric potential differences (EPDs) between cancerous and cancerous, cancerous and noncancerous, and noncancerous and noncancerous lesions at approximately 1-cm intervals following resection were evaluated. Pathological evaluation identified 30 of these samples as pancreatic invasive ductal carcinoma (PIDC, 10 without preoperative chemotherapy and 20 after chemotherapy), seven other pancreatic cancers, three tumours of Vater’s ampulla (VA), and eight extrahepatic cholangiocarcinoma (EHCC) samples. We also evaluated the differences in pH for cancerous and noncancerous lesions in nine PIDC samples. Our data suggest that the EP of pancreatic cancerous tissues is higher than that of noncancerous tissues, especially in PIDCs. We also noted that EPD was the highest when comparing cancerous and noncancerous lesions. Additionally, PIDC tissues presented with low pH; the pH difference between cancerous and noncancerous sites was significantly correlated with EPD (P = 0.011). These EPDs were also correlated with tumour size in PIDCs and inversely correlated with their response to chemotherapy. The EP values for both the cancerous and noncancerous sites in both the VA tumours and EHCC samples were not significantly different, whereas EPD in PIDC correlated with tumour extension and viable tumour content, suggesting that EPD might be useful for evaluating the viability and effectiveness of neoadjuvant chemotherapy., This research was partially supported by a Grant-in-Aid for Scientific Research (A) (Nos. 15H02567 and 17H05102) from the Ministry of Education, Culture, Sports, Science, and Technology, and the Ministry of Health, Labour, and Welfare for Japan.

Identifying the electrical properties of cancer relies on the understanding of the electric potential (EP) of cancer tissues. This study aimed to investigate the EP properties in 49 pancreatic head cancer tissues using a digital multimetre. The anode was placed at the central side of the tumour, and the electric potential differences (EPDs) between cancerous and cancerous, cancerous and noncancerous, and noncancerous and noncancerous lesions at approximately 1-cm intervals following resection were evaluated. Pathological evaluation identified 30 of these samples as pancreatic invasive ductal carcinoma (PIDC, 10 without preoperative chemotherapy and 20 after chemotherapy), seven other pancreatic cancers, three tumours of Vater’s ampulla (VA), and eight extrahepatic cholangiocarcinoma (EHCC) samples. We also evaluated the differences in pH for cancerous and noncancerous lesions in nine PIDC samples. Our data suggest that the EP of pancreatic cancerous tissues is higher than that of noncancerous tissues, especially in PIDCs. We also noted that EPD was the highest when comparing cancerous and noncancerous lesions. Additionally, PIDC tissues presented with low pH; the pH difference between cancerous and noncancerous sites was significantly correlated with EPD (P = 0.011). These EPDs were also correlated with tumour size in PIDCs and inversely correlated with their response to chemotherapy. The EP values for both the cancerous and noncancerous sites in both the VA tumours and EHCC samples were not significantly different, whereas EPD in PIDC correlated with tumour extension and viable tumour content, suggesting that EPD might be useful for evaluating the viability and effectiveness of neoadjuvant chemotherapy., This research was partially supported by a Grant-in-Aid for Scientific Research (A) (Nos. 15H02567 and 17H05102) from the Ministry of Education, Culture, Sports, Science, and Technology, and the Ministry of Health, Labour, and Welfare for Japan.

Identifying the electrical properties of cancer relies on the understanding of the electric potential (EP) of cancer tissues. This study aimed to investigate the EP properties in 49 pancreatic head cancer tissues using a digital multimetre. The anode was placed at the central side of the tumour, and the electric potential differences (EPDs) between cancerous and cancerous, cancerous and noncancerous, and noncancerous and noncancerous lesions at approximately 1-cm intervals following resection were evaluated. Pathological evaluation identified 30 of these samples as pancreatic invasive ductal carcinoma (PIDC, 10 without preoperative chemotherapy and 20 after chemotherapy), seven other pancreatic cancers, three tumours of Vater’s ampulla (VA), and eight extrahepatic cholangiocarcinoma (EHCC) samples. We also evaluated the differences in pH for cancerous and noncancerous lesions in nine PIDC samples. Our data suggest that the EP of pancreatic cancerous tissues is higher than that of noncancerous tissues, especially in PIDCs. We also noted that EPD was the highest when comparing cancerous and noncancerous lesions. Additionally, PIDC tissues presented with low pH; the pH difference between cancerous and noncancerous sites was significantly correlated with EPD (P = 0.011). These EPDs were also correlated with tumour size in PIDCs and inversely correlated with their response to chemotherapy. The EP values for both the cancerous and noncancerous sites in both the VA tumours and EHCC samples were not significantly different, whereas EPD in PIDC correlated with tumour extension and viable tumour content, suggesting that EPD might be useful for evaluating the viability and effectiveness of neoadjuvant chemotherapy., This research was partially supported by a Grant-in-Aid for Scientific Research (A) (Nos. 15H02567 and 17H05102) from the Ministry of Education, Culture, Sports, Science, and Technology, and the Ministry of Health, Labour, and Welfare for Japan.

Introduction: Hepatoblastoma (HB) is the most common paediatric liver tumour, and epigenetic aberrations may be important in HB development. Recently, the Children's He-patic Tumors International Collaboration-Hepatoblastoma Stratification (CHIC-HS) devel-oped risk stratification based on clinicopathological factors. This study aimed to construct a more accurate model by integrating CHIC-HS with molecular factors based on DNA methylation. Methods: HB tumour specimens (N = 132) from patients treated with the Japanese Pediatric Liver Tumors Group-2 protocol were collected and subjected to methylation analysis by bisul-fite pyrosequencing. Associations between methylation status and clinicopathological factors, overall survival (OS), and event-free survival (EFS) were retrospectively analysed. We inves-tigated the effectiveness of the evaluation of methylation status in each CHIC-HS risk group and generated a new risk stratification model. Results: Most specimens (82%) were from post-chemotherapy tissue. Hypermethylation in > 2 of the four genes (RASSF1A, PARP6, OCIAD2, and MST1R) was significantly associated with poorer OS and EFS. Multivariate analysis indicated that > 2 methylated genes was an in-dependent prognostic factor (hazard ratios of 6.014 and 3.684 for OS and EFS, respectively). Two or more methylated genes was also associated with poorer OS in the CHIC-very low (VL)-/low (L)-risk and CHIC-intermediate (I) risk groups (3-year OS rates were 83% vs. 98% and 50% vs. 95%, respectively). The 3-year OS rates of the VL/L, I, and high-risk groups in the new stratification model were 98%, 90%, and 62% (vs. CHIC-HS [96%, 82%, and 65%, respectively]), optimising CHIC-HS. Conclusions: Our proposed stratification system considers individual risk in HB and may improve patient clinical management. (C) 2022 Elsevier Ltd. All rights reserved.

Introduction: Hepatoblastoma (HB) is the most common paediatric liver tumour, and epigenetic aberrations may be important in HB development. Recently, the Children's He-patic Tumors International Collaboration-Hepatoblastoma Stratification (CHIC-HS) devel-oped risk stratification based on clinicopathological factors. This study aimed to construct a more accurate model by integrating CHIC-HS with molecular factors based on DNA methylation. Methods: HB tumour specimens (N = 132) from patients treated with the Japanese Pediatric Liver Tumors Group-2 protocol were collected and subjected to methylation analysis by bisul-fite pyrosequencing. Associations between methylation status and clinicopathological factors, overall survival (OS), and event-free survival (EFS) were retrospectively analysed. We inves-tigated the effectiveness of the evaluation of methylation status in each CHIC-HS risk group and generated a new risk stratification model. Results: Most specimens (82%) were from post-chemotherapy tissue. Hypermethylation in > 2 of the four genes (RASSF1A, PARP6, OCIAD2, and MST1R) was significantly associated with poorer OS and EFS. Multivariate analysis indicated that > 2 methylated genes was an in-dependent prognostic factor (hazard ratios of 6.014 and 3.684 for OS and EFS, respectively). Two or more methylated genes was also associated with poorer OS in the CHIC-very low (VL)-/low (L)-risk and CHIC-intermediate (I) risk groups (3-year OS rates were 83% vs. 98% and 50% vs. 95%, respectively). The 3-year OS rates of the VL/L, I, and high-risk groups in the new stratification model were 98%, 90%, and 62% (vs. CHIC-HS [96%, 82%, and 65%, respectively]), optimising CHIC-HS. Conclusions: Our proposed stratification system considers individual risk in HB and may improve patient clinical management. (C) 2022 Elsevier Ltd. All rights reserved.

type:text, Efforts to identify biomarkers for neuroblastoma (NB) have been ongoing, but no definite biomarker has been identified in peripheral blood. We proposed the use of plasma exosomal miRNAs as biomarkers of unfavorable NB patient outcomes. Exosomal miRNAs isolated from 31 plasma and 37 tissue samples, many from the same NB patients, were sequenced using a next-generation sequencing instrument. We analyzed the correlation between miRNA expression levels in plasma and tissue samples with International Neuroblastoma Risk Group staging system (INRGSS) outcome and MYCN status. We chose differentially expressed miRNAs with similar expression patterns in plasma and tissue samples in each of the three analysis groups and combined those miRNAs to find the optimal combination with the potential to be considered as a biomarker. MicroRNA-92a-3p was found to be significantly upregulated in deceased patients (p = 0.017), miR-375 was upregulated in INRGSS stage M patients (p = 0.002), and plasma miR-92a-3p and miR-99a-5 levels were upregulated in patients with MYCN amplification (p = 0.007 and 0.006). The combination of miR-92a-3p, miR-375, and miR-99a-5p levels was shown to be a statistically significant predictor of NB patient outcomes (AUC = 0.726, p = 0.001, 95% CI = 0.612–0.841, sensitivity = 77%, specificity = 56.7%). Thus, the combination of miR-92a3p, miR-375, and miR-99a-5p may potentially be used as a biomarker for unfavorable NB patient outcomes. However, further validation is required in a larger number of NB patients., This research was partially supported by a Grant-in-Aid for Scientific Research (A) (No.19H0105600) from the Ministry of Education, Culture, Sports, Science and Technology.

Efforts to identify biomarkers for neuroblastoma (NB) have been ongoing, but no definite biomarker has been identified in peripheral blood. We proposed the use of plasma exosomal miRNAs as biomarkers of unfavorable NB patient outcomes. Exosomal miRNAs isolated from 31 plasma and 37 tissue samples, many from the same NB patients, were sequenced using a next-generation sequencing instrument. We analyzed the correlation between miRNA expression levels in plasma and tissue samples with International Neuroblastoma Risk Group staging system (INRGSS) outcome and MYCN status. We chose differentially expressed miRNAs with similar expression patterns in plasma and tissue samples in each of the three analysis groups and combined those miRNAs to find the optimal combination with the potential to be considered as a biomarker. MicroRNA-92a-3p was found to be significantly upregulated in deceased patients (p = 0.017), miR-375 was upregulated in INRGSS stage M patients (p = 0.002), and plasma miR-92a-3p and miR-99a-5 levels were upregulated in patients with MYCN amplification (p = 0.007 and 0.006). The combination of miR-92a-3p, miR-375, and miR-99a-5p levels was shown to be a statistically significant predictor of NB patient outcomes (AUC = 0.726, p = 0.001, 95% CI = 0.612–0.841, sensitivity = 77%, specificity = 56.7%). Thus, the combination of miR-92a3p, miR-375, and miR-99a-5p may potentially be used as a biomarker for unfavorable NB patient outcomes. However, further validation is required in a larger number of NB patients., This research was partially supported by a Grant-in-Aid for Scientific Research (A) (No.19H0105600) from the Ministry of Education, Culture, Sports, Science and Technology.

Cell-free DNA (cfDNA) analysis may provide a non-invasive diagnostic approach for lung adenocarcinoma patients. Recently, droplet digital PCR (ddPCR) has been developed as a highly sensitive detection method for a low mutant allele percentage. The ddPCR detection limit for epithelial growth factor receptor (EGFR) mutations was evaluated using cell lines, NCI-H1975 for EGFR L858R point mutation and PC-9 for EGFR E746-A750del. Subsequently, detection of EGFR mutations by ddPCR was performed in tumor DNA (tDNA) and cfDNA samples of 19 lung adenocarcinoma patients whose tumor biopsies were already evaluated for EGFR mutations by clamp PCR (13 of L858R, 3 of E746-750del, and 3 of EGFR negative). In 12 cases, immunohistochemical analysis was performed to quantify the number of EGFR L858R-positive cells rate. EGFR point mutation or deletion were detected in 16 tumor DNA samples. In the measurable cfDNA samples, the rate of detection by ddPCR in cfDNA was 61.5% (8/13) for L858R and 100% (3/3) for E746-A750del. A relative correlation was found between the allele fraction (AF) of tDNA and the number of EGFR L858R-positive cells rate. No correlation was found between the AF of tDNA and AF of cfDNA. In our study, cfDNA mutation detection was not associated with clinicopathological features, but cases with high AF of cfDNA did have metastatic lesions. Our study shows that ddPCR enables cfDNA analysis for EGFR L858R and E746-A750del, with a high detection rate. Therefore, cfDNA analysis using ddPCR may complement to tumor biopsy and is beneficial for precision medicine in lung adenocarcinoma patients.

type:text, Cell-free DNA (cfDNA) analysis may provide a non-invasive diagnostic approach for lung adenocarcinoma patients. Recently, droplet digital PCR (ddPCR) has been developed as a highly sensitive detection method for a low mutant allele percentage. The ddPCR detection limit for epithelial growth factor receptor (EGFR) mutations was evaluated using cell lines, NCI-H1975 for EGFR L858R point mutation and PC-9 for EGFR E746-A750del. Subsequently, detection of EGFR mutations by ddPCR was performed in tumor DNA (tDNA) and cfDNA samples of 19 lung adenocarcinoma patients whose tumor biopsies were already evaluated for EGFR mutations by clamp PCR (13 of L858R, 3 of E746-750del, and 3 of EGFR negative). In 12 cases, immunohistochemical analysis was performed to quantify the number of EGFR L858R-positive cells rate. EGFR point mutation or deletion were detected in 16 tumor DNA samples. In the measurable cfDNA samples, the rate of detection by ddPCR in cfDNA was 61.5% (8/13) for L858R and 100% (3/3) for E746-A750del. A relative correlation was found between the allele fraction (AF) of tDNA and the number of EGFR L858R-positive cells rate. No correlation was found between the AF of tDNA and AF of cfDNA. In our study, cfDNA mutation detection was not associated with clinicopathological features, but cases with high AF of cfDNA did have metastatic lesions. Our study shows that ddPCR enables cfDNA analysis for EGFR L858R and E746-A750del, with a high detection rate. Therefore, cfDNA analysis using ddPCR may complement to tumor biopsy and is beneficial for precision medicine in lung adenocarcinoma patients.

Cell-free DNA (cfDNA) analysis may provide a non-invasive diagnostic approach for lung adenocarcinoma patients. Recently, droplet digital PCR (ddPCR) has been developed as a highly sensitive detection method for a low mutant allele percentage. The ddPCR detection limit for epithelial growth factor receptor (EGFR) mutations was evaluated using cell lines, NCI-H1975 for EGFR L858R point mutation and PC-9 for EGFR E746-A750del. Subsequently, detection of EGFR mutations by ddPCR was performed in tumor DNA (tDNA) and cfDNA samples of 19 lung adenocarcinoma patients whose tumor biopsies were already evaluated for EGFR mutations by clamp PCR (13 of L858R, 3 of E746-750del, and 3 of EGFR negative). In 12 cases, immunohistochemical analysis was performed to quantify the number of EGFR L858R-positive cells rate. EGFR point mutation or deletion were detected in 16 tumor DNA samples. In the measurable cfDNA samples, the rate of detection by ddPCR in cfDNA was 61.5% (8/13) for L858R and 100% (3/3) for E746-A750del. A relative correlation was found between the allele fraction (AF) of tDNA and the number of EGFR L858R-positive cells rate. No correlation was found between the AF of tDNA and AF of cfDNA. In our study, cfDNA mutation detection was not associated with clinicopathological features, but cases with high AF of cfDNA did have metastatic lesions. Our study shows that ddPCR enables cfDNA analysis for EGFR L858R and E746-A750del, with a high detection rate. Therefore, cfDNA analysis using ddPCR may complement to tumor biopsy and is beneficial for precision medicine in lung adenocarcinoma patients.

Cell-free DNA (cfDNA) analysis may provide a non-invasive diagnostic approach for lung adenocarcinoma patients. Recently, droplet digital PCR (ddPCR) has been developed as a highly sensitive detection method for a low mutant allele percentage. The ddPCR detection limit for epithelial growth factor receptor (EGFR) mutations was evaluated using cell lines, NCI-H1975 for EGFR L858R point mutation and PC-9 for EGFR E746-A750del. Subsequently, detection of EGFR mutations by ddPCR was performed in tumor DNA (tDNA) and cfDNA samples of 19 lung adenocarcinoma patients whose tumor biopsies were already evaluated for EGFR mutations by clamp PCR (13 of L858R, 3 of E746-750del, and 3 of EGFR negative). In 12 cases, immunohistochemical analysis was performed to quantify the number of EGFR L858R-positive cells rate. EGFR point mutation or deletion were detected in 16 tumor DNA samples. In the measurable cfDNA samples, the rate of detection by ddPCR in cfDNA was 61.5% (8/13) for L858R and 100% (3/3) for E746-A750del. A relative correlation was found between the allele fraction (AF) of tDNA and the number of EGFR L858R-positive cells rate. No correlation was found between the AF of tDNA and AF of cfDNA. In our study, cfDNA mutation detection was not associated with clinicopathological features, but cases with high AF of cfDNA did have metastatic lesions. Our study shows that ddPCR enables cfDNA analysis for EGFR L858R and E746-A750del, with a high detection rate. Therefore, cfDNA analysis using ddPCR may complement to tumor biopsy and is beneficial for precision medicine in lung adenocarcinoma patients.

This study aimed to elucidate retrospectively the correlations between the genome and phenotype in clinical methicillin-resistant Staphylococcus aureus (MRSA) gentamicin (GEN), clindamycin (CLI), and minocycline (MIN) susceptibility using next-generation sequencing (NGS) technology. Ninety two MRSA strains were isolated from individual inpatients treated in Hiroshima University Hospital, Hiroshima, Japan, extracted for their genomic DNA, and sequenced using an Illumina® MiSeq sequencer to obtain their de novo whole-genome assembly. An in silico analysis using ResFinder was performed to obtain the genomic antimicrobial susceptibility profile which was analyzed together with GEN, CLI, and MIN minimum inhibitory concentration (MIC) levels. This study found aac(6’)aph(2”)+, spc+, ermA+, tetM+ MRSA strains were predominant (42/92) and were shown to exhibit >16 mg/L GEN (40/42), >4 mg/L CLI (26/42), and >8 mg/L MIN MIC levels (30/42). Associations between aac(6’)aph(2”) detections and GEN MIC levels (p <0.001), ermA detections and CLI MIC levels (p <0.001), and tetM detections and MIN MIC levels (p <0.001) were revealed in this study. Correlations between simultaneous detections of aac(6’) aph(2”)-spc-ermA-tetM and GEN MIC levels (φc= 0.398, p <0.001), CLI MIC levels (φc= 0.448, p <0.001), and MIN MIC levels (φc= 0.515, p <0.001) were revealed in this study. The genomicphenotypic correlation analyses in this study provided an insight of a rapid antimicrobial detection in MRSA using in silico genomic antimicrobial susceptibility profiling., This research was partially supported by a Grant-in-Aid for Scientific Research (A) (No.15H02567) from the Ministry of Education, Culture, Sports, Science, and Technology and those from Ministry of Health, Labor, and Welfare of the Government of Japan.

type:text, A congenital portosystemic shunt of the portal vein is a very rare vascular anomaly associated with the liver. We report the case of a 5-year-old girl with a patent ductus venosus and her 31-year-old mother with a congenital portosystemic shunt. The child presented with a history of an extremely low birth weight in addition to an atrial septal defect and a patent ductus venosus. At the age of 2, she underwent ligation of the ductus venosus. Her mother was also diagnosed with a congenital vascular anomaly at the age of 16. We have followed up and evaluated her asymptomatic mother for 15 years. To our knowledge, this is the first report describing the occurrence of a congenital portosystemic shunt in both a mother and her child.

A congenital portosystemic shunt of the portal vein is a very rare vascular anomaly associated with the liver. We report the case of a 5-year-old girl with a patent ductus venosus and her 31-year-old mother with a congenital portosystemic shunt. The child presented with a history of an extremely low birth weight in addition to an atrial septal defect and a patent ductus venosus. At the age of 2, she underwent ligation of the ductus venosus. Her mother was also diagnosed with a congenital vascular anomaly at the age of 16. We have followed up and evaluated her asymptomatic mother for 15 years. To our knowledge, this is the first report describing the occurrence of a congenital portosystemic shunt in both a mother and her child.

A congenital portosystemic shunt of the portal vein is a very rare vascular anomaly associated with the liver. We report the case of a 5-year-old girl with a patent ductus venosus and her 31-year-old mother with a congenital portosystemic shunt. The child presented with a history of an extremely low birth weight in addition to an atrial septal defect and a patent ductus venosus. At the age of 2, she underwent ligation of the ductus venosus. Her mother was also diagnosed with a congenital vascular anomaly at the age of 16. We have followed up and evaluated her asymptomatic mother for 15 years. To our knowledge, this is the first report describing the occurrence of a congenital portosystemic shunt in both a mother and her child.

We previously developed a method for determining MYCN gene amplification status using cell-free DNA fragments released from cancer cells into the blood of patients with neuroblastoma (NB). Here, we analyzed the relationship between MYCN amplification (MNA) status and neuroblastoma prognosis. We screened serum samples from 151 patients with NB for MNA, using real-time quantitative PCR, and compared the results with MYCN status determined using paired tumor samples. We additionally investigated whether MNA status correlates with patient survival. When a cut-off value of 5 was used, serum-based MNA analysis was found to show good sensitivity (86%) and very high specificity (95%). The sensitivities for stage 1 and 2 might be acceptable, even though it is not as good as for stage 3 and 4 (67% for stage 1 and 2, 92% for stage 3, and 87% for stage 4). MNA status correlated with overall survival in our cohort of 82 patients, with survival data available (p < 0.01). The hazard ratio of MNA status was 4.98 in patients diagnosed at less than 18 months of age (95% confidence interval, 1.00–24.78), and 1.41 (95% confidence interval, 0.63–3.14) for those diagnosed at 18 months of age or older. Serum-based MNA analysis is rapid and non-invasive compared with tumor-based MNA analysis, and has potential to predict tumor MNA status. There is still a room to improve the sensitivity of the test for tumors of stages 1 and 2, nonetheless this assay might help to determine therapeutic strategies prior to tumor biopsy, especially for patients with a life-threatening condition, as well as for patients of less than 18 months of age whose risk-grouping and treatment allocation depends on their MNA status.

Research team head, Research team member, Publisher, PAX3-FKHRまたはPAX7-FKHRキメラ遺伝子は胞巣型・混合型横紋筋肉腫の76%で陽性なのに対し胎児型では検出されず, 生存率はキメラ遺伝子陽性の胞巣型で44.2%に対してキメラ遺伝子陰性の胞巣型では75.0%と有意に高く, 胎児型の76.3%と同様だった。幹細胞マーカー発現は全ての横紋筋肉腫細胞株でCD44陽性, CD44v陰性で, CD133表出率はRh30, KYM-1, RMS-YM株では2%以下, RD株では25%で, CD133陽性細胞は抗癌剤抵抗性で免疫不全マウスにおいて高い造腫瘍能を示した。Genotypeや腫瘍幹細胞のマーカー発現に基づく新治療戦略の可能性が検討された。 Either PAX3-FKHR or PAX7-FKHR gene was positive in 76% of alveolar type rhabomyosarcoma, whereas never detected in embryonal type tumor. Survival rate was significantly lower in alveolar type cases with positive chimeric gene (44.2±7.3%) compared to those with negative chimeric gene (75.0±12.5%), which was similar to that of embryonal cases (76.3±4.9%). ALL mixed type cases were alive regardless of chimeric gene positivity. CD44, a stem cell marker, was positive in all rhabdomyosarcoma cell line examined, whereas, CD44v was negative in all the cell lines. Positivity rate of CD133 was 25% in RD cell line, whereas, less than 2% in other cell lines such as Rh30, KYM-1, RMS-YM. However, CD133 positive cells are chemotherapy resistant, and rapidly formed tumor after administrated in mice. Novel treatment strategy may be stratified according to the genotype and expression of the stem cell makers in rhabdomyosarcoma., 研究種目 : 挑戦的萌芽研究 研究期間 : 2014～2015 課題番号 : 26670766 研究分野 : 小児外科学

Hepatoblastoma (HB) is very rare but the most common malignant neoplasm of the liver occurring in children. Despite improvements in therapy, outcomes for patients with advanced HB that is refractory to standard preoperative chemotherapy remain unsatisfactory. To improve the survival rate among this group, identification of novel prognostic markers and therapeutic targets is needed. We have previously reported that altered DNA methylation patterns are of biological and clinical importance in HB. In the present study, using genome-wide methylation analysis and bisulfite pyrosequencing with specimens from HB tumors, we detected nine methylated genes. We then focused on four of those genes, GPR180, MST1R, OCIAD2, and PARP6, because they likely encode tumor suppressors and their increase of methylation was associated with a poor prognosis. The methylation status of the four genes was also associated with age at diagnosis, and significant association with the presence of metastatic tumors was seen in three of the four genes. Multivariate analysis revealed that the presence of metastatic tumors and increase of methylation of GPR180 were independent prognostic factors affecting event-free survival. These findings indicate that the four novel tumor suppressor candidates are potentially useful molecular markers predictive of a poor outcome in HB patients, which may serve as the basis for improved therapeutic strategies when clinical trials are carried out.

Hepatoblastoma (HB) is very rare but the most common malignant neoplasm of the liver occurring in children. Despite improvements in therapy, outcomes for patients with advanced HB that is refractory to standard preoperative chemotherapy remain unsatisfactory. To improve the survival rate among this group, identification of novel prognostic markers and therapeutic targets is needed. We have previously reported that altered DNA methylation patterns are of biological and clinical importance in HB. In the present study, using genome-wide methylation analysis and bisulfite pyrosequencing with specimens from HB tumors, we detected nine methylated genes. We then focused on four of those genes, GPR180, MST1R, OCIAD2, and PARP6, because they likely encode tumor suppressors and their increase of methylation was associated with a poor prognosis. The methylation status of the four genes was also associated with age at diagnosis, and significant association with the presence of metastatic tumors was seen in three of the four genes. Multivariate analysis revealed that the presence of metastatic tumors and increase of methylation of GPR180 were independent prognostic factors affecting event-free survival. These findings indicate that the four novel tumor suppressor candidates are potentially useful molecular markers predictive of a poor outcome in HB patients, which may serve as the basis for improved therapeutic strategies when clinical trials are carried out.

Hepatoblastoma (HB) is very rare but the most common malignant neoplasm of the liver occurring in children. Despite improvements in therapy, outcomes for patients with advanced HB that is refractory to standard preoperative chemotherapy remain unsatisfactory. To improve the survival rate among this group, identification of novel prognostic markers and therapeutic targets is needed. We have previously reported that altered DNA methylation patterns are of biological and clinical importance in HB. In the present study, using genome-wide methylation analysis and bisulfite pyrosequencing with specimens from HB tumors, we detected nine methylated genes. We then focused on four of those genes, GPR180, MST1R, OCIAD2, and PARP6, because they likely encode tumor suppressors and their increase of methylation was associated with a poor prognosis. The methylation status of the four genes was also associated with age at diagnosis, and significant association with the presence of metastatic tumors was seen in three of the four genes. Multivariate analysis revealed that the presence of metastatic tumors and increase of methylation of GPR180 were independent prognostic factors affecting event-free survival. These findings indicate that the four novel tumor suppressor candidates are potentially useful molecular markers predictive of a poor outcome in HB patients, which may serve as the basis for improved therapeutic strategies when clinical trials are carried out.

Hepatoblastoma (HB) is very rare but the most common malignant neoplasm of the liver occurring in children. Despite improvements in therapy, outcomes for patients with advanced HB that is refractory to standard preoperative chemotherapy remain unsatisfactory. To improve the survival rate among this group, identification of novel prognostic markers and therapeutic targets is needed. We have previously reported that altered DNA methylation patterns are of biological and clinical importance in HB. In the present study, using genome-wide methylation analysis and bisulfite pyrosequencing with specimens from HB tumors, we detected nine methylated genes. We then focused on four of those genes, GPR180, MST1R, OCIAD2, and PARP6, because they likely encode tumor suppressors and their increase of methylation was associated with a poor prognosis. The methylation status of the four genes was also associated with age at diagnosis, and significant association with the presence of metastatic tumors was seen in three of the four genes. Multivariate analysis revealed that the presence of metastatic tumors and increase of methylation of GPR180 were independent prognostic factors affecting event-free survival. These findings indicate that the four novel tumor suppressor candidates are potentially useful molecular markers predictive of a poor outcome in HB patients, which may serve as the basis for improved therapeutic strategies when clinical trials are carried out.

type:text, While single nucleotide polymorphisms (SNP) have been extensively researched in atherosclerotic aortic aneurysms, there are too few data about acute aortic dissection (AAD). The matrix metalloproteinase regulation system has shown a high biological relevance to the development of aortic aneurysms and AAD. The TIMP-1 c.T372C (rs4898, nt+434) SNP was previously associated with the onset of abdominal aortic and other aneurysms. Therefore, we chose this SNP to search for a connection with AAD and to find its place among the other risk factors. 115 patients with AAD were studied for their TIMP-1 c.T372C genotype by means of conventional restrictase analysis. To confirm the biological relevance of our findings, immunohistochemistry for TIMP-1 was performed on tissue samples from the same patients with AAD and compared with a control group of 23 autopsy cases. The TIMP-1 c.T372C showed a significant difference in allele frequency in the AAD patients compared to the general population (p < 0.0001 for both sexes). This genotype did not show any association with any other co-morbid condition, nor with age. The immunohistochemistry results also showed significantly lower TIMP-1 expression in the dissected aortas. The C allele of TIMP-1 c.T372C shows a strong association with the onset of AAD.

While single nucleotide polymorphisms (SNP) have been extensively researched in atherosclerotic aortic aneurysms, there are too few data about acute aortic dissection (AAD). The matrix metalloproteinase regulation system has shown a high biological relevance to the development of aortic aneurysms and AAD. The TIMP-1 c.T372C (rs4898, nt+434) SNP was previously associated with the onset of abdominal aortic and other aneurysms. Therefore, we chose this SNP to search for a connection with AAD and to find its place among the other risk factors. 115 patients with AAD were studied for their TIMP-1 c.T372C genotype by means of conventional restrictase analysis. To confirm the biological relevance of our findings, immunohistochemistry for TIMP-1 was performed on tissue samples from the same patients with AAD and compared with a control group of 23 autopsy cases. The TIMP-1 c.T372C showed a significant difference in allele frequency in the AAD patients compared to the general population (p < 0.0001 for both sexes). This genotype did not show any association with any other co-morbid condition, nor with age. The immunohistochemistry results also showed significantly lower TIMP-1 expression in the dissected aortas. The C allele of TIMP-1 c.T372C shows a strong association with the onset of AAD.

type:text, Background: Microarray technology is a high-throughput method for measuring the expression levels of thousand of genes simultaneously. The observed intensities combine a non-specific binding, which is a major disadvantage with microarray data. The Affymetrix GeneChip assigned a mismatch (MM) probe with the intention of measuring nonspecific binding, but various opinions exist regarding usefulness of MM measures. It should be noted that not all observed intensities are associated with expressed genes and many of those are associated with unexpressed genes, of which measured values express mere noise due to non-specific binding, cross-hybridization, or stray signals. The implicit assumption that all genes are expressed leads to poor performance of microarray data analyses. We assume two functional states of a gene - expressed or unexpressed - and propose a robust method to estimate gene expression states using an order relationship between PM and MM measures. Results: An indicator 'probability of a gene being expressed' was obtained using the number of probe pairs within a probe set where the PM measure exceeds the MM measure. We examined the validity of the proposed indicator using Human Genome U95 data sets provided by Affymetrix. The usefulness of 'probability of a gene being expressed' is illustrated through an exploration of candidate genes involved in neuroblastoma prognosis. We identified the candidate genes for which expression states differed (un-expressed or expressed) when compared between two outcomes. The validity of this result was subsequently confirmed by quantitative RT-PCR. Conclusion: The proposed qualitative evaluation, 'probability of a gene being expressed', is a useful indicator for improving microarray data analysis. It is useful to reduce the number of false discoveries. Expression states - expressed or unexpressed - correspond to the most fundamental gene function 'On' and 'Off', which can lead to biologically meaningful results.

Background: Microarray technology is a high-throughput method for measuring the expression levels of thousand of genes simultaneously. The observed intensities combine a non-specific binding, which is a major disadvantage with microarray data. The Affymetrix GeneChip assigned a mismatch (MM) probe with the intention of measuring nonspecific binding, but various opinions exist regarding usefulness of MM measures. It should be noted that not all observed intensities are associated with expressed genes and many of those are associated with unexpressed genes, of which measured values express mere noise due to non-specific binding, cross-hybridization, or stray signals. The implicit assumption that all genes are expressed leads to poor performance of microarray data analyses. We assume two functional states of a gene - expressed or unexpressed - and propose a robust method to estimate gene expression states using an order relationship between PM and MM measures. Results: An indicator 'probability of a gene being expressed' was obtained using the number of probe pairs within a probe set where the PM measure exceeds the MM measure. We examined the validity of the proposed indicator using Human Genome U95 data sets provided by Affymetrix. The usefulness of 'probability of a gene being expressed' is illustrated through an exploration of candidate genes involved in neuroblastoma prognosis. We identified the candidate genes for which expression states differed (un-expressed or expressed) when compared between two outcomes. The validity of this result was subsequently confirmed by quantitative RT-PCR. Conclusion: The proposed qualitative evaluation, 'probability of a gene being expressed', is a useful indicator for improving microarray data analysis. It is useful to reduce the number of false discoveries. Expression states - expressed or unexpressed - correspond to the most fundamental gene function 'On' and 'Off', which can lead to biologically meaningful results.

Background: Microarray technology is a high-throughput method for measuring the expression levels of thousand of genes simultaneously. The observed intensities combine a non-specific binding, which is a major disadvantage with microarray data. The Affymetrix GeneChip assigned a mismatch (MM) probe with the intention of measuring nonspecific binding, but various opinions exist regarding usefulness of MM measures. It should be noted that not all observed intensities are associated with expressed genes and many of those are associated with unexpressed genes, of which measured values express mere noise due to non-specific binding, cross-hybridization, or stray signals. The implicit assumption that all genes are expressed leads to poor performance of microarray data analyses. We assume two functional states of a gene - expressed or unexpressed - and propose a robust method to estimate gene expression states using an order relationship between PM and MM measures. Results: An indicator 'probability of a gene being expressed' was obtained using the number of probe pairs within a probe set where the PM measure exceeds the MM measure. We examined the validity of the proposed indicator using Human Genome U95 data sets provided by Affymetrix. The usefulness of 'probability of a gene being expressed' is illustrated through an exploration of candidate genes involved in neuroblastoma prognosis. We identified the candidate genes for which expression states differed (un-expressed or expressed) when compared between two outcomes. The validity of this result was subsequently confirmed by quantitative RT-PCR. Conclusion: The proposed qualitative evaluation, 'probability of a gene being expressed', is a useful indicator for improving microarray data analysis. It is useful to reduce the number of false discoveries. Expression states - expressed or unexpressed - correspond to the most fundamental gene function 'On' and 'Off', which can lead to biologically meaningful results.

type:text, The log rank test and the Cox regression, or modifications thereof, emphasize the effect of covariates on survival rate parameter. In some cases, cured individuals, i.e., individuals who may not experience the event of interest may exist in the population of interest. In this situation, we may wish to examine the effect of covariates on both survival rate and cured fraction parameters. Motivated by the Japanese neuroblastoma dataset, we consider a cure model that accounted for the effect of covariates on both of the abovementioned parameters. To deal with heterogeneity that is not explained by covariates, as well as individual random heterogeneity, we perform a frailty variable. Moreover, some nested models are fitted to deal with the principle of parsimony. The effect of covariates was then evaluated by the best nested model. From a statistical point of view, we found that the model of analysis is flexible and adequate to describe the abovementioned dataset. From a medical point of view, we confirmed AGE and STAGE to be the most dominant prognosis factor of neuroblastoma. We also conclude that NMYC and FERRITIN are the other most important prognosis factors. The analysis designated that some of the prognosis factors of neuroblastoma probably just affected the median life of patients and some others are the fatal prognosis factor indicated by their effect which significance on both of survival rate and cured fraction parameters. The present model of analysis is also potentially extendable to facilitate other aspects of inferences.

The log rank test and the Cox regression, or modifications thereof, emphasize the effect of covariates on survival rate parameter. In some cases, cured individuals, i.e., individuals who may not experience the event of interest may exist in the population of interest. In this situation, we may wish to examine the effect of covariates on both survival rate and cured fraction parameters. Motivated by the Japanese neuroblastoma dataset, we consider a cure model that accounted for the effect of covariates on both of the abovementioned parameters. To deal with heterogeneity that is not explained by covariates, as well as individual random heterogeneity, we perform a frailty variable. Moreover, some nested models are fitted to deal with the principle of parsimony. The effect of covariates was then evaluated by the best nested model. From a statistical point of view, we found that the model of analysis is flexible and adequate to describe the abovementioned dataset. From a medical point of view, we confirmed AGE and STAGE to be the most dominant prognosis factor of neuroblastoma. We also conclude that NMYC and FERRITIN are the other most important prognosis factors. The analysis designated that some of the prognosis factors of neuroblastoma probably just affected the median life of patients and some others are the fatal prognosis factor indicated by their effect which significance on both of survival rate and cured fraction parameters. The present model of analysis is also potentially extendable to facilitate other aspects of inferences.

The log rank test and the Cox regression, or modifications thereof, emphasize the effect of covariates on survival rate parameter. In some cases, cured individuals, i.e., individuals who may not experience the event of interest may exist in the population of interest. In this situation, we may wish to examine the effect of covariates on both survival rate and cured fraction parameters. Motivated by the Japanese neuroblastoma dataset, we consider a cure model that accounted for the effect of covariates on both of the abovementioned parameters. To deal with heterogeneity that is not explained by covariates, as well as individual random heterogeneity, we perform a frailty variable. Moreover, some nested models are fitted to deal with the principle of parsimony. The effect of covariates was then evaluated by the best nested model. From a statistical point of view, we found that the model of analysis is flexible and adequate to describe the abovementioned dataset. From a medical point of view, we confirmed AGE and STAGE to be the most dominant prognosis factor of neuroblastoma. We also conclude that NMYC and FERRITIN are the other most important prognosis factors. The analysis designated that some of the prognosis factors of neuroblastoma probably just affected the median life of patients and some others are the fatal prognosis factor indicated by their effect which significance on both of survival rate and cured fraction parameters. The present model of analysis is also potentially extendable to facilitate other aspects of inferences.

type:text, Tumor hypoxia has been reported to cause a functional loss in DNA mismatch repair (MMR) system as a result of down-regulation of MMR genes, although the precise molecular mechanisms remain unclear. In this study, we focused on the down-regulation of a key MMR gene, MLH1, and demonstrated that hypoxia-inducible transcription repressors, DEC1 and DEC2, participated in its transcriptional regulation via their bindings to E-box-like motif(s) in MLH1 promoter region. In all cancer cell lines examined, hypoxia increased expression of DEC1 and DEC2, known as hypoxia-inducible genes, but decreased MLH1 expression in an exposure time-dependent manner at both the mRNA and protein levels. Co-transfection reporter assay revealed that DEC1 and, to greater extent, DEC2 as well as hypoxia repressed MLH1 promoter activity. We further found that the action was remarkably inhibited by trichostatin A, and identified a possible DEC-response element in the MLH1 promoter. In vitro electrophoretic gel mobility shift and chromatin immunoprecipitation assays demonstrated that DEC1 or DEC2 directly bounds to the suggested element, and transient transfection assay revealed that overexpression of DEC2 repressed endogenous MLH1 expression in the cells. Hypoxia-induced DEC may impair MMR function through repression of MLH1 expression, possibly via the histone deacethylase (HDAC)-mediated mechanism in cancer cells.

type:text, Regulation mechanism of lung fibroblast proliferation remains unknown. To elucidate the key molecules in it, we here established mortal and immortal non-transformed lung fibroblast cell line/strains with elongated lifespan by telomerase reversetranscriptase gene transfection. Comparing the expression profiles of them, 51 genes were explored to be the candidates responsible for regulation of cellular proliferation of lung fibroblasts. This set of fibrobrast strains of same origin with different proliferative capacities may become useful model cells for research on lung fibroblast growth regulation and the candidate genes explored in this study may provide biomarkers or therapeutic targets of pulmonary fibrosis.

Tumor hypoxia has been reported to cause a functional loss in DNA mismatch repair (MMR) system as a result of down-regulation of MMR genes, although the precise molecular mechanisms remain unclear. In this study, we focused on the down-regulation of a key MMR gene, MLH1, and demonstrated that hypoxia-inducible transcription repressors, DEC1 and DEC2, participated in its transcriptional regulation via their bindings to E-box-like motif(s) in MLH1 promoter region. In all cancer cell lines examined, hypoxia increased expression of DEC1 and DEC2, known as hypoxia-inducible genes, but decreased MLH1 expression in an exposure time-dependent manner at both the mRNA and protein levels. Co-transfection reporter assay revealed that DEC1 and, to greater extent, DEC2 as well as hypoxia repressed MLH1 promoter activity. We further found that the action was remarkably inhibited by trichostatin A, and identified a possible DEC-response element in the MLH1 promoter. In vitro electrophoretic gel mobility shift and chromatin immunoprecipitation assays demonstrated that DEC1 or DEC2 directly bounds to the suggested element, and transient transfection assay revealed that overexpression of DEC2 repressed endogenous MLH1 expression in the cells. Hypoxia-induced DEC may impair MMR function through repression of MLH1 expression, possibly via the histone deacethylase (HDAC)-mediated mechanism in cancer cells.

Regulation mechanism of lung fibroblast proliferation remains unknown. To elucidate the key molecules in it, we here established mortal and immortal non-transformed lung fibroblast cell line/strains with elongated lifespan by telomerase reversetranscriptase gene transfection. Comparing the expression profiles of them, 51 genes were explored to be the candidates responsible for regulation of cellular proliferation of lung fibroblasts. This set of fibrobrast strains of same origin with different proliferative capacities may become useful model cells for research on lung fibroblast growth regulation and the candidate genes explored in this study may provide biomarkers or therapeutic targets of pulmonary fibrosis.

Regulation mechanism of lung fibroblast proliferation remains unknown. To elucidate the key molecules in it, we here established mortal and immortal non-transformed lung fibroblast cell line/strains with elongated lifespan by telomerase reversetranscriptase gene transfection. Comparing the expression profiles of them, 51 genes were explored to be the candidates responsible for regulation of cellular proliferation of lung fibroblasts. This set of fibrobrast strains of same origin with different proliferative capacities may become useful model cells for research on lung fibroblast growth regulation and the candidate genes explored in this study may provide biomarkers or therapeutic targets of pulmonary fibrosis.

Tumor hypoxia has been reported to cause a functional loss in DNA mismatch repair (MMR) system as a result of down-regulation of MMR genes, although the precise molecular mechanisms remain unclear. In this study, we focused on the down-regulation of a key MMR gene, MLH1, and demonstrated that hypoxia-inducible transcription repressors, DEC1 and DEC2, participated in its transcriptional regulation via their bindings to E-box-like motif(s) in MLH1 promoter region. In all cancer cell lines examined, hypoxia increased expression of DEC1 and DEC2, known as hypoxia-inducible genes, but decreased MLH1 expression in an exposure time-dependent manner at both the mRNA and protein levels. Co-transfection reporter assay revealed that DEC1 and, to greater extent, DEC2 as well as hypoxia repressed MLH1 promoter activity. We further found that the action was remarkably inhibited by trichostatin A, and identified a possible DEC-response element in the MLH1 promoter. In vitro electrophoretic gel mobility shift and chromatin immunoprecipitation assays demonstrated that DEC1 or DEC2 directly bounds to the suggested element, and transient transfection assay revealed that overexpression of DEC2 repressed endogenous MLH1 expression in the cells. Hypoxia-induced DEC may impair MMR function through repression of MLH1 expression, possibly via the histone deacethylase (HDAC)-mediated mechanism in cancer cells.

type:text, Telomeres, guanine-rich tandem DNA repeats of the chromosomal end, provide chromosomal stability, and cellular replication causes their loss. In somatic cells, the activity of telomerase, a reverse transcriptase that can elongate telomeric repeats, is usually diminished after birth so that the telomere length is gradually shortened with cell divisions, and triggers cellular senescence. In embryonic stem cells, telomerase is activated and maintains telomere length and cellular immortality; however, the level of telomerase activity is low or absent in the majority of stem cells regardless of their proliferative capacity. Thus, even in stem cells, except for embryonal stem cells and cancer stem cells, telomere shortening occurs during replicative aging, possibly at a slower rate than that in normal somatic cells. Recently, the importance of telomere maintenance in human stem cells has been highlighted by studies on dyskeratosis congenital, which is a genetic disorder in the human telomerase component. The regulation of telomere length and telomerase activity is a complex and dynamic process that is tightly linked to cell cycle regulation in human stem cells. Here we review the role of telomeres and telomerase in the function and capacity of the human stem cells.

Telomeres, guanine-rich tandem DNA repeats of the chromosomal end, provide chromosomal stability, and cellular replication causes their loss. In somatic cells, the activity of telomerase, a reverse transcriptase that can elongate telomeric repeats, is usually diminished after birth so that the telomere length is gradually shortened with cell divisions, and triggers cellular senescence. In embryonic stem cells, telomerase is activated and maintains telomere length and cellular immortality; however, the level of telomerase activity is low or absent in the majority of stem cells regardless of their proliferative capacity. Thus, even in stem cells, except for embryonal stem cells and cancer stem cells, telomere shortening occurs during replicative aging, possibly at a slower rate than that in normal somatic cells. Recently, the importance of telomere maintenance in human stem cells has been highlighted by studies on dyskeratosis congenital, which is a genetic disorder in the human telomerase component. The regulation of telomere length and telomerase activity is a complex and dynamic process that is tightly linked to cell cycle regulation in human stem cells. Here we review the role of telomeres and telomerase in the function and capacity of the human stem cells.

Telomeres, guanine-rich tandem DNA repeats of the chromosomal end, provide chromosomal stability, and cellular replication causes their loss. In somatic cells, the activity of telomerase, a reverse transcriptase that can elongate telomeric repeats, is usually diminished after birth so that the telomere length is gradually shortened with cell divisions, and triggers cellular senescence. In embryonic stem cells, telomerase is activated and maintains telomere length and cellular immortality; however, the level of telomerase activity is low or absent in the majority of stem cells regardless of their proliferative capacity. Thus, even in stem cells, except for embryonal stem cells and cancer stem cells, telomere shortening occurs during replicative aging, possibly at a slower rate than that in normal somatic cells. Recently, the importance of telomere maintenance in human stem cells has been highlighted by studies on dyskeratosis congenital, which is a genetic disorder in the human telomerase component. The regulation of telomere length and telomerase activity is a complex and dynamic process that is tightly linked to cell cycle regulation in human stem cells. Here we review the role of telomeres and telomerase in the function and capacity of the human stem cells.

type:text, Catecholamines are biogenic amines that play an important role in the nervous system. Some catecholamines have been used as tumor makers of phenochromocytoma, paraganglioma and neuroblastoma. The analysis of total catecholamine metabolites should be useful for one-shot screening of multiple aspects of diseases; however, it is difficult to do this, because the catecholamine metabolites are divided into three groups: five amines, one amino acid and three carbonic acids. Catecholamines and small molecules were separated from plasma proteins by an internal-surface reversed-phase column (protein-coated octadeyclsilica column) and were analyzed by liquid chromatography (LC)/mass spectrometry (MS) using electrospray ionization time-of-flight MS. Using a reversed-phase column and hydrophilic mobile phases, we succeeded in the separation of nine catecholamines, all of which had similar structures. These nine substances were eluted in the following order: norepinephrine, epinephrine, normetanephrine, dopamine, metanephrine, 3,4-dihydroxyphenylalanine, vanillomandelic acid, 3,4-dihydroxyphenylacetic acid and homovanillic acid. The reproducibility of this method was acceptable. The highest coefficient of variation was 7.4%.0 In addition, various types of compounds were separated from and detected in plasma proteins by applying LC/MS. The plasma direct injection method, which uses an internal-surface reversed-phase column and an ion-pair reagent, allowed us to separate small molecules from plasma proteins. MS detected some compounds that high-performance LC could not succeed in separating and detecting with UV detection. We think that the method can be applied to find new markers in neuroblastoma, by comparing the plasma of patients with that of normal infants. The method can be also used to help in making a diagnosis of other diseases and finding their new makers.

Catecholamines are biogenic amines that play an important role in the nervous system. Some catecholamines have been used as tumor makers of phenochromocytoma, paraganglioma and neuroblastoma. The analysis of total catecholamine metabolites should be useful for one-shot screening of multiple aspects of diseases; however, it is difficult to do this, because the catecholamine metabolites are divided into three groups: five amines, one amino acid and three carbonic acids. Catecholamines and small molecules were separated from plasma proteins by an internal-surface reversed-phase column (protein-coated octadeyclsilica column) and were analyzed by liquid chromatography (LC)/mass spectrometry (MS) using electrospray ionization time-of-flight MS. Using a reversed-phase column and hydrophilic mobile phases, we succeeded in the separation of nine catecholamines, all of which had similar structures. These nine substances were eluted in the following order: norepinephrine, epinephrine, normetanephrine, dopamine, metanephrine, 3,4-dihydroxyphenylalanine, vanillomandelic acid, 3,4-dihydroxyphenylacetic acid and homovanillic acid. The reproducibility of this method was acceptable. The highest coefficient of variation was 7.4%.0 In addition, various types of compounds were separated from and detected in plasma proteins by applying LC/MS. The plasma direct injection method, which uses an internal-surface reversed-phase column and an ion-pair reagent, allowed us to separate small molecules from plasma proteins. MS detected some compounds that high-performance LC could not succeed in separating and detecting with UV detection. We think that the method can be applied to find new markers in neuroblastoma, by comparing the plasma of patients with that of normal infants. The method can be also used to help in making a diagnosis of other diseases and finding their new makers.

Catecholamines are biogenic amines that play an important role in the nervous system. Some catecholamines have been used as tumor makers of phenochromocytoma, paraganglioma and neuroblastoma. The analysis of total catecholamine metabolites should be useful for one-shot screening of multiple aspects of diseases; however, it is difficult to do this, because the catecholamine metabolites are divided into three groups: five amines, one amino acid and three carbonic acids. Catecholamines and small molecules were separated from plasma proteins by an internal-surface reversed-phase column (protein-coated octadeyclsilica column) and were analyzed by liquid chromatography (LC)/mass spectrometry (MS) using electrospray ionization time-of-flight MS. Using a reversed-phase column and hydrophilic mobile phases, we succeeded in the separation of nine catecholamines, all of which had similar structures. These nine substances were eluted in the following order: norepinephrine, epinephrine, normetanephrine, dopamine, metanephrine, 3,4-dihydroxyphenylalanine, vanillomandelic acid, 3,4-dihydroxyphenylacetic acid and homovanillic acid. The reproducibility of this method was acceptable. The highest coefficient of variation was 7.4%.0 In addition, various types of compounds were separated from and detected in plasma proteins by applying LC/MS. The plasma direct injection method, which uses an internal-surface reversed-phase column and an ion-pair reagent, allowed us to separate small molecules from plasma proteins. MS detected some compounds that high-performance LC could not succeed in separating and detecting with UV detection. We think that the method can be applied to find new markers in neuroblastoma, by comparing the plasma of patients with that of normal infants. The method can be also used to help in making a diagnosis of other diseases and finding their new makers.

In major systemic inflammation such as severe peritonitis, various pro-inflammatory cytokines, such as TNF-α, IL-1/) and IL-6, play important roles in the development of multiple organ dysfunction syndrome (MODS). The purpose of this study was to investigate the outflow of pro-inflammatory and anti-inflammatory cytokines from the efferent mesenteric lymphatic vessels under peritonitis. Mesenteric lymph samples were collected from adult male rats at 2, 4, 6, 8 and 10 hr after an intraperitoneal injection of zymosan at a dosage of 0.1 mg/g (non-lethal dose) or 0.5 mg/g (lethal dose). Blood samples were obtained at 10 hr after zymosan administration. The amounts of drained TNF-α and IL-6 in the lymph peaked at 2-4 hr and 4-8 hr after zymosan administration, respectively. The amounts of drained IL-10 in the lymph gradually increased until 10 hr. The amounts of drained TNF-αand IL-10 in the mesenteric lymph were significantly correlated with the dosage of zymosan. In conclusion, under intraperitoneal inflammation, pro-inflammatory cytokines (TNF-αand IL-6) increased in the mesenteric lymph and were drained into circulation. IL-10, one of the anti-inflammatory cytokines, also increased in the mesenteric lymph after several hours' delay and its increase was remarkable in severe inflammations. These findings suggested that the gut might be one of the pro-inflammatory and anti-inflammatory cytokine-generating organs under peritonitis. The lymph-drained amounts of each cytokine under peritonitis are considered to differ with the time or severity of inflammation, which may cause different conditions in patients due to the imbalance of pro-inflammatory and anti-inflammatory cytokines., This work was supported by a Grant-in Aid from the Ministry of Education, Science and Culture.

Some genetic studies have shown a linkage between malignant hyperthermia susceptibility (MHS) and chromosome 19q or the skeletal muscle ryanodine receptor (RYR1) gene. Some types of MHS seem to be caused by an abnormality of calcium-induced calcium release (CICR). We analyzed the linkage of RYR 1 gene polymorphisms in Japanese MHS families and investigated the correlation between genetic evidence of RYR1 gene mutations and an accelerated rate of CICR. We studied 63 subjects who were referred to our institute for investigation of MHS. CICR rates were measured by the skinned fiber method in 23 subjects. DNA samples were collected from 63 individuals belonging to 22 unrelated families. Restriction fragment length polymorphism (RFLP) analyses on the RYR1 locus and hypervariable microsatellite analysis were performed. We found one family with a linkage between acceleration of the CICR mechanism and a group of RFLPs. In CICR tests, ten of the 11 patients who had presented with fulminant MH showed accelerated rates of CICR. Analysis for the mutation C1840T, which was performed in 63 samples, did not demonstrate an alteration in any of the patients. Although we found heterozygotes in RFLP studies, we did not recognize a specific relationship between the acceleration of CICR and the RFLPs. We suggest a linkage between the acceleration of CICR and an abnormal human RYR1 gene in MHS. These results also suggest that heterogeneity exists for MH. We conclude that genetic tests cannot replace CICR tests or caffeine-halothane contracture tests with muscle biopsy as a diagnosing test for MH in the near future., This work was partly supported by grants-in-aid from the Ministry of Education, Science and Culture of Japan (No.08407052: Osafumi Yuge, No.09771157: Yasuhiro Maehara) and by a research grant from the Labor Welfare Corporation of Japan.

The intrinsic resistance of methicillin-resistant Staphylococcus aureus (MRSA) is frequently explained by the production of an additional penicillin-binding protein (PEP), which is encoded by the mecA gene. The mec regulator genes, mecR1 and mecI, was identified in mecA-carrying Staphylococcus aureus N315. Between February and March, 1993, 179 clinical MRSA isolates were collected from institutions in Hiroshima prefecture. According to serological types of coagulase, enterotoxins, and toxic shock syndrome toxin-1 (TSST-1) productions, these strains were classified into 6 groups. In 53 strains chosen from all groups, mec regulatory gene distributions were divided into two groups; one with whole regulatory genes and another with the lacking region, including 3'-partial region of the mecR1 gene and mecI gene. This same deletion was detected across the different groups, suggesting that the deletion occurred at the ancestral strain before branching according to coagulase or enterotoxin productions. The strains with this lacking region showed a high-level of resistance to methicillin, while the strains with whole regulatory genes consisted of low and high levels of resistant strains. The highly resistant strains with whole regulatory genes were found to harbor a point mutation in the mecI gene. The basal levels of mecA gene transcription were elevated in the strains with the lacking region or the mecI point mutations. These data suggest that deletion or mutation of the mecI gene, the repressor on the mecA gene, might play an important role in methicillin resistance in clinical isolates of MRSA., This work was supported by a Grant-in-Aid for Cancer Research from the Ministry of Education, Science and Culture.

type:text, The intrinsic resistance of methicillin-resistant Staphylococcus aureus (MRSA) is frequently explained by the production of an additional penicillin-binding protein (PEP), which is encoded by the mecA gene. The mec regulator genes, mecR1 and mecI, was identified in mecA-carrying Staphylococcus aureus N315. Between February and March, 1993, 179 clinical MRSA isolates were collected from institutions in Hiroshima prefecture. According to serological types of coagulase, enterotoxins, and toxic shock syndrome toxin-1 (TSST-1) productions, these strains were classified into 6 groups. In 53 strains chosen from all groups, mec regulatory gene distributions were divided into two groups; one with whole regulatory genes and another with the lacking region, including 3'-partial region of the mecR1 gene and mecI gene. This same deletion was detected across the different groups, suggesting that the deletion occurred at the ancestral strain before branching according to coagulase or enterotoxin productions. The strains with this lacking region showed a high-level of resistance to methicillin, while the strains with whole regulatory genes consisted of low and high levels of resistant strains. The highly resistant strains with whole regulatory genes were found to harbor a point mutation in the mecI gene. The basal levels of mecA gene transcription were elevated in the strains with the lacking region or the mecI point mutations. These data suggest that deletion or mutation of the mecI gene, the repressor on the mecA gene, might play an important role in methicillin resistance in clinical isolates of MRSA., This work was supported by a Grant-in-Aid for Cancer Research from the Ministry of Education, Science and Culture.

The intrinsic resistance of methicillin-resistant Staphylococcus aureus (MRSA) is frequently explained by the production of an additional penicillin-binding protein (PEP), which is encoded by the mecA gene. The mec regulator genes, mecR1 and mecI, was identified in mecA-carrying Staphylococcus aureus N315. Between February and March, 1993, 179 clinical MRSA isolates were collected from institutions in Hiroshima prefecture. According to serological types of coagulase, enterotoxins, and toxic shock syndrome toxin-1 (TSST-1) productions, these strains were classified into 6 groups. In 53 strains chosen from all groups, mec regulatory gene distributions were divided into two groups; one with whole regulatory genes and another with the lacking region, including 3'-partial region of the mecR1 gene and mecI gene. This same deletion was detected across the different groups, suggesting that the deletion occurred at the ancestral strain before branching according to coagulase or enterotoxin productions. The strains with this lacking region showed a high-level of resistance to methicillin, while the strains with whole regulatory genes consisted of low and high levels of resistant strains. The highly resistant strains with whole regulatory genes were found to harbor a point mutation in the mecI gene. The basal levels of mecA gene transcription were elevated in the strains with the lacking region or the mecI point mutations. These data suggest that deletion or mutation of the mecI gene, the repressor on the mecA gene, might play an important role in methicillin resistance in clinical isolates of MRSA., This work was supported by a Grant-in-Aid for Cancer Research from the Ministry of Education, Science and Culture.