Ab-initio study of pyrrole ring deformation in the indole group of 5-HT interacting with water molecules.

5-Hydroxytryptamine (5-HT; serotonin) regulates metabolism and various homeostatic mechanisms in the body, and is involved in depression. These complicated functions of 5-HT are supported by several 5-HT receptor and 5-HT transporter subtypes. The development of agonists/antagonists and activators/inhibitors of 5-HT receptors and transporters is a strong target for drug studies. Toward this purpose, we calculated the conformations and electrical states of ionized 5-HT in aqueous solution using ab-initio methods. When we assumed an ionized 5-HT molecule and three surrounding water molecules, the hydrogen bond network for these four molecules formed a ring shape, resulting in deformation of the pyrrole ring in the indole group of 5-HT. To our knowledge, this is the first finding demonstrating deformation of the indole skeleton. The findings suggest that the direct involvement of water in the binding between 5-HT and its receptors and transporters should be taken account when designing candidate 5-HT active compounds.

A prospective evaluation of liquid biopsy for detecting MYCN amplification in neuroblastoma patients.

BACKGROUND: Our previous study reported a method for determining MYCN gene amplification (MNA) status using cell-free DNA in serum. We prospectively analyzed the serum MNA status using sera obtained before the initial diagnosis from patients with neuroblastoma and evaluated the utility of this method. METHODS: Eighty patients were enrolled in the study. The serum MYCN/NAGK ratio was assessed for all cases. RESULTS: Fifteen cases showed serum MNA, while 65 did not. Of the 80 total patients, tumor samples for a genetic analysis were not obtained from 27 due to the patients' condition or other reasons. For the 43 of 80 cases that had both serum and tumor samples analyzed, the serum-based MNA status matched to tumor-based MNA status (P < 0.001). The sensitivity and the specificity were 100%, respectively. Seven of 15 cases who diagnosed as MNA by serum-based MNA status were <18 months of age, and tumor samples were not obtained from 4 of these cases. Based on the serum MNA status, these cases were able to start treatment immediately. The 4-year event-free survival rates of cases with and without MNA in sera were 37.5% and 84.8%, respectively (P < 0.001). CONCLUSION: The serum-based MNA status was useful for precisely predicting the MNA status in tumor and it has clinical benefits for predicting risk stratification in patients for whom obtaining tumor samples is difficult.

Rare MYC-amplified Neuroblastoma With Large Cell Histology.

Background Although MYCN (aka N-myc) amplification is reported in ∼20% of neuroblastomas, MYC (aka C-myc) amplification appears to be a rare event in this disease. As of today, only 2 MYC-amplified neuroblastomas have been briefly mentioned in the literature. Methods We studied here the clinicopathological features of 3 MYC-amplified neuroblastomas. Results All 3 patients (2 females and 1 male) had stage 4 disease. One female is currently alive and well 52 months after the diagnosis, while the other female and male patients died of disease 24 and 20 months after the diagnosis, respectively. Further analysis on 2 tumors revealed unfavorable histology with MYC protein overexpression but with neither MYCN amplification nor MYCN protein overexpression. Both of these tumors exhibited "large cell neuroblastoma" histology with enlarged, uniquely open nuclei and nucleolar hypertrophy, along with "aberrant" desmin expression. Conclusions MYC-amplified neuroblastomas are extremely rare and seem to present with distinct clinicopathological features.

Development of rhabdomyolysis in a child after norovirus gastroenteritis.

BACKGROUND: In children, the most significant cause of rhabdomyolysis or muscle breakdown is viral infection. However, there are no reports that norovirus, a gastroenteric virus that commonly infects children, specifically causes rhabdomyolysis. Here, we report the first pediatric case of norovirus-associated rhabdomyolysis. CASE PRESENTATION: The patient, a 2-year-old boy with fever, diarrhea, and vomiting, was referred to our hospital with dysstasia and transaminitis. He was diagnosed with rhabdomyolysis. Additionally, norovirus genogroup GII was detected from stool samples by real-time quantitative reverse transcription Polymerase Chain Reaction, and thereafter, the norovirus GII.4 variant was identified. CONCLUSION: However, the association between rhabdomyolysis and the isolated norovirus variant was not clarified. After treatment the patient recovered without renal failure or disseminated intravascular coagulation. Rhabdomyolysis is a disease for which there is a need for early detection and treatment. If abnormal posture or muscle weakness is observed during the course of gastroenteritis, blood and urinary tests should be performed to rule out rhabdomyolysis.

Serum-Based Quantification of MYCN Gene Amplification in Young Patients with Neuroblastoma: Potential Utility as a Surrogate Biomarker for Neuroblastoma.

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.

Residual tumor in cases of intermediate-risk neuroblastoma did not influence the prognosis.

BACKGROUND: It remains unclear whether a residual tumor mass following therapy influences the prognosis of neuroblastoma. METHODS: We retrospectively reviewed 20 patients with intermediate-risk tumors treated at our institution between 1993 and 2012 to elucidate whether additional treatment is required for residual tumors. RESULTS: The patient ages at diagnosis ranged from 0 days to 7 years. The 5-year overall survival rate was 94.4%. Thirteen patients had Stage 3 disease and seven patients had Stage 4 disease. Nine patients showed intraspinal extension. Twelve patients had a residual tumor mass at the completion of therapy, and eight showed intraspinal extension. Five of these 12 patients showed metaiodobenzylguanidine (MIBG) uptake at the end of treatment, but the uptake disappeared during the follow-up period. Except for one patient who died due to treatment complications, the rest are all alive, and nine are alive with a residual mass. We examined the residual mass in four patients and found that these tissues had differentiated into a ganglioneuroma or changed to a necrotic tissue. For the three patients with neurological symptoms at the end of treatment, some slight neurological symptoms still remained during the follow-up. Five patients with an intraspinal mass eventually presented with new symptoms. CONCLUSIONS: The presence of a residual mass at the end of treatment did not influence the patients' prognosis. Therefore, an invasive radical surgical resection and additional treatment may not be necessary. Cases with a residual intraspinal mass also require a long-term follow-up to assess the neurological prognosis.The presence of a residual mass in cases of intermediate-risk neuroblastoma at the end of treatment did not influence the patients' prognosis.

Boron neutron capture therapy (BNCT) as a new approach for clear cell sarcoma (CCS) treatment: Trial using a lung metastasis model of CCS.

Clear cell sarcoma (CCS) is a rare malignant tumor with a poor prognosis. In the present study, we established a lung metastasis animal model of CCS and investigated the therapeutic effect of boron neutron capture therapy (BNCT) using p-borono-L-phenylalanine (L-BPA). Biodistribution data revealed tumor-selective accumulation of (10)B. Unlike conventional gamma-ray irradiation, BNCT significantly suppressed tumor growth without damaging normal tissues, suggesting that it may be a potential new therapeutic option to treat CCS lung metastases.

Potential of boron neutron capture therapy (BNCT) for malignant peripheral nerve sheath tumors (MPNST).

Malignant peripheral nerve sheath tumors (MPNST) are relatively rare neoplasms with poor prognosis. At present there is no effective treatment for MPNST other than surgical resection. Nonetheless, the anti-tumor effect of boron neutron capture therapy (BNCT) was recently demonstrated in two patients with MPNST. Subsequently, tumor-bearing nude mice subcutaneously transplanted with a human MPNST cell line were injected with p-borono-L-phenylalanine (L-BPA) and subjected to BNCT. Pathological studies then revealed that the MPNST cells were selectively destroyed by BNCT.

Boron neutron capture therapy as new treatment for clear cell sarcoma: trial on different animal model.

Clear cell sarcoma (CCS) is a rare malignant tumor with a poor prognosis. In our previous study, the tumor disappeared under boron neutron capture therapy (BNCT) on subcutaneously-transplanted CCS-bearing animals. In the present study, the tumor disappeared under this therapy on model mice intramuscularly implanted with three different human CCS cells. BNCT led to the suppression of tumor-growth in each of the different model mice, suggesting its potentiality as an alternative to, or integrative option for, the treatment of CCS.

Boron neutron capture therapy (BNCT) selectively destroys human clear cell sarcoma in mouse model.

Clear cell sarcoma of tendons and aponeuroses (CCS) is a rare malignant tumor with no effective treatment. This study demonstrates the efficacy of BNCT with the use of human CCS-bearing nude mice. Groups A and C were administered saline, and groups B and D were injected with p-borono-L-phenylalanine-fructose complex. Groups C and D were then irradiated with thermal neutrons. The tumors in only group D disappeared, demonstrating that BNCT is a potentially new option for the treatment of human CCS.

A MYCN-amplified cell line derived from a long-term event-free survivor among our sixteen established neuroblastoma cell lines.

Although more than 110 neuroblastoma (NB) cell lines have been established, there have been neither reports on the rate of success to establish NB cell lines, nor well-documented NB cell lines from long-term-survivors. We attempted to establish NB cell lines from 114 patients. Sixteen NB cell lines were established from 12 patients. The success rates to establish cell lines were 1.4% (1/70) from patients in early stages, 25.0% (11/44) from those in advanced stages, and 10.5% (12/114) from those in all stages respectively. Eleven of these 12 patients eventually died. The surviving patient, who was in stage 4 with MYCN-amplification, has been event-free for 19 years after completing therapy. The serum MYCN DNA level in patient TK was very high before therapy, decreased after chemotherapy, and has remained at the normal levels until now. The gene expression profiling of the primary tumor and the K-N-TK cell line was analyzed with an NB-specific cDNA microarray, and indicated that the probability of 5-year survival was extremely low. Microarray-based comparative genomic hybridization (CGH) analysis indicated that genomic aberration profiles of the cell line were uncommon, with MYCN amplification, 17q gain and 11q loss. A unique KP-N-TK cell line, established from an event-free survivor, will be a useful tool for investigating how a patient can survive a tumor with an extremely poor prognosis.

Successful treatment of infants with localized neuroblastoma based on their MYCN status.

BACKGROUND: The aim of this study was to evaluate the effectiveness of post-surgical chemotherapy for infants with localized neuroblastoma without MYCN amplification (MNA), and determine whether risk classification using MNA is reasonable. METHODS: Four hundred and fourteen eligible patients were registered between 1998 and 2004. Resectable patients in stage 1 and 2A/2B were treated by surgical resection only. Unresectable patients in stage 3 without MNA received either 6 cycles of regimen A or 3 cycles of regimen A plus 3 cycles of regimen C2; regimen A consisted of low doses of cyclophosphamide and vincristine and regimen C consisted of cyclophosphamide, vincristine and pirarubicin before surgical resection. The resectable and unresectable patients were randomly selected to receive post-surgical chemotherapy. The patients with MNA received intensive chemotherapy regimen D2, consisting of cyclophosphamide, vincristine, pirarubicin and cisplatin, and some of them received high-dose chemotherapy with stem cell transplantation. RESULTS: The 5-year event-free survival (5-EFS) rates of stage 1 and 2A/2B patients without MNA were 97.2 and 89.0% respectively (p = 0.02). A total of 31 patients in stage 3 without MNA received post-surgical chemotherapy, and 30 patients did not. The 5-EFS rates of these two groups (96.0 and 96.2%, respectively) were not significantly different (p = 0.869). The 5-EFS rate for localized patients with MNA (n = 6) was 50.0%, and that of patients without MNA was 95.0% (p < 0.001). CONCLUSION: Post-surgical chemotherapy was therefore unnecessary for localized patients without MNA. This treatment strategy using MNA is considered to be appropriate in infants.

Is the prognosis of stage 4s neuroblastoma in patients 12 months of age and older really excellent?

PURPOSE: In the International Neuroblastoma Risk Group (INRG) classification system, stage 4s was changed into stage MS in children less than 18 months of age. Stage MS is defined as a metastatic disease with skin, liver and bone marrow, similar to INSS stage 4s. To evaluate the outcome of stage 4s cases in patients 12 months of age and over and to determine the appropriate treatment strategy. METHOD: We performed a retrospective review of 3834 patients registered with the Japanese Society of Pediatric Oncology and Japanese Society of Pediatric Surgeons between 1980 and 1998. RESULTS: The rates of stage 4s patients were 10.7%, 6.3% and 3.3% in patients of ≥ 11 months of age, from ≥ 12 to ≤ 17 months of age, ≥ 18 months of age, respectively. The 5 year event-free survival rates were 89.4%, 100% and 53.1%, respectively. The rates of MYCN amplification and unfavourable histology were smaller in stage 4s groups than stage 4 groups in all ages. CONCLUSION: In the children 12 months of age and older, stage 4s cases are markedly different from stage 4 cases in regard to the clinical features and prognosis. The prognosis of stage 4s cases from ≥ 12 to ≤ 17months of age is excellent. The concept of stage MS appears to be appropriate.

Molecular dynamics simulations for glutamate-binding and cleft-closing processes of the ligand-binding domain of GluR2.

The gating of ion channel of ionotropic glutamate receptors is controlled by the structural change of the ligand-binding domain of GluR2. We examined the roles of residues in the glutamate-binding and cleft-closing mechanisms by molecular dynamics (MD) simulations. A glutamate entered the cleft deeply within the order of nanoseconds and the cleft locked the glutamate completely at 15 ns in an MD run. TYR450 seemed to regulate the orientation of the glutamate upon binding by cation-π interaction. A semi-open state was identified in the free energy profile evaluated with the structures on the spontaneously glutamate-bound and cleft-closed pathway by the unbiased MD simulations for the first time to our knowledge. In the semi-open state, the two sub-domains were bridged by two hydrogen bonds of GLU705 in the sub-domain 2 with TYR732 in the sub-domain 1 and with the glutamate bound to the sub-domain 1 until the transition to the closed state.

Prenatal and lactational exposure to low-doses of bisphenol A alters adult mice behavior.

Bisphenol A (BPA) is an endocrine-disrupting chemical, widely used in dentistry and various industries. We previously reported that BPA affected murine neocortical development by accelerating neuronal differentiation/migration, resulting in abnormal neocortical architecture as well as aberrant thalamocortical connections in the brains of adult mice. The aim of this study was to investigate whether prenatal and lactational BPA exposure affected behavior in adult mice. Pregnant mice were injected subcutaneously with 20µg/kg of BPA daily from embryonic day 0 (E0) until postnatal day 21 (P21). Control animals received a vehicle alone. Behavioral tests (n=15-20) were conducted at postnatal 3weeks (P3W) and P10-15W. After an open-field test, an elevated plus maze and Morris water maze tests were performed. The total distance in the elevated plus maze test at P3W and in the open-field test at P10W was significantly decreased in the BPA-exposed group, compared with the control group. Significant sex differences were observed in the time spent in the central area in the open-field test at P3W and in the total distance in the elevated plus maze test at P11W. These results indicated that prenatal and lactational BPA exposure disturbed the murine behavior in the postnatal development period and the adult mice.

Sensitivity of malignant rhabdoid tumor cell lines to PD 0332991 is inversely correlated with p16 expression.

Malignant rhabdoid tumor (MRT) is a rare and highly aggressive neoplasm of young children. MRT is characterized by inactivation of integrase interactor 1 (INI1). Cyclin-dependent kinase 4 (CDK4), which acts downstream of INI1, is required for the proliferation of MRT cells. Here we investigated the effects of PD 0332991 (PD), a potent inhibitor of CDK4, against five human MRT cell lines (MP-MRT-AN, KP-MRT-RY, G401, KP-MRT-NS, KP-MRT-YM). In all of the cell lines except KP-MRT-YM, PD inhibited cell proliferation >50%, (IC(50) values 0.01 to 0.6 µM) by WST-8 assay, and induced G1-phase cell cycle arrest, as shown by flow cytometry and BrdU incorporation assay. The sensitivity of the MRT cell lines to PD was inversely correlated with p16 expression (r=0.951). KP-MRT-YM cells overexpress p16 and were resistant to the growth inhibitory effect of PD. Small interfering RNA against p16 significantly increased the sensitivity of KP-MRT-YM cells to PD (p<0.05). These results suggest that p16 expression in MRT could be used to predict its sensitivity to PD. PD may be an attractive agent for patients with MRT whose tumors express low levels of p16.

Preoperative analysis of 11q loss using circulating tumor-released DNA in serum: a novel diagnostic tool for therapy stratification of neuroblastoma.

Allelic deletion of the long arm of chromosome 11 (11q loss) is closely associated with the prognosis of neuroblastoma (NB). Here we examined 11q loss using tumor-released DNA fragments in the sera of 24 cases. The allelic intensity score of a panel of polymorphic markers in 11q23 in serum DNA was significantly different between the 11q loss-positive group and the11q loss-negative group. The 11q loss-positive and -negative groups did not overlap when a cut-off value of 0.5 was chosen for the allelic intensity score. Our serum-based 11q loss analysis could predict the allelic status of 11q in tumors.

Identification of therapy-sensitive and therapy-resistant neuroblastoma subtypes in stages III, IVs and IV.

The aim was to present a new model of risk stratification with high predictive sensitivity for non-localized neuroblastomas (NBs). "MYCN amplification", "unfavorable histology of the International Neuroblastoma Pathology Classification (INPC) system" and "low Ha-ras/trk A expression" could be defined as an independent predictor for high-risk NBs. A risk stratification flow chart was applied to 103 advanced NBs in which all three factors were examined and 69 were grouped as high-risk NBs of which 38 patients died. The predictive sensitivity for poor patient outcome was 86%, which included 38 of the 44 total deaths in this analysis. Using the number of the three independent risk factors in each tumor, the 69 high-risk NBs were classified into three subgroups. NBs with the three risk factors (triple risk) represented the most aggressive character and survival of the affected patients was only 10% ("therapy-resistant NBs"). Survivals of the patients with NBs possessed the two (double) risk factors or the one (single) risk factor were 29% and 66%, respectively. This stratification also elucidated a subgroup in which patient survival was 90% ("therapy-sensitive"). There were 21 NBs with "high Ha-ras/trk A expression", "favorable INPC histology" and "unamplified MYCN" (no risk NBs). Among the four subgroups without a risk factor, with a single risk factor, with double risk and with triple risk, Kaplan-Meier analysis showed a significant difference in NB patient outcome (p<0.0001). Risk stratification might improve the therapeutic efficacy for the high-risk NBs and might decrease therapy-related sequelae in the lower risk NBs.

Prenatal and lactational exposure to low-doses of bisphenol A alters brain monoamine concentration in adult mice.

Bisphenol A (BPA) is an endocrine-disrupting chemical, widely used in industry and dentistry. We have previously reported that BPA affects murine neocortical development by accelerating neuronal differentiation/migration resulting in abnormal neocortical architecture as well as apparent thalamocortical connections in adult mice brains. The aim of this study was to investigate whether or not prenatal and lactational BPA-exposure affects the level of neurotransmitters in mice brains. Pregnant mice were injected subcutaneously with 20µg/kg of BPA daily from embryonic day 0 (E0) until postnatal day 21 (P21). Control animals received a vehicle alone. The brains were removed and dissected into six regions for biochemical assays (n=7-8) at postnatal 3 weeks (P3W) and P10-15W. The concentration of the neurotransmitters was determined by high-performance liquid chromatography. The levels of dopamine and its metabolite significantly increased in the caudate/putamen and dorsal raphe nucleus, whereas serotonin and its metabolite increased in the caudate/putamen, dorsal raphe nucleus, thalamus and Substantia nigra in the BPA-exposure group at both P3W and/or P14-15W. These results suggested that prenatal and lactational BPA-exposure might perturb the neurotransmitter system in adult mice.

Lysosomal-associated protein multispanning transmembrane 5 gene (LAPTM5) is associated with spontaneous regression of neuroblastomas.

BACKGROUND: Neuroblastoma (NB) is the most frequently occurring solid tumor in children, and shows heterogeneous clinical behavior. Favorable tumors, which are usually detected by mass screening based on increased levels of catecholamines in urine, regress spontaneously via programmed cell death (PCD) or mature through differentiation into benign ganglioneuroma (GN). In contrast, advanced-type NB tumors often grow aggressively, despite intensive chemotherapy. Understanding the molecular mechanisms of PCD during spontaneous regression in favorable NB tumors, as well as identifying genes with a pro-death role, is a matter of urgency for developing novel approaches to the treatment of advanced-type NB tumors. PRINCIPAL FINDINGS: We found that the expression of lysosomal associated protein multispanning transmembrane 5 (LAPTM5) was usually down-regulated due to DNA methylation in an NB cell-specific manner, but up-regulated in degenerating NB cells within locally regressing areas of favorable tumors detected by mass-screening. Experiments in vitro showed that not only a restoration of its expression but also the accumulation of LAPTM5 protein, was required to induce non-apoptotic cell death with autophagic vacuoles and lysosomal destabilization with lysosomal-membrane permeabilization (LMP) in a caspase-independent manner. While autophagy is a membrane-trafficking pathway to degrade the proteins in lysosomes, the LAPTM5-mediated lysosomal destabilization with LMP leads to an interruption of autophagic flux, resulting in the accumulation of immature autophagic vacuoles, p62/SQSTM1, and ubiqitinated proteins as substrates of autophagic degradation. In addition, ubiquitin-positive inclusion bodies appeared in degenerating NB cells. CONCLUSIONS: We propose a novel molecular mechanism for PCD with the accumulation of autophagic vacuoles due to LAPTM5-mediated lysosomal destabilization. LAPTM5-induced cell death is lysosomal cell death with impaired autophagy, not cell death by autophagy, so-called autophagic cell death. Thus LAPTM5-mediated PCD is closely associated with the spontaneous regression of NBs and opens new avenues for exploring innovative clinical interventions for this tumor.