Rapid detection of human herpesvirus 8 DNA using loop-mediated isothermal amplification

The reliability of a loop-mediated isothermal amplification (LAMP) method for the detection of human herpesvirus 8 (HHV-8) DNA was evaluated. Although LAMP products were produced with the DNA sample extracted from BCP-1 cells, LAMP products were not produced with the DNAs from seven other human herpesviruses. The detection limit of the HHV-8 LAMP method was 100 copies of target sequence/tube. To determine whether the HHV-8 LAMP method could be used to quantify viral DNA, threshold times, which are defined as the time (in s) it takes to reach the threshold turbidity level (0.1), were measured for the amplification of serial dilutions of a DNA plasmid containing the target sequence. The standard curve possessed a correlation coefficient of 0.9428 with a slope of -84.079 and y-intercept value of 1936.2. Additionally, an attempt was made to detect viral DNA in 17 specimens collected from Kaposi's sarcomas and two cell lines obtained from primary effusion lymphomas. HHV-8 DNA was detected in 14 of the 17 Kaposi's sarcoma tissue samples and both of the primary effusion lymphoma cell lines. Viral DNA was not detected in HHV-8 LAMP-negative samples using the real-time PCR method.     

Structures of triterpenoids from the leaves of Lansium domesticum

Journal of Natural Medicines - From the methanolic extract of the leaves of Lansium domesticum, three new onoceranoid-type triterpenoids, lansium acids X–XII and a new cycloartane-type...     

High linear-energy-transfer radiation can overcome radioresistance of glioma stem-like cells to low linear-energy-transfer radiation

Ionizing radiation is applied as the standard treatment for glioblastoma multiforme (GBM). However, radiotherapy remains merely palliative, not curative, because of the existence of glioma stem cells (GSCs), which are regarded as highly radioresistant to low linear-energy-transfer (LET) photons. Here we analyzed whether or not high-LET particles can overcome the radioresistance of GSCs. Glioma stem-like cells (GSLCs) were induced from the GBM cell line A172 in stem cell culture medium. The phenotypes of GSLCs and wild-type cells were confirmed using stem cell markers. These cells were irradiated with ⁶⁰Co gamma rays or reactor neutron beams. Under neutron-beam irradiation, high-LET proton particles can be produced through elastic scattering or nitrogen capture reaction. Radiosensitivity was assessed by a colony-forming assay, and the DNA double-strand breaks (DSBs) were assessed by a histone gamma-H2AX focus detection assay. In stem cell culture medium, GSLCs could form neurosphere-like cells and express neural stem cell markers (Sox2 and Musashi) abundantly in comparison with their parental cells. GSLCs were significantly more radioresistant to gamma rays than their parental cells, but neutron beams overcame this resistance. There were significantly fewer gamma-H2AX foci in the A172 GSLCs 24 h after irradiation with gamma rays than in their parental cultured cells, while there was no apparent difference following neutron-beam irradiation. High-LET radiation can overcome the radioresistance of GSLCs by producing unrepairable DNA DSBs. High-LET radiation therapy might have the potential to overcome GBM''s resistance to X-rays in a clinical setting.     

Vesicular nucleotide transporter-immunoreactive type I cells associated with P2X3-immunoreactive nerve endings in the rat carotid body

ATP is the major excitatory transmitter from chemoreceptor type I cells to sensory nerve endings in the carotid body, and has been suggested to be released by exocytosis from these cells. We investigated the mRNA expression and immunohistochemical localization of vesicular nucleotide transporter (VNUT) in the rat carotid body. RT-PCR detected mRNA expression of VNUT in extracts of the tissue. Immunoreactivity for VNUT was localized in a part of type I cells immunoreactive for synaptophysin (SYN), but not in glial-like type II cells immunoreactive for S100 and S100B. Among SYN-immunoreactive type I cells, VNUT immunoreactivity was selectively localized in the sub-population of tyrosine hydroxylase (TH)-immunorective type I cells associated with nerve endings immunoreactive for the P2X3 purinoceptor; however, it was not detected in the sub-population of type I cells immunoreactive for dopamine beta-hydroxylase. Multi-immunolabeling for VNUT, P2X3, and Bassoon revealed that Bassoon-immunoreactive products were localized in type I cells with VNUT immunoreactivity, and accumulated on the contact side of P2X3-immunoreactive nerve endings. These results revealed the selective localization of VNUT in the subpopulation of TH-immunoreactive type I cells attached to sensory nerve endings and suggested that these cells release ATP by exocytosis for chemosensory transmission in the carotid body.     

Japanese version of The Cancer Genome Atlas,JCGA, established using fresh frozen tumors obtained from 5143 cancer patients

This study aimed to establish the Japanese Cancer Genome Atlas (JCGA) using data from fresh frozen tumor tissues obtained from 5143 Japanese cancer patients, including those with colorectal cancer (31.6%), lung cancer (16.5%), gastric cancer (10.8%) and other cancers (41.1%). The results are part of a single‐center study called “High‐tech Omics‐based Patient Evaluation” or “Project HOPE” conducted at the Shizuoka Cancer Center, Japan. All DNA samples and most RNA samples were analyzed using whole‐exome sequencing, cancer gene panel sequencing, fusion gene panel sequencing and microarray gene expression profiling, and the results were annotated using an analysis pipeline termed “Shizuoka Multi‐omics Analysis Protocol” developed in‐house. Somatic driver alterations were identified in 72.2% of samples in 362 genes (average, 2.3 driver events per sample). Actionable information on drugs that is applicable in the current clinical setting was associated with 11.3% of samples. When including those drugs that are used for investigative purposes, actionable information was assigned to 55.0% of samples. Germline analysis revealed pathogenic mutations in hereditary cancer genes in 9.2% of samples, among which 12.2% were confirmed as pathogenic mutations by confirmatory test. Pathogenic mutations associated with non–cancerous hereditary diseases were detected in 0.4% of samples. Tumor mutation burden (TMB) analysis revealed 5.4% of samples as having the hypermutator phenotype (TMB ≥ 20). Clonal hematopoiesis was observed in 8.4% of samples. Thus, the JCGA dataset and the analytical procedures constitute a fundamental resource for genomic medicine for Japanese cancer patients.