Blood is a commonly used biofluid for biomarker discovery. Although blood lipid metabolites are considered to be potential biomarker candidates, their fundamental properties are not well characterized. We aimed to (1) investigate the matrix type (serum vs. plasma) that may be preferable for lipid biomarker exploration, (2) elucidate age- and gender-associated differences in lipid metabolite levels, and (3) examine the stability of lipid metabolites in matrix samples subjected to repeated freeze-thaw cycles. Using liquid chromatography-mass spectrometry, we performed lipidomic analyses for fasting plasma and serum samples for four groups (15 subjects/group) of young and elderly (25–34 and 55–64 years old, respectively) males and females and for an additional aliquot of samples from young males, which were subjected to repeated freeze-thaw cycles. Lysophosphatidylcholine and diacylglycerol levels were higher in serum than in plasma samples, suggesting that the clotting process influences serum lipid metabolite levels. Gender-associated differences highlighted that the levels of many sphingomyelin species were significantly higher in females than in males, irrespective of age and matrix (plasma and serum). Age-associated differences were more prominent in females than in males, and in both matrices, levels of many triacylglycerols were significantly higher in elderly females than in young females. Plasma and serum levels of most lipid metabolites were reduced by freeze-thawing. Our results indicate that plasma is an optimal matrix for exploring lipid biomarkers because it represents the original properties of an individual’s blood sample. In addition, the levels of some blood lipid species of healthy adults showed gender- and age-associated differences; thus, this should be considered during biomarker exploration and its application in diagnostics. Our fundamental findings on sample selection and handling procedures for measuring blood lipid metabolites is important for ensuring the quality of biomarkers identified and its qualification process.

Renal cell carcinoma (RCC) is the most common histological type of adult kidney cancer. In this study, we obtained lipidomic profiles of clear cell RCC (ccRCC), a major RCC subtype, by performing a lipidomic analysis of specimens of cancerous tissue and the surrounding normal renal cortex obtained from the same patients (N = 49). We also compared the lipidomic profiles with the lipogenic transcriptome of specimens of cancerous tissue and the surrounding normal renal cortex for an additional set of patient samples (N = 95). Overall, we detected 326 lipids, including phospholipids, sphingolipids, neutral lipids, and eicosanoids. The levels of more than 70% of the detected lipids were significantly different (P < 0.01, corrected by the false discovery rate). The cancerous tissue was distinguished by higher levels of ether-type phospholipids, cholesterol esters, and triacylglycerols, as well as by lower levels of phospholipids (except for phosphatidylcholines) and polyunsaturated fatty acids. Characteristic changes in the levels of mRNAs and metabolites suggested that the phosphatidylethanolamine (PE) synthesis pathway is suppressed in ccRCC and associated with cell proliferation. The present study represents the lipidomic profiles of ccRCC, which provides novel information about the metabolic changes in renal cancerous tissue and RCC pathophysiology.

Pyrrole-imidazole (PI) polyamides are small synthetic molecules that recognize and attach to the minor groove of DNA, thereby inhibiting gene transcription by blocking transcription factor binding. These derivatives can act as gene silencers inhibiting target gene expression under stimulatory conditions such as disease. To evaluate PI polyamides as treatments for the progression of renal diseases, we examined morphological effects, pharmacological properties, and the specificity of PI polyamides targeted to the transforming growth factor (TGF)-β1 promoter during salt-induced hypertensive nephrosclerosis in Dahl salt-sensitive rats. The targeted PI polyamide markedly reduced glomerulosclerosis and interstitial fibrosis without side effects. PI polyamide significantly decreased expression of TGF-β1 and extracellular matrix in the renal cortex. Microarray analysis found that only 3% of the transcripts were affected by PI polyamide, but this included decreased expression of extracellular matrix, TGF-β1-related cytokines, angiogenic, and cell stabilizing factors, proteinases, and renal injury-related factors. Thus, targeted PI polyamides are potential gene silencers for diseases not treatable by current remedies.

Although the "gold standard" for diagnosis of tuberculous meningitis (TBM) is bacterial isolation of Mycobacterium tuberculosis, there are still several complex issues. Recently, we developed an internally controlled novel wide-range quantitative nested real-time PCR (WR-QNRT-PCR) assay for M. tuberculosis DNA in order to rapidly diagnose TBM. For use as an internal control calibrator to measure the copy number of M. tuberculosis DNA, an original new-mutation plasmid (NM-plasmid) was developed. Due to the development of the NM-plasmid, the WR-QNRT-PCR assay demonstrated statistically significant accuracy over a wide detection range (1 to 10 5 copies). In clinical applications, the WR-QNRT-PCR assay revealed sufficiently high sensitivity (95.8%) and specificity (100%) for 24 clinically suspected TBM patients. In conditional logistic regression analysis, a copy number of M. tuberculosis DNA (per 1 ml of cerebrospinal fluid) of >8,000 was an independent risk factor for poor prognosis for TBM (i.e., death) (odds ratio, 16.142; 95% confidence interval, 1.191 to 218.79; P value, 0.0365). In addition, the copy numbers demonstrated by analysis of variance statistically significant alterations (P < 0.01) during the clinical treatment course for 10 suspected TBM patients. In simple regression analysis, the significant correlation (R 2 ‫؍‬ 0.597; P < 0.0001) was demonstrated between copy number and clinical stage of TBM. We consider the WR-QNRT-PCR assay to be a useful and advanced assay technique for assessing the clinical treatment course of TBM.Tuberculous meningitis (TBM) is the severest form of infection of Mycobacterium tuberculosis, causing death or severe neurological defects in more than half of those affected in spite of antituberculosis treatment (ATT) (1,2,8,18). The diagnosis of TBM remains a complex issue, because the most widely used conventional bacteriological detection methods, such as direct smear for acid-fast bacilli (AFB) and culture for M. tuberculosis, are unable to rapidly detect M. tuberculosis with sufficient sensitivity in the acute phase of TBM (3-13, 18, 19). In 2006, we designed a novel internally controlled quantitative nested real-time PCR (QNRT-PCR) assay based on TaqMan PCR (Applied Biosystems) (15). Moreover, based on this original QNRT-PCR (OR-QNRT-PCR) assay, an improved wide-range QNRT-PCR (WR-QNRT-PCR) assay was developed (17). For use as a "calibrator" in WR-QNRT-PCR assay, a new internal control was constructed (17).In the preliminary experiments, the WR-QNRT-PCR assay demonstrated significantly improved quantitative accuracy and had a wide detection range (1 to 10 5 copies) compared to what was seen for the OR-QNRT-PCR assay (17).In this study, we tried to quantitatively detect M. tuberculosis DNA in actual cerebrospinal fluid (CSF) samples by using the WR-QNRT-PCR assay. In addition, the clinical usefulness of this novel assay technique for the rapid and accurate diagnosis of TBM and for assessing the clinical course of TBM was examined. MATERIALS AND METHODSThis study was approv...

Non-thermal atmospheric gas plasma (AGP) exhibits cytotoxicity against malignant cells with minimal cytotoxicity toward normal cells. However, the mechanisms of its tumor-selective cytotoxicity remain unclear. Here we report that AGP-activated medium increases caspase-independent cell death and mitochondrial network collapse in a panel of human cancer cells, but not in non-transformed cells. AGP irradiation stimulated reactive oxygen species (ROS) generation in AGP-activated medium, and in turn the resulting stable ROS, most likely hydrogen peroxide (H2O2), activated intracellular ROS generation and mitochondrial ROS (mROS) accumulation. Culture in AGP-activated medium resulted in cell death and excessive mitochondrial fragmentation and clustering, and these responses were inhibited by ROS scavengers. AGP-activated medium also increased dynamin-related protein 1-dependent mitochondrial fission in a tumor-specific manner, and H2O2 administration showed similar effects. Moreover, the vulnerability of tumor cells to mitochondrial network collapse appeared to result from their higher sensitivity to mROS accumulation induced by AGP-activated medium or H2O2. The present findings expand our previous observations on death receptor-mediated tumor-selective cell killing and reinforce the importance of mitochondrial network remodeling as a powerful target for tumor-selective cancer treatment.

Nonalcoholic steatohepatitis (NASH) is a major health problem since it often leads to hepatocellular carcinoma. However, the underlying mechanisms of NASH development and subsequent fibrosis have yet to be clarified. We compared comprehensive lipidomic profiles between mice with high fat diet (HFD)-induced steatosis and STAM mice with NASH and subsequent fibrosis. The STAM mouse is a model that demonstrates NASH progression resembling the disease in humans: STAM mice manifest NASH at 8 weeks, which progresses to fibrosis at 12 weeks, and finally develop hepatocellular carcinoma. Overall, 250 lipid molecules were detected in the liver using liquid chromatography-mass spectrometry. We found that STAM mice with NASH presented a significantly higher abundance of sphingolipids and lower levels of triacylglycerols than the HFD-fed control mice. The abundance of certain fatty acids in phospholipid side chains was also significantly different between STAM and control mice, although global levels of phosphatidylcholines and phosphatidylethanolamines were comparable. Finally, increase in levels of acylcarnitines and some diacylglycerols was observed in STAM mice toward the fibrosis stage, but not in age-matched control mice. Our study provides insights into the lipid status of the steatotic, NASH, and fibrotic liver that would help elucidate the molecular pathophysiology of NASH progression.

We have demonstrated that mesenchymal cells from spontaneously hypertensive rats genetically express complement 3 (C3). Mature tubular epithelial cells can undergo epithelial-to-mesenchymal transition (EMT) that is linked to the pathogenesis of renal fibrosis and injury. In this study, we investigated the contribution of C3 in EMT and in the renal renin-angiotensin (RA) systems associated with hypertension. C3a induced EMT in mouse TCMK-1 epithelial cells, which displayed increased expression of renin and Krüppel-like factor 5 (KLF5) and nuclear localization of liver X receptor α (LXRα). C3 and renin were strongly stained in the degenerated nephrotubulus and colocalized with LXRα and prorenin receptor in unilateral ureteral obstruction (UUO) kidneys from wild-type mice. In C3-deficient mice, hydronephrus and EMT were suppressed, with no expression of renin and C3. After UUO, systolic blood pressure was increased in wild-type but not C3-deficient mice. In wild-type mice, intrarenal angiotensin II (ANG II) levels were markedly higher in UUO kidneys than normal kidneys and decreased with aliskiren. There were no increases in intrarenal ANG II levels after UUO in C3-deficient mice. Thus C3 induces EMT and dedifferentiation of epithelial cells, which produce renin through induction of LXRα. These data indicate for the first time that C3 may be a primary factor to activate the renal RA systems to induce hypertension.

Previously, we designed an internally controlled quantitative nested real-time (QNRT) PCR assay forTuberculous meningitis (TBM) is the severest form of infection of Mycobacterium tuberculosis, causing death or severe neurological defects in more than half of those affected in spite of antituberculosis treatment (25). The diagnosis of TBM remains a complex issue because the most widely used conventional bacteriological detection methods, such as direct smear for acid-fast bacilli and culture for M. tuberculosis, are unable to rapidly detect M. tuberculosis with sufficient sensitivity in the acute phase of TBM (7,8,11,12,18,19,21,22,23,25). At present, the detection of M. tuberculosis DNA in cerebrospinal fluid (CSF) by use of PCR is widely used as a more rapid, sensitive, and specific diagnostic method (1,7,8,10,11,12,15,17,18,19,21,22,23,26). Recently, we designed a novel internally controlled quantitative nested real-time PCR (QNRT-PCR) assay based on TaqMan PCR (Applied Biosystems) (22). This novel assay technique combines the high sensitivity of nested PCR with the accurate quantification of real-time PCR (22, 23). However, this original QNRT-PCR (OR-QNRT-PCR) assay is still unstable and has many points that should be improved (22,23).In this study, to reliably detect M. tuberculosis DNA in CSF samples with a wider detection range, we attempted to improve on the OR-QNRT-PCR technique; therefore, a new internal control for use as a "calibrator" was prepared. We named this improved assay technique wide-range QNRT-PCR (WR-QNRT-PCR) and examined its ability to quantitatively detect M. tuberculosis DNA in samples. In this paper, the development and methodology of the WR-QNRT-PCR assay are stated. MATERIALS AND METHODSThis study was approved by the Nihon University Institutional Review Board. Preparation of the new internal control (plasmid) for use as a calibrator. For the WR-QNRT-PCR assay, two types of the original plasmid, wild plasmids (W-plasmids) and new-mutation plasmids (NM-plasmids), were prepared for a quantitative detection of M. tuberculosis DNA, and this was done as well for the OR-QNRT-PCR assay (22, 23).W-plasmid, which was inserted into a 239-bp DNA fragment of the gene sequence encoding the MPB64 protein of M. tuberculosis (MPT64; GenBank accession no. NC_000962) (22, 23) into pCR 2.1 vector (Invitrogen Corp., San Diego, CA) was constructed for use as the standard template by the previously reported procedure (22,23).NM-plasmid was developed based on the previously reported M-plasmid (22, 23) for use as a new internal-control "calibrator" in the WR-QNRT-PCR assay. In NM-plasmid, a total of four regions, where two pairs of (outer and inner) forward and reverse primers annealed, were replaced with the artificial random nucleotides added to the TaqMan probe annealing region in the M-plasmid (Fig. 1). The sequences of the artificial random nucleotides were set to have the same nucleotide composition as MPT64 of wild M. tuberculosis. Replacing procedures