Sensitive and specific quantification of antisense oligonucleotides using probe alteration-linked self-assembly reaction technology.

Quantitative bioanalysis is essential when establishing pharmacokinetic properties during the drug development process. To overcome challenges of sensitivity, specificity and process complexity associated with the conventional analysis of antisense oligonucleotides (ASOs), a new approach to nonenzymatic hybridization assays using probe alteration-linked self-assembly reaction (PALSAR) technology as a signal amplifier was evaluated. PALSAR quantification of ASOs in mouse tissue and plasma was able to achieve a high sensitivity ranging from 1.5 to 6 pg/ml, intra-/interday accuracies in the range of 86.8-119.1% and 88.1-113.1%, respectively, and precision of ≤17.2%. Furthermore, crossreactivity of 3'n-1, a metabolite with a single base difference, was <1%. Our approach provides an auspicious method for distinguishing metabolites and detecting ASOs with high sensitivity and specificity.

The effectiveness of small changes for pressure redistribution; using the air mattress for small changes.

Observing small changes (SCs) at specific sites is a new form of managing changes in position. We investigated SCs at specific sites considering interface pressure, contact area, body alignment and physical sensation in nine healthy female adults and evaluated SCs using the air mattress that was divided into six cells (A-F). Thirty-three SC combinations at one or several sites were evaluated. Pressure in the sacral region significantly decreased in 28 SC combinations compared with the supine position (p < 0.05), and the effect of pressure redistribution was greater when SCs were applied at several instead of a single site. The contact area at 17 of the 28 SC combinations significantly increased (p < 0.05). Among sites ranked based on interface pressure, body alignment and physical sensation, SCs at sites BCE, AE and BD were the most favorable. The common feature among these three combinations was that they involved tilting the buttock region and one other site. The findings suggested that SCs at the buttock region could reduce disruptions in alignment as well as the impact on physical sensation caused by the body sinking into the mattress and improve interface pressure redistribution via increased contact area with the mattress.

Self-assembly formed by a short DNA probe pair: Application for highly sensitive mRNA species detection without reverse transcription.

We describe a novel technology for detecting nucleic acids: Probe Alteration Link Self-Assembly Reactions (PALSAR). PALSAR comprises DNA self-assembly of pairs of short DNA probes formed by alternate hybridization of three complementary regions in a pair of honeycomb probes (HCPs). Self-assembly occurs at designated salt concentrations and reaction temperatures and requires no enzymes. We prepared pairs of HCPs to detect mRNAs encoded by the GAPDH gene ß-actin (BA) gene, CD3D gene, CD4 gene, major vault protein (MV) gene and the signalling lymphocytic activation molecule-associated protein (SAP) gene, and succeeded in quantitatively detecting these mRNAs. PALSAR could detect mRNA directly without synthesizing cDNA. Moreover, multiple mRNAs could be detected simultaneously in a single reaction tube and there was a good correlation between the results obtained PALSAR and those by real-time PCR.

A hydroxymethylglutaryl coenzyme A reductase inhibitor improves endothelial function within 7 days in patients with chronic hemodialysis.

BACKGROUND: Atherosclerosis-related diseases are leading causes of morbidity among patients undergoing hemodialysis. The effects of hydroxymethylglutaryl coenzyme A reductase inhibitors (statins) on the endothelial function of hemodialyzed patients are not known. METHODS AND RESULTS: For 16 weeks, we prescribed simvastatin (low dose: 5 mg or moderate dose: 10 mg) to 28 patients (low dose: n=14, 61.2 ± 8.6 years, moderate dose: n=14, 60.8 ± 10.2 years) and chose 9 patients (61.5 ± 5.2 years) without prescriptions as controls. We compared the effects of statin on lipids, flow-mediated endothelium-dependent and nitroglycerin-induced endothelium-independent dilatation (%FMD, %NTD), and markers of oxidant stress and atherosclerosis. Serum HDL-cholesterol and triglycerides did not change significantly in any of the three groups; however, LDL-cholesterol was decreased at 16 weeks in both simvastatin groups. The %FMD and plasma NOx increased at 1 and 16 weeks in both statin groups, but not in the control group (P<0.01). The %NTD did not change. Oxidized LDL, VCAM-1, and 8-isoprostane decreased significantly after 16 weeks in both statin groups; however, TNF-α and interleukin 6 did not change. In the control group, no significant changes in these parameters were observed. Multiple regression analyses showed that the (short) period of hemodialysis and (young) age are significant factors associated with %FMD improvement. CONCLUSIONS: A statin improved impaired endothelial function in the arteries of chronic dialysis patients, in part by enhancing NO bioavailability within one week. Improved endothelial function is in line with the anti-atherosclerotic effects observed in patients undergoing chronic hemodialysis.

Nitric oxide metabolites are associated with survival in older patients.

OBJECTIVES: To assess the efficacy of various vascular endocrinological substances, such as plasma nitric oxide metabolites (NOx), as surrogate markers of survival in older patients. DESIGN: Prospective cohort, observational. SETTING: Nagoya University Hospital and related hospitals, Japan. PARTICIPANTS: One hundred fifty patients aged 70 and older, recruited consecutively from the outpatient clinics of Nagoya University Hospital and related hospitals. MEASUREMENT: Serum biochemical analyses such as albumin and total cholesterol, various prognostic markers, such as tumor necrosis factor (TNF)-alpha, NOx, activities of daily living (ADLs), and instrumental ADLs (IADLs) were evaluated on enrollment. ADLs, IADLs, and comorbidities, especially depression and impaired cognition, were evaluated on enrollment. The main outcome was survival rate over 2.75 years. RESULTS: Forty-nine patients died during the follow-up period. Mann-Whitney U-test showed that hemoglobin, total protein, serum albumin, total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, high sensitive c-reactive protein, NOx, B-type natriuretic peptide, interleukin-6, and TNF-alpha levels; ADLs; cognitive impairment; and depressive status were significantly different for subjects who survived and those who died. Of the dependent variables in the Cox proportional hazards regression analyses, only ADLs, NOx, and albumin were significantly different. In the Kaplan-Meier analyses of mortality, the prognosis of patients in the third and fourth quartiles of NOx was significantly worse than that of patients in the first or second quartile. The prognosis of patients with impaired ADLs was worse than that of other patients for the overall period. CONCLUSION: Lower levels of NOx may be associated with survival in older patients. It may be an effective marker, like ADLs, which is a well-known marker.

Nitric oxide (NO) is a new clinical biomarker of survival in the elderly patients and its efficacy might be nearly equal to albumin.

BACKGROUND: For elderly patients, the consideration of prognostic factors is very important, but there have been few reports about the potential use of vasoactive substances as prognostic markers in the elderly. OBJECTIVE: We assessed endocrinological substances, such as plasma NO(x) (metabolites of NO), as the prognostic marker in elderly. We compared their efficacy with that of such well-known markers as albumin and pro-inflammatory cytokines such as IL-6. METHODS: The patients were recruited consequently from the clinics of Nagoya University Hospital or related home care services facilities. One hundred and twenty seven elderly aged 65 and older were registered. Biochemical analyses such as albumin, total cholesterol, BNP, and NO(x) were measured upon enrollment. The main outcome was the survival rate. RESULTS: Forty-six patients died during the follow-up period. Mann-Whitney's U-test showed that the levels of age, hemoglobin, total protein, serum albumin, serum creatinine, total cholesterol, HDL-cholesterol, LDL-cholesterol, high sensitive CRP, NO(x), IL-6, and TNF-alpha were significantly different between the living and deceased subjects. Among the dependent variables in the logistic regression analyses, only albumin and NO(x) were significantly different. In the Kaplan-Meier analyses of mortality, the prognosis of patients in 3rd and 4th quartile of NO(x) was significantly worse than that in 1st or 2nd quartile. CONCLUSION: NO(x) has potential both as a vascular marker and as a marker for predicting survival in elderly. In the latter role, it may be as effective as albumin.

A new HMG-CoA reductase inhibitor, pitavastatin remarkably retards the progression of high cholesterol induced atherosclerosis in rabbits.

BACKGROUND: The remarkable anti-atherosclerotic effects of 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitor have not been demonstrated in diet induced severe hyperlipidemia in rabbit model. OBJECTIVE: We have investigated the effect of pitavastatin, a newly developed statin, on atherosclerosis in rabbits. METHODS AND RESULTS: Oophorectomized female NZW rabbits were fed 0.3% cholesterol chow for 12 weeks with or without pitavastatin (0.1mg/kg per day) (Gp.NK and HCD). The level of serum cholesterol was decreased in Gp.NK compared with Gp.HCD (772.8 +/- 70.2 versus 1056.9 +/- 108.3 mg/d), whereas no significant alterations were observed in triglyceride and HDL-cholesterol. NO dependent response stimulated by acetylcholine and calcium ionophore A23187 and tone related basal NO response induced by N(G)-monomethyl-l-arginine acetate were all improved by pitavastatin treatment. Pitavastatin treatment increased the level of cyclic GMP in the aorta of cholesterol fed rabbits. In the aorta, the expression of eNOS mRNA was significantly up regulated and O(2)(-) production was slightly reduced in Gp.NK animals. Atherosclerotic area was significantly decreased in aortic arch and thoracic aorta from Gp.NK compared with those from Gp.HCD ( 15.1 +/- 5.3 versus 41.9 +/- 10.2%, 3.1 +/- 1.1versus 7.9 +/- 1.2% in Gp.NK and Gp.HCD aortic arch and thoracic aorta). Anti-macrophage staining area, the MMP1 or 2 and the nitrotyrosine positive area were decreased in Gp.NK. CONCLUSION: Pitavastatin retards the progression of atherosclerosis formation and it improves NO bioavailability by eNOS up-regulation and decrease of O(2)(-).