Extraction of urinary cell-free DNA by using triamine-modified silica particles for liquid biopsy.

The presence of approximately 200-bp cell-free DNA (cfDNA) in the urine has attracted attention as a biomarker for liquid biopsy. However, it is currently useful only for diagnoses of cancers in which a large amount of cfDNA is excreted in the urine. Therefore, the development of an efficient method for extracting cfDNA existing in small amounts in the urine is essential for diagnosing many other diseases. We examined the effect of particle size, small pore size (surface area), and surface modification of porous silica particles on the efficiency of DNA extraction. Our observations suggested that cfDNA could be captured by tertiary amine-modified particles and then removed from the particles by repeatedly washing with sodium bicarbonate (pH 11). Using this method with 30 mg of triamine-modified particles, we succeeded in extracting a few hundred nanograms of cfDNA from 15 mL urine. Furthermore, we could detect ~ 67 fg/mL caries DNA (211 bp) in 15 mL urine sample, suggesting that this method may be suitable for the extraction of genetic biomarkers for cfDNA-based liquid biopsy.

Enrichment of liposomal nanomedicines using monolithic solid phase extraction discs following preactivation with bivalent metal ion solutions.

Silicate is an excellent adsorbent because of its large surface area and amenability to surface modification. In this study, the representative liposome nanomedicines DOXIL® and AmBisome® were enriched using a silica monolith disc (diameter 4.2 mm, length 1.5 mm) with bimodal pores. Although the nanoparticles passed through the disc without retention when water was used as the preactivation solution, they were strongly retained by the disc when a 1 M bivalent metal (such as Mg2+, Ca2+, and Ni2+) solution was used. Notably, strong affinity was observed to DOXIL, a pegylated liposomal nanoparticle, by the disc composed of 5 µm and 10 nm through- and meso pores, respectively, and nearly 100% of DOXIL was recovered from a 40× diluted solution. Overall, the results demonstrate that monolithic discs are effective for the enrichment of liposomal nanomedicines.

Development and clinical application of a bioluminescence enzyme immunoassay for oxytocin.

The development of a highly sensitive analytical method for oxytocin could be useful in the diagnosis and treatment of autistic spectrum disorder. We previously developed a colorimetric enzyme immunoassay (EIA) for plasma oxytocin measurement. In this study, we developed a method to measure oxytocin concentrations using a higher sensitivity bioluminescent EIA. Biotinylated oxytocin bridged with five lysine residues was used in a competitive format. The standard curve range for oxytocin was 1.0 to 1000 pg/assay. In addition, there was good correlation between the colorimetric and bioluminescent immunoassays in terms of measured oxytocin concentration (r = 0.9665, n = 48). The bioluminescent EIA for plasma oxytocin was more rapid and provided higher sensitivity than the colorimetric immunoassay, making it suitable for clinical application.

Development of a highly specific enzyme immunoassay for oxytocin and its use in plasma samples.

Background The peptide hormone oxytocin acts in the central nervous system and plays an important role in various complex social behaviours. We report the production of a high affinity and specificity antibody for oxytocin and its use in a highly sensitive enzyme immunoassay. Biotin that was chemically bound to oxytocin derivative containing zero to six lysines as bridge was the labelled antigen. Seven labelled antigens were used to develop a highly sensitive enzyme immunoassay. Methods Antioxytocin antiserum was obtained by immunization of oxytocin-bovine thyrogloblin conjugate to rabbit. Oxytocin sample was added to the second antibody-coated microtitre plate and allowed to react overnight at 4℃, then biotinylated oxytocin was added 1 h at 4℃, and horseradish peroxidase-labelled avidin was added and incubated for 1 h at room temperature. The plate was then washed. Horseradish peroxidase activity was measured by a colorimetric method using o-phenylenediamine (490 nm). Results The sensitivity of the enzyme immunoassay improved as the number of lysine residues increased; consequently, biotinylated oxytocin bridged with five lysines was used. A standard curve for oxytocin ranged from 1.0 to 1000 pg/assay. The detection limit of the assay was 2.36 pg, and the reproducibility was 3.6% as CV% ( n = 6). Cross-reactivity with vasopressin and vasotocin was less than 0.01%. Conclusion The sensitivity of the enzyme immunoassay could be improved by increasing the number of lysine residues on the biotin-labelled antigen. The proposed method is sensitive and more specific than conventional immunoassays for oxytocin and can be used to determine plasma oxytocin concentrations.

Development of RNA aptamer and its ligand binding assay on microchip electrophoresis.

Microchip electrophoresis (ME) coupled with fluorescence detection was used to estimate the binding activity of aptamer in each systematic evolution of ligands by exponential enrichment (SELEX) round for a target molecule. This approach is a non-radioisotopic, rapid and simple platform, and electrophoretic separation appears to be an effective technique for aptamers of oligonucleotide molecules. We tried to obtain gonadotropin-specific RNA aptamer by the above approach. As a result, the peaks of aptamers based on the conformational differences between them were separated and detected on the electropherograms. Moreover, the intensity of peak of unbound aptamer was decreased with progression through the SELEX rounds, suggesting that RNA aptamer with high affinity was obtained by the proposed method.

Quality determination of nickel-loaded silica prepared from poaceous biomass.

Klason lignin or preacid hydrolysate of a poaceous biomass such as rice husk, rice straw ( Oryza sativa ), and wheat straw ( Triticum aestivum ) became a good source of highly pure silica by simple calcinations in the testing process for application of high-boiling solvent (HBS) pulping of agricultural byproduct. Especially, Klason lignin or preacid hydrolysis residue of rice husks offered highly purified silica, which was converted to an excellent Ni/SiO(2) catalyst for methanation of carbon dioxide. The Ni/SiO(2) catalyst showed superior properties after the following sequential treatments; preacid hydrolysis of rice husks, HBS pulping of the resultant residue, cellulase hydrolysis of the HBS pulp, calcinations of the resultant powder, impregnation of the silica sample with an aqueous solution of nickel nitrate, and final calcinations.

Development of a novel fluorometric assay for nitric oxide utilizing sesamol and its application to analysis of nitric oxide-releasing drugs.

Nitric oxide (NO) is related to various physiological effects as well as to numerous diseases caused by accentuation of NO production. Measurement of NO in cells and tissues is difficult as NO readily reacts with other molecules; furthermore, its half-life as a radical is fleeting. Currently, many NO pharmaceuticals are marketed as therapeutic agents for ischemic disease. Consequently, the identification of NO radicals and determination of generation rate from pharmaceuticals is very important when the effect of the medicinal supply is estimated. In this study, we developed a fluorometric assay for NO employing sesamol (3,4-methylenedioxyphenol) as a fluorometric substrate. Sesamol is converted to a fluorescent derivative (ex. 365 nm, em. 447 nm), which is dimmer in the presence of NO. The detection limit of NO with this method is 400 fmol; moreover, NO generated from drugs can be measured.

Increase of the trophoblast giant cells with prolactin-releasing peptide (PrRP) receptor expression in p53-null mice.

Trophoblast giant cells in the mouse placentas are polyploid cells that form as a result of endoreduplication. The giant cells form the outermost layer of the extraembryonic compartment and produce a number of pregnancy-specific hormones, including prolactin family members. Here we demonstrate that trophoblast giant cells are increased, and display upregulation of prolactin releasing peptide (PrRP) receptor in the p53-null (p53(-/-)) embryonic placentas. At day 13.5 of gestation, the weight of p53(-/-) placentas was less than that of both wild-type and p53(+/-) placentas. In p53(-/-) placentas, the spongiotrophoblast layer was significantly decreased in thickness, and the trophoblast giant cells were observed not only in the outer layer of placentas but in both the spongiotrophoblast layer and the labyrinthine layer. The giant cells spread over the spongiotrophoblast and labyrinthine layer in p53(-/-) placentas displayed more intensive expression of immunoreactive PrRP receptor than in wild-type placentas. Previous studies indicated that the association between PrRP and PrRP receptor physiologically involves in the expression and secretion of the peptide hormones, including prolactin and growth hormones. These results suggest that p53 may regulate the differentiation of trophoblast giant cells, and may control the physiological PrRP stimuli in mouse placentas.

Adiponectin plays an important role in efficient energy usage under energy shortage.

Adiponectin is an adipose tissue-specific secretory protein known to be an insulin-sensitizing protein. In this study, we generated adiponectin sense and antisense transgenic (Tg) mice to investigate whether adiponectin plays a role in the regulation of energy homeostasis during the growth stage. Spontaneous motor activity of antisense Tg mice were markedly reduced during fasting, particularly in young female mice, compared with wild type (Wt) and sense Tg mice. Furthermore, both body weight and adipose tissue mass of the antisense female Tg mice drastically reduced during fasting. To examine the relationship between the collapse of abdominal white adipose tissue (WAT) and serum adiponectin level, we measured the expression of genes related to energy expenditure, such as uncoupling protein (UCP). Notably, the mRNA of UCP1 in the WAT of antisense Tg female mice was markedly less than that of Wt mice and the UCP1 mRNA was strongly increased during fasting. These findings suggest that the serum adiponectin is important to maintaining energy homeostasis under energy shortage conditions, such as over female pubertal development.

A novel 21-kDa cytochrome c-releasing factor is generated upon treatment of human leukemia U937 cells with geranylgeraniol.

Geranylgeraniol (GGO) induces apoptosis in various lines of human tumor cells through a mitochondrion-dependent pathway. The present study describes identification of a 21-kDa cytochrome c-releasing factor that appears in the cytosolic fraction after treatment of human leukemia U937 cells with GGO. Incubation of isolated mitochondria with a lysate of U937 cells that had been treated with GGO resulted in the release of cytochrome c from the mitochondria. Utilizing this cell-free system, we purified a 21-kDa protein that induced the release of cytochrome c from mitochondria and appeared to be involved in the apoptosis that is induced in U937 cells by GGO. We designated this protein cytochrome c-releasing factor 21 (CRF21). Overexpression of CRF21 in HeLa cells induced the release of cytochrome c from mitochondria, with subsequent apoptosis. Our results suggest that CRF21 might play an important role in the induction of apoptosis by GGO in leukemia U937 cells.

Fractionation of wheat straw by atmospheric acetic acid process.

Fractionation of wheat straw was investigated using an atmospheric acetic acid process. Under the typical conditions of 90% (v/v) aqueous AcOH, 4% H(2)SO(4) (w/w, on straw), ratio of liquor to straw (L/S) 10 (v/w), pulping temperature 105 degrees C, and pulping time 3h, wheat straw was fractionated to pulp (cellulose), lignin and monosaccharides mainly from hemicellulose with yields of approximately 50%, 15% and 35%, respectively. Acetic acid pulp from the straw had an acceptable strength for paper and could be bleached to a high brightness over 85% with a short bleaching sequence. Acetic acid pulp was also a potential feedstock for fuels and chemicals. The acetic acid process separated pentose and hexose in wheat straw to a large extent. Most of the pentose (xylan) was dissolved, whereas the hexose (glucan) remained in the pulp. Approximately 30% of carbohydrates in wheat straw were hydrolyzed to monosaccharides during acetic acid pulping, of which xylose accounted for 70% and glucose for 12%. The acetic acid lignin from wheat straw showed relatively lower molecular weight and fusibility, which made the lignin a promising raw material for many products, such as adhesive and molded products.

Lignin gel with unique swelling property.

Lignin gels were prepared from acetic acid lignin by use of polyethylene glycol diglycidyl ether as cross-linker. The gels were found to swell in aqueous ethanol solution, in particular 50% (v/v) solution. In addition, they also swelled in alkaline solution and shrank upon heating. A literature search showed that investigation on gel swelling in aqueous ethanol has not been reported so far. Gels prepared from the cross-linker alone and its analogues did not show such swelling characteristics in aqueous ethanol. Therefore, the unique swelling property must be attributable to an intrinsic property of lignin.