Haruki Shibata

New CETP Inhibitor K-312 Reduces PCSK9 Expression: A Potential Effect on LDL Cholesterol Metabolism

Despite significant reduction of cardiovascular events by statin treatment, substantial residual risk persists, driving emerging needs for the development of new therapies. We identified a novel cholesteryl ester transfer protein (CETP) inhibitor, K-312, that raises HDL and lowers LDL cholesterol levels in animals. K-312 also suppresses hepatocyte expression of proprotein convertase subtilisin/kexin 9 (PCSK9), a molecule that increases LDL cholesterol. We explored the underlying mechanism for the reduction of PCSK9 expression by K-312. K-312 inhibited in vitro human plasma CETP activity (IC50; 0.06 μM). Administration of K-312 to cholesterol-fed New Zealand White rabbits for 18 wk raised HDL cholesterol, decreased LDL cholesterol, and attenuated aortic atherosclerosis. Our search for additional beneficial characteristics of this compound revealed that K-312 decreases PCSK9 expression in human primary hepatocytes and in the human hepatoma cell line HepG2. siRNA silencing of CETP in HepG2 did not compromise the suppression of PCSK9 by K-312, suggesting a mechanism independent of CETP. In HepG2 cells, K-312 treatment decreased the active forms of sterol regulatory element-binding proteins (SREBP-1 and -2) that regulate promoter activity of PCSK9. Chromatin immunoprecipitation assays demonstrated that K-312 decreased the occupancy of SREBP-1 and SREBP-2 on the sterol regulatory element of the PCSK9 promoter. PCSK9 protein levels decreased by K-312 treatment in the circulating blood of cholesterol-fed rabbits, as determined by two independent mass spectrometry approaches, including the recently developed, highly sensitive parallel reaction monitoring method. New CETP inhibitor K-312 decreases LDL cholesterol and PCSK9 levels, serving as a new therapy for dyslipidemia and cardiovascular disease.

A glass fiber sheet-based electroosmotic lateral flow immunoassay for point-of-care testing.

We have developed a quantitative immunoassay chip targeting point-of-care testing. To implement a lateral flow immunoassay, a glass fiber sheet was chosen as the material for the microfluidic channel in which the negative charge on the fiber surfaces efficiently generates the electroosmotic flow (EOF). The EOF, in turn, allows controllable bound/free separation of antigen/antibody interactions on the chip and enables precise determination of the antigen concentration. In addition, the defined size of the porous matrix was suitable for the filtration of undesired large particles. We confirmed the linear relationship between the concentration of analyte and the resulting fluorescence intensity from the immunoassay of two model analytes, C-reactive protein (CRP) and insulin, demonstrating that analyte concentration was quantitatively determined within the developed chip in 20 min. The limits of detection were 8.5 ng mL(-1) and 17 ng mL(-1) for CRP and insulin, respectively.

Effects of intramuscular administration of tiletamine-zolazepam with and without sedative pretreatment on plasma and serum biochemical values and glucose tolerance test results in Japanese black bears (Ursus thibetanus japonicus)

OBJECTIVE To establish a safe anesthetic protocol with little effect on blood biochemical values and IV glucose tolerance test (IVGTT) results in Japanese black bears (Ursus thibetanus japonicus). ANIMALS 16 captive female Japanese black bears (5 to 17 years of age). PROCEDURES Bears were randomly assigned to 4 treatment groups (4 bears/group) in which various treatment combinations were administered via blow dart: tiletamine HCl and zolazepam HCl (9 mg/kg) alone (TZ), TZ (6 mg/kg) and acepromazine maleate (0.1 mg/kg), TZ (6 mg/kg) and butorphanol tartrate (0.3 mg/kg), or TZ (3 mg/kg) and medetomidine HCl (40 μg/kg). Glucose injection for the IVGTT was started 130 minutes after TZ administration. Blood samples were obtained before, at, and intermittently after glucose injection for measurement of biochemical variables as well as plasma glucose and serum insulin concentrations during the IVGTT. Rectal temperature, pulse rate, and respiratory rate were assessed every 15 minutes during the experiment. RESULTS Induction and maintenance of anesthesia were safely achieved with little adverse effect on cardiopulmonary function when each of the 4 anesthetic regimens was used, although mild hypothermia was induced. No difference was evident between treatment groups in blood biochemical values. Blood glucose and insulin concentration profiles during the IVGTT were similar among the bears given TZ, with or without acepromazine or butorphanol, but hyperglycemia and hypoinsulinemia developed in bears given TZ with medetomidine. CONCLUSIONS AND CLINICAL RELEVANCE All 4 anesthetic regimens yielded chemical restraint without affecting clinical and biochemical values in bears, but medetomidine appeared to affect IVGTT results. For this reason, medetomidine should not be used when anesthetizing bears for IVGTTs.

Seasonality and fasting effect in raccoon dog Nyctereutes procyonoides serum leptin levels determined by canine leptin-specific enzyme-linked immunosorbent assay

Leptin is an adipocyte-derived peptide hormone that acts on the brain and regulates food intake and energy balance. Several previous reports have suggested that overwintering raccoon dogs Nyctereutes procyonoides are able to control their adiposity efficiently, but the contribution of leptin to weight regulation in these animals remains unclear. To study the seasonality of overwintering raccoon dogs as well as the effects of fasting on them, serum leptin levels were investigated using a newly established canine leptin-specific enzyme-linked immunosorbent assay (ELISA) kit. Of the nine animals studied, five were fed and four were fasted (deprived of food for 2 months in winter). Blood samples and body fat weights were monitored once a month throughout the experimental period (July 2007-March 2008). Leptin concentrations obtained by ELISA were significantly higher than and had a positive correlation with those obtained by previously used multispecies radioimmunoassay (RIA) kits. Moreover, ELISA showed a clearer correlation between the body fat weight and leptin levels compared with RIA, suggesting the efficacy of canine leptin-specific ELISA kit for leptin estimation in raccoon dogs. Autumnal fattening was observed in both groups of animals, but the wintertime loss of adipose tissue was more obvious in the fasted group. Serum leptin concentrations determined by ELISA showed seasonal changes without significant differences between the fed and fasted animals. Therefore, high levels of leptin may be responsible for the suppression of feeding behavior in raccoon dogs before winter.

Simple molding fabrication for poly acrylamide hydrogel

We present a simple fabrication method based on nano-molding and capillary force lithography to achieve micro/nano structures of poly-acrylamide (PAAm) hydrogel. We fabricated various patterns, such as line and space (L&S), posts, and grid with 140–2,000 nm in width or diameter using nano-molding method. Also, we could fabricate fiber and mesh structures of 2 µm in width using capillary force lithography. The nano L&S structure allowed the cells to align along the line and have long and narrow shape. The hydrogel micromesh could trap cells inside of mesh. Therefore, the present hydrogel molding method provides easy, fast, and flexible micro/nano fabrication of PAAm hydrogel, indicating the potential to functional surface for chemical and biomedical applications.

Nano-patterned hydrogel reduced inflammatory effects in subcutaneous tissue

We present a nano-patterned poly-acrylamide (PAAm) hydrogel that can reduce inflammatory effects after subcutaneous implantation. Although hydrogel is considered as an excellent biomaterial for implants due to its biocompatibility, hydrogel still induces inflammation after implantation. To enhance biocompatibility for subcutaneous implantable sensors, we modified hydrogel surface with nano-patterns using simple molding process. To test the anti-inflammatory effect, we implanted the samples to rats back. Since macrophages play an important role in the immune response and development of encapsulation after inflammation reaction, we counted macrophages neighboring the implanted nano-patterned hydrogels after 3 and 7 days and measured thickness of encapsulation after 21 days from implantation. We found that the sample with the line-and-space pattern of 600 nm in space successfully suppressed the inflammation reaction. Therefore, nano-patterned hydrogel is promising for long-term subcutaneous implantable sensors.

Fluorescent hydrogel fibers for long-term in vivo glucose monitoring

Fluorescence-based implantable glucose sensors can provide continuous glucose monitoring (CGM) with wireless transdermal transmission and long-lasting activity in vivo. Here, we present a long-term in vivo glucose monitoring method using glucose-responsive fluorescent hydrogel fibers. The fiber structure enables the sensor to remain at an implantation site for a long period and be removed from the implantation site after use. We found that the polyethylene glycol (PEG)-bonded polyacrylamide (PAAm) hydrogel fibers reduced inflammation, transdermally glowed and continuously responded to blood glucose concentration changes after 2.5 months, showing their potential application for long-term in vivo continuous glucose monitoring.

Implantable fluorescent hydrogel for continous blood glucose monitoring

We present a continuous glucose monitoring sensors using glucose-sensitive fluorescent dyes. We prepared microgels by copolymerizing acrylamide with the glucose sensitive fluorescent dyes. This fluorescent gel shows a high quantifiability and rapid response in the physiological glucose concentration range of 62.5 to 1000 mg/dL. Our in vivo study revealed that fluorescent intensity of the gels changed in response to in vivo blood-glucose levels. We expect this method to be useful in the development of implantable continuous monitoring systems based on fluorescent gels.