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The present study uses a novel approach to gene therapy in which plasmid DNA is targeted to the pancreas in vivo using ultrasound targeted microbubble destruction (UTMD) to achieve islet regeneration. Intravenous microbubbles carrying plasmids are destroyed within the pancreatic microcirculation by ultrasound, achieving local gene expression that is further targeted to beta-cells by a modified rat insulin promoter (RIP3.1). A series of genes implicated in endocrine development were delivered to rats 2 days after streptozotocin-induced diabetes. The genes PAX4, Nkx2.2, Nkx6.1, Ngn3, and Mafa produced alpha cell hyperplasia, but no significant improvement in beta cell mass or blood glucose 30 days after UTMD. In contrast, RIP3.1-NeuroD1 promoted islet regeneration from surviving beta-cells, with normalization of glucose, insulin, and C-peptide at 30 days. In a longer-term experiment, 4 of 6 rats had return of diabetes at 90 days, accompanied by beta cell apoptosis on Tunel staining. Pre-treatment with the JNK inhibitor SP600125 successfully blocked beta-cell apoptosis and resulted in restoration of beta cell mass and normalization of blood glucose for up to 90 days. This technique allows in vivo islet regeneration, restoration of beta cell mass, and normalization of blood sugar, insulin, and C-peptide in rats without viruses.

This study was done to improve efficiency and islet specificity of the rat insulin promoter (RIP). Various rat insulin promoter lengths were prepared and tested in vitro to drive luciferase reporter gene expression in INS1-cells, alpha-cells, acinar cells, ductal cells, and fibroblasts. The CMV promoter was used as a positive control. In addition, the DsRed reporter gene was administered in vivo to rat pancreas by ultrasound-targeted microbubble destruction (UTMD). Confocal microscopy was used to detect the presence and distribution of DsRed within the pancreas after UTMD. A modified RIP3.1 promoter, which includes portions of the insulin gene after its transcription start site is 5-fold more active in INS-1 cells than the full length RIP promoter or the CMV promoter. RIP3.1 is regulated by glucose level and various islet transcription factors in vitro, and exhibits activity in alpha-cells, but not exocrine cells. In vivo delivery of RIP3.1-DsRed resulted in expression of DsRed protein in beta-cells, and to a lesser extent alpha cells under normal glucose conditions. No DsRed signal was present in exocrine pancreas under RIP3.1. A modified rat insulin promoter, RIP3.1, efficiently and specifically directs gene expression to endocrine pancreas.

Contains fulltext : 80646.pdf (Publisher’s version ) (Closed access) We performed high-resolution in vitro proton nuclear magnetic resonance spectroscopy on cerebrospinal fluid and urine samples of 44 patients with leukodystrophies of unknown cause. Free sialic acid concentration was increased in cerebrospinal fluid of two siblings with mental retardation and mild hypomyelination. By contrast, urinary excretion of free sialic acid in urine was normal on repeated testing by two independent methods. Both patients were homozygous for the K136E mutation in SLC17A5, the gene responsible for the free sialic acid storage diseases. Our findings demonstrate that mutations in the SLC17A5 gene have to be considered in patients with hypomyelination, even in the absence of sialuria.

Ultrasound-targeted microbubble destruction (UTMD) was used to direct betacellulin (BTC) and pancreatic duodenal homeobox-1 (PDX1) to rat pancreas 48 h after islet destruction by streptozotocin (STZ). Sprague-Dawley rats were rendered diabetic by STZ injection. Controls included normal rats, STZ only without UTMD, and UTMD with DsRed reporter gene. Blood glucose increased dramatically in all rats 48 h after STZ, and continued to rise after UTMD with BTC alone. Blood glucose declined from day 3 to day 10 after UTMD with PDX1, but remained elevated (261+/-8 mg/dl). However, in rats treated with both BTC and PDX1, blood glucose remained below 200 mg/dl throughout day 10. This was accompanied by normalization of blood insulin and C-peptide. Histology demonstrated islet-like clusters of glucagon-staining cells in the rats treated with BTC and PDX1, but these clusters disappeared by 30 days after UTMD treatment. Although regeneration of insulin-producing islets was not seen, diabetes was reversed for up to 15 days after a single UTMD treatment by ectopic insulin production by pancreatic acinar cells. These cells co-expressed amylase and insulin and demonstrated several beta-cell markers by reverse transcription-PCR. Gene therapy by UTMD can reverse diabetes in vivo in adult rats by restoring pancreatic insulin production.

Myocardial angiogenesis mediated by human vascular endothelial growth factor 165 (hVEGF165) cDNA was promoted in rat myocardium using an in vivo-targeted gene delivery system known as ultrasound-targeted microbubble destruction (UTMD). Microbubbles carrying plasmids encoding hVEGF165, or control solutions were infused intravenously during ultrasonic destruction of the microbubbles within the myocardium. Biochemical and histological assessment of gene expression and angiogenesis were performed 5, 10, and 30 days after UTMD. UTMD-treated myocardium contained hVEGF165 protein and mRNA. The myocardium of UTMD-treated animals showed hypercellular foci associated with hVEGF165 expression and endothelial cell markers. Capillary density in UTMD-treated rats increased 18% at 5 days and 33% at 10 days, returning to control levels at 30 days (P

Melamine, a widely used industry chemical, has been detected in pet foods and some protein-based food commodities, such as milk as a fraudulent substitute for protein since 2007. Melamine with concentrations higher than the safety limits can cause kidney injury. Therefore there is growing needs for a low cost portable sensor that can detect melamine in food. Here, we demonstrate two assay methods of using functionalized Si nanowire transistors (SiNW FETs) to detect melamine with high sensitivity. The direct detection using antibody coated SiNW FETs shows selective detection of melamine down to 50 ppb level (or 2 μM). Another competitive assay takes advantage of competition reaction between the BSASM2-antibody vs. melamine-antibody to indirectly detect the concentration of melamine selectively down to 20 pM level in buffer solutions. The results show that SiNW has the potential to become a portable low cost sensor for melamine detection.

Here we demonstrate the use of multiple Si nanochannel (NC) or nanograting (NG) instead of the conventional single nanochannel or nanowire design in biosensors. The NG devices can significantly reduce device-to-device variation, and improve device performance, e.g. higher current, higher ON/OFF ratio, smaller subthreshold slope, lower threshold voltage Vt in buffer solution. NG devices also result in higher sensor stability in buffer and diluted human serum. We believe such improvements are due to reduced discrete dopant fluctuation in the Si nanowires and biochemical noise in the solution because of the multiple-channel design. The improved devices allow us to sense pH linearly with 3-aminopropyltriethoxysilane coated devices, and to selectively detect insulin with limit of detection down to 10 fM in both buffer solution and diluted human serum without pre-purification.

Here we demonstrate the use of multiple Si nanochannel (NC) or nanograting (NG) instead of the conventional single channel or nanowire design in biosensors can significantly reduce device to device variation, and improve device performance (higher current, higher ON/OFF ratio, smaller SS, lower Vt) in buffer solution. NG devices also result in higher sensor stability (repeated sensing over tens of hours) in buffer and human serum. We believe such improvements are due to reduced discrete dopant fluctuation and biochemical noise in the solution. The improved devices allow us to sense pH linearly with 3-aminopropyltriethoxysilane (APTES) coated devices, and to selectively detect insulin with limit of detection down to 10fM in both buffer solution and human serum without pre-purification.

Summary Aromatase is the enzyme that catalyzes the conversion of androgen to oestrogen. Aromatase expression in extra-gonadal sites and local oestrogen synthesis play an important role in the physiological conditions and in the growth of certain neoplasms. Objective The purpose of this study was to investigate aromatase expression in oral keratinocytes and oral squamous cell carcinoma (SCC). Design Immunocytochemistry and RT-nested PCR were used to detect aromatase protein and mRNA expression in primary human oral epithelial cell culture and in an oral SCC cell line. Immunohistochemistry was used to detect aromatase protein expression in frozen and archival human tissue sections of normal oral epithelium and oral SCC. Results Cytoplasmic immunostaining was found in normal oral keratinocytes and SCC cells in culture. The common coding region of aromatase mRNA was detected in the oral keratinocytes derived from five different normal individuals and in the SCC cell line. However, there were variations in aromatase exon 1 expression among normal oral keratinocyte samples. Cytoplasmic staining was found in normal oral epithelium and well-differentiated oral SCC but not in poorly differentiated oral SCC by immunohistochemistry. Conclusion Aromatase was expressed in normal oral keratinocytes and oral SCC both in cell culture and in tissues, indicating local oestrogen synthesis in normal and neoplastic conditions of oral epithelium.