Children with hypoglycemia due to recessive loss of function mutations of the ␤-cell ATP-sensitive potassium (K ATP ) channel can develop hypoglycemia in response to protein feeding. We hypothesized that amino acids might stimulate insulin secretion by unknown mechanisms, because the K ATP channel-dependent pathway of insulin secretion is defective. We therefore investigated the effects of amino acids on insulin secretion and intracellular calcium in islets from normal and sulfonylurea receptor 1 knockout (SUR1؊/؊) mice. Even though SUR1؊/؊ mice are euglycemic, their islets are considered a suitable model for studies of the human genetic defect. SUR1؊/؊ islets, but not normal islets, released insulin in response to an amino acid mixture ramp. This response to amino acids was decreased by 60% when glutamine was omitted. Insulin release by SUR1؊/؊ islets was also stimulated by a ramp of glutamine alone. Glutamine was more potent than leucine or dimethyl glutamate. Basal intracellular calcium was elevated in SUR1؊/؊ islets and was increased further by glutamine. In normal islets, methionine sulfoximine, a glutamine synthetase inhibitor, suppressed insulin release in response to a glucose ramp. This inhibition was reversed by glutamine or by 6-diazo-5-oxo-L-norleucine, a non-metabolizable glutamine analogue. High glucose doubled glutamine levels of islets. Methionine sulfoximine inhibition of glucose stimulated insulin secretion was associated with accumulation of glutamate and aspartate. We hypothesize that glutamine plays a critical role as a signaling molecule in amino acid-and glucosestimulated insulin secretion, and that ␤-cell depolarization and subsequent intracellular calcium elevation are required for this glutamine effect to occur.

Separate primary and validating clinical studies concur that tumor VEGF level is the most important prognostic parameter among several markers of tumor angiogenesis and proteolysis.

Glutamate dehydrogenase (GDH) plays an important role in insulin secretion as evidenced in children by gain of function mutations of this enzyme that cause a hyperinsulinism-hyperammonemia syndrome (GDH-HI) and sensitize ␤-cells to leucine stimulation. GDH transgenic mice were generated to express the human GDH-HI H454Y mutation and human wild-type GDH in islets driven by the rat insulin promoter. H454Y transgene expression was confirmed by increased GDH enzyme activity in islets and decreased sensitivity to GTP inhibition. The H454Y GDH transgenic mice had hypoglycemia with normal growth rates. H454Y GDH transgenic islets were more sensitive to leucine-and glutamine-stimulated insulin secretion but had decreased response to glucose stimulation. The fluxes via GDH and glutaminase were measured by tracing 15 N flux from [2-15 N]glutamine. The H454Y transgene in islets had higher insulin secretion in response to glutamine alone and had 2-fold greater GDH flux. High glucose inhibited both glutaminase and GDH flux, and leucine could not override this inhibition.15 NH 4 Cl tracing studies showed 15 N was not incorporated into glutamate in either H454Y transgenic or normal islets. In conclusion, we generated a GDH-HI disease mouse model that has a hypoglycemia phenotype and confirmed that the mutation of H454Y is disease causing. Stimulation of insulin release by the H454Y GDH mutation or by leucine activation is associated with increased oxidative deamination of glutamate via GDH. This study suggests that GDH functions predominantly in the direction of glutamate oxidation rather than glutamate synthesis in mouse islets and that this flux is tightly controlled by glucose.Glucose, fatty acids, and amino acids are fuels that stimulate pancreatic ␤-cell insulin secretion. Congenital hyperinsulinism (HI), 2 a group of disorders arising from mutations of genes encoding ␤-cell function, illustrates this basic phenomenon. For instance, gain of function mutations of glucokinase cause HI by lowering the threshold for glucosestimulated insulin secretion (GSIS) and highlight the role of glucokinase as the glucosensor of the ␤-cell (1, 2). Recently a form of HI due to loss of function mutations in the enzyme short-chain 3-hydroxyacyl-CoA dehydrogenase has been identified (3-6). Although the biochemical mechanisms of short-chain 3-hydroxyacyl-CoA dehydrogenase-HI are unknown, this disorder provides evidence of a role for fatty acid metabolism in insulin secretion. The ATP-dependent potassium channel (K ATP ), encoded by the sulfonylurea receptor 1 (SUR1) and Kir 6.2, transduces the energy state of the ␤-cell. Loss of function mutations in the ATP-dependent potassium channel cause the most common form of HI (K ATP -HI) and confirm that the channel plays a key role in triggering insulin release (7-9). In 1998, we identified mutations of glutamate dehydrogenase (GDH) in children with a dominant form of hyperinsulinism (GDH-HI) and implicated this enzyme as a mediator of leucine-stimulated insulin secretion (LSIS) (10 -12). GDH-HI mut...

Chemically stabilized small interfering RNA (siRNA) can be delivered systemically by intravenous injection of lipid nanoparticles (LNPs) in rodents and primates. The biodistribution and kinetics of LNP–siRNA delivery in mice at organ and cellular resolution have been studied using immunofluorescence (IF) staining and quantitative polymerase chain reaction (qPCR). At 0.5 and 2 hr post tail vein injection of Cy5-labeled siRNA encapsulated in LNP, the organ rank-order of siRNA levels is liver > spleen > kidney, with only negligible accumulation in duodenum, lung, heart, and brain. Similar conclusions were drawn by using qPCR to measure tissue siRNA levels as a secondary end point. siRNA levels in these tissues decreased by more than 10-fold after 24 hr. Within the liver, LNPs delivered siRNA to hepatocytes, Kupffer cells, and sinusoids in a time-dependent manner, as revealed by IF staining and signal quantitation methods established using OPERA/Columbus software. siRNA first accumulated in liver sinusoids and trafficked to hepatocytes by 2 hr post dose, corresponding to the onset of target mRNA silencing. Fluorescence in situ hybridization methods were used to detect both strands of siRNA in fixed tissues. Collectively, the authors have implemented a platform to evaluate biodistribution of siRNA across cell types and across tissues in vivo, with the objective of elucidating the pharmacokinetic and pharmacodynamic relationship to guide optimization of delivery vehicles.

BackgroundWhole transcriptome sequencing (RNA-seq) represents a powerful approach for whole transcriptome gene expression analysis. However, RNA-seq carries a few limitations, e.g., the requirement of a significant amount of input RNA and complications led by non-specific mapping of short reads. The Ion AmpliSeq™ Transcriptome Human Gene Expression Kit (AmpliSeq) was recently introduced by Life Technologies as a whole-transcriptome, targeted gene quantification kit to overcome these limitations of RNA-seq. To assess the performance of this new methodology, we performed a comprehensive comparison of AmpliSeq with RNA-seq using two well-established next-generation sequencing platforms (Illumina HiSeq and Ion Torrent Proton). We analyzed standard reference RNA samples and RNA samples obtained from human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs).ResultsUsing published data from two standard RNA reference samples, we observed a strong concordance of log2 fold change for all genes when comparing AmpliSeq to Illumina HiSeq (Pearson’s r = 0.92) and Ion Torrent Proton (Pearson’s r = 0.92). We used ROC, Matthew’s correlation coefficient and RMSD to determine the overall performance characteristics. All three statistical methods demonstrate AmpliSeq as a highly accurate method for differential gene expression analysis. Additionally, for genes with high abundance, AmpliSeq outperforms the two RNA-seq methods. When analyzing four closely related hiPSC-CM lines, we show that both AmpliSeq and RNA-seq capture similar global gene expression patterns consistent with known sources of variations.ConclusionsOur study indicates that AmpliSeq excels in the limiting areas of RNA-seq for gene expression quantification analysis. Thus, AmpliSeq stands as a very sensitive and cost-effective approach for very large scale gene expression analysis and mRNA marker screening with high accuracy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2270-1) contains supplementary material, which is available to authorized users.

A gene cluster responsible for the biosynthesis of anticancer agent FK228 has been identified, cloned, and partially characterized in Chromobacterium violaceum no. 968. First, a genome-scanning approach was applied to identify three distinctive C. violaceum no. 968 genomic DNA clones that code for portions of nonribosomal peptide synthetase and polyketide synthase. Next, a gene replacement system developed originally for Pseudomonas aeruginosa was adapted to inactivate the genomic DNA-associated candidate natural product biosynthetic genes in vivo with high efficiency. Inactivation of a nonribosomal peptide synthetase-encoding gene completely abolished FK228 production in mutant strains. Subsequently, the entire FK228 biosynthetic gene cluster was cloned and sequenced. This gene cluster is predicted to encompass a 36.4-kb DNA region that includes 14 genes. The products of nine biosynthetic genes are proposed to constitute an unusual hybrid nonribosomal peptide synthetase-polyketide synthase-nonribosomal peptide synthetase assembly line including accessory activities for the biosynthesis of FK228. In particular, a putative flavin adenine dinucleotidedependent pyridine nucleotide-disulfide oxidoreductase is proposed to catalyze disulfide bond formation between two sulfhydryl groups of cysteine residues as the final step in FK228 biosynthesis. Acquisition of the FK228 biosynthetic gene cluster and acclimation of an efficient genetic system should enable genetic engineering of the FK228 biosynthetic pathway in C. violaceum no. 968 for the generation of structural analogs as anticancer drug candidates. FK228 (C 24 H 36 N 4 O 6 S 2 ; molecular weight, 540.2) ( Fig. 1), also known as FR901228 or depsipeptide and registered as NSC 630176 or romidepsin, is a natural product discovered in the fermentation broth of Chromobacterium violaceum no. 968 in a screening program for agents that reverse the malignant phenotype of a Ha-ras oncogene-transformed NIH 3T3 cell line (51, 52). It exhibited outstanding anticancer activities against an array of tumor cell lines, including many members of a standard panel of 60 cell lines from the U.S. National Cancer Institute (18, 53). FK228 has entered extensive clinical trials and has shown promising properties as a new type of anticancer drug (5,30,35,36,41). A multinational pivotal trial of FK228 for the treatment of cutaneous T-cell lymphoma has been launched by Gloucester Pharmaceuticals, Inc., and the company plans to file for U.S. Food and Drug Administration approval in late 2007.Structurally, FK228 is a bicyclic depsipeptide that features a 16-membered macrolactone ring containing an ester linkage and a 17-membered ring containing the same ester linkage and a disulfide bond, the latter of which endows FK228 with an unprecedented molecular scaffold (Fig. 1). Its structure was determined by spectroscopic and X-ray crystallographic analyses (45) and was confirmed by total synthesis (27). A close examination of the FK228 structure identified building blocks of three amino acids (D-cy...

Cardiac hypertrophy is an independent risk factor for cardiovascular disease and heart failure. There is increasing evidence that microRNAs (miRNAs) play an important role in the regulation of messenger RNA (mRNA) and the pathogenesis of various cardiovascular diseases. However, the ability to comprehensively study cardiac hypertrophy on a gene regulatory level is impacted by the limited availability of human cardiomyocytes. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer the opportunity for disease modeling. Here we utilize a previously established in vitro model of cardiac hypertrophy to interrogate the regulatory mechanism associated with the cardiac disease process. We perform miRNA sequencing and mRNA expression analysis on endothelin 1 (ET-1) stimulated hiPSC-CMs to describe associated RNA expression profiles. MicroRNA sequencing revealed over 250 known and 34 predicted novel miRNAs to be differentially expressed between ET-1 stimulated and unstimulated control hiPSC-CMs. Messenger RNA expression analysis identified 731 probe sets with significant differential expression. Computational target prediction on significant differentially expressed miRNAs and mRNAs identified nearly 2000 target pairs. A principal component analysis approach comparing the in vitro data with human myocardial biopsies detected overlapping expression changes between the in vitro samples and myocardial biopsies with Left Ventricular Hypertrophy. These results provide further insights into the complex RNA regulatory mechanism associated with cardiac hypertrophy.

Short interfering RNAs (siRNAs) are a valuable tool for gene silencing with applications in both target validation and therapeutics. Many advances have recently been made to improve potency and specificity, and reduce toxicity and immunostimulation. However, siRNA delivery to a variety of tissues remains an obstacle for this technology. To date, siRNA delivery to muscle has only been achieved by local administration or by methods with limited potential use in the clinic. We report systemic delivery of a highly chemically modified cholesterol-conjugated siRNA targeting muscle-specific gene myostatin (Mstn) to a full range of muscles in mice. Following a single intravenous injection, we observe 85–95% knockdown of Mstn mRNA in skeletal muscle and >65% reduction in circulating Mstn protein sustained for >21 days. This level of Mstn knockdown is also accompanied by a functional effect on skeletal muscle, with animals showing an increase in muscle mass, size, and strength. The cholesterol-conjugated siRNA platform described here could have major implications for treatment of a variety of muscle disorders, including muscular atrophic diseases, muscular dystrophy, and type II diabetes.