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BackgroundDysregulation of microRNA (miRNA) expression in various tissues and body fluids has been demonstrated to be associated with several diseases, including Type 2 Diabetes mellitus (T2D). Here, we compare miRNA expression profiles in different tissues (pancreas, liver, adipose and skeletal muscle) as well as in blood samples from T2D rat model and highlight the potential of circulating miRNAs as biomarkers of T2D. In parallel, we have examined the expression profiles of miRNAs in blood samples from Impaired Fasting Glucose (IFG) and T2D male patients.Methodology/principal findingsEmploying miRNA microarray and stem-loop real-time RT-PCR, we identify four novel miRNAs, miR-144, miR-146a, miR-150 and miR-182 in addition to four previously reported diabetes-related miRNAs, miR-192, miR-29a, miR-30d and miR-320a, as potential signature miRNAs that distinguished IFG and T2D. Of these microRNAs, miR-144 that promotes erythropoiesis has been found to be highly up-regulated. Increased circulating level of miR-144 has been found to correlate with down-regulation of its predicted target, insulin receptor substrate 1 (IRS1) at both mRNA and protein levels. We could also experimentally demonstrate that IRS1 is indeed the target of miR-144.ConclusionWe demonstrate that peripheral blood microRNAs can be developed as unique biomarkers that are reflective and predictive of metabolic health and disorder. We have also identified signature miRNAs which could possibly explain the pathogenesis of T2D and the significance of miR-144 in insulin signaling.

Aquaporin, a transmembrane protein that functions as a highly selective water channel, has intrigued membrane researchers around the world who have attempted to synthesize membranes embedded with aquaporin for water purification. We have molecularly designed pore-suspending biomimetic membranes embedded with Aquaporin Z (AQPz) based on different lipid protein ratios and investigated their local mechanical stabilities by force indentation using atomic force microscopy (AFM). The compatibility between 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and AQPz was proved by stop-flow experiments, in which the DMPC-AQPz (a lipid to protein molar ratio of 2000 : 1) exhibited 3000-fold higher permeability than the pristine DMPC vesicles. A series of vesicles were then ruptured on pristine porous alumina and carboxylated polyethylene glycol (PEG) coated porous alumina. The carboxylated-PEG polymer cushion significantly enhances the flexibility of the DMPC bilayer on the substrate and also gives rise to higher breakthrough forces from AFM when AQPz is reconstituted into the pore-suspending membrane on the carboxylated-PEG coated substrate. The local membrane mechanical stability is further improved by increasing the AQPz content, suggesting that AQPz increases the energy barrier required for a normal force to punch through the resultant biomimetic membrane. Therefore, the carboxylated-PEG grafted porous alumina exhibits a promising platform for the fabrication of pore-suspending biomimetic membranes. Nevertheless, to achieve the highest performance of DMPC-AQPz biomimetic membranes, a compromise may be needed to optimize the DMPC-AQPz ratio.

We have developed a new method for quantification of promoter activity in cell lines transfected with recombinant plasmids containing the reporter gene encoding chloramphenicol acetyl transferase (CAT) by real-time PCR. As the efficiency of transfection has a direct influence on the total mRNA produced, we have used the neomycin-resistance gene present within the same vector DNA to normalize the measurement of mRNA levels. Three promoters from genes encoding toxins (pre-synaptic neurotoxin phospholipase A2, post-synaptic α neurotoxin and cardiotoxin), believed to have evolved from the same ancestor but exhibiting different promoter activities, have been employed in this study to demonstrate the feasibility and accuracy of the method in CAT gene reporter analysis.

1. 1. The lethalities, anticoagulant effects, hemorrhagic, thrombin-like enzyme, hyaluronidase, protease, arginine ester hydrolase, 5′-nucleotidase, l -amino acid oxidase, alkaline phosphomonoesterase, phosphodiesterase and phospholipase A activities of twenty-three samples of venoms from twelve species of Asian lance-headed pit vipers (genus Trimeresurus) were examined. 2. 2. The results indicate that notwithstanding individual variations in venom properties, the differences in biological properties of the Trimeresurus venoms can be used for the differentiation of venoms from different species of Trimeresurus. 3. 3. The results also suggest that differences in the biological properties of snake venoms are useful parameters in the classification of snake species. 4. 4. Our results indicate that venoms from the species T. okinavensis exhibited biological properties markedly different from other Trimeresurus venoms examined. This observation supports the recently proposed reclassification of T. okinavensis as a member of the genus Ovophis, rather than the genus Trimeresurus.

The venom of the spitting cobra, Naja naja sputatrix contains highly potent alpha-neurotoxins (NTXs) in addition to phospholipase A2 (PLA2) and cardiotoxin (CTX). In this study, we report the complete characterization of three genes that are responsible for the synthesis of three isoforms of alpha-NTX in the venom of a single spitting cobra. DNA amplification by long-distance polymerase chain reaction (LD-PCR) and genome walking have provided information on the gene structure including their promoter and 5' and 3' UTRs. Each NTX isoform is approximately 4 kb in size and contains three exons and two introns. The sequence homology among these isoforms was found to be 99%. Two possible transcription sites were identified by primer extension analysis and they corresponded to the adenine (A) nucleotide at positions +1 and -45. The promoter also contains two TATA boxes and a CCAAT box. Putative binding sites for transcriptional factors AP-2 and GATA are also present. The high percentage of similarity observed among the NTX gene isoforms of N. n. sputatrix as well as with the alpha-NTX and kappa-NTX genes from other land snakes suggests that the NTX gene has probably evolved from a common ancestral gene.