Enhanced snoMEN Vectors Facilitate Establishment of GFP–HIF-1α Protein Replacement Human Cell Lines

The snoMEN (snoRNA Modulator of gene ExpressioN) vector technology was developed from a human box C/D snoRNA, HBII-180C, which contains an internal sequence that can be manipulated to make it complementary to RNA targets, allowing knock-down of targeted genes. Here we have screened additional human nucleolar snoRNAs and assessed their application for gene specific knock-downs to improve the efficiency of snoMEN vectors. We identify and characterise a new snoMEN vector, termed 47snoMEN, that is derived from box C/D snoRNA U47, demonstrating its use for knock-down of both endogenous cellular proteins and G/YFP-fusion proteins. Using multiplex 47snoMEM vectors that co-express multiple 47snoMEN in a single transcript, each of which can target different sites in the same mRNA, we document >3-fold increase in knock-down efficiency when compared with the original HBII-180C based snoMEN. The multiplex 47snoMEM vector allowed the construction of human protein replacement cell lines with improved efficiency, including the establishment of novel GFP–HIF-1α replacement cells. Quantitative mass spectrometry analysis confirmed the enhanced efficiency and specificity of protein replacement using the 47snoMEN-PR vectors. The 47snoMEN vectors expand the potential applications for snoMEN technology in gene expression studies, target validation and gene therapy

Adiponectin-Mediated Analgesia and AntiInflammatory Effects in Rat

The adipose tissue-derived protein, adiponectin, has significant anti-inflammatory properties in a variety of disease conditions. Recent evidence that adiponectin and its receptors (AdipoR1 and AdipoR2) are expressed in central nervous system, suggests that it may also have a central modulatory role in pain and inflammation. This study set out to investigate the effects of exogenously applied recombinant adiponectin (via intrathecal and intraplantar routes; 10–5000 ng) on the development of peripheral inflammation (paw oedema) and pain hypersensitivity in the rat carrageenan model of inflammation. Expression of adiponectin, AdipoR1 and AdipoR2 mRNA and protein was characterised in dorsal spinal cord using real-time polymerase chain reaction (PCR) and Western blotting. AdipoR1 and AdipoR2 mRNA and protein were found to be constitutively expressed in dorsal spinal cord, but no change in mRNA expression levels was detected in response to carrageenan-induced inflammation. Adiponectin mRNA, but not protein, was detected in dorsal spinal cord, although levels were very low. Intrathecal administration of adiponectin, both pre- and 3 hours post-carrageenan, significantly attenuated thermal hyperalgesia and mechanical hypersensitivity. Intrathecal administration of adiponectin post-carrageenan also reduced peripheral inflammation. Intraplantar administration of adiponectin pre-carrageenan dose-dependently reduced thermal hyperalgesia but had no effect on mechanical hypersensitivity and peripheral inflammation. These results show that adiponectin functions both peripherally and centrally at the spinal cord level, likely through activation of AdipoRs to modulate pain and peripheral inflammation. These data suggest that adiponectin receptors may be a novel therapeutic target for pain modulation

Effects of NaOH treatment and NaOH treatment conditions on the mechanical properties of coir fibres for use in composites manufacture

The influences of NaOH treatment and NaOH treatment conditions (NaOH concentration, treatment temperature and duration) on the mechanical properties of coir fibres were examined. The experiment was designed on a Minitab Software with a view to determining the optimal NaOH treatment conditions that would reveal the variable(s) that exerted the most significant influence on the tensile properties of the NaOH-treated coir fibres. NaOH treatment brought about increase in the tensile strength of coir fibres. The highest tensile strength and Young’s modulus were observed at the same treatment condition of; 20% NaOH concentration, 24 h duration and at 50 °C. Pareto Chart confirmed concentration as the parameter that exerted the highest influence on the material’s tensile properties. Observations from the microstructure showed that removal of tyloses was more as a result of treatment temperature and duration than NaOH concentration. The microstructure of the NaOH-treated coir fibres showed rough surfaces which can lead to improved adhesion between the fibres and the matrix during composites manufacture, thus improved mechanical properties of the composites.</p

Deformation of sample pans used in differential scanning calorimeters

The authors have previously reported on an optical technique to enable the simultaneous and non-contact acquisition of spectral, thermal and physical information of a sample in a differential scanning calorimeter (DSC). This was achieved using a simple bifurcated fibre optic probe to link the DSC to a conventional Fourier transform infrared spectrometer and an optical spectrum analyser. The fibre optic probe was located over the sample and reference compartments of the DSC. In the current study, a series of experiments were designed to investigate the stability of DSC pans during heating from ambient to 230 °C. During the first heating cycle, the base of the aluminium pans used in these experiments was found to deform in a non-linear manner. The deformation characteristics of pans manufactured from copper and steel were also investigated. Annealing the aluminium pans was found to improve significantly the deformation or expansion characteristics.</p