Randomised clinical study: GR-MD-02, a galectin-3 inhibitor, vs. placebo in patients having non-alcoholic steatohepatitis with advanced fibrosis.

BACKGROUND: Non-alcoholic steatohepatitis (NASH) and resultant liver fibrosis is a major health problem without approved pharmacotherapy. Pre-clinical results of GR-MD-02, a galectin-3 inhibitor, suggested potential efficacy in NASH with advanced fibrosis/cirrhosis and prompted initiation of a clinical development programme in NASH with advanced fibrosis. AIM: To evaluate the safety, pharmacokinetics and exploratory pharmacodynamic markers of GR-MD-02 in subjects having NASH with bridging fibrosis. METHODS: The GT-020 study was a first-in-human, sequential dose-ranging, placebo controlled, double-blinded study with the primary objective to assess the safety, tolerability and dose limiting toxicity of GR-MD-02, in subjects with biopsy-proven NASH with advanced fibrosis (Brunt stage 3). The secondary objectives were to characterise first-dose and multiple-dose pharmacokinetic profiles and to evaluate changes in potential serum biomarkers and liver stiffness as assessed by FibroScan. RESULTS: GR-MD-02 single and three weekly repeated of 2, 4 and 8 mg/kg revealed no meaningful clinical differences in treatment emergent adverse events, vital signs, electrocardiographic findings or laboratory tests. Pharmokinetic parameters showed a dose-dependent relationship with evidence of drug accumulation following 8 mg/kg (~twofold). CONCLUSIONS: GR-MD-02 doses were in the upper range of the targeted therapeutic dose determined from pre-clinical data and were safe and well tolerated with evidence of a pharmacodynamic effect. These results provide support for a Phase 2 development programme in advanced fibrosis due to NASH.

Family-centred music therapy to promote social engagement in young children with severe autism spectrum disorder: a randomized controlled study.

BACKGROUND: Limited capacity for social engagement is a core feature of autism spectrum disorder (ASD), often evident early in the child's development. While these skills are difficult to train, there is some evidence that active involvement in music-making provides unique opportunities for social interaction between participants. Family-centred music therapy (FCMT) endeavours to support social engagement between child and parent within active music-making, yet the extent of benefits provided is unknown. AIM: This study investigated the impacts of FCMT on social engagement abilities. METHODS: Twenty-three children (36-60 months) with severe ASD received either 16 weeks of FCMT in addition to their early intervention programmes (n = 12), or their early intervention programme only (n = 11). Change in social engagement was measured with standardized parent-report assessments, parent interviews and clinician observation. RESULTS: Intention-to-treat analysis for the Vineland Social Emotional Early Childhood Scale indicated a significant effect in favour of FCMT. Thematic qualitative analysis of the parent interviews showed that the parent-child relationship grew stronger. CONCLUSION: FCMT improves social interactions in the home and community and the parent-child relationship, but not language skills or general social responsiveness. This study provides preliminary support for the use of FCMT to promote social engagement in children with severe ASD.

Influence of relative layer height and testing method on the failure mode and origin in a bilayered dental ceramic composite.

OBJECTIVES: The purpose of this study was to examine the influence of testing method (uniaxial and biaxial) and relative layer heights on the failure origin and failure mode of bilayered ceramic composite beams and disks composed of In-Ceram and Vitadur Alpha porcelain. METHODS: Beams and disks were fabricated, with relative layer heights of 1:2, 1:1, and 2:1, respectively, for In-Ceram and Vitadur Alpha porcelain. Ninety specimens each (thirty 1:2, thirty 1:1, and thirty 2:1) were tested in 3-point, 4-point-1/4-point, and biaxial ring-on-ring testing apparatuses. Fractography was used to categorize failure origins as either surface or interfacial, and failure modes as delamination or nondelamination. RESULTS: Surface and interfacial failure origins were observed in 3-point and biaxial disk test specimens, but not 4-point-1/4-point specimens where only surface failures occurred. None of the "clinically similar" specimens (1:2) failed at the interface. All testing methods resulted in delamination of Vitadur Alpha from the In-Ceram, while only 3-point and biaxial disk testing methods resulted in crack propagation through the composite interface without delamination. SIGNIFICANCE: Varying relative layer heights or varying testing method in laminate composite tensile specimens can affect failure mode and failure origin.

Identification of an ARF type low molecular mass GTP-binding protein in pea (Pisum sativum).

The ARF class of low molecular mass GTP-binding proteins, originally detected as cholera toxin-activated ADP-ribosylation factors (ARF) in mammalian cells, is now known to participate in intracellular membrane vesicle trafficking. We identified a GTP-binding protein in pea plumules which resembles ARF in several respects. Like mammalian ARF, the pea protein had an apparent molecular mass of 21 kDa and was distributed mainly (approximately 97%) in the cytosol, with 1% and 1.5% occurring in the Golgi and microsomal fractions, respectively. In comparison, small GTP-binding proteins in the 26-30 kDa range were enriched in membranous fractions. The 21 kDa protein crossreacted strongly with an ARF class I antibody prepared against a mammalian ARF, but did not crossreact with ARF 5 (of class II) antibodies. An anti-Volvox yptl antibody did not show immunoreactivity to the 21 kDa pea protein, although it crossreacted strongly with a 28 kDa pea plumule protein. Our results strongly suggest that the 21 kDa pea protein is an analog of the ARF protein localized in the cytosol of mammalian cells. As far as we are aware, this is the first observation of an ARF protein in plants.

Modification of Brassica seed oil by antisense expression of a stearoyl-acyl carrier protein desaturase gene.

Molecular gene transfer techniques have been used to engineer the fatty acid composition of Brassica rapa and Brassica napus (canola) oil. Stearoyl-acyl carrier protein (stearoyl-ACP) desaturase (EC 1.14.99.6) catalyzes the first desaturation step in seed oil biosynthesis, converting stearoyl-ACP to oleoyl-ACP. Seed-specific antisense gene constructs of B. rapa stearoyl-ACP desaturase were used to reduce the protein concentration and enzyme activity of stearoyl-ACP desaturase in developing rapeseed embryos during storage lipid biosynthesis. The resulting transgenic plants showed dramatically increased stearate levels in the seeds. A continuous distribution of stearate levels from 2% to 40% was observed in seeds of a transgenic B. napus plant, illustrating the potential to engineer specialized seed oil compositions.

The mechanism of membrane response to chilling. Effect of temperature on phospholipid deacylation and reacylation reactions in the cell surface membrane.

The ciliary membrane of Tetrahymena pyriformis is physically and metabolically remote from the main centers of lipid metabolism. Nevertheless, it possesses an independent capacity to modify its phospholipid molecular species composition rapidly under stress. The role of ciliary phospholipid deacylating and reacylating enzymes in this phenomenon has been evaluated. Isolated cilia showed substantial phospholipase A (combined A1 and A2), acyl-CoA synthetase and acyltransferase activities. Activities of all the three enzymes of cilia from 39 degrees C-grown cells were greatly reduced when the cilia were incubated at 15 degrees C. In contrast, the phospholipase A and acyltransferase activities in cilia from 15 degrees C-grown cells were surprisingly high at 15 degrees C and twice as high at 37 degrees C as were the equivalent activities in preparations from 39 degrees C-grown cells. While the in vivo substrate specificity of phospholipase A could not be meaningfully assessed, the acyltransferases exhibited a temperature-dependent substrate specificity in vivo. Growth temperature also affected the positional distribution of fatty acids incorporated into ciliary phospholipids in vivo. The ability of acyltransferases to utilize added [14C] acyl-CoA could be markedly stimulated, and their lipid class specificity could be significantly altered in vitro by supplementing the incubation mixture with exogenous lysophospholipid acceptors. These findings suggest that the rate-limiting factor in acyl chain turnover is not the activity of acyltransferases per se but rather the availability of suitable substrates and acceptors. Therefore, we postulate that temperature alters the rate and specificity of ciliary membrane phospholipid retailoring primarily by controlling the in situ phospholipase A activity.

The effects of high concentrations of sodium or calcium ions on the lipid composition and properties of Tetrahymena membranes.

Tetrahymena pyriformis cells have been grown in media varying in NaCl concentration from 3.7 mM (normal medium) to 0.3 M and varying in CaCl2 from 0.2 mM (normal medium) to 0.1 M. Tetrahymena grown in 0.3 M NaCl showed relatively few alterations in phospholipid composition, with significant changes being found only in the cell surface membranes (pellicle), which incrased in phosphatidylethanolamine content from 39% (low Na+) to 48% (high Na+) of the total phospholipids. The small decrease in fatty acid unsaturation and increase in shorter chain fatty acids in pellicle phospholipids were not statistically significant. No significant changes in phospholipid head group composition or fatty acid distribution were observed in high Ca2+-grown cells. Complementary studies of membrane fluidity, as inferred from freeze-fracture electron microscopy analysis, indicated that membranes of high Na+-acclimated cells were similar to those of control cells, when each was measured in its respective medium. However, the outer alveolar membrane of the pellicle and the food vacuolar membrane were considerably less fluid in high-Ca2+ cells. The lower fluidity in vacuolar membranes may have been responsible for alterations in the cells' capacity to form food vacuoles.

Self-regulation of membrane fluidity. The effect of saturated normal and methoxy fatty acid supplementation on Tetrahymena membrane physical properties and lipid composition.

Tetrahymena cells elongated and desaturated massive supplements of palmitic or lauric acid at nearly twice the rates employed by unfed cells, thereby maintaining constant the physical properties of their membrane lipids. However, when a mixture of the 9- and 10-monomethoxy derivatives of stearic acid was administered, these compounds were incorporated without further metabolism. The marked fluidizing effect of the phospholipid-bound methoxy-fatty acids elicited an immediate reduction in fatty acid desaturase activity, the pattern of change being very similar to that induced by supplements of polyunsaturated fatty acids. The modulation of fatty acid desaturase activity by methoxy-acids clearly seems to be governed by membrane fluidity rather than by some form of end product inhibition of the type which might have been postulated to explain the similar effect caused by polyunsaturated fatty acids.

Molecular control of membrane properties during temperature acclimation. Membrane fluidity regulation of fatty acid desaturase action?

Further studies on the molecular mechanisms of temperature acclimation have been carried out using the ciliate Tetrahymena pyriformis. The most prominent change in lipid metabolism during acclimation to high temperature--depression of fatty acid desaturase activity--could be simulated by supplementing the growth medium of isothermally-grown cells with polyunsaturated fatty acids. Such cells resisted the membrane-fluidizing effect of the incorporated exogenous acids by increased use of de novo synthesized saturated acids in their phospholipids. The data support the conclusions arising from earlier experiments with temperature-shifted cells (Martin, C.E., Hiramitsu, K., Kitajima, Y., Nozawa, Y., Skriver, L., and Thompson, G.A., Jr. (1976), Biochemistry 15), showing that, when membrane fluidity increased to a superoptimal level, the activity of membrane-associated fatty acid desaturases was decreased. Since the reaction is controlled by membrane fluidity, rather than temperature per se, we postulate that it is the general mechnaism employed by cells adjusting to any fluidity-modifying factor, such as cations, drugs, etc.