A Unique Gene-Silencing Approach, Using an Intelligent RNA Expression Device (iRed), Results in Minimal Immune Stimulation When Given by Local Intrapleural Injection in Malignant Pleural Mesothelioma

Background: We have recently introduced an intelligent RNA expression device (iRed), comprising the minimum essential components needed to transcribe short hairpin RNA (shRNA) in cells. Use of iRed efficiently produced shRNA molecules after transfection into cells and alleviated the innate immune stimulation following intravenous injection. Methods: To study the usefulness of iRed for local injection, the engineered iRed encoding luciferase shRNA (Luc iRed), complexed with cationic liposomes (Luc iRed/liposome-complexes), was intrapleurally injected into an orthotopic mesothelioma mouse model. Results: Luc iRed/liposome-complexes markedly suppressed the expression of a luciferase marker gene in pleurally disseminated mesothelioma cells. The suppressive efficiency was correlated with the expression level of shRNA within the mesothelioma cells. In addition, intrapleural injection of iRed/liposome-complexes did not induce IL-6 production in the pleural space and consequently in the blood compartment, although plasmid DNA (pDNA) or dsDNA (the natural construct for iRed) in the formulation did. Conclusion: Local delivery of iRed could augment the in vivo gene silencing effect without eliciting pronounced innate immune stimulation. Our results might hold promise for widespread utilization of iRed as an RNAi-based therapeutic for intracelial malignant cancers

Effects of Triacontanol on Mycelial Growth, Amylase Activity and Lipid Compostion of Zygomycetes

The growth of mycelia and the production of amylase in a liquid medium were accelerated in the presence of 1-triacontanol(TRIA) added to the medium and the most effective amount of TRIA to be added was 1 ppm. The ratio of triglycerides(TG) was high in the lipid compositions of the mycelia well grown in this medium and having high amylase activity

Gene Silencing Using 4′-thioDNA as an Artificial Template to Synthesize Short Hairpin RNA Without Inducing a Detectable Innate Immune Response

The development of a versatile technique to induce RNA interference (RNAi) without immune stimulation in vivo is of interest as existing approaches to trigger RNAi, such as small interfering RNA (siRNA) and plasmid DNA (pDNA) expressing short hairpin RNA (shRNA), present drawbacks arising from innate immune stimulation. To overcome them, an intelligent shRNA expression device (iRed) designed to induce RNAi was developed. The minimum sequence of iRed encodes only the U6 promoter and shRNA. A series of iRed comprises a polymerase chain reaction (PCR)-amplified 4′-thioDNA in which any one type of adenine (A), guanine (G), cytosine (C), or thymine (T) nucleotide unit was substituted by each cognate 4′-thio derivatives, i.e., dSA iRed, dSG iRed, dSC iRed, and ST iRed respectively. Each modified iRed acted as a template to transcribe shRNA with RNAi activity. The highest shRNA yield was generated using dSC iRed that exerted gene silencing activity in an orthotopic mouse model of mesothelioma. Reducing the minimal structure required to transcribe shRNA and the presence of the 4′-thiomodification synergistically function to abrogate innate immune response induced by dsDNA. The iRed will introduce a new approach to induce RNAi without inducing a detectable innate immune response

Dysregulation of the proteasome increases the toxicity of ALS-linked mutant SOD1.

A hallmark of protein conformational disease, exemplified by neurodegenerative disorders, is the expression of misfolded and aggregated proteins. The relationship between protein aggregation and cellular toxicity is complex, and various models of experimental pathophysiology have often yielded conflicting or controversial results. In this study, we examined the biophysical properties of amyotrophic lateral sclerosis (ALS)-linked mutations of Cu/Zn superoxide dismutase 1 (SOD1) expressed in human tissue culture cells. Fluorescence correlation spectroscopy (FCS) and Förster resonance energy transfer (FRET) analyses revealed that changes in proteasome activity affected both the expression of FCS- and FRET-detected oligomers and cellular toxicity. Under normal conditions, highly aggregation-prone mutant SOD1 exhibited very little toxicity. However, when the activity of the proteasome was transiently inhibited, only upon recovery did we observe the appearance of ordered soluble oligomers, which were closely correlated with cellular toxicity. These results shed light on the importance of balance in proteostasis and suggest that transient shifts of activity in the cellular machinery can alter the course of protein conformational transitions and dysregulate modulation of proteasome activity. In neurodegenerative disorders including ALS, such changes may be a risk factor for pathogenesis

Nighttime Cooling Is an Effective Method for Improving Milk Production in Lactating Goats Exposed to Hot and Humid Environment

Heat production in ruminants follows a diurnal pattern over the course of a day peaking 3 hours following afternoon feeding and then gradually declining to its lowest point prior to morning feeding. In order to clarify the cooling period most effective in reducing decreases in feed intake and milk production, experiments were carried out based on the diurnal rhythm of heat production and heat dissipation. In experiment 1, the effects of hot environment on milk production were investigated. The animals were kept first in a thermoneutral environment (20.0°C, 80.0%) for 12 days, they were then transitioned to a hot environment (32°C, 80.0%) for 13 days before being returned to second thermoneutral environment for a further 12 days. In experiment 2, the effectiveness of daytime cooling or nighttime cooling for improving milk production in hot environment was compared. While ten lactating Japanese Saanen goats (aged 2 years, weighing 41.0 kg) during early lactation were used in experiment 1, ten lactating goats (aged 2 years, weighing 47.5 kg) during mid-lactation were used in experiment 2. The animals were fed 300 g of concentrated feed and excessive amounts of crushed alfalfa hay cubes twice daily. Water was given ad libitum. The animals were milked twice daily. When exposed to a hot environment, milk yield and composition decreased significantly (p<0.05). Milk yield in the hot environment did not change with daytime cooling, but tended to increase with nighttime cooling. Compared to the daytime cooling, milk components percentages in the nighttime cooling were not significantly different but the milk components yields in the nighttime cooling were significantly higher (p<0.05). The results indicate that nighttime cooling is more effective than daytime cooling in the reduction of milk production declines in lactating goats exposed to a hot environment