Combination of active and inactive siRNA targeting the mitotic kinesin Eg5 impairs silencing efficiency in several cancer cell lines

Gene silencing by RNA interference (RNAi) has proven to be a powerful tool for investigating gene function in mammalian cells. Combination of several short interfering RNA (siRNA) targeting the same gene is commonly used to improve RNA interference. However, in contrary to the well-described mechanism of RNAi, efficiency of single siRNA compared to pool remains poorly documented. We addressed this issue using several active and inactive siRNA targeting Eg5, a kinesin-related motor involved in mitotic spindle assembly. These siRNA, used alone or in combination, were tested for their silencing efficiency in several cancer cell lines. Here we show that presence of inactive Eg5 siRNA in a pool dramatically decreases knockdown efficacy in a cell line- and dose-dependent manner. Lack of inhibition by unrelated siRNA suggests that a competition may occur during siRNA incorporation into RNA-induced silencing complexes (RISCs) along with the target mRNA. Altogether, our results, which need to be confirmed with additional inactive siRNA, indicate that combination of siRNA may not increase but instead decrease silencing efficiency.     

Characterization of Bacterial Isolates Collected from a Sheep Model of Osseointegration

Percutaneous osseointegrated implant technology provides a potential alternative to current socket prosthetics for individuals with limb loss. However, similar to other percutaneous devices, there remain concerns of periprosthetic infection. To understand this process of infection, bacterial isolates were collected and characterized from a sheep model of osseointegration. CSA-13, a novel cationic steroid antimicrobial, was used at the skin/implant interface in an attempt to reduce the rate of infection. Results indicated that in this application, normal flora and environmental organisms continued to colonize the skin/implant interface as well as cause infection in the presence of CSA-13. Two factors are believed to have contributed to this outcome: the delivery of CSA-13 and the lack of a skin seal at the skin/implant interface, which would create a biological barrier to infection.     

Mechanical integrin stress and magnetic forces induce biological responses in mesenchymal stem cells which depend on environmental factors

The control of mesenchymal stem cells (MSC) by physical cues is of great interest in regenerative medicine. Because integrin receptors function as mechanotransducers, we applied drag forces to β1 integrins on the apical surface of adherent human MSC. In addition to mechanical forces, the technique we used involved also the exposure of the cells to an inhomogeneous magnetic field. In order to assess the influence of the substrate on cell adhesion, cells were cultured on plain tissue culture polystyrene (TCP) or on coated well plates, which allowed only adhesion to embedded fibronectin or RGD peptides. We found that the expression of collagen I, which is involved in osteogenesis, and VEGF, a factor which stimulates angiogenesis, increased as a result of short-term mechanical integrin stress. Whereas, collagen I expression was stimulated by mechanical forces when the cells were cultured on fibronectin and RGD peptides but not on TCP, VEGF expression was enhanced by physical stimulation on TCP. The study further revealed that magnetic forces enhanced Sox 9 expression, a marker of chondrogenesis, and reduced the expression of ALP. Concerning the intracellular mechanisms involved, we found that the expression of VEGF induced by physical forces depended on Akt activation. Together, the results implicate that biological functions of MSC can be stimulated by integrin-mediated mechanical forces and a magnetic field. However, the responses of cells depend strongly on the substrate to which they adhere and on the cross-talk between integrin-mediated signals and soluble factors.     

Iron defecation by sperm whales stimulates carbon export in the Southern Ocean

The iron-limited Southern Ocean plays an important role in regulating atmospheric CO₂ levels. Marine mammal respiration has been proposed to decrease the efficiency of the Southern Ocean biological pump by returning photosynthetically fixed carbon to the atmosphere. Here, we show that by consuming prey at depth and defecating iron-rich liquid faeces into the photic zone, sperm whales (Physeter macrocephalus) instead stimulate new primary production and carbon export to the deep ocean. We estimate that Southern Ocean sperm whales defecate 50 tonnes of iron into the photic zone each year. Molar ratios of C_export ∶Fe_added determined during natural ocean fertilization events are used to estimate the amount of carbon exported to the deep ocean in response to the iron defecated by sperm whales. We find that Southern Ocean sperm whales stimulate the export of 4 × 10⁵ tonnes of carbon per year to the deep ocean and respire only 2 × 10⁵ tonnes of carbon per year. By enhancing new primary production, the populations of 12 000 sperm whales in the Southern Ocean act as a carbon sink, removing 2 × 10⁵ tonnes more carbon from the atmosphere than they add during respiration. The ability of the Southern Ocean to act as a carbon sink may have been diminished by large-scale removal of sperm whales during industrial whaling.     

Factors Likely to Affect the Long-term Results of Ventricular Stimulation After Myocardial Infarction

Background

The results of programmed ventricular stimulation (PVS) may change after myocardial infarction (MI). The objective was to study the factors that could predict the results of a second PVS.

Methods

Left ventricular ejection fraction (LVEF) and QRS duration were determined and PVS performed within 3 to 14 years of one another (mean 7.5±5) in 50 patients studied systematically between 1 and 3 months after acute MI.

Results

QRS duration increased from 120±23 ms to 132±29 (p 0.04). LVEF did not decrease significantly (36±12 % vs 37±13 %). Ventricular tachycardia with cycle length (CL) > 220ms (VT) was induced in 11 patients at PVS 1, who had inducible VT with a CL > 220 ms (8) or < 220 ms (ventricular flutter, VFl) (3) at PVS 2. VFl or fibrillation (VF) was induced in 14 patients at PVS 1 and remained inducible in 5; 5 patients had inducible VT and 4 had a negative 2nd PVS. 2. 25 patients had initially negative PVS; 7 had secondarily inducible VT, 4 a VFl/VF, 14 a negative PVS. Changes of PVS were related to initially increasing QRS duration and secondarily changes in LVEF and revascularization but not to the number of extrastimuli required to induce VFl.

Conclusions

In patients without induced VT at first study, changes of PVS are possible during the life. Patients with initially long QRS duration and those who developed decreased LVEF are more at risk to have inducible monomorphic VT at 2nd study, than other patients.