A Flexible Method to Study Neuronal Differentiation of Mouse Embryonic Stem Cells

Embryonic Stem Cells (ESCs) represent an invaluable tool for the study of early mammalian development, for regenerative medicine and for drug discovery. To fulfill these promises, efficient and easy protocols to differentiate ESCs have to be developed. Most of these protocols results in low efficiency of neural induction and/or requires extended in vitro culture. Here we describe in detail an easy and efficient method to differentiate ESCs into neurons, that can be used to identify molecules required for proper neuronal differentiation. Moreover, we present a modification of this method that allows to clearly evaluate the ability of some molecules to favor neuron formation in vitro. These methods can represent an efficient platform for studying the molecular mechanisms underlying early events of neural induction and differentiation in ESCs, as well as for testing molecule efficacy in the pharmaceutical testing.     

Semi‐field evaluation of a granulovirus and Bacillus thuringiensis ssp. kurstaki for season‐long control of the potato tuber moth,Phthorimaea operculella

There are few insecticidal options for potato tuber moth (PTM), Phthorimaea operculella Zeller (Lepidoptera: Gelechiidae), control late in the growing season. We evaluated the PTM granulovirus (PoGV) and Bacillus thuringiensis Berliner ssp. kurstaki (Btk) for season‐long control of PTM on potato foliage in 2006 and 2007. Compared to untreated controls, 10 weekly applications of PoGV (10¹³ occlusion bodies/ha) reduced PTM populations in replicated 1‐m³ field cages by 86–96% on pre‐harvest foliage and 90–97% on tubers added to cages shortly before harvest. Infection rates of 82–95% of L4 larvae by PoGV were noted within individual larval cohorts. Equivalently timed Btk treatments (1.12 kg product/ha) were significantly less effective at population suppression, with a 36–76% reduction in larvae recovered from tubers added to cages. A PoGV/Btk alternation was significantly more effective than Btk alone and as effective as PoGV in 2007, but not in 2006. There was some evidence that reduced rate PoGV treatments (10% rate or 50% application frequency) were less effective than the standard program. There were no treatment effects on percentage of tubers growing in the ground that were infested at harvest, which remained comparatively low at ≤8.1%. Bioassays were conducted to evaluate the residual activities of foliar deposits. Early‐season applications were highly effective for the first 24 h (≥93% mortality) with a steady decline in activity over 10 days. A second application, applied later in the season, showed similar patterns, although in this case Btk was less persistent than PoGV, whereas both agents provided significant larval mortality compared with controls over 14 days. Both PoGV and Btk provide alternatives to manage field infestations of PTM prior to harvest, thus reducing the risk of tuber infestations in storage.     

Trade-off between mobility and fitness in Cydia pomonella L. (Lepidoptera: Tortricidae)

Abstract.  1. The codling moth Cydia pomonella , one of the most important pest insects of apple fruit orchards worldwide, is regarded as a 'sedentary' insect, but the natural populations consist of both mobile and sedentary genotypes that display different dispersal capacity in the field. The current study investigated whether there is a fitness consequence of being mobile in this species by comparing two strains with contrasting levels of mobility obtained through bi-directional selection.
2. Female moths of the sedentary strain were significantly larger, laid more eggs during their lifespan, and both male and female moths of this strain lived longer, when compared with the mobile strain.
3. The age-specific fecundity of female moths from the sedentary strain was significantly greater, in particular between 3 and 5 days after emergence when the peaks of both oviposition and flight capacity occur, relative to the mobile moths, thereby leading to a greater reproductive function.
4. These differences resulted in different demographic parameters between the two strains. The sedentary strain had a significantly greater net reproductive rate ( R _o ) and a higher intrinsic rate of natural increase ( r _m ), although the generation time ( T ) was similar, hence requiring a shorter time to double the population size, in comparison to the mobile strain.
5. This study provides empirical evidence for the cost of being mobile in C. pomonella . Trade-offs between mobility and fitness might have contributed to the existence of genetic variance for dispersal behaviour in the natural populations and the maintenance of relative sedentariness in the species.     

Painting protein misfolding in the cell in real time with an atomic-scale brush

The direct observation of specific biochemical events in living cells is now possible as a result of combined advances in molecular biology and fluorescence microscopy. By genetically encoding the source of a unique spectroscopic signal, target proteins can be selectively detected within the complex cellular environment, with limited interference from background signals. A recent study takes advantage of arsenical reagent-based methodologies to monitor in vivo protein misfolding and inclusion body formation in real time. This approach promises to yield important information on the kinetics of aggregate formation in living cells and its relation to the time-course of protein expression and post-translational processing. The ability to follow protein self-association in real time accurately from its early stages is unique to this method, and has far-reaching implications for both biotechnology and misfolding-based disease.     

Transmembrane topogenesis of a tail-anchored protein is modulated by membrane lipid composition

A large class of proteins with cytosolic functional domains is anchored to selected intracellular membranes by a single hydrophobic segment close to the C-terminus. Although such tail-anchored (TA) proteins are numerous, diverse, and functionally important, the mechanism of their transmembrane insertion and the basis of their membrane selectivity remain unclear. To address this problem, we have developed a highly specific, sensitive, and quantitative in vitro assay for the proper membrane-spanning topology of a model TA protein, cytochrome b5 (b5). Selective depletion from membranes of components involved in cotranslational protein translocation had no effect on either the efficiency or topology of b5 insertion. Indeed, the kinetics of transmembrane insertion into protein-free phospholipid vesicles was the same as for native ER microsomes. Remarkably, loading of either liposomes or microsomes with cholesterol to levels found in other membranes of the secretory pathway sharply and reversibly inhibited b5 transmembrane insertion. These results identify the minimal requirements for transmembrane topogenesis of a TA protein and suggest that selectivity among various intracellular compartments can be imparted by differences in their lipid composition.     

Computational studies of shape memory alloy behavior in biomedical applications

BACKGROUND: Nowadays, shape memory alloys (SMAs) and in particular Ni-Ti alloys are commonly used in bioengineering applications as they join important qualities as resistance to corrosion, biocompatibility, fatigue resistance, MR compatibility, kink resistance with two unique thermo-mechanical behaviors: the shape memory effect and the pseudoelastic effect. They allow Ni-Ti devices to undergo large mechanically induced deformations and then to recover the original shape by thermal loading or simply by mechanical unloading. METHOD OF APPROACH: A numerical model is developed to catch the most significant SMA macroscopic thermo-mechanical properties and is implemented into a commercial finite element code to simulate the behavior of biomedical devices. RESULTS: The comparison between experimental and numerical response of an intravascular coronary stent allows to verify the model suitability to describe pseudo-elasticity. The numerical study of a spinal vertebrae spacer where the effects of different geometries and material characteristic temperatures are investigated, allows to verify the model suitability to describe shape memory effect. CONCLUSION: the results presented show the importance of computational studies in designing and optimizing new biomedical devices.     

Possible repair action of Vitamin C on DNA damage induced by methyl methanesulfonate, cyclophosphamide, FeSO4 and CuSO4 in mouse blood cells in vivo

Interaction between Vitamin C (VitC) and transition metals can induce the formation of reactive oxygen species (ROS). VitC may also act as an ROS scavenger and as a metal chelant. To examine these possibilities, we tested in vivo the effect of two doses of VitC (1 and 30 mg/kg of mouse body weight) on the genotoxicity of known mutagens and transition metals. We used the alkaline version of the comet assay to assess DNA damage in peripheral white blood cells of mice. Animals were orally given either water (control), cyclophosphamide (CP), methyl methanesulfonate (MMS), cupric sulfate or ferrous sulfate. A single treatment with each VitC dose was administered after treatment with the mutagens or the metal sulfates. Both doses of VitC enhanced DNA damage caused by the metal sulfates. DNA damage caused by MMS was significantly reduced by the lower dose, but not by the higher dose of VitC. For CP, neither post-treatment dose of VitC affected the DNA damage level. These results indicate a modulatory role of Vitamin C in the genotoxicity/repair effect of these compounds. Single treatment with either dose of VitC showed genotoxic effects after 24 h but not after 48 h, indicating repair. Double treatment with VitC (at 0 and 24 h) induced a cumulative genotoxic response at 48 h, more intense for the higher dose. The results suggest that VitC can be either genotoxic or a repair stimulant, since the alkaline version of the comet assay does not differentiate "effective" strand breaks from those generated as an intermediate step in excision repair (incomplete excision repair sites). Further data is needed to shed light upon the beneficial/noxious effects of VitC.