Laminin induced local axonal translation of β-actin mRNA is impaired in SMN-deficient motoneurons

Reduced levels of the SMN (survival of motoneuron) protein cause spinal muscular atrophy, the main form of motoneuron disease in children and young adults. In cultured motoneurons, reduced SMN levels lead to disturbed axon growth that correlates with reduced actin mRNA and protein in growth cones, indicating that anterograde transport and local translation of β-actin mRNA are altered in this disease. However, it is not fully understood how local translation of the β-actin mRNA is regulated in SMN-deficient motoneurons. Here, we established a lentiviral GFP-based reporter construct to monitor local translation of β-actin mRNA. Time-lapse imaging of fluorescence recovery after photobleaching (FRAP) in living motoneurons revealed that β-actin is locally translated in the growth cones of embryonic motoneurons. Interestingly, local translation of the β-actin reporter construct was differentially regulated by various Laminin isoforms, indicating that Laminins provide extracellular cues for the regulation of local translation in growth cones. Notably, local translation of β-actin mRNA was deregulated in motoneurons from a mouse model for the most severe form of SMA (Smn ^?/? ;SMN2). Taken together our findings suggest that local translation of β-actin in growth cones of motoneurons is regulated by Laminin signalling and that this signalling is disturbed in SMA.     

Progressive degradation of serum samples limits proteomic biomarker discovery

Preanalytical variables play a key role in discovery of biomarkers. Although the effect of several preanalytical variables on the mass spectral profiles has been studied extensively, little is known about long-term storage of serum samples. This is important because samples used in case-control or epidemiological studies are usually stored for a long time before analysis. Here we evaluated long-term storage effects on mass spectral peak patterns of serum peptides extracted using weak cation exchange magnetic beads. For this, 20 serum samples stored at −80 °C were divided equally into two groups based on their storage time. We found that intensities of 26 mass spectral peaks significantly varied between these two groups. Intensities of these peaks significantly correlated with storage time. Genetic algorithm-based models generated using these 26 peaks could classify 63 additional samples into these two groups with 100% and 96% accuracy, respectively. We also show that storing samples for 10 months at −80 and −20 °C results in the appearance/disappearance or intensity variation of peaks, some of which were previously reported as disease biomarkers.     

Application of ultrasound to selectively localize nanodroplets for targeted imaging and therapy

Lipid-coated perfluorocarbon nanodroplets are submicrometer-diameter liquid-filled droplets with proposed applications in molecularly targeted therapeutics and ultrasound (US) imaging. Ultrasonic molecular imaging is unique in that the optimal application of these agents depends not only on the surface chemistry, but also on the applied US field, which can increase receptor-ligand binding and membrane fusion. Theory and experiments are combined to demonstrate the displacement of perfluorocarbon nanoparticles in the direction of US propagation, where a traveling US wave with a peak pressure on the order of megapascals and frequency in the megahertz range produces a particle translational velocity that is proportional to acoustic intensity and increases with increasing center frequency. Within a vessel with a diameter on the order of hundreds of micrometers or larger, particle velocity on the order of hundreds of micrometers per second is produced and the dominant mechanism for droplet displacement is shown to be bulk fluid streaming. A model for radiation force displacement of particles is developed and demonstrates that effective particle displacement should be feasible in the microvasculature. In a flowing system, acoustic manipulation of targeted droplets increases droplet retention. Additionally, we demonstrate the feasibility of US-enhanced particle internalization and therapeutic delivery.     

Nuclear localization and in situ DNA damage by Mycobacterium tuberculosis nucleoside-diphosphate kinase

Nucleoside-diphosphate kinase of Mycobacterium tuberculosis (mNdK) is a secretory protein, but the rationale behind secreting an enzyme involved in the maintenance of cellular pool of nucleoside triphosphates is not clearly understood. To elucidate the biological significance of mNdK secretion, we expressed mNdK fused to green fluorescent protein in HeLa and COS-1 cells. Interestingly, mNdK was detected in the nuclei of HeLa and COS-1 cells. Incubation of mNdK with nuclei isolated from HeLa and COS-1 cells led to in situ damage of chromosomal DNA. Surface plasmon resonance studies demonstrated that mNdK binds supercoiled plasmid DNA lacking apurinic/apyrimidinic sites with a dissociation constant of 30 +/- 3.2 mum. Plasmid cleavage by mNdK was found to be dependent on the specific divalent metal ion and inhibited by a metal ion chelator. Moreover, the metal ion-dependent DNA cleavage by mNdK was mediated by superoxide radicals as detected by electron paramagnetic resonance. The cleavage reaction was inhibited under nitrogen atmosphere confirming the necessity of molecular oxygen for DNA cleavage. In view of the findings that mNdK is secreted by intracellular mycobacteria and damages the nuclear DNA, it can be postulated that mNdK may cause cell death that could help in the dissemination of the pathogen.     

Nutritional evaluation of structured lipid containing omega 6 fatty acid synthesized from coconut oil in rats

Coconut oil is rich in medium chain fatty acids, but deficient in polyunsaturated fatty acids (PUFA). Structured lipids (SL) enriched with omega 6 PUFA were synthesized from coconut oil triglycerides by employing enzymatic acidolysis with free fatty acids obtained from safflower oil. Rats were fed a diet containing coconut oil, coconut oil-safflower oil blend (1:0.7 w/w) or structured lipid at 10% levels for a period of 60 days. The SL lowered serum cholesterol levels by 10.3 and 10.5% respectively in comparison with those fed coconut oil and blended oil. Similarly the liver cholesterol levels were also decreased by 35.9 and 26.6% respectively in animals fed structured lipids when compared to those fed on coconut oil or the blended oil. Most of the decrease observed in serum cholesterol levels of animals fed structured lipids was found in LDL fraction. The triglyceride levels in serum showed a decrease by 17.5 and 17.4% while in the liver it was reduced by 45.8 and 23.5% in the structured lipids fed animals as compared to those fed coconut oil or blended oil respectively. Differential scanning calorimetric studies indicated that structured lipids had lower melting points and solid fat content when compared to coconut oil or blended oils. These studies indicated that enrichment of coconut oil triglycerides with omega 6 fatty acids lowers its solid fat content. The omega 6 PUFA enriched structured lipids also exhibited hypolipidemic activity.     

Phosphoprotein phosphatase of Mycobacterium tuberculosis dephosphorylates serine-threonine kinases PknA and PknB

The regulation of cellular processes by the modulation of protein phosphorylation/dephosphorylation is fundamental to a large number of processes in living organisms. These processes are carried out by specific protein kinases and phosphatases. In this study, a previously uncharacterized gene (Rv0018c) of Mycobacterium tuberculosis, designated as mycobacterial Ser/Thr phosphatase (mstp), was cloned, expressed in Escherichia coli, and purified as a histidine-tagged protein. Purified protein (Mstp) dephosphorylated the phosphorylated Ser/Thr residues of myelin basic protein (MBP), histone, and casein but failed to dephosphorylate phospho-tyrosine residue of these substrates, suggesting that this phosphatase is specific for Ser/Thr residues. It has been suggested that mstp is a part of a gene cluster that also includes two Ser/Thr kinases pknA and pknB. We show that Mstp is a trans-membrane protein that dephosphorylates phosphorylated PknA and PknB. Southern blot analysis revealed that mstp is absent in the fast growing saprophytes Mycobacterium smegmatis and Mycobacterium fortuitum. PknA has been shown, whereas PknB has been proposed to play a role in cell division. The presence of mstp in slow growing mycobacterial species, its trans-membrane localization, and ability to dephosphorylate phosphorylated PknA and PknB implicates that Mstp may play a role in regulating cell division in M. tuberculosis.