Transfection of primary brain capillary endothelial cells for protein synthesis and secretion of recombinant erythropoietin: a strategy to enable protein delivery to the brain

Treatment of chronic disorders affecting the central nervous system (CNS) is complicated by the inability of drugs to cross the blood–brain barrier (BBB). Non-viral gene therapy applied to brain capillary endothelial cells (BCECs) denotes a novel approach to overcome the restraints in this passage, as turning BCECs into recombinant protein factories by transfection could result in protein secretion further into the brain. The present study aims to investigate the possibility of transfecting primary rat brain endothelial cells (RBECs) for recombinant protein synthesis and secretion of the neuroprotective protein erythropoietin (EPO). We previously showed that 4% of RBECs with BBB properties can be transfected without disrupting the BBB integrity in vitro, but it can be questioned whether this is sufficient to enable protein secretion at therapeutic levels. The present study examined various transfection vectors, with regard to increasing the transfection efficiency without disrupting the BBB integrity. Lipofectamine 3000? was the most potent vector compared to polyethylenimine (PEI) and Turbofect. When co-cultured with astrocytes, the genetically modified RBECs secreted recombinant EPO into the cell culture medium both luminally and abluminally, and despite lower levels of EPO reaching the abluminal chamber, the amount of recombinant EPO was sufficient to evolve a biological effect on astrocytes cultured at the abluminal side in terms of upregulated gene expression of brain-derived neurotropic factor (BDNF). In conclusion, non-viral gene therapy to RBECs leads to protein secretion and signifies a method for therapeutic proteins to target cells inside the CNS otherwise omitted due to the BBB.     

Low-Mach-number turbulence in interstellar gas revealed by radio polarization gradients

The interstellar medium of the Milky Way is multiphase, magnetized and turbulent. Turbulence in the interstellar medium produces a global cascade of random gas motions, spanning scales ranging from 100 parsecs to 1,000 kilometres (ref. 4). Fundamental parameters of interstellar turbulence such as the sonic Mach number (the speed of sound) have been difficult to determine, because observations have lacked the sensitivity and resolution to image the small-scale structure associated with turbulent motion. Observations of linear polarization and Faraday rotation in radio emission from the Milky Way have identified unusual polarized structures that often have no counterparts in the total radiation intensity or at other wavelengths, and whose physical significance has been unclear. Here we report that the gradient of the Stokes vector (Q, U), where Q and U are parameters describing the polarization state of radiation, provides an image of magnetized turbulence in diffuse, ionized gas, manifested as a complex filamentary web of discontinuities in gas density and magnetic field. Through comparison with simulations, we demonstrate that turbulence in the warm, ionized medium has a relatively low sonic Mach number, M(s)???2. The development of statistical tools for the analysis of polarization gradients will allow accurate determinations of the Mach number, Reynolds number and magnetic field strength in interstellar turbulence over a wide range of conditions.     

A nuclear Argonaute promotes multigenerational epigenetic inheritance and germline immortality

Epigenetic information is frequently erased near the start of each new generation. In some cases, however, epigenetic information can be transmitted from parent to progeny (multigenerational epigenetic inheritance). A particularly notable example of this type of epigenetic inheritance is double-stranded RNA-mediated gene silencing in Caenorhabditis elegans. This RNA-mediated interference (RNAi) can be inherited for more than five generations. To understand this process, here we conduct a genetic screen for nematodes defective in transmitting RNAi silencing signals to future generations. This screen identified the heritable RNAi defective 1 (hrde-1) gene. hrde-1 encodes an Argonaute protein that associates with small interfering RNAs in the germ cells of progeny of animals exposed to double-stranded RNA. In the nuclei of these germ cells, HRDE-1 engages the nuclear RNAi defective pathway to direct the trimethylation of histone H3 at Lys?9 (H3K9me3) at RNAi-targeted genomic loci and promote RNAi inheritance. Under normal growth conditions, HRDE-1 associates with endogenously expressed short interfering RNAs, which direct nuclear gene silencing in germ cells. In hrde-1- or nuclear RNAi-deficient animals, germline silencing is lost over generational time. Concurrently, these animals exhibit steadily worsening defects in gamete formation and function that ultimately lead to sterility. These results establish that the Argonaute protein HRDE-1 directs gene-silencing events in germ-cell nuclei that drive multigenerational RNAi inheritance and promote immortality of the germ-cell lineage. We propose that C. elegans use the RNAi inheritance machinery to transmit epigenetic information, accrued by past generations, into future generations to regulate important biological processes.     

Changes in Greenland ice sheet elevation attributed primarily to snow accumulation variability

The response of grounded ice sheets to a changing climate critically influences possible future changes in sea level. Recent satellite surveys over southern Greenland show little overall elevation change at higher elevations, but large spatial variability. Using satellite studies alone, it is not possible to determine the geophysical processes responsible for the observed elevation changes and to decide if recent rates of change exceed the natural variability. Here we derive changes in ice-sheet elevation in southern Greenland, for the years 1978-88, using a physically based model of firn densification and records of annual snow accumulation reconstructed from 12 ice cores at high elevation. Our patterns of accumulation-driven elevation change agree closely with contemporaneous satellite measurements of ice-sheet elevation change, and we therefore attribute the changes observed in 1978-88 to variability in snow accumulation. Similar analyses of longer ice-core records show that in this decade the Greenland ice sheet exhibited typical variability at high elevations, well within the long-term natural variability. Our results indicate that a better understanding of ice-sheet mass changes will require long-term measurements of both surface elevation and snow accumulation.