Manipulation of cell cycle progression can counteract the apparent loss of correction frequency following oligonucleotide-directed gene repair

BACKGROUND: Single-stranded oligonucleotides (ssODN) are used routinely to direct specific base alterations within mammalian genomes that result in the restoration of a functional gene. Despite success with the technique, recent studies have revealed that following repair events, correction frequencies decrease as a function of time, possibly due to a sustained activation of damage response signals in corrected cells that lead to a selective stalling. In this study, we use thymidine to slow down the replication rate to enhance repair frequency and to maintain substantial levels of correction over time. RESULTS: First, we utilized thymidine to arrest cells in G1 and released the cells into S phase, at which point specific ssODNs direct the highest level of correction. Next, we devised a protocol in which cells are maintained in thymidine following the repair reaction, in which the replication is slowed in both corrected and non-corrected cells and the initial correction frequency is retained. We also present evidence that cells enter a senescence state upon prolonged treatment with thymidine but this passage can be avoided by removing thymidine at 48 hours. CONCLUSION: Taken together, we believe that thymidine may be used in a therapeutic fashion to enable the maintenance of high levels of treated cells bearing repaired genes

The Vice President-More than an Afterthought?

A round-table discussion among former U.S. Vice President Richard B. Cheney, Caruso Family Professor of Law and retired U.S. Ambassador Douglas Kmiec, and former U.S. Attorney General Edwin Meese III considered the practical implications of conceiving the Vice President as a legislative officer, an executive officer, or both. It was noted that until the second half of the twentieth century, the Office of the Vice President was conceived as legislative. Funding for the Office appeared in budget lines relating to Congress and physically, the Vice President’s office was in the Capitol. Beginning with Walter Mondale’s service as Vice President, presidents have been delegating increasing executive authority, seeing the Vice President as a “deputy president.” Perhaps the most aggressive and influential of the modern “deputy presidents” was Vice President Cheney himself. Attorney General Meese concurred and saw this as positive. Ambassador Kmiec was less approving, encouraging Vice President Cheney and Attorney General Meese to contemplate the benefits that a dual-natured legislative–executive Vice President supplies to maintaining a workable government. The capacity of the Vice President to assert independence, as late Justice Scalia explained in an Office of Legal Counsel opinion, is unique. Unlike members of the Cabinet, the Vice President is not removable by the President, and thus, the Vice President can use his dual nature to advance executive–legislative compromise. Vice President Cheney’s reliance upon his significant, but personal, legislative experience prior to his vice presidency to facilitate executive–legislative bargaining suggests qualities that presidential nominees might consider more directly in vice presidential selection, and not just geographic complementarity and ideological compatibility. While it has been commonplace to think of the vice presidential office as “an afterthought” borrowed from state charters at the time of the founding, this dialogue suggests how a vice president with a foot in each of the Legislative and Executive Branches can assist in overcoming dysfunctional periods when partisan division is great

Proliferation of genetically modified human cells on electrospun nanofiber scaffolds.

Gene editing is a process by which single base mutations can be corrected, in the context of the chromosome, using single-stranded oligodeoxynucleotides (ssODNs). The survival and proliferation of the corrected cells bearing modified genes, however, are impeded by a phenomenon known as reduced proliferation phenotype (RPP); this is a barrier to practical implementation. To overcome the RPP problem, we utilized nanofiber scaffolds as templates on which modified cells were allowed to recover, grow, and expand after gene editing. Here, we present evidence that some HCT116-19, bearing an integrated, mutated enhanced green fluorescent protein (eGFP) gene and corrected by gene editing, proliferate on polylysine or fibronectin-coated polycaprolactone (PCL) nanofiber scaffolds. In contrast, no cells from the same reaction protocol plated on both regular dish surfaces and polylysine (or fibronectin)-coated dish surfaces proliferate. Therefore, growing genetically modified (edited) cells on electrospun nanofiber scaffolds promotes the reversal of the RPP and increases the potential of gene editing as an ex vivo gene therapy application.Molecular Therapy - Nucleic Acids (2012) 1, e59; doi:10.1038/mtna.2012.51; published online 4 December 2012

Liquid Metal-Elastomer Soft Composites with Independently Controllable and Highly Tunable Droplet Size and Volume Loading

Soft composites are critical for soft and flexible materials in energy harvesting, actuators, and multifunctional devices. One emerging approach to create multifunctional composites is through the incorporation of liquid metal (LM) droplets such as eutectic gallium indium (EGaIn) in highly deformable elastomers. The microstructure of such systems is critical to their performance, however, current materials lack control of particle size at diverse volume loadings. Here, we present a fabrication approach to create liquid metal-elastomer composites with independently controllable and highly tunable droplet size (100 nm ≦ D ≦ 80 μm) and volume loading (0 ≦ φ ≦ 80%). This is achieved through a combination of shear mixing and sonication of concentrated LM/elastomer emulsions to control droplet size and subsequent dilution and homogenization to tune LM volume loading. These materials are characterized utilizing dielectric spectroscopy supported by analytical modeling which shows a high relative permittivity of 60 (16x the unfilled elastomer) in a composite with φ = 80%, a low tan δ of 0.02, and a significant dependence on φ and minor dependence on droplet size. Temperature response and stability are determined using dielectric spectroscopy through temperature and frequency sweeps and with DSC. These results demonstrate a wide temperature stability of the liquid metal phase (crystallizing \u3c -85 °C for D \u3c 20 μm). Additionally, all composites are electrically insulating across a wide frequency (0.1 Hz - 10 MHz) and temperature (-70°C to 100°C) range even up to φ = 80%. We highlight the benefit of LM microstructure control by creating all soft matter stretchable capacitive sensors with tunable sensitivity. These sensors are further integrated into a wearable sensing glove where we identify different objects during grasping motions. This work enables programmable LM composites for soft robotics and stretchable electronics where flexibility and tunable functional response are critical

All-In-One: Advanced preparation of Human Parenchymal and Non-Parenchymal Liver Cells

BACKGROUND & AIMS: Liver cells are key players in innate immunity. Thus, studying primary isolated liver cells is necessary for determining their role in liver physiology and pathophysiology. In particular, the quantity and quality of isolated cells are crucial to their function. Our aim was to isolate a large quantity of high-quality human parenchymal and non-parenchymal cells from a single liver specimen. METHODS: Hepatocytes, Kupffer cells, liver sinusoidal endothelial cells, and stellate cells were isolated from liver tissues by collagenase perfusion in combination with low-speed centrifugation, density gradient centrifugation, and magnetic-activated cell sorting. The purity and functionality of cultured cell populations were controlled by determining their morphology, discriminative cell marker expression, and functional activity. RESULTS: Cell preparation yielded the following cell counts per gram of liver tissue: 2.0+/-0.4x107 hepatocytes, 1.8+/-0.5x106 Kupffer cells, 4.3+/-1.9x105 liver sinusoidal endothelial cells, and 3.2+/-0.5x105 stellate cells. Hepatocytes were identified by albumin (95.5+/-1.7%) and exhibited time-dependent activity of cytochrome P450 enzymes. Kupffer cells expressed CD68 (94.5+/-1.2%) and exhibited phagocytic activity, as determined with 1mum latex beads. Endothelial cells were CD146+ (97.8+/-1.1%) and exhibited efficient uptake of acetylated low-density lipoprotein. Hepatic stellate cells were identified by the expression of alpha-smooth muscle actin (97.1+/-1.5%). These cells further exhibited retinol (vitamin A)-mediated autofluorescence. CONCLUSIONS: Our isolation procedure for primary parenchymal and non-parenchymal liver cells resulted in cell populations of high purity and quality, with retained physiological functionality in vitro. Thus, this system may provide a valuable tool for determining liver function and disease

Dynamics in sub-monolayer Fe-films

Abstract The properties of thin films are directly connected with the atomic structure. At elevated temperatures this structure is determined by atomic dynamics. Pronounced effects are expected for thin films of low coverage. We have investigated electronic and dynamical properties of a submonolayer Fe film on a W(1 1 0) substrate with nuclear resonance scattering (NRS) in grazing-incidence geometry. This atomistic technique is best suited for such investigations due to its element (isotopic) and submonolayer sensitivity as demonstrated in the model system of Fe/W(1 1 0). A simple relaxation model was used to explain the temperature dependence of the NRS spectra. The relaxation rates and diffusion coefficients have been calculated

Classification and evolutionary history of the single-strand annealing proteins, RecT, Redβ, ERF and RAD52

BACKGROUND: The DNA single-strand annealing proteins (SSAPs), such as RecT, Redβ, ERF and Rad52, function in RecA-dependent and RecA-independent DNA recombination pathways. Recently, they have been shown to form similar helical quaternary superstructures. However, despite the functional similarities between these diverse SSAPs, their actual evolutionary affinities are poorly understood. RESULTS: Using sensitive computational sequence analysis, we show that the RecT and Redβ proteins, along with several other bacterial proteins, form a distinct superfamily. The ERF and Rad52 families show no direct evolutionary relationship to these proteins and define novel superfamilies of their own. We identify several previously unknown members of each of these superfamilies and also report, for the first time, bacterial and viral homologs of Rad52. Additionally, we predict the presence of aberrant HhH modules in RAD52 that are likely to be involved in DNA-binding. Using the contextual information obtained from the analysis of gene neighborhoods, we provide evidence of the interaction of the bacterial members of each of these SSAP superfamilies with a similar set of DNA repair/recombination protein. These include different nucleases or Holliday junction resolvases, the ABC ATPase SbcC and the single-strand-binding protein. We also present evidence of independent assembly of some of the predicted operons encoding SSAPs and in situ displacement of functionally similar genes. CONCLUSIONS: There are three evolutionarily distinct superfamilies of SSAPs, namely the RecT/Redβ, ERF, and RAD52, that have different sequence conservation patterns and predicted folds. All these SSAPs appear to be primarily of bacteriophage origin and have been acquired by numerous phylogenetically distant cellular genomes. They generally occur in predicted operons encoding one or more of a set of conserved DNA recombination proteins that appear to be the principal functional partners of the SSAPs

Postnatal Development of Hepatic Innate Immune Response

The liver is an immunocompetent organ that plays a key role in the immune response to infections, and the development of hepatic immune function during early postnatal stages has not been thoroughly characterized. This study analyzed the constitutive expression of complement factors, namely C3 and C9, and pattern recognition receptors, namely CD14, toll-like receptor (TLR)-4, and lipopolysaccharide binding protein (LBP), in the liver of postnatal day (P)1, P21, and P70 rats, and compared the kinetics of induction of cytokines and chemokines in the liver of P 1 and P 21 animals. Our studies found that while the mRNA expression of C3, C9, CD14, and TLR-4 was lower in P1 animals, the mRNA level of LBP was higher in P1 animals as compared to older animals, and that the kinetics of induction of cytokines and chemokines was significantly delayed in P1 as compared to P21 liver following LPS stimulation. Our data suggest that hepatic innate immunity is deficient in the neonates and undergo significant development during early postnatal life