Alleviation of Murine Leukemia Virus Repression in Embryonic Carcinoma Cells by Genetically Engineered Primer Binding Sites and Artificial tRNA Primers

AbstractThe primer binding site (PBS) plays pivotal roles during reverse transcription of retroviruses and also is the target of a cellular host defense impeding the transcription of murine leukemia virus (MLV) harboring a proline (pro) PBS in embryonic cells. Both the PBS and the tRNA primer are copied during reverse transcription and anneal as complementary DNA sequences creating the PBS of the integrated provirus. The pro PBS of MLV can be exchanged by PBS sequences matching endogenous or engineered tRNAs to allow replication of Akv MLV-derived vectors in fibroblasts. Here we use the PBS escape mutant B2 to demonstrate the capacity of the synthetic tRNAB2 to function in reverse transcription in competition with endogenous tRNAs in fibroblasts and embryonic carcinoma (EC) cells. We further show symmetry between PBS and the primer by the ability of the synthetic tRNAB2 to confer escape from EC repression of a PBS-Pro vector. Of a panel of vectors with the repressed pro PBS substituted for other natural or artificial PBS sequences, all except one efficiently expressed the neo marker gene when transferred to NIH/3T3 and EC cells, hence avoiding PBS-mediated silencing in EC cells. A non-natural PBS matching an artificially designed tRNA molecule conferred no further relief from repression than that attained with the B2 escape mutant or the natural alternative PBSs. Interestingly, a vector harboring a PBS matching tRNALys1.2 suffered repression similar to the wild-type PBS-Pro but was partially rescued by a single point mutation of the PBS

Correlating microbial community profiles with geochemical data in highly stratified sediments from the Arctic Mid-Ocean Ridge

Microbial communities and their associated metabolic activity in marine sediments have a profound impact on global biogeochemical cycles. Their composition and structure are attributed to geochemical and physical factors, but finding direct correlations has remained a challenge. Here we show a significant statistical relationship between variation in geochemical composition and prokaryotic community structure within deep-sea sediments. We obtained comprehensive geochemical data from two gravity cores near the hydrothermal vent field Loki’s Castle at the Arctic Mid-Ocean Ridge, in the Norwegian- Greenland Sea. Geochemical properties in the rift valley sediments exhibited strong centimeter-scale stratigraphic variability. Microbial populations were profiled by pyrosequencing from 15 sediment horizons (59,364 16S rRNA gene tags), quantitatively assessed by qPCR, and phylogenetically analyzed. Although the same taxa were generally present in all samples, their relative abundances varied substantially among horizons and fluctuated between Bacteria- and Archaea-dominated communities. By independently summarizing covariance structures of the relative abundance data and geochemical data, using principal components analysis, we found a significant correlation between changes in geochemical composition and changes in community structure. Differences in organic carbon and mineralogy shaped the relative abundance of microbial taxa. We used correlations to build hypotheses about energy metabolisms, particularly of the Deep Sea Archaeal Group, specific Deltaproteobacteria, and sediment lineages of potentially anaerobic Marine Group I Archaea. We demonstrate that total prokaryotic community structure can be directly correlated to geochemistry within these sediments, thus enhancing our understanding of biogeochemical cycling and our ability to predict metabolisms of uncultured microbes in deep-sea sediments

Magneto-structural correlations in a family of ReIVCuII chains based on the hexachlororhenate(IV) metalloligand

Six novel one-dimensional chloro-bridged ReIVCuII complexes of formula {[Cu(L)4][ReCl6]}n are characterised structurally, magnetically and theoretically.</p

Magneto-structural correlations in dirhenium(IV) complexes possessing magnetic pathways with even or odd numbers of atoms.

Spin-polarization enforces either ferro- or antiferromagnetic exchange interactions between ReIV ions with odd or even numbers of intervening atoms.</p

Flexible Stamp for Nanoimprint Lithography

The design, fabrication and performance of a flexible silicon stamp for homogenous large area nanoimprint lithography (NIL) are presented. The flexible stamp is fabricated by bulk semiconductor micro machining of a 4-inch silicon wafer and consists of thick anchor like imprint areas connected by membranes. The bending stiffness difference between the imprint areas and the membranes ensures that the deformation of the stamp during the imprint process mainly takes place in the membranes, leaving the imprint structures unaffected. By this design the strong demand to the parallelism between stamp and substrate in the imprint situation is decoupled from the pressing tool and the wafer quality. The stamp consist of 1562 imprint areas (1 mm × 1 mm) containing the patterns to be replicated. The imprinted patterns are characterized with respect to the imprint depth and the polymer residual layer thickness. It is found that within a 50 mm diameter the polymer residual layer thickness is 18.8 nm with a standard deviation of 6.6 nm

Alterations in hypothalamic gene expression following Roux-en-Y gastric bypass

Objective: The role of the central nervous system in mediating metabolic effects of Roux-en-Y gastric bypass (RYGB) surgery is poorly understood. Using a rat model of RYGB, we aimed to identify changes in gene expression of key hypothalamic neuropeptides known to be involved in the regulation of energy balance. Methods: Lean male Sprague-Dawley rats underwent either RYGB or sham surgery. Body weight and food intake were monitored bi-weekly for 60 days post-surgery. In situ hybridization mRNA analysis of hypothalamic AgRP, NPY, CART, POMC and MCH was applied to RYGB and sham animals and compared with ad libitum fed and food-restricted rats. Furthermore, in situ hybridization mRNA analysis of dopaminergic transmission markers (TH and DAT) was applied in the midbrain. Results: RYGB surgery significantly reduced body weight and intake of a highly palatable diet but increased chow consumption compared with sham operated controls. In the arcuate nucleus, RYGB surgery increased mRNA levels of orexigenic AgRP and NPY, whereas no change was observed in anorexigenic CART and POMC mRNA levels. A similar pattern was seen in food-restricted versus ad libitum fed rats. In contrast to a significant increase of orexigenic MCH mRNA levels in food-restricted animals, RYGB did not change MCH expression in the lateral hypothalamus. In the VTA, RYGB surgery induced a reduction in mRNA levels of TH and DAT, whereas no changes were observed in the substantia nigra relative to sham surgery. Conclusion: RYGB surgery increases the mRNA levels of hunger-associated signaling markers in the rat arcuate nucleus without concomitantly increasing downstream MCH expression in the lateral hypothalamus, suggesting that RYGB surgery puts a brake on orexigenic hypothalamic output signals. In addition, down-regulation of midbrain TH and DAT expression suggests that altered dopaminergic activity also contributes to the reduced intake of palatable food in RYGB rats. Keywords: Roux-en-Y gastric bypass, Energy homeostasis, Hypothalamus, Hedonic, Mesolimbic pathwa

Molecular Basis of Enhanced Activity in Factor VIIa-Trypsin Variants Conveys Insights into Tissue Factor-mediated Allosteric Regulation of Factor VIIa Activity

The complex of coagulation factor VIIa (FVIIa), a trypsin-like serine protease, and membrane-bound tissue factor (TF) initiates blood coagulation upon vascular injury. Binding of TF to FVIIa promotes allosteric conformational changes in the FVIIa protease domain and improves its catalytic properties. Extensive studies have revealed two putative pathways for this allosteric communication. Here we provide further details of this allosteric communication by investigating FVIIa loop swap variants containing the 170 loop of trypsin that display TF-independent enhanced activity. Using x-ray crystallography, we show that the introduced 170 loop from trypsin directly interacts with the FVIIa active site, stabilizing segment 215–217 and activation loop 3, leading to enhanced activity. Molecular dynamics simulations and novel fluorescence quenching studies support that segment 215–217 conformation is pivotal to the enhanced activity of the FVIIa variants. We speculate that the allosteric regulation of FVIIa activity by TF binding follows a similar path in conjunction with protease domain N terminus insertion, suggesting a more complete molecular basis of TF-mediated allosteric enhancement of FVIIa activity

Tumor–associated antigens identified by mRNA expression profiling as tumor rejection epitopes

Thirteen H-2(b)-binding peptides derived from six potentially overexpressed proteins in p53(-/- )thymoma (SM7) cells were studied for immunogenecity and vaccine-induced prevention of tumor growth in mice inoculated with SM7 tumor cells. Six of the peptides generated specific CTL responses after immunization, but only two of these peptides (RAD(23–31 )and RAD(24–31)) were capable of generating a weak vaccination-induced protection against adoptive tumor growth. SM7 inoculated mice treated with a blocking antibody against the inhibitory T cell signal transducing molecule CTLA4 appeared to delay tumor take, suggesting that SM7 thymoma cells are recognized by the adaptive immune system of the host. However, prophylactic vaccination with RAD(23–31 )and RAD(24–31 )peptides combined with anti-CTLA4 Ab treatment and did not improve tumor resistance. Our data would indicate that vaccination with immunogenic peptides derived from potentially overexpressed tumor proteins, as identified by mRNA expression profiling of p53(-/- )thymoma cells, at best results in a weak tumor protection thus questioning this way of detection of new tumor rejection epitopes