Generation of cell lines to complement Adenovirus vectors using recombination-mediated cassette exchange

Background Adenovirus serotype 5 (Ad5) has many favourable characteristics for development as a gene therapy vector. However, the utility of current Ad5 vectors is limited by transient transgene expression, toxicity and immunogenicity. The most promising form of vector is the high capacity type, which is deleted for all viral genes. However, these vectors can only be produced to relatively low titres and with the aid of helper virus. Therefore a continuing challenge is the generation of more effective Ad5 vectors that can still be grown to high titres. Our approach is to generate complementing cell lines to support the growth of Ad5 vectors with novel late gene deficiencies. Results We have used LoxP/Cre recombination mediated cassette exchange (RMCE) to generate cell lines expressing Ad5 proteins encoded by the L4 region of the genome, the products of which play a pivotal role in the expression of Ad5 structural proteins. A panel of LoxP parent 293 cell lines was generated, each containing a GFP expression cassette under the control of a tetracycline-regulated promoter inserted at a random genome location; the cassette also contained a LoxP site between the promoter and GFP sequence. Clones displayed a variety of patterns of regulation, stability and level of GFP expression. Clone A1 was identified as a suitable parent for creation of inducible cell lines because of the tight inducibility and stability of its GFP expression. Using LoxP-targeted, Cre recombinase-mediated insertion of an L4 cassette to displace GFP from the regulated promoter in this parent clone, cell line A1-L4 was generated. This cell line expressed L4 100K, 22K and 33K proteins at levels sufficient to complement L4-33K mutant and L4-deleted viruses. Conclusions RMCE provides a method for rapid generation of Ad5 complementing cell lines from a pre-selected parental cell line, chosen for its desirable transgene expression characteristics. Parent cell lines can be selected for high or low gene expression, and for tight regulation, allowing viral protein expression to mirror that found during infection. Cell lines derived from a single parent will allow the growth of different vectors to be assessed without the complication of varying complementing protein expression

Accessing Rare Heterocycles from Alkynyl Ethers and Nitrogenous Electrophiles & The Development of Small Molecule Inhibitors Against Naegleria \u3ci\u3efowleri\u3c/i\u3e infection

https://tigerprints.clemson.edu/csrp/1002/thumbnail.jp

Diffusion measurements to understand dynamics and structuring in solutions involving a homologous series of ionic liquids

The self-diffusion coefficients of each of the components in mixtures containing pyridine and each of the homologous series 1-alkyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imides in acetonitrile were determined using NMR diffusometry (i. e., Pulsed Gradient Spin Echo). The nature of solvation was found to change significantly with the proportion of salt in the mixtures. Increased diffusion coefficients (when corrected for viscosity) for the molecular components were observed with increasing proportion of ionic liquid and with increasing alkyl chain length on the cation. Comparison of the molecular solvents suggests increased interactions in solution of the pyridine with other components of the mixture, consistent with the proposed interactions shown previously to drive changes in reaction kinetics. Discontinuities were seen in the diffusion data for each species in solution across different ionic liquids between the hexyl and octyl derivatives, suggesting a change in the structuring in solution as the alkyl chain on the cation changes and demonstrating the importance of such when considering homologous series

Oil and Gas Map of the Middlesboro 30 x 60 Minute Quadrangle, Kentucky

The purpose of the oil and gas map series is to portray the distribution of oil and gas resources in a manner useful to the oil and gas industry and to others interested in subsurface research

Drug Interactions with Glutaredoxin Orthologues

Glutaredoxin, an enzymatic protein, is an important component of cell viability and function. It catalyzes reactions involved in DNA synthesis and innate immunity [1,4]. Glutaredoxin is also essential in antibiotic resistance in pathogenic bacterial species. Pseudomonas aeruginosa in particular is responsible for infecting the lung tissue of its human hosts, resulting in the development of pneumonia and cystic fibrosis [3]. Because glutaredoxin is pertinent in cell proliferation of eukaryotic and bacterial cells alike, medicinal fragments that take advantage of the subtle differences in protein structure of the orthologous proteins can be synthesized and enhanced to bind bacterial glutaredoxins, without inhibiting the function of the human form. This can be accomplished by exploiting the mechanisms of fragment based drug discovery using NMR techniques. A library of small potential medicinal fragments are screened against each protein to determine which interact, or bind most efficiently to the bacterial orthologues with little to no interaction with eukaryotic cells. To confirm the ability of select fragments hits to kill bacterial cells without harming human cells, MTT and MIC assays are performed to determine what concentration of fragment is needed to obtain desired therapeutic results [6,7]. These assays were performed against selected lead fragments, particularly RK207 and RK395, which can be structurally enhanced to bind even more to the bacterial orthologues. This research can potentially lead to the development of drug targets against bacterial orthologues of glutaredoxin to treat life threatening diseases caused by pathogenic species

Open access series of imaging studies: Longitudinal MRI data in nondemented and demented older adults

The Open Access Series of Imaging Studies (OASIS) is a series of neuroimaging data sets that is publicly available for study and analysis. The present MRI data set consists of a longitudinal collection of 150 subjects aged 60 to 96 all acquired on the same scanner using identical sequences. Each subject was scanned on two or more visits, separated by at least one year for a total of 373 imaging sessions. Subjects were characterized using the Clinical Dementia Rating (CDR) as either nondemented or with very mild to mild Alzheimer‘s disease (AD). 72 of the subjects were characterized as nondemented throughout the study. 64 of the included subjects were characterized as demented at the time of their initial visits and remained so for subsequent scans, including 51 individuals with CDR 0.5 similar level of impairment to individuals elsewhere considered to have ‘mild cognitive impairment’. Another 14 subjects were characterized as nondemented at the time of their initial visit (CDR 0) and were subsequently characterized as demented at a later visit (CDR > 0). The subjects were all right-handed and include both men (n=62) and women (n=88). For each scanning session, 3 or 4 individual T1-weighted MRI scans were obtained. Multiple within-session acquisitions provide extremely high contrast-to-noise making the data amenable to a wide range of analytic approaches including automated computational analysis. Automated calculation of whole brain volume is presented to demonstrate use of the data for measuring differences associated with normal aging and AD

Revealing the Population of Isolated, Massive Stars in the Central Molecular Zone

We report on the current census of isolated massive stars in the Galactic center region, including recently discovered objects. The latest discoveries were selected for their hard X-ray counterparts, detected with the Chandra X-ray Observatory; or for their Paschen-α emission-line excess, detected in narrowband images with HST/NICMOS. The confirmed stars span a wide range of massive star evolutionary stages: OIa, Of, Ofpe, LBV, WNh, WN, WNE, WC, and WCd. We suspect that the majority of the hard X-ray-emitting, massive stars we have identified are colliding-wind binaries, although some may be HMXBs. Most of the confirmed massive stars have no obvious association with the known, young stellar clusters, but some may have escaped from them. Extrapolation of their numbers suggests the existence of a massive star population, comparable in size to that contained within the clusters collectively, which could account for the integrated far-IR luminosity emerging from the central half-kiloparsec. This additional massive-star population may have been supplemented by the tidal disruption of stellar clusters, or suggests an alternate mode of isolated massive star formation operating in the Galactic center region. Future experiments will constrain their kinematics, binary characteristics, and mode of formation