372 research outputs found
Characterisation of the DNA Binding Domain of the Transcriptional Regulator Encoded by VZV Gene 62
Get PDFThe understanding of gene regulation during varicella-zoster virus (VZV) infection is still rudimentary, although insight can be gained from the more extensively studied system of the genetically related herpes simplex virus type 1 (HSV-1). Upon infection, HSV-1 expresses three main classes of genes in a coordinately regulated temporal cascade; the immediate-early (IE), early (E) and late (L) genes. The product of the HSV-1 IE3 gene, Vmwl75, is considered to be the major regulatory protein of HSV-1 because functional Vmwl75 is an absolute requirement for transactivation of E and L gene expression and for the down-regulation of its own IE3 promoter. The polypeptide encoded by VZV gene 62 (VZV 140k) is the sequence homologue of HSV-1 Vmwl75. Transient transfection experiments have demonstrated that VZV 140k is a potent and promiscuous transactivator of gene expression, and that it can also negatively autoregulate its own gene 62 promoter. In addition, VZV 140k can complement HSV-1 viruses lacking functional Vmwl75. Therefore, by analogy to its HSV-1 counterpart, VZV 140k is likely to play a critical role in the regulation of VZV gene expression. The research described in this Thesis aimed to investigate the properties and activities of the DNA binding domain of the VZV 140k protein, in order to gain a better understanding of the mechanisms of 140k-mediated gene regulation during infection. The primary sequences of VZV 140k and HSV-1 Vmwl75 have been divided into five co-linear regions on the basis of the extent of their predicted amino acid identity. The region 2 sequences are particularly highly conserved between the two proteins and the regulatory functions of Vmwl75 require the integrity of region 2. Furthermore, sequences spanning HSV-1 Vmwl75 region 2 constitute a physically separable, stable DNA binding domain. Many of the experiments in this study stemmed from the initial demonstration that the corresponding region of the VZV 140k protein also yields a sequence-specific DNA binding domain when expressed as a non-fusion polypeptide in bacteria. The bacterially expressed VZV 140k DNA binding domain specifically recognised multiple DNA sequences in the vicinity of its target promoters; the 140k binding sites identified within its own VZV gene 62 promoter may have a role in the negative autoregulatory function of the 140k protein. Several of the 140k recognition sites showed sequence similarity to the Vmwl75 consensus binding sequence, although gel retardation assays with a systematically mutagenised binding site found the VZV 140k DNA binding domain peptide to be less sequence-specific than the corresponding domain of HSV-1 Vmwl75. These and further differences that have been identified between the DNA binding activities of the VZV 140k and HSV-1 Vmwl75 DNA binding domains may explain the previously reported variations between the regulatory functions of 140k and Vmwl75. The minimal region of the VZV 140k protein required for sequence-specific DNA binding has been defined by a deletion analysis to within 162 residues, essentially comprising the highly conserved region 2. However, additional sequences from the C-terminal end of region 1 were necessary for the full DNA binding interaction, as determined by DNase I footprinting analysis. As such, the DNA binding domain of VZV 140k is larger than those reported for other transcriptional regulators (which are often less than 80 amino acids) and therefore it may constitute a novel type of DNA binding structure. A short sequence that exhibits intriguing homology to the conserved DNA recognition helix of the homeodomain DNA binding motif is found at the centre of the VZV 140k DNA binding domain. The alterations to the DNA binding interaction that resulted from the substitution of single amino acids within this region indicate its probable involvement in the DNA recognition by VZV 140k. Of particular note, mutation of a single lysine residue drastically reduced the DNA binding activity and destroyed the transactivation function of VZV 140k; this result emphasises the importance of DNA binding for VZV 140k-mediated gene regulation. The highly purified VZV 140k DNA binding domain was a stable dimer in solution, as demonstrated by gel filtration, glycerol gradient centrifugation and cross-linking techniques. The fact that the VZV 140k DNA binding domain binds to DNA as a dimer was indicated by gel retardation assays that showed novel protein-DNA complexes of intermediate mobility following in vitro co-translation of pairs of VZV 140k peptides of differing sizes. In addition, the VZV 140k and HSV-1 Vmwl75 DNA binding domain peptides readily heterodimerised on co-translation, which indicated that these two related domains have similar modes of dimerisation. In vitro co-translation of a wide range of truncation and insertion mutation versions of the VZV 140k and HSV-1 Vmwl75 DNA binding domains, followed by analysis of their dimerisation by co-immunoprecipitation and their DNA binding ability by gel retardation, has indicated that the structural requirements for dimerisation are lower than for the DNA binding interaction. Finally, construction of a hybrid Vmwl75 protein with the VZV 140k DNA binding domain replacing that of HSV-1 Vmwl75 has allowed the relatedness of the two domains to be assessed directly. The hybrid protein was functional in tissue culture transient transfection assays and furthermore, supported viral growth when expressed from a recombinant HSV-1 genome. The details of the regulatory activities of the hybrid protein in the transient assays implied that the characteristic DNA binding activities of the VZV 140k DNA binding domain play an important part in determining its specific regulatory functions
AFRL Recurve Mission Success, Loss, Diagnosis, and Lessons Learned
Get PDFSmall satellites are making leaps in capability and are more often successful than even 5 years ago. However, there are many practices and approaches that are fairly new and deserve consideration for their efficacy. AFRL’s Small Satellite Portfolio (SSP) has been researching both first-of-their-kind technology demonstrations and the practices/approaches for their implementation. This paper discusses those practices and their results on the recent flight mission Recurve.
The Portfolio, somewhat uniquely, has the ability to execute flight demonstrations starting from concept and ending in operations, literally under one roof. Recurve was comprised of in-house developed and vendor supplied subsystems; in-house flight software and ground data systems; vendor supplied ground antennas; and an in-house assembly, test, and operations campaign. The team had significant influence over the implementation details and mission practices. This influence led to visibility into how decisions impacted execution. This mission execution enabled not only on-orbit experimentation, but also direct application of past lessons learned, which allowed for experimental systems engineering and project management approaches. Experimenting with in-house practices and methodologies resulted in a successful mission campaign and validated many design philosophies. One such philosophy is the Portfolio’s approach towards risk management, operating under a higher risk acceptance posture than many DoD missions. This includes a scalable scope based upon budget/schedule constraints and a higher risk tolerance with specific regards to component selection and iterative testing campaigns.
Advantages, lessons learned, and improvements to the practices employed are discussed. Further, the paper also discusses how some of those decisions led to the early end of the on-orbit mission, likely from radiation effects, after about 2 months of operations. Finally, this paper balances preliminary alterations to those practices against risk acceptance
Myogenic Progenitor Cells Control Extracellular Matrix Production by Fibroblasts during Skeletal Muscle Hypertrophy
Get PDFSatellite cells, the predominant stem cell population in adult skeletal muscle, are activated in response to hypertrophic stimuli and give rise to myogenic progenitor cells (MPCs) within the extracellular matrix (ECM) that surrounds myofibers. This ECM is composed largely of collagens secreted by interstitial fibrogenic cells, which influence satellite cell activity and muscle repair during hypertrophy and aging. Here we show that MPCs interact with interstitial fibrogenic cells to ensure proper ECM deposition and optimal muscle remodeling in response to hypertrophic stimuli. MPC-dependent ECM remodeling during the first week of a growth stimulus is sufficient to ensure long-term myofiber hypertrophy. MPCs secrete exosomes containing miR-206, which represses Rrbp1, a master regulator of collagen biosynthesis, in fibrogenic cells to prevent excessive ECM deposition. These findings provide insights into how skeletal stem and progenitor cells interact with other cell types to actively regulate their extracellular environments for tissue maintenance and adaptation
Downtown Providence Farmers Market Design
Get PDFThis market essentially combines a marketplace with an existing or new industrial building. The hybrid created results in space that has daily retail sales and hours, as well as other goods available at other times. The industrial aspect typically acts as the anchor, as the market’s activities help attract a different group of consumers at other business times. An example of this would be a seafood supply warehouse that also provides dining and entertainment as a nightlife destination
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