Development of new plasmid DNA vaccine vectors with R1-based replicons

ABSTRACT: BACKGROUND: There has been renewed interest in biopharmaceuticals based on plasmid DNA (pDNA) in recent years due to the approval of several veterinary DNA vaccines, on-going clinical trials of human pDNA-based therapies, and significant advances in adjuvants and delivery vehicles that have helped overcome earlier efficacy deficits. With this interest comes the need for high-yield, cost-effective manufacturing processes. To this end, vector engineering is one promising strategy to improve plasmid production. RESULTS: In this work, we have constructed a new DNA vaccine vector, pDMB02-GFP, containing the runaway R1 origin of replication. The runaway replication phenotype should result in plasmid copy number amplification after a temperature shift from 30degreesC to 42degreesC. However, using Escherichia coli DH5alpha as a host, we observed that the highest yields of pDMB02-GFP were achieved during constant-temperature culture at 30degreesC, with a maximum yield of approximately 19 mg pDNA/g DCW being observed. By measuring mRNA and protein levels of the R1 replication initiator protein, RepA, we determined that RepA may be limiting pDMB02-GFP yield at 42degreesC. A mutant plasmid, pDMB-ATG, was constructed by changing the repA start codon from the sub-optimal GTG to ATG. In cultures of DH5alpha[pDMB-ATG], temperature-induced plasmid amplification was more dramatic than that observed with pDMB02-GFP, and RepA protein was detectable for several hours longer than in cultures of pDMB02-GFP at 42degreesC. CONCLUSIONS: Overall, we have demonstrated that R1-based plasmids can produce high yields of high-quality pDNA without the need for a temperature shift, and have laid the groundwork for further investigation of this class of vectors in the context of plasmid DNA production.     

Mitochondrial DNA structure and colony expansion dynamics of New Zealand fur seals (Arctocephalus forsteri) around Banks Peninsula

New Zealand fur seals are one of many pinniped species that survived the commercial sealing of the eighteenth and nineteenth centuries in dangerously low numbers. After the enforcement of a series of protection measures in the early twentieth century, New Zealand fur seals began to recover from the brink of extinction. We examined the New Zealand fur seal populations of Banks Peninsula, South Island, New Zealand using the mitochondrial DNA control region. We identified a panmictic population structure around Banks Peninsula. The most abundant haplotype in the area showed a slight significant aggregated structure. The Horseshoe Bay colony showed the least number of shared haplotypes with other colonies, suggesting a different origin of re-colonisation of this specific colony. The effective population size of the New Zealand fur seal population at Banks Peninsula was estimated at approximately 2500 individuals. The exponential population growth rate parameter for the area was 35, which corresponds to an expanding population. In general, samples from adjacent colonies shared 4.4 haplotypes while samples collected from colonies separated by between five and eight bays shared 1.9 haplotypes. The genetic data support the spill-over dynamics of colony expansion already suggested for this species. Approximate Bayesian computations analysis suggests re-colonisation of the area from two main clades identified across New Zealand with a most likely admixture coefficient of 0.41 to form the Banks Peninsula population. Approximate Bayesian computations analysis estimated a founder population size of approximately 372 breeding individuals for the area, which then rapidly increased in size with successive waves of external recruitment. The population of fur seals in the area is probably in the late phase of maturity in the colony expansion dynamic.     

Role for the Epidermal Growth Factor Receptor in Chemotherapy-Induced Alopecia

Treatment of cancer patients with chemotherapeutics like cyclophosphamide often causes alopecia as a result of premature and aberrant catagen. Because the epidermal growth factor receptor (EGFR) signals anagen hair follicles to enter catagen, we hypothesized that EGFR signaling may be involved in cyclophosphamide-induced alopecia. To test this hypothesis, skin-targeted Egfr mutant mice were generated by crossing floxed Egfr and Keratin 14 promoter-driven Cre recombinase mice. Cyclophosphamide treatment of control mice resulted in alopecia while Egfr mutant skin was resistant to cyclophosphamide-induced alopecia. Egfr mutant skin entered catagen normally, as indicated by dermal papilla condensation and decreased follicular proliferation, but did not progress to telogen as did Egfr wild type follicles. Egfr mutant follicles responded with less proliferation, apoptosis, and fewer p53-positive cells after cyclophosphamide. Treatment of control mice with the EGFR inhibitors erlotinib or gefitinib similarly suppressed alopecia and catagen progression by cyclophosphamide. Secondary analysis of clinical trials utilizing EGFR-targeted therapies and alopecia-inducing chemotherapy also revealed evidence for involvement of EGFR in chemotherapy-induced alopecia. Taken together, our results demonstrated the involvement of EGFR signaling in chemotherapy-induced alopecia, which will help in the design of novel therapeutic regimens to minimize chemotherapy-induced alopecia.     

Biogeography of the Purple Nonsulfur Bacterium Rhodopseudomonas palustris

The biogeography of the purple nonsulfur bacterium Rhodopseudomonas palustris on a local scale was investigated. Thirty clones of phototrophic bacteria were isolated from each of five unevenly spaced sampling locations in freshwater marsh sediments along a linear 10-m transect, and a total of 150 clones were characterized by BOX-PCR genomic DNA fingerprinting. Cluster analysis of 150 genomic fingerprints yielded 26 distinct genotypes, and 106 clones constituted four major genotypes that were repeatedly isolated. Representatives of these four major genotypes were tentatively identified as R. palustris based on phylogentic analyses of 16S rRNA gene sequences. The differences in the genomic fingerprint patterns among the four major genotypes were accompanied by differences in phenotypic characteristics. These phenotypic differences included differences in the kinetics of carbon source use, suggesting that there may be functional differences with possible ecological significance among these clonal linages. Morisita-Horn similarity coefficients (C_MH), which were used to compare the numbers of common genotypes found at pairs of sampling locations, showed that there was substantial similarity between locations that were 1 cm apart (C_MH, ≥0.95) but there was almost no similarity between locations that were ≥9 m apart (C_MH, ≤0.25). These calculations showed there was a gradual decrease in similarity among the five locations as a function of distance and that clones of R. palustris were lognormally distributed along the linear 10-m transect. These data indicate that natural populations of R. palustris are assemblages of genetically distinct ecotypes and that the distribution of each ecotype is patchy.     

Compartmentation of glutamate metabolism in brain. Evidence for the existence of two different tricarboxylic acid cycles in brain

1. (14)C from [1-(14)C]glucose injected intraperitoneally into mice is incorporated into glutamate, aspartate and glutamine in the brain to a much greater extent than (14)C from [2-(14)C]glucose. This difference for [1-(14)C]glucose and [2-(14)C]glucose increases with time. The amount of (14)C in C-1 of glutamate increases steadily with time with both precursors. It is suggested that a large part of the glutamate and aspartate pools in brain are in close contact with intermediates of a fast-turning tricarboxylic acid cycle. 2. (14)C from [1-(14)C]acetate and [2-(14)C]acetate is incorporated to a much larger extent into glutamine than into glutamate. An examination of the time-course of (14)C incorporated into glutamine and glutamate reveals that glutamine is not formed from the glutamate pool, labelled extensively by glucose, but from a small glutamate pool. This small glutamate pool is not derived from an intermediate of a fast-turning tricarboxylic acid cycle. 3. It is proposed that two different tricarboxylic acid cycles exist in brain.     

Spatial and temporal changes in microbial community structure associated with recharge-influenced chemical gradients in a contaminated aquifer

In a contaminated water-table aquifer, we related microbial community structure on aquifer sediments to gradients in 24 geochemical and contaminant variables at five depths, under three recharge conditions. Community amplified ribsosomal DNA restriction analysis (ARDRA) using universal 16S rDNA primers and denaturing gradient gel electrophoresis (DGGE) using bacterial 16S rDNA primers indicated: (i). communities in the anoxic, contaminated central zone were similar regardless of recharge; (ii). after recharge, communities at greatest depth were similar to those in uncontaminated zones; and (iii). after extended lack of recharge, communities at upper and lower aquifer margins differed from communities at the same depths on other dates. General aquifer geochemistry was as important as contaminant or terminal electron accepting process (TEAP) chemistry in discriminant analysis of community groups. The Shannon index of diversity (H) and the evenness index (E), based on DGGE operational taxonomic units (OTUs), were statistically different across community groups and aquifer depths. Archaea or sulphate-reducing bacteria 16S rRNA abundance was not clearly correlated with TEAP chemistry indicative of methanogenesis or sulphate reduction. Eukarya rRNA abundance varied by depth and date from 0 to 13% of the microbial community. This contaminated aquifer is a dynamic ecosystem, with complex interactions between physical, chemical and biotic components, which should be considered in the interpretation of aquifer geochemistry and in the development of conceptual or predictive models for natural attenuation or remediation.     

Inferring Cetacean Population Densities from the Absolute Dynamic Topography of the Ocean in a Hierarchical Bayesian Framework

We inferred the population densities of blue whales (Balaenoptera musculus) and short-beaked common dolphins (Delphinus delphis) in the Northeast Pacific Ocean as functions of the water-column’s physical structure by implementing hierarchical models in a Bayesian framework. This approach allowed us to propagate the uncertainty of the field observations into the inference of species-habitat relationships and to generate spatially explicit population density predictions with reduced effects of sampling heterogeneity. Our hypothesis was that the large-scale spatial distributions of these two cetacean species respond primarily to ecological processes resulting from shoaling and outcropping of the pycnocline in regions of wind-forced upwelling and eddy-like circulation. Physically, these processes affect the thermodynamic balance of the water column, decreasing its volume and thus the height of the absolute dynamic topography (ADT). Biologically, they lead to elevated primary productivity and persistent aggregation of low-trophic-level prey. Unlike other remotely sensed variables, ADT provides information about the structure of the entire water column and it is also routinely measured at high spatial-temporal resolution by satellite altimeters with uniform global coverage. Our models provide spatially explicit population density predictions for both species, even in areas where the pycnocline shoals but does not outcrop (e.g. the Costa Rica Dome and the North Equatorial Countercurrent thermocline ridge). Interannual variations in distribution during El Niño anomalies suggest that the population density of both species decreases dramatically in the Equatorial Cold Tongue and the Costa Rica Dome, and that their distributions retract to particular areas that remain productive, such as the more oceanic waters in the central California Current System, the northern Gulf of California, the North Equatorial Countercurrent thermocline ridge, and the more southern portion of the Humboldt Current System. We posit that such reductions in available foraging habitats during climatic disturbances could incur high energetic costs on these populations, ultimately affecting individual fitness and survival.