Phylogeographic genomics of mitochondrial DNA: Highly-resolved patterns of intraspecific evolution and a multi-species, microarray-based DNA sequencing strategy for biodiversity studies

Phylogeographic genomics, based on multiple complete mtDNA genome sequences from within individual vertebrate species, provides highly-resolved intraspecific trees for the detailed study of evolutionary biology. We describe new biogeographic and historical insights from our studies of the genomes of codfish, wolffish, and harp seal populations in the Northwest Atlantic, and from the descendants of the founding human population of Newfoundland. Population genomics by conventional sequencing methods remains laborious. A new biotechnology, iterative DNA “re-sequencing”, uses a DNA microarray to recover 30–300 kb of contiguous DNA sequence in a single experiment. Experiments with a single-species mtDNA microarray show that the method is accurate and efficient, and sufficiently species-specific to discriminate mtDNA genomes of moderately-divergent taxa. Experiments with a multi-species DNA microarray (the “ArkChip”) show that simultaneous sequencing of species in different orders and classes detects SNPs within each taxon with equal accuracy as single-species-specific experiments. Iterative DNA sequencing offers a practical method for high-throughput biodiversity genomics that will enable standardized, coordinated investigation of multiple species of interest to Species at Risk and conservation biologists.     

The effect of Fc glycan forms on human IgG2 antibody clearance in humans

Several studies using a variety of approaches have investigated the impact of the Fc glycan structure on IgG clearance rates. Most, but not all, of these studies have concluded that glycan structural differences do not affect clearance. Here we investigated the impact of glycan on the clearance of a human antibody in humans. To monitor glycan-dependent changes, a human IgG2 was affinity purified in a single step from serum samples from a human pharmacokinetic study. The glycan profile from the purified antibody samples was determined by RP-HPLC/MS analysis of the 2-aminobenzamide-labeled glycans. Relative levels of high-mannose species (M6-M9) decreased over circulation time. Differences in the individual high-mannose structural isoform clearance rates were measured from extracted ion current profiles. Similar changes to the glycan profile could be achieved through incubation of the antibody in serum in vitro, suggesting that the changes observed in vivo were the result of glycan cleavage, not differential antibody clearance. These results confirm that antibody clearance is not significantly affected by the Fc glycan structure and provide evidence for the presence of circulating mannosidase activity in humans.     

Studies on a series of potent,orally bioavailable, 5-HT1 receptor ligands—Part II

A series of 5-(piperidinylethyloxy)quinoline 5-HT₁ receptor ligands have been studied by elaboration of the series of dual 5-HT₁-SSRIs reported previously. These new compounds display a different in vitro pharmacological profile with potent affinity across the 5-HT_1A, 5-HT_1B and 5-HT_1D receptors and selectivity against the serotonin transporter. Furthermore, they have improved pharmacokinetic profiles and CNS penetration.     

Pipeline transport and simultaneous saccharification of corn stover

Pipeline transport of corn stover delivered by truck from the field is evaluated against a range of truck transport costs. Corn stover transported by pipeline at 20% solids concentration (wet basis) or higher could directly enter an ethanol fermentation plant, and hence the investment in the pipeline inlet end processing facilities displaces comparable investment in the plant. At 20% solids, pipeline transport of corn stover costs less than trucking at capacities in excess of 1.4 M drytonnes/yr when compared to a mid range of truck transport cost (excluding any credit for economies of scale achieved in the ethanol fermentation plant from larger scale due to multiple pipelines). Pipelining of corn stover gives the opportunity to conduct simultaneous transport and saccharification (STS). If current enzymes are used, this would require elevated temperature. Heating of the slurry for STS, which in a fermentation plant is achieved from waste heat, is a significant cost element (more than 5 cents/l of ethanol) if done at the pipeline inlet unless waste heat is available, for example from an electric power plant located adjacent to the pipeline inlet. Heat loss in a 1.26 m pipeline carrying 2 M drytonnes/yr is about 5 degrees C at a distance of 400 km in typical prairie clay soils, and would not likely require insulation; smaller pipelines or different soil conditions might require insulation for STS. Saccharification in the pipeline would reduce the need for investment in the fermentation plant, saving about 0.2 cents/l of ethanol. Transport of corn stover in multiple pipelines offers the opportunity to develop a large ethanol fermentation plant, avoiding some of the diseconomies of scale that arise from smaller plants whose capacities are limited by issues of truck congestion.     

Technical note: Modeling primate occlusal topography using geographic information systems technology

Most functional analyses of primate tooth form have been limited to linear or area measurements. Such studies have offered but a limited glimpse at differences in occlusal relief among taxa. Such differences in dental topography may relate to tooth function and, so, have considerable implications for the inference of diet from fossil teeth. In this article, we describe a technique to model and compare primate molars in three dimensions using Geographic Resources Analysis Support System (GRASS) software. We examine unworn lower second molars of three extant hominoids with known differences in diet (Gorilla gorilla, Pan troglodytes, and Pongo pygmaeus), and two fossil forms, (Afropithecus turkanesis and Dryopithecus laietanus). First, we obtained approximately 400 landmarks on the occlusal surfaces of each tooth using an electromagnetic digitizer. Raster “terrain models” of occlusal surfaces were then created by interpolation of the coordinate data. We used GRASS terrain analysis automated techniques to quantify the volumes and slopes of individual cusps. We also used the GRASS watershed technique to identify the volume of liquid that would accumulate in each tooth's basin (a measure of basin area), and the directions and intensity of drainage over the occlusal surface. In sum, GRASS shows considerable potential for the characterization and comparison of tooth surfaces. Furthermore, techniques described here are not limited to the study of teeth, but may be broadly applicable to studies of skulls, joints, and other biological structures. Am J Phys Anthropol 107:137–142, 1998. © 1998 Wiley-Liss, Inc.     

Zinc status in human immunodeficiency virus infection

Plasma zinc and copper concentrations, erythrocyte zinc concentration, copper-zinc superoxide dismutase activity and urinary zinc concentrations were determined for control subjects and individuals with AIDS, ARC, or asymptomatic HIV infection. Significant differences among the population groups were not noted for the above parameters with the exception of plasma copper which was higher in the AIDS group than in other patient groups. These results do not support the idea that zinc deficiency is a common contributory factor of HIV infectivity or clinical expression, nor that HIV infection induces a zinc deficiency.     

A comprehensive internal quality control system for a large cytology laboratory

The British Columbia Cervical Cytology Program is operated through the Central Laboratory at the Cancer Control Agency in Vancouver and processes all of the gynecologic Papanicolaou smears collected by 3,200 physicians throughout the province of British Columbia. The laboratory receives approximately 2,400 smears per day, and the program currently processes in excess of 500,000 smears annually. This article describes the methods that have been developed for ensuring that adequate quality control is present in the screening and interpretation of half a million smears per year, both at the cytotechnologist and at the cytopathologist level. The results of the quality control program, which was considerably modified in 1985, are also presented. The modified program shows a significant improvement in the number of undercalled and overcalled cases detected in two comparable six-month periods.     

PC phosphorylation increases the ability of AFAP-110 to cross-link actin filaments

The actin filament-associated protein and Src-binding partner, AFAP-110, is an adaptor protein that links signaling molecules to actin filaments. AFAP-110 binds actin filaments directly and multimerizes through a leucine zipper motif. Cellular signals downstream of Src(527F) can regulate multimerization. Here, we determined recombinant AFAP-110 (rAFAP-110)-bound actin filaments cooperatively, through a lateral association. We demonstrate rAFAP-110 has the capability to cross-link actin filaments, and this ability is dependent on the integrity of the carboxy terminal actin binding domain. Deletion of the leucine zipper motif or PKC phosphorylation affected AFAP-110's conformation, which correlated with changes in multimerization and increased the capability of rAFAP-110 to cross-link actin filaments. AFAP-110 is both a substrate and binding partner of PKC. On PKC activation, stress filament organization is lost, motility structures form, and AFAP-110 colocalizes strongly with motility structures. Expression of a deletion mutant of AFAP-110 that is unable to bind PKC blocked the effect of PMA on actin filaments. We hypothesize that upon PKC activation, AFAP-110 can be cooperatively recruited to newly forming actin filaments, like those that exist in cell motility structures, and that PKC phosphorylation effects a conformational change that may enable AFAP-110 to promote actin filament cross-linking at the cell membrane.     

AFAP120 regulates actin organization during neuronal differentiation

During development, dynamic changes in the actin cytoskeleton determine both cell motility and morphological differentiation. In most mature tissues, cells are generally minimally motile and have morphologies specialized to their functions. In metastatic cancer, cells generally lose their specialized morphology and become motile. Therefore, proteins that regulate the transition between the motile and morphologically differentiated states can play important roles in determining cancer outcomes. AFAP120 is a neuronal-specific protein that binds Src kinase and protein kinase C (PKC) and cross-links actin filaments. Here we report that expression and tyrosine phosphorylation of AFAP120 are developmentally regulated in the cerebellum. In cerebellar cultures, PKC activation induces Src kinase-dependent phosphorylation of AFAP120, indicating that AFAP120 may be a downstream effector of Src. In neuroblastoma cells induced to differentiate by treatment with a PKC activator, tyrosine phosphorylation of AFAP120 appears to regulate the formation of the lamellar actin structures and subsequent neurite initiation. Together, these results indicate that AFAP120 plays a role in organizing dynamic actin structures during neuronal differentiation and suggest that AFAP120 may help regulate the transition from motile precursor to morphologically differentiated neurons.     

Neurotrophin Gene Therapy for Sustained Neural Preservation after Deafness

The cochlear implant provides auditory cues to profoundly deaf patients by electrically stimulating the residual spiral ganglion neurons. These neurons, however, undergo progressive degeneration after hearing loss, marked initially by peripheral fibre retraction and ultimately culminating in cell death. This research aims to use gene therapy techniques to both hold and reverse this degeneration by providing a sustained and localised source of neurotrophins to the deafened cochlea. Adenoviral vectors containing green fluorescent protein, with or without neurotrophin-3 and brain derived neurotrophic factor, were injected into the lower basal turn of scala media of guinea pigs ototoxically deafened one week prior to intervention. This single injection resulted in localised and sustained gene expression, principally in the supporting cells within the organ of Corti. Guinea pigs treated with adenoviral neurotrophin-gene therapy had greater neuronal survival compared to contralateral non-treated cochleae when examined at 7 and 11 weeks post injection. Moreover; there was evidence of directed peripheral fibre regrowth towards cells expressing neurotrophin genes after both treatment periods. These data suggest that neurotrophin-gene therapy can provide sustained protection of spiral ganglion neurons and peripheral fibres after hearing loss.