Epidemiological Panel Studies of Older Adults: New Frontiers in the Research on Human– Animal Interaction

ABSTRACT

This paper discusses epidemiological studies of older adults and human–animal interaction (HAI), and potential relevance to future research on HAI and animal-assisted interventions (AAI) in aging populations. Key issues in epidemiological work are discussed, including target populations, treatment/exposures, follow up, and endpoint measures as they relate to interacting with or owning companion animals. In outlining the limits in our current state of knowledge, we also make recommendations for the design and analysis of epidemiological studies to advance research on HAI and aging.     

Multiple causation of phylogeographical pattern as revealed by nested clade analysis of the bamboo viper (Trimeresurus stejnegeri) within Taiwan

In order to assess the utility of nested clade analysis, both standard phylogenetic algorithms and nested clade analysis were performed on a geographically widespread survey of mitochondrial DNA haplotypes of the bamboo viper, Trimeresurus stejnegeri, within Taiwan. Gross tree topologies were congruent for all analyses and indicated the presence of two geographically overlapping clades within Taiwan. The smaller lineage was restricted to the north and east coasts, whereas the larger lineage occupied all but the northern range of the species within Taiwan including the Pacific offshore populations of Green and Orchid Islands. The phylogeographical pattern supports the existence of at least one colonization event from the continent since the initial isolation of Taiwan from the mainland in the Pliocene. However, determining the exact number of colonization events was not possible due to the simultaneous vicariant forces of hypothesized continental landbridge connections and the occurrence of dramatic in situ orogenesis throughout the Pleistocene. Nested clade analysis provided multiple temporal and spatial population historical inferences that are not possible with standard analyses and therefore should become widely applied to future phylogeographical studies.     

The use of amplified fragment length polymorphism in determining species trees at fine taxonomic levels: analysis of a medically important snake, Trimeresurus albolabris

There are a huge number of phylogenetic studies based on mitochondrial DNA (mtDNA); however, these may represent gene trees that may not be congruent with the species tree. A solution to this problem is to include additional, independent, loci from the nuclear genome. At fine taxonomic levels, i.e. between populations and closely related species, previously suggested nuclear markers such as intron sequence data may not be appropriate. In this study we investigate the use of amplified fragment length polymorphisms (AFLP) to aid determination of the species tree for 24 specimens of a medically important snake, Trimeresurus albolabris. This is of particular importance for many venomous snakes as venom often varies intraspecifically. Five different primer combinations produced 434 bands and were analysed by constructing a phylogenetic tree using neighbour joining and principal component analysis. Results were similar across all methods and found distinct groupings. The results were compared with mtDNA data and a reconciled tree was constructed in order to determine the species tree for T. albolabris. We found that T. albolabris (sensu lato) is not monophyletic. Specimens from the Indonesian islands (except West Java) form a distinct clade and we propose elevation to species level. A specimen from Nepal is also distinct and suggests that this population also deserves specific status. We suggest that AFLPs may prove a valuable aid in determining species trees as opposed to gene trees at fine taxonomic levels and this should facilitate the incorporation of molecular data into such activities as antivenom production and conservation management.     

Offshore insular variation in the diet of the Taiwanese bamboo viper Trimeresurus stejnegeri (Schmidt)

Dietary data were ascertained for 229 T. stejnegeri (snout vent length >300mm) from 36 localities throughout the main island of Taiwan and the outlying Orchid (Lanyu) and Green (Ludau) Islands. Twenty nine percent of the snakes were devoid of any prey, and of the snakes containing prey, 43% of the cases were unidentifiable. This relatively large proportion of unidentifiable prey items (observed in the hindgut) may reflect either rapid digestion of amphibian prey and/or rapid venting of feces as an evolutionary adaptation to arboreal life. Trimeresurus stejnegeri appears euryphagous, taking primarily amphibians, but additionally reptilian, mammalian and insect prey. There was no discrepancy in prey composition based on comparisons of where the prey item was recorded in the digestive tract. No sexual variation in diet composition was evident, although males were more likely to contain prey than females, indicating the utilisation of different foraging strategies on similar sympatric prey items. Variation in diet composition was observed between mainland Taiwan and offshore islands, which is most likely the result of differences in prey availability.     

Enhancing the catalytic repertoire of nucleic acids: a systematic study of linker length and rigidity

The incorporation of potentially catalytic groups in DNA is of interest for the in vitro selection of novel deoxyribozymes. A series of 10 C5-modified analogues of 2'-deoxyuridine triphosphate have been synthesised that possess side chains of differing flexibility and bearing a primary amino or imidazole functionality. For each series of nucleotide analogues differing degrees of flexibility of the C5 side chain was achieved through the use of alkynyl, alkenyl and alkyl moieties. The imidazole function was conjugated to these C5-amino-modified nucleotides using either imidazole 4-acetic acid or imidazole 4-acrylic acid (urocanic acid). The substrate properties of the nucleotides (fully replacing dTTP) with TAQ polymerase during PCR have been investigated in order to evaluate their potential applications for in vitro selection experiments. 5-(3-Aminopropynyl)dUTP and 5-(E-3-aminopropenyl)dUTP and their imidazole 4-acetic acid- and urocanic acid-modified conjugates were found to be substrates. In contrast, C5-amino-modified dUTPs with alkane or Z-alkene linkers and their corresponding conjugates were not substrates. The incorporation of these analogues during PCR has been confirmed by inhibition of restriction enzyme digestion using XBAI and by mass spectrometry of the PCR products.     

Control of directionality in the site-specific recombination system of the Streptomyces phage phiC31

The genome of the Streptomyces temperate phage phiC31 integrates into the host chromosome via a recombinase belonging to a novel group of phage integrases related to the resolvase/invertase enzymes. Previously, it was demonstrated that, in an in vitro recombination assay, phiC31 integrase catalyses integration (attP/attB recombination) but not excision (attL/attR). The mechanism responsible for this recombination site selectivity was therefore investigated. Purified integrase was shown to bind with similar apparent binding affinities to between 46 bp and 54 bp of DNA at each of the attachment sites, attP, attB, attL and attR. Assays using recombination sites of 50 bp and 51 bp for attP and attB, respectively, showed that these fragments were functional in attP/attB recombination and maintained strict site selectivity, i.e. no recombination between non-permissive sites, such as attP/attP, attB/attL, etc., was observed. Using bandshifts and supershift assays in which permissive and non-permissive combinations of att sites were used in the presence of integrase, only the attP/attB combination could generate supershifts. Recombination products were isolated from the supershifted complexes. It was concluded that these supershifted complexes contained the recombination synapse and that site specificity, and therefore directionality, is determined at the level of stable synapse formation.     

4-Hydroxycinnamoyl-CoA: an ionizable probe of the active site of the medium chain acyl-CoA dehydrogenase

4-OH-Cinnamoyl-CoA has been synthesized as a probe of the active site in the medium chain acyl-CoA dehydrogenase. The protonated form of the free ligand (lambda(max) = 336 nm) yields the corresponding phenolate (lambda(max) = 388 nm) with a pK of 8.9. 4-OH-Cinnamoyl-CoA binds tightly (K(d) = 47 nM, pH 6) to the pig kidney dehydrogenase with a prominent new band at 388 nm, suggesting ionization of the bound ligand. However, this spectrum reflects polarization, not deprotonation, of the neutral form of the ligand. Thus, the 388 nm band is abolished as the pH is raised (not lowered), and analogous spectral and pH behavior is observed with the nonionizable analogue 4-methoxycinnamoyl-CoA. Studies with wild type, E99G, and E376Q mutants of the human medium chain acyl-CoA dehydrogenase showed that these two active site carboxylates strongly suppress ionization of the 4-OH ligand. Binding to the double mutant E99G/E376Q gives an intense new band as the pH is raised (pK = 7.8), with an absorbance maximum at 498 nm resembling the natural 4-OH-cinnamoyl-thioester chromophore of the photoactive yellow protein. Raman difference spectroscopy in water and D(2)O, using the free ligand and wild-type and double-mutant enzyme.ligand complexes, confirms that the 4-OH group of the thioester is ionized only when bound to the double mutant. These data demonstrate the strong electrostatic coupling between ligand and enzyme, and the critical role Glu376 plays in modulating thioester polarization in the medium chain acyl-CoA dehydrogenase.