Pleiotrophin Gene Therapy for Peripheral Ischemia: Evaluation of Full-Length and Truncated Gene Variants

Pleiotrophin (PTN) is a growth factor with both pro-angiogenic and limited pro-tumorigenic activity. We evaluated the potential for PTN to be used for safe angiogenic gene therapy using the full length gene and a truncated gene variant lacking the domain implicated in tumorigenesis. Mouse myoblasts were transduced to express full length or truncated PTN (PTN or T-PTN), along with a LacZ reporter gene, and injected into mouse limb muscle and myocardium. In cultured myoblasts, PTN was expressed and secreted via the Golgi apparatus, but T-PTN was not properly secreted. Nonetheless, no evidence of uncontrolled growth was observed in cells expressing either form of PTN. PTN gene delivery to myocardium, and non-ischemic skeletal muscle, did not result in a detectable change in vascularity or function. In ischemic hindlimb at 14 days post-implantation, intramuscular injection with PTN-expressing myoblasts led to a significant increase in skin perfusion and muscle arteriole density. We conclude that (1) delivery of the full length PTN gene to muscle can be accomplished without tumorigenesis, (2) the truncated PTN gene may be difficult to use in a gene therapy context due to inefficient secretion, (3) PTN gene delivery leads to functional benefit in the mouse acute ischemic hindlimb model.     

Delimitation of the maritime boundary in the gulf of Maine area

The compulsory dispute settlement regime included in the 1982 Law of the Sea Convention is recognized as one of the most comprehensive in a modern international convention. Yet, in the recent application of this regime, the question has arisen as to whether the procedural prerequisites associated with the LOS Convention's compulsory dispute settlement mechanism are so arduous as to avoid binding and compulsory jurisdiction in most instances. This article addresses that question by examining, in particular, the reasoning of the Southern Bluefin Tuna arbitration tribunal, which found Article 281 of Section 1 of the LOS Convention to bar jurisdiction to the compulsory dispute settlement mechanism prescribed by the Convention, and offers suggestions as to how states might distinguish or overcome the barriers imposed by the Southern Bluefin Tuna tribunal in future cases.     

Recent intron gain in elongation factor-1alpha of colletid bees (Hymenoptera: Colletidae)

We discovered the presence of a unique spliceosomal intron in the F1 copy of elongation factor-1alpha (EF-1alpha) restricted to the bee family Colletidae (Hymenoptera: Apoidae). The intron ranges in size from 101 to 1044 bp and shows no positional sliding. Our data also demonstrate the complete absence of this intron from exemplars representing all other bee families, as well as from close hymenopteran relatives. A review of the literature finds that this intron is likewise absent from all other arthropods for which data are available. This provides unambiguous evidence for a relatively recent intron insertion event in the colletid common ancestor and, at least in this specific instance, lends support to the introns-late hypothesis. The comparative distribution of this novel intron also supports the monophyly of Colletidae and the exclusion of the Stenotritidae from this family, providing an example of the potential of some introns to act as robust markers of shared descent.     

Molecular phylogenetics and evolution of maternal care in Membracine treehoppers

The treehopper subfamily Membracinae (Insecta: Hemiptera: Membracidae) comprises the majority of genera and species diversity in the New World tropics. These treehoppers exhibit a wide range of social behaviors, making them an excellent group for studying patterns of social evolution in insects. However, to date the tribal and generic relationships have remained unclear. We reconstructed the phylogeny of the Membracinae using a combined mitochondrial (COI, COII, tRNA-Leu, and 12S) and nuclear (Wg) gene data set. A total of 2608 aligned nucleotide sites were obtained for 112 species, representing 25 of 38 currently recognized genera and all four tribes. A strict consensus of five equally parsimonious trees recovered the subfamily and three of its four tribes. The majority rule consensus tree derived from the Bayesian analyses based on the GTR+I+G and mixed-models recovered many clades shared with the parsimony trees and is identical to the single best tree inferred from maximum likelihood analysis, aside from the rearrangement of one node. A comparison of mitochondrial and nuclear genes indicated that Wg provided higher consistency index (CI), data decisiveness (DD), partitioned Bremer support (PBS) than any of the mitochondrial genes analyzed. The combined mitochondrial and nuclear DNA provide strong support for the monophyly of the subfamily and three of its four tribes (Aconophorini, Hoplophorionini, and Hypsoprorini). Membracini is paraphyletic with respect to Hoplophorionini and contains two lineages, the Membracini sensu strictu and the newly resurrected tribe Bolbonotini. Our analyses show that there is a strong phylogenetic component to the evolution of maternal care. Given the widespread occurrence of maternal care within the subfamily, this trait is estimated to have < or = 3 origins, two reversals, and one loss. Our results suggest that the evolution of maternal care in insects may not be as evolutionarily labile as previously thought.     

Elongation factor-1 alpha occurs as two copies in bees: implications for phylogenetic analysis of EF-1 alpha sequences in insects

We report the complete sequence of a paralogous copy of elongation factor-1 alpha (EF-1 alpha) in the honeybee, Apis mellifera (Hymenoptera: Apidae). This copy differs from a previously described copy in the positions of five introns and in 25% of the nucleotide sites in the coding regions. The existence of two paralogous copies of EF-1 alpha in Drosophila and Apis suggests that two copies of EF-1 alpha may be widespread in the holometabolous insect orders. To distinguish between a single, ancient gene duplication and parallel, independent fly and bee gene duplications, we performed a phylogenetic analysis of hexapod EF-1 alpha sequences. Unweighted parsimony analysis of nucleotide sequences suggests an ancient gene duplication event, whereas weighted parsimony analysis of nucleotides and unweighted parsimony analysis of amino acids suggests the contrary: that EF-1 alpha underwent parallel gene duplications in the Diptera and the Hymenoptera. The hypothesis of parallel gene duplication is supported both by congruence among nucleotide and amino acid data sets and by topology-dependent permutation tail probability (T-PTP) tests. The resulting tree topologies are also congruent with current views on the relationships among the holometabolous orders included in this study (Diptera, Hymenoptera, and Lepidoptera). More sequences, from diverse orders of holometabolous insects, will be needed to more accurately assess the historical patterns of gene duplication in EF-1 alpha.      

Characterizing the Google Books Corpus: Strong Limits to Inferences of Socio-Cultural and Linguistic Evolution

It is tempting to treat frequency trends from the Google Books data sets as indicators of the “true” popularity of various words and phrases. Doing so allows us to draw quantitatively strong conclusions about the evolution of cultural perception of a given topic, such as time or gender. However, the Google Books corpus suffers from a number of limitations which make it an obscure mask of cultural popularity. A primary issue is that the corpus is in effect a library, containing one of each book. A single, prolific author is thereby able to noticeably insert new phrases into the Google Books lexicon, whether the author is widely read or not. With this understood, the Google Books corpus remains an important data set to be considered more lexicon-like than text-like. Here, we show that a distinct problematic feature arises from the inclusion of scientific texts, which have become an increasingly substantive portion of the corpus throughout the 1900s. The result is a surge of phrases typical to academic articles but less common in general, such as references to time in the form of citations. We use information theoretic methods to highlight these dynamics by examining and comparing major contributions via a divergence measure of English data sets between decades in the period 1800–2000. We find that only the English Fiction data set from the second version of the corpus is not heavily affected by professional texts. Overall, our findings call into question the vast majority of existing claims drawn from the Google Books corpus, and point to the need to fully characterize the dynamics of the corpus before using these data sets to draw broad conclusions about cultural and linguistic evolution.     

Low nuclear DNA variation supports a recent origin of Hawaiian Hylaeus bees (Hymenoptera: Colletidae)

Previous phylogenetic work on the Hawaiian bees of the genus Hylaeus, based on mitochondrial DNA and morphology, appeared to support a recent origin for the group, but support for the resulting tree was weak. Four nuclear genes with varying evolutionary rates -- arginine kinase, EF-1alpha, opsin, and wingless -- were sequenced for a reduced taxon set in an attempt to find one or more data set that would provide better support. All showed very low variation (<2%) in the ingroup. Comparison among genes revealed a much higher than expected rate of evolution in mtDNA, especially at first and second positions. While the data from the nuclear genes showed insufficient variation for phylogenetic analysis, the strong sequence similarity among the Hawaiian species supports the previous hypothesis of a recent origin for the group.     

Rooting phylogenies using gene duplications: an empirical example from the bees (Apoidea)

The placement of the root node in a phylogeny is fundamental to characterizing evolutionary relationships. The root node of bee phylogeny remains unclear despite considerable previous attention. In order to test alternative hypotheses for the location of the root node in bees, we used the F1 and F2 paralogs of elongation factor 1-alpha (EF-1α) to compare the tree topologies that result when using outgroup versus paralogous rooting. Fifty-two taxa representing each of the seven bee families were sequenced for both copies of EF-1α. Two datasets were analyzed. In the first (the "concatenated" dataset), the F1 and F2 copies for each species were concatenated and the tree was rooted using appropriate outgroups (sphecid and crabronid wasps). In the second dataset (the "duplicated" dataset), the F1 and F2 copies were aligned to each another and each copy for all taxa were treated as separate terminals. In this dataset, the root was placed between the F1 and F2 copies (e.g., paralog rooting). Bayesian analyses demonstrate that the outgroup rooting approach outperforms paralog rooting, recovering deeper clades and showing stronger support for groups well established by both morphological and other molecular data. Sequence characteristics of the two copies were compared at the amino acid level, but little evidence was found to suggest that one copy is more functionally conserved. Although neither approach yields an unambiguous root to the tree, both approaches strongly indicate that the root of bee phylogeny does not fall near Colletidae, as has been previously proposed. We discuss paralog rooting as a general strategy and why this approach performs relatively poorly with our particular dataset.     

A spatial analytical approach for selecting reintroduction sites for burbot in English rivers

1. Availability of suitable habitat is a prerequisite for species reintroduction success, and to ensure population persistence, investigations of a species’ habitat utilisation throughout its life history should be conducted as part of a feasibility study. 2. Habitat utilisation models for burbot, Lota lota, developed using data from field studies conducted in France and Germany and information from the literature were used to assess the feasibility of reintroducing burbot into rivers of its former native range in eastern England. 3. Per cent tree roots, aquatic vegetation and flow types were important predictors of adult burbot abundance. Furthermore, the habitat utilisation models were supplemented with information from the literature, which suggested that off‐channel habitat such as wetlands and backwaters is important for spawning and nursery stages. 4. An assessment of the habitat availability in the rivers of the burbot’s former native range using variables related to spawning and nursery and adult life stages showed that although adult habitat was widely distributed, the availability of spawning and nursery habitat was less abundant, potentially limiting successful reestablishment. 5. Potential suitable habitat was concentrated in the central and southern areas of the species’ former English distribution. Overall, rivers of the burbot’s former range potentially afford suitable habitat to sustain a reintroduced population. However, sites should be preferentially selected on the basis of having appropriate spawning and nursery areas.     

Immunostimulating complexes incorporating Eimeria tenella antigens and plant saponins as effective delivery system for coccidia vaccine immunization

Immunostimulating complexes (ISCOMs) are unique, multimolecular structures formed by encapsulating antigens, lipids, and triterpene saponins of plant origin, and are an effective delivery system for various kinds of antigens. The uses of ISCOMs formulated with saponins from plants collected in Kazakhstan, with antigens from the poultry coccidian parasite Eimeria tenella, were evaluated for their potential use in developing a vaccine for control of avian coccidiosis. Saponins isolated from the plants Aesculus hippocastanum and Glycyrrhiza glabra were partially purified by HPLC. The saponin fractions obtained from HPLC were evaluated for toxicity in chickens and chicken embryos. The HPLC saponin fractions with the least toxicity, compared to a commercial saponin Quil A, were used to assemble ISCOMs. When chicks were immunized with ISCOMs prepared with saponins from Kazakhstan plants and E. tenella antigens, and then challenged with E. tenella oocysts, significant protection was conveyed compared to immunization with antigen alone. The results of this study indicate that ISCOMs formulated with saponins isolated from plants indigenous to Kazakhstan are an effective antigen delivery system which may be successfully used, with low toxicity, for preparation of highly immunogenic coccidia vaccine.