The phylogeny of Dendropsophini (Anura: Hylidae: Hylinae)

The relationships of the hyline tribe Dendropsophini remain poorly studied, with most published analyses dealing with few of the species groups of Dendropsophus. In order to test the monophyly of Dendropsophini, its genera, and the species groups currently recognized in Dendropsophus, we performed a total evidence phylogenetic analysis. The molecular dataset included sequences of three mitochondrial and five nuclear genes from 210 terminals, including 12 outgroup species, the two species of Xenohyla, and 93 of the 108 recognized species of Dendropsophus. The phenomic dataset includes 46 terminals, one per species (34 Dendropsophus, one Xenohyla, and 11 outgroup species). Our results corroborate the monophyly of Dendropsophini and the reciprocal monophyly of Dendropsophus and Xenohyla. Some species groups of Dendropsophus are paraphyletic (the D. microcephalus, D. minimus, and D. parviceps groups, and the D. rubicundulus clade). On the basis of our results, we recognize nine species groups; for three of them (D. leucophyllatus, D. microcephalus, and D. parviceps groups) we recognize some nominal clades to highlight specific morphology or relationships and facilitate species taxonomy. We further discuss the evolution of oviposition site selection, where our results show multiple instances of independent evolution of terrestrial egg clutches during the evolutionary history of Dendropsophus.     

Convergent evolution of a complex fruit structure in the tribe Brassiceae (Brassicaceae)

? Premise of study: Many angiosperms have fruit morphologies that result in seeds from the same plant having different dispersal capabilities. A prime example is found in the Brassiceae (Brassicaceae), which has many members with segmented or heteroarthrocarpic fruits. Since only 40% of the genera are heteroarthrocarpic, this tribe provides an opportunity to study the evolution of an ecologically significant novelty and its variants. ? Methods: We analyzed nuclear (PHYA) and plastid (matK) sequences from 66 accessions using maximum parsimony, maximum likelihood, and Bayesian inference approaches. The evolution of heteroarthrocarpy and its variants was evaluated using maximum parsimony and maximum likelihood ancestral state reconstructions. ? Key results: Although nuclear and plastid phylogenies are incongruent with each other, the following findings are consistent: (1) Cakile, Crambe, Vella, and Zilla lineages are monophyletic; (2) the Nigra lineage is not monophyletic; and (3) within the Cakile clade, Cakile, Didesmus, and Erucaria are paraphyletic. Despite differences in the matK and PHYA topologies at both deep and shallow nodes, similar patterns of morphological evolution emerge. Heteroarthrocarpy, a complex morphological trait, has evolved multiple times across the tribe. Moreover, there are convergent transitions in dehiscence capabilities and fruit disarticulation across the tribe. ? Conclusions: We present the first explicit analysis of fruit evolution within the Brassiceae, which exemplifies evolutionary lability. The repeated loss and gain of segment dehiscence and disarticulation suggests conservation in the genetic pathway controlling abscission with differential expression across taxa. This study provides a strong foundation for future studies of mechanisms underlying variation in dispersal capabilities of Brassiceae.     

Intrinsically de-sialylated CD103+ CD8 T cells mediate beneficial anti-glioma immune responses

Background

Cancer vaccines reproducibly cure laboratory animals and reveal encouraging trends in brain tumor (glioma) patients. Identifying parameters governing beneficial vaccine-induced responses may lead to the improvement of glioma immunotherapies. CD103⁺ CD8 T cells dominate post-vaccine responses in human glioma patients for unknown reasons, but may be related to recent thymic emigrant (RTE) status. Importantly, CD8 RTE metrics correlated with beneficial immune responses in vaccinated glioma patients.

Methods

We show by flow cytometry that murine and human CD103⁺ CD8 T cells respond better than their CD103^? counterparts to tumor peptide-MHC I (pMHC I) stimulation in vitro and to tumor antigens on gliomas in vivo.

Results

Glioma responsive T cells from mice and humans both exhibited intrinsic de-sialylation-affecting CD8 beta. Modulation of CD8 T cell sialic acid with neuraminidase and ST3Gal-II revealed de-sialylation was necessary and sufficient for promiscuous binding to and stimulation by tumor pMHC I. Moreover, de-sialylated status was required for adoptive CD8 T cells and lymphocytes to decrease GL26 glioma invasiveness and increase host survival in vivo. Finally, increased tumor ST3Gal-II expression correlated with clinical vaccine failure in a meta-analysis of high-grade glioma patients.

Conclusions

Taken together, these findings suggest that de-sialylation of CD8 is required for hyper-responsiveness and beneficial anti-glioma activity by CD8 T cells. Because CD8 de-sialylation can be induced with exogenous enzymes (and appears particularly scarce on human T cells), it represents a promising target for clinical glioma vaccine improvement.     

A Novel Humanized GLP-1 Receptor Model Enables Both Affinity Purification and Cre-LoxP Deletion of the Receptor

Class B G protein-coupled receptors (GPCRs) are important regulators of endocrine physiology, and peptide-based therapeutics targeting some of these receptors have proven effective at treating disorders such as hypercalcemia, osteoporosis, and type 2 diabetes mellitus (T2DM). As next generation efforts attempt to develop novel non-peptide, orally available molecules for these GPCRs, new animal models expressing human receptor orthologs may be required because small molecule ligands make fewer receptor contacts, and thus, the impact of amino acid differences across species may be substantially greater. The objective of this report was to generate and characterize a new mouse model of the human glucagon-like peptide-1 receptor (hGLP-1R), a class B GPCR for which established peptide therapeutics exist for the treatment of T2DM. hGLP-1R knock-in mice express the receptor from the murine Glp-1r locus. Glucose tolerance tests and gastric emptying studies show hGLP-1R mice and their wild-type littermates display similar physiological responses for glucose metabolism, insulin secretion, and gastric transit, and treatment with the GLP-1R agonist, exendin-4, elicits similar responses in both groups. Further, ex vivo assays show insulin secretion from humanized islets is glucose-dependent and enhanced by GLP-1R agonists. To enable additional utility, the targeting construct of the knock-in line was engineered to contain both flanking LoxP sites and a C-terminal FLAG epitope. Anti-FLAG affinity purification shows strong expression of hGLP-1R in islets, lung, and stomach. We crossed the hGLP-1R line with Rosa26Cre mice and generated global Glp-1r^−/− animals. Immunohistochemistry of pancreas from humanized and knock-out mice identified a human GLP-1R-specific antibody that detects the GLP-1R in human pancreas as well as in the pancreas of hGLP-1r knock-in mice. This new hGLP-1R model will allow tissue-specific deletion of the GLP-1R, purification of potential GLP-1R partner proteins, and testing of novel therapeutic agents targeting the hGLP-1R.     

Transport of neutral amino acids by human erythrocytes

The transport of several neutral amino acids by human erythrocytes in vitro was studied. The measurements made included steady-state distributions, kinetics of initial rates of uptake, effects of monovalent cations and anions, general mutual inhibitory interactions, kinetics of inhibitions, effluxes, ability to produce accelerative exchange diffusion, and the inhibitory action of the thiol reagent N-ethylmaleimide. The results are interpreted as showing that the human erythrocyte membrane possesses several distinct transport systems for these amino acids, including one Na⁺-dependent system and one dependent on both Na⁺ and a suitable anion, that are qualitatively similar to those systems previously described in pigeon erythrocytes and mammalian reticulocytes. Quantitatively, however, the systems differ among the different kinds of red cell and a major difference lies in their abilities to produce accelerative exchange diffusion.     

On testing biological data for the presence of a boundary

Under the boundary line model for a biological data set, where one variable is a biological response (e.g. crop yield) to an independent variable (e.g. available water content of the soil), we interpret the upper (or lower) boundary on a plot of the dependent variable (ordinate) against the independent variable (abscissa) as representing the maximum (or minimum) possible response for a given value of the independent variable. This concept has been widely used in soil science, agronomy and plant physiology; but it has been subject to criticism. In particular, no methods that are used to analyse the boundary line quantify the evidence that the envelope of the plot represents a boundary (in the sense of some limiting response to the independent variable) rather than simply being a fringe of extreme values of no intrinsic biological interest. In this article, we present a novel procedure that tests a data set for evidence of a boundary by considering its statistical properties in the region of the proposed boundary. The method is demonstrated using both simulated and real data sets.     

Plate 463. Hibbertia Cuneiformis Dilleniaceae

The shrubby Hibbertia cuneiformis (Labill.) Smith ( Dilleniaceae ) is described and illustrated. Its history and cultivation are discussed.     

Lung Epithelial Cells Induce Both Phenotype Alteration and Senescence in Breast Cancer Cells

Purpose

The lung is one of the most common sites of breast cancer metastasis. While metastatic seeding is often accompanied by a dormancy-promoting mesenchymal to epithelial reverting transitions (MErT), we aimed to determine whether lung epithelial cells can impart this phenotype on aggressive breast cancer cells.

Methods

Co-culture experiments of normal lung epithelial cell lines (SAEC, NHBE or BEAS-2B) and breast cancer cell lines (MCF-7 or MDA-MB-231) were conducted. Flow cytometry analysis, immunofluorescence staining for E-cadherin or Ki-67 and senescence associated beta-galactosidase assays assessed breast cancer cell outgrowth and phenotype.

Results

Co-culture of the breast cancer cells with the normal lung cells had different effects on the epithelial and mesenchymal carcinoma cells. The epithelial MCF-7 cells were increased in number but still clustered even if in a slightly more mesenchymal-spindle morphology. On the other hand, the mesenchymal MDA-MB-231 cells survived but did not progressively grow out in co-culture. These aggressive carcinoma cells underwent an epithelial shift as indicated by cuboidal morphology and increased E-cadherin. Disruption of E-cadherin expressed in MDA-MB-231 using shRNA prevented this phenotypic reversion in co-culture. Lung cells limited cancer cell growth kinetics as noted by both (1) some of the cells becoming larger and positive for senescence markers/negative for proliferation marker Ki-67, and (2) Ki-67 positive cells significantly decreasing in MDA-MB-231 and MCF-7 cells after co-culture.

Conclusions

Our data indicate that normal lung epithelial cells can drive an epithelial phenotype and suppress the growth kinetics of breast cancer cells coincident with changing their phenotypes.     

The Response of the Alpine Dwarf Shrub Salix herbacea to Altered Snowmelt Timing: Lessons from a Multi-Site Transplant Experiment

Climate change is altering spring snowmelt patterns in alpine and arctic ecosystems, and these changes may alter plant phenology, growth and reproduction. To predict how alpine plants respond to shifts in snowmelt timing, we need to understand trait plasticity, its effects on growth and reproduction, and the degree to which plants experience a home-site advantage. We tested how the common, long-lived dwarf shrub Salix herbacea responded to changing spring snowmelt time by reciprocally transplanting turfs of S. herbacea between early-exposure ridge and late-exposure snowbed microhabitats. After the transplant, we monitored phenological, morphological and fitness traits, as well as leaf damage, during two growing seasons. Salix herbacea leafed out earlier, but had a longer development time and produced smaller leaves on ridges relative to snowbeds. Longer phenological development times and smaller leaves were associated with reduced sexual reproduction on ridges. On snowbeds, larger leaves and intermediate development times were associated with increased clonal reproduction. Clonal and sexual reproduction showed no response to altered snowmelt time. We found no home-site advantage in terms of sexual and clonal reproduction. Leaf damage probability depended on snowmelt and thus exposure period, but had no short-term effect on fitness traits. We conclude that the studied populations of S. herbacea can respond to shifts in snowmelt by plastic changes in phenology and leaf size, while maintaining levels of clonal and sexual reproduction. The lack of a home-site advantage suggests that S. herbacea may not be adapted to different microhabitats. The studied populations are thus unlikely to react to climate change by rapid adaptation, but their responses will also not be constrained by small-scale local adaptation. In the short term, snowbed plants may persist due to high stem densities. However, in the long term, reduction in leaf size and flowering, a longer phenological development time and increased exposure to damage may decrease overall performance of S. herbacea under earlier snowmelt.