Incorporating Field Studies into Species Distribution and Climate Change Modelling: A Case Study of the Koomal Trichosurus vulpecula hypoleucus (Phalangeridae)

Species distribution models (SDMs) are an effective way of predicting the potential distribution of species and their response to environmental change. Most SDMs apply presence data to a relatively generic set of predictive variables such as climate. However, this weakens the modelling process by overlooking the responses to more cryptic predictive variables. In this paper we demonstrate a means by which data gathered from an intensive animal trapping study can be used to enhance SDMs by combining field data with bioclimatic modelling techniques to determine the future potential distribution for the koomal (Trichosurus vulpecula hypoleucus). The koomal is a geographically isolated subspecies of the common brushtail possum, endemic to south-western Australia. Since European settlement this taxon has undergone a significant reduction in distribution due to its vulnerability to habitat fragmentation, introduced predators and tree/shrub dieback caused by a virulent group of plant pathogens of the genus Phytophthora. An intensive field study found: 1) the home range for the koomal rarely exceeded 1 km in in length at its widest point; 2) areas heavily infested with dieback were not occupied; 3) gap crossing between patches (>400 m) was common behaviour; 4) koomal presence was linked to the extent of suitable vegetation; and 5) where the needs of koomal were met, populations in fragments were demographically similar to those found in contiguous landscapes. We used this information to resolve a more accurate SDM for the koomal than that created from bioclimatic data alone. Specifically, we refined spatial coverages of remnant vegetation and dieback, to develop a set of variables that we combined with selected bioclimatic variables to construct models. We conclude that the utility value of an SDM can be enhanced and given greater resolution by identifying variables that reflect observed, species-specific responses to landscape parameters and incorporating these responses into the model.     

OCTAL: Optimal Completion of gene trees in polynomial time

Background

For a combination of reasons (including data generation protocols, approaches to taxon and gene sampling, and gene birth and loss), estimated gene trees are often incomplete, meaning that they do not contain all of the species of interest. As incomplete gene trees can impact downstream analyses, accurate completion of gene trees is desirable.

Results

We introduce the Optimal Tree Completion problem, a general optimization problem that involves completing an unrooted binary tree (i.e., adding missing leaves) so as to minimize its distance from a reference tree on a superset of the leaves. We present OCTAL, an algorithm that finds an optimal solution to this problem when the distance between trees is defined using the Robinson–Foulds (RF) distance, and we prove that OCTAL runs in \(O(n^2)\) time, where n is the total number of species. We report on a simulation study in which gene trees can differ from the species tree due to incomplete lineage sorting, and estimated gene trees are completed using OCTAL with a reference tree based on a species tree estimated from the multi-locus dataset. OCTAL produces completed gene trees that are closer to the true gene trees than an existing heuristic approach in ASTRAL-II, but the accuracy of a completed gene tree computed by OCTAL depends on how topologically similar the reference tree (typically an estimated species tree) is to the true gene tree.

Conclusions

OCTAL is a useful technique for adding missing taxa to incomplete gene trees and provides good accuracy under a wide range of model conditions. However, results show that OCTAL’s accuracy can be reduced when incomplete lineage sorting is high, as the reference tree can be far from the true gene tree. Hence, this study suggests that OCTAL would benefit from using other types of reference trees instead of species trees when there are large topological distances between true gene trees and species trees.

     

Copper-dependent Recycling of hCTR1, the Human High Affinity Copper Transporter

Copper is an essential co-factor in many important physiological processes, but at elevated levels it is toxic to cells. Thus at both the organism and cellular level mechanisms have evolved to finely tune copper homeostasis. The protein responsible for copper entry from the circulation in most human cells is hCTR1, a small protein (190 amino acid residues) that functions as a trimer in the plasma membrane. In the present work we employ cell surface biotinylation and isotopic copper uptake studies of overexpressed hCTR1 in HEK293 cells to examine the acute (minutes) response of hCTR1 to changes in extracellular copper. We show that within 10 min of exposure to copper at 2.5 μm or higher, plasma membrane hCTR1 levels are reduced (by ∼40%), with a concomitant reduction in copper uptake rates. We are unable to detect any degradation of internalized hCTR1 in the presence of cycloheximide after up to 2 h of exposure to 0–100 μm copper. Using a reversible biotinylation assay, we quantified internalized hCTR1, which increased upon the addition of copper and corresponded to the hCTR1 lost from the surface. In addition, when extracellular copper is then removed, internalized hCTR1 is promptly (within 30 min) recycled to the plasma membrane. We have shown that in the absence of added extracellular copper, there is a small but detectable amount of internalized hCTR1 that is increased in the presence of copper. Similar studies on endogenous hCTR1 show a cell-specific response to elevated extracellular copper. Copper-dependent internalization and recycling of hCTR1 provides an acute and reversible mechanism for the regulation of cellular copper entry.Copper is an essential micronutrient and plays an important function as a co-factor for a number of cellular processes including oxidative phosphorylation, free radical detoxification, neurotransmitter synthesis, iron metabolism, and maturation of connective tissue (1). Copper in excess of cellular requirements is toxic; therefore cells have developed sophisticated mechanisms for regulating copper acquisition and secretion, thus maintaining a critical copper homeostasis (2, 3). In eukaryotes a family of transporters known as the copper transporter (Ctr) proteins mediate cellular copper uptake (4). Ctr proteins are integral membrane proteins that are structurally conserved with three membrane-spanning domains and a number of methionine rich motifs in the N terminus (5). They contain a sequence of conserved cysteine and histidine residues at or close to the C terminus and are predominantly located at the plasma membrane (6). In the yeast, Saccharomyces cerevisiae, the first high affinity copper transporters, yCtr1 and yCtr3, were identified (7, 8), and this facilitated the identification of the human copper transporter gene, hCTR1,² by functional complementation of yeast high affinity copper uptake mutant, ctr1 (9). The mouse CTR1 is 92% identical to hCTR1 (10), and the deletion of mCTR1 results in early embryonic lethality, suggesting an essential role for the high affinity copper transporter in mammalian growth and development (11).hCTR1 has 190 amino acid residues, three membrane-spanning domains, an extracellular N terminus (of 66 amino acids), a large cytoplasmic loop (of 46 amino acid residues), and a short C-terminal tail (of 15 amino acids) and has been shown to form stable dimers and trimers (12–14). The hCTR1 protein has been shown in ⁶⁴Cu uptake experiments to mediate copper transport with a K_m of 1–5 μm and is thought to transport the reduced form, Cu(I) (12, 13, 15). The extracellular N terminus has both N- and O-linked glycosylation at residues Asn¹⁵ and Thr²⁷, respectively (12, 16, 17), and contains two histidine-rich regions and two methionine motifs that are thought to function in copper binding/sensing. Recent studies showed that mutation or deletion of the methionine residues closest to the first transmembrane domain (Met⁴³ and Met⁴⁵) and the conserved methionine residues in the second transmembrane domain (Met¹⁵⁰ and Met¹⁵⁴) had a large inhibitory effect on ⁶⁴Cu uptake (18, 19). Mutational analysis provided no evidence for the tight binding of copper at any specific residues, and it was proposed that hCTR1 provided a pore for the permeation of copper across the membrane (18). Structural confirmation of such a mechanism was provided in the low resolution structure obtained by cryo-electron microscopy studies on recombinant protein (20, 21).Considerable progress has been made in understanding the biochemical, structure-functional, and molecular aspects of hCTR1-mediated copper transport, although many questions remain unanswered (22). It is also important to determine whether or not hCTR1 has a regulatory role preventing the accumulation of toxic levels of copper and maintaining cellular copper homeostasis. Previous reports on whether or not hCTR1 is involved in an acute response to elevated copper have been somewhat controversial. It has been reported that elevated extracellular copper (1–100 μm) stimulates rapid endocytosis and degradation of hCTR1-Myc-tagged protein in HEK293 cells (23), but also high copper levels had no effect on endogenous hCTR1 localization in both HeLa and Caco-2 cells (14). In a study of overexpressed hCTR1 in insect cells, no evidence was seen of internalization in response to elevated copper (24). Imaging studies have shown that the cellular location of hCTR1 varies among cell lines, CTR1 in MDCK and HEK293 cells resides mainly at the plasma membrane (13, 15, 23, 24). Endogenous hCTR1 is located in cytoplasmic vesicular compartments in HeLa, Caco-2, and HepG2 cell lines with some plasma membrane staining in Caco-2 (14). In intestinal sections, basolateral and subapical staining is seen (15).Previous studies (see above) have utilized internalization of prebound antibody (23) or imaging methods (14) to characterize the response of hCTR1 to elevated copper. In the present work we employed HEK cells overexpressing hCTR1 and used cell surface biotinylation, a sensitive and quantitative measure of CTR1 at the cell surface (15, 17). We have combined this with measurements of hCTR1-mediated ⁶⁴Cu uptake as a functional measure of plasma membrane hCTR1 levels. We find that a fraction (∼40%) of hCTR1 is rapidly internalized in the presence of elevated copper and that there is a concomitant reduction in the hCTR1-mediated copper uptake rate. The internalized transporter is not degraded and can be detected in the cytosol. On removal of extracellular copper, the transporter is recycled promptly to the plasma membrane. Internalization of endogenous CTR1 is also observed in MDCK and HepG2 cells, and no reduction is seen in T47D cells. This is, to our knowledge, the first such report of copper-dependent recycling of hCTR1 in response to copper and represents an acute regulatory mechanism that reversibly modulates cellular copper entry.     

Ascaris lumbricoides pseudocoelomic body fluid induces a partially activated dendritic cell phenotype with Th2 promoting ability in vivo

Dendritic cells (DCs) matured with helminth-derived molecules that promote Th2 immune responses do not follow conventional definitions of DC maturation processes. While a number of models of DC maturation by Th2 stimuli are postulated, further studies are required if we are to clearly define DC maturation processes that lead to Th2 immune responses. In this study, we examine the interaction of Th2-inducing molecules from the parasitic helminth Ascaris lumbricoides with the maturation processes and function of DCs. Here we show that murine bone marrow-derived DCs are partially matured by A. lumbricoides pseudocoelomic body fluid (ABF) as characterised by the production of IL-6, IL-12p40 and macrophage inflammatory protein 2 (MIP-2) but no enhanced expression of cluster of differentiation (CD)-14, T-cell co-stimulatory markers CD80, CD86, CD40, OX40L and major histocompatibility complex class II was observed. Despite these phenotypic characteristics, ABF-stimulated DCs displayed the functional hallmarks of fully matured cells, enhancing DC phagocytosis and promoting Th2-type responses in skin-draining lymph node cells in vivo. ABF activated Th2-associated extracellular signal-regulated kinase-1 and nuclear factor-kB intracellular signalling pathways independently of toll-like receptor 4. Taken together, we believe this is the first paper to demonstrate A. lumbricoides murine DC-Th cell-driven responses shedding further light on DC maturation processes by helminth antigens.     

The discovery of a potent, intracellular, orally bioavailable, long duration inhibitor of human neutrophil elastase--GW311616A a development candidate

The discovery of a potent intracellular inhibitor of human neutrophil elastase which is orally active and has a long duration of action is described. The pharmacodynamic and pharmacokinetic properties of a trans-lactam development candidate, GW311616A, are described.     

Structure-based design, synthesis and SAR of a novel series of thiopheneamidine urokinase plasminogen activator inhibitors

The serine protease urokinase plasminogen activator (uPA) is thought to play a central role in tumor metastasis and angiogenesis. Molecular modeling studies suggest that 5-thiomethylthiopheneamidine inhibits uPA by binding at the S1 pocket of the active site. Further structure based elaboration of this residue resulted in a novel class of potent and selective inhibitors of uPA.