Identification of Thioaptamer Ligand against E-Selectin: Potential Application for Inflamed Vasculature Targeting

Active targeting of a drug carrier to a specific target site is crucial to provide a safe and efficient delivery of therapeutics and imaging contrast agents. E-selectin expression is induced on the endothelial cell surface of vessels in response to inflammatory stimuli but is absent in the normal vessels. Thus, E-selectin is an attractive molecular target, and high affinity ligands for E-selectin could be powerful tools for the delivery of therapeutics and/or imaging agents to inflamed vessels. In this study, we identified a thiophosphate modified aptamer (thioaptamer, TA) against E-selectin (ESTA-1) by employing a two-step selection strategy: a recombinant protein-based TA binding selection from a combinatorial library followed by a cell-based TA binding selection using E-selectin expressing human microvascular endothelial cells. ESTA-1 selectively bound to E-selectin with nanomolar binding affinity (K_D = 47 nM) while exhibiting minimal cross reactivity to P- and L-selectin. Furthermore, ESTA-1 binding to E-selectin on the endothelial cells markedly antagonized the adhesion (over 75% inhibition) of sLe^x positive HL-60 cells at nanomolar concentration. ESTA-1 also bound specifically to the inflamed tumor-associated vasculature of human carcinomas derived from breast, ovarian, and skin but not to normal organs, and this binding was highly associated with the E-selectin expression level. Similarly, intravenously injected ESTA-1 demonstrated distinct binding to the tumor vasculature in a breast cancer xenograft model. Together, our data substantiates the discovery of a thioaptamer (ESTA-1) that binds to E-selectin with high affinity and specificity, thereby highlighting the potential application of ESTA-1 for E-selectin targeted delivery.     

Emerging Infectious Disease Leads to Rapid Population Declines of Common British Birds

Emerging infectious diseases are increasingly cited as threats to wildlife, livestock and humans alike. They can threaten geographically isolated or critically endangered wildlife populations; however, relatively few studies have clearly demonstrated the extent to which emerging diseases can impact populations of common wildlife species. Here, we report the impact of an emerging protozoal disease on British populations of greenfinch Carduelis chloris and chaffinch Fringilla coelebs, two of the most common birds in Britain. Morphological and molecular analyses showed this to be due to Trichomonas gallinae. Trichomonosis emerged as a novel fatal disease of finches in Britain in 2005 and rapidly became epidemic within greenfinch, and to a lesser extent chaffinch, populations in 2006. By 2007, breeding populations of greenfinches and chaffinches in the geographic region of highest disease incidence had decreased by 35% and 21% respectively, representing mortality in excess of half a million birds. In contrast, declines were less pronounced or absent in these species in regions where the disease was found in intermediate or low incidence. Also, populations of dunnock Prunella modularis, which similarly feeds in gardens, but in which T. gallinae was rarely recorded, did not decline. This is the first trichomonosis epidemic reported in the scientific literature to negatively impact populations of free-ranging non-columbiform species, and such levels of mortality and decline due to an emerging infectious disease are unprecedented in British wild bird populations. This disease emergence event demonstrates the potential for a protozoan parasite to jump avian host taxonomic groups with dramatic effect over a short time period.     

HOW DOES SPATIAL DISPERSAL NETWORK AFFECT THE EVOLUTION OF PARASITE LOCAL ADAPTATION?

Studying patterns of parasite local adaptation can provide insights into the spatiotemporal dynamics of host–parasite coevolution. Many factors, both biotic and abiotic, have been identified that influence parasite local adaptation. In particular, dispersal and population structuring are considered important determinants of local adaptation. We investigated how the shape of the spatial dispersal network within experimental landscapes affected local adaptation of a bacteriophage parasite to its bacterial host. Regardless of landscape topology, dispersal always led to the evolution of phages with broader infectivity range. However, when the spatial dispersal network resulted in spatial variation in the breadth of phage infectivity range, significant levels of parasite local adaptation and local maladaptation were detected within the same landscape using the local versus foreign definition of local adaptation. By contrast, local adaptation was not detected using the home versus away or local versus global definitions of local adaptation. This suggests that spatial dispersal networks may play an important role in driving parasite local adaptation, particularly when the shape of the dispersal network generates nonuniform levels of host resistance or parasite infectivity throughout a species’ range.     

Mutations in MAP3K1 cause 46,XY disorders of sex development and implicate a common signal transduction pathway in human testis determination

Investigations of humans with disorders of sex development (DSDs) resulted in the discovery of many of the now-known mammalian sex-determining genes, including SRY, RSPO1, SOX9, NR5A1, WT1, NR0B1, and WNT4. Here, the locus for an autosomal sex-determining gene was mapped via linkage analysis in two families with 46,XY DSD to the long arm of chromosome 5 with a combined, multipoint parametric LOD score of 6.21. A splice-acceptor mutation (c.634-8T>A) in MAP3K1 segregated with the phenotype in the first family and disrupted RNA splicing. Mutations were demonstrated in the second family (p.Gly616Arg) and in two of 11 sporadic cases (p.Leu189Pro, p.Leu189Arg)-18% prevalence in this cohort of sporadic cases. In cultured primary lymphoblastoid cells from family 1 and the two sporadic cases, these mutations altered the phosphorylation of the downstream targets, p38 and ERK1/2, and enhanced binding of RHOA to the MAP3K1 complex. Map3k1 within the syntenic region was expressed in the embryonic mouse gonad prior to, and after, sex determination. Thus, mutations in MAP3K1 that result in 46,XY DSD with partial or complete gonadal dysgenesis implicate this pathway in normal human sex determination.     

Seasonal shifts in giant panda feeding behavior: relationships to bamboo plant part consumption

The giant panda (Ailuropoda melanoleuca) is classified as a carnivore, yet subsists on a diet comprised almost exclusively of bamboo. Wild and captive giant pandas use highly selective foraging behaviors for processing and consuming bamboo. These behaviors are for the first time quantified in captive giant pandas over a 5‐year period of time showing highly specific seasonal trends. Giant panda feeding behavior was recorded using live video observations of two giant pandas housed at the Memphis Zoo from November 2003 to June 2008. Leaf was the primary plant part consumed from June to December, whereas culm was consumed primarily from February to May, with both bears displaying similar seasonal shifts in plant part consumption. From May to June, leaf consumption increased significantly (P‐values<0.001); from June to August, leaf consumption remained high and stable. From December to March, leaf consumption decreased significantly (P‐values<0.001). Specific behaviors for bamboo leaf and culm consumption were also observed. Both bears formed wads of leaves before ingestion while feeding on leaf, but the male employed this feeding behavior more often than the female (54 and 33%, respectively). Both bears used similar culm‐stripping behavior (26 and 25%), used to remove the outer layer and isolate the pith for consumption. This study indicates that unique seasonal foraging behaviors observed in wild pandas are also apparent in captive animals in relation to plant part selectivity and feeding behaviors. Zoo Biol 29:470–483, 2010. © 2009 Wiley‐Liss, Inc.     

Crystal Structures of an Oligopeptide-Binding Protein from the Biosynthetic Pathway of the β-Lactamase Inhibitor Clavulanic Acid

Clavulanic acid (CA) is a clinically important β-lactamase inhibitor that is produced by fermentation of Streptomyces clavuligerus. The CA biosynthesis pathway starts from arginine and glyceraldehyde-3-phosphate and proceeds via (3S,5S)-clavaminic acid, which is converted to (3R,5R)-clavaldehyde, the immediate precursor of (3R,5R)-CA. Open reading frames 7 (orf7) and 15 (orf15) of the CA biosynthesis cluster encode oligopeptide-binding proteins (OppA1 and OppA2), which are essential for CA biosynthesis. OppA1/2 are proposed to be involved in the binding and/or transport of peptides across the S. clavuligerus cell membrane. Peptide binding assays reveal that recombinant OppA1 and OppA2 bind di-/tripeptides containing arginine and certain nonapeptides including bradykinin. Crystal structures of OppA2 in its apo form and in complex with arginine or bradykinin were solved to 1.45, 1.7, and 1.7 Å resolution, respectively. The overall fold of OppA2 consists of two lobes with a deep cavity in the center, as observed for other oligopeptide-binding proteins. The large cavity creates a peptide/arginine binding cleft. The crystal structures of OppA2 in complex with arginine or bradykinin reveal that the C-terminal arginine of bradykinin binds similarly to arginine. The results are discussed in terms of the possible roles of OppA1/2 in CA biosynthesis.     

Sulbutiamine Counteracts Trophic Factor Deprivation Induced Apoptotic Cell Death in Transformed Retinal Ganglion Cells

Sulbutiamine is a highly lipid soluble synthetic analogue of vitamin B₁ and is used clinically for the treatment of asthenia. The aim of our study was to demonstrate whether sulbutiamine is able to attenuate trophic factor deprivation induced cell death to transformed retinal ganglion cells (RGC-5). Cells were subjected to serum deprivation for defined periods and sulbutiamine at different concentrations was added to the cultures. Various procedures (e.g. cell viability assays, apoptosis assay, reactive oxygen species analysis, Western blot analysis, flow cytometric analysis, glutathione (GSH) and glutathione-S-transferase (GST) measurement) were used to demonstrate the effect of sulbutiamine. Sulbutiamine dose-dependently attenuated apoptotic cell death induced by serum deprivation and stimulated GSH and GST activity. Moreover, sulbutiamine decreased the expression of cleaved caspase-3 and AIF. This study demonstrates for the first time that sulbutiamine is able to attenuate trophic factor deprivation induced apoptotic cell death in neuronal cells in culture.     

Reduced versican cleavage due to Adamts9 haploinsufficiency is associated with cardiac and aortic anomalies

Here, we demonstrate that ADAMTS9, a highly conserved versican-degrading protease, is required for correct cardiovascular development and adult homeostasis. Analysis of Adamts9^+/LacZ adult mice revealed anomalies in the aortic wall, valvulosinus and valve leaflets. Abnormal myocardial projections and ‘spongy’ myocardium consistent with non-compaction of the left ventricle were also found in Adamts9^+/LacZ mice. During development, Adamts9 was expressed in derivatives of the Secondary Heart Field, vascular smooth muscle cells in the arterial wall, mesenchymal cells of the valves, and non-myocardial cells of the ventricles, but expression also continued in the adult heart and ascending aorta. Thus, the adult cardiovascular anomalies found in Adamts9^+/LacZ hearts could result from subtle developmental alterations in extracellular matrix remodeling or defects in adult homeostasis. The valvular and aortic anomalies of Adamts9^+/LacZ hearts were associated with accumulation of versican and a decrease in cleaved versican relative to WT littermates. These data suggest a potentially important role for ADAMTS9 cleavage of versican, or other, as yet undefined substrates in development and allostasis of cardiovascular extracellular matrix. In addition, these studies identify ADAMTS9 as a potential candidate gene for congenital cardiac anomalies. Mouse models of ADAMTS9 deficiency may be useful to study myxomatous valve degeneration.