Structural model of a2-subunit N-terminus and its binding interface for Arf-GEF CTH2: Implication for regulation of V-ATPase, CTH2 function and rational drug design

We have previously identified the interaction between mammalian V-ATPase a2-subunit isoform and cytohesin-2 (CTH2) and studied molecular details of binding between these proteins. In particular, we found that six peptides derived from the N-terminal cytosolic domain of a2 subunit (a2N1–402) are involved in interaction with CTH2 (Merkulova, Bakulina, Thaker, Grüber, & Marshansky, 2010). However, the actual 3D binding interface was not determined in that study due to the lack of high-resolution structural information about a-subunits of V-ATPase. Here, using a combination of homology modeling and NMR analysis, we generated the structural model of complete a2N1–402 and uncovered the CTH2-binding interface. First, using the crystal-structure of the bacterial M. rubber Icyt-subunit of A-ATPase as a template (Srinivasan, Vyas, Baker, & Quiocho, 2011), we built a homology model of mammalian a2N1–352 fragment. Next, we combined it with the determined NMR structures of peptides a2N368–395 and a2N386–402 of the C-terminal section of a2N1–402. The complete molecular model of a2N1–402 revealed that six CTH2 interacting peptides are clustered in the distal and proximal lobe sub-domains of a2N1–402. Our data indicate that the proximal lobe sub-domain is the major interacting site with the Sec7 domain of first CTH2 protein, while the distal lobe sub-domain of a2N1–402 interacts with the PH-domain of second CTH2. Indeed, using Sec7/Arf-GEF activity assay we experimentally confirmed our model. The interface formed by peptides a2N1–17 and a2N35–49 is involved in specific interaction with Sec7 domain and regulation of GEF activity. These data are critical for understanding of the cross-talk between V-ATPase and CTH2 as well as for the rational drug design to regulate their function

Development of a Fully Human Anti-PDGFRβ Antibody That Suppresses Growth of Human Tumor Xenografts and Enhances Antitumor Activity of an Anti-VEGFR2 Antibody

Platelet-derived growth factor receptor β (PDGFRβ) is upregulated in most of solid tumors. It is expressed by pericytes/smooth muscle cells, fibroblast, macrophage, and certain tumor cells. Several PDGF receptor-related antagonists are being developed as potential antitumor agents and have demonstrated promising antitumor activity in both preclinical and clinical settings. Here, we produced a fully human neutralizing antibody, IMC-2C5, directed against PDGFRβ from an antibody phage display library. IMC-2C5 binds to both human and mouse PDGFRβ and blocks PDGF-B from binding to the receptor. IMC-2C5 also blocks ligand-stimulated activation of PDGFRβ and downstream signaling molecules in tumor cells. In animal studies, IMC-2C5 significantly delayed the growth of OVCAR-8 and NCI-H460 human tumor xenografts in nude mice but failed to show antitumor activities in OVCAR-5 and Caki-1 xenografts. Our results indicate that the antitumor efficacy of IMC-2C5 is primarily due to its effects on tumor stroma, rather than on tumor cells directly. Combination of IMC-2C5 and DC101, an anti-mouse vascular endothelial growth factor receptor 2 antibody, resulted in significantly enhanced antitumor activity in BxPC-3, NCI-H460, and HCT-116 xenografts, compared with DC101 alone, and the trend of additive effects to DC101 treatment in several other tumor models. ELISA analysis of NCI-H460 tumor homogenates showed that IMC-2C5 attenuated protein level of vascular endothelial growth factor and basic fibroblast growth factor elevated by DC101 treatment. Finally, IMC-2C5 showed a trend of additive effects when combined with DC101/chemotherapy in MIA-PaCa-2 and NCI-H460 models. Taken together, these results lend great support to the use of PDGFRβ antagonists in combination with other antiangiogenic agents in the treatment of a broad range of human cancers

Promoting Diversity and Inclusivity in DH in Ireland and the UK. Fourth Discussion Paper of the UK-Ireland DH Network

The UK-Ireland Digital Humanities Network is an AHRC/IRC-funded project to undertake research and consultation towards the implementation of a permanent Digital Humanities association for the UK and Ireland. This is the fourth discussion paper produced by the Network, in consultation with the wider Digital Humanities community in the two countries and beyond. It summarises the findings of the fourth workshop organised by the Network, and offers recommendations based on these findings

Capacity Enhancement in Digital Humanities in the United Kingdom and Ireland: Training and Beyond

This first discussion paper, produced by the UK-Ireland Digital Humanities Network in consultation with the wider Digital Humanities (DH) community in the two countries and beyond, summarises the findings of a) the first workshop organised by the network and b) the post-workshop survey and offers recommendations based on these findings

Communicating the Value and Impact of Digital Humanities in Teaching, Research, and Infrastructure Development

This is the second discussion paper produced by the UK-Ireland Digital Humanities Network in consultation with the wider Digital Humanities (DH) Community in the two countries and beyond. It summarises the findings of the second workshop organised by the network, and offers recommendations based on these findings

r84, a Novel Therapeutic Antibody against Mouse and Human VEGF with Potent Anti-Tumor Activity and Limited Toxicity Induction

Vascular endothelial growth factor (VEGF) is critical for physiological and pathological angiogenesis. Within the tumor microenvironment, VEGF functions as an endothelial cell survival factor, permeability factor, mitogen, and chemotactic agent. The majority of these functions are mediated by VEGF-induced activation of VEGF receptor 2 (VEGFR2), a high affinity receptor tyrosine kinase expressed by endothelial cells and other cell types in the tumor microenvironment. VEGF can also ligate other cell surface receptors including VEGFR1 and neuropilin-1 and -2. However, the importance of VEGF-induced activation of these receptors in tumorigenesis is still unclear. We report the development and characterization of r84, a fully human monoclonal antibody that binds human and mouse VEGF and selectively blocks VEGF from interacting with VEGFR2 but does not interfere with VEGF∶VEGFR1 interaction. Selective blockade of VEGF binding to VEGFR2 by r84 is shown through ELISA, receptor binding assays, receptor activation assays, and cell-based functional assays. Furthermore, we show that r84 has potent anti-tumor activity and does not alter tissue histology or blood and urine chemistry after chronic high dose therapy in mice. In addition, chronic r84 therapy does not induce elevated blood pressure levels in some models. The ability of r84 to specifically block VEGF∶VEGFR2 binding provides a valuable tool for the characterization of VEGF receptor pathway activation during tumor progression and highlights the utility and safety of selective blockade of VEGF-induced VEGFR2 signaling in tumors

Prealternate molt-migration in Rusty Blackbirds and its implications for stopover biology

To achieve greater understanding of the full annual cycles of birds, it is critical to describe the spatial nature of little-understood phases. One of the least understood aspects of avian annual cycles is the ecology of molt: the periodic replacement of feathers. While work on the spatial nature of molt in migratory passerines has increasingly found incidences of species and populations completing molt during migration, this work has been limited entirely to prebasic flight feather molt. We examined the prevalence and progression of contour feather molt in a migratory songbird, the Rusty Blackbird (Euphagus carolinus), during spring stopover. We found that 98% of birds exhibited a partial prealternate molt during stopover, primarily in the head region. Furthermore, molt intensity peaked in the middle of the migration period and was negatively associated with fat score. This is the first evidence in the passerine literature of an obligate prealternate molt completed during migration, which is in many ways similar to the molt strategy of a variety of shorebirds (Families Charadriidae and Scolopacidae). These findings could prove crucial to understanding the constraints on spring migration in this declining species. Furthermore, we argue that molt schedules such as those of the Rusty Blackbird and shorebirds should be referred to as “prealternate molt-migration,” broadening the traditional definition of molt-migration beyond prebasic flight feather molt