Peptide Ligands for Pro-survival Protein Bfl-1 from Computationally Guided Library Screening

Pro-survival members of the Bcl-2 protein family inhibit cell death by binding short helical BH3 motifs in pro-apoptotic proteins. Mammalian pro-survival proteins Bcl-x[subscript L], Bcl-2, Bcl-w, Mcl-1, and Bfl-1 bind with varying affinities and specificities to native BH3 motifs, engineered peptides, and small molecules. Biophysical studies have determined interaction patterns for these proteins, particularly for the most-studied family members Bcl-x[subscript L] and Mcl-1. Bfl-1 is a pro-survival protein implicated in preventing apoptosis in leukemia, lymphoma, and melanoma. Although Bfl-1 is a promising therapeutic target, relatively little is known about its binding preferences. We explored the binding of Bfl-1 to BH3-like peptides by screening a peptide library that was designed to sample a high degree of relevant sequence diversity. Screening using yeast-surface display led to several novel high-affinity Bfl-1 binders and to thousands of putative binders identified through deep sequencing. Further screening for specificity led to identification of a peptide that bound to Bfl-1 with K[subscript d] < 1 nM and very slow dissociation from Bfl-1 compared to other pro-survival Bcl-2 family members. A point mutation in this sequence gave a peptide with ~50 nM affinity for Bfl-1 that was selective for Bfl-1 in equilibrium binding assays. Analysis of engineered Bfl-1 binders deepens our understanding of how the binding profiles of pro-survival proteins differ and may guide the development of targeted Bfl-1 inhibitors.National Institute of General Medical Sciences (U.S.) (Award GM084181)National Institute of General Medical Sciences (U.S.) (Award P50-GM68762

Indigenous Children’s Language Practices in Australia

While the documentation of Australian Aboriginal and Torres Strait Islander languages has attracted considerable research attention, the use of these languages by children has only recently emerged as a field of research. Building on the small number of early studies of these children’s language acquisition, development, and practices, we review the now considerable variety of studies which have explored Australian Aboriginal children’s early language learning environments and processes. In this ecologically complex linguistic environment, studies investigate children’s acquisition of some remaining traditional languages—often in multilingual contexts, child-directed speech styles and practices, and the development of new and emerging contact languages—both mixed languages and creoles, and the ways that children and young people are altering and innovating the language ecologies. The studies focus particularly on those children who are being raised in remote settings where, while English is taught in school, it is neither the language the children learn as their first language nor the language of the community in which the children live

Borrowing contextual inflection: evidence from northern Australia

Gurindji Kriol is a north Australian mixed language which combines lexical and structural elements from Gurindji (Pama-Nyungan), and Kriol (English-lexifier). One of the more striking features of the grammar of Gurindji Kriol is the presence of the Gurindji case paradigm including ergative and dative case-markers within a Kriol verbal frame. Given the fragility of inflectional morphology in other language contact situations, particularly contextual inflections such as structural case markers, this situation bears closer scrunity. This paper argues that the presence of Gurindji case morphology is the result of pervasive code-switching practices which immediately preceded the genesis of the mixed language. As the code-switching stabilised into a mixed language, case-marking was integrated into predicate argument structure of Gurindji Kriol via nominal adjunct structures. Yet, these case markers were not absorbed unscathed. Although the Gurindji Kriol case paradigm bears a close resemblance to its Gurindji source in form, these case markers have not been perfectly replicated in function and distribution. Contact with Kriol functional equivalents such as prepositions and word order have altered the function and distribution of these case markers. The last part of this paper examines the shift that has occurred in Gurindji-derived case morphology in Gurindji Kriol. © 2010 Springer Science+Business Media B.V

Comparison of binding characteristics and in vitro activities of three inhibitors of vascular endothelial growth factor A.

The objectives of this study were to evaluate the relative binding and potencies of three inhibitors of vascular endothelial growth factor A (VEGF), used to treat neovascular age-related macular degeneration, and assess their relevance in the context of clinical outcome. Ranibizumab is a 48 kDa antigen binding fragment, which lacks a fragment crystallizable (Fc) region and is rapidly cleared from systemic circulation. Aflibercept, a 110 kDa fusion protein, and bevacizumab, a 150 kDa monoclonal antibody, each contain an Fc region. Binding affinities were determined using Biacore analysis. Competitive binding by sedimentation velocity analytical ultracentrifugation (SV-AUC) was used to support the binding affinities determined by Biacore of ranibizumab and aflibercept to VEGF. A bovine retinal microvascular endothelial cell (BREC) proliferation assay was used to measure potency. Biacore measurements were format dependent, especially for aflibercept, suggesting that biologically relevant, true affinities of recombinant VEGF (rhVEGF) and its inhibitors are yet to be determined. Despite this assay format dependency, ranibizumab appeared to be a very tight VEGF binder in all three formats. The results are also very comparable to those reported previously.1-3 At equivalent molar ratios, ranibizumab was able to displace aflibercept from preformed aflibercept/VEGF complexes in solution as assessed by SV-AUC, whereas aflibercept was not able to significantly displace ranibizumab from preformed ranibizumab/VEGF complexes. Ranibizumab, aflibercept, and bevacizumab showed dose-dependent inhibition of BREC proliferation induced by 6 ng/mL VEGF, with average IC50 values of 0.088 ± 0.032, 0.090 ± 0.009, and 0.500 ± 0.091 nM, respectively. Similar results were obtained with 3 ng/mL VEGF. In summary Biacore studies and SV-AUC solution studies show that aflibercept does not bind with higher affinity than ranibizumab to VEGF as recently reported,4 and both inhibitors appeared to be equipotent with respect to their ability to inhibit VEGF function