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Two-pore physiologically-based pharmacokinetic (PBPK) models can be expected to describe the tissue distribution and elimination kinetics of soluble proteins, endogenous or dosed, as function of their size. In this work, we amalgamated our previous two-pore PBPK model for an inert domain antibody (dAb) in mice with the cross-species platform PBPK model for monoclonal antibodies described in literature into a unified two-pore platform that describes protein modalities of different sizes and includes neonatal Fc receptor (FcRn) mediated recycling. This unified PBPK model was parametrized for organ-specific lymph flow rates and the endosomal recycling rate constant using an extended tissue distribution time-course dataset that included an inert dAb, albumin and IgG in rats and mice. The model was evaluated by comparing the ab initio predictions for the tissue distribution and elimination properties of albumin-binding dAbs (AlbudAbsTM) in mice and rats with the experimental observations. Due to the large number of molecular species and reactions involved in large-scale PBPK models, we have also developed and deployed a MatlabTM script for automating the assembly of SimBiologyTM-based two-pore biologics PBPK models which drastically cuts the time and effort required for model building.

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Although a fairly large number of binary group 15/16 element cations have been reported, no example involving phosphorus in combination with a group 16 element has been synthesized and characterized to date. In this contribution is reported the synthesis and structural characterization of the first example of such a cation, namely a nortricyclane-type [P3Se4](+). This cation has been independently discovered by three groups through three different synthetic routes, as described herein. The molecular and electronic structure of the [P3Se4](+) cage and its crystal properties in the solid state have been characterized comprehensively by using X-ray diffraction, Raman, and nuclear magnetic resonance spectroscopies, as well as quantum chemical calculations.

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A new structural arrangement Te3 (RP(III) )3 and the first crystal structures of organophosphorus(III)-tellurium heterocycles are presented. The heterocycles can be stabilized and structurally characterized by the appropriate choice of substituents in Tem (P(III) R)n (m=1: n=2, R=OMes* (Mes*=supermesityl or 2,4,6-tri-tert-butylphenyl); n=3, R=adamantyl (Ad); n=4, R=ferrocene (Fc); m=n=3: R=trityl (Trt), Mesor by the installation of a P(V) 2 N2 anchor in RP(III) [TeP(V) (tBuN)(µ-NtBu)]2 (R=Ad, tBu).

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Cellular therapies that either use modifications of a patient's own cells or allogeneic cell lines are becoming in vogue. Besides the technical issues of optimal isolation, cultivation and modification, quality control of the generated cellular products are increasingly being considered to be more important. This is not only relevant for the cell's therapeutic application but also for cell science in general. Recent changes in editorial policies of respected journals, which now require proof of authenticity when cell lines are used, demonstrate that the subject of the present paper is not a virtual problem at all. In this article we provide 2 examples of contaminated cell lines followed by a review of the recent developments used to verify cell lines, stem cells and modifications of autologous cells. With relative simple techniques one can now prove the authenticity and the quality of the cellular material of interest and therefore improve the scientific basis for the development of cells for therapeutic applications. The future of advanced cellular therapies will require production and characterization of cells under GMP and GLP conditions, which include proof of identity, safety and functionality and absence of contamination.

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Four new salts of the P2Se8(2-) anion have been prepared, starting from easily available reagents using different reaction strategies including reaction of the elements, oxidation of P4Se3 with alkalimetal diselenides and elemental selenium, and the use of an ionic liquid as a reaction medium. Multinuclear NMR investigations show the presence of both chair-P2Se8(2-) and twist-P2Se8(2-) in solution, with twist-P2Se8(2-) being the predominant conformer. The interconversion between the two conformers is slow on the NMR time scale. Structural investigations of the new salts by single-crystal X-ray diffraction show that chair-P2Se8(2-) is the conformer mostly found in the solid state. A first structural characterization of twist-P2Se8(2-) is reported. The bonding situation in the P2Se8(2-) anion as well as the relative stability of the chair, twist, and boat conformers was elucidated by quantum chemical calculations.

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We describe a method for selecting aggregation-resistant proteins by heat denaturation. This is illustrated with antibody heavy chain variable domains (dAbs), which are prone to aggregate. The dAbs were displayed multivalently at the infective tip of filamentous bacteriophage, and heated transiently to induce unfolding and to promote aggregation of the dAbs. After cooling, the dAbs were selected for binding to protein A (a ligand common to these folded dAbs). Phage displaying dAbs that unfold reversibly were thereby enriched with respect to those that do not. From a repertoire of phage dAbs, six dAbs were characterized after selection; they all resisted aggregation, and were soluble, well expressed in bacteria and could be purified in good yields. The method should be useful for making aggregation-resistant proteins and for helping to identify features that promote or prevent protein aggregation, including those responsible for misfolding diseases.

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The antigen binding site of antibodies usually comprises associated heavy (V(H)) and light (V(L)) chain variable domains, but in camels and llamas, the binding site frequently comprises the heavy chain variable domain only (referred to as V(HH)). In contrast to reported human V(H) domains, V(HH) domains are well expressed from bacteria and yeast, are readily purified in soluble form and refold reversibly after heat-denaturation. These desirable properties have been attributed to highly conserved substitutions of the hydrophobic residues of V(H) domains, which normally interact with complementary V(L) domains. Here, we describe the discovery and characterisation of an isolated human V(H) domain (HEL4) with properties similar to those of V(HH) domains. HEL4 is highly soluble at concentrations of > or =3 mM, essentially monomeric and resistant to aggregation upon thermodenaturation at concentrations as high as 56 microM. However, in contrast to V(HH) domains, the hydrophobic framework residues of the V(H):V(L) interface are maintained and the only sequence changes from the corresponding human germ-line segment (V3-23/DP-47) are located in the loops comprising the complementarity determining regions (CDRs). The crystallographic structure of HEL4 reveals an unusual feature; the side-chain of a framework residue (Trp47) is flipped into a cavity formed by Gly35 of CDR1, thereby increasing the hydrophilicity of the V(H):V(L) interface. To evaluate the specific contribution of Gly35 to domain properties, Gly35 was introduced into a V(H) domain with poor solution properties. This greatly enhanced the recovery of the mutant from a gel filtration matrix, but had little effect on its ability to refold reversibly after heat denaturation. Our results confirm the importance of a hydrophilic V(H):V(L) interface for purification of isolated V(H) domains, and constitute a step towards the design of isolated human V(H) domains with practical properties for immunotherapy.

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We have used NMR to study the effects of peptide binding on the N-terminal p53-binding domain of human MDM2 (residues 25-109). There were changes in HSQC-chemical shifts throughout the domain on binding four different p53-derived peptide ligands that were significantly large to be indicative of global conformational changes. Large changes in chemical shift were observed in two main regions: the peptide-binding cleft that directly binds the p53 ligands; and the hinge regions connecting the beta-sheet and alpha-helical structures that form the binding cleft. These conformational changes reflect the adaptation of the cleft on binding peptide ligands that differ in length and amino acid composition. Different ligands may induce different conformational transitions in MDM2 that could be responsible for its function. The dynamic nature of MDM2 might be important in the design of anti-cancer drugs that are targeted to its p53-binding site.

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The mechanism of assembly of multiprotein complexes and the subsequent organization of activity are not well understood. Here we report the application of biophysical tools to investigate the relationship between structure and function in protein assemblies. We used as a model system the SCF(Skp2) complex that targets p27(Kip1) for ubiquitination and subsequent degradation; this process requires an adapter protein, Cks1. By dissecting the interactions between the different subunits we show that the properties of Cks1 are highly context dependent, and its activity is acquired only when the complex is fully assembled. The results provide insights into the central role of small adapters in macromolecular assembly and explain their high sequence conservation. Simultaneous and synergistic binding of multiple subunits in a complex provides the specificity and control required before the key cell-cycle regulator p27 is committed to degradation.

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The FF domain is a 60 amino acid residue phosphopeptide-binding module found in a variety of eukaryotic proteins including the transcription elongation factor CA150, the splicing factor Prp40 and p190RHOGAP. We have determined the structure of an FF domain from HYPA/FBP11. The domain is composed of three alpha helices arranged in an orthogonal bundle with a 3(10) helix in the loop between the second and third alpha helices. The structure differs from those of other phosphopeptide-binding domains and represents a novel phosphopeptide-binding fold.

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We have investigated the kinetic and thermodynamic basis of the p53-MDM2 interaction using a set of peptides based on residues 15-29 of p53. Wild-type p53 peptide bound MDM2 with a dissociation constant of 580nM. Phosphorylation of S15 and S20 did not affect binding, but T18 phosphorylation weakened binding tenfold, indicating that phosphorylation of only T18 is responsible for abrogating p53-MDM2 binding. Truncation to residues 17-26 increased affinity 13-fold, but further truncation to 19-26 abolished binding. NMR studies of the binding of the p53-derived peptides revealed global conformational changes of the overall structure of MDM2, stretching far beyond the binding cleft, indicating significant changes in the domain dynamics of MDM2 upon ligand binding.

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