We present a novel structure determination approach that exploits the global orientational restraints from RDCs to resolve ambiguous NOE assignments. Unlike traditional approaches that bootstrap the initial fold from ambiguous NOE assignments, we start by using RDCs to compute accurate secondary structure element (SSE) backbones at the beginning of structure calculation. Our structure determination package, called RDC-PANDA (RDC-based SSE PAcking with NOEs for Structure Determination and NOE Assignment), consists of three modules: (1) RDC-EXACT; (2) PACKER; and (3) HANA (HAusdorff-based NOE Assignment). RDC-EXACT computes the global optimal solution of backbone dihedral angles for each secondary structure element by exactly solving a system of quartic RDC equations derived by Wang and Donald (2004a,b), and systematically searching over the roots, each of which is a backbone dihedral ϕ-or ψ-angle consistent with the RDC data. Using a small number of unambiguous inter-SSE NOEs extracted using only chemical shift information, PACKER performs a systematic search for the core structure, including all SSE backbone conformations. HANA uses a Hausdorff-based scoring function to measure the similarity between the experimental spectra and the back-computed NOE pattern for each side-chain from a statistically-diverse rotamer library, and drives the selection of optimal position-specific rotamers for filtering ambiguous NOE assignments. Finally, a local minimization approach is used to compute the loops and refine side-chain conformations by fixing the core structure as a rigid body while allowing movement of loops and side-chains. RDC-PANDA was applied to NMR data for the FF Domain 2 of human transcription elongation factor CA150 (RNA polymerase II C-terminal domain interacting protein), human ubiquitin, the ubiquitin-binding zinc finger domain of the human Y-family DNA polymerase Eta (pol η UBZ), and the human Set2-Rpb1 interacting domain (hSRI). These results demonstrated the efficiency and accuracy of our algorithm, and show that RDC-PANDA can be successfully applied for highresolution protein structure determination using only a limited set of NMR data by first computing RDC-defined backbones.

The mechanical response of a multiwalled carbon nanospring was examined with an atomic force microscope. Cantilever deflection, oscillation amplitude, and resonance were simultaneously monitored during the cycled movement of the scanner. A nonlinear response of the nanospring was observed, consistent with compression and buckling of the nanospring. This is the first reported measurement of a shift in the cantilever resonance frequency as a result of the interaction of a nanospring-tipped cantilever with the substrate.

Transforming growth factor-alpha (TGFA) has been shown to play a role in experimental chronic kidney disease associated with nephron reduction, while its role in diabetic kidney disease (DKD) is unknown. We show here that intrarenal mRNA expression, as well as urine and serum TGFA, are increased in human DKD. We used a TGFA neutralizing antibody to determine the role of TGFA in two models of renal disease, the remnant surgical reduction model and the uninephrectomized (uniNx) DKD model. In addition, the contribution of TGFA to DKD progression was examined using an adeno-associated virus approach to increase circulating TGFA in experimental DKD. In vivo blockade of TGFA attenuated kidney disease progression in both nondiabetic nephron reduction and Type 2 diabetic uniNx models, whereas overexpression of TGFA in uniNx model accelerated renal disease. Therapeutic activity of the TGFA antibody was enhanced with renin angiotensin system inhibition with further improvement in renal parameters. These findings suggest a pathologic contribution of TGFA in DKD and support the possibility that therapeutic administration of neutralizing antibodies could provide a novel treatment for the disease.

At least seven distinct epidermal growth factor (EGF) ligands bind to and activate the EGF receptor (EGFR). This activation plays an important role in the embryo and in the maintenance of adult tissues. Importantly, pharmacologic EGFR inhibition also plays a critical role in the pathophysiology of diverse disease states, especially cancer. The roles of specific EGFR ligands are poorly defined in these disease states. Accumulating evidence suggests a role for transforming growth factor a (TGFa) in skin, lung, and kidney disease. To explore the role of Tgfa, we generated a monoclonal antibody (mAb41) that binds to and neutralizes human Tgfa with high affinity (K D 5 36.5 pM). The antibody also binds human epiregulin (Ereg) (K D 5 346.6 pM) and inhibits ligand induced myofibroblast cell proliferation (IC 50 values of 0.52 and 1.12 nM for human Tgfa and Ereg, respectively). In vivo, a single administration of the antibody to pregnant mice (30 mg/kg s.c. at day 14 after plug) or weekly administration to neonate mice (20 mg/kg s.c. for 4 weeks) phenocopy Tgfa knockout mice with curly whiskers, stunted growth, and expansion of the hypertrophic zone of growth plate cartilage. Humanization of this monoclonal antibody to a human IgG4 antibody (LY3016859) enables clinical development. Importantly, administration of the humanized antibody to cynomolgus monkeys is absent of the skin toxicity observed with current EGFR inhibitors used clinically and no other pathologies were noted, indicating that neutralization of Tgfa could provide a relatively safe profile as it advances in clinical development.