Identification of FAK substrate peptides via colorimetric screening of a one‐bead one‐peptide combinatorial library

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/110814/1/psc2751.pd

Nanoparticle Diffusion in Methycellulose Thermoreversible Association Polymer

Solutions of aqueous methylcellulose, a hydro-phobically modified polymer (molecular weight approximate to 270 kg/mol, methyl content,approximate to 130%), are mixed with either dilute coumarin fluorescent dye or carboxylated latex (20 nm diameter), and the tracer diffusion is contrasted as a function of temperature and polymer concentration (from dilute to 36 times the overlap concentration) in deionized water. From two-photon fluorescence correlation spectroscopy (FCS), mean-square displacement is inferred. At room temperature, which is the fluid state, we observe Fickian diffusion provided that the tracer particle size is less than the polymer mesh size, whereas tighter meshes produce subdiffusion followed by Fickian diffusion at long times. At elevated temperature, which is the gel state, subdiffusion is observed over the entire experimental time window. To quantify subdiffusion, the data are described equally well as two discrete relaxations or a stretched exponential, and the former is analyzed in detail as it is considered to be more meaningful physically. These measurements allow us to discuss the structure and degree of inhomogeneity of methylcellulose in the gel state. This industrially relevant polymer produces simple, physically meaningful diffusion patterns that we find to be repeatable, obeying systematic patterns described quantitatively in this paper

Solid-State Nuclear Magnetic Resonance (NMR) Spectroscopy of Human Immunodeficiency Virus gp41 Protein That Includes the Fusion Peptide: NMR Detection of Recombinant Fgp41 in Inclusion Bodies in Whole Bacterial Cells and Structural Characterization of Purified and Membrane-Associated Fgp41

Human immunodeficiency virus (HIV) infection of a host cell begins with fusion of the HIV and host cell membranes and is mediated by the gp41 protein, a single-pass integral membrane protein of HIV. The 175 N-terminal residues make up the ectodomain that lies outside the virus. This work describes the production and characterization of an ectodomain construct containing the 154 N-terminal gp41 residues, including the fusion peptide (FP) that binds to target cell membranes. The Fgp41 sequence was derived from one of the African clade A strains of HIV-1 that have been less studied than European/North American clade B strains. Fgp41 expression at a level of ∼100 mg/L of culture was evidenced by an approach that included amino acid type ¹³CO and ¹⁵N labeling of recombinant protein and solid-state NMR (SSNMR) spectroscopy of lyophilized whole cells. The approach did not require any protein solubilization or purification and may be a general approach for detection of recombinant protein. The purified Fgp41 yield was ∼5 mg/L of culture. SSNMR spectra of membrane-associated Fgp41 showed high helicity for the residues C-terminal of the FP. This was consistent with a “six-helix bundle” (SHB) structure that is the final gp41 state during membrane fusion. This observation and negligible Fgp41-induced vesicle fusion supported a function for SHB gp41 of membrane stabilization and fusion arrest. SSNMR spectra of residues in the membrane-associated FP provided evidence of a mixture of molecular populations with either helical or β-sheet FP conformation. These and earlier SSNMR data strongly support the existence of these populations in the SHB state of membrane-associated gp41

Feasibility Investigation of Cellulose Polymers for Mucoadhesive Nasal Drug Delivery Applications

The feasibility of various cellulose polymer derivatives, including methylcellulose (MC), hydroxypropyl methylcellulose (HPMC), sodium-carboxymethylcellulose (sodium-CMC), and cationic-hydroxyethylcellulose (cationic-HEC), for use as an excipient to enhance drug delivery in nasal spray formulations was investigated. Three main parameters for evaluating the polymers in nasal drug delivery applications include rheology, ciliary beat frequency (CBF), and permeation across nasal tissue. Reversible thermally induced viscosity enhancement was observed at near nasal physiological temperature when cellulose derivatives were combined with an additional excipient, poly­(vinyl caprolactam)–poly­(vinyl acetate)–poly­(ethylene glycol) graft copolymer (PVCL–PVA–PEG). Cationic-HEC was shown to enhance acyclovir permeation across the nasal mucosa. None of the tested cellulosic polymers caused any adverse effects on porcine nasal tissues and cells, as assessed by alterations in CBF. Upon an increase in polymer concentration, a reduction in CBF was observed when ciliated cells were immersed in the polymer solution, and this decrease returned to baseline when the polymer was removed. While each cellulose derivative exhibited unique advantages for nasal drug delivery applications, none stood out on their own to improve more than one of the performance characteristics examined. Hence, these data may be useful for the development of new cellulose derivatives in nasal drug formulations