Molecular mechanics of cartilage : quantification of GAG electrostatic interactions via high-resolution force spectroscopy

Thesis (Sc. D.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2003.Includes bibliographical references (leaves 121-135).Intermolecular repulsion forces between negatively charged glycosaminoglycan (CS-GAG) macromolecules are a major determinant of cartilage biomechanical properties. It is thought that the electrostatic component of the total intermolecular interaction is responsible for 50-75% of the equilibrium elastic modulus of cartilage in compression, while other forces (e.g., steric, hydration, van der Waals, etc.) may also play a role. To investigate these forces, radiolabeled CS-GAG polymer chains were chemically end-grafted to a planar surface to form model biomimetic polyelectrolyte "brush" layers whose environment was varied to mimic physiological conditions. The total intersurface force (<[or equal to] nN) between the CS-GAG brushes and chemically modified probe tips (SO₃⁻ and OH) was measured as a function of tip-substrate separation distance in aqueous solution using the technique of high-resolution force spectroscopy (HRFS). These experiments showed long-range, nonlinear, purely repulsive forces that decreased in magnitude and range with increasing ionic strength and decreasing pH. In order to estimate the contribution of the electrostatic component to the total intersurface force, the data were compared to a theoretical model of electrical double layer repulsion based on the Poisson-Boltzmann formulation. The CS-GAG brush layer was approximated as either a flat surface charge density or a smoothed volume of known fixed charge density and the probe tip was modeled as a smooth hemisphere of constant surface charge density.(cont.) To further closely mimic physiological condition of the cartilage, the CS-GAG molecules were successfully attached to the AFM probe tip using electric field. The CS-GAG modified tip was characterized by measuring force at various environments and its parking density was also estimated using newly developed molecular level model. The measured force between CS-GAG modified tip and CS-GAG modified substrate showed a long-range interaction that significantly dependent on the ionic strength and pH, indicating the significant role of Coulombic interaction between CS-GAG layers. The equilibrium brush height measured using ellipsometry showed that CS-GAG behaves as an annealed polyelectrolyte that reached its maximum brush height around 0.1 M salt concentration. The equilibrium brush height was compared with the onset of the force increase to obtain further insight on the CS-GAG brush behavior during the force measurement.by Joonil Seog.Sc.D

Comprehensive molecular characterization of gastric adenocarcinoma

Gastric cancer is a leading cause of cancer deaths, but analysis of its molecular and clinical characteristics has been complicated by histological and aetiological heterogeneity. Here we describe a comprehensive molecular evaluation of 295 primary gastric adenocarcinomas as part of The Cancer Genome Atlas (TCGA) project. We propose a molecular classification dividing gastric cancer into four subtypes: tumours positive for Epstein–Barr virus, which display recurrent PIK3CA mutations, extreme DNA hypermethylation, and amplification of JAK2, CD274 (also known as PD-L1) and PDCD1LG2 (also knownasPD-L2); microsatellite unstable tumours, which show elevated mutation rates, including mutations of genes encoding targetable oncogenic signalling proteins; genomically stable tumours, which are enriched for the diffuse histological variant and mutations of RHOA or fusions involving RHO-family GTPase-activating proteins; and tumours with chromosomal instability, which show marked aneuploidy and focal amplification of receptor tyrosine kinases. Identification of these subtypes provides a roadmap for patient stratification and trials of targeted therapies

A comparative study of biomolecule and polymer surface modifications by a surface microdischarge

Cold atmospheric plasma (CAP) sources are attractive sources of reactive species with promising industrial and biomedical applications, but an understanding of underlying surface mechanisms is lacking. A kHz-powered surface microdischarge (SMD) operating with N2/O2 mixtures was used to study the biological deactivation of two immune-stimulating biomolecules: lipopolysaccharide (LPS) and peptidoglycan (PGN), found in bacteria such as Escherichia coli and Staphylococcus aureus, respectively. Model polymers were also studied to isolate specific functional groups. Changes in the surface chemistry were measured to understand which plasma-generated species and surface modifications are important for biological deactivation. The overall goal of this work is to determine which effects of CAP treatment are generic and which bonds are susceptible to attack. CAP treatment deactivated biomolecules, oxidized surfaces, and introduced surface bound NO3. These effects can be controlled by the N2 fraction in O2 and applied voltage and vary among different target surfaces. The SMD was compared with an Ar/O2/N2-fed kHz-powered atmospheric pressure plasma jet and showed much higher surface modifications and surface chemistry tunability compared to the jet. Possible mechanisms are discussed and findings are compared with recent computational investigations. Our results demonstrate the importance of long-lived plasma-generated species and advance an atomistic understanding of CAP-surface interactions

Direct Observation of Amyloid Nucleation under Nanomechanical Stretching

Self-assembly of amyloid nanofiber is associated with both functional biological and pathological processes such as those in neurodegenerative diseases. Despite intensive studies, the stochastic nature of the process has made it difficult to elucidate a molecular mechanism for the key amyloid nucleation event. Here we investigated nucleation of the silk-elastin-like peptide (SELP) amyloid using time-lapse lateral force microscopy (LFM). By repeated scanning of a single line on a SELP-coated mica surface, we observed a sudden stepwise height increase. This corresponds to nucleation of an amyloid fiber, which subsequently grew perpendicular to the scanning direction. The lateral force profiles followed either a worm-like chain model or an exponential function, suggesting that the atomic force microscopy (AFM) tip stretches a single or multiple SELP molecules along the scanning direction. The probability of nucleation correlated with the maximum stretching force and extension, implying that stretching of SELP molecules is a key molecular event for amyloid nucleation. The mechanically induced nucleation allows for positional and directional control of amyloid assembly <i>in vitro,</i> which we demonstrate by generating single nanofibers at predetermined nucleation sites