An essential saccharide binding domain for the mAb 2C7 established for Neisseria gonorrhoeae LOS by ES-MS and MSn

A study of bacterial surface oligosaccharides were investigated among different strains of Neisseria gonorrhoeae to correlate structural features essential for binding to the MAb 2C7. This epitope is widely expressed and conserved in gonococcal isolates, characteristics essential to an effective candidate vaccine antigen. Sample lipooligosaccharides (LOS), was prepared by a modification of the hot phenol-water method from which de-O-acetylated LOS and oligosaccharide (OS) components were analyzed by ES-MS-CID-MS and ES-MSnin a triple quadrupole and an ion trap mass spectrometer, respectively. Previously documented natural heterogeneity was apparent from both LOS and OS preparations which was admixed with fragments induced by hydrazine and mild acid treatment. Natural heterogeneity was limited to phosphorylation and antenni extensions to the alpha-chain. Mild acid hydrolysis to release OS also hydrolyzed the beta(1-->6) glycosidic linkage of lipid A. OS structures were determined by collisional and resonance excitation combined with MS and multistep MSn which provided sequence information from both neutral loss, and nonreducing terminal fragments. A comparison of OS structures, with earlier knowledge of MAb binding, enzyme treatment, and partial acid hydrolysis indicates a generic overlapping domain for 2C7 binding. Reoccurring structural features include a Hepalpha(1-->3)Hepbeta(1-->5)KDO trisaccharide core branched on the nonreducing terminus (Hep-2) with an alpha(1-->2) linked GlcNAc (gamma-chain), and an alpha-linked lactose (beta-chain) residue. From the central heptose (Hep-1), a beta(1-->4) linked lactose (alpha-chain), moiety is required although extensions to this residue appear unnecessary.      

Intrinsic Protein Fluorescence Interferes with Detection of Tear Glycoproteins in SDS-Polyacrylamide Gels Using Extrinsic Fluorescent Dyes

Intrinsic protein fluorescence may interfere with the visualization of proteins after SDS-polyacrylamide electrophoresis. In an attempt to analyze tear glycoproteins in gels, we ran tear samples and stained the proteins with a glycoprotein-specific fluorescent dye. The fluorescence detected was not limited to glycoproteins. There was strong intrinsic fluorescence of proteins normally found in tears after soaking the gels in 40% methanol plus 1-10% acetic acid and, to a lesser extent, in methanol or acetic acid alone. Nanograms of proteins gave visible native fluorescence and interfere with extrinsic fluorescent dye detection. Poly-L-lysine, which does not contain intrinsically fluorescent amino acids, did not fluoresce.     

Measurement of microstructural strain in cortical bone

It is well known that mechanical factors affect bone remodeling such that increased mechanical demand results in net bone formation, whereas decreased demand results in net bone resorption. Current theories suggest that bone modeling and remodeling is controlled at the cellular level through signals mediated by osteocytes. The objective of this study was to investigate how macroscopically applied bone strains similar in magnitude to those that occur in vivo are manifest at the microscopic level in the bone matrix. Using a digital image correlation strain measurement technique, experimentally determined bone matrix strains around osteocyte lacuna resulting from macroscopic strains of approximately 2,000 microstrain (0.2%) reach levels of over 30,000 microstrain (3%) over fifteen times greater than the applied macroscopic strain. Strain patterns were highly heterogeneous and in some locations similar to observed microdamage around osteocyte lacuna indicating the resulting strains may represent the precursors to microdamage. This information may lead to a better understanding of how bone cells are affected by whole bone functional loading.     

Tissue strain amplification at the osteocyte lacuna: a microstructural finite element analysis

A parametric finite element model of an osteocyte lacuna was developed to predict the microstructural response of the lacuna to imposed macroscopic strains. The model is composed of an osteocyte lacuna, a region of perilacunar tissue, canaliculi, and the surrounding bone tissue. A total of 45 different simulations were modeled with varying canalicular diameters, perilacunar tissue material moduli, and perilacunar tissue thicknesses. Maximum strain increased with a decrease in perilacunar tissue modulus and decreased with an increase in perilacunar tissue modulus, regardless of the thickness of the perilacunar region. An increase in the predicted maximum strain was observed with an increase in canalicular diameter from 0.362 to 0.421 microm. In response to the macroscopic application of strain, canalicular diameters increased 0.8% to over 1.0% depending on the perilacunar tissue modulus. Strain magnification factors of over 3 were predicted. However, varying the size of the perilacunar tissue region had no effect on the predicted perilacunar tissue strain. These results indicate that the application of average macroscopic strains similar to strain levels measured in vivo can result in significantly greater perilacunar tissue strains and canaliculi deformations. A decrease in the perilacunar tissue modulus amplifies the perilacunar tissue strain and canaliculi deformation while an increase in the local perilacunar tissue modulus attenuates this effect.     

Development of a parametric finite element model of the proximal femur using statistical shape and density modelling

Skeletal fractures associated with bone mass loss are a major clinical problem and economic burden, and lead to significant morbidity and mortality in the ageing population. Clinical image-based measures of bone mass show only moderate correlative strength with bone strength. However, engineering models derived from clinical image data predict bone strength with significantly greater accuracy. Currently, image-based finite element (FE) models are time consuming to construct and are non-parametric. The goal of this study was to develop a parametric proximal femur FE model based on a statistical shape and density model (SSDM) derived from clinical image data. A small number of independent SSDM parameters described the shape and bone density distribution of a set of cadaver femurs and captured the variability affecting proximal femur FE strength predictions. Finally, a three-dimensional FE model of an 'unknown' femur was reconstructed from the SSDM with an average spatial error of 0.016 mm and an average bone density error of 0.037 g/cm(3).     

Machine vision photogrammetry: a technique for measurement of microstructural strain in cortical bone.

Understanding local microstructural deformations and strains in cortical bone may lead to a better understanding of cortical bone damage development, fracture, and remodeling. Traditional experimental techniques for measuring deformation and strain do not allow characterization of these quantities at the microstructural level in cortical bone. This study describes a technique based on digital stereoimaging used to measure the microstructural strain fields in cortical bone. The technique allows the measurement of material surface displacements and strains by comparing images acquired from a specimen at two distinct stress states. The accuracy of the system is investigated by analyzing an undeformed image set; the test image is identical to the reference image but translated by a known pixel amount. An increase in the correlation sub-image train parameter results in an increase in displacement measurement accuracy from 0.049 to 0.012 pixels. Errors in strain calculated from the measured displacement field were between 39 and 564 microstrain depending upon the sub-image train size and applied image displacement. The presence of a microcrack in cortical bone results in local strain at the crack tip reaching 0.030 (30,000 microstrain) and 0.010 (10,000 microstrain) near osteocyte lacunae. It is expected that the use of this technique will allow a greater understanding of bone strength and fracture as well as bone mechanotransduction.