Gross Cystic Disease Fluid Protein-15/Prolactin-Inducible Protein as a Biomarker for Keratoconus Disease

Keratoconus (KC) is a bilateral degenerative disease of the cornea characterized by corneal bulging, stromal thinning, and scarring. The etiology of the disease is unknown. In this study, we identified a new biomarker for KC that is present in vivo and in vitro. In vivo, tear samples were collected from age-matched controls with no eye disease (n = 36) and KC diagnosed subjects (n = 17). Samples were processed for proteomics using LC-MS/MS. In vitro, cells were isolated from controls (Human Corneal Fibroblasts-HCF) and KC subjects (Human Keratoconus Cells-HKC) and stimulated with a Vitamin C (VitC) derivative for 4 weeks, and with one of the three transforming growth factor-beta (TGF-β) isoforms. Samples were analyzed using real-time PCR and Western Blots. By using proteomics analysis, the Gross cystic disease fluid protein-15 (GCDFP-15) or prolactin-inducible protein (PIP) was found to be the best independent biomarker able to discriminate between KC and controls. The intensity of GCDFP-15/PIP was significantly higher in healthy subjects compared to KC-diagnosed. Similar findings were seen in vitro, using a 3D culture model. All three TGF-β isoforms significantly down-regulated the expression of GCDFP-15/PIP. Zinc-alpha-2-glycoprotein (AZGP1), a protein that binds to PIP, was identified by proteomics and cell culture to be highly regulated. In this study by different complementary techniques we confirmed the potential role of GCDFP-15/PIP as a novel biomarker for KC disease. It is likely that exploring the GCDFP-15/PIP-AZGP1 interactions will help better understand the mechanism of KC disease.     

Subcellular Metabolite and Lipid Analysis of Xenopus laevis Eggs by LAESI Mass Spectrometry

Xenopus laevis eggs are used as a biological model system for studying fertilization and early embryonic development in vertebrates. Most methods used for their molecular analysis require elaborate sample preparation including separate protocols for the water soluble and lipid components. In this study, laser ablation electrospray ionization (LAESI), an ambient ionization technique, was used for direct mass spectrometric analysis of X. laevis eggs and early stage embryos up to five cleavage cycles. Single unfertilized and fertilized eggs, their animal and vegetal poles, and embryos through the 32-cell stage were analyzed. Fifty two small metabolite ions, including glutathione, GABA and amino acids, as well as numerous lipids including 14 fatty acids, 13 lysophosphatidylcholines, 36 phosphatidylcholines and 29 triacylglycerols were putatively identified. Additionally, some proteins, for example thymosin β4 (Xen), were also detected. On the subcellular level, the lipid profiles were found to differ between the animal and vegetal poles of the eggs. Radial profiling revealed profound compositional differences between the jelly coat vitelline/plasma membrane and egg cytoplasm. Changes in the metabolic profile of the egg following fertilization, e.g., the decline of polyamine content with the development of the embryo were observed using LAESI-MS. This approach enables the exploration of metabolic and lipid changes during the early stages of embryogenesis.     

Detection of True IgE-expressing Mouse B Lineage Cells

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH isotypes, such as IgA, IgE and IgG. Measurement of IgH CSR in vitro is a key method for the study of a number of biologic processes ranging from DNA recombination and repair to aspects of molecular and cellular immunology. In vitro CSR assay involves the flow cytometric measurement surface Ig expression on activated B cells. While measurement of IgA and IgG subclasses is straightforward, measurement of IgE by this method is problematic due to soluble IgE binding to FcεRII/CD23 expressed on the surface of activated B cells. Here we describe a unique procedure for accurate measurement of IgE-producing mouse B cells that have undergone CSR in culture. The method is based on trypsin-mediated cleavage of IgE-CD23 complexes on cell surfaces, allowing for detection of IgE-producing B lineage cells by cytoplasmic staining. This procedure offers a convenient solution for flow cytometric analysis of CSR to IgE.     

Land use impacts on physical properties of 28 years old reclaimed mine soils in Ohio

Reclamation enhances soil quality by improving physical and chemical properties, which helps in restoration of mine soils. Evaluation of the effects of post-reclamation land uses on physical and chemical properties of mine soils helps to identify suitable land uses for mining companies. The objectives of this study were to evaluate the effects of post-reclamation land uses (e.g., forest, hay and pasture) on selected physical properties of soil in relation to undisturbed forest and agricultural land use. Soil samples were collected from the 0- to 5-, 5- to 15- and 15- to 30-cm depths in order to determine particle size distribution, bulk density, water-stable aggregates, mean-weight diameter and soil moisture retention. Cone index and infiltration rate were determined at soil surface. After 28 years of reclamation, bulk density in the surface layer of all land uses in the reclaimed mine soil (RMS) was similar to that of undisturbed forest (1.1 Mg m⁻³) but lower than that of agricultural soils (1.3 Mg m⁻³). However, soil bulk density at lower depths was not affected. The cone index was higher in the RMS-pasture (2.6 MPa) than the RMS-forest (1.4 MPa) and RMS-hay (1.5 MPa) due to the trampling effect of grazing animals. The water-stable aggregates (>2 mm), of 5–8 mm aggregates, were higher in RMS-forest by 24%, 90%, 66%, and under RMS-hay by 13%, 74%, 43% for the 0- to 5-, 5- to 15-, and 15- to 30-cm depths, respectively, than that under undisturbed forest. The mean-weight diameter (0- to 30-cm) of aggregates under RMS-forest and RMS-hay were higher than that under undisturbed forest by 41% and 27%, respectively. The initial infiltration rates at 5 min in RMS under forest, hay and pasture were less by 20%, 53% and 85%, respectively, than that under undisturbed forest (19.3 cm min⁻¹). The reclamation of mine soils with forest and hay improved surface soil bulk density and cone index, and enhanced water infiltration capacity and water-stable aggregates at the lower depths. Therefore, establishment of forest and hay should be encouraged in the RMS.