Developmental and Radiobiologic Characteristics of Canine Multinucleated, Osteoclast-Like Cells Generated in Vitro from Canine Bone Marrow

We report here our initial observations on the growth and morphology, and developmental radiosensitivity of giant, multinucleated, osteoclast-like cells (MN-OS} generated through in vitro cultivation of hematopoietic progenitor-enriched canine bone marrow samples. Maximum cell densities of 5.5 x 103 to 6.5 x 103 MN-OS per cm2 of growth area were achieved following 10 to 14 days of culture at 37°C. Acute gamma irradiation of the initial marrow inocula resulted in significant, dose-dependent perturbations of MN-OS formation, growth, and development. Attempts to estimate radiosensitivity of MN-OS progenitors from canine marrow yielded a range of DO values from a low of 212 cGy measured at six days of culture to higher values of 405 to 542 cGy following 10 to 22 days of culture. At the intermediate times of culture (10 to 14 days}, the radiation-induced responses were clearly biphasic, reflecting either (a) the presence of multiple subpopulations of MN-OS progenitors with varying degrees of radiosensitivity or (b) the inherent biphasic nature of MN-OS development involving early progenitor cell proliferation followed by maturation and subsequent fusion. Morphologically, MN-OS generated from irradiated marrow inocula appeared only marginally altered, with alterations expressed largely in a biphasic, dose-dependent fashion in terms of smaller cell size, reduced number of nuclei, increased expression of both surface microprojections, and a unique set of crystalloid cytoplasmic inclusions. Functionally, MN-OS appeared to be impaired by irradiation of marrow progenitors, as evidenced by failure to initiate resorptive attachments to devitalized bone spicules in vitro

Modifying the stereochemistry of an enzyme-catalyzed reaction by directed evolution

Aldolases have potential as tools for the synthesis of stereochemically complex carbohydrates. Here, we show that directed evolution can be used to alter the stereochemical course of the reaction catalyzed by tagatose-1,6-bisphosphate aldolase. After three rounds of DNA shuffling and screening, the evolved aldolase showed an 80-fold improvement in k-cat/K-m toward the non-natural substrate fructose 1,6-bisphosphate, resulting in a 100-fold change in stereospecificity. (31)P NMR spectroscopy was used to show that, in the synthetic direction, the evolved aldolase catalyzes the formation of carbon—carbon bonds with unnatural diastereoselectivity, where the >99:<1 preference for the formation of tagatose 1,6-bisphosphate was switched to a 4:1 preference for the diastereoisomer, fructose 1,6-bisphosphate. This demonstration is of considerable significance to synthetic chemists requiring efficient syntheses of complex stereoisomeric products, such as carbohydrate mimetics

Extracellular superoxide dismutase (SOD3) regulates oxidative stress at the vitreoretinal interface

Oxidative stress is a pathogenic feature in vitreoretinal disease. However, the ability of the inner retina to manage metabolic waste and oxidative stress is unknown. Proteomic analysis of antioxidants in the human vitreous, the extracellular matrix opposing the inner retina, identified superoxide dismutase-3 (SOD3) that localized to a unique matrix structure in the vitreous base and cortex. To determine the role of SOD3, Sod3-/- mice underwent histological and clinical phenotyping. Although the eyes were structurally normal, at the vitreoretinal interface Sod3-/- mice demonstrated higher levels of 3-nitrotyrosine, a key marker of oxidative stress. Pattern electroretinography also showed physiological signaling abnormalities within the inner retina. Vitreous biopsies and epiretinal membranes collected from patients with diabetic vitreoretinopathy (DVR) and a mouse model of DVR showed significantly higher levels of nitrates and/or 3-nitrotyrosine oxidative stress biomarkers suggestive of SOD3 dysfunction. This study analyzes the molecular pathways that regulate oxidative stress in human vitreous substructures. The absence or dysregulation of the SOD3 antioxidant at the vitreous base and cortex results in increased oxidative stress and tissue damage to the inner retina, which may underlie DVR pathogenesis and other vitreoretinal diseases

Galleria mellonella as an alternative in vivo model to study bacterial biofilms on stainless steel and titanium implants

The purpose of this study was to establish an infection model of Galleria mellonella larvae as an alternative in vivo model for biofilm-associated infections on stainless steel and titanium implants. First, the model was established with bacteria-free implants to evaluate the biocompatibility of implants in the larvae. Titanium or stainless steel implants were implanted without any adverse effects over the entire observation period of 5 days compared to controls. Then, stainless steel and titanium implants pre-incubated with Staphylococcus aureus were implanted into the larvae to mimic biofilm-associated infection. For both materials, pre-incubation of the implant with S. aureus led to significantly reduced survival of the larvae compared to bacteria-free implants. Survival rates of the larvae could not be improved in this biofilm infection situation by the addition of gentamicin, whereas gentamicin could significantly improve the survival of the larvae in case of planktonic infection of the larvae with S. aureus without an implant, confirming the typical characteristics of reduced antibiotic susceptibility of biofilm infections. Additionally, biofilm formation and various stages of biofilm maturation were confirmed by surface electron microscopy and by measuring gene expression of biofilm-related genes with the pre-incubated implant, which showed strong biofilm formation and upregulation of autolysin (atl) and sarA genes. In conclusion, G. mellonella can be used as an alternative in vivo model to study biofilm-associated infections on stainless steel and titanium implants, which may help to reduce animal infection experiments with vertebrates in the future

The Key Events Dose-Response Framework: Its Potential for Application to Foodborne Pathogenic Microorganisms

The Key Events Dose-Response Framework (KEDRF) is an analytical approach that facilitates the use of currently available data to gain insight regarding dose-response relationships. The use of the KEDRF also helps identify critical knowledge gaps that once filled, will reduce reliance on assumptions. The present study considers how the KEDRF might be applied to pathogenic microorganisms, using fetal listeriosis resulting from maternal ingestion of food contaminated with L. monocytogenes as an initial example. Major biological events along the pathway between food ingestion and the endpoint of concern are systematically considered with regard to dose (i.e., number of organisms), pathogen factors (e.g., virulence), and protective host mechanisms (e.g., immune response or other homeostatic mechanisms). It is concluded that the KEDRF provides a useful structure for systematically evaluating the complex array of host and pathogen factors that influence the dose-response relationship. In particular, the KEDRF supports efforts to specify and quantify the sources of variability, a prerequisite to strengthening the scientific basis for food safety decision making

Evaluation of natural and tracer fluorescent emission methods for droplet size measurements in a diesel spray

The final publication is available at Springer via http://dx.doi.org/10.1007/s12239-012-0070-zSpray sizing that records fluorescent emission and scattered light has been widely applied to spray diagnostics over the last two decades. Different experimental strategies have been developed, but comparing the different solutions offered has remained of interest to experimentalists. In this work, a comparison of two fluorescence strategies for measuring droplet size in the liquid phase of a last-generation DI diesel spray is conducted. The natural fluorescent emission of a commercial diesel fuel and the fluorescence emitted by a tracer (Rhodamine B) are compared using theoretical and experimental approaches. The LIF/Mie ratio commonly called Planar Droplet Sizing (PDS) technique is applied in two different ways to elucidate the possible advantages of using a fluorescent dopant. The sprays were injected under non-evaporative conditions into a constant pressure vessel that simulates densities present at the moment of injection in currently used passenger car diesel engines. Characterization of the signal properties was performed by measuring the absorption coefficient, fluorescence emission spectrum, quantum yield and lifetime of both configurations. The scattered light and fluorescence intensities were calculated to verify the dependencies of the droplet surface and volume. When applying the two techniques to quantify droplet size in dense diesel sprays, the results show that signal weakness and lack of control over the properties of natural fluorescence produce distortion in the shape of the spray and cause measurements to be unreliable. © 2012 The Korean Society of Automotive Engineers and Springer-Verlag Berlin Heidelberg.This research has been funded in the frame of the project PROFUEL reference TRA2011-26293 from Ministerio de Ciencia e Innovacion. 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Gene expression profiles in human gastric cancer: expression of maspin correlates with lymph node metastasis

To seek for a candidate gene that would regulate tumour progression and metastasis in gastric cancer, we investigated gene expression profiles by using DNA microarray. Tumour tissue and adjacent normal tissue were obtained from 21 patients with gastric cancer and then examined for their gene expression profiles by the Gene Chip® Human U95Av2 array, which includes 12 000 human genes and EST sequences. A total of 25 genes were upregulated and two genes were downregulated by at least four-fold in the tumour tissue. In a further analysis according to lymph node metastasis, the expressed levels of maspin, as well as carcinoembryonic antigen and nonspecific crossreacting antigen were significantly higher in tumours with lymph node metastasis than in those without it. Maspin expression in 85 gastric cancer patients was further investigated by using immunohistochemistry. Maspin expression was not observed in normal gastric epithelia without intestinal metaplasia. In contrast, maspin was expressed in 74 of 85 tumour tissues. There was a significant correlation between the incidence of maspin-positive tumour staining and lymph node metastasis. These results suggest that maspin has a potential role for tumour metastasis in gastric cancer

DNA methylation changes in ovarian cancer are cumulative with disease progression and identify tumor stage

<p>Abstract</p> <p>Background</p> <p>Hypermethylation of promoter CpG islands with associated loss of gene expression, and hypomethylation of CpG-rich repetitive elements that may destabilize the genome are common events in most, if not all, epithelial cancers.</p> <p>Methods</p> <p>The methylation of 6,502 CpG-rich sequences spanning the genome was analyzed in 137 ovarian samples (ten normal, 23 low malignant potential, 18 stage I, 16 stage II, 54 stage III, and 16 stage IV) ranging from normal tissue through to stage IV cancer using a sequence-validated human CpG island microarray. The microarray contained 5' promoter-associated CpG islands as well as CpG-rich satellite and Alu repetitive elements.</p> <p>Results</p> <p>Results showed a progressive de-evolution of normal CpG methylation patterns with disease progression; 659 CpG islands showed significant loss or gain of methylation. Satellite and Alu sequences were primarily associated with loss of methylation, while promoter CpG islands composed the majority of sequences with gains in methylation. Since the majority of ovarian tumors are late stage when diagnosed, we tested whether DNA methylation profiles could differentiate between normal and low malignant potential (LMP) compared to stage III ovarian samples. We developed a class predictor consisting of three CpG-rich sequences that was 100% sensitive and 89% specific when used to predict an independent set of normal and LMP samples versus stage III samples. Bisulfite sequencing confirmed the NKX-2-3 promoter CpG island was hypermethylated with disease progression. In addition, 5-aza-2'-deoxycytidine treatment of the ES2 and OVCAR ovarian cancer cell lines re-expressed NKX-2-3. Finally, we merged our CpG methylation results with previously published ovarian expression microarray data and identified correlated expression changes.</p> <p>Conclusion</p> <p>Our results show that changes in CpG methylation are cumulative with ovarian cancer progression in a sequence-type dependent manner, and that CpG island microarrays can rapidly discover novel genes affected by CpG methylation in clinical samples of ovarian cancer.</p

Maspin is a tumour suppressor that inhibits breast cancer tumour metastasis in vivo

Maspin is a member of the serpin family of serine proteases and functions as a tumour suppressor. A study using a new syngeneic mouse model for breast cancer suggests that maspin can inhibit metastasis in vivo