Parametric study of the potential for BWR ECCS strainer blockage due to LOCA generated debris. Final report

This report documents a plant-specific study for a BWR/4 with a Mark I containment that evaluated the potential for LOCA generated debris and the probability of losing long term recirculation capability due ECCS pump suction strainer blockage. The major elements of this study were: (1) acquisition of detailed piping layouts and installed insulation details for a reference BWR; (2) analysis of plant specific piping weld failure probabilities to estimate the LOCA frequency; (3) development of an insulation and other debris generation and drywell transport models for the reference BWR; (4) modeling of debris transport in the suppression pool; (5) development of strainer blockage head loss models for estimating loss of NPSH margin; (6) estimation of core damage frequency attributable to loss of ECCS recirculation capability following a LOCA. Elements 2 through 5 were combined into a computer code, BLOCKAGE 2.3. A point estimate of overall DEGB pipe break frequency (per Rx-year) of 1.59E-04 was calculated for the reference plant, with a corresponding overall ECCS loss of NPSH frequency (per Rx-year) of 1.58E-04. The calculated point estimate of core damage frequency (per Rx-year) due to blockage related accident sequences for the reference BWR ranged from 4.2E-06 to 2.5E-05. The results of this study show that unacceptable strainer blockage and loss of NPSH margin can occur within the first few minutes after ECCS pumps achieve maximum flows when the ECCS strainers are exposed to LOCA generated fibrous debris in the presence of particulates (sludge, paint chips, concrete dust). Generic or unconditional extrapolation of these reference plant calculated results should not be undertaken

Targeting of Pseudorabies Virus Structural Proteins to Axons Requires Association of the Viral Us9 Protein with Lipid Rafts

The pseudorabies virus (PRV) Us9 protein plays a central role in targeting viral capsids and glycoproteins to axons of dissociated sympathetic neurons. As a result, Us9 null mutants are defective in anterograde transmission of infection in vivo. However, it is unclear how Us9 promotes axonal sorting of so many viral proteins. It is known that the glycoproteins gB, gC, gD and gE are associated with lipid raft microdomains on the surface of infected swine kidney cells and monocytes, and are directed into the axon in a Us9-dependent manner. In this report, we determined that Us9 is associated with lipid rafts, and that this association is critical to Us9-mediated sorting of viral structural proteins. We used infected non-polarized and polarized PC12 cells, a rat pheochromocytoma cell line that acquires many of the characteristics of sympathetic neurons in the presence of nerve growth factor (NGF). In these cells, Us9 is highly enriched in detergent-resistant membranes (DRMs). Moreover, reducing the affinity of Us9 for lipid rafts inhibited anterograde transmission of infection from sympathetic neurons to epithelial cells in vitro. We conclude that association of Us9 with lipid rafts is key for efficient targeting of structural proteins to axons and, as a consequence, for directional spread of PRV from pre-synaptic to post-synaptic neurons and cells of the mammalian nervous system

ChemBank: a small-molecule screening and cheminformatics resource database

ChemBank (http://chembank.broad.harvard.edu/) is a public, web-based informatics environment developed through a collaboration between the Chemical Biology Program and Platform at the Broad Institute of Harvard and MIT. This knowledge environment includes freely available data derived from small molecules and small-molecule screens and resources for studying these data. ChemBank is unique among small-molecule databases in its dedication to the storage of raw screening data, its rigorous definition of screening experiments in terms of statistical hypothesis testing, and its metadata-based organization of screening experiments into projects involving collections of related assays. ChemBank stores an increasingly varied set of measurements derived from cells and other biological assay systems treated with small molecules. Analysis tools are available and are continuously being developed that allow the relationships between small molecules, cell measurements, and cell states to be studied. Currently, ChemBank stores information on hundreds of thousands of small molecules and hundreds of biomedically relevant assays that have been performed at the Broad Institute by collaborators from the worldwide research community. The goal of ChemBank is to provide life scientists unfettered access to biomedically relevant data and tools heretofore available primarily in the private sector

Over-expression of adenosine deaminase in mouse podocytes does not reverse puromycin aminonucleoside resistance

<p>Abstract</p> <p>Background</p> <p>Edema in nephrotic syndrome results from renal retention of sodium and alteration of the permeability properties of capillaries. Nephrotic syndrome induced by puromycin aminonucleoside (PAN) in rats reproduces the biological and clinical signs of the human disease, and has been widely used to identify the cellular mechanisms of sodium retention. Unfortunately, mice do not develop nephrotic syndrome in response to PAN, and we still lack a good mouse model of the disease in which the genetic tools necessary for further characterizing the pathophysiological pathway could be used. Mouse resistance to PAN has been attributed to a defect in glomerular adenosine deaminase (ADA), which metabolizes PAN. We therefore attempted to develop a mouse line sensitive to PAN through induction of normal adenosine metabolism in their podocytes.</p> <p>Methods</p> <p>A mouse line expressing functional ADA under the control of the podocyte-specific podocin promoter was generated by transgenesis. The effect of PAN on urinary excretion of sodium and proteins was compared in rats and in mice over-expressing ADA and in littermates.</p> <p>Results</p> <p>We confirmed that expression of ADA mRNAs was much lower in wild type mouse than in rat glomerulus. Transgenic mice expressed ADA specifically in the glomerulus, and their ADA activity was of the same order of magnitude as in rats. Nonetheless, ADA transgenic mice remained insensitive to PAN treatment in terms of both proteinuria and sodium retention.</p> <p>Conclusions</p> <p>Along with previous results, this study shows that adenosine deaminase is necessary but not sufficient to confer PAN sensitivity to podocytes. ADA transgenic mice could be used as a background strain for further transgenesis.</p

Impact of Transmammary-Delivered Meloxicam on Biomarkers of Pain and Distress in Piglets after Castration and Tail Docking

To investigate a novel route for providing analgesia to processed piglets via transmammary drug delivery, meloxicam was administered orally to sows after farrowing. The objectives of the study were to demonstrate meloxicam transfer from sows to piglets via milk and to describe the analgesic effects in piglets after processing through assessment of pain biomarkers and infrared thermography (IRT). Ten sows received either meloxicam (30 mg/kg) (n = 5) or whey protein (placebo) (n = 5) in their daily feedings, starting four days after farrowing and continuing for three consecutive days. During this period, blood and milk samples were collected at 12-hour intervals. On Day 5 after farrowing, three boars and three gilts from each litter were castrated or sham castrated, tail docked, and administered an iron injection. Piglet blood samples were collected immediately before processing and at predetermined times over an 84-hour period. IRT images were captured at each piglet blood collection point. Plasma was tested to confirm meloxicam concentrations using a validated high-performance liquid chromatography-mass spectrometry method. Meloxicam was detected in all piglets nursing on medicated sows at each time point, and the mean (± standard error of the mean) meloxicam concentration at castration was 568.9±105.8 ng/mL. Furthermore, ex-vivo prostaglandin E2(PGE2) synthesis inhibition was greater in piglets from treated sows compared to controls (p = 0.0059). There was a time-by-treatment interaction for plasma cortisol (p = 0.0009), with meloxicam-treated piglets demonstrating lower cortisol concentrations than control piglets for 10 hours after castration. No differences in mean plasma substance P concentrations between treatment groups were observed (p = 0.67). Lower cranial skin temperatures on IRT were observed in placebo compared to meloxicam-treated piglets (p = 0.015). This study demonstrates the successful transfer of meloxicam from sows to piglets through milk and corresponding analgesia after processing, as evidenced by a decrease in cortisol and PGE2levels and maintenance of cranial skin temperature

Drug-mediated inhibition of Fli-1 for the treatment of leukemia

The Ets transcription factor, Fli-1 is activated in murine erythroleukemia and overexpressed in various human malignancies including Ewing's sarcoma, induced by the oncogenic fusion protein EWS/Fli-1. Recent studies by our group and others have demonstrated that Fli-1 plays a key role in tumorigenesis, and disrupting its oncogenic function may serve as a potential treatment option for malignancies associated with its overexpression. Herein, we describe the discovery of 30 anti-Fli-1 compounds, characterized into six functional groups. Treatment of murine and human leukemic cell lines with select compounds inhibits Fli-1 protein or mRNA expression, resulting in proliferation arrest and apoptosis. This anti-cancer effect was mediated, at least in part through direct inhibition of Fli-1 function, as anti-Fli-1 drug treatment inhibited Fli-1 DNA binding to target genes, such as SHIP-1 and gata-1, governing hematopoietic differentiation and proliferation. Furthermore, treatment with select Fli-1 inhibitors revealed a positive relationship between the loss of DNA-binding activity and Fli-1 phosphorylation. Accordingly, anti-Fli-1 drug treatment significantly inhibited leukemogenesis in a murine erythroleukemia model overexpressing Fli-1. This study demonstrates the ability of this drug-screening strategy to isolate effective anti-Fli-1 inhibitors and highlights their potential use for the treatment of malignancies overexpressing this oncogene

Acatalasemic mice are mildly susceptible to adriamycin nephropathy and exhibit increased albuminuria and glomerulosclerosis

Background: Catalase is an important antioxidant enzyme that regulates the level of intracellular hydrogen peroxide and hydroxyl radicals. The effects of catalase deficiency on albuminuria and progressive glomerulosclerosis have not yet been fully elucidated. The adriamycin (ADR) nephropathy model is considered to be an experimental model of focal segmental glomerulosclerosis. A functional catalase deficiency was hypothesized to exacerbate albuminuria and the progression of glomerulosclerosis in this model. Methods: ADR was intravenously administered to both homozygous acatalasemic mutant mice (C3H/AnLCs(b)Cs(b)) and control wild-type mice (C3H/AnLCs(a)Cs(a)). The functional and morphological alterations of the kidneys, including albuminuria, renal function, podocytic, glomerular and tubulointerstitial injuries, and the activities of catalase were then compared between the two groups up to 8 weeks after disease induction. Moreover, the presence of a mutation of the toll-like receptor 4 (tlr4) gene, which was previously reported in the C3H/HeJ strain, was investigated in both groups. Results: The ADR-treated mice developed significant albuminuria and glomerulosclerosis, and the degree of these conditions in the ADR-treated acatalasemic mice was higher than that in the wild-type mice. ADR induced progressive renal fibrosis, renal atrophy and lipid peroxide accumulation only in the acatalasemic mice. In addition, the level of catalase activity was significantly lower in the kidneys of the acatalasemic mice than in the wild-type mice during the experimental period. The catalase activity increased after ADR injection in wild-type mice, but the acatalasemic mice did not have the ability to increase their catalase activity under oxidative stress. The C3H/AnL strain was found to be negative for the tlr4 gene mutation. Conclusions: These data indicate that catalase deficiency plays an important role in the progression of renal injury in the ADR nephropathy model

Incompatibilities Involving Yeast Mismatch Repair Genes: A Role for Genetic Modifiers and Implications for Disease Penetrance and Variation in Genomic Mutation Rates

Genetic background effects underlie the penetrance of most genetically determined phenotypes, including human diseases. To explore how such effects can modify a mutant phenotype in a genetically tractable system, we examined an incompatibility involving the MLH1 and PMS1 mismatch repair genes using a large population sample of geographically and ecologically diverse Saccharomyces cerevisiae strains. The mismatch repair incompatibility segregates into naturally occurring yeast strains, with no strain bearing the deleterious combination. In assays measuring the mutator phenotype conferred by different combinations of MLH1 and PMS1 from these strains, we observed a mutator phenotype only in combinations predicted to be incompatible. Surprisingly, intragenic modifiers could be mapped that specifically altered the strength of the incompatibility over a 20-fold range. Together, these observations provide a powerful model in which to understand the basis of disease penetrance and how such genetic variation, created through mating, could result in new mutations that could be the raw material of adaptive evolution in yeast populations

Recombinant Human Endostatin Endostar Inhibits Tumor Growth and Metastasis in a Mouse Xenograft Model of Colon Cancer

To investigate the effects of recombinant human endostatin Endostar on metastasis and angiogenesis and lymphangiogenesis of colorectal cancer cells in a mouse xenograft model. Colon cancer cells SW620 were injected subcutaneously into the left hind flank of nude mice to establish mouse xenograft models. The mice were treated with normal saline or Endostar subcutaneously every other day. The growth and lymph node metastasis of tumor cells, angiogenesis and lymphangiogenesis in tumor tissue were detected. Apoptosis and cell cycle distribution were studied by flow cytometry. The expression of VEGF-A, -C, or -D in SW620 cells was determined by immunoblotting assays. Endostar inhibited tumor growth and the rate of lymph node metastasis (P < 0.01). The density of blood vessels in or around the tumor area was 12.27 ± 1.21 and 22.25 ± 2.69 per field in Endostar-treated mice and controls (P < 0.05), respectively. Endostar also decreased the density of lymphatic vessels in tumor tissues (7.84 ± 0.81 vs. 13.83 ± 1.08, P < 0.05). Endostar suppresses angiogenesis and lymphangiogenesis in the lymph nodes with metastases, simultaneously. The expression of VEGF-A, -C and -D in SW620 cells treated with Endostar was substantially lower than that of controls. Endostar inhibited growth and lymph node metastasis of colon cancer cells by inhibiting angiogenesis and lymphangiogenesis in a mouse xenograft model of colon cancer

A Genome-Wide Analysis Reveals No Nuclear Dobzhansky-Muller Pairs of Determinants of Speciation between S. cerevisiae and S. paradoxus, but Suggests More Complex Incompatibilities

The Dobzhansky-Muller (D-M) model of speciation by genic incompatibility is widely accepted as the primary cause of interspecific postzygotic isolation. Since the introduction of this model, there have been theoretical and experimental data supporting the existence of such incompatibilities. However, speciation genes have been largely elusive, with only a handful of candidate genes identified in a few organisms. The Saccharomyces sensu stricto yeasts, which have small genomes and can mate interspecifically to produce sterile hybrids, are thus an ideal model for studying postzygotic isolation. Among them, only a single D-M pair, comprising a mitochondrially targeted product of a nuclear gene and a mitochondrially encoded locus, has been found. Thus far, no D-M pair of nuclear genes has been identified between any sensu stricto yeasts. We report here the first detailed genome-wide analysis of rare meiotic products from an otherwise sterile hybrid and show that no classic D-M pairs of speciation genes exist between the nuclear genomes of the closely related yeasts S. cerevisiae and S. paradoxus. Instead, our analyses suggest that more complex interactions, likely involving multiple loci having weak effects, may be responsible for their post-zygotic separation. The lack of a nuclear encoded classic D-M pair between these two yeasts, yet the existence of multiple loci that may each exert a small effect through complex interactions suggests that initial speciation events might not always be mediated by D-M pairs. An alternative explanation may be that the accumulation of polymorphisms leads to gamete inviability due to the activities of anti-recombination mechanisms and/or incompatibilities between the species' transcriptional and metabolic networks, with no single pair at least initially being responsible for the incompatibility. After such a speciation event, it is possible that one or more D-M pairs might subsequently arise following isolation