Determination of the cell tropism of serotype 1 feline infectious peritonitis virus using the spike affinity histochemistry in paraffin‐embedded tissues

Unlike for serotype II feline coronaviruses (FCoV II), the cellular receptor for serotype I FCoV (FCoV I), the most prevalent FCoV serotype, is unknown. To provide a platform for assessing the pattern by which FCoV I attaches to its host receptor(s), HEK293 cell lines that stably express the ectodomains of the spike (S) proteins derived from a FCoV I feline enteric coronavirus strain UU7 (FECV UU7) and a feline infectious peritonitis virus strain UU4 (FIPV UU4) were established. Using the recombinant S proteins as probes to perform S protein affinity histochemistry in paraffin‐embedded tissues, although no tissue or enteric binding of FECV UU7 S protein was detected, it was found that by immunohistochemistry that the tissue distribution of FIPV UU4 S protein‐bound cells correlated with that of FIPV antigen‐positive cells and lesions associated with FIP and that the affinity binding of FIPV UU4 S protein on macrophages was not affected by enzymatic removal of host cell‐surface sialic acid with neuraminidase. These findings suggest that a factor(s) other than sialic acid contribute(s) to the macrophage tropism of FIPV strain UU4. This approach allowed obtaining more information about both virus–host cell interactions and the biological characteristics of the unidentified cellular receptor for FCoV I.

     

Correlation of F4-neuroprostanes levels in cerebrospinal fluid with outcome of aneurysmal subarachnoid hemorrhage in humans

Aneurysmal subarachnoid hemorrhage (aSAH) is one type of hemorrhagic stroke in humans. F₂-isoprostanes (F₂-IsoPs) and F₄-neuroprostanes (F₄-NPs), derived from arachidonic acid and docosahexaenoic acid (DHA), respectively, are specific markers of lipid peroxidation. We previously demonstrated that F₂-IsoPs levels in cerebrospinal fluid (CSF) of aSAH patients positively correlated with poor clinical conditions. In this work, we refined F₄-NPs analysis and investigated the role of potential oxidative damage to neurons in aSAH patients by detecting F₄-NPs in CSF. [²H₄]-15-F_2t-IsoP, rather than [¹⁸O₂]-17-F_4c-NP or [²H₄]-PGF_2α, was used as the internal standard for F₄-NPs analysis. One problem of the use of [¹⁸O₂]-17-F_4c-NP was the potential interference resulting from F₂-dihomo-IsoPs in CSF. CSF specimens of 15 aSAH patients for up to 10 days and those of 12 non-aSAH controls were analyzed. First day, mean, and peak levels of F₄-NPs were all significantly higher in aSAH patients than in controls and correlated with the Fisher Scale and 3-month Glasgow Outcome Scale, but only mean levels of F₄-NPs correlated with Hunt and Hess Grade. The results first demonstrate oxidative damage to DHA in brain tissue following aSAH and suggest that F₄-NPs in CSF could be a better predictor for outcome of aSAH than F₂-IsoPs at early time points.     

Oxidative stress, DNA damage and osmolality in the Pacific white shrimp, Litopenaeus vannamei exposed to acute low temperature stress

To evaluate the genotoxic, physiological and immunological effects of short-term acute low temperature stress on the Pacific white shrimp, Litopenaeus vannamei, we rapidly transferred shrimp from tanks at 23±2 °C to aquaria at the same temperature (controls) or 12±2 °C for 12 h. Changes in the shrimp hemocyte respiratory burst activity and DNA damage were examined during and after exposure to the temperature stress using flow cytometry and the comet assay, respectively. We also monitored changes in the total hemocyte count, malondialdehyde levels, total protein concentration and osmolality in shrimp plasma. The results show that hemocyte respiratory burst activity, malondialdehydes levels and hemocyte DNA damage in the plasma all increased significantly after exposure to 12±2 °C for 3 h. In contrast, total hemocyte count, total protein concentration and osmolality in the plasma decreased compared to the controls. We conclude that acute low temperature can induce oxidative stress, DNA damage, lipid peroxidation and changes in osmolality in L. vannamei.     

Detection of the inferred interaction network in hepatocellular carcinoma from EHCO (Encyclopedia of Hepatocellular Carcinoma genes Online)

Background

An adequate and expressive ontological representation of biological organisms and their parts requires formal reasoning mechanisms for their relations of physical aggregation and containment.

Results

We demonstrate that the proposed formalism allows to deal consistently with "role propagation along non-taxonomic hierarchies", a problem which had repeatedly been identified as an intricate reasoning problem in biomedical ontologies.

Conclusion

The proposed approach seems to be suitable for the redesign of compositional hierarchies in (bio)medical terminology systems which are embedded into the framework of the OBO (Open Biological Ontologies) Relation Ontology and are using knowledge representation languages developed by the Semantic Web community.     

Sam68 regulates a set of alternatively spliced exons during neurogenesis

Sam68 (Src-associated in mitosis, 68 kDa) is a KH domain RNA binding protein implicated in a variety of cellular processes, including alternative pre-mRNA splicing, but its functions are not well understood. Using RNA interference knockdown of Sam68 expression and splicing-sensitive microarrays, we identified a set of alternative exons whose splicing depends on Sam68. Detailed analysis of one newly identified target exon in epsilon sarcoglycan (Sgce) showed that both RNA elements distributed across the adjacent introns and the RNA binding activity of Sam68 are necessary to repress the Sgce exon. Sam68 protein is upregulated upon neuronal differentiation of P19 cells, and many Sam68 RNA targets change in expression and splicing during this process. When Sam68 is knocked down by short hairpin RNAs, many Sam68-dependent splicing changes do not occur and P19 cells fail to differentiate. We also found that the differentiation of primary neuronal progenitor cells from embryonic mouse neocortex is suppressed by Sam68 depletion and promoted by Sam68 overexpression. Thus, Sam68 controls neurogenesis through its effects on a specific set of RNA targets.     

Depletion and refilling of intracellular calcium pools in bovine chromaffin cells

To investigate Ca²⁺ uptake by Ca²⁺-depleted bovine chromaffin cells we depleted these cells of Ca²⁺ by incubating them in Ca²⁺-free buffer, then measured changes in cytoplasmic Ca²⁺ concentration ([Ca²⁺ ₁)⁴⁵Ca²⁺ uptake, and Mn²⁺ uptake in response to added Ca²⁺ or MN²⁺. In depleted cells, the increase in [Ca²⁺]_i after Ca²⁺ addition, and the Mn²⁺ and⁴⁵Ca²⁺ uptakes were higher than in control cells, and were inhibited by verapamil. The size of the intracellular Ca²⁺ pools in depleted cells increased after Ca²⁺ addition. The times for [Ca²⁺]_i rise and Mn²⁺ entry to reach plateau levels were much shorter than the time for refilling of intracellular Ca²⁺ stores. In Ca²⁺-depleted cells and cells which had been loaded with BAPTA,⁴⁵Ca²⁺ uptake was much higher than in control cells. These results suggest that extracellular Ca²⁺ enters the cytoplasm first before refilling the intracellular stores. The rate of Mn²⁺ influx depended on the level of filling of the Ca²⁺ stores, suggesting that some signalling takes place between the intracellular stores and Ca²⁺ entry pathways through the plasma membrane.Abbreviations used BAPTA 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid - BAPTA/AM acetoxymethyl ester of BAPTA - [Ca²⁺]_i cytosolic Ca²⁺ concentration - IP₃ inositol 1,4,5-trisphosphate - tBHQ 2,5-di-(t-butyl)-1,4-benzohydroquinone This work was included in a thesis submitted by A.-L. Sui to the Department of Biochemistry, National Yang-Ming Medical College, in partial fulfillment of the requirements for the degree of Doctor of Philosophy