The Role of Viral Population Diversity in Adaptation of Bovine Coronavirus to New Host Environments

The high mutation rate of RNA viruses enables a diverse genetic population of viral genotypes to exist within a single infected host. In-host genetic diversity could better position the virus population to respond and adapt to a diverse array of selective pressures such as host-switching events. Multiple new coronaviruses, including SARS, have been identified in human samples just within the last ten years, demonstrating the potential of coronaviruses as emergent human pathogens. Deep sequencing was used to characterize genomic changes in coronavirus quasispecies during simulated host-switching. Three bovine nasal samples infected with bovine coronavirus were used to infect human and bovine macrophage and lung cell lines. The virus reproduced relatively well in macrophages, but the lung cell lines were not infected efficiently enough to allow passage of non lab-adapted samples. Approximately 12 kb of the genome was amplified before and after passage and sequenced at average coverages of nearly 950×(454 sequencing) and 38,000×(Illumina). The consensus sequence of many of the passaged samples had a 12 nucleotide insert in the consensus sequence of the spike gene, and multiple point mutations were associated with the presence of the insert. Deep sequencing revealed that the insert was present but very rare in the unpassaged samples and could quickly shift to dominate the population when placed in a different environment. The insert coded for three arginine residues, occurred in a region associated with fusion entry into host cells, and may allow infection of new cell types via heparin sulfate binding. Analysis of the deep sequencing data indicated that two distinct genotypes circulated at different frequency levels in each sample, and support the hypothesis that the mutations present in passaged strains were “selected” from a pre-existing pool rather than through de novo mutation and subsequent population fixation.     

Pre-clinical and clinical significance of heparanase in Ewing's sarcoma

Heparanase is an endoglycosidase that specifically cleaves heparan sulphate side chains of heparan sulphate proteoglycans, activity that is strongly implicated in cell migration and invasion associated with tumour metastasis, angiogenesis and inflammation. Heparanase up-regulation was documented in an increasing number of human carcinomas, correlating with reduced post-operative survival rate and enhanced tumour angiogenesis. Expression and significance of heparanase in human sarcomas has not been so far reported. Here, we applied the Ewing's sarcoma cell line TC71 and demonstrated a potent inhibition of cell invasion in vitro and tumour xenograft growth in vivo upon treatment with a specific inhibitor of heparanase enzymatic activity (compound SST0001, non-anticoagulant N-acetylated, glycol split heparin). Next, we examined heparanase expression and cellular localization by immunostaining of a cohort of 69 patients diagnosed with Ewing's sarcoma. Heparanase staining was noted in all patients. Notably, heparanase staining intensity correlated with increased tumour size (P = 0.04) and with patients' age (P = 0.03), two prognostic factors associated with a worse outcome. Our study indicates that heparanase expression is induced in Ewing's sarcoma and associates with poor prognosis. Moreover, it encourages the inclusion of heparanase inhibitors (i.e. SST0001) in newly developed therapeutic modalities directed against Ewing's sarcoma and likely other malignancies.     

pH-sensitive pHLIP® coated niosomes

Nanomedicine is becoming very popular over conventional methods due to the ability to tune physico-chemical properties of nano vectors, which are used for encapsulation of therapeutic and diagnostic agents. However, the success of nanomedicine primarily relies on how specifically and efficiently nanocarriers can target pathological sites to minimize undesirable side effects and enhance therapeutic efficacy. Here, we introduce a novel class of targeted nano drug delivery system, which can be used as an effective nano-theranostic for cancer. We formulated pH-sensitive niosomes (80–90?nm in diameter) using nonionic surfactants Span20 (43–45?mol%), cholesterol (50?mol%) and 5?mol% of pH (Low) insertion peptide (pHLIP) conjugated with DSPE lipids (DSPE-pHLIP) or hydrophobic fluorescent dye, pyrene, (Pyr-pHLIP). In coating of niosomes, pHLIP was used as an acidity sensitive targeting moiety. We have demonstrated that pHLIP coated niosomes sense the extracellular acidity of cancerous cells. Intravenous injection of fluorescently labeled (R18) pHLIP-coated niosomes into mice bearing tumors showed significant accumulation in tumors with minimal targeting of kidney, liver and muscles. Tumor-targeting niosomes coated with pHLIP exhibited 2–3 times higher tumor uptake compared to the non-targeted niosomes coated with PEG polymer. Long circulation time and uniform bio-distribution throughout the entire tumor make pHLIP-coated niosomes to be an attractive novel delivery system.     

High-dose,unlabeled, nonspecific antibody pretreatment: influence on specific antibody localization to human melanoma xenografts

Summary Nonspecific uptake of radiolabeled monoclonal antibodies in normal tissues is a significant problem for tumor imaging. A potential means of decreasing nonspecific antibody binding is to blockade nonspecific antibody binding sites by predosing with cold, nonspecific isotypematched antibody, before injecting specific antibody. Nontumor-specific murine monoclonal antibody LK2H10 (IgG1) or Ab-1 (IgG2a) was given i.v. at doses of 0 to 3.5 mg to nude mice with xenografts of human melanoma. These mice were then given i.v. 4 g of ¹³¹I anti-high molecular weight antigen of melanoma (HMWMAA) monoclonal antibody 763.24T (IgG1) or 225.28S (IgG2a), respectively. These mice were also given a tracer dose of ¹²⁵I LK2H10 or Ab-1, respectively. Specific tumor uptake of anti-HMWMAA antibodies was see in all cases. No drop in tumor or nontumor uptake was demonstrated for either of the tumor-specific or nonspecific monoclonal antibodies due to nonspecific monoclonal antibody pretreatment. These data suggest that high doses of isotype-matched unlabeled nonspecific monoclonal antibody given before ¹³¹I tumor-specific monoclonal antibody, will not enhance tumor imaging. Present address: Hybritech, San Diego, CA, USA     

Exposure and Multiple Lines of Evidence Assessment of Risk for PCBs Found in the Diets of Passerine Birds at the Kalamazoo River Superfund Site,Michigan

Dietary exposures of passerine birds at the Kalamazoo River, Michigan, were examined due to the presence of polychlorinated biphenyls (PCBs) in the terrestrial and aquatic food webs. Average potential daily doses in diets were 6- to 29-fold and 16- to 35-fold greater at a contaminated location than at a reference location for PCB exposures quantified as total PCBs and 2,3,7,8–tetrachlorodibenzo-p-dioxin equivalents (TEQs), respectively. Birds with diets comprised of primarily aquatic insects had greater dietary exposure than birds with diets of primarily terrestrial insects. Risk associated with dietary exposure varied with the selection of the threshold for effects including hazard quotients, which exceeded 1 in instances where the most conservative toxicity reference values were utilized. Risk based on concentrations of PCBs in the tissues indicated little risk to avian species, and co-located studies evaluating reproductive health did not suggest that observed incidences of diminished reproductive success were related to PCB exposure. Measures of risk based on comparison to toxicity reference values (TRVs) were consistent with direct measures of ecologically relevant endpoints of reproductive fitness, but uncertainty exists in the selection of threshold values for effects in these species especially based on TEQs. This is largely due to the absence of species-specific, dose-response relationships. Therefore, the best estimate of risk is through the application of multiple lines of evidence.     

Silencing of Ribosomal Protein S9 Elicits a Multitude of Cellular Responses Inhibiting the Growth of Cancer Cells Subsequent to p53 Activation

Background

Disruption of the nucleolus often leads to activation of the p53 tumor suppressor pathway through inhibition of MDM2 that is mediated by a limited set of ribosomal proteins including RPL11 and RPL5. The effects of ribosomal protein loss in cultured mammalian cells have not been thoroughly investigated. Here we characterize the cellular stress response caused by depletion of ribosomal protein S9 (RPS9).

Methodology/Principal Findings

Depletion of RPS9 impaired production of 18S ribosomal RNA and induced p53 activity. It promoted p53-dependent morphological differentiation of U343MGa Cl2:6 glioma cells as evidenced by intensified expression of glial fibrillary acidic protein and profound changes in cell shape. U2OS osteosarcoma cells displayed a limited senescence response with increased expression of DNA damage response markers, whereas HeLa cervical carcinoma cells underwent cell death by apoptosis. Knockdown of RPL11 impaired p53-dependent phenotypes in the different RPS9 depleted cell cultures. Importantly, knockdown of RPS9 or RPL11 also markedly inhibited cell proliferation through p53-independent mechanisms. RPL11 binding to MDM2 was retained despite decreased levels of RPL11 protein following nucleolar stress. In these settings, RPL11 was critical for maintaining p53 protein stability but was not strictly required for p53 protein synthesis.

Conclusions

p53 plays an important role in the initial restriction of cell proliferation that occurs in response to decreased level of RPS9. Our results do not exclude the possibility that other nucleolar stress sensing molecules act upstream or in parallel to RPL11 to activate p53. Inhibiting the expression of certain ribosomal proteins, such as RPS9, could be one efficient way to reinitiate differentiation processes or to induce senescence or apoptosis in rapidly proliferating tumor cells.     

Spectral studies of the Ca2+-dependent interaction of trifluoperazine with S100b

S100b is a calcium-binding protein that will bind to many calmodulin target molecules in a Ca²⁺-dependent manner. In order to study the Ca²⁺-dependent binding properties of S100b, its interaction with a calmodulin antagonist, trifluoperazine (TFP), was investigated using [¹⁹F]- and [¹H]-NMR and UV-difference spectroscopy. It was estimated from [¹⁹F]-NMR that in the absence of Ca²⁺, thek _1/2 value of TFP was 130 µM, while itsk _1/2 value decreased to 28 µM in the presence of Ca²⁺. The addition of KCl was not antagonistic to the Ca²⁺-dependent interaction of TFP to S100b. The chemical exchange rate of TFP with Ca²⁺-bound S100b was estimated to be 9×10² sec^?1. By comparison with TFP-calmodulin exchange rates, it is suggested that the TFP-binding site on S100b is structurally different from its binding sites on calmodulin. Proton NMR resonance broadening in the range 6.8–7.2 ppm, corresponding to phenylalanine nuclei of S100b, indicates that these residues may be involved in TFP binding. Addition of Ca²⁺ to a 1:1 mixture of S100b and TFP resulted in a red-shifted UV-difference spectrum, while no significant difference spectrum was detected when Mg²⁺ was added to a S100b-TFP solution. Thus, we suggest that Ca²⁺ induces the exposure of a hydrophobic domain on S100b containing one or more phenylalanine residues that will bind TFP but that this domain is different from the hydrophobic domain on calmodulin.     

Expression Study on D123 Gene Product: Evidence for High Positivity in Testis

A novel 44-kDa gene product (D123) has been proposed as necessary for S-phase entry of the cell cycle: a point mutation resulted in a temperature-sensitive arrest in G1-phase. From human fibrosarcoma cDNA library, we have isolated an identical gene and studied its sequence and mRNA and protein expression. Compared withD123,three nucleotide differences within the human coding sequence, plus others, result in a change of two amino acids. A partial sequence similarity has been found with a yeast gene of unknown function. The protein has several potential phosphorylation sites, is highly hydrophilic, and may be highly structured in α-helix. The mRNA is abundantly expressed by a variety of normal and transformed cells and by all tissues examined, being most highly expressed in testis. Specific antibodies, raised against a rhD123 polypeptide, recognize a major 42- to 44-kDa molecule in crude extract of various human cell lines. Immunohistochemistry reveals that D123 protein is not homogeneously expressed: it is detected, often in granular vescicles, in the cytoplasm of some epithelial, stromal, and sperm cells and in varicosities lining nervous fibers, while it appears to be absent in nuclei, endothelial, and smooth muscle cells. The precise link between cytoplasmic occurrence of D123 and cell cycle progression still remains to be clarified.     

Anxiolytic Properties of New Chemical Entity, 5TIO1

2-[(2,6-dichlorobenzylidene)amino]-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carbonitrile), 5TIO1, is a new 2-aminothiophene derivative with a promising pharmacological activity. The aim of this work was to evaluate the potential anxiolytic effect of 5TIO1 in animal models. In the elevated plus-maze test, 5TIO1 (0.1, 1.0 and 10.0 mg/kg, i.p) increased the time of permanence and the number of entries in the open arms. In the light/dark box test, 5TIO1 at dose of 0.1 mg/kg (i.p) also showed anxiolytic-like effect indicated by an increase in the time spent in the light box, similar to diazepam 2.0 mg/kg (i.p). 5TIO1 groups did not change locomotor and coordination activities in open field and rotarod tests, respectively, when compared to vehicle. Dose dependent process was not observed and the anxiolytic effects demonstrated were not completely reversed by flumazenil 25 mg/kg (i.p). Our results suggest that 5TIO1 can bind with other receptors, besides the benzodiazepine site of the GABA receptor in mouse brain.