Extensive Variation in the Activities of Pseudocerastes and Eristicophis Viper Venoms Suggests Divergent Envenoming Strategies Are Used for Prey Capture.

Snakes of the genera Pseudocerastes and Eristicophis (Viperidae: Viperinae) are known as the desert vipers due to their association with the arid environments of the Middle East. These species have received limited research attention and little is known about their venom or ecology. In this study, a comprehensive analysis of desert viper venoms was conducted by visualising the venom proteomes via gel electrophoresis and assessing the crude venoms for their cytotoxic, haemotoxic, and neurotoxic properties. Plasmas sourced from human, toad, and chicken were used as models to assess possible prey-linked venom activity. The venoms demonstrated substantial divergence in composition and bioactivity across all experiments. Pseudocerastes urarachnoides venom activated human coagulation factors X and prothrombin and demonstrated potent procoagulant activity in human, toad, and chicken plasmas, in stark contrast to the potent neurotoxic venom of P. fieldi. The venom of E. macmahonii also induced coagulation, though this did not appear to be via the activation of factor X or prothrombin. The coagulant properties of P. fieldi and P. persicus venoms varied among plasmas, demonstrating strong anticoagulant activity in the amphibian and human plasmas but no significant effect in that of bird. This is conjectured to reflect prey-specific toxin activity, though further ecological studies are required to confirm any dietary associations. This study reinforces the notion that phylogenetic relatedness of snakes cannot readily predict venom protein composition or function. The significant venom variation between these species raises serious concerns regarding antivenom paraspecificity. Future assessment of antivenom is crucial.

Comparison of initial feasibility of host cell lines for viral vaccine production.

In order to reduce the time required for the development and production of viral vaccines, host cell lines should be available as expression systems for production of viral vaccines against groups of viral pathogens. A selection of cell lines was compared for their initial feasibility as expression system for the replication of polioviruses, influenza A viruses and respiratory syncytial virus (wild type strain A2). Six adherent cell lines (Vero, HEK-293, MRC-5, CHO-K1, BHK-21 c13, MDCK) and six single cell suspension cell lines (CAP, AGE1.CR.HS, sCHO-K1, BHK-21 c13 2p, MDCK SFS) were studied for their ability to propagate viruses. First, maximum cell densities were determined. Second, virus receptor expression and polarization of the cell lines regarding receptor distribution of eight different viruses were monitored using flow cytometry and immunocytochemistry. Organization of the actin cytoskeleton was studied by transfection of the cells with Lifeact™, a construct coding for actin-EGFP. Finally, the ability to produce virus progeny of the viruses studied was assayed for each cell line. The results suggest that single cell suspension cell lines grown on serum free medium are the best candidates to serve as host cell lines for virus replication.

Naphthalimide gold(I) phosphine complexes as anticancer metallodrugs.

Gold(I) phosphine complexes exhibit promising properties for anticancer drug development. Here we report on a series of gold(I) phosphine complexes containing a naphthalimide ligand. Strong antiproliferative effects were observed in MCF-7 breast cancer cells as well as in HT-29 colon carcinoma cells. The cellular and nuclear gold levels were increased compared to analogues, in which the naphthalimide ligand was replaced by a chloro ligand. Compound 4a was selected for more detailed biochemical and biological studies, which revealed solvent dependent fluorescence emission, uptake of the compound into the organelles of tumor cells as well as antiangiogenic effects concerning angiogenesis and tumor-induced angiogenesis in vivo. Antiangiogenic properties of 4a were observed in two different zebrafish angiogenesis models, including a tumor-cell induced neovascularization assay.

Retinoic acid receptor antagonists inhibit miR-10a expression and block metastatic behavior of pancreatic cancer.

BACKGROUND & AIMS: The infiltrating ductal adenocarcinoma of the pancreas is among the most lethal of all solid malignancies, largely owing to a high frequency of early metastasis. We identified microRNA-10a (miR-10a) as an important mediator of metastasis formation in pancreatic tumor cells and investigated the upstream and downstream regulatory mechanisms of miR-10a. METHODS: Northern blot analysis revealed increased expression levels of miR-10a in metastatic pancreatic adenocarcinoma. The role of miR-10a was analyzed by Morpholino and short interfering RNA transfection of pancreatic carcinoma cell lines and resected specimens of human pancreatic carcinoma. Metastatic behavior of primary pancreatic tumors and cancer cell lines was tested in xenotransplantation experiments in zebrafish embryos. RESULTS: We show that miR-10a expression promotes metastatic behavior of pancreatic tumor cells and that repression of miR-10a is sufficient to inhibit invasion and metastasis formation. We further show that miR-10a is a retinoid acid target and that retinoic acid receptor antagonists effectively repress miR-10a expression and completely block metastasis. This antimetastatic activity can be prevented by specific knockdown of HOX genes, HOXB1 and HOXB3. Interestingly, suppression of HOXB1 and HOXB3 in pancreatic cancer cells is sufficient to promote metastasis formation. CONCLUSIONS: These findings suggest that miR-10a is a key mediator of metastatic behavior in pancreatic cancer, which regulates metastasis via suppression of HOXB1 and HOXB3. Inhibition of miR-10a expression (with retinoic acid receptor antagonists) or function (with specific inhibitors) is a promising starting point for antimetastatic therapies.

A critical role for myoglobin in zebrafish development.

The globin family, including hemoglobin, myoglobin, neuroglobin and cytoglobin, plays an important role in oxygen storage and delivery. Myoglobin has been shown to be necessary for cardiac function during development, but no information is currently available on the developmental regulation of myoglobin gene expression during embryogenesis. In this study, we used whole mount in situ hybridization to visualize myoglobin mRNA expression during zebrafish development. Our results show for the first time the spatial and temporal gene expression pattern of myoglobin during embryogenesis. Myoglobin was expressed as a maternal RNA and ubiquitous expression was observed until the end of gastrulation. At later stages of development, we discovered novel expression domains for myoglobin, including several non-muscular ones. Environmental stresses, like low oxygen tension (hypoxia) can lead to a developmental delay in zebrafish embryos. We show here that hypoxic stress induces myoglobin expression in skeletal muscle cells of anterior somites and in the dorsal aorta of zebrafish larvae. Finally, we analyzed the role of myoglobins in development by targeted gene knock-down. Silencing myoglobin in zebrafish embryos with gene-specific morpholinos led to a dose dependent curvature, vascular defects, enlarged pericardia and reduction of the gut. In conclusion, our results indicate that myoglobin plays a crucial role in zebrafish development and is important for angiogenesis and gut development.

Metastatic behaviour of primary human tumours in a zebrafish xenotransplantation model.

BACKGROUND: Aberrant regulation of cell migration drives progression of many diseases, including cancer cell invasion and metastasis formation. Analysis of tumour invasion and metastasis in living organisms to date is cumbersome and involves difficult and time consuming investigative techniques. For primary human tumours we establish here a simple, fast, sensitive and cost-effective in vivo model to analyse tumour invasion and metastatic behaviour. METHODS: We fluorescently labelled small explants from gastrointestinal human tumours and investigated their metastatic behaviour after transplantation into zebrafish embryos and larvae. The transparency of the zebrafish embryos allows to follow invasion, migration and micrometastasis formation in real-time. High resolution imaging was achieved through laser scanning confocal microscopy of live zebrafish. RESULTS: In the transparent zebrafish embryos invasion, circulation of tumour cells in blood vessels, migration and micrometastasis formation can be followed in real-time. Xenografts of primary human tumours showed invasiveness and micrometastasis formation within 24 hours after transplantation, which was absent when non-tumour tissue was implanted. Furthermore, primary human tumour cells, when organotopically implanted in the zebrafish liver, demonstrated invasiveness and metastatic behaviour, whereas primary control cells remained in the liver. Pancreatic tumour cells showed no metastatic behaviour when injected into cloche mutant embryos, which lack a functional vasculature. CONCLUSION: Our results show that the zebrafish is a useful in vivo animal model for rapid analysis of invasion and metastatic behaviour of primary human tumour specimen.

A gold(I) phosphine complex containing a naphthalimide ligand functions as a TrxR inhibiting antiproliferative agent and angiogenesis inhibitor.

The novel luminescent gold(I) complex [N-(N',N'-dimethylaminoethyl)-1,8-naphthalimide-4-sulfide](triethylphosphine)gold(I) was prepared and investigated for its primary biological properties. Cell culture experiments revealed strong antiproliferative effects and induction of apoptosis via mitochondrial pathways. Biodistribution studies by fluorescence microscopy and atomic absorption spectroscopy showed the uptake into cell organelles, an accumulation in the nuclei of tumor cells, and a homogeneous distribution in zebrafish embryos. In vivo monitoring of vascularisation in developing zebrafish embryos revealed a significant anti-angiogenic potency of the complex. Mechanistic experiments indicated that the inhibition of thioredoxin reductase (based on the covalent binding of a gold triethylphosphine fragment) might be involved in the pharmacodynamic behavior of this novel gold species.

LIMK1 and LIMK2 are important for metastatic behavior and tumor cell-induced angiogenesis of pancreatic cancer cells.

The two LIM kinases (LIMKs) LIMK1 and LIMK2 are members of the PDZ/LIM family. These serine/threonine protein kinases are involved in actin cytoskeleton reorganization through phosphorylation and inactivation of ADF/cofilin. Different subcellular localizations of LIMK1 and LIMK2 suggest different functions. LIMK1 is implicated in microtubule disassembly in endothelial- and cancer cells, whereas LIMK2 plays a role in cell cycle progression. To compare the role of the two LIMKs in cancer-related processes, we used a cell-based in vitro migration assay, as well as two zebrafish xenograft assays. We analyzed here the metastatic behavior and tumor cell-induced neovascularization of pancreatic cancer cells in which both LIMK genes were silenced by siRNAs. Both LIMK1 and LIMK2 single knock down led to a reduction of invasion and metastatic behavior in the zebrafish xenograft metastasis assay. Interestingly, the double knock down completely blocked invasion and formation of micrometastasis in vivo. Moreover, in the zebrafish xenograft angiogenesis assay, we observed a reduction of pancreatic cancer cell-induced angiogenesis for both the LIMK1 and LIMK2 knockdowns. Our results demonstrate similar functions for the two LIMKs in pancreatic cancer cells and suggest an important role for both LIMK1 and LIMK2 in tumor progression and metastasis formation.