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Many advanced snakes possess a unique venom delivery system which they utilise to subdue prey and for defence. Despite extensive efforts, the evolutionary differences in this key system between advanced snake families remains enigmatic. The current study has investigated the development of the venom delivery system using two oviparous Elapidae models, Naja siamensis and Oxyuranus microlepidotus. The development stages of the embryos in both models were detailed using previously standardised characterisation. Variations in the days post-oviposition between these stages was observed, despite a continuous development trajectory. These differences also translated to the development of the venom delivery system. Refereed/Peer-reviewed

Tumour vasculature supports the growth and progression of solid cancers with both angiogenesis (endothelial cell proliferation) and vasculogenic mimicry (VM, the formation of vascular structures by cancer cells themselves) predictors of poor patient outcomes. Increased circulating platelet counts also predict poor outcome for cancer patients but the influence of platelets on tumour vasculature is incompletely understood. Herein, we show with in vitro assays that platelets did not influence angiogenesis but did actively inhibit VM formation by cancer cell lines. Both platelet sized beads and the releasates from platelets were partially effective at inhibiting VM formation suggesting that direct contact maximises the effect. Platelets also promoted cancer cell invasion in vitro. B16F10 melanomas in Bcl-xPlt20/Plt20 thrombocytopenic mice showed a higher content of VM than their wildtype counterparts while angiogenesis did not differ. In a xenograft mouse model of breast cancer with low-dose aspirin to inactivate the platelets, the burden of MDA-MB-231-LM2 breast cancer cells was reduced and the gene expression profile of the cancer cells was altered; but no effect on tumour vasculature was observed. Taken together, this study provides new insights into the action of platelets on VM formation and their involvement in cancer progression.

Trocarin D (TroD), a venom prothrombin activator from Tropidechis carinatus, shares similar structure and function with blood coagulation factor Xa [Tropidechis carinatus FX (TrFX) a]. Their distinct physiologic roles are due to their distinct expression patterns. The genes of TroD and TrFX are highly similar, except for promoter and intron 1, indicating that TroD has probably evolved by duplication of FX, the plasma counterpart. The promoter insertion in TroD accounts for the elevated but not venom gland-specific expression. Here we examined the roles of 3 insertions and 2 deletions in intron 1 of TroD in the regulation of expression using luciferase as a reporter. By systematic deletions, we showed that a 209 bp region within the second insertion silences expression in mammalian and unmilked venom gland cells. Through bioinformatics analysis, we identified 5 AG-rich motifs in this region. All except the 5th motif are important for silencing function. YY1, Sp3 and HMGB2 were identified to bind these AG-rich motifs and silence gene expression in mammalian cells. Similar AG-rich motif clusters are also found in other toxin genes but not in their physiologic counterparts. Thus, AG-rich motifs contribute to regulation of expression of TroD, and probably other toxin genes.Han, S. X., Kwong, S., Ge, R., Kolatkar, P. R., Woods, A. E., Blanchet, G., Kini, R. M. Regulation of expression of venom toxins: silencing of prothrombin activator trocarin D by AG-rich motifs. Refereed/Peer-reviewed

Snakes are limbless predators, and many species use venom to help overpower relatively large, agile prey. Snake venoms are complex protein mixtures encoded by several multilocus gene families that function synergistically to cause incapacitation. To examine venom evolution, we sequenced and interrogated the genome of a venomous snake, the king cobra (Ophiophagus hannah), and compared it, together with our unique transcriptome, microRNA, and proteome datasets from this species, with data from other vertebrates. In contrast to the platypus, the only other venomous vertebrate with a sequenced genome, we find that snake toxin genes evolve through several distinct co-option mechanisms and exhibit surprisingly variable levels of gene duplication and directional selection that correlate with their functional importance in prey capture. The enigmatic accessory venom gland shows a very different pattern of toxin gene expression from the main venom gland and seems to have recruited toxin-like lectin genes repeatedly for new nontoxic functions. In addition, tissue- specific microRNA analyses suggested the co-option of core genetic regulatory components of the venom secretory system from a pancreatic origin. Although the king cobra is limbless, we recovered coding sequences for all Hox genes involved in amniote limb development, with the exception of Hoxd12. Our results provide a unique view of the origin and evolution of snake venom and reveal multiple genome-level adaptive responses to natural selection in this complex biological weapon system. More generally, they provide insight into mechanisms of protein evolution under strong selection. Refereed/Peer-reviewed

SummaryTrocarin D is a prothrombin activator from the Tropidechis carinatus venom. It is a functional and structural homologue to mammalian blood coagulation factor Xa.Trocarin D is hypothesised to have evolved from its factor X counterpart (TrFX) through gene duplication and recruitment.The genes of trocarin D and TrFX have significant sequence identities, except for insertions/deletions in their intron 1 and promoter regions. In trocarin D intron 1 region, there are three insertions and two deletions. In trocarin D promoter region, there is a novel 264 bp insertion which has potential cis-elements.This insertion is termed as Venom Recruitment/Switch Element (VERSE) and is hypothesised to account for switching the low-level constitutive expression of factor X in the liver to the high-level inducible expression of trocarin D in the venom gland. To understand the role of VERSE in the trocarin D expression,its cis-elements were characterised by luciferase assays in mammalian cell lines as well as snake venom gland cells. The ability of VERSE to drive luciferase expression is comparable to that of the trocarin D promoter. The predicted cis-elements are important in promoting expression as their mutagenesis resulted in lower luciferase expression.VERSE minimal core promoter and three novel cis-elements (two up-regulatory and one suppressor elements) were identified using deletion/site-directed mutagenesis studies. VERSE is primarily responsible for the increase of trocarin D expression. The insertions/deletions within trocarin D intron 1 need to be characterised for their role in tissue-specific and inducible expression of trocarin D.

T cells are in general tolerant of prostate-specific tumor antigens. That prostate tumor tissue makes transforming growth factor-beta (TGFbeta) is thought to play a role in the induction of T-cell tolerance within the host and to contribute to tumor progression itself. Here we sought to investigate the influence of TGFbeta signaling on prostate antigen-specific T-cell responses as well as prostate tumorogenesis in an autochthonous murine model of the disease. The response of naive and activated ovalbumin (OVA) antigen-specific T cells, which had been rendered incapable of responding to TGFbeta through T-cell-specific transgenic expression of a dominant-negative variant of the TGFbeta receptor II (dnTGFRII), was analyzed after adoptive transfer into prostate OVA-expressing transgenic (POET) mice. However, ex vivo activation facilitated entry of TGFbeta-insensitive T cells into the prostate and was associated with prostate tissue damage. Early tumor progression was delayed in TRAMP mice that carried endogenous TGFbeta-insensitive T cells. Together, these results suggest that TGFbeta-signaling represses CD8(+) T-cell responses to a prostate-specific antigen. TGFbeta-mediated repression of T-cell function may include production of IFNgamma, which is known to contribute to tumor immunosurveillance. The role of TGFbeta signaling in endogenous T cells in mice, which spontaneously form tumors, was also assessed by monitoring prostate tumor formation and progression in F1 progeny of productive matings between transgenic adenocarcinoma of the mouse prostate (TRAMP) and dnTGFRII mice. TGFbeta-resistant CD8(+) T cells proliferated more and produced IFNgamma more readily after OVA stimulation in vitro. OVA-specific T cells did not damage the prostate gland of POET mice irrespective of TGFbeta responsiveness. Refereed/Peer-reviewed