We purified from activated T lymphocytes a novel, highly conserved, 116-kDa, intracellular protein that occurred at high levels in the large, dividing cells of the thymus, was up-regulated when resting T or B lymphocytes or hemopoietic progenitors were activated, and was down-regulated when a monocytic leukemia, M1, was induced to differentiate. Expression of the protein was highest in the thymus and spleen and lowest in tissues with a low proportion of dividing cells such as kidney or muscle, although expression was high in the brain. The protein was localized to the cytosol and was phosphorylated, which is consistent with a previous report that the Xenopus laevis ortholog was phosphorylated by a mitotically activated kinase (1 ). The cDNA was previously mischaracterized as encoding p137, a 137-kDa GPI-linked membrane protein (2 ). We propose that the authentic protein encoded by this cDNA be called cytoplasmic activation/proliferation-associated protein-1 (caprin-1), and show that it is the prototype of a novel family of proteins characterized by two novel protein domains, termed homology regions-1 and -2 (HR-1, HR-2). Although we have found evidence for caprins only in urochordates and vertebrates, two insect proteins exhibit well-conserved HR-1 domains. The HR-1 and HR-2 domains have no known function, although the HR-1 of caprin-1 appeared necessary for formation of multimeric complexes of caprin-1. Overexpression of a fusion protein of enhanced green fluorescent protein and caprin-1 induced a specific, dose-dependent suppression of the proliferation of NIH-3T3 cells, consistent with the notion that caprin-1 plays a role in cellular activation or proliferation.

The androgen receptor (AR) plays a critical role in the progression of prostate cancer. Silencing this protein using shorthairpin RNA (shRNA) has been correlated with tumor growth inhibition and decreases in serum prostate specific antigen (PSA). In our study, we have investigated the ability of lipid nanoparticle (LNP) formulations of small-interfering RNA (siRNA) to silence AR in human prostate tumor cell lines in vitro and in LNCaP xenograft tumors following intravenous (i.v.) injection. In vitro screening studies using a panel of cationic lipids showed that LNPs containing the ionizable cationic lipid 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA) exhibited the most potent AR silencing effects in LNCaP cells. This is attributed to an optimized ability of DLin-KC2-DMA-containing LNP to be taken up into cells and to release the siRNA into the cell cytoplasm following endocytotic uptake. DLin-KC2-DMA LNPs were also effective in silencing the AR in a wild-type AR expressing cell line, LAPC-4, and a variant AR expressing cell line, CWR22Rv1. Importantly, it is demonstrated that LNP ARsiRNA systems containing DLin-KC2-DMA can silence AR gene expression in distal LNCaP xenograft tumors and decrease serum PSA levels following i.v. injection. To our knowledge, this is the first report demonstrating the feasibility of LNP delivery of siRNA for silencing AR gene expression in vivo.Prostate cancer is the most frequently diagnosed nonskin cancer and the second-leading cause of cancer-related deaths in males.

Betulinic acid (BA), a pentacyclic triterpene of natural origin, has been demonstrated to have varied biologic activities including anti-viral, anti-inflammatory, and anti-malarial effects; it has also been found to induce apoptosis in many types of cancer. However, little is known about the effect of BA on normal cells. In this study, the effects of BA on normal neuronal cell apoptosis and the mechanisms involved were studied using differentiated PC12 cells as a model. Treatment with 50 μM BA for 24 h apparently induced PC12 cell apoptosis. In the early stage of apoptosis, the level of intracellular reactive oxygen species (ROS) increased. Afterwards, the loss of the mitochondrial membrane potential, the release of cytochrome c and the activation of caspase-3 occurred. Treatment with antioxidants could significantly reduce BA-induced PC12 cell apoptosis. In conclusion, we report for the first time that BA induced the mitochondrial apoptotic pathway in differentiated PC12 cells through ROS.

Orthotopic bladder cancer xenografts are essential for testing novel therapies and molecular manipulations of cell lines in vivo. Current xenografts rely on tumor cell inoculation by intravesical instillation or direct injection into the bladder wall. Instillation is limited by the lack of cell lines that are tumorigenic when delivered in this manner. The invasive model inflicts morbidity on the mice by the need for laparotomy and mobilization of the bladder. Furthermore this procedure is complex and time-consuming. Three bladder cancer cell lines (UM-UC1, UM-UC3, UM-UC13) were inoculated into 50 athymic nude mice by percutaneous injection under ultrasound guidance. PBS was first injected between the muscle wall and the mucosa to separate these layers, and tumor cells were subsequently injected into this space. Bioluminescence and ultrasound were used to monitor tumor growth. Contrast-enhanced ultrasound was used to study changes in tumor perfusion after systemic gemcitabine/cisplatin treatment. To demonstrate proof of principle that therapeutic agents can be injected into established xenografts under ultrasound guidance, oncolytic virus (VSV) was injected into UM-UC3 tumors. Xenograft tissue was harvested for immunohistochemistry after 23–37 days. Percutaneous injection of tumor cells into the bladder wall was performed efficiently (mean time: 5.7 min) and without complications in all 50 animals. Ultrasound and bioluminescence confirmed presence of tumor in the anterior bladder wall in all animals 3 days later. The average tumor volumes increased steadily over the study period. UM-UC13 tumors showed a marked decrease in volume and perfusion after chemotherapy. Immunohistochemical staining for VSV-G demonstrated virus uptake in all UM-UC3 tumors after intratumoral injection. We have developed a novel method for creating orthotopic bladder cancer xenograft in a minimally invasive fashion. In our hands this has replaced the traditional model requiring laparotomy, because this model is more time efficient, more precise and associated with less morbidity for the mice.

Bladder cancer is the second most common genitourinary malignancy. At initial diagnosis, f70% of cases are nonmuscle-invasive; however, current treatment options for superficial disease are of limited efficacy because many patients will develop recurrent tumors. The purpose of this study was to examine two replication-competent oncolytic vesicular stomatitis virus (VSV) strains as intravesical agents in an orthotopic murine model of high-grade bladder cancer. Four human bladder cancer cell lines (RT4, MGH-U3, UM-UC3, and KU-7) were treated with either wild-type VSV or a mutant #51M variant (AV3) in vitro. Both wild-type VSV and AV3, which has an impaired ability to shutdown innate immunity, preferentially killed the more aggressive, IFN-nonresponsive cell lines UM-UC3 and KU-7, whereas IFN-responsive RT4 and MGH-U3 cells were less susceptible. In vivo, KU-7-luc bladder tumor cells, which stably express firefly luciferase, were inoculated into nude mice by intravesical instillation and tumor growth was quantified using bioluminescence imaging. Mice with established xenografts were administered VSV intravesically on days 4, 9, and 14, and necropsy was performed after 3 weeks. AV3 as well as wild-type VSV significantly inhibited KU-7-luc tumor growth by 90% (AV3) and 98% (wild-type), respectively, as compared with controls treated with UV-inactivated VSV. Despite using immunocompromised hosts, there was no evidence of toxicity in either group. In conclusion, VSV instillation therapy showed promising antitumor activity and safety in an orthotopic model of bladder cancer. These findings provide preclinical proof-ofprinciple for the intravesical use of VSV against non-muscleinvasive bladder cancer, especially in IFN-refractory patients.

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The probing of physiological processes in living organisms is a grand challenge that requires bespoke analytical tools. Optical fiber probes offer a minimally invasive approach to report physiological signals from specific locations inside the body. This perspective article discusses a wide range of such fiber probes developed at the Australian Research Council Centre of Excellence for Nanoscale BioPhotonics. Our fiber platforms use a range of sensing modalities, including embedded nanodiamonds for magnetometry, interferometric fiber cavities for refractive index sensing, and tailored metal coatings for surface plasmon resonance sensing. Other fiber probes exploit molecularly sensitive Raman scattering or fluorescence where optical fibers have been combined with chemical and immunosensors. Fiber imaging probes based on interferometry and computational imaging are also discussed as emerging in vivo diagnostic devices. We provide examples to illustrate how the convergence of multiple scientific disciplines generates opportunities for the fiber probes to address key challenges in real-time in vivo diagnostics. These future fiber probes will enable the asking and answering of scientific questions that were never possible before.

PEDOT–Boc–Leu and PEDOT–Leu exhibit excellent electrochromic performance, and are promising optoelectronic materials.