Ultrasensitive gold micro-structured electrodes enabling the detection of extra-cellular long-lasting potentials in astrocytes populations

Ultra-sensitive electrodes for extracellular recordings were fabricated and electrically characterized. A signal detection limit defined by a noise level of 0.3-0.4 mu V for a bandwidth of 12.5 Hz was achieved. To obtain this high sensitivity, large area (4 mm(2)) electrodes were used. The electrode surface is also micro-structured with an array of gold mushroom-like shapes to further enhance the active area. In comparison with a flat gold surface, the micro-structured surface increases the capacitance of the electrode/electrolyte interface by 54%. The electrode low impedance and low noise enable the detection of weak and low frequency quasi-periodic signals produced by astrocytes populations that thus far had remained inaccessible using conventional extracellular electrodes. Signals with 5 mu V in amplitude and lasting for 5-10 s were measured, with a peak-to-peak signal-to-noise ratio of 16. The electrodes and the methodology developed here can be used as an ultrasensitive electrophysiological tool to reveal the synchronization dynamics of ultra-slow ionic signalling between non-electrogenic cells.Portuguese Foundation for Science and Technology (FCT), through the project "Implantable organic devices for advanced therapies" (INNOVATE) [PTDC/EEI-AUT/5442/2014]; Instituto de Telecomunicacoes [UID/Multi/04326/2013]; Associated Laboratory - Institute of Nanoscience and Nanotechnology [POCI-01-0145-FEDER-016623]; [PTDC/CTM-NAN/3146/2014

A High-Temporal Resolution Technology for Dynamic Proteomic Analysis Based on 35S Labeling

As more and more research efforts have been attracted to dynamic or differential proteomics, a method with high temporal resolution and high throughput is required. In present study, a 35S in vivo Labeling Analysis for Dynamic Proteomics (SiLAD) was designed and tested by analyzing the dynamic proteome changes in the highly synchronized A549 cells, as well as in the rat liver 2/3 partial hepatectomy surgery. The results validated that SiLAD technique, in combination with 2-Dimensional Electrophoresis, provided a highly sensitivity method to illustrate the non-disturbed endogenous proteins dynamic changes with a good temporal resolution and high signal/noise ratio. A significant number of differential proteins can be discovered or re-categorized by this technique. Another unique feature of SiLAD is its capability of quantifying the rate of protein expression, which reflects the cellular physiological turn points more effectively. Finally, the prescribed SiLAD proteome snapshot pattern could be potentially used as an exclusive symbol for characterizing each stage in well regulated biological processes

PDGF induced microRNA alterations in cancer cells

Platelet derived growth factor (PDGF) regulates gene transcription by binding to specific receptors. PDGF plays a critical role in oncogenesis in brain and other tumors, regulates angiogenesis, and remodels the stroma in physiologic conditions. Here, we show by using microRNA (miR) arrays that PDGFs regulate the expression and function of miRs in glioblastoma and ovarian cancer cells. The two PDGF ligands AA and BB affect expression of several miRs in ligand-specific manner; the most robust changes consisting of let-7d repression by PDGF-AA and miR-146b induction by PDGF-BB. Induction of miR-146b by PDGF-BB is modulated via MAPK-dependent induction of c-fos. We demonstrate that PDGF regulates expression of some of its known targets (e.g. cyclin D1) through miR alterations and identify the epidermal growth factor receptor (EGFR) as a new PDGF-BB target. We show that its expression and function are repressed by PDGF-induced miR-146b and that mir-146b and EGFR correlate inversely in human glioblastomas. We propose that PDGF-regulated gene transcription involves alterations in non-coding RNAs and provide evidence for a miR-dependent feedback mechanism balancing growth factor receptor signaling in cancer cells

Regulation of cell cycle proteins by chemokine receptors: A novel pathway in human immunodeficiency virus neuropathogenesis?

In order to test the hypothesis that alteration of cell cycle proteins are involved in the neuronal damage caused by human immunodeficiency virus (HIV), the authors have been studying the effect of chemokines on the CDK/Rb/E2F-1 pathway--which is involved in neuronal apoptosis and differentiation. First, they have asked whether CXCR4, the specific receptor for the chemokine SDF-1 and X4-using gp120s, can regulate Rb and E2F-1 activity in cultures of differentiated rat neurons. Although CCR3 and CCR5 are known to mediate infection of microglia by HIV-1, recent evidence indicate that CXCR4 also play important roles in HIV-induced neuronal injury, and dual-tropic isolates that use CXCR4 to infect macrophages have recently been reported. The authors have focused on two specific brain areas in which CXCR4 is physiologically relevant, i.e., the cerebellum and the hippocampus. So far, the data indicate that changes in the nuclear and cytosolic levels of Rb, which result in the functional loss of this protein, are associated with apoptosis in these neurons, and that SDF-1alpha and gp120IIIB affect this pathway. A summary of the findings are presented

Abstract 5804: Heterogeneity in androgen receptor and IL-1beta expression by prostate cancer cells in skeletal metastases

Abstract Bone metastases are a prevalent complication of advanced prostate cancer (PCa). The current standard of care for advanced PCa patients is androgen deprivation therapy (ADT), which fails within approximately two years and patients progress to castration resistant prostate cancer (CRPC). CRPC presents with additional metastasis in soft tissues and is incurable. We have found that in PCa patients, bone metastases have heterogeneous Androgen Receptor (AR) expression, and that cells lacking AR are the only PCa cell type that secretes the cytokine Interleukin-1 beta (IL-1β). Furthermore, pre-clinical work in our lab has revealed the role for IL-1β in promoting bone colonization by AR+ PCa cells and generation of heterogeneous skeletal metastases. A significant barrier to studying tumor heterogeneity in PCa metastasis has been the lack of an animal model to study the interaction between multiple PCa phenotypes in the bone microenvironment. By engrafting a suspension containing both AR− and AR+ cells directly into the left cardiac ventricle of mice, our lab has developed an in vivo model that allows us to study the progression of both AR− and AR+ cells in skeletal and soft tissue metastases. AR− and AR+ cells were genetically engineered to express two different Luciferase constructs (630 nm Red-shifted Luc and 540 nm Luc2 respectively) thereby allowing the progression of AR− and AR+ cells to be independently tracked and quantified. We used this model in combination with histological and radiographic analyses to assimilate the pathological features of our in vivo mixed AR+/AR− metastases to the human clinical scenario. Finally, we used castrated mice to determine whether IL-1β secreting PCa cells could support the growth and survival of IL-1β–/AR+ cells in androgen deprived conditions. Co-injection of AR− (PC3-ML) and AR+ cells (LNCaP, VCaP, and 22Rv1) leads to increased establishment of soft tissue metastases as compared to injecting AR− cells alone. Unlike the metastases generated by PC3-ML cells alone, which show exclusively osteolytic activity, AR−/AR+ mixed metastases are both osteosclerotic and osteolytic, thereby recapitulating clinical pathology. Finally, preliminary results suggest that co-injection with IL-1β-secreting AR− cells enables the growth of androgen-dependent AR+ cells in castrated mice. This animal model of metastasis heterogeneity recapitulates human pathology and enables further interrogation of the mechanism by which IL-1β supports survival of AR+ cells and the potential role for AR− in evading AR-targeted therapy during castration resistant prostate cancer (CRPC). Citation Format: Asurayya A. Worrede-Mahdi, Melisa Diaz, Alessandro Fatatis. Heterogeneity in androgen receptor and IL-1beta expression by prostate cancer cells in skeletal metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5804. doi:10.1158/1538-7445.AM2017-5804</jats:p