Early glioma is associated with abnormal electrical events in cortical cultures.

The prodromal stages of some neurological diseases have a distinct electrical profile which can potentially be leveraged for early diagnosis, predicting disease recurrence, monitoring of disease progression, and better understanding of the disease pathology. Gliomas are tumors that originate from glial cells present in the brain and spinal cord. Healthy glial cells support normal neuronal function and play an important role in modulating the regular electrical activity of neurons. However, gliomas can disrupt the normal electrical dynamics of the brain. Though experimental and clinical studies suggest that glioma and injury to glial cells disrupt electrical dynamics of the brain, whether these disruptions are present during the earliest stages of glioma and glial injury are unclear. The primary aim of this study is to investigate the effect of early in vitro glial pathology (glioma and glial injury in specific) on neuronal electrical activity. In particular, we investigated the effect of glial pathology on neural synchronization: an important phenomenon that underlies several central neurophysiological processes (ScienceDirect, 2018 ). We used two in vitro disease samples: (a) a sample in which cortical cultures were treated with anti-mitotic agents that deplete glial cells and (b) a glioma sample in which healthy cortical cells were cultured with CRL-2303 (an aggressive glioma cell line). Healthy cortical culture samples were used as controls. Cultures were established over a glass dish embedded with microelectrodes that permits simultaneous measurement of extracellular electrical activity from multiple sites of the culture. We observed that healthy cortical cultures produce spontaneous and synchronized oscillations which were attenuated in the absence of glial cells. The presence of glioma was associated with the emergence of two types of "abnormal electrical activity" each with distinct amplitude and frequency profile. Our results indicate that even early stages of glioma and glial injury are associated with distinct changes in neuronal electrical activity. Graphical abstract.

Human papillomavirus type 16 E5-mediated upregulation of Met in human keratinocytes.

Human papillomaviruses (HPVs) cause benign lesions that can lead to malignancy. How cellular changes induced by viral oncogenes contribute to the progeny virion production is not always clear. Stromally-derived growth factors and their receptors are critical for development of malignancy, but their impact on the pre-malignant HPV life cycle is unknown. We show that HPV16 increases levels of Met, a growth factor receptor critical for tumor cell invasion, motility, and cancer metastasis. The viral oncogene E5 is primarily responsible for Met upregulation, with E6 playing a minor role. Met induction by E5 requires the epidermal growth factor receptor, which is also increased by E5 at the mRNA level. E5-induced Met contributes motility of HPV-containing cells. Finally, Met signaling is necessary for viral gene expression, particularly in the differentiation-dependent phase of the viral life cycle. These studies show a new role for E5 in epithelial-stromal interactions, with implications for cancer development.

A Novel High Content Imaging-Based Screen Identifies the Anti-Helminthic Niclosamide as an Inhibitor of Lysosome Anterograde Trafficking and Prostate Cancer Cell Invasion.

Lysosome trafficking plays a significant role in tumor invasion, a key event for the development of metastasis. Previous studies from our laboratory have demonstrated that the anterograde (outward) movement of lysosomes to the cell surface in response to certain tumor microenvironment stimulus, such as hepatocyte growth factor (HGF) or acidic extracellular pH (pHe), increases cathepsin B secretion and tumor cell invasion. Anterograde lysosome trafficking depends on sodium-proton exchanger activity and can be reversed by blocking these ion pumps with Troglitazone or EIPA. Since these drugs cannot be advanced into the clinic due to toxicity, we have designed a high-content assay to discover drugs that block peripheral lysosome trafficking with the goal of identifying novel drugs that inhibit tumor cell invasion. An automated high-content imaging system (Cellomics) was used to measure the position of lysosomes relative to the nucleus. Among a total of 2210 repurposed and natural product drugs screened, 18 "hits" were identified. One of the compounds identified as an anterograde lysosome trafficking inhibitor was niclosamide, a marketed human anti-helminthic drug. Further studies revealed that niclosamide blocked acidic pHe, HGF, and epidermal growth factor (EGF)-induced anterograde lysosome redistribution, protease secretion, motility, and invasion of DU145 castrate resistant prostate cancer cells at clinically relevant concentrations. In an effort to identify the mechanism by which niclosamide prevented anterograde lysosome movement, we found that this drug exhibited no significant effect on the level of ATP, microtubules or actin filaments, and had minimal effect on the PI3K and MAPK pathways. Niclosamide collapsed intralysosomal pH without disruption of the lysosome membrane, while bafilomycin, an agent that impairs lysosome acidification, was also found to induce JLA in our model. Taken together, these data suggest that niclosamide promotes juxtanuclear lysosome aggregation (JLA) via modulation of pathways involved in lysosome acidification. In conclusion, we have designed a validated reproducible high-content assay to screen for drugs that inhibit lysosome trafficking and reduce tumor invasion and we summarize the action of one of these drugs.

Generation of Scalable, Metallic High-Aspect Ratio Nanocomposites in a Biological Liquid Medium.

The goal of this protocol is to describe the synthesis of two novel biocomposites with high-aspect ratio structures. The biocomposites consist of copper and cystine, with either copper nanoparticles (CNPs) or copper sulfate contributing the metallic component. Synthesis is carried out in liquid under biological conditions (37 °C) and the self-assembled composites form after 24 hr. Once formed, these composites are highly stable in both liquid media and in a dried form. The composites scale from the nano- to micro- range in length, and from a few microns to 25 nm in diameter. Field emission scanning electron microscopy with energy dispersive X-ray spectroscopy (EDX) demonstrated that sulfur was present in the NP-derived linear structures, while it was absent from the starting CNP material, thus confirming cystine as the source of sulfur in the final nanocomposites. During synthesis of these linear nano- and micro-composites, a diverse range of lengths of structures is formed in the synthesis vessel. Sonication of the liquid mixture after synthesis was demonstrated to assist in controlling average size of the structures by diminishing the average length with increased time of sonication. Since the formed structures are highly stable, do not agglomerate, and are formed in liquid phase, centrifugation may also be used to assist in concentrating and segregating formed composites.