Primary Cell Culture of Live Neurosurgically Resected Aged Adult Human Brain Cells and Single Cell Transcriptomics.

Investigation of human CNS disease and drug effects has been hampered by the lack of a system that enables single-cell analysis of live adult patient brain cells. We developed a culturing system, based on a papain-aided procedure, for resected adult human brain tissue removed during neurosurgery. We performed single-cell transcriptomics on over 300 cells, permitting identification of oligodendrocytes, microglia, neurons, endothelial cells, and astrocytes after 3 weeks in culture. Using deep sequencing, we detected over 12,000 expressed genes, including hundreds of cell-type-enriched mRNAs, lncRNAs and pri-miRNAs. We describe cell-type- and patient-specific transcriptional hierarchies. Single-cell transcriptomics on cultured live adult patient derived cells is a prime example of the promise of personalized precision medicine. Because these cells derive from subjects ranging in age into their sixties, this system permits human aging studies previously possible only in rodent systems.

Effects of the expression level of epidermal growth factor receptor on the ligand-induced restructuring of focal adhesions: a QCM-D study.

Epidermal growth factor receptor (EGFR) plays a major role in cell migration and invasion and is considered to be the primary source of activation of various malignant tumors. To gain insight into how elevated levels of EGFR influence cellular function, particularly cell motility, we used a quartz crystal microbalance with dissipation monitoring (QCM-D) to examine restructuring of focal adhesions in MCF-10A cells induced by epidermal growth factor. Engineered cells that overexpress epidermal growth factor receptor (EGFR) exhibited a very different kinetic profile from wildtype MCF-10A cells that have a lower level of EGFR with a higher rate for the initial disassembly of focal adhesion and a much lower rate for the later reassembly of focal adhesions. It is conceivable that these effects exhibited by EGFR-overexpressing cells may promote the initiation and maintenance of a more favorable adhesion state for cell migration. This study has demonstrated the capability of the dissipation monitoring function of the QCM-D to quantitatively assess kinetic aspects of cellular processes with a high temporal resolution and sensitivity.

[Population exposure to mercury in the municipality of San Marcos (Sucre department) due to eating contaminated rice (Oryza sativa)]. / Exposición a mercurio en habitantes del municipio de San Marcos (Departamento de Sucre) debida a la ingesta de arroz (Oryza sativa) contaminado.

OBJECTIVE: Determining the magnitude of mercury exposure in the population living in the municipality of San Marcos due to eating contaminate drice (Oryza sativa). METHODS: Twenty people (representative of the population) were selected, as were food (raw rice) and hair samples for determining total mercury and methyl mercury by cold vapour atomic absorption spectrophotometry. Student's t-test was used for comparing different samples (p<0.05 significance level) and correlation was analysed for determining the relationship between consumption habits and mercury concentration in humans. RESULTS: Rice sold loose (i.e. unpackaged San Marcos white rice) was the only sample having 0.021 mg/g minimum total mercury concentration, whilst rice sold in packaged form yielded no measurable value. Only 5% of the population sample exceeded the US Environmental Protection Agency's (EPA) 0.1 mg/kg bw/day reference dose (RfD) for Me Hg ingestion (RfD). CONCLUSIONS: The HgT exposure of people living in and around San Marcos concerning rice consumption was low and did not involve great risks to their health. However, frequent consumption of other types of contaminated food could pose a potential threat to the consumers' health, meaning that ongoing environmental monitoring is necessary.

Dissipation monitoring for assessing EGF-induced changes of cell adhesion.

Epidermal growth factor (EGF)-induced cell de-adhesion has been implicated as a critical step of normal embryonic development, wound repair, inflammatory response, and tumor cell metastasis. Like many other cellular processes, this cell de-adhesion exhibits a complex, time-dependent pattern. A comprehensive understanding of this process requires a quantitative, real-time assessment of cell-substrate interactions at the molecular level. We employed the quartz crystal microbalance with dissipation monitoring (QCM-D) to successfully track the EGF-induced changes in energy dissipation factor, ΔD, of a monolayer of MCF10A cells in real time. This time-dependent ΔD response correlates well both qualitatively and quantitatively with sequential events of a rapid disassembly, transition, and slow reassembly of focal adhesions of the cells in response to EGF exposure. Based on this strong correlation, we utilized the QCM-D to demonstrate that this dynamic focal-adhesion restructuring is regulated temporally by the downstream pathways of EGFR signaling such as the PI3K, MAPK/ERK, and PLC pathways. Because the QCM-D is a noninvasive technique, this novel approach potentially has a broad range of applications in the fundamental study of cellular processes, such as cell signaling and trafficking and mechanotransduction, and holds promise for drug and biomarker screening.

Evaluating Inhibition of the Epidermal Growth Factor (EGF)-Induced Response of Mutant MCF10A Cells with an Acoustic Sensor.

Many cancer treatments rely on inhibition of epidermal growth factor (EGF)-induced cellular responses. Evaluating drug effects on such responses becomes critical to the development of new cancer therapeutics. In this report, we have employed a label-free acoustic sensor, the quartz crystal microbalance with dissipation monitoring (QCM-D), to track the EGF-induced response of mutant MCF10A cells under various inhibitory conditions. We have identified a complex cell de-adhesion process, which can be distinctly altered by inhibitors of signaling pathways and cytoskeleton formation in a dose-dependent manner. The dose dependencies of the inhibitors provide IC50 values which are in strong agreement with the values reported in the literature, demonstrating the sensitivity and reliability of the QCM-D as a screening tool. Using immunofluorescence imaging, we have also verified the quantitative relationship between the ΔD-response (change in energy dissipation factor) and the level of focal adhesions quantified with the areal density of immunostained vinculin under those inhibitory conditions. Such a correlation suggests that the dynamic restructuring of focal adhesions can be assessed based on the time-dependent change in ΔD-response. Overall, this report has shown that the QCM-D has the potential to become an effective sensing platform for screening therapeutic agents that target signaling and cytoskeletal proteins.