Direct Observation and Analysis of the Dynamics of the Photoresponsive Transcription Factor GAL4.

Herein, the direct visualization of the dynamic interaction between a photoresponsive transcription factor fusion, GAL4-VVD, and DNA using high-speed atomic force microscopy (HS-AFM) is reported. A series of different GAL4-VVD movements, such as binding, sliding, stalling, and dissociation, was observed. Inter-strand jumping on two double-stranded (ds) DNAs was also observed. Detailed analysis using a long substrate DNA strand containing five GAL4-binding sites revealed that GAL4-VVD randomly moved on the dsDNA using sliding and hopping to rapidly find specific binding sites, and then stalled to the specific sites to form a stable complex formation. These results suggest the existence of different conformations of the protein to enable sliding and stalling. This single-molecule imaging system with nanoscale resolution provides an insight into the searching mechanism used by DNA-binding proteins.

Whole-Cell Scale Dynamic Organization of Lysosomes Revealed by Spatial Statistical Analysis.

In eukaryotic cells, lysosomes are distributed in the cytoplasm as individual membrane-bound compartments to degrade macromolecules and to control cellular metabolism. A fundamental yet unanswered question is whether and, if so, how individual lysosomes are organized spatially to coordinate and integrate their functions. To address this question, we analyzed their collective behavior in cultured cells using spatial statistical techniques. We found that in single cells, lysosomes maintain non-random, stable, yet distinct spatial distributions mediated by the cytoskeleton, the endoplasmic reticulum (ER), and lysosomal biogenesis. Throughout the intracellular space, lysosomes form dynamic clusters that significantly increase their interactions with endosomes. Cluster formation is associated with local increases in ER spatial density but does not depend on fusion with endosomes or spatial exclusion by mitochondria. Taken together, our findings reveal whole-cell scale spatial organization of lysosomes and provide insights into how organelle interactions are mediated and regulated across the entire intracellular space.

Graphene Microelectrode Arrays for Electrical and Optical Measurements of Human Stem Cell-Derived Cardiomyocytes.

INTRODUCTION: Cell-cell communication plays a pivotal role in biological systems' coordination and function. Electrical properties have been linked to specification and differentiation of stem cells into targeted progeny, such as neurons and cardiomyocytes. Currently, there is a critical need in developing new ways to complement fluorescent indicators, such as Ca2+-sensitive dyes, for direct electrophysiological measurements of cells and tissue. Here, we report a unique transparent and biocompatible graphene-based electrical platform that enables electrical and optical investigation of human embryonic stem cell-derived cardiomyocytes' (hESC-CMs) intracellular processes and intercellular communication. METHODS: Graphene, a honeycomb sp2 hybridized two-dimensional carbon lattice, was synthesized using low pressure chemical vapor deposition system, and was tested for biocompatibility. Au and graphene microelectrode arrays (MEAs) were fabricated using well-established microfabrication methods. Au and graphene MEAs were interfaced with hESC-CMs to perform both optical and electrical recordings. RESULTS: Optical imaging and Raman spectroscopy confirmed the presence of monolayer graphene. Viability assay showed biocompatibility of graphene. Electrochemical characterization proved graphene's functional activity. Nitric acid treatment further enhanced the electrochemical properties of graphene. Graphene electrodes' transparency enabled both optical and electrical recordings from hESC-CMs. Graphene MEA detected changes in beating frequency and field potential duration upon ß-adrenergic receptor agonist treatment. CONCLUSION: The transparent graphene platform enables the investigation of both intracellular and intercellular communication processes and will create new avenues for bidirectional communication (sensing and stimulation) with electrically active tissues and will set the ground for investigations reported diseases such as Alzheimer, Parkinson's disease and arrhythmias.

Feasibility of spatial frequency-domain imaging for monitoring palpable breast lesions.

In breast cancer diagnosis and therapy monitoring, there is a need for frequent, noninvasive disease progression evaluation. Breast tumors differ from healthy tissue in mechanical stiffness as well as optical properties, which allows optical methods to detect and monitor breast lesions noninvasively. Spatial frequency-domain imaging (SFDI) is a reflectance-based diffuse optical method that can yield two-dimensional images of absolute optical properties of tissue with an inexpensive and portable system, although depth penetration is limited. Since the absorption coefficient of breast tissue is relatively low and the tissue is quite flexible, there is an opportunity for compression of tissue to bring stiff, palpable breast lesions within the detection range of SFDI. Sixteen breast tissue-mimicking phantoms were fabricated containing stiffer, more highly absorbing tumor-mimicking inclusions of varying absorption contrast and depth. These phantoms were imaged with an SFDI system at five levels of compression. An increase in absorption contrast was observed with compression, and reliable detection of each inclusion was achieved when compression was sufficient to bring the inclusion center within ∼12 mm of the phantom surface. At highest compression level, contrasts achieved with this system were comparable to those measured with single source-detector near-infrared spectroscopy.

Effect of Graphene on Nonneuronal and Neuronal Cell Viability and Stress.

In recent years graphene has drawn considerable research interest for biomedical applications. However, applications of graphene in biological systems also raise concerns about its possible toxicity. Here, by using live cell imaging techniques, we investigate the effect of pristine graphene on the viability as well as stress of both nonneuronal and neuronal cells under physiological conditions. We find that graphene promotes cell adhesion and proliferation. Furthermore, we find that graphene has no detectable adverse effect on mitochondrial membrane potential and morphology, or autophagy levels in the cell, indicating that graphene does not induce cell stress. Our results highlight the potential of graphene to be used in biomedical applications by providing long-term and stable nonneural and neural interfaces.