Microfluidic approaches to synchrotron radiation-based Fourier transform infrared (SR-FTIR) spectral microscopy of living biosystems

Author(s):

Kevin Loutherback, Giovanni Birarda, Liang Chen and Hoi-Ying N. HolmanPages 273-282 (10)

Abstract:

A long-standing desire in biological and biomedical sciences is to be able to probe cellular chemistry as biological processes are happening inside living cells. Synchrotron radiation-based Fourier transform infrared (SR-FTIR) spectral microscopy is a label-free and nondestructive analytical technique that can provide spatiotemporal distributions and relative abundances of biomolecules of a specimen by their characteristic vibrational modes. Despite great progress in recent years, SR-FTIR imaging of living biological systems remains challenging because of the demanding requirements on environmental control and strong infrared absorption of water. To meet this challenge, microfluidic devices have emerged as a method to control the water thickness while providing a hospitable environment to measure cellular processes and responses over many hours or days. This paper will provide an overview of microfluidic device development for SR-FTIR imaging of living biological systems, provide contrast between the various techniques including closed and open-channel designs, and discuss future directions of development within this area. Even as the fundamental science and technological demonstrations develop, other ongoing issues must be addressed; for example, choosing applications whose experimental requirements closely match device capabilities, and developing strategies to efficiently complete the cycle of development. These will require imagination, ingenuity and collaboration.

Keywords:

FTIR, live cells, microfabrication, microfluidics, synchrotron radiation.

Affiliation:

Berkeley Synchrotron Infrared Structural Biology Program, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

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Kinetics for Zinc Ion Induced Sepia Pharaonis Arginine Kinase Inactivation and Aggregation

Author(s):

Yue-Xiu Si, Jinhyuk Lee, Juan-Ge Cheng, Shang-Jun Yin, Yong-Doo Park, Guo-Ying Qian and Xia-Min JiangPages 508-517 (10)

Abstract:

Arginine kinase is an essential enzyme which is closely related to energy metabolism in marine invertebrates. Arginine kinase provides a significant role in quick response to environmental change and stress. In this study, we simulated a tertiary structure of Sepia pharaonis arginine kinase (SPAK) based on the gene sequence and conducted the molecular dynamics simulations between SPAK and Zn2+. Using these results, the Zn2+ binding sites were predicted and the initial effect of Zn2+ on the SPAK structure was elucidated. Subsequently, the experimental kinetic results were compared with the simulation results. Zn2+ markedly inhibited the activity of SPAK in a manner of non-competitive inhibitions for both arginine and ATP. We also found that Zn2+ binding to SPAK resulted in tertiary conformational change accompanying with the hydrophobic residues exposure. These changes caused SPAK aggregation directly. We screened two protectants, glycine and proline, which effectively prevented SPAK aggregation and recovered the structure and activity. Overall, our study suggested the inhibitory effect of Zn2+ on SPAK and Zn2+ can trigger SPAK aggregation after exposing large extent of hydrophobic surface. The protective effects of glycine and proline against Zn2+ on SPAK folding were also demonstrated.

Keywords:

Aggregation, arginine kinase, Zn2+, inhibition, molecular dynamics simulation, Sepia pharaonis.

Affiliation:

College of Biological and Environmental Sciences, Zhejiang Wanli University, Ningbo 315100, P.R China., School of Marine Sciences, Ningbo University, Ningbo 315211, P.R. China.

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The Influence of Ionizing Radiation on Exosome Composition, Secretion and Intercellular Communication

Author(s):

Karol Jelonek, Piotr Widlak and Monika PietrowskaPages 656-663 (8)

Abstract:

A large variety of vesicles is actively secreted into the extracellular space by most type of cells. The smallest nanoparticles (30-120 nm), called exosomes, are known to transport their cargo (nucleic acids, proteins and lipids) between diverse locations in the body. Specific content of exosomes and their influence on recipient cells depends primarily on the type of the secretory (donor) cell, yet several studies highlight the importance of environmental stress on which the donor cells are exposed. Ionizing radiation, which induces damage to DNA and other structures of a target cell, is one of well-recognized stress conditions influencing behavior of affected cells. A few recent studies have evidenced radiationinduced changes in composition of exosomes released from irradiated cells and their involvement in radiation-related communication between cells. Inducible pathways of exosome secretion activated in irradiated cells are regulated by TSAP6 protein (the transmembrane protein tumor suppressor-activated pathway 6), which is transcriptionally regulated by p53, hence cellular status of this major DNA damage response factor affects composition and secretion rate of exosomes released from target cells. Moreover, exosomes released from irradiated cells have been shown to mediate the radiation-induced bystander effect. Understanding radiation-related mechanisms involved in exosome formation and “makeup” of their cargo would shed light on the role of exosomes in systemic response of cells, tissues and organisms to ionizing radiation which may open new perspectives in translational medicine and anticancer-treatment.

Keywords:

Exosome, ionizing radiation, intercellular communication, bystander effect.

Affiliation:

Center for Translational Research and Molecular Biology of Cancer, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Wybrzeze Armii Krajowej 15, 44-100 Gliwice, Poland.

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Cell-Free Technologies for Proteomics and Protein Engineering

Author(s):

Hiroyuki Ohashi and Etsuko Miyamoto-SatoPages 819-827 (9)

Abstract:

Cell-free translation systems facilitate rapid production of specific proteins and are particularly suited as high-throughput methods for whole-genome protein synthesis. Moreover, these systems do not rely on living cells, thereby allowing the synthesis of unstable or cytotoxic proteins in vitro. In this review, we describe the principles and potential applications of cell-free protein translation systems and the future prospects of proteomics approaches using next-generation sequencing and cell-free expression technologies.

Keywords:

Cell-free, IVV (in vitro virus), mRNA display, PPI (protein-protein interaction), Proteomics, PURE system.

Affiliation:

Division of Molecular Biology, Tokyo University of Science Research Institute for Biomedical Science, 2669 Yamazaki, Noda, Chiba 278-0022, Japan.

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