Sungsu Park

Unique sandwich stacking of pyrene-adenine-pyrene for selective and ratiometric fluorescent sensing of ATP at physiological pH.

A pincer-like benzene-bridged sensor 1 with a pyrene excimer as a signal source and imidazolium as a phosphate anion receptor was synthesized and investigated for ATP sensing. A unique switch of excimer vs monomer pyrene fluorescence of 1 is observed in the presence of ATP due to the charcteristic sandwich pi-pi stacking of pyrene-adenine-pyrene. On the other hand, four other bases of nucleoside triphosphates such as GTP, CTP, UTP, and TTP can interact only from the outside with the already stabilized stacked pyrene-pyrene dimer of 1, resulting in excimer fluorescence quenching. The fluorescent intensity ratio of monomer-to-excimer for 1 upon binding with ATP (I(375)/I(487)) is much larger than that upon binding with ADP and AMP. This difference is large enough to discriminate ATP from ADP and AMP. As one of the biological applications, sensor 1 is successfully applied to the ATP staining experiments. Sensor 1 is also applied to monitor the hydrolysis of ATP and ADP by apyrase. The results indicate that 1 is a useful fluorescent sensor for investigations of ATP-relevant biological processes.

A highly selective ratiometric near-infrared fluorescent cyanine sensor for cysteine with remarkable shift and its application in bioimaging

We developed a highly selective ratiometric near-infrared cyanine-based probe CyAC for cysteine (Cys) over homocysteine (Hcy) and glutathione (GSH). Upon the addition of Cys to the solution of CyAC, remarkable shifts in the spectra of CyAC can be monitored (from 770 nm to 515 nm in absorption spectra and from 780 nm to 570 nm in emission spectra). For the first time, the novel strategy that reversibly modulates the polymethine π-electron system by conjugation and removal of the specific trigger moiety was implemented for the generation of a ratiometric cyanine-based sensor. Hydroxy cyanine CyAE was chosen as the flurophore scaffold because the tautomerism (CyAE and CyAK or CyAD) can cause the reversible change in the π-conjugation system of the dyes with large shifts in the spectra. An acrylate group containing a α, β-unsaturated ketone as a functional trigger moiety was incorporated with CyAK to form the sensor CyAC. This specific response for Cys was based on the differences of the kinetics of intramolecular adduct/cyclizations. Moreover, CyAC was successfully applied for bioimaging Cys in living cancer cells. This paradigm by modulation of the polymethine π-electron system in the cyanine dye provides a promising methodology for the design of ratiometric cyanine-based sensors.

A near-infrared fluorescent sensor for detection of cyanide in aqueous solution and its application for bioimaging

A new NIR fluorescent sensor based on an amine-substituted heptamethine cyanine dye displayed a highly selective fluorescence enhancement with cyanide in aqueous solutions, and was applied for the imaging of anthropogenic and biogenic cyanide.

Salicylimine-Based Fluorescent Chemosensor for Aluminum Ions and Application to Bioimaging

In this study, an assay to quantify the presence of aluminum ions using a salicylimine-based receptor was developed utilizing turn-on fluorescence enhancement. Upon treatment with aluminum ions, the fluorescence of the sensor was enhanced at 510 nm due to formation of a 1:1 complex between the chemosensor and the aluminum ions at room temperature. As the concentration of Al(3+) was increased, the fluorescence gradually increased. Other metal ions, such as Na(+), Ag(+), K(+), Ca(2+), Mg(2+), Hg(2+), Mn(2+), Co(2+), Ni(2+), Cu(2+), Zn(2+), Cd(2+), Pb(2+), Cr(3+), Fe(3+), and In(3+), had no such significant effect on the fluorescence. In addition, we show that the probe could be used to map intracellular Al(3+) distribution in live cells by confocal microscopy.

A gel-free 3D microfluidic cell culture system

3D microfluidic cell culture systems offer a biologically relevant model to conduct micro-scale mammalian cell-based research and applications. Various natural and synthetic hydrogels have been successfully incorporated into microfluidic systems to support mammalian cells in 3D. However, embedment of cells in hydrogels introduces operational complexity, potentially hinders mass transfer, and is not suitable for establishing cell-dense, ECM-poor constructs. We present here a gel-free method for seeding and culturing mammalian cells three-dimensionally in a microfluidic channel. A combination of transient inter-cellular polymeric linker and micro-fabricated pillar arrays was used for the in situ formation and immobilization of 3D multi-cellular aggregates in a microfluidic channel. 3D cellular constructs formed this way are relieved of hydrogel embedment for cellular support. Two mammalian cell lines (A549 and C3A) and a primary mammalian cell (bone marrow mesenchymal stem cells) were cultured in the gel-free 3D microfluidic cell culture system. The cells displayed 3D cellular morphology, cellular functions and differentiation capability, affirming the versatility of the system as a 3D cell perfusion culture platform for anchorage-dependent mammalian cells.

Influence of topology on bacterial social interaction

The environmental topology of complex structures is used by Escherichia coli to create traveling waves of high cell density, a prelude to quorum sensing. When cells are grown to a moderate density within a confining microenvironment, these traveling waves of cell density allow the cells to find and collapse into confining topologies, which are unstable to population fluctuations above a critical threshold. This was first observed in mazes designed to mimic complex environments, then more clearly in a simpler geometry consisting of a large open area surrounding a square (250 × 250 μm) with a narrow opening of 10–30 μm. Our results thus show that under nutrient-deprived conditions bacteria search out each other in a collective manner and that the bacteria can dynamically confine themselves to highly enclosed spaces.

Bacterial metapopulations in nanofabricated landscapes.

We have constructed a linear array of coupled, microscale patches of habitat. When bacteria are inoculated into this habitat landscape, a metapopulation emerges. Local bacterial populations in each patch coexist and weakly couple with neighbor populations in nearby patches. These spatially distributed bacterial populations interact through local extinction and colonization processes. We have further built heterogeneous habitat landscapes to study the adaptive dynamics of the bacterial metapopulations. By patterning habitat differences across the landscape, our device physically implements an adaptive landscape. In landscapes with higher niche diversity, we observe rapid adaptation to large-scale, low-quality (high-stress) areas. Our results illustrate the potential lying at the interface between nanoscale biophysics and landscape evolutionary ecology.

Enhanced Caenorhabditis elegans Locomotion in a Structured Microfluidic Environment

Background Behavioral studies of Caenorhabditis elegans traditionally are done on the smooth surface of agar plates, but the natural habitat of C. elegans and other nematodes is the soil, a complex and structured environment. In order to investigate how worms move in such environments, we have developed a technique to study C. elegans locomotion in microstructures fabricated from agar. Methodology/Principal Findings When placed in open, liquid-filled, microfluidic chambers containing a square array of posts, we discovered that worms are capable of a novel mode of locomotion, which combines the fast gait of swimming with the more efficient movements of crawling. When the wavelength of the worms matched the periodicity of the post array, the microstructure directed the swimming and increased the speed of C. elegans ten-fold. We found that mutants defective in mechanosensation (mec-4, mec-10) or mutants with abnormal waveforms (unc-29) did not perform this enhanced locomotion and moved much more slowly than wild-type worms in the microstructure. Conclusion/Significance These results show that the microstructure can be used as a behavioral screen for mechanosensory and uncoordinated mutants. It is likely that worms use mechanosensation in the movement and navigation through heterogeneous environments.

Cyclic stretching of soft substrates induces spreading and growth

In the body, soft tissues often undergo cycles of stretching and relaxation that may affect cell behaviour without changing matrix rigidity. To determine whether transient forces can substitute for a rigid matrix, we stretched soft pillar arrays. Surprisingly, 1-5% cyclic stretching over a frequency range of 0.01-10 Hz caused spreading and stress fibre formation (optimum 0.1 Hz) that persisted after 4 h of stretching. Similarly, stretching increased cell growth rates on soft pillars comparative to rigid substrates. Of possible factors linked to fibroblast growth, MRTF-A (myocardin-related transcription factor-A) moved to the nucleus in 2 h of cyclic stretching and reversed on cessation; but YAP (Yes-associated protein) moved much later. Knockdown of either MRTF-A or YAP blocked stretch-dependent growth. Thus, we suggest that the repeated pulling from a soft matrix can substitute for a stiff matrix in stimulating spreading, stress fibre formation and growth.

Nictation, a dispersal behavior of the nematode Caenorhabditis elegans, is regulated by IL2 neurons

Many nematodes show a stage-specific behavior called nictation in which a worm stands on its tail and waves its head in three dimensions. Here we show that nictation is a dispersal behavior regulated by a specific set of neurons, the IL2 cells, in C. elegans. We established assays for nictation and showed that cholinergic transmission was required for nictation. Cell type–specific rescue experiments and genetic ablation experiments revealed that the IL2 ciliated head neurons were essential for nictation. Intact cilia in IL2 neurons, but not in other ciliated head neurons, were essential, as the restoration of the corresponding wild-type gene activity in IL2 neurons alone in cilia-defective mutants was sufficient to restore nictation. Optogenetic activation of IL2 neurons induced nictation, suggesting that signals from IL2 neurons are sufficient for nictation. Finally, we demonstrated that nictation is required for transmission of C. elegans to a new niche using flies as artificial carriers, suggesting a role of nictation as a dispersal and survival strategy under harsh conditions.