Dynamic Properties of Heart Fragments from Different Regions and Their Synchronization.

The dynamic properties of the heart differ based on the regions that effectively circulate blood throughout the body with each heartbeat. These properties, including the inter-beat interval (IBI) of autonomous beat activity, are retained even in in vitro tissue fragments. However, details of beat dynamics have not been well analyzed, particularly at the sub-mm scale, although such dynamics of size are important for regenerative medicine and computational studies of the heart. We analyzed the beat dynamics in sub-mm tissue fragments from atria and ventricles of hearts obtained from chick embryos over a period of 40 h. The IBI and contraction speed differed by region and atrial fragments retained their values for a longer time. The major finding of this study is synchronization of these fragment pairs physically attached to each other. The probability of achieving this and the time required differ for regional pairs: atrium-atrium, ventricle-ventricle, or atrium-ventricle. Furthermore, the time required to achieve 1:1 synchronization does not depend on the proximity of initial IBI of paired fragments. Various interesting phenomena, such as 1:n synchronization and a reentrant-like beat sequence, are revealed during synchronization. Finally, our observation of fragment dynamics indicates that mechanical motion itself contributes to the synchronization of atria.

Biochemical study of type I collagen purified from skin of warm sea teleost Mahi mahi (Coryphaena hippurus), with a focus on thermal and physical stability.

Acid- and pepsin-soluble collagen were purified from the skin of mahi mahi (mmASC and mmPSC). The Pro+Hyp content of the latter (185/1,000 residues) was highest among all marine teleost fishes. Fourier transform infrared spectroscopy and Circular Dichroism (CD) analysis showed the typical structure of type I collagen. The ratio of positive over negative peak intensity calculated from the CD spectrum was approximately 1.19 in mmPSC, which is remarkably high, and indicates the stability of the triple helix. The denaturation temperatures (Td ) of mmASC and mmPSC were the highest (29.5 and 28.8°C, respectively) among the marine teleost fishes previously studied. atomic force microscope and scanning electron microscope images showed that even after pretreatment, the fibrils presented their structure and fiber orientation. These results indicate the robustness of both collagens, which can be attributed to the high value of Pro+Hyp stabilizing the helix structure of the collagen molecule. Practical applications While Mahi mahi is highly valuable for its meat, other parts such as skin is not fully utilized in seafood industry. On the contrary, it has been empirically shown that the skin of Mahi mahi has high thermal stability, thus, the skin has been used for leather products in some areas located in the tropical and subtropical zones. In this study, we focused on collagen a major component in skin and investigated the structure and the biochemical characteristics of it. Some results showed that collagen from skin has high physical stability. The collagen from skin of Mahi mahi will be a new fishery resource which could be used as a material for collagen products.

Clog and Release, and Reverse Motions of DNA in a Nanopore.

Motions of circular and linear DNA molecules of various lengths near a nanopore of 100 or 200 nm diameter were experimentally observed and investigated by fluorescence microscopy. The movement of DNA molecules through nanopores, known as translocation, is mainly driven by electric fields near and inside the pores. We found significant clogging of nanopores by DNA molecules, particularly by circular DNA and linear T4 DNA (165.65 kbp). Here, the probabilities of DNA clogging events, depending on the DNA length and shape-linear or circular-were determined. Furthermore, two distinct DNA motions were observed: clog and release by linear T4 DNA, and a reverse direction motion at the pore entrance by circular DNA, after which both molecules moved away from the pore. Finite element method-based numerical simulations were performed. The results indicated that DNA molecules with pores 100⁻200 nm in diameter were strongly influenced by opposing hydrodynamic streaming flow, which was further enhanced by bulky DNA configurations.

Involvement of selective epithelial cell death in the formation of feather buds on a bioengineered skin.

Selective cell death by apoptosis plays important roles in organogenesis. Apoptotic cells are observed in the developmental and homeostatic processes of several ectodermal organs, such as hairs, feathers, and mammary glands. In chick feather development, apoptotic events have been observed during feather morphogenesis, but have not been investigated during early feather bud formation. Previously, we have reported a method for generating feather buds on a bioengineered skin from dissociated skin epithelial and mesenchymal cells in three-dimensional culture. During the development of the bioengineered skin, epithelial cavity formation by apoptosis was observed in the epithelial tissue. In this study, we examined the selective epithelial cell death during the bioengineered skin development. Histological analyses suggest that the selective epithelial cell death in the bioengineered skin was induced by caspase-3-related apoptosis. The formation of feather buds of the bioengineered skin was disturbed by the treatment with a pan-caspase inhibitor. The pan-caspase inhibitor treatment suppressed the rearrangement of the epithelial layer and the formation of dermal condensation, which are thought to be essential step to form feather buds. The suppression of the formation of feather buds on the pan-caspase inhibitor-treated skin was partially compensated by the addition of a GSK-3ß inhibitor, which activates Wnt/ß-catenin signaling. These results suggest that the epithelial cell death is involved in the formation of feather buds of the bioengineered skin. These observations also suggest that caspase activities and Wnt/ß-catenin signaling may contribute to the formation of epithelial and mesenchymal components in the bioengineered skin.

In vitro formation of the Merkel cell-neurite complex in embryonic mouse whiskers using organotypic co-cultures.

A Merkel cell-neurite complex is a touch receptor composed of specialized epithelial cells named Merkel cells and peripheral sensory nerves in the skin. Merkel cells are found in touch-sensitive skin components including whisker follicles. The nerve fibers that innervate Merkel cells of a whisker follicle extend from the maxillary branch of the trigeminal ganglion. Whiskers as a sensory organ attribute to the complicated architecture of the Merkel cell-neurite complex, and therefore it is intriguing how the structure is formed. However, observing the dynamic process of the formation of a Merkel cell-neurite complex in whiskers during embryonic development is still difficult. In this study, we tried to develop an organotypic co-culture method of a whisker pad and a trigeminal ganglion explant to form the Merkel cell-neurite complex in vitro. We initially developed two distinct culture methods of a single whisker row and a trigeminal ganglion explant, and then combined them. By dissecting and cultivating a single row from a whisker pad, the morphogenesis of whisker follicles could be observed under a microscope. After the co-cultivation of the whisker row with a trigeminal ganglion explant, a Merkel cell-neurite complex composed of Merkel cells, which were positive for both cytokeratin 8 and SOX2, Neurofilament-H-positive trigeminal nerve fibers and Schwann cells expressing Nestin, SOX2 and SOX10 was observed via immunohistochemical analyses. These results suggest that the process for the formation of a Merkel cell-neurite complex can be observed under a microscope using our organotypic co-culture method.

Gate-Voltage-Controlled Threading DNA into Transistor Nanopores.

We present a simple method for DNA translocation driven by applying AC voltages, such as square and sawtooth waves, on an embedded thin film as a gate electrode inside of a dielectric nanopore, without applying a conventional bias voltage externally across the pore membrane. Square waveforms on a gate can drive a single DNA molecule into a nanopore, which often returns from the pore, causing an oscillation across the membrane. An optimized sawtooth-like negative voltage pulse on the gate can thread a fraction of a DNA molecule into a pore after a single pulse. This trapped DNA molecule continues to finish its translocation slowly through the pore. The DNA's slow speed was comparable to previous findings of the escaping DNA speed from a nanopore estimated by the Smoluchowski equation with excluded-volume interactions of a long-chain molecule and electrophoresis by extremely low electric fields. This simple scheme, controlling DNA molecules only by gate potential modulation at a nanopore, will provide an additional method to thread, translocate, or oscillate a single biomolecule at a gated nanopore.

Role of the boundary in feather bud formation on one-dimensional bioengineered skin.

The role of a boundary in pattern formation from a homogenous state in Turing's reaction-diffusion equations is important, particularly when the domain size is comparable to the pattern scale. Such experimental conditions may be achieved for in vitro regeneration of ectodermal appendages such as feathers, via reconstruction of embryonic single cells. This procedure can eliminate a predefined genetic map, such as the midline of chick feather bud formation, leaving uniformly distributed identical cells as a bioengineered skin. Here, the self-organizing nature of multiple feather bud formation was examined in bioengineered 1D-skin samples. Primal formation of feather buds occurred at a fixed length from the skin edge. This formation was numerically recapitulated by a standard two-component reaction-diffusion model, suggesting that the boundary effect caused this observation. The proper boundary conditions were nonstandard, either mixed Dirichlet-Neumann or partial-flux. In addition, the model implies imperfect or hindered bud formation as well as nearly equal distances between buds. In contrast, experimental observations indicated that the skin curvature, which was not included in our model, also strongly affected bud formation. Thus, bioengineered skin may provide an ideal template for modeling a self-organized process from a homogenous state. This study will examine the possible diffusion activities of activator or inhibitor molecular candidates and mechanical activities during cell aggregation, which will advance our understanding of skin appendage regeneration from pluripotent or embryonic stem cells.

Generation of bioengineered feather buds on a reconstructed chick skin from dissociated epithelial and mesenchymal cells.

Various kinds of in vitro culture systems of tissues and organs have been developed, and applied to understand multicellular systems during embryonic organogenesis. In the research field of feather bud development, tissue recombination assays using an intact epithelial tissue and mesenchymal tissue/cells have contributed to our understanding the mechanisms of feather bud formation and development. However, there are few methods to generate a skin and its appendages from single cells of both epithelium and mesenchyme. In this study, we have developed a bioengineering method to reconstruct an embryonic dorsal skin after completely dissociating single epithelial and mesenchymal cells from chick skin. Multiple feather buds can form on the reconstructed skin in a single row in vitro. The bioengineered feather buds develop into long feather buds by transplantation onto a chorioallantoic membrane. The bioengineered bud sizes were similar to those of native embryo. The number of bioengineered buds was increased linearly with the initial contact length of epithelial and mesenchymal cell layers where the epithelial-mesenchymal interactions occur. In addition, the bioengineered bud formation was also disturbed by the inhibition of major signaling pathways including FGF (fibroblast growth factor), Wnt/ß-catenin, Notch and BMP (bone morphogenetic protein). We expect that our bioengineering technique will motivate further extensive research on multicellular developmental systems, such as the formation and sizing of cutaneous appendages, and their regulatory mechanisms.

DNA motion induced by electrokinetic flow near an Au coated nanopore surface as voltage controlled gate.

We used fluorescence microscopy to investigate the diffusion and drift motion of λ DNA molecules on an Au-coated membrane surface near nanopores, prior to their translocation through solid-state nanopores. With the capability of controlling electric potential at the Au surface as a gate voltage, Vgate, the motions of DNA molecules, which are presumably generated by electrokinetic flow, vary dramatically near the nanopores in our observations. We carefully investigate these DNA motions with different values of Vgate in order to alter the densities and polarities of the counterions, which are expected to change the flow speed or direction, respectively. Depending on Vgate, our observations have revealed the critical distance from a nanopore for DNA molecules to be attracted or repelled-DNA's anisotropic and unsteady drifting motions and accumulations of DNA molecules near the nanopore entrance. Further finite element method (FEM) numerical simulations indicate that the electrokinetic flow could qualitatively explain these unusual DNA motions near metal-collated gated nanopores. Finally, we demonstrate the possibility of controlling the speed and direction of DNA motion near or through a nanopore, as in the case of recapturing a single DNA molecule multiple times with alternating current voltages on the Vgate.

Raman and autofluorescence spectrum dynamics along the HRG-induced differentiation pathway of MCF-7 cells.

Cellular differentiation proceeds along complicated pathways, even when it is induced by extracellular signaling molecules. One of the major reasons for this complexity is the highly multidimensional internal dynamics of cells, which sometimes causes apparently stochastic responses in individual cells to extracellular stimuli. Therefore, to understand cell differentiation, it is necessary to monitor the internal dynamics of cells at single-cell resolution. Here, we used a Raman and autofluorescence spectrum analysis of single cells to detect dynamic changes in intracellular molecular components. MCF-7 cells are a human cancer-derived cell line that can be induced to differentiate into mammary-gland-like cells with the addition of heregulin (HRG) to the culture medium. We measured the spectra in the cytoplasm of MCF-7 cells during 12 days of HRG stimulation. The Raman scattering spectrum, which was the major component of the signal, changed with time. A multicomponent analysis of the Raman spectrum revealed that the dynamics of the major components of the intracellular molecules, including proteins and lipids, changed cyclically along the differentiation pathway. The background autofluorescence signals of Raman scattering also provided information about the differentiation process. Using the total information from the Raman and autofluorescence spectra, we were able to visualize the pathway of cell differentiation in the multicomponent phase space.

Directly observing the motion of DNA molecules near solid-state nanopores.

We investigate the diffusion and the drift motion of λ DNA molecules near solid-state nanopores prior to their translocation through the nanopores using fluorescence microscopy. The radial dependence of the electric field near a nanopore generated by an applied voltage in ionic solution can be estimated quantitatively in 3D by analyzing the motion of negatively charged DNA molecules. We find that the electric field is approximately spherically symmetric around the nanopore under the conditions investigated. In addition, DNA clogging at the nanopore was directly observed. Surprisingly, the probability of the clogging event increases with increasing external bias voltage. We also find that DNA molecules clogging the nanopore reduce the electric field amplitude at the nanopore membrane surface. To better understand these experimental results, analytical method with Ohm's law and computer simulation with Poisson and Nernst-Planck (PNP) equations are used to calculate the electric field near the nanopore. These results are of great interest in both experimental and theoretical considerations of the motion of DNA molecules near voltage-biased nanopores. These findings will also contribute to the development of solid-state nanopore-based DNA sensing devices.

Metal- and hydrogen-bonding competition during water adsorption on Pd(111) and Ru(0001).

The initial stages of water adsorption on the Pd(111) and Ru(0001) surfaces have been investigated experimentally by scanning tunneling microscopy in the temperature range between 40 and 130 K, and theoretically with density functional theory (DFT) total energy calculations and scanning tunneling microscopy (STM) image simulations. Below 125 K, water dissociation does not occur at any appreciable rate, and only molecular films are formed. Film growth starts by the formation of flat hexamer clusters where the molecules bind to the metal substrate through the O-lone pair while making H-bonds with neighboring molecules. As coverage increases, larger networks of linked hexagons are formed with a honeycomb structure, which requires a fraction of the water molecules to have their molecular plane perpendicular to the metal surface with reduced water-metal interaction. Energy minimization favors the growth of networks with limited width. As additional water molecules adsorb on the surface, they attach to the periphery of existing islands, where they interact only weakly with the metal substrate. These molecules hop along the periphery of the clusters at intermediate temperatures. At higher temperatures, they bind to the metal to continue the honeycomb growth. The water-Ru interaction is significantly stronger than the water-Pd interaction, which is consistent with the greater degree of hydrogen-bonded network formation and reduced water-metal bonding observed on Pd relative to Ru.

Nanoscale volcanoes: accretion of matter at ion-sculpted nanopores.

We demonstrate the formation of nanoscale volcano-like structures induced by ion-beam irradiation of nanoscale pores in freestanding silicon nitride membranes. Accreted matter is delivered to the volcanoes from micrometer distances along the surface. Volcano formation accompanies nanopore shrinking and depends on geometrical factors and the presence of a conducting layer on the membrane's back surface. We argue that surface electric fields play an important role in accounting for the experimental observations.

Rapid and sensitive determination of morphine in street opium samples by thermal desorption gas chromatography using a microfurnace pyrolyzer.

Thermal desorption of the alkaloids in opium samples at 300 degrees C using a vertical microfurnace pyrolyzer was followed by their on-line gas chromatographic (GC) analysis on a large-bore glass capillary column. This method permitted rapid and sensitive determination of the content of the main alkaloid, morphine, in the small (ca. 100 microg) opium samples with a relative standard deviation within 4% for 5 runs. The observed morphine contents of about 12 to 15 w/w% in the given opium samples were in fairly good agreement with those estimated by a conventional GC-MS method.

Atomic Layer Deposition to Fine-Tune the Surface Properties and Diameters of Fabricated Nanopores.

Atomic layer deposition of alumina enhanced the molecule sensing characteristics of fabricated nanopores by fine-tuning their surface properties, reducing 1/f noise, neutralizing surface charge to favor capture of DNA and other negative polyelectrolytes, and controlling the diameter and aspect ratio of the pores with near single Ångstrom precision. The control over the chemical and physical nature of the pore surface provided by atomic layer deposition produced a higher yield of functional nanopore detectors.