Lysophosphatidic acid signaling through LPA receptor subtype 1 induces colony scattering of gastrointestinal cancer cells

Purpose  Lysophosphatidic acid (LPA) is a multifunctional lipid mediator involved in triggering tumor cell invasion and metastasis, as well as malignant cell growth. LPA is also known to modulate the colony scattering of epithelial cancers, which is a prerequisite for cell invasion. However, the underlying details of how this is accomplished are not clear. Here we have investigated the roles of specific LPA receptor subtypes in cell scattering. Methods  Gastrointestinal carcinoma cell lines were examined for cell scattering activity in response to LPA, and the expression of LPA receptor subtypes was determined by RT-PCR. The effect of down regulation of each LPA receptor in DLD1 cells was determined using a shRNA-lentivirus system. In addition, the effect of overexpression of LPA receptors on cell scattering was investigated using lentivirus expression constructs. Results  The colonies of AGS and DLD1, but not MKN74, cells were dispersed in response to LPA. RT-PCR analysis revealed that the mRNAs of LPA1, LPA2, and LPA3 were present in AGS and DLD1 cells, but only LPA2 mRNA was detected in MKN74 cells. In DLD1 cells, the scattering activity induced by LPA was partially blocked by pretreatment with PP2 and PD98059, inhibitors of src kinase and MEK, respectively. LPA1 knockdown with shRNA decreased the degree of cell scattering induced by LPA. Knockdown of LPA2 or LPA3 had no effect on LPA-induced scattering. In addition, overexpression of LPA1 in DLD1 cells slightly decreased the response time of LPA-induced cell scattering. On the contrary, MKN74 cells expressing exogenous LPA1 did not respond to LPA by scattering. Conclusion  These results demonstrate that LPA1 mediates LPA-stimulated cell scattering of gastrointestinal carcinomas, but that activation of other intracellular pathways, besides those contributing to ERK phosphorylation, is also necessary for cell scattering in response to LPA. Y. L. Kim and K.-J. Shin equally contributed to this work.     

Metamaterials and the Landau–Lifshitz permeability argument: Large permittivity begets high-frequency magnetism

Homogeneous composites, or metamaterials, made of dielectric or metallic particles are known to show magnetic properties that contradict arguments by Landau and Lifshitz [Landau LD, Lifshitz EM (1960) Electrodynamics of Continuous Media (Pergamon, Oxford, UK), p 251], indicating that the magnetization and, thus, the permeability, loses its meaning at relatively low frequencies. Here, we show that these arguments do not apply to composites made of substances with ImεS ≫ λ/ℓ or ReεS ∼ λ/ℓ (ε_S and ℓ are the complex permittivity and the characteristic length of the particles, and λ ≫ ℓ is the vacuum wavelength). Our general analysis is supported by studies of split rings, one of the most common constituents of electromagnetic metamaterials, and spherical inclusions. An analytical solution is given to the problem of scattering by a small and thin split ring of arbitrary permittivity. Results reveal a close relationship between ε_S and the dynamic magnetic properties of metamaterials. For |εS | ≪ λ/a (a is the ring cross-sectional radius), the composites exhibit very weak magnetic activity, consistent with the Landau–Lifshitz argument and similar to that of molecular crystals. In contrast, large values of the permittivity lead to strong diamagnetic or paramagnetic behavior characterized by susceptibilities whose magnitude is significantly larger than that of natural substances. We compiled from the literature a list of materials that show high permittivity at wavelengths in the range 0.3–3000 μm. Calculations for a system of spherical inclusions made of these materials, using the magnetic counterpart to Lorentz–Lorenz formula, uncover large magnetic effects the strength of which diminishes with decreasing wavelength.     

Depression of reactivity by the collision energy in the single barrier H?+?CD4?→?HD?+?CD3 reaction

Crossed molecular beam experiments and accurate quantum scattering calculations have been carried out for the polyatomic H + CD₄ → HD + CD₃ reaction. Unprecedented agreement has been achieved between theory and experiments on the energy dependence of the integral cross section in a wide collision energy region that first rises and then falls considerably as the collision energy increases far over the reaction barrier for this simple hydrogen abstraction reaction. Detailed theoretical analysis shows that at collision energies far above the barrier the incoming H-atom moves so quickly that the heavier D-atom on CD₄ cannot concertedly follow it to form the HD product, resulting in the decline of reactivity with the increase of collision energy. We propose that this is also the very mechanism, operating in many abstraction reactions, which causes the differential cross section in the backward direction to decrease substantially or even vanish at collision energies far above the barrier height.     

Giant birefringence in optical antenna arrays with widely tailorable optical anisotropy

The manipulation of light by conventional optical components such as lenses, prisms, and waveplates involves engineering of the wavefront as it propagates through an optically thick medium. A unique class of flat optical components with high functionality can be designed by introducing abrupt phase shifts into the optical path, utilizing the resonant response of arrays of scatterers with deeply subwavelength thickness. As an application of this concept, we report a theoretical and experimental study of birefringent arrays of two-dimensional (V- and Y-shaped) optical antennas which support two orthogonal charge-oscillation modes and serve as broadband, anisotropic optical elements that can be used to locally tailor the amplitude, phase, and polarization of light. The degree of optical anisotropy can be designed by controlling the interference between the waves scattered by the antenna modes; in particular, we observe a striking effect in which the anisotropy disappears as a result of destructive interference. These properties are captured by a simple, physical model in which the antenna modes are treated as independent, orthogonally oriented harmonic oscillators.     

Optical spectroscopy shows that the normal state of URu2Si2 is an anomalous Fermi liquid

Fermi showed that, as a result of their quantum nature, electrons form a gas of particles whose temperature and density follow the so-called Fermi distribution. As shown by Landau, in a metal the electrons continue to act like free quantum mechanical particles with enhanced masses, despite their strong Coulomb interaction with each other and the positive background ions. This state of matter, the Landau–Fermi liquid, is recognized experimentally by an electrical resistivity that is proportional to the square of the absolute temperature plus a term proportional to the square of the frequency of the applied field. Calculations show that, if electron-electron scattering dominates the resistivity in a Landau–Fermi liquid, the ratio of the two terms, b, has the universal value of b = 4. We find that in the normal state of the heavy Fermion metal URu₂Si₂, instead of the Fermi liquid value of 4, the coefficient b = 1 ± 0.1. This unexpected result implies that the electrons in this material are experiencing a unique scattering process. This scattering is intrinsic and we suggest that the uranium f electrons do not hybridize to form a coherent Fermi liquid but instead act like a dense array of elastic impurities, interacting incoherently with the charge carriers. This behavior is not restricted to URu₂Si₂. Fermi liquid-like states with b ≠ 4 have been observed in a number of disparate systems, but the significance of this result has not been recognized.     

Microscopic identification of the order parameter governing liquid–liquid transition in a molecular liquid

A liquid–liquid transition (LLT) in a single-component substance is an unconventional phase transition from one liquid to another. LLT has recently attracted considerable attention because of its fundamental importance in our understanding of the liquid state. To access the order parameter governing LLT from a microscopic viewpoint, here we follow the structural evolution during the LLT of an organic molecular liquid, triphenyl phosphite (TPP), by time-resolved small- and wide-angle X-ray scattering measurements. We find that locally favored clusters, whose characteristic size is a few nanometers, are spontaneously formed and their number density monotonically increases during LLT. This strongly suggests that the order parameter of LLT is the number density of locally favored structures and of nonconserved nature. We also show that the locally favored structures are distinct from the crystal structure and these two types of orderings compete with each other. Thus, our study not only experimentally identifies the structural order parameter governing LLT, but also may settle a long-standing debate on the nature of the transition in TPP, i.e., whether the transition is LLT or merely microcrystal formation.Liquid-liquid transition (LLT) is an intriguing phenomenon in which a liquid transforms into another one via a first-order transition. This means that there can be more than two liquid states for a single-component substance. Despite its counterintuitive nature, there have recently been many pieces of experimental and numerical evidence for the existence of LLT, for various liquids such as water (1–5), aqueous solutions (6–8), triphenyl phosphite (9–12), l-butanol (13), phosphorus (14), silicon (15, 16), germanium (17), and Y₂O₃–Al₂O₃ (18, 19). This suggests that the LLT may be rather universally observed for various types of liquids. However, none of the LLTs reported so far is free from criticisms (20, 21), mainly because these LLTs take place under experimentally difficult conditions [e.g., at high temperature and pressure (14, 15, 17–19)] or in a supercooled state below the melting point (1–3, 5–7, 9, 10), where the transition is inevitably contaminated by microcrystal formation. The latter is not limited to experiments but arises in numerical simulations, often causing many controversies [LLT (22–25) vs. crystallization (26–28)]. For ST2 water, however, this issue has recently been settled by an extensive simulation study by Palmer et al. (4).One of the hottest and long-standing debates is on the nature of the transition found in a molecular liquid, triphenyl phosphite (TPP), by Kivelson and his coworkers (29). The transition is very easy to access experimentally, because it takes place at ambient pressure and at a temperature range between 230 and 210 K and the transformation speed is slow enough to follow the kinetics. Since the finding of this transition (29, 30), many researchers thus have been interested in this intriguing phenomenon and there have been hot discussions on the nature of the transition (20, 21). Some people interpreted this as a liquid-associated phenomenon (9, 10, 31, 32), but others interpret it differently. All of the controversies come from the fact that this transition accompanies microcrystal formation and thus the final state, which is called “glacial phase,” often contains microcrystallites. This led many researchers to explain the transition by non-LLT scenarios, which include a defect-ordered phase scenario predicted by a frustration limited domain theory (29, 30, 33, 34), a microcrystallization scenario (35–38), and a liquid-crystal or plastic-crystal phase scenario (39). Each scenario captures a certain feature of the glacial phase, but fails in explaining all of the experimental results in a consistent manner. Similar situations are often seen in other candidates of LLTs, such as l-butanol [LLT (13) vs. microcrystallization (40–43)], confined water [LLT (5) vs. other phenomena (44–46)], and aqueous solutions [LLT (6, 7) vs. microcrystallization (8, 28, 47, 48)]. For TPP, however, some pieces of experimental evidence supportive of the LLT scenario rather than the microcrystallization scenario have recently been reported (11, 12).We propose a two-order-parameter (TOP) model of a liquid to explain LLT (20, 49). The main point of this model is that it is necessary to consider the spatiotemporal hierarchical nature of a liquid to understand LLT. More specifically, we argue that in addition to density order parameter ρ describing a gas–liquid transition, we need an additional scalar order parameter S, which is the number density of locally favored structures (LFS). In this model, LLT is a consequence of the cooperative ordering of the scalar nonconserved order parameter S, i.e., the cooperative formation of LFS. In other words, LLT is regarded as a gas–liquid-like transition of LFS: one liquid is a gas state of LFS (low-S state), and the other is its liquid state (high-S state). Recently, it was proposed by Anisimov and coworkers (50, 51) that the thermodynamic ordering field conjugate to the order parameter is the conversion equilibrium constant, which further characterizes the nature of LLT. We explained our experimental observation of LLT in TPP in terms of this model (9, 10). We also studied the phase transition dynamics and the physical and chemical properties of the second liquid state (liquid II), which were also explained by the model (20, 21).However, we have not had any direct experimental evidence for the formation of such LFS up to now; thus, an open question is, what is the relevant order parameter governing LLT, although the link of the order parameter to the enthalpy (9, 10), the refractive index (or, density) (9, 10, 29, 30), and the polarity associated with local molecular ordering (12) has been suggested for LLT in TPP. There have been structural studies on LLT by X-ray and neutron scattering measurements, focusing on local liquid structures at an inter- and intramolecular scale (36, 38, 52–54) and mesoscopic structures (34, 55). However, there has been no experimental evidence for the presence of locally favored structures, which characterize the liquid state uniquely, or the order parameter has still not been identified from a microscopic viewpoint.Here we study the structural change of TPP during LLT by time-resolved small- and wide-angle X-ray scattering measurements, which cover a length scale from a single molecule size ( ～  1 nm) to more than tens of nanometers. We show, to our knowledge, the first direct evidence for the presence of LFS and the temporal increase upon the liquid I-to-liquid II transformation. Furthermore, we also find an indication of the formation of microcrystallites during LLT. However, we reveal that LFS and microcrystallites have different sizes and growth kinetics, indicating that although they sometimes appear simultaneously during the process of LLT, LLT itself is driven by the formation of LFS and not by that of microcrystallites. We also discover that LFS are destroyed upon crystallization, clearly indicating not only that these two types of orderings are competing with each other but also that LFS is a structure unique to the liquid state. Our findings provide a comprehensive view on the long-standing controversy on the origin of the glacial phase, which was discovered by Kivelson and his coworkers (29, 30), and show that the fraction of LFS may be the relevant order parameter of LLT. This suggests that a liquid can have a spatiotemporal hierarchical structure at a low temperature, contrary to the common picture of a high-temperature liquid where the structure is random and homogeneous beyond the molecular size.     

Label-free DNA imaging in vivo with stimulated Raman scattering microscopy

Label-free DNA imaging is highly desirable in biology and medicine to perform live imaging without affecting cell function and to obtain instant histological tissue examination during surgical procedures. Here we show a label-free DNA imaging method with stimulated Raman scattering (SRS) microscopy for visualization of the cell nuclei in live animals and intact fresh human tissues with subcellular resolution. Relying on the distinct Raman spectral features of the carbon-hydrogen bonds in DNA, the distribution of DNA is retrieved from the strong background of proteins and lipids by linear decomposition of SRS images at three optimally selected Raman shifts. Based on changes on DNA condensation in the nucleus, we were able to capture chromosome dynamics during cell division both in vitro and in vivo. We tracked mouse skin cell proliferation, induced by drug treatment, through in vivo counting of the mitotic rate. Furthermore, we demonstrated a label-free histology method for human skin cancer diagnosis that provides comparable results to other conventional tissue staining methods such as H&E. Our approach exhibits higher sensitivity than SRS imaging of DNA in the fingerprint spectral region. Compared with spontaneous Raman imaging of DNA, our approach is three orders of magnitude faster, allowing both chromatin dynamic studies and label-free optical histology in real time.In vivo imaging of chromatin or chromosome structures and dynamics during vital cellular processes, such as cell division, differentiation, apoptosis, and carcinogenesis, generally relies on the use of either exogenous or endogenous fluorescent labels, the latter of which often involves complicated transgenic organisms (1, 2). A label-free approach, however, allows the visualization of these processes in a noninvasive way in live organisms. In medicine, visualization of nuclear morphology, architecture, size, shape, and mitotic figures provide the most important cytologic features for rendering histologic diagnosis (3, 4). Conventional histology is heavily reliant on tissue biopsies and staining (such as H&E or immunohistochemistry), whereas label-free imaging is able to reveal similar information as that from the stained tissue, and in addition, it allows for a noninvasive characterization and diagnosis of human tissue in real time in vivo.Stimulated Raman scattering (SRS) microscopy offers a contrast mechanism based on Raman spectroscopy, probing the intrinsic vibrational frequencies of chemical bonds or groups (5–8). In SRS microscopy, the collinear pump and Stokes laser beams, at frequencies of ω_p and ω_s, respectively, are tightly focused onto the sample (Fig. 1A). When the frequency difference, ω_p − ω_s, matches a Raman-active molecular vibration, the SRS signal (attenuation to the pump beam or increase on the Stokes beam) is generated through a nonlinear process similar to the stimulated emission. With a highly sensitive detection scheme, involving megahertz modulation transfer, SRS microscopy exhibits orders of magnitude of shorter acquisition time than conventional Raman microscopy (5). Being a nonlinear optical microscopy, it offers 3D sectioning capability with a diffraction-limited spatial resolution. SRS microscopy has been extensively applied to image biomolecules in cells and tissues (9–15).Open in a separate windowFig. 1.Label-free SRS imaging of DNA (magenta), protein (blue), and lipids (green) in live cells. SRS images at three selected Raman shifts in the CH stretching vibrational band were acquired. Linear decomposition was performed with a premeasured calibration matrix to retrieve the distribution of DNA, protein, and lipids. (A) Setup of the SRS microscopy, capable of automatically acquiring images at multiple Raman shifts. This was achieved by synchronizing the tuning of the laser frequency (Lyot filter) to the imaging frame trigger of the microscope. (Inset) Time-lapse images of a HeLa cell undergoing cell division (Movie S1). (B) Raman spectra of DNA, cellular protein, and cellular lipids extracted from HeLa cells. (C) Raman spectrum of the cell pellet. Linear fitting demonstrated that the three compounds in B accounted for ∼90% of the total CH stretching vibration of the cells. (D) SRS images of a live cell in mitotic phase (prophase) at 2,967, 2,926, and 2,850 cm⁻¹, respectively, and the decomposed distribution of DNA, protein, lipids, and the overlay. Chromosomes were visualized with both high contrast and high signal-to-noise ratio. (E) SRS images of a live cell in interphase and the decomposed distribution of DNA, protein, lipids, and the overlay. Detailed internal nuclear features were revealed clearly. (F) Images with SRS and TPEF of a mitotic cell stained with DRAQ5, correlated very well with each other. (Scale bar, 10 μm.)SRS imaging was initially carried out at one Raman shift at a time (5). Recent developments on multiplex detection allow for distinguishing various chemical species with overlapping Raman bands by either broadband excitation (16, 17) or narrowband scanning (18, 19). SRS at two specific Raman shifts within the broadband of the carbon-hydrogen (CH) stretching vibrational mode (2,800–3,050 cm⁻¹) has been used to simultaneously map protein and lipid distribution in cells and tissues (20, 21). In particular, protein and lipid imaging has been applied to delineate brain tumor margins, providing images similar to conventional H&E staining (11). However, SRS does not offer detailed nuclear morphology and architecture, compared with the conventional histology, due to the lack of imaging contrast for DNA.SRS has been demonstrated to be valuable for DNA imaging in cultured cells based on detection of the phosphate peaks within the fingerprint spectral region (22). However, imaging of DNA in this spectral region is difficult for cells in interphase because of the lower DNA density, especially in live tissue. This challenge is also the case for spontaneous Raman imaging (SI Text) (23).Here we demonstrate that, relying on the unique and distinct spectral features of DNA in the CH stretching vibrational region (the high wavenumber range), the distribution of DNA, together with those of protein and lipids, can be mapped by the linear decomposition of images at three optimally selected Raman shifts. This approach offers much higher sensitivity than that of DNA imaging in the fingerprint region, making dynamic imaging of DNA feasible for both mitotic phase and interphase cells in vitro and in vivo.     

知母标准汤剂的质量评价方法及指标成分的量值传递规律探索

目的：建立知母标准汤剂的质量评价方法,明确10批知母标准汤剂中指标成分的含量及转移率范围,探索从饮片到标准汤剂,指标成分的量值传递规律。方法：制备10批知母标准汤剂,采用超高效液相色谱-二极管阵列检测器法(UPLC-PDA)对指标成分芒果苷进行含量测定,以0.1%甲酸水(A)-乙腈(B)为流动相进行梯度洗脱(0～3 min,10%～13%B;3～6 min,13%～14%B;6～7 min,14%～90%B),检测波长为258 nm;采用高效液相色谱-蒸发光散射检测器法(HPLC-ELSD)对知母皂苷BⅡ进行含量测定,以乙腈-水(25∶75)为流动相进行等度洗脱,载气流速为2.8 L·min^–1,漂移管温度为104℃。结果：建立的指标成分含量测定方法的方法学验证均良好,能用于知母标准汤剂中2个指标性成分的定量检测。10批知母标准汤剂中芒果苷、知母皂苷BⅡ的质量分数分别为0.88%～1.12%和0.88%～2.53%,芒果苷含量位于其均值的±30%范围内;转移率分别为45.83%～57.27%和26.94%～43.49%,均在其均值的±30%范围内。结论：建立了知母...     

Hepatocyte growth factor and c-Met (HGF/c-Met) in adenoid cystic carcinoma of the human salivary gland

BACKGROUND: Hepatocyte growth factor (HGF) enhances cell growth, morphogenesis, and scattering of various epithelial cells. The aim of this study was to evaluate the hypothesis that HGF/c-Met plays a biological role in the invasive growth of adenoid cystic carcinoma (ACC). METHODS: Immunohistochemically, expression of HGF and its receptor c-Met was examined in 15 cases of ACC. To examine the direct effects of HGF on ACC, cell line derived from of ACC (ACC3) was used. The expression of HGF and c-met in ACC3 was investigated by RT-PCR. Analysis of mechanisms of invasion was done by performing scattering assay and matrigel invasion chamber assay. RESULTS: Positive staining of HGF was found in all cases, and that of c-Met was 67%. In ACC3, c-met was expressed, but not HGF. Stimulation of ACC3 by rhHGF induced scattering and promoted invasion. CONCLUSION: The present results suggest that HGF/c-Met increases tumor cell scattering and may play a part in invasiveness of ACC.