Molecular Gradients Research Articles

Chemokine Signaling Facilitates Early-Stage Breast Cancer Survival and Invasion through Fibroblast-Dependent Mechanisms.

Ductal carcinoma in situ (DCIS) is the most common form of breast cancer, with 50,000 cases diagnosed every year in the United States. Overtreatment and undertreatment remain significant clinical challenges in patient care. Identifying key mechanisms associated with DCIS progression could uncover new biomarkers to better predict patient prognosis and improve guided treatment. Chemokines are small soluble molecules that regulate cellular homing through molecular gradients. CCL2-mediated recruitment of CCR2+ macrophages are a well-established mechanism for metastatic progression. Although the CCL2/CCR2 pathway is a therapeutic target of interest, little is known about the role of CCR2 expression in breast cancer. Here, using a mammary intraductal injection (MIND) model to mimic DCIS formation, the role of CCR2 was explored in minimally invasive SUM225 and highly invasive DCIS.com breast cancer cells. CCR2 overexpression increased SUM225 breast cancer survival and invasion associated with accumulation of CCL2 expressing fibroblasts. CCR2-deficient DCIS.com breast cancer cells formed fewer invasive lesions with fewer CCL2+ fibroblasts. Cografting CCL2-deficient fibroblasts with DCIS.com breast cancer cells in the subrenal capsule model inhibited tumor invasion and survival associated with decreased expression of aldehyde dehydrogenase (ALDH1), a proinvasive factor, and decreased expression of HTRA2, a proapoptotic serine protease. Through data mining analysis, high expression of CCR2 and ALDH1 and low HTRA2 expression were correlated with poor prognosis of breast cancer patients.Implications: This study demonstrates that CCR2 overexpression in breast cancer drives early-stage breast cancer progression through stromal-dependent expression of CCL2 with important insight into prognosis and treatment of DCIS. Mol Cancer Res; 16(2); 296-308. ©2017 AACR.

High Speed Motors Driven by a Molecular Tension Gradient

High Speed Motors Driven by a Molecular Tension Gradient

Extracellular Processing of Molecular Gradients by Eukaryotic Cells Can Improve Gradient Detection Accuracy.

Eukaryotic cells sense molecular gradients by measuring spatial concentration variation through the difference in the number of occupied receptors to which molecules can bind. They also secrete enzymes that degrade these molecules, and it is presently not well understood how this affects the local gradient perceived by cells. Numerical and analytical results show that these enzymes can substantially increase the signal-to-noise ratio of the receptor difference and allow cells to respond to a much broader range of molecular concentrations and gradients than they would without these enzymes.

Regeneration of cortical tissue from brain injury by implantation of defined molecular gradient of semaphorin 3A

Despite great efforts in the exploration of therapeutic strategies for treating brain injuries, it is still challenging to regenerate neural tissues and to restore the lost function within an injured brain. In this report, we employed a tissue engineering approach to regenerate cortical tissue from brain injury by implantation of defined semaphorin 3A (Sema3A) gradient packaged in a hydrogel based device. Over a thirty-day recovery period, the implanted Sema3A gradient was sufficient to induce substantial migration of neural progenitor cells to the hydrogel and to promote differentiation of these cells for neuroregeneration at the injury site. As revealed by molecular characterization and RNA transcriptome analysis, the regenerated tissues induced by Sema3A gradient exhibited significant similarity to normal cortical tissues. Many genes associated with neuronal migration and stem cell differentiation were significantly up-regulated. In addition, our result suggested a crosstalk between Sema3A and Wnt/β-catenin pathways in course of induced brain regeneration. This study demonstrated an innovative strategy to regenerate brain tissue after traumatic injury by controlling the in vivo chemotactic environment with unprecedented sophistication, and also resolved new insights about Sema3A's role in adult neurogenesis.

Preparation of Adult Rat Sensory Neuron Cultures and Their Application to Growth Cone Turning Assays.

The directional trajectory of growing peripheral nerve axons in the adult impacts their successful regeneration to denervated target tissues. Misdirected axons in neuromas, injured nerve trunks, or nerves with attempted repair diminish the success of regeneration. The behavior of adult rodent peripheral sensory neurons in vitro, in turn, is helpful in predicting axonal behavior in vivo. Here, we describe the adaptation of embryonic neuron growth cone turning assays, an important technique in developmental neurobiology, to adult rat sensory neurons. With some key modifications, and selection of subtypes of neurons likely to respond to a purported growth factor, short-term responses to molecular gradients can be analyzed using routine dorsal root ganglion neuronal cell culture techniques. The caveats are that short-term turning does not necessarily reflect on the overall tropic impact of a molecule, particularly if it alters growth cones through intra-axonal translation. Similarly, to understand the trajectory of an axon, it must be in a growth mode, such as that associated with preconditioning from previous injury.

Activity-dependent formation of a vesicular inhibitory amino acid transporter gradient in the superior olivary complex of NMRI mice

BackgroundIn the mammalian superior olivary complex (SOC), synaptic inhibition contributes to the processing of binaural sound cues important for sound localization. Previous analyses demonstrated a tonotopic gradient for postsynaptic proteins mediating inhibitory neurotransmission in the lateral superior olive (LSO), a major nucleus of the SOC. To probe, whether a presynaptic molecular gradient exists as well, we investigated immunoreactivity against the vesicular inhibitory amino acid transporter (VIAAT) in the mouse auditory brainstem.ResultsImmunoreactivity against VIAAT revealed a gradient in the LSO and the superior paraolivary nucleus (SPN) of NMRI mice, with high expression in the lateral, low frequency processing limb and low expression in the medial, high frequency processing limb of both nuclei. This orientation is opposite to the previously reported gradient of glycine receptors in the LSO. Other nuclei of the SOC showed a uniform distribution of VIAAT-immunoreactivity. No gradient was observed for the glycine transporter GlyT2 and the neuronal protein NeuN. Formation of the VIAAT gradient was developmentally regulated and occurred around hearing-onset between postnatal days 8 and 16. Congenital deaf Claudin14−/− mice bred on an NMRI background showed a uniform VIAAT-immunoreactivity in the LSO, whereas cochlear ablation in NMRI mice after hearing-onset did not affect the gradient. Additional analysis of C57Bl6/J, 129/SvJ and CBA/J mice revealed a strain-specific formation of the gradient.ConclusionsOur results identify an activity-regulated gradient of VIAAT in the SOC of NRMI mice. Its absence in other mouse strains adds a novel layer of strain-specific features in the auditory system, i.e. tonotopic organization of molecular gradients. This calls for caution when comparing data from different mouse strains frequently used in studies involving transgenic animals. The presence of strain-specific differences offers the possibility of genetic mapping to identify molecular factors involved in activity-dependent developmental processes in the auditory system. This would provide an important step forward concerning improved auditory rehabilitation in cases of congenital deafness.

Enhanced Stability of Surface-Tethered Diblock Copolymer Brushes with a Neutral Polymer Block and a Weak Polyelectrolyte Block: Effects of Molecular Weight and Hydrophobicity of the Neutral Block

We study the stability of diblock copolymer brushes featuring a bottom neutral block, poly(methyl methacrylate) (PMMA) or poly(poly(ethylene glycol) methacrylate) (PPEGMA), and a top poly(acrylic acid) (PAA) block on flat silicon substrate. The polymer brushes are prepared by surface-initiated atom transfer radical polymerization (SI-ATRP). We use a combinatorial design featuring a molecular weight gradient in the bottom neutral block to investigate systematically the effect of the molecular weight of that block on the stability of the copolymer brush. We measure variations in dry thickness of the diblock copolymer brush by ellipsometry after different incubation times in aqueous buffer (pH = 9.0) as a function of thickness of the neutral block, indicating degrafting of the mechanically activated copolymer chains via hydrolysis of ester groups in the initiator and/or Si–O bonds that attach the polymer to the substrate. The stability of the diblock copolymer brushes is higher than that of PAA homopolymer b...

Numerical analysis and model development for laminar flame speed of stratified methane/air mixtures

Numerical simulations of stratified methane/air flames with various stratification configurations are conducted using an 1-D unsteady reacting flow solver. Among all the stratified flame cases investigated, rich-to-lean stratified flames show significant departures from homogeneous flames, i.e., up to 50% increase in fuel consumption speeds, primarily due to preferential diffusion of lighter species and radicals from rich burnt products. A sensitivity study of transport properties further reveals that preferential diffusion of molecular hydrogen H2 along with radicals H, OH is the dominant factor in increasing stratified flame speeds, compared to the influence of heat diffusion and diffusion of H2O. Larger departure of stratified flames from homogeneous flames is observed in those cases with higher degree of stratification. In order to model transient behaviors of stratified flames with arbitrary stratification configurations, a local stratification level (LSL) model is proposed. LSL incorporates the effect of preferential diffusion by introducing a transfer function from molecular hydrogen concentration gradients to equivalence ratio gradients, and the memory effect by solving the transient model equation of LSL. The model results match well with directly simulated results quantitatively with error less than ∼10% for both lean and rich conditions.

Sperm chemorepulsion, a supplementary mechanism to regulate fertilization.

Are human spermatozoa able of chemorepulsive behaviour? Capacitated human spermatozoa are able to be chemorepelled by synthetic Progesterone Receptor Ligands (sPRL, known as contraceptives) and zinc (a cation released by the oocyte upon fertilization). Moving cells can be oriented towards or against a molecular gradient, processes called chemoattraction and chemorepulsion, respectively, which have been described in unicellular organisms such as amoebas and bacteria, to organismic cells such macrophages and developmental cells. In the case of spermatozoa, chemoattraction may help the finding of an oocyte and has been widely studied in various invertebrate and mammalian species; however, chemorepulsion has not yet been verified in spermatozoa. This is an in vitro study involving human, rabbit and mouse spermatozoa which were used to perform 3-30 experiments per treatment. Human sperm samples were obtained by masturbation from healthy donors who gave written consent. Only those samples exhibiting normal semen parameters according to current WHO criteria were included in the study. Rabbit spermatozoa were obtained by artificial vagina whereas mice spermatozoa were obtained from epididymis. The sperm selection assay (SSA), originally designed to evaluate sperm chemoattraction towards progesterone (P), and a video-microscopy and computer motion analysis system were used to test sperm chemorepulsion. Additional kinetic parameters were also determined by video-microscopy and computer motion analysis. In some experiments, the level of induced acrosome-reacted spermatozoa was determined. Rabbit mating manipulation was achieved to perform the sperm-oocyte co-incubation assay. Sperm accumulation in the well containing 100 pg/ml of sPRL was lower than the culture medium negative control (P < 0.05). The percentage of sperm persistence against the well containing 100 pg/ml ulipristal acetate (UPA) (P = 0.001), and the percentage of sperm showing a repulsive pattern of movement (a linear trajectory followed by a transitional one after turning against the UPA), were higher than the culture medium negative control (P = 0.049). Sperm accumulation was diminished when spermatozoa where exposed to a homogeneous distribution of 100 pg/ml sPRL combined with a chemotactic gradient of progesterone (P), with respect to the culture medium negative control (P < 0.05). These results were reverted when non-capacitated spermatozoa were used to perform the same experimental settings. The accumulation of spermatozoa against 100 pg/ml sPRL was lower than the culture medium negative control also in rabbits and mice (P < 0.05). The relative number of rabbit spermatozoa arriving to the vicinity of the oocyte was diminished under the presence of 100 pg/ml UPA (P = 0.004). Sperm accumulation in the well containing zinc was decreased compared to the culture medium negative control (P < 0.05). A homogeneous distribution of zinc combined with a gradient of 10 pM P, was lower than the culture medium negative control (P = 0.016). The results were quite reproducible with two different methodologies (accumulation assay and video-microscopy combined with computer motion analysis), in three mammalian species. The experiments were performed in vitro. Even though a quite complete characterization of sperm chemorepulsion was provided, the molecular mechanism that governs sperm repulsion is currently under investigation. Since the chemorepelled spermatozoa are those physiologically ready to fertilize the oocyte, these findings may have both biological and clinical implications, preventing either polyspermy under natural conditions or fertilization under pharmacological treatment with sPRL. The study was financed by the Universidad Nacional de Cordoba (Argentina). The authors declare that they do not have competing financial interests. N/A.

Modulation of Wetting Gradients by Tuning the Interplay between Surface Structuration and Anisotropic Molecular Layers with Bipolar Electrochemistry.

A new simple and versatile method for the preparation of surface-wetting gradients is proposed. It is based on the combination of electrode surface structuration introduced by a sacrificial template approach and the formation of a tunable molecular gradient by bipolar electrochemistry. The gradient involves the formation of a self-assembled monolayer on a gold surface by selecting an appropriate thiol molecule and subsequent reductive desorption by means of bipolar electrochemistry. Under these conditions, completion of the reductive desorption process evolves along the bipolar surface with a maximum strength localized at the cathodic edge and a decreasing driving force towards the middle of the surface. The remaining quantity of surface-immobilized thiol, therefore, varies as a function of the axial position, resulting in the formation of a molecular gradient. The surface of the bipolar electrode is characterized at each step of the modification by recording heterogeneous electron transfer. Also, the evolution of static contact angles measured with a water droplet deposited on the surface directly reveals the presence of the wetting gradient, which can be modulated by changing the properties of the thiol. This is exemplified with a long, hydrophobic alkane-thiol and a short, hydrophilic mercaptan.

Generating Large Thermally Stable Marangoni-Driven Topography in Polymer Films by Stabilizing the Surface Energy Gradient

Marangoni forces drive a fluid to flow in response to positional differences in surface energy. In thin polymer films, a difference in surface energy between two coincident liquid polymers could offer a useful route to manufacture topographically patterned surfaces via the Marangoni effect. Previously, we have demonstrated a photochemical method using the Marangoni effect for patterning thin polystyrene films. To generalize the approach, a theoretical model that gives the underlying physics of this process was also developed, which further revealed that low viscosities, low diffusivities, and large surface energy gradients favor rapid evolution of large film thickness variations. However, as described by the Stokes−Einstein equation or the Rouse model, low viscosity is generally correlated with high diffusivity in a single-component system. Herein, we report a strategy to decouple film viscosity and diffusivity by co-casting a high molecular weight surface energy gradient creating copolymer (low diffusivi...

Combining frozen-density embedding with the conductor-like screening model using Lagrangian techniques for response properties.

We present the explicit derivation of an approach to the multiscale description of molecules in complex environments that combines frozen-density embedding (FDE) with continuum solvation models, in particular the conductor-like screening model (COSMO). FDE provides an explicit atomistic description of molecule-environment interactions at reduced computational cost, while the outer continuum layer accounts for the effect of long-range isotropic electrostatic interactions. Our treatment is based on a variational Lagrangian framework, enabling rigorous derivations of ground- and excited-state response properties. As an example of the flexibility of the theoretical framework, we derive and discuss FDE + COSMO analytical molecular gradients for excited states within the Tamm-Dancoff approximation (TDA) and for ground states within second-order Møller-Plesset perturbation theory (MP2) and a second-order approximate coupled cluster with singles and doubles (CC2). It is shown how this method can be used to describe vertical electronic excitation (VEE) energies and Stokes shifts for uracil in water and carbostyril in dimethyl sulfoxide (DMSO), respectively. In addition, VEEs for some simplified protein models are computed, illustrating the performance of this method when applied to larger systems. The interaction terms between the FDE subsystem densities and the continuum can influence excitation energies up to 0.3 eV and, thus, cannot be neglected for general applications. We find that the net influence of the continuum in presence of the first FDE shell on the excitation energy amounts to about 0.05 eV for the cases investigated. The present work is an important step toward rigorously derived ab initio multilayer and multiscale modeling approaches. © 2017 Wiley Periodicals, Inc.

Semi-convective layer formation

Semi-convective mixing, as an example of double-diffusive convection, is of general importance in multi-component fluid mixing processes. In astrophysics it occurs when the mean molecular weight gradient caused by a mixture of light material on top of heavier one counteracts the convective instability caused by a temperature gradient. Direct numerical simulations of double-diffusive fluid flows in a realistic stellar or planetary parameter space are currently non-feasible. Hence, a model describing incompressible semi-convection was developed, which allows to investigate semi-convective layer formation. A detailed parameter study with varying Rayleigh number and stability parameter has been performed for the giant planet case. We conclude that semi-convective layering may not play that important role as suggested in earlier works for the planetary case.

Nonequilibrium chemo-electronic conversion of water on the nanosized YSZ: experiment and Molecular Dynamics modelling problem formulation

The exothermic heterogeneous electrochemical energy conversion to the electric energy through interaction of the ZrO2 based nanopowder system with atmospheric moisture have been explored within this work. Electrical properties of the experimental samples were investigated during humidification at the conditions of molecular flux density gradient. The morphological features of the surface cross-section and aggregates of 3 mol% Y2O3 doped ZrO2 nanopowder systems were investigated. Initial conditions for molecular dynamics modelling of the adsorption processes were obtained. A novel approach for developing of chemo-electronic converters based on nanoscale processes and materials with dielectric conductivity type proposed.

Thermochemistry and vertical mixing in the tropospheres of Uranus and Neptune: How convection inhibition can affect the derivation of deep oxygen abundances

Thermochemical models have been used in the past to constrain the deep oxygen abundance in the gas and ice giant planets from tropospheric CO spectroscopic measurements. Knowing the oxygen abundance of these planets is a key to better understand their formation. These models have widely used dry and/or moist adiabats to extrapolate temperatures from the measured values in the upper troposphere down to the level where the thermochemical equilibrium between H2O and CO is established. The mean molecular mass gradient produced by the condensation of H2O stabilizes the atmosphere against convection and results in a vertical thermal profile and H2O distribution that departs significantly from previous estimates. We revisit O/H estimates using an atmospheric structure that accounts for the inhibition of the convection by condensation. We use a thermochemical network and the latest observations of CO in Uranus and Neptune to calculate the internal oxygen enrichment required to satisfy both these new estimates of the thermal profile and the observations. We also present the current limitations of such modeling.

Sensitivity and Robustness in an Axon Guidance Signaling System

Correct functioning of the brain requires the orderly wiring of billions of cells during development. A major mechanism that mediates this is the guidance of axons to their targets by extracellular chemical gradients. Although well studied, the computational rules by which even simple guidance decisions are made are not yet understood at a biophysical level. The task of a growing axon to estimate a direction signal is immense, as unavoidable biological noise corrupts the measurement at all levels of processing. Here, we show how the modulation of growth by a balance of positive and negative feedback can explain the remarkable chemotactic sensitivity observed in vitro. We perform a detailed analysis of the experimental data of ref. [1] which characterizes the neurite growth of ∼2500 rat dorsal root ganglia explants in very shallow gradients of nerve growth factor. Constrained by these data, we construct a model chemical signaling system for growth and guidance in the developing brain. In the model, amplification of the gradient signal occurs via paracrine signalling between cell bodies within the ganglion, while robustness is conferred by the dynamics of receptor trafficking. The model gives a unified and quantitative account of experimentally observed behavior, and yields testable predictions with implications for understanding brain development and repair after injury.[1] Mortimer, D. et al (2009) A Bayesian model predicts the response of axons to molecular gradients. Proc. Natl. Acad. Sci. U.S.A, 106(25), 10296-10301.

A New Generation of Standard Solar Models

Abstract We compute a new generation of standard solar models (SSMs) that includes recent updates on some important nuclear reaction rates and a more consistent treatment of the equation of state. Models also include a novel and flexible treatment of opacity uncertainties based on opacity kernels, required in light of recent theoretical and experimental works on radiative opacity. Two large sets of SSMs, each based on a different canonical set of solar abundances with high and low metallicity (Z), are computed to determine model uncertainties and correlations among different observables. We present detailed comparisons of high- and low-Z models against different ensembles of solar observables, including solar neutrinos, surface helium abundance, depth of the convective envelope, and sound speed profile. A global comparison, including all observables, yields a p-value of 2.7σ for the high-Z model and 4.7σ for the low-Z one. When the sound speed differences in the narrow region of are excluded from the analysis, results are 0.9σ and 3.0σ for high- and low-Z models respectively. These results show that high-Z models agree well with solar data but have a systematic problem right below the bottom of the convective envelope linked to steepness of molecular weight and temperature gradients, and that low-Z models lead to a much more general disagreement with solar data. We also show that, while simple parametrizations of opacity uncertainties can strongly alleviate the solar abundance problem, they are insufficient to substantially improve the agreement of SSMs with helioseismic data beyond that obtained for high-Z models due to the intrinsic correlations of theoretical predictions.

Topology of molecular electron density and electrostatic potential with DAMQT

A new version of the DAMQT package incorporating topological analysis of the molecular electron density and electrostatic potential is reported. Evaluation of electron density, electrostatic potential and their first and second derivatives within DAM partition–expansion is discussed, and the pertaining equations are reported. An efficient algorithm for the search of critical points, gradient paths, atomic basins and Hessian analysis is implemented using these equations. 3D viewer built in DAMQT incorporates new facilities for visualization of these properties, as well as for distance and angle measurements. Full control of projection mode is also added to the viewer in DAMQT. Some examples are provided showing the excellent performance for large molecular systems. Program summaryProgram Title: DAMQT_2.1Program Files doi: http://dx.doi.org/10.17632/2rxvgbsnhx.1Licensing provisions: GPLv3Programming language: Fortran90 and C++Supplementary material: Quick-start guide and User’s manual in PDF format included in the package. User’s manual is also accessible from the GUI.Nature of problem: Analysis and visualization of the molecular electron density, electrostatic potential, critical points, gradient paths, atomic basins, electric field and Hellmann–Feynman forces on nuclei.Solution method: The method of Deformed Atoms in Molecules, reported elsewhere[1], is used for partitioning the molecular electron density into atomic fragments, which are further expanded in spherical harmonics times radial factors. The partition is used for defining molecular density deformations and for the fast calculation of several properties associated with density, including topological analysis of electron density and electrostatic potential.Restrictions: Density must come from a LCAO calculation (any computational level) with spherical (not Cartesian) Slater or Gaussian functions.Unusual features: The program contains an OPEN statement to binary files (stream) in several files. This statement does not have a standard syntax in Fortran 90. Two possibilities are considered in conditional compilation: Intel’s ifort and Fortran2003 standard. This latter is applied to compilers other than ifort (gfortran uses this one, for instance).External routines/libraries: Qt (4.8 or higher), OpenGL (3.x or higher), freeGLUT 2.8.x

Transcriptomic, proteomic, and metabolomic landscape of positional memory in the caudal fin of zebrafish

Regeneration requires cells to regulate proliferation and patterning according to their spatial position. Positional memory is a property that enables regenerating cells to recall spatial information from the uninjured tissue. Positional memory is hypothesized to rely on gradients of molecules, few of which have been identified. Here, we quantified the global abundance of transcripts, proteins, and metabolites along the proximodistal axis of caudal fins of uninjured and regenerating adult zebrafish. Using this approach, we uncovered complex overlapping expression patterns for hundreds of molecules involved in diverse cellular functions, including development, bioelectric signaling, and amino acid and lipid metabolism. Moreover, 32 genes differentially expressed at the RNA level had concomitant differential expression of the encoded proteins. Thus, the identification of proximodistal differences in levels of RNAs, proteins, and metabolites will facilitate future functional studies of positional memory during appendage regeneration.

Analysis of 3D multi-layer microfluidic gradient generator.

We developed a three-dimensional (3D) simple multi-layer microfluidic gradient generator to create molecular gradients on the centimeter scale with a wide range of flow rates. To create the concentration gradients, a main channel (MC) was orthogonally intersected with vertical side microchannel (SC) in a 3D multi-layer microfluidic device. Through sequential dilution from the SC, a spatial gradient was generated in the MC. Two theoretical models were created to assist in the design of the 3D multi-layer microfluidic gradient generator and to compare its performance against a two-dimensional equivalent. A first mass balance model was used to predict the steady-state concentrations reached, while a second computational fluid dynamic model was employed to predict spatial development of the gradient by considering convective as well as diffusive mass transport. Furthermore, the theoretical simulations were verified through experiments to create molecular gradients in a 3D multi-layer microfluidic gradient generator.