Molecular Gradients Research Articles

Lipoprotein separation in a novel iodixanol density gradient, for composition, density, and phenotype analysis

Separation of lipoproteins by traditional sequential salt density floatation is a prolonged process ( approximately 72 h) with variable recovery, whereas iodixanol-based, self-generating density gradients provide a rapid ( approximately 4 h) alternative. A novel, three-layered iodixanol gradient was evaluated for its ability to separate lipoprotein fractions in 63 subjects with varying degrees of dyslipidemia. Lipoprotein cholesterol, triglycerides, and apolipoproteins were measured in 21 successive iodixanol density fractions. Iodixanol fractionation was compared with sequential floatation ultracentrifugation. Iodixanol gradient formation showed a coefficient of variation of 0.29% and total lipid recovery from the gradient of 95.4% for cholesterol and 84.7% for triglyceride. Recoveries for VLDL-, LDL-, and HDL-cholesterol, triglycerides, and apolipoproteins were approximately 10% higher with iodixanol compared with sequential floatation. The iodixanol gradient effectively discriminated classic lipoproteins and their subfractions, and there was evidence for improved resolution of lipoproteins with the iodixanol gradient. LDL particles subfractionated by the gradient showed good correlation between density and particle size with small, dense LDL (<25.5 nm) separated in fractions with density >1.028 g/dl. The new iodixanol density gradient enabled rapid separation with improved resolution and recovery of all lipoproteins and their subfractions, providing important information with regard to LDL phenotype from a single centrifugation step with minimal in-vitro modification of lipoproteins.

Extraction of green fluorescent proteins with sonoporation

The mechanism of permeabilization with sonoporation is not understood but the recurrent hypothesis assumes the formation of pores in the cell membrane. The aim of our study is to comfort this assumption by investigating whether ultrasound (US) and microbubbles could also facilitate outward transport of molecules across the plasma membrane through these probable pore formations. Stably transfected Hela-GFP cells were used and insonified with a 1 MHz unfocused transducer in presence of BR14 microbubbles (Bracco Research, Geneva) at different acoustic parameters. The percentage of Hela-GFP cells and the mean cell fluorescence intensity (FI) were measured by flow cytometry to evaluate the GFP release. While US alone did not affect the cells, the addition of BR14 microbubbles induced a significant decrease of FI and the percentage of Hela-GFP cells. A reduction of more than 50% of FI and GFP + cells was achieved between 400-600 kPa and 40-75 % of duty cycle during 2 minutes insonation. However a progressive recovery of Hela-GFP cells percentage and FI post-insonation has been observed which indicates a viable situation of Hela-GFP permeabilized cells allowing a restoration of GFP cell content. These results comfort pore formation hypothesis allowing transmembrane transport following molecular concentration gradient.

Multi-Molecular Gradients of Permissive and Inhibitory Cues Direct Neurite Outgrowth

Correct development of neuronal tracts requires the coordination of multiple permissive and inhibitory signals. By generating an in vitro microenvironment using soft lithography and microfluidic techniques, multiple guidance cues can be presented in a spatially defined way. Here we evaluated how neurites of dorsal root ganglia neurons responded to permissive and inhibitory cues presented by substrate-bound molecular gradients. Linear gradients containing inhibitory chondroitin sulfate proteoglycan (CSPG) and/or permissive laminin-1 (LN) were generated as single-cue gradients, parallel double-cue gradients, and opposing double-cue gradients with varying slopes. Neurite growth was analyzed using circular statistical methods, and for all gradients examined, neurons extended neurites toward regions of lower CSPG and higher LN concentrations. Single-cue gradients elicited similarly directed neurite growth responses at the higher concentrations tested for both LN and CSPG, and both gradient slope and fractional concentration change affected neurite growth. When the two contrasting molecular cues were presented together, neurites responded differently depending on the directions of the gradients. Neurite growth on LN-CSPG double gradients of opposite direction was strongly directed, while neurite growth on LN-CSPG double gradients of parallel direction was uniform. These results represent an important step toward understanding how neurite growth is guided by complex microenvironments containing multiple molecular cues.

Instabilities and mixing in stellar radiation zones

AbstractThe standard model of stellar structure is unable to account for various observational facts, and there is now a large consensus that some ‘extra mixing’ must occur in the radiation zones. The possible causes for such mixing are briefly reviewed. The most efficient among them is probably the shear-turbulence generated by the differential rotation, which itself results from the transport of angular momentum that can be mediated through the large-scale circulation induced by structural adjustments or by the applied torques (stellar wind, accretion, tides). In solar-type stars this angular momentum transport is ensured mainly by internal gravity waves that are excited at the boundary with convection zones. Another cause of mixing manifests itself in the red giant phase, namely the thermohaline instability due to an inversion of the molecular weight gradient. The implementation of these processes in stellar evolution codes is giving rise to a new generation of stellar models, which are in much better agreement with the observational constraints.

Formation of Molecular Gradients on Bipolar Electrodes

Intelligent surfaces: Electrochemical reactions can be induced on a conducting surface placed in an electric field. In this way, a bipolar electrode is formed, and this effect can be used to create molecular gradients (see picture). The major advantage of the technique is that the electrode can be of virtually any thickness, shape, and material—as long as it is conductive.

A cadherin‐based code for the divisions of the mouse basal ganglia

The expression of 12 different classic cadherins and delta-protocadherins was mapped in consecutive series of sections through the basal ganglia of the postnatal and adult mouse by in situ hybridization. A particular focus was the caudoputamen, which consists of patches (striosomes) and a surrounding matrix that is histologically uniform. The different areas within the caudoputamen are connected specifically to other parts of the basal ganglia and to other brain regions, for example, the substantia nigra. The molecules regulating the morphogenesis and functional connectivity of the basal ganglia are largely unknown. Previous studies suggested that cadherins, a large family of adhesion molecules, are involved in basal ganglia development. In the present work, we study the expression of 12 cadherins and show that the patch and matrix compartments of the caudoputamen express the cadherins differentially, although partial overlap is observed. Moreover, the cadherins are expressed in multiple and diverse gradients within the caudoputamen and other parts of the basal ganglia. The persistence of the expression patterns in the adult basal ganglia suggests the possibility that cadherins also play a role at adult stages. Our results suggest that cadherins provide a code of potentially adhesive cues that specify not only patch and matrix compartments but also multiple molecular gradients within the basal ganglia. This code may relate to patterns of connectivity.

Molecular properties with dual basis set methods

The dual basis set approach has proven to be very successful for accurately estimating total energies with large basis sets. This study extends the applications of this technique to the calculation of molecular properties, including energy derivatives with respect to nuclear positions and to an external electric field. All energy derivatives have been calculated numerically via finite-differences. Molecular gradients and Hessians as well as dipole moments and polarizabilites have been calculated at the HF and MP2 levels using two alternative versions of the dual basis set method. The accuracy of these approaches is discussed in the context of quality of basis sets used in calculations. It is shown that even quite poor results obtained with the 6-311G basis set are significantly improved in dual basis set calculations with the 6-311G(d,p) and 6-311G(3df,3dp) basis sets.

Role of IGF Signaling in Olfactory Sensory Map Formation and Axon Guidance

Olfactory neurons project their axons to spatially invariant glomeruli in the olfactory bulb, forming an ordered pattern of innervation comprising the olfactory sensory map. A mirror symmetry exists within this map, such that neurons expressing a given receptor typically project to one glomerulus on the medial face and one glomerulus on the lateral face of the bulb. The mechanisms underlying an olfactory neuron's choice to project medially versus laterally remain largely unknown, however. Here we demonstrate that insulin-like growth factor (IGF) signaling is required for sensory innervation of the lateral olfactory bulb. Mutations that eliminate IGF signaling cause axons destined for targets in the lateral bulb to shift to ectopic sites on the ventral-medial surface. Using primary cultures of olfactory and cerebellar neurons, we further show that IGF is a chemoattractant for axon growth cones. Together these observations reveal a role of IGF signaling in sensory map formation and axon guidance.

What Prevents Internal Gravity Waves from Disturbing the Solar Uniform Rotation?

Internal gravity waves (IGWs) are naturally produced by convection in stellar envelopes, and they could be an important mechanism for transporting angular momentum in the radiative interiors of stars. Prior work has established that they could operate over a short enough timescale to explain the internal solar rotation as a function of depth. We demonstrate that the natural action of IGWs is to produce large-scale oscillations in the solar rotation as a function of depth, which is in marked contrast to the nearly uniform rotation in the outer radiative envelope of the Sun. An additional angular momentum transport mechanism is therefore required, and neither molecular nor shear-induced turbulent viscosity is sufficient to smooth out the profile. Magnetic processes, such as the Tayler-Spruit dynamo, could flatten the rotation profile. We therefore conclude that IGWs must operate in conjunction with magnetic angular momentum transport processes if they operate at all. Furthermore, both classes of mechanisms must be inhibited to some degree by mean molecular weight gradients in order to explain the recent evidence for a rapidly rotating embedded core in the Sun, should it be confirmed by a further analysis of solar g-mode oscillations.

SLC25A12 expression is associated with neurite outgrowth and is upregulated in the prefrontal cortex of autistic subjects

Autism is a neurodevelopmental disorder with a strong genetic component, probably involving several genes. Genome screens have provided evidence of linkage to chromosome 2q31-q33, which includes the SLC25A12 gene. Association between autism and single-nucleotide polymorphisms in SLC25A12 has been reported in various studies. SLC25A12 encodes the mitochondrial aspartate/glutamate carrier functionally important in neurons with high-metabolic activity. Neuropathological findings and functional abnormalities in autism have been reported for Brodmann's area (BA) 46 and the cerebellum. We found that SLC25A12 was expressed more strongly in the post-mortem brain tissues of autistic subjects than in those of controls, in the BA46 prefrontal cortex but not in cerebellar granule cells. SLC25A12 expression was not modified in brain subregions of bipolar and schizophrenic patients. SLC25A12 was expressed in developing human neuronal tissues, including neocortical regions containing excitatory neurons and neocortical progenitors and the ganglionic eminences that generate neocortical inhibitory interneurons. At mid-gestation, when gyri and sulci start to develop, SLC25A12 molecular gradients were identified in the lateral prefrontal and ventral temporal cortex. These fetal structures generate regions with abnormal activity in autism, including the dorsolateral prefrontal cortex (BA46), the pars opercularis of the inferior frontal cortex and the fusiform gyrus. SLC25A12 overexpression or silencing in mouse embryonic cortical neurons also modified dendrite length and the mobility of dendritic mitochondria. Our findings suggest that SLC25A12 overexpression may be involved in the pathophysiology of autism, modifying neuronal networks in specific subregions, such as the dorsolateral prefrontal cortex and fusiform gyrus, at both pre- and postnatal stages.

Towards the elaboration of a QM method to describe molecular solutes under the effect of a very high pressure

In this paper, we present a new computational QM method for the study of structural properties (i.e. equilibrium geometry) of molecular systems at very high pressure. The procedure is based on the polarizable continuum model (PCM) method, usually used to study molecular solutes under standard pressure conditions. The paper considers two critical items: the definition of the pressure and the elaboration of an analytical code for the calculation of molecular gradients. The method has been developed at HF and DFT levels, with computational costs comparable with those of similar calculations in vacuo. The numerical examples regarding the equilibrium geometries and conformational energies of 1,3-butadiene under high pressure give an indication of the potentialities of the approach and of the problems to which it may be applied.

Photo-deprotection patterning of self-assembled monolayers

Photo-deprotectable self-assembled monolayers (SAMs) provide a versatile platform for creating functional patterned surfaces. In this study, we present nanoscale photo-patterning, multi-component patterning, and a method for producing molecular gradients using photo-deprotectable SAMs. Nanoscale patterning of photo-deprotectable SAMs was achieved by coupling a UV laser (365 nm) through a scanning near field probe to produce nanoscale lines of ∼40 nm, i.e. λ/9. Multi-component patterning was achieved by a two-stage method combining both microcontact printing and soft-UV photo-patterning. The example demonstrated in this study produced a three-component patterned surface with regions of CF3, CH3 and COOH/CF3 functionality. The versatility of these photocleavable SAMs is further demonstrated by creating linear molecular gradients of two functionalities along a distance of ∼25 mm. The use of ‘soft’ UV gives several advantages including the ability to pattern SAMs with micron-scale features over large areas quickly, with greater control over the photochemical reactions, and compatibility with existing lithographic facilities thus offering an effective alternative to other patterning methods such microcontact-printing or deep UV patterning.

Can Pulsational Instabilities Impact a Massive Star's Rotational Evolution?

AbstractWe investigate whether angular momentum transport due to unstable pulsation modes can play a significant role in the rotational evolution of massive stars. We find that these modes can redistribute appreciable angular momentum, and moreover trigger shear-instability mixing in the molecular weight gradient zone adjacent to stellar cores, with significant evolutionary impact.

Master equation simulation analysis of immunostained Bicoid morphogen gradient

BackgroundThe concentration gradient of Bicoid protein which determines the developmental pathways in early Drosophila embryo is the best characterized morphogen gradient at the molecular level. Because different developmental fates can be elicited by different concentrations of Bicoid, it is important to probe the limits of this specification by analyzing intrinsic fluctuations of the Bicoid gradient arising from small molecular number. Stochastic simulations can be applied to further the understanding of the dynamics of Bicoid morphogen gradient formation at the molecular number level, and determine the source of the nucleus-to-nucleus expression variation (noise) observed in the Bicoid gradient.ResultsWe compared quantitative observations of Bicoid levels in immunostained Drosophila embryos with a spatially extended Master Equation model which represents diffusion, decay, and anterior synthesis. We show that the intrinsic noise of an autonomous reaction-diffusion gradient is Poisson distributed. We demonstrate how experimental noise can be identified in the logarithm domain from single embryo analysis, and then separated from intrinsic noise in the normalized variance domain of an ensemble statistical analysis. We show how measurement sensitivity affects our observations, and how small amounts of rescaling noise can perturb the noise strength (Fano factor) observed. We demonstrate that the biological noise level in data can serve as a physical constraint for restricting the model's parameter space, and for predicting the Bicoid molecular number and variation range. An estimate based on a low variance ensemble of embryos suggests that the steady-state Bicoid molecular number in a nucleus should be larger than 300 in the middle of the embryo, and hence the gradient should extend to the posterior end of the embryo, beyond the previously assumed background limit. We exhibit the predicted molecular number gradient together with measurement effects, and make a comparison between conditions of higher and lower variance respectively.ConclusionQuantitative comparison of Master Equation simulations with immunostained data enabled us to determine narrow ranges for key biophysical parameters, which for this system can be independently validated. Intrinsic noise is clearly detectable as well, although the staining process introduces certain limits in resolution.

Behavior of Surface-Anchored Poly(acrylic acid) Brushes with Grafting Density Gradients on Solid Substrates: 1. Experiment

We describe experiments pertaining to the formation of surface-anchored poly(acrylic acid) (PAA) brushes with a gradual variation of the PAA grafting densities on flat surfaces and provide detailed analysis of their properties. The PAA brush gradients are generated by first covering the substrate with a molecular gradient of the polymerization initiator, followed by the “grafting from” polymerization of tert-butyl acrylate (tBA) from these substrate-bound initiator centers, and finally converting the PtBA into PAA. We use spectroscopic ellipsometry to measure the wet thickness of the grafted PAA chains in aqueous solutions at three different pH values (4, 5.8, and 10) and a series of ionic strengths (IS). Our measurements reveal that at low grafting densities, σ, the wet thickness of the PAA brush (H) remains relatively constant, the polymers are in the mushroom regime. Beyond a certain value of σ, the macromolecules enter the brush regime, where H increases with increasing σ. For a given σ, H exhibits a ...

Strategies for controlled placement of nanoscale building blocks.

The capability of placing individual nanoscale building blocks on exact substrate locations in a controlled manner is one of the key requirements to realize future electronic, optical, and magnetic devices and sensors that are composed of such blocks. This article reviews some important advances in the strategies for controlled placement of nanoscale building blocks. In particular, we will overview template assisted placement that utilizes physical, molecular, or electrostatic templates, DNA-programmed assembly, placement using dielectrophoresis, approaches for non-close-packed assembly of spherical particles, and recent development of focused placement schemes including electrostatic funneling, focused placement via molecular gradient patterns, electrodynamic focusing of charged aerosols, and others.

Generation of Stable Complex Gradients Across Two-Dimensional Surfaces and Three-Dimensional Gels

Many chemical and biological processes are dependent on molecular gradients. We describe a new microfluidic approach that can be used to produce spatiotemporal gradients across two-dimensional surfaces and three-dimensional gels under flow-free conditions. Free diffusion between dynamically replenished flow channels acting as a sink and source is utilized to give rise to stable steady-state gradient profiles. The gradient profile is dictated by the engineered design of the device's gradient-generating region. Different designs can yield both linear and non-linear gradients of varying profiles. More complex gradients can be made by juxtaposing different designs within a single gradient-generating region. By fabricating an array of designs along the gradient-generating region, different gradient profiles can be generated simultaneously, allowing for parallel analysis. Additionally, simple methods of localizing gels into microdevices are demonstrated. The device was characterized by experimentally obtained gradient profiles of fluorescent molecules that corroborated closely with a simulated finite element model.

Microbial Communities in Long-Term Heavy Metal Contaminated Ombrotrophic Peats

High concentrations of heavy metals are known to be toxic to many soil organisms. The effects of long-term exposure to lower levels of metals on the soil microbial community are, however, less well understood. The southern Pennines of the U.K. are characterised by expanses of ombrotrophic peat soils that have experienced deposition of high levels of heavy metals since the mid to late 1800s. Concentrations of metals in the peat remain high but the effect of the contamination on the in-situ microbial communities is unknown. Geochemical and molecular polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and sequencing techniques were used to derive new information on the metal chemistry and microbial populations in peat soils from six locations in the southern Pennines. All sites were highly acidic (pH 3.00–3.14) with high concentrations of potentially toxic heavy metals, particularly porewater Zn and particulate-associated Pb. The results also reveal a split in site characteristics between the most polluted sites with the highest levels of bioavailable metals (Bleaklow, FeatherBed Moss and White Hill) and those with much lower bioavailable metals (Cowms Moor, Holme Moss and Round Hill). There was no difference in the number of dominant bacterial species between the sites but there were significant differences in the species composition. At the three sites with the highest levels of bioavailable metals, bacterial species with a high similarity to acidophilic sulphur- and iron-oxidizing bacteria and those from high metal environments were detected. The transformations carried out by these metal mobilising and acid producing bacteria may make heavy metals more bioavailable and therefore more toxic to higher organisms. Bacteria with similarity to those typically found in forest and grassland soils were documented at the three sites with the lowest levels of bioavailable metals. The data highlight the need for further studies to elucidate the species diversity and functionality of bacteria in heavy metal contaminated peats in order to assess implications for moorland restoration.

Genetic analysis of molecular oscillators in mammalian somitogenesis: Clues for studies of human vertebral disorders

The repeating pattern of the human vertebral column is shaped early in development, by a process called somitogenesis. In this embryonic process, pairs of mesodermal segments called somites are serially laid down along the developing neural tube. Somitogenesis is an iterative process, repeating at regular time intervals until the last somite is formed. This process lays down the vertebrate body axis from head to tail, making for a progression of developmental steps along the rostral-caudal axis. In this review, the roles of the Notch, Wnt, fibroblast growth factor, retinoic acid and other pathways are described during the following key steps in somitogenesis: formation of the presomitic mesoderm (PSM) and establishment of molecular gradients; prepatterning of the PSM by molecular oscillators; patterning of rostral-caudal polarity within the somite; formation of somite borders; and maturation and resegmentation of somites to form musculoskeletal tissues. Disruption of somitogenesis can lead to severe vertebral birth defects such as spondylocostal dysostosis (SCD). Genetic studies in the mouse have been instrumental in finding mutations in this disorder, and ongoing mouse studies should provide functional insights and additional candidate genes to help in efforts to identify genes causing human spinal birth defects.

Directed Electroless Growth of Metal Nanostructures on Patterned Self-Assembled Monolayers

The directed placement of Cu nanostructures on surfaces has been studied using a combination of scanning probe lithography and electroless metal deposition onto nanopatterned SAMs of 16-mercaptohexadecanoic acid (16-MHA) on Au. In situ studies using nanoscale molecular gradients reveal how controlling the areal density of the 16-MHA molecules dictates the nucleation and growth of the metal nanostructures. The influence of controlling pattern line spacing and tip path on pattern feature fidelity is also discussed.