High-resolution-scanning waveguide microscopy: spatial refractive index and topography quantification.

We report on a high-resolution metal-clad waveguide scanning microscopic method with a diffraction-limited resolution. This microscope can be operated in both TM and TE waveguide modes with radially and azimuthally polarized beams, respectively, and allows both refractive index and topography of dielectric objects to be evaluated at high resolution and sensitivity. We emphasize the performance of this microscopic method from calibrated 3D polymer microstructures with rectangular, disk, and ring shapes.

Dynamical study of Νaν channel excitability under mechanical stress.

Alteration of [Formula: see text] channel functions (channelopathies) has been encountered in various hereditary muscle diseases. [Formula: see text] channel mutations lead to aberrant excitability in skeletal muscle myotonia and paralysis. In general, these mutations disable inactivation of the [Formula: see text] channel, producing either repetitive action potential firing (myotonia) or electrical dormancy (flaccid paralysis) in skeletal muscles. These "sick-excitable" cell conditions were shown to correlate with a mechanical stretch-driven left shift of the conductance factors of the two gating mechanisms of a fraction of [Formula: see text] channels, which make them firing at inappropriate hyperpolarised (left-shifted) voltages. Here we elaborate on a variant of the Hodgkin-Huxley model that includes a stretch elasticity energy component in the activation and inactivation gate kinetic rates. We show that this model reproduces fairly well sick-excitable cell behaviour and can be used to predict the parameter domains where aberrant excitability or paralysis may occur. By allowing us to separate the incidences of activation and inactivation gate impairments in [Formula: see text] channel excitability, this model could be a strong asset for diagnosing the origin of excitable cell disorders.

Revealing stiffening and brittling of chronic myelogenous leukemia hematopoietic primary cells through their temporal response to shear stress.

Cancer cell transformation is often accompanied by a modification of their viscoelastic properties. When capturing the stress-to-strain response of primary chronic myelogenous leukemia (CML) cells, from two data sets of CD34+ hematopoietic cells isolated from healthy and leukemic bone marrows, we show that the mean shear relaxation modulus increases upon cancer transformation. This stiffening of the cells comes along with local rupture events, detected as reinforced sharp local maxima of this modulus, suggesting that these cancer cells respond to a local mechanical stress by a cascade of local brittle failure events.

Deciphering DNA replication dynamics in eukaryotic cell populations in relation with their averaged chromatin conformations.

We propose a non-local model of DNA replication that takes into account the observed uncertainty on the position and time of replication initiation in eukaryote cell populations. By picturing replication initiation as a two-state system and considering all possible transition configurations, and by taking into account the chromatin's fractal dimension, we derive an analytical expression for the rate of replication initiation. This model predicts with no free parameter the temporal profiles of initiation rate, replication fork density and fraction of replicated DNA, in quantitative agreement with corresponding experimental data from both S. cerevisiae and human cells and provides a quantitative estimate of initiation site redundancy. This study shows that, to a large extent, the program that regulates the dynamics of eukaryotic DNA replication is a collective phenomenon that emerges from the stochastic nature of replication origins initiation.

SWDreader: a wavelet-based algorithm using spectral phase to characterize spike-wave morphological variation in genetic models of absence epilepsy.

BACKGROUND: Spike-wave discharges (SWD) found in neuroelectrical recordings are pathognomonic to absence epilepsy. The characteristic spike-wave morphology of the spike-wave complex (SWC) constituents of SWDs can be mathematically described by a subset of possible spectral power and phase values. Morlet wavelet transform (MWT) generates time-frequency representations well-suited to identifying this SWC-associated subset. NEW METHOD: MWT decompositions of SWDs reveal spectral power concentrated at harmonic frequencies. The phase relationships underlying SWC morphology were identified by calculating the differences between phase values at SWD fundamental frequency from the 2nd, 3rd, and 4th harmonics, then using the three phase differences as coordinates to generate a density distribution in a {360°×360°×360°} phase difference space. Strain-specific density distributions were generated from SWDs of mice carrying the Gria4, Gabrg2, or Scn8a mutations to determine whether SWC morphological variants reliably mapped to the same regions of the distribution, and if distribution values could be used to detect SWD. COMPARISON WITH EXISTING METHODS: To the best of our knowledge, this algorithm is the first to employ spectral phase to quantify SWC morphology, making it possible to computationally distinguish SWC morphological subtypes and detect SWDs. RESULTS/CONCLUSIONS: Proof-of-concept testing of the SWDfinder algorithm shows: (1) a major pattern of variation in SWC morphology maps to one axis of the phase difference distribution, (2) variability between the strain-specific distributions reflects differences in the proportions of SWC subtypes generated during SWD, and (3) regularities in the spectral power and phase profiles of SWCs can be used to detect waveforms possessing SWC-like morphology.

Revealing long-range interconnected hubs in human chromatin interaction data using graph theory.

We use graph theory to analyze chromatin interaction (Hi-C) data in the human genome. We show that a key functional feature of the genome--"master" replication origins--corresponds to DNA loci of maximal network centrality. These loci form a set of interconnected hubs both within chromosomes and between different chromosomes. Our results open the way to a fruitful use of graph theory concepts to decipher DNA structural organization in relation to genome functions such as replication and transcription. This quantitative information should prove useful to discriminate between possible polymer models of nuclear organization.

Wavelet-based decomposition of high resolution surface plasmon microscopy V(Z) curves at visible and near infrared wavelengths.

Surface plasmon resonance is conventionally conducted in the visible range and, during the past decades, it has proved its efficiency in probing molecular scale interactions. Here we elaborate on the first implementation of a high resolution surface plasmon microscope that operates at near infrared (IR) wavelength for the specific purpose of living matter imaging. We analyze the characteristic angular and spatial frequencies of plasmon resonance in visible and near IR lights and how these combined quantities contribute to the V(Z) response of a scanning surface plasmon microscope (SSPM). Using a space-frequency wavelet decomposition, we show that the V(Z) response of the SSPM for red (632.8 nm) and near IR (1550 nm) lights includes the frequential response of plasmon resonance together with additional parasitic frequencies induced by the objective pupil. Because the objective lens pupil profile is often unknown, this space-frequency decomposition turns out to be very useful to decipher the characteristic frequencies of the experimental V(Z) curves. Comparing the visible and near IR light responses of the SSPM, we show that our objective lens, primarily designed for visible light microscopy, is still operating very efficiently in near IR light. Actually, despite their loss in resolution, the SSPM images obtained with near IR light remain contrasted for a wider range of defocus values from negative to positive Z values. We illustrate our theoretical modeling with a preliminary experimental application to blood cell imaging.

Linking the DNA strand asymmetry to the spatio-temporal replication program: II. Accounting for neighbor-dependent substitution rates.

In paper I, we addressed the impact of the spatio-temporal program on the DNA composition evolution in the case of time homogeneous and neighbor-independent substitution rates. But substitution rates do depend on the flanking nucleotides as exemplified in vertebrates where CpG sites are hypermutable so that the substitution rate C --> T depends dramatically (ten fold) on whether the cytosine belongs to a CG dinucleotide or not. With the specific goal to account for neighbor-dependence, we revisit our minimal modeling of neutral substitution rates in the human genome. When assuming that r = CpG --> TpG and its reverse complement r(c) = CpG --> CpA are (by far) the main neighbor-dependent substitution rates, we demonstrate, using perturbative analysis, that neighbor-dependence does not affect the decomposition of the compositional asymmetry into a transcription- and a replication-associated components, the former increases in magnitude with transcription rate and changes sign with gene orientation, whereas the latter is proportional to the replication fork polarity. Indeed the neighbor dependence case differs from the neighbor-independent model by an additional source term related to the CG dinucleotide content in both the transcription and replication-associated components. We finally discuss the case of time-dependent substitution rates confirming as a very general result the fact that the skew can still be decomposed into a transcription- and a replication-associated components.

Linking the DNA strand asymmetry to the spatio-temporal replication program. I. About the role of the replication fork polarity in genome evolution.

Two key cellular processes, namely transcription and replication, require the opening of the DNA double helix and act differently on the two DNA strands, generating different mutational patterns (mutational asymmetry) that may result, after long evolutionary time, in different nucleotide compositions on the two DNA strands (compositional asymmetry). We elaborate on the simplest model of neutral substitution rates that takes into account the strand asymmetries generated by the transcription and replication processes. Using perturbation theory, we then solve the time evolution of the DNA composition under strand-asymmetric substitution rates. In our minimal model, the compositional and substitutional asymmetries are predicted to decompose into a transcription- and a replication-associated components. The transcription-associated asymmetry increases in magnitude with transcription rate and changes sign with gene orientation while the replication-associated asymmetry is proportional to the replication fork polarity. These results are confirmed experimentally in the human genome, using substitution rates obtained by aligning the human and chimpanzee genomes using macaca and orangutan as outgroups, and replication fork polarity determined in the HeLa cell line as estimated from the derivative of the mean replication timing. When further investigating the dynamics of compositional skew evolution, we show that it is not at equilibrium yet and that its evolution is an extremely slow process with characteristic time scales of several hundred Myrs.

Inferring where and when replication initiates from genome-wide replication timing data.

Based on an analogy between DNA replication and one dimensional nucleation-and-growth processes, various attempts to infer the local initiation rate I(x,t) of DNA replication origins from replication timing data have been developed in the framework of phase transition kinetics theories. These works have all used curve-fit strategies to estimate I(x,t) from genome-wide replication timing data. Here, we show how to invert analytically the Kolmogorov-Johnson-Mehl-Avrami model and extract I(x,t) directly. Tests on both simulated and experimental budding-yeast data confirm the location and firing-time distribution of replication origins.

Revisiting polymer statistical physics to account for the presence of long-range-correlated structural disorder in 2D DNA chains.

We elaborate on a generalization of the 2D wormlike chain (WLC) model that accounts for the presence of long-range correlations (LRC) in the intrinsic curvature distribution of eukaryotic DNA. This model predicts some decrease of the DNA persistence length resulting from some large-scale intrinsic curvature induced by sequence-dependent persistent random distribution of local bending sites. When assisting exact analytical calculations by numerical DNA simulations, we show that the conjugated contributions of i) the thermal curvature fluctuations characterized by the "dynamic" persistence length ℓ(p)(d) = 2A, where A is the elastic bending modulus, and ii) the intrinsic LRC curvature disorder of amplitude σ(o) and Hurst exponent H > 1/2, characterized by a "static" persistence length ℓ(p)(H) = A(1/2H)σ(o)(-1/H) Γ(1/2H + 1), can be described by a continuum of generalized WLC (GWLC) models parametrized by the LRC exponent H. We use perturbation analysis to investigate the two limiting cases of weak static disorder (w(H) << 1 and weak dynamical fluctuations (1/w (H) << 1), where w(H) = l(p)(d)/l(p)(H) is a dimensionless parameter. From a quantitative point of view, our study demonstrates that even for a small value of the LRC (H approximately equal 0.6-0.8) static disorder amplitude σ(o) ~ 10(-2), as previously reported for genomic DNA, the decrease of the persistence length from the WLC prediction l(p)(d) can be very significant, up to twofold. The implications of these results on the first steps of compaction of DNA in eukaryotic cells are discussed.

Thermodynamics of intragenic nucleosome ordering.

The nucleosome ordering observed in vivo along yeast genes is described by a thermodynamical model of nonuniform fluid of 1D hard rods confined by two excluding energy barriers at gene extremities. For interbarrier distances L less than or approximately equal to 1.5 kbp, nucleosomes equilibrate into a crystal-like configuration with a nucleosome repeat length (NRL) L/n approximately 165 bp, where n is the number of regularly positioned nucleosomes. We also observe "bistable" genes with a fuzzy chromatin resulting from a statistical mixing of two crystal states, one with an expanded chromatin (NRL approximately L/n) and the other with a compact one (NRL approximately L/(n+1)). By means of single nucleosome switching, bistable genes may drastically alter their expression level as suggested by their higher transcriptional plasticity. These results enlighten the role of the intragenic chromatin on gene expression regulation.

Spontaneous emergence of sequence-dependent rosettelike folding of chromatin fiber.

In the crowded environment of the eukaryotic nucleus, the presence of intrinsic structural defects is shown to predispose chromatin fiber to spontaneously form rosettelike structures. These multilooped patterns self-organize through entropy-driven clustering of sequence-induced fiber defects by depletive forces prior to any external factors coming into play. They provide an attractive description of replication foci that are observed in interphase mammalian nuclei as stable chromatin domains of autonomous DNA replication and gene expression. Experimental perspectives for in vivo visualization of rosettelike organization of the chromatin fiber via the clustering of recently identified putative replication initiation zones are discussed.

Universal intermittent properties of particle trajectories in highly turbulent flows.

We present a collection of eight data sets from state-of-the-art experiments and numerical simulations on turbulent velocity statistics along particle trajectories obtained in different flows with Reynolds numbers in the range R{lambda}in[120:740]. Lagrangian structure functions from all data sets are found to collapse onto each other on a wide range of time lags, pointing towards the existence of a universal behavior, within present statistical convergence, and calling for a unified theoretical description. Parisi-Frisch multifractal theory, suitably extended to the dissipative scales and to the Lagrangian domain, is found to capture the intermittency of velocity statistics over the whole three decades of temporal scales investigated here.

Chromosome territories have a highly nonspherical morphology and nonrandom positioning.

Interphase chromosomes are organized into discrete chromosome territories (CTs) that may occupy preferred sub-nuclear positions. While chromosome size and gene density appear to influence positioning, the biophysical mechanisms behind CT localization, especially the relationship between morphology and positioning, remain obscure. One reason for this has been the difficulty in imaging, segmenting, and analyzing structures with variable or imprecise boundaries. This prompted us to develop a novel approach, based on the two-dimensional (2D) wavelet-transform modulus maxima (WTMM) method, adapted to perform objective and rigorous CT segmentation from nuclear background. The wavelet transform acts as a mathematical microscope to characterize spatial image information over a continuous range of size scales. This multiresolution nature, combined with full objectivity of the formalism, makes it more accurate than intensity-based segmentation algorithms and more appropriate than manual intervention. Using the WTMM method in combination with numerical simulation models, we show that CTs have a highly nonspherical 3D morphology, that CT positioning is nonrandom, and favors heterologous CT groupings. We discuss potential relationships between morphology, positioning, chromosomal function, and instability.

Bifractality of human DNA strand-asymmetry profiles results from transcription.

We use the wavelet transform modulus maxima method to investigate the multifractal properties of strand-asymmetry DNA walk profiles in the human genome. This study reveals the bifractal nature of these profiles, which involve two competing scale-invariant (up to repeat-masked distances less, or similar 40 kbp) components characterized by Hölder exponents h{1}=0.78 and h{2}=1, respectively. The former corresponds to the long-range-correlated homogeneous fluctuations previously observed in DNA walks generated with structural codings. The latter is associated with the presence of jumps in the original strand-asymmetry noisy signal S. We show that a majority of upward (downward) jumps co-locate with gene transcription start (end) sites. Here 7228 human gene transcription start sites from the refGene database are found within 2 kbp from an upward jump of amplitude DeltaS > or = 0.1 which suggests that about 36% of annotated human genes present significant transcription-induced strand asymmetry and very likely high expression rate.

Probing persistence in DNA curvature properties with atomic force microscopy.

We elaborate on a mean-field extension of the wormlike chain model that accounts for the presence of long-range correlations (LRC) in the intrinsic curvature disorder of genomic DNA, the stronger the LRC, the smaller the persistence length. The comparison of atomic force microscopy imaging of straight, uncorrelated virus and correlated human DNA fragments with DNA simulations confirms that the observed decrease in persistence length for human DNA more likely results from a sequence-induced large-scale intrinsic curvature than from some increased flexibility.

Experiments confirm the influence of genome long-range correlations on nucleosome positioning.

From the statistical analysis of nucleosome positioning data for chromosome III of S. cerevisiae, we demonstrate that long-range correlations (LRC) in the genomic sequence strongly influence the organization of nucleosomes. We present a physical explanation of how LRC may significantly condition the overall formation and positioning of nucleosomes including the nucleosome-free regions observed at gene promoters. From grand canonical Monte Carlo simulations based upon a simple sequence-dependent nucleosome model, we show that LRC induce a patchy nucleosome occupancy landscape with alternation of "crystal-like" phases of confined regularly spaced nucleosomes and "fluidlike" phases of rather diluted nonpositioned nucleosomes.

DNA replication timing data corroborate in silico human replication origin predictions.

We develop a wavelet-based multiscale pattern recognition methodology to disentangle the replication- from the transcription-associated compositional strand asymmetries observed in the human genome. Comparing replication skew profiles to recent high-resolution replication timing data reveals that most of the putative replication origins that border the so-identified replication domains are replicated earlier than their surroundings whereas the central regions replicate late in the S phase. We discuss the implications of this first experimental confirmation of these replication origin predictions that are likely to be early replicating and active in most tissues.

Formation and positioning of nucleosomes: effect of sequence-dependent long-range correlated structural disorder.

The understanding of the long-range correlations (LRC) observed in DNA sequences is still an open and very challenging problem. In this paper, we start reviewing recent results obtained when exploring the scaling properties of eucaryotic, eubacterial and archaeal genomic sequences using the space-scale decomposition provided by the wavelet transform (WT). These results suggest that the existence of LRC up to distances approximately 20-30 kbp is the signature of the nucleosomal structure and dynamics of the chromatin fiber. Actually the LRC are mainly observed in the DNA bending profiles obtained when using some structural coding of the DNA sequences that accounts for the fluctuations of the local double-helix curvature within the nucleosome complex. Because of the approximate planarity of nucleosomal DNA loops, we then study the influence of the LRC structural disorder on the thermodynamical properties of 2D elastic chains submitted locally to mechanical/topological constraint as loops. The equilibrium properties of the one-loop system are derived numerically and analytically in the quite realistic weak-disorder limit. The LRC are shown to favor the spontaneous formation of small loops, the larger the LRC, the smaller the size of the loop. We further investigate the dynamical behavior of such a loop using the mean first passage time (MFPT) formalism. We show that the typical short-time loop dynamics is superdiffusive in the presence of LRC. For displacements larger than the loop size, we use large-deviation theory to derive a LRC-dependent anomalous-diffusion rule that accounts for the lack of disorder self-averaging. Potential biological implications on DNA loops involved in nucleosome positioning and dynamics in eucaryotic chromatin are discussed.