Andre Khalil

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.

Muscle Development Is Disrupted in Zebrafish Embryos Deficient for Fibronectin

After somitogenesis, skeletal muscle precursors elongate into muscle fibers that anchor to the somite boundary, which becomes the myotome boundary. Fibronectin (Fn) is a major component of the extracellular matrix in both boundaries. Although Fn is required for somitogenesis, effects of Fn disruption on subsequent muscle development are unknown. We show that fn knockdown disrupts myogenesis. Muscle morphogenesis is more disrupted in fn morphants than in a mutant where initial somite boundaries did not form, aei/deltaD. We quantified this disruption using the two‐dimensional Wavelet‐Transform Modulus Maxima method, which uses the variation of intensity in an image with respect to the direction considered to characterize the structure in a cell lattice. We show that fibers in fn morphants are less organized than in aei/deltaD mutant embryos. Fast‐ and slow‐twitch muscle lengths are also more frequently uncoupled. These data suggest that fn may function to regulate fiber organization and limit fast‐twitch muscle fiber length. Developmental Dynamics 237:2542–2553, 2008.

Nrk2b-mediated NAD+ production regulates cell adhesion and is required for muscle morphogenesis in vivo: Nrk2b and NAD+ in muscle morphogenesis.

Cell-matrix adhesion complexes (CMACs) play fundamental roles during morphogenesis. Given the ubiquitous nature of CMACs and their roles in many cellular processes, one question is how specificity of CMAC function is modulated. The clearly defined cell behaviors that generate segmentally reiterated axial skeletal muscle during zebrafish development comprise an ideal system with which to investigate CMAC function during morphogenesis. We found that Nicotinamide riboside kinase 2b (Nrk2b) cell autonomously modulates the molecular composition of CMACs in vivo. Nrk2b is required for normal Laminin polymerization at the myotendinous junction (MTJ). In Nrk2b-deficient embryos, at MTJ loci where Laminin is not properly polymerized, muscle fibers elongate into adjacent myotomes and are abnormally long. In yeast and human cells, Nrk2 phosphorylates Nicotinamide Riboside and generates NAD+ through an alternative salvage pathway. Exogenous NAD+ treatment rescues MTJ development in Nrk2b-deficient embryos, but not in laminin mutant embryos. Both Nrk2b and Laminin are required for localization of Paxillin, but not beta-Dystroglycan, to CMACs at the MTJ. Overexpression of Paxillin in Nrk2b-deficient embryos is sufficient to rescue MTJ integrity. Taken together, these data show that Nrk2b plays a specific role in modulating subcellular localization of discrete CMAC components that in turn plays roles in musculoskeletal development. Furthermore, these data suggest that Nrk2b-mediated synthesis of NAD+ is functionally upstream of Laminin adhesion and Paxillin subcellular localization during MTJ development. These results indicate a previously unrecognized complexity to CMAC assembly in vivo and also elucidate a novel role for NAD+ during morphogenesis.

NAD+ biosynthesis ameliorates a zebrafish model of muscular dystrophy.

NAD+ improves muscle tissue structure and function in dystrophic zebrafish by increasing basement membrane organization.

Time-Lapse Analysis and Mathematical Characterization Elucidate Novel Mechanisms Underlying Muscle Morphogenesis

Skeletal muscle morphogenesis transforms short muscle precursor cells into long, multinucleate myotubes that anchor to tendons via the myotendinous junction (MTJ). In vertebrates, a great deal is known about muscle specification as well as how somitic cells, as a cohort, generate the early myotome. However, the cellular mechanisms that generate long muscle fibers from short cells and the molecular factors that limit elongation are unknown. We show that zebrafish fast muscle fiber morphogenesis consists of three discrete phases: short precursor cells, intercalation/elongation, and boundary capture/myotube formation. In the first phase, cells exhibit randomly directed protrusive activity. The second phase, intercalation/elongation, proceeds via a two-step process: protrusion extension and filling. This repetition of protrusion extension and filling continues until both the anterior and posterior ends of the muscle fiber reach the MTJ. Finally, both ends of the muscle fiber anchor to the MTJ (boundary capture) and undergo further morphogenetic changes as they adopt the stereotypical, cylindrical shape of myotubes. We find that the basement membrane protein laminin is required for efficient elongation, proper fiber orientation, and boundary capture. These early muscle defects in the absence of either lamininβ1 or lamininγ1 contrast with later dystrophic phenotypes in lamininα2 mutant embryos, indicating discrete roles for different laminin chains during early muscle development. Surprisingly, genetic mosaic analysis suggests that boundary capture is a cell-autonomous phenomenon. Taken together, our results define three phases of muscle fiber morphogenesis and show that the critical second phase of elongation proceeds by a repetitive process of protrusion extension and protrusion filling. Furthermore, we show that laminin is a novel and critical molecular cue mediating fiber orientation and limiting muscle cell length.

Chromosome neighborhood composition determines translocation outcomes after exposure to high-dose radiation in primary cells

Radiation exposure is an occupational hazard for military personnel, some health care professionals, airport security screeners, and medical patients, with some individuals at risk for acute, high-dose exposures. Therefore, the biological effects of radiation, especially the potential for chromosome damage, are major occupational and health concerns. However, the biophysical mechanisms of chromosome instability subsequent to radiation-induced DNA damage are poorly understood. It is clear that interphase chromosomes occupy discrete structural and functional subnuclear domains, termed chromosome territories (CT), which may be organized into ‘neighborhoods’ comprising groups of specific CTs. We directly evaluated the relationship between chromosome positioning, neighborhood composition, and translocation partner choice in primary lymphocytes, using a cell-based system in which we could induce multiple, concentrated DNA breaks via high-dose irradiation. We critically evaluated mis-rejoining profiles and tested whether breaks occurring nearby were more likely to fuse than breaks occurring at a distance. We show that CT neighborhoods comprise heterologous chromosomes, within which inter-CT distances directly relate to translocation partner choice. These findings demonstrate that interphase chromosome arrangement is a principal factor in genomic instability outcomes in primary lymphocytes, providing a structural context for understanding the biological effects of radiation exposure, and the molecular etiology of tumor-specific translocation patterns.

Characterizing Complexity in Solar Magnetogram Data Using a Wavelet-based Segmentation Method

The multifractal nature of solar photospheric magnetic structures is studied using the two-dimensional wavelet transform modulus maxima (WTMM) method. This relies on computing partition functions from the wavelet transform skeleton defined by the WTMM method. This skeleton provides an adaptive space-scale partition of the fractal distribution under study, from which one can extract the multifractal singularity spectrum. We describe the implementation of a multiscale image processing segmentation procedure based on the partitioning of the WT skeleton, which allows the disentangling of the information concerning the multifractal properties of active regions from the surrounding quiet-Sun field. The quiet Sun exhibits an average Holder exponent ~–0.75, with observed multifractal properties due to the supergranular structure. On the other hand, active region multifractal spectra exhibit an average Holder exponent ~0.38, similar to those found when studying experimental data from turbulent flows.

Wavelet-based 3D reconstruction of microcalcification clusters from two mammographic views: new evidence that fractal tumors are malignant and Euclidean tumors are benign.

The 2D Wavelet-Transform Modulus Maxima (WTMM) method was used to detect microcalcifications (MC) in human breast tissue seen in mammograms and to characterize the fractal geometry of benign and malignant MC clusters. This was done in the context of a preliminary analysis of a small dataset, via a novel way to partition the wavelet-transform space-scale skeleton. For the first time, the estimated 3D fractal structure of a breast lesion was inferred by pairing the information from two separate 2D projected mammographic views of the same breast, i.e. the cranial-caudal (CC) and mediolateral-oblique (MLO) views. As a novelty, we define the “CC-MLO fractal dimension plot”, where a “fractal zone” and “Euclidean zones” (non-fractal) are defined. 118 images (59 cases, 25 malignant and 34 benign) obtained from a digital databank of mammograms with known radiologist diagnostics were analyzed to determine which cases would be plotted in the fractal zone and which cases would fall in the Euclidean zones. 92% of malignant breast lesions studied (23 out of 25 cases) were in the fractal zone while 88% of the benign lesions were in the Euclidean zones (30 out of 34 cases). Furthermore, a Bayesian statistical analysis shows that, with 95% credibility, the probability that fractal breast lesions are malignant is between 74% and 98%. Alternatively, with 95% credibility, the probability that Euclidean breast lesions are benign is between 76% and 96%. These results support the notion that the fractal structure of malignant tumors is more likely to be associated with an invasive behavior into the surrounding tissue compared to the less invasive, Euclidean structure of benign tumors. Finally, based on indirect 3D reconstructions from the 2D views, we conjecture that all breast tumors considered in this study, benign and malignant, fractal or Euclidean, restrict their growth to 2-dimensional manifolds within the breast tissue.

Multifractal analysis of dynamic infrared imaging of breast cancer

The wavelet transform modulus maxima (WTMM) method was used in a multifractal analysis of skin breast temperature time-series recorded using dynamic infrared (IR) thermography. Multifractal scaling was found for healthy breasts as the signature of a continuous change in the shape of the probability density function (pdf) of temperature fluctuations across time scales from to 3 s. In contrast, temperature time-series from breasts with malignant tumors showed homogeneous monofractal temperature fluctuations statistics. These results highlight dynamic IR imaging as a very valuable non-invasive technique for preliminary screening in asymptomatic women to identify those with risk of breast cancer.

MULTI-SCALE MORPHOLOGICAL ANALYSIS OF SDSS DR5 SURVEY USING THE METRIC SPACE TECHNIQUE

Following the novel development and adaptation of the Metric Space Technique (MST), a multi-scale morphological analysis of the Sloan Digital Sky Survey (SDSS) Data Release 5 (DR5) was performed. The technique was adapted to perform a space-scale morphological analysis by filtering the galaxy point distributions with a smoothing Gaussian function, thus giving quantitative structural information on all size scales between 5 and 250 Mpc. The analysis was performed on a dozen slices of a volume of space containing many newly measured galaxies from the SDSS DR5 survey. Using the MST, observational data were compared to galaxy samples taken from N-body simulations with current best estimates of cosmological parameters and from random catalogs. By using the maximal ranking method among MST output functions, we also develop a way to quantify the overall similarity of the observed samples with the simulated samples.