Fractal analysis of STM images of lignin polymer obtained by in vitro synthesis

Lignin, the structural polymer of the plant cell walls, is produced by free radical polymerization of phenolic alcohols, catalyzed by different peroxidases. The mechanism and the structural organization of lignin in the cell have not been completely understood. In this study we applied fractal analysis to images of lignin polymer obtained using scanning tunneling microscope. The analysis showed the regularity of the polymer at different levels of organization. According to the results obtained, at the 95% confidence level, there is no significant difference in the fractal dimension between images representing different organizational levels of lignin. In other words, lignin produced in in vitro conditions has fractal structural organization and, consequently the polymer can be expected to be regular in in vivo conditions. The value of the fractal dimension 1.929 +/- 0.021 is in good agreement with the theoretically predicted value for polyaddition and polycondensation mechanism of polymerization. The mechanism of in vivo lignin synthesis is discussed in terms of various experimental and theoretical evidences. In this paper, we could show that fractal analysis of the lignin polymer is a useful complementary approach to the experimental data collection in structural and phenomenological studies.     

Microstructural characterization of chitosan and alginate films by microscopy techniques and texture image analysis

The aim of this work is to characterize the microstructure of chitosan and alginate edible films by microscopy techniques and texture image analysis. Edible films were obtained by solution casting and solvent evaporation. The microscopy techniques used in this work were: light, environmental scanning electron and atomic force microscopy. Textural features and fractal dimension were extracted from the images. Entropy and fractal dimension were more useful to evaluate the complexity and roughness of films. The highest values of entropy and fractal dimension corresponded to alginate/chitosan, followed of alginate and chitosan films. An entropy/fractal dimension ratio, proposed here, was useful to characterize the degree of image complexity and roughness of edible films at different magnifications. It was possible to postulate that microscopy techniques combined with texture image analysis are efficient tools to quantitatively evaluate the surface morphology of edible films made of chitosan and alginate.     

A study of lignin formation at the molecular level by scanning tunneling microscopy.

A scanning tunneling microscope (STM) was used to observe the temporal formation and organization of dehydrogenative polymer (DHP) synthesized from coniferyl alcohol. The images obtained elucidate this structure for the first time. The structure of DHP, as seen from STM images, shows long-range order. It appears that DHP consists of building units or modules assembled into larger assemblies called supermodules. Supermodules are interconnected into the overall lattice-like polymer structure with or without spherical regions. One module consists of about 20 monomers, while the supermodule contains about 500 monomers. Calculated molecular weights for modules and supermodules agree with DHP molecular weight distribution peaks. Samples prepared at two different pH values, 6.4 and 7.6, have the same characteristics. The results presented demonstrate that the process of lignification, even in in vitro conditions, is highly ordered, and as such contribute to our understanding of the structure of lignin, a significant constitutive and functional element of cell walls.     

The Quasi-Fractal Structure of Fish Brain Neurons

The box-counting method for calculating the fractal dimension (D) with the ImageJ 1.20s software is used as a tool for quantitative analysis of the neuronal morphology in the fish brain. The fractal dimension was determined for several types of neurons in the brain of two teleost species, Pholidapus dybowskii and Oncorhynchus keta. These results were compared with those obtained for some neurons of the human brain. The fractal (fractional) dimension (D), as a quantitative index of filling of two-dimensional space by the black and white image of a cell, is shown to vary from 1.22 to 1.72 depending on the type of neuron. The fractal dimension reaches its maximum in less specialized neurons that carry out a number of different functions. On the other hand, highly specialized neurons display a relatively low fractal dimension. Thus, the fractal dimension serves as a numerical measure of the spatial complexity of the neuron and correlates with the morphofunctional organization of the cell.     

Fractal dimension in human cerebellum measured by magnetic resonance imaging

Fractal dimension has been used to quantify the structures of a wide range of objects in biology and medicine. We measured fractal dimension of human cerebellum (CB) in magnetic resonance images of 24 healthy young subjects (12 men and 12 women). CB images were resampled to a series of image sets with different 3D resolutions. At each resolution, the skeleton of the CB white matter was obtained and the number of pixels belonging to the skeleton was determined. Fractal dimension of the CB skeleton was calculated using the box-counting method. The results indicated that the CB skeleton is a highly fractal structure, with a fractal dimension of 2.57 +/- 0.01. No significant difference in the CB fractal dimension was observed between men and women.     

Topographical characterization and surface force spectroscopy of the photochemical lignin model compound.

By combining the results from atomic force microscopy (AFM) and environmental scanning electron microscopy (ESEM), herein we investigate properties of photochemical lignin model compounds. We provide evidence that photochemical lignin forms random, probably non-functional structures. The topography of such structures is explored using ESEM. Non-functionality of such structures is proved by AFM and atomic force spectroscopy experiments wherein the photochemical lignin functionalized tip is approached to the substrate covered with photochemical lignin. There was no evidence of existence of any kind of host-guest interaction during the approach/retraction experiments. These results provide evidence for our previously stated hypothesis that photochemical lignin polymerization may be one of the degrading effects of UV radiation to the plant cell.     

The role of of laccase in lignification

The enzymatic mechanism of monolignol polymerization in lignin biosynthesis is not known, although it has been the subject of significant interest for more than 60 years. Peroxidase had been considered to be the exclusive plant enzyme involved in the oxidative polymerization of lignin precursors. Recently, laccase and laccase-like oxidase activities have been associated with lignification. Laccase is bound to lignifying plant cell walls and can polymerize lignin precursors in vitro . Strong circumstantial evidence from different species implicates this enzyme in the polymerization of lignin precursors. Lignin has a complex structure and it has been difficult to analyze the heterogeneity of lignin by chemical and physical techniques. If lignin precursors are polymerized by enzymes that differ in their catalytic properties, then lignin heterogeneity could be produced by differential expression of multiple enzymes during plant development. When laccase genes are correctly identified, these ideas can be tested in genetic experiments where gain or loss of function can be predicted by the presence or absence of the functional gene.     

Fractal analysis of the uterine contractions.

The fractal dimension D may be calculated in many ways, since its strict definition, the Hausdorff definition is too complicated for practical estimation. In this paper we perform a comparative study often methods of fractal analysis of time series. In Benoit, a commercial program for fractal analysis, five methods of computing fractal dimension of time series (rescaled range analysis, power spectral analysis, roughness-length, variogram methods and wavelet method) are available. We have implemented some other algorithms for calculating D: Higuchi's fractal dimension, relative dispersion analysis, running fractal dimension, method based on mathematical morphology and method based on intensity differences. For biomedical signals results obtained by means of different algorithms are different, but consistent.     

A Fractal Nature for Polymerized Laminin

Polylaminin (polyLM) is a non-covalent acid-induced nano- and micro-structured polymer of the protein laminin displaying distinguished biological properties. Polylaminin stimulates neuritogenesis beyond the levels achieved by ordinary laminin and has been shown to promote axonal regeneration in animal models of spinal cord injury. Here we used confocal fluorescence microscopy (CFM), scanning electron microscopy (SEM) and atomic force microscopy (AFM) to characterize its three-dimensional structure. Renderization of confocal optical slices of immunostained polyLM revealed the aspect of a loose flocculated meshwork, which was homogeneously stained by the antibody. On the other hand, an ordinary matrix obtained upon adsorption of laminin in neutral pH (LM) was constituted of bulky protein aggregates whose interior was not accessible to the same anti-laminin antibody. SEM and AFM analyses revealed that the seed unit of polyLM was a flat polygon formed in solution whereas the seed structure of LM was highly heterogeneous, intercalating rod-like, spherical and thin spread lamellar deposits. As polyLM was visualized at progressively increasing magnifications, we observed that the morphology of the polymer was alike independently of the magnification used for the observation. A search for the Hausdorff dimension in images of the two matrices showed that polyLM, but not LM, presented fractal dimensions of 1.55, 1.62 and 1.70 after 1, 8 and 12 hours of adsorption, respectively. Data in the present work suggest that the intrinsic fractal nature of polymerized laminin can be the structural basis for the fractal-like organization of basement membranes in the neurogenic niches of the central nervous system.     

Fractal analysis and its applications in human palaentology

Fractal analysis was applied in human palaentology by Gibert and Palmqvist to estimate the value of the fractal dimension obtained from the cranial sutures preserved in the fragment of occipital bone (VM-0) found at the Venta Micena site. This paper also estimated the values of fractal dimension for different specimens in order to establish a taxonomy. Although that initial study demonstrated that the technique could be useful in human palaentology, the large variability of sutures observed in the VM-0 sample requires a mechanism that makes it possible to automatically obtain an objective plot of the suture to be analyzed.     

Supramolecular organization of heteroxylan-dehydrogenation polymers (synthetic lignin) nanoparticles

The supramolecular organization of particles composed of heteroxylans (HX) and synthetic lignin (dehydrogenation polymer, DHPs) was studied by light scattering (LS), atomic force microscopy (AFM), and fluorescent probes. Results from static and quasi-elastic light scattering indicate a dense core surrounded by a soft corona. Such organization is also supported by AFM images of the particles that display Gaussian height profiles when a low tapping force is applied, whereas the shape of the profile obtained at a higher mechanical solicitation is irregular and sharp due to deformation of the particles resulting from the tip indentation. This suggests a difference in mechanical behavior between the inner and outer parts of the particles. The formation of local chemical heterogeneities was demonstrated by use of two fluorescent polarity probes (pyrene and methyl-amino-pyrene) to be induced by the core-corona organization.     

Fractal dimension of K1735 mouse melanoma clones and spheroid invasion in vitro

An in vitro tumour-host confrontation method to investigate the invasion behaviour of cancer has been applied to K1735 mouse melanomas. Fluorescently labelled spheroids of cancer cells and host cells were confronted and the temporal course of cancer invasion into the host was investigated using confocal laser scanning microscopy. To improve the quantitative data of this method, the boundary images of the fluorescently labelled confrontation pairs were treated as fractals. The physical and mathematical framework for determination of the fractal capacity dimension is widely used in biology and medicine and has proved to be a very useful tool for describing the cancer invasion process. The fractal capacity dimension determination was carried out by dilation of the binary boundaries of the objects, which were treated as an estimate of the Minkowski-Bouligand dimension. The fractal dimension correlated well with the degree of invasion of the K1735-M2 clone. Control experiments, with host-host confrontations and various K1735 clones with reduced invasiveness, support these results.     

Fractal properties of forest spatial structure

The definition of fractal dimension of natural objects, which enables to deal with scale dependence of fractal dimension is discussed. Abrupt changes of fractal dimension of spatial structure of terrestrial ecosystems are considered in the context of hierarchical paradigm. On this ground the procedure is proposed for segmentation of a territory, which takes into account the scale dependence of spatial variability of ecological parameters. Using remotely sensed data — normalized difference vegetation index (NDVI) and thermal radiation in the infrared band — fractal dimensions and critical scales are evaluated for different forest types with the help of software, developed for this purpose. The results obtained corroborate the potentialities of fractal approach in ecology. These methods and results can be used for discrimination of remotely sensed data; but further investigations, including detailed comparison of fractal characteristics of remotely sensed forest images with results of on-site field studies are necessary to validate them.     

Analysis of fractal dimension of O2A glial cells differentiating in vitro

Fractal dimension is a quantitative measure of morphological complexity. Glial cells of the oligodendrocyte-type 2 astrocyte (O2A) lineage exhibit increasing morphological complexity as they differentiate in vitro. Enriched populations of O2A progenitor cells isolated from neonatal rat cerebral hemispheres or optic nerves were allowed to differentiate in vitro, and their fractal dimensions were measured over time. The fractal dimensions of the maturing cells correlated with perceived complexity; cells with elaborate process branching had larger fractal dimensions than cells with a simpler morphology. An analysis of changes in fractal dimension revealed distinct rates of growth for both oligodendrocytes and type 2 astrocytes. The fractal dimension remained constant over a 10-fold range in optical magnification, demonstrating that cultured O2A glial cells exhibit self-similarity, a defining characteristic of fractal objects. These results illustrate that fractal dimension analysis of maturing cell populations is a useful method for quantitatively describing the process of cell differentiation.     

Lignin dehydrogenative polymerization mechanism: a poplar cell wall peroxidase directly oxidizes polymer lignin and produces in vitro dehydrogenative polymer rich in beta-O-4 linkage

An investigation was performed to determine whether lignin dehydrogenative polymerization proceeds via radical mediation or direct oxidation by peroxidases. It was found that coniferyl alcohol radical transferred quickly to sinapyl alcohol. The transfer to syringaresinol was slower, however, the transfer to polymeric lignols occurred very slightly. This result suggests that the radical mediator theory does not sufficiently explain the mechanism for dehydrogenative polymerization of lignin. A cationic cell wall peroxidase (CWPO-C) from poplar (Populus alba L.) callus showed a strong substrate preference for sinapyl alcohol and the sinapyl alcohol dimer, syringaresinol. Moreover, CWPO-C was capable of oxidizing high-molecular-weight sinapyl alcohol polymers and ferrocytochrome c. Therefore, the CWPO-C characteristics are important to produce polymer lignin. The results suggest that CWPO-C may be a peroxidase isoenzyme responsible for the lignification of plant cell walls.     

Fractal geometric analysis of lung cancer angiogenic patterns

For medical images, the fractal dimension D may be used as an index of irregularity. The angiogenesis patterns of lung cancer were analysed by means of the perimeter-area and box counting algorithms. The fractal nature of all images in the sense of the perimeter-area method and of 68% images in the sense of the box-counting method suggest the possibility to use the fractal dimension as a new non-morphometric parameter evaluating angiogenic processes in neoplasms.     

Fractal nature of chromatin organization in interphase chicken erythrocyte nuclei: DNA structure exhibits biphasic fractal properties

Arrangement of chromatin in intact chicken erythrocyte nuclei was investigated by small angle neutron scattering. The scattering spectra have revealed that on the scales between 15 nm and 1.5 microm the interior of the nucleus exhibited properties of a mass fractal. The fractal dimension of the protein component of cell nucleus held constant at approximately 2.5, while the DNA organization was biphasic, with the fractal dimension slightly higher than 2 on the scales smaller than 300 nm and approaching 3 on the larger scales.     

An engineered monolignol 4-o-methyltransferase depresses lignin biosynthesis and confers novel metabolic capability in Arabidopsis

Although the practice of protein engineering is industrially fruitful in creating biocatalysts and therapeutic proteins, applications of analogous techniques in the field of plant metabolic engineering are still in their infancy. Lignins are aromatic natural polymers derived from the oxidative polymerization of primarily three different hydroxycinnamyl alcohols, the monolignols. Polymerization of lignin starts with the oxidation of monolignols, followed by endwise cross-coupling of (radicals of) a monolignol and the growing oligomer/polymer. The para-hydroxyl of each monolignol is crucial for radical generation and subsequent coupling. Here, we describe the structure-function analysis and catalytic improvement of an artificial monolignol 4-O-methyltransferase created by iterative saturation mutagenesis and its use in modulating lignin and phenylpropanoid biosynthesis. We show that expressing the created enzyme in planta, thus etherifying the para-hydroxyls of lignin monomeric precursors, denies the derived monolignols any participation in the subsequent coupling process, substantially reducing lignification and, ultimately, lignin content. Concomitantly, the transgenic plants accumulated de novo synthesized 4-O-methylated soluble phenolics and wall-bound esters. The lower lignin levels of transgenic plants resulted in higher saccharification yields. Our study, through a structure-based protein engineering approach, offers a novel strategy for modulating phenylpropanoid/lignin biosynthesis to improve cell wall digestibility and diversify the repertories of biologically active compounds.