Since the first half of the twentieth century, evolutionary theory has been dominated by the idea that mutations occur randomly with respect to their consequences
. Here we test this assumption with ...large surveys of de novo mutations in the plant Arabidopsis thaliana. In contrast to expectations, we find that mutations occur less often in functionally constrained regions of the genome-mutation frequency is reduced by half inside gene bodies and by two-thirds in essential genes. With independent genomic mutation datasets, including from the largest Arabidopsis mutation accumulation experiment conducted to date, we demonstrate that epigenomic and physical features explain over 90% of variance in the genome-wide pattern of mutation bias surrounding genes. Observed mutation frequencies around genes in turn accurately predict patterns of genetic polymorphisms in natural Arabidopsis accessions (r = 0.96). That mutation bias is the primary force behind patterns of sequence evolution around genes in natural accessions is supported by analyses of allele frequencies. Finally, we find that genes subject to stronger purifying selection have a lower mutation rate. We conclude that epigenome-associated mutation bias
reduces the occurrence of deleterious mutations in Arabidopsis, challenging the prevailing paradigm that mutation is a directionless force in evolution.
On the basis of structural and experimental data, it was previously demonstrated that the snake integument consists of a hard, robust, inflexible outer surface (Oberhäutchen and β-layer) and softer, ...flexible inner layers (α-layers). It is not clear whether this phenomenon is a general adaptation of snakes to limbless locomotion or only to specific conditions, such as habitat and locomotion. The aim of the present study was to compare the structure and material properties of the outer scale layers (OSLs) and inner scale layers (ISLs) of the exuvium epidermis in four snake species specialized to live in different habitats: Lampropeltis getula californiae (terrestrial), Epicrates cenchria cenchria (generalist), Morelia viridis (arboreal) and Gongylophis colubrinus (sand-burrowing). Scanning electron microscopy (SEM) of skin cross sections revealed a strong variation in the epidermis structure between species. The nanoindentation experiments clearly demonstrated a gradient of material properties along the epidermis in the integument of all the species studied. The presence of such a gradient is a possible adaptation to locomotion and wear minimization on natural substrates. In general, the difference in both the effective elastic modulus and hardness of the OSL and ISL between species was not large compared with the difference in epidermis thickness and architecture.
Electric field induced second harmonic generation (E-FISH) has recently demonstrated significant potential as a method for making absolute electric field measurements in non-equilibrium plasmas and ...gas discharges. Previous studies have relied on the plane-wave approximation in quantifying these measurements, while in reality, focused laser beams are almost always used. In this work, we perform a theoretical and experimental study using focused Gaussian beams, and examine the consequent effects on the E-FISH signal. We show that in addition to important parameters such as the external electric field strength, wave vector mismatch and Rayleigh range, the signal is strongly influenced by the full length and shape of this external field profile. We attribute this to the Gouy phase shift associated with focused beams, and note that analogous effects have been previously observed in second and third harmonic generation microscopy. This dependence of the E-FISH signal on the spatial profile of the external field is worth highlighting since it is often not easily determined a priori in a plasma, and neglecting its influence could lead to an incorrect electric field measurement. To minimize any inaccuracies associated with this issue, we propose several recommendations to consider when using the E-FISH diagnostic with focused beams.
Collagen is a triple-helical protein that forms various macromolecular organizations in tissues and is responsible for the biomechanical and physical properties of most organs. Second-harmonic ...generation (SHG) microscopy is a valuable imaging technique to probe collagen fibrillar organization. In this article, we use a multiscale nonlinear optical formalism to bring theoretical evidence that anisotropy of polarization-resolved SHG mostly reflects the micrometer-scale disorder in the collagen fibril distribution. Our theoretical expectations are confirmed by experimental results in rat-tail tendon. To that end, we report what to our knowledge is the first experimental implementation of polarization-resolved SHG microscopy combined with mechanical assays, to simultaneously monitor the biomechanical response of rat-tail tendon at macroscopic scale and the rearrangement of collagen fibrils in this tissue at microscopic scale. These experiments bring direct evidence that tendon stretching corresponds to straightening and aligning of collagen fibrils within the fascicle. We observe a decrease in the SHG anisotropy parameter when the tendon is stretched in a physiological range, in agreement with our numerical simulations. Moreover, these experiments provide a unique measurement of the nonlinear optical response of aligned fibrils. Our data show an excellent agreement with recently published theoretical calculations of the collagen triple helix hyperpolarizability.
The transparency and mechanical strength of the cornea are related to the highly organized three-dimensional distribution of collagen fibrils. It is of great interest to develop specific and ...contrasted in vivo imaging tools to probe these collagenous structures, which is not available yet. Second Harmonic Generation (SHG) microscopy is a unique tool to reveal fibrillar collagen within unstained tissues, but backward SHG images of cornea fail to reveal any spatial features due to the nanometric diameter of stromal collagen fibrils. To overcome this limitation, we performed polarization-resolved SHG imaging, which is highly sensitive to the sub-micrometer distribution of anisotropic structures. Using advanced data processing, we successfully retrieved the orientation of the collagenous fibrils at each depth of human corneas, even in backward SHG homogenous images. Quantitative information was also obtained about the submicrometer heterogeneities of the fibrillar collagen distribution by measuring the SHG anisotropy. All these results were consistent with numerical simulation of the polarization-resolved SHG response of cornea. Finally, we performed in vivo SHG imaging of rat corneas and achieved structural imaging of corneal stroma without any labeling. Epi-detected polarization-resolved SHG imaging should extend to other organs and become a new diagnosis tool for collagen remodeling.
Polarization resolved second-harmonic generation (pSHG) microscopy is increasingly used for mapping organized arrays of noncentrosymmetric proteins such as collagen, myosin, and tubulin, and holds ...potential for probing their molecular structure and supramolecular organization in intact tissues. However, the contrast mechanism of pSHG is complex and the development of applications in the life sciences is hampered by the lack of models accurately relating the observed pSHG signals to the underlying molecular and macromolecular organization. In this work, we establish a general multiscale numerical framework relating the micrometer-scale SHG measurements to the atomic-scale and molecular structure of the proteins under study and their supramolecular arrangement. We first develop a new method to automatically analyze pSHG signals independently of the protein type and fiber orientation. We then characterize experimentally pSHG signals in live zebrafish larvae and show that they can be used to distinguish collagen, myosin, and tubulin structures in intact tissues. We then introduce a numerical model that considers the peptide bond (PB) as the elementary SHG source in proteins and takes into account the three-dimensional (3D) distribution of PBs to predict the second-order hyperpolarizability tensor β of proteins, as well as the SHG efficiency and pSHG response of an arbitrary macromolecular assembly. We show that this model accurately reproduces pSHG measurements obtained from collagen, myosin, microtubule, and actin structures, revealing the precise dependence of SHG signals on the 3D distribution of PBs within protein assemblies. We then use our model to analyze pSHG from a 3D distribution of microtubule assemblies as a function of out-of-plane angles, angular disorder, and polarity. Finally, we demonstrate that our model predicts SHG from different molecular conformations of tubulin that are highly relevant from a biomedical point of view as associated with microtubules (de)polymerization. By bridging scales from the molecular bonds to the optical wavelength, our model provides an accurate interpretation of SHG signals in terms of protein structure and supramolecular organization.
The quantification of collagen fibril size is a major issue for the investigation of pathological disorders associated with structural defects of the extracellular matrix. Second-harmonic generation ...microscopy is a powerful technique to characterize the macromolecular organization of collagen in unstained biological tissues. Nevertheless, due to the complex coherent building of this nonlinear optical signal, it has never been used to measure fibril diameter so far. Here we report absolute measurements of second-harmonic signals from isolated fibrils down to 30 nm diameter, via implementation of correlative second-harmonic-electron microscopy. Moreover, using analytical and numerical calculations, we demonstrate that the high sensitivity of this technique originates from the parallel alignment of collagen triple helices within fibrils and the subsequent constructive interferences of second-harmonic radiations. Finally, we use these absolute measurements as a calibration for ex vivo quantification of fibril diameter in the Descemet's membrane of a diabetic rat cornea.
Bio-engineering technologies are currently used to produce biomimetic artificial corneas that should present structural, chemical, optical, and biomechanical properties close to the native tissue. ...These properties are mainly supported by the corneal stroma which accounts for 90% of corneal thickness and is mainly made of collagen type I. The stromal collagen fibrils are arranged in lamellae that have a plywood-like organization. The fibril diameter is between 25 and 35 nm and the interfibrillar space about 57 nm. The number of lamellae in the central stroma is estimated to be 300. In the anterior part, their size is 10–40 μm. They appear to be larger in the posterior part of the stroma with a size of 60–120 μm. Their thicknesses also vary from 0.2 to 2.5 μm. During development, the acellular corneal stroma, which features a complex pattern of organization, serves as a scaffold for mesenchymal cells that invade and further produce the cellular stroma. Several pathways including Bmp4, Wnt/β-catenin, Notch, retinoic acid, and TGF-β, in addition to EFTFs including the mastering gene Pax-6, are involved in corneal development. Besides, retinoic acid and TGF- β seem to have a crucial role in the neural crest cell migration in the stroma. Several technologies can be used to produce artificial stroma. Taking advantage of the liquid-crystal properties of acid-soluble collagen, it is possible to produce transparent stroma-like matrices with native-like collagen I fibrils and plywood-like organization, where epithelial cells can adhere and proliferate. Other approaches include the use of recombinant collagen, cross-linkers, vitrification, plastically compressed collagen or magnetically aligned collagen, providing interesting optical and mechanical properties. These technologies can be classified according to collagen type and origin, presence of telopeptides and native-like fibrils, structure, and transparency. Collagen matrices feature transparency >80% for the appropriate 500-μm thickness. Non-collagenous matrices made of biopolymers including gelatin, silk, or fish scale have been developed which feature interesting properties but are less biomimetic. These bioengineered matrices still need to be colonized by stromal cells to fully reproduce the native stroma.
•During development, mesenchymal cells invade the organized acellular corneal stroma and further produce the cellular stroma.•Collagen liquid-crystal properties permit producing transparent matrices with collagen I fibrils and plywood organization.•Other approaches include recombinant collagen, cross-linkers, compressed collagen, or magnetically aligned collagen.•Bioengineered matrices still need to be colonized by stromal cells to fully reproduce the native stroma.
Interfaces provide the structural basis of essential bone functions. In the hierarchical structure of bone tissue, heterogeneities such as porosity or boundaries are found at scales ranging from ...nanometers to millimeters, all of which contributing to macroscopic properties. To date, however, the complexity or limitations of currently used imaging methods restrict our understanding of this functional integration. Here we address this issue using label-free third-harmonic generation (THG) microscopy. We find that the porous lacuno-canalicular network (LCN), revealing the geometry of osteocytes in the bone matrix, can be directly visualized in 3D with submicron precision over millimetric fields of view compatible with histology. THG also reveals interfaces delineating volumes formed at successive remodeling stages. Finally, we show that the structure of the LCN can be analyzed in relation with that of the extracellular matrix and larger-scale structures by simultaneously recording THG and second-harmonic generation (SHG) signals relating to the collagen organization.
We thoroughly analyze the linear propagation effects that affect polarization-resolved Second Harmonic Generation imaging of thick anisotropic tissues such as collagenous tissues. We develop a ...theoretical model that fully accounts for birefringence and diattenuation along the excitation propagation, and polarization scrambling upon scattering of the harmonic signal. We obtain an excellent agreement with polarizationresolved SHG images at increasing depth within a rat-tail tendon for both polarizations of the forward SHG signal. Most notably, we observe interference fringes due to birefringence in the SHG depth profile when excited at π/4 angle from the tendon axis. We also measure artifactual decrease of ρ = Χxxx/Χxyy with depth due to diattenuation of the excitation. We therefore derive a method that proves reliable to determine both ρ and the tendon birefringence and diattenuation.
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