Hyper Rayleigh scattering from DNA nucleotides in aqueous solution.

Nucleotides are organic compounds consisting of a phosphate group, a nitrogenous base, namely adenine (A), thymine (T), cytosine (C), or guanine (G), and a sugar, here deoxyribose. The magnitude of the first hyperpolarizability ß of these four DNA nucleotides was determined in aqueous solution with the nonlinear optical technique of hyper rayleigh scattering under non resonant conditions at a fundamental wavelength of 800 nm. The smallest value is found to be 1.67 ± 0.15 × 10-30 esu for thymidine-5'-monophosphate and the highest is 1.76 ± 0.16 × 10-30 esu for 2'-guanosine-5'-monophosphate. Polarization resolved studies were also performed to question the symmetry of the first hyperpolarizability tensor and access the ratio of some elements of the first hyperpolarizability tensor. These experimental results were then compared to the theoretical values of these first hyperpolarizabilities obtained with the density functional theory at the level of the PCM-B3LYP/6-31G+(d) basis and taking into account the solvent.

Organization of collagen fibers and tissue hardening: Markers of fibrotic scarring after spinal cord injury in mice revealed by multiphoton-atomic force microscopy imaging.

Spinal cord injury is a dramatic disease leading to severe motor, sensitive and autonomic impairments. After injury the axonal regeneration is partly inhibited by the glial scar, acting as a physical and chemical barrier. The scarring process involves microglia, astrocytes and extracellular matrix components, such as collagen, constructing the fibrotic component of the scar. To investigate the role of collagen, we used a multimodal label-free imaging approach combining multiphoton and atomic force microscopy. The second harmonic generation signal exhibited by fibrillar collagen enabled to specifically monitor it as a biomarker of the lesion. An increase in collagen density and the formation of more tortuous fibers over time after injury are observed. Nano-mechanical investigations revealed a noticeable hardening of the injured area, correlated with collagen fibers' formation. These observations indicate the concomitance of important structural and mechanical modifications during the fibrotic scar evolution.

Revealing Capillarity in AFM Indentation of Cells by Nanodiamond-Based Nonlocal Deformation Sensing.

Nanoindentation based on atomic force microscopy (AFM) can measure the elasticity of biomaterials and cells with high spatial resolution and sensitivity, but relating the data to quantitative mechanical properties depends on information on the local contact, which is unclear in most cases. Here, we demonstrate nonlocal deformation sensing on biorelevant soft matters upon AFM indentation by using nitrogen-vacancy centers in nanodiamonds, providing data for studying both the elasticity and capillarity without requiring detailed knowledge about the local contact. Using fixed HeLa cells for demonstration, we show that the apparent elastic moduli of the cells would have been overestimated if the capillarity was not considered. In addition, we observe that both the elastic moduli and the surface tensions are reduced after depolymerization of the actin cytoskeleton in cells. This work demonstrates that the nanodiamond sensing of nonlocal deformation with nanometer precision is particularly suitable for studying mechanics of soft biorelevant materials.

MorphoScript: a dedicated analysis to assess the morphology and contractile structures of cardiomyocytes derived from stem cells.

MOTIVATION: Cardiomyocytes derived from stem cells are closely followed, notably since the discovery in 2007 of human induced pluripotent stem cells (hiPSC). Cardiomyocytes (hiPSC-CM) derived from hiPSC are indeed more and more used to study specific cardiac diseases as well as for developing novel applications such as drug safety experiments. Robust dedicated tools to characterize hiPSC-CM are now required. The hiPSC-CM morphology constitutes an important parameter since these cells do not demonstrate the expected rod shape, characteristic of native human cardiomyocytes. Similarly, the presence, the density and the organization of contractile structures would be a valuable parameter to study. Precise measurements of such characteristics would be useful in many situations: for describing pathological conditions, for pharmacological screens or even for studies focused on the hiPSC-CM maturation process. RESULTS: For this purpose, we developed a MATLAB based image analysis toolbox, which gives accurate values for cellular morphology parameters as well as for the contractile cell organization. AVAILABILITY AND IMPLEMENTATION: To demonstrate the power of this automated image analysis, we used a commercial maturation medium intended to promote the maturation status of hiPSC-CM, and compare the parameters with the ones obtained with standard culture medium, and with freshly dissociated mouse cardiomyocytes. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Cytotoxic CD8+ T lymphocytes expressing ALS-causing SOD1 mutant selectively trigger death of spinal motoneurons.

Adaptive immune response is part of the dynamic changes that accompany motoneuron loss in amyotrophic lateral sclerosis (ALS). CD4+ T cells that regulate a protective immunity during the neurodegenerative process have received the most attention. CD8+ T cells are also observed in the spinal cord of patients and ALS mice although their contribution to the disease still remains elusive. Here, we found that activated CD8+ T lymphocytes infiltrate the central nervous system (CNS) of a mouse model of ALS at the symptomatic stage. Selective ablation of CD8+ T cells in mice expressing the ALS-associated superoxide dismutase-1 (SOD1)G93A mutant decreased spinal motoneuron loss. Using motoneuron-CD8+ T cell coculture systems, we found that mutant SOD1-expressing CD8+ T lymphocytes selectively kill motoneurons. This cytotoxicity activity requires the recognition of the peptide-MHC-I complex (where MHC-I represents major histocompatibility complex class I). Measurement of interaction strength by atomic force microscopy-based single-cell force spectroscopy demonstrated a specific MHC-I-dependent interaction between motoneuron and SOD1G93A CD8+ T cells. Activated mutant SOD1 CD8+ T cells produce interferon-γ, which elicits the expression of the MHC-I complex in motoneurons and exerts their cytotoxic function through Fas and granzyme pathways. In addition, analysis of the clonal diversity of CD8+ T cells in the periphery and CNS of ALS mice identified an antigen-restricted repertoire of their T cell receptor in the CNS. Our results suggest that self-directed immune response takes place during the course of the disease, contributing to the selective elimination of a subset of motoneurons in ALS.

Effects of green light photobiomodulation on Dental Pulp Stem Cells: enhanced proliferation and improved wound healing by cytoskeleton reorganization and cell softening.

Photobiomodulation (PBM) has been shown to improve cell proliferation and cell migration. Many cell types have been investigated, with most studies using deep penetrating red light irradiation. Considering the interest of surface biostimulation of oral mesenchymal cells after surgical wound, the present study aimed to assess green light irradiation effects on Dental Pulp Stem Cells' (DPSC) proliferation and migration. To understand the mechanisms underlying these effects, we investigated cytoskeleton organization and subsequent cell shape and stiffness. A 532-nm wavelength Nd:YAG laser (30 mW) was applied between 30 and 600 s on DPSC in vitro. Cell proliferation was analyzed at 24, 48, and 72 h after irradiation, by cell counting and enzymatic activity quantification (paranitrophenylphosphate phosphatase (pNPP) test). A wound healing assay was used to study cell migration after irradiation. Effects of PBM on cytoskeleton organization and cell shape were assessed by actin filaments staining. Elasticity changes after irradiation were quantified in terms of Young's modulus measured using Atomic Force Microscopy (AFM) force spectroscopy. Green light significantly improved DPSC proliferation with a maximal effect obtained after 300-s irradiation (energy fluence 5 J/cm2). This irradiation had a significant impact on cell migration, improving wound healing after 24 h. These results were concomitant with a decrease of cells' Young's modulus after irradiation. This cell softening was explained by actin cytoskeleton reorganization, with diminution of cell circularity and more abundant pseudopodia. This study highlights the interest of green laser PMB for the proliferation and migration of mesenchymal stem cells, with encouraging results for clinical application, especially for surgical wound healing procedures.

Strategies for Chalcogenide Thin Film Functionalization.

We report the functionalization of chalcogenide thin films with biotinylated 12-mer peptides SVSVGMKPSPRP and LLADTTHHRPWT exhibiting a high binding affinity toward inorganic surfaces, on the one hand, and with (3-aminopropyl)triethoxysilane (APTES), on the other hand. The specific biotin moieties were used to bind streptavidin proteins and demonstrate the efficacy of the biofunctionalizated chalcogenide thin films to capture biomolecules. Atomic force microscopy provided high-resolution images of the interfaces, and water contact angle measurements gave insight into the interaction mechanisms. Fourier transform infrared spectroscopy in attenuated total reflection mode provided information about the secondary structure of the bound proteins, thanks to the deconvolution of the amide I band (1700-1600 cm-1). Following adsorption of the biotinylated peptides or APTES immobilization, a homogenous coverage of the biotin layer exhibiting very low roughness was obtained, also rendering more hydrophilic Ge-Se-Te surfaces. Subsequent capture of streptavidin depends on the functionalization approach, permitting more or less an optimal orientation of the biotin to bind streptavidin. The molecular interface layer formed on Ge-Se-Te is crucial also for retaining the native secondary structure of the protein. Altogether, our results demonstrate that both peptides and APTES were appropriate linkers to build a favorable interface on chalcogenide materials to capture proteins, opening hereby promising biosensing applications.

Relationship between Changes in Chemical Composition of Enamel Subsurface Lesions and the Emitted Nonlinear Optical Signals: An in vitro Study.

The development of new diagnostic technologies based on the light scattering and autofluorescence properties of dental tissues is required to improve the diagnostic ability of initial caries lesions earlier than previously done and promoting the potential of treatment without surgical intervention. The aim of this study is to correlate fluorescence-based results provided by multiphoton microscopy (MPM) with confocal Raman microscopy records using phosphate level at 960 cm-1 and the organic matrix at ∼2,931 cm-1 in healthy and demineralized human enamel. Measurements on 14 teeth were made using two incident lights of different wavelengths, released by confocal Raman microscopy and MPM. Raman phosphate peak intensity at 960 cm-1 along with organic to mineral ratio at (2,931/430 cm-1) and nonlinear optical signals (second harmonic generation [SHG] and intrinsic two-photon excited fluorescence [I2PEF]) were recorded from the demineralized and healthy enamel sites. Raman spectral maps showed that the higher the organic/mineral ratio in the demineralized enamel, the lower the intensity of mineral component in the same zone. MPM revealed new optical indicators of carious lesion as shown by the presence of a red-shifted fluorescence peak in the 650- to 750-nm area of the fluorescence spectrum of demineralized enamel. Moreover, on sample regions with insignificant autofluorescence, the emergence of the SHG signal could be noted. By comparing I2PEF images with the structural motifs observed by the confocal Raman imaging system, the morphological similarity of the acquired images was quite evident. Any change in the I2PEF spectra reflects alterations in the chemical composition of enamel. These findings may provide an important basis for potentially valuable applications of photonic tools in the clinical diagnosis of tooth pathological conditions, besides exposing the fundamental role of organic matrix in enamel integrity and reparation.

Internal structure and remodeling in dystrophin-deficient cardiomyocytes using second harmonic generation.

Duchenne muscular dystrophy (DMD) is a debilitating disorder related to dystrophin encoding gene mutations, often associated with dilated cardiomyopathy. However, it is still unclear how dystrophin deficiency affects cardiac sarcomere remodeling and contractile dysfunction. We employed second harmonic generation (SHG) microscopy, a nonlinear optical imaging technique that allows studying contractile apparatus organization without histologic fixation and immunostaining. Images were acquired on alive DMD (mdx) and wild type cardiomyocytes at different ages and at various external calcium concentrations. An automated image processing was developed to identify individual myofibrils and extract data about their organization. We observed a structural aging-dependent remodeling in mdx cardiomyocytes affecting sarcomere sinuosity, orientation and length that could not be anticipated from standard optical imaging. These results revealed for the first time the interest of SHG to evaluate the intracellular and sarcomeric remodeling of DMD cardiac tissue in an age-dependent manner that could participate in progressive contractile dysfunction.

De-adhesion dynamics of melanoma cells from brain endothelial layer.

Metastasis formation is a complex and not entirely understood process. The poorest prognosis and the most feared complications are associated to brain metastases. Melanoma derived brain metastases show the highest prevalence. Due to the lack of classical lymphatic drainage, in the process of brain metastases formation the haematogenous route is of primordial importance. The first and crucial step in this multistep process is the establishment of firm adhesion between the blood travelling melanoma cells and the tightly connected layer of the endothelium, which is the fundamental structure of the blood-brain barrier. This study compares the de-adhesion properties and dynamics of three melanoma cells types (WM35, A2058 and A375) to a confluent layer of brain micro-capillary endothelial cells. Cell type dependent adhesion characteristics are presented, pointing towards the existence of metastatic potential related nanomechanical aspects. Apparent mechanical properties such as elasticity, maximal adhesion force, number, size and distance of individual rupture events showed altered values pointing towards cell type dependent aspects. Our results underline the importance of mechanical details in case of intercellular interactions. Nevertheless, it suggests that in adequate circumstances elastic and adhesive characterizations might be used as biomarkers.

Multiphoton Microscopy for Caries Detection with ICDAS Classification.

Dentin carious lesion is a dynamic process that involves demineralization and collagen denaturation. Collagen type I is the major protein in dentin and it has been investigated based on its optical properties. Multiphoton microscopy (MPM) is a nonlinear imaging technique that reveals the caries process using the collagen two-photon excitation fluorescence (2PEF) and its second-harmonic generation (SHG). Combining the histological and the International Caries Detection and Assessment System (ICDAS) classifications with nonlinear optical spectroscopy (NLOS), 2PEF and SHG intensities of enamel and dentin were highly altered during the caries process. It has been proven that the ratio SHG/2PEF is a relevant indicator of the organic matrix denaturation [Terrer et al.: J Dent Res 2016; 96: 574-579]. In the present study, a series of measurable signals is made to detect early stages of carious lesion according to the ICDAS classification and to explore the relationship between these measures and the ICDAS scale. Comparison of the efficiency of nonlinear optical signals for caries detection with the ICDAS classification is essential to evaluate their potential for clinical application. In our study, the use of the NLOS measured by MPM allowed us to monitor a quantitative parameter (SHG/2PEF ratio) according to the dentin carious lesion state (ICDAS and histological examination). Three coherent new groups were defined (ICDAS 0/1; ICDAS 2/3; ICDAS 4/5/6), where the carious process can be clearly described with a statistically significant decrease of the SHG/2PEF ratio.

Atomic Force Microscopy Study of the Topography and Nanomechanics of Casein Micelles Captured by an Antibody.

Casein micelles (CMs) are colloidal phospho-protein-mineral complexes naturally present in milk. This study used atomic force microscopy (AFM) in a liquid environment to evaluate the topography and nanomechanics of single native CMs immobilized by a novel capture method. The proposed immobilization method involves weak interactions with the antiphospho-Ser/Thr/Tyr monoclonal antibody covalently bound to a carboxylic acid self-assembled monolayer (SAM) on a gold surface. This capture strategy was compared to the commonly used covalent immobilization method of CMs via carbodiimide chemistry. With this conventional method, CMs remained mainly mobile during AFM measurements in liquid, disturbing the evaluation of their average size and elastic properties. Conversely, when captured by the specific antibody, they were successfully immobilized and their integrity was preserved during the AFM measurement. The characterization of both CM topography and elastic properties was carried out in a liquid ionic environment at native pH 6.6. The CMs' capture efficiency via antibody was concurrently proved by surface plasmon resonance. The calculation of casein micelles' width, height, and contact angle was carried out from the recorded 2D AFM images. CMs were characterized by a mean width of 148 ± 8 nm and a mean height of 42 ± 1 nm. Weak forces were applied to single captured CMs. The obtained force versus indentation curves were fitted using the Hertz model in order to evaluate their elastic properties. The elasticity distribution of native CMs exhibited a unimodal trend with a peak centered at 269 ± 14 kPa.

Interactions at the Peptide/Silicon Surfaces: Evidence of Peptide Multilayer Assembly.

Selective deposition of peptides from liquid solutions to n- and p-doped silicon has been demonstrated. The selectivity is governed by peptide/silicon adhesion differences. A noninvasive, fast characterization of the obtained peptide layers is required to promote their application for interfacing silicon-based devices with biological material. In this study we show that spectroscopic ellipsometry-a method increasingly used for the investigation of biointerfaces-can provide essential information about the amount of adsorbed peptide material and the degree of coverage on silicon surfaces. We observed the formation of peptide multilayers for a strongly binding adhesion peptide on p-doped silicon. Application of the patterned layer ellipsometric evaluation method combined with Sellmeier dispersion led to physically consistent results, which describe well the optical properties of peptide layers in the visible spectral range. This evaluation allowed the estimation of surface coverage, which is an important indicator of adsorption quality. The ellipsometric findings were well supported by atomic force microscopy results.

Engineered Adhesion Peptides for Improved Silicon Adsorption.

Engineering peptides that present selective recognition and high affinity for a material is a major challenge for assembly-driven elaboration of complex systems with wide applications in the field of biomaterials, hard-tissue regeneration, and functional materials for therapeutics. Peptide-material interactions are of vital importance in natural processes but less exploited for the design of novel systems for practical applications because of our poor understanding of mechanisms underlying these interactions. Here, we present an approach based on the synthesis of several truncated peptides issued from a silicon-specific peptide recovered via phage display technology. We use the photonic response provided by porous silicon microcavities to evaluate the binding efficiency of 14 different peptide derivatives. We identify and engineer a short peptide sequence (SLVSHMQT), revealing the highest affinity for p(+)-Si. The molecular recognition behavior of the obtained peptide fragment can be revealed through mutations allowing identification of the preferential affinity of certain amino acids toward silicon. These results constitute an advance in both the engineering of peptides that reveal recognition properties for silicon and the understanding of biomolecule-material interactions.

Characterization of C69R variant HBsAg: effect on binding to anti-HBs and the structure of virus-like particles.

Several variants of the major "a" determinant of the HBsAg, the main target of HBV neutralization by antibodies, have been described. However, mutations outside this region have not been as thoroughly investigated. During the genotyping of HBV from Tunisian patients with chronic hepatitis B, we identified a variant with a C69R substitution in the cytosolic loop of the S protein, resulting in a change in the hydrophobicity profile compared to the wild-type HBsAg. Wild-type and mutant HBsAgs were produced in Saccharomyces cerevisiae and recombinant proteins were tested for their ability to correctly self-assemble into virus-like particles (VLPs), and their ability to bind to HBs antibodies. The C69R substitution resulted in a decrease in binding to commercial anti-HBs antibodies, and although the variant appeared to assemble properly into VLPs, the average size of the particles was larger than that of the wild-type HBsAg. Prediction of the tertiary structure of the C69R mutant revealed a change in the first (aa 60-70) and the second loop (aa 110 to 120) compared to the wild-type protein. Furthermore, we showed by an isothermal titration calorimetry assay that the interaction between the wild-type HBsAg and the anti-HBs antibody was exothermic, whereas that with the mutant C69R was endothermic, indicating an effect on the binding affinity.

Design rules for metal binding biomolecules: understanding of amino acid adsorption on platinum crystallographic facets from density functional calculations.

Understanding the mechanism of biomolecules' interaction with inorganic surfaces might pave the way for the design of material interfaces with controlled and highly predictable properties. Here we have focused on the adsorption mechanism of facet-specific amino acids in the sequence of peptides selected for programmed synthesis of Pt(111) and Pt(100) nanocrystals. Using the first principles calculations we have demonstrated that the specific surface recognition of amino acid side chains occurs due to the combination of multiple processes: electron exchange, partial charge transfer and/or dispersive effects providing a high binding affinity to both polar and non-polar residues against both Pt facets. Our approach points towards promising novel routes for controlled design of material-specific linkers for future materials engineering.

Molecular mechanism of selective binding of peptides to silicon surface.

Despite extensive recent research efforts on material-specific peptides, the fundamental problem to be explored yet is the molecular interactions between peptides and inorganic surfaces. Here we used computer simulations (density functional theory and classical molecular dynamics) to investigate the adsorption mechanism of silicon-binding peptides and the role of individual amino acids in the affinity of peptides for an n-type silicon (n(+)-Si) semiconductor. Three silicon binding 12-mer peptides previously elaborated using phage display technology have been studied. The peptides' conformations close to the surface have been determined and the best-binding amino acids have been identified. Adsorption energy calculations explain the experimentally observed different degrees of affinity of the peptides for n(+)-Si. Our residual scanning analysis demonstrates that the binding affinity relies on both the identity of the amino acid and its location in the peptide sequence.

Insights on the facet specific adsorption of amino acids and peptides toward platinum.

Engineering shape-controlled bionanomaterials requires comprehensive understanding of interactions between biomolecules and inorganic surfaces. We explore the origin of facet-selective binding of peptides adsorbed onto Pt(100) and Pt(111) crystallographic planes. Using molecular dynamics simulations, we show that upon adsorption the peptides adopt a predictable conformation. We compute the binding energies of the amino acids constituting two adhesion peptides for Pt, S7, and T7 and demonstrate that peptides' surface recognition behavior that makes them unique among populations originates from differential adsorption of their building blocks. We find that the degree of peptide binding is mainly due to polar amino acids and the molecular architecture of the peptides close to the Pt facets. Our analysis is a first step in the prediction of enhanced affinity between inorganic materials and a peptides, toward the synthesis of novel nanomaterials with programmable shape, structure, and properties.

Cardiomyocyte sarcomere length variability: Membrane fluorescence versus second harmonic generation myosin imaging.

Sarcomere length (SL) and its variation along the myofibril strongly regulate integrated coordinated myocyte contraction. It is therefore important to obtain individual SL properties. Optical imaging by confocal fluorescence (for example, using ANEPPS) or transmitted light microscopy is often used for this purpose. However, this allows for the visualization of structures related to Z-disks only. In contrast, second-harmonic generation (SHG) microscopy visualizes A-band sarcomeric structures directly. Here, we compared averaged SL and its variability in isolated relaxed rat cardiomyocytes by imaging with ANEPPS and SHG. We found that SL variability, evaluated by several absolute and relative measures, is two times smaller using SHG vs. ANEPPS, while both optical methods give the same average (median) SL. We conclude that optical methods with similar optical spatial resolution provide valid estimations of average SL, but the use of SHG microscopy for visualization of sarcomeric A-bands may be the "gold standard" for evaluation of SL variability due to the absence of optical interference between the sarcomere center and non-sarcomeric structures. This contrasts with sarcomere edges where t-tubules may not consistently colocalize to Z-disks. The use of SHG microscopy instead of fluorescent imaging can be a prospective tool to map sarcomere variability both in vitro and in vivo conditions and to reveal its role in the functional behavior of living myocardium.

Biomechanical characterization of a fibrinogen-blood hydrogel for human dental pulp regeneration.

In dental practice, Regenerative Endodontic Treatment (RET) is applied as an alternative to classical endodontic treatments of immature necrotic teeth. This procedure, also known as dental pulp revitalization, relies on the formation of a blood clot inside the root canal leading to the formation of a reparative vascularized tissue similar to dental pulp, which would provide vitality to the affected tooth. Despite the benefit of this technique, it lacks reproducibility due to the fast degradation and poor mechanical properties of blood clots. This work presents a method for constructing a fibrinogen-blood hydrogel that mimics the viscoelastic properties of human dental pulp while preserving the biological properties of blood for application in RET. By varying the blood and fibrinogen concentrations, gels with different biomechanical and biological properties were obtained. Rheology and atomic force microscopy (AFM) were combined to study the viscoelastic properties. AFM was used to evaluate the elasticity of human dental pulp. The degradation and swelling rates were assessed by measuring weight changes. The biomimetic properties of the gels were demonstrated by studying the cell survival and proliferation of dental pulp cells (DPCs) for 14 days. The formation of an extracellular matrix (ECM) was assessed by multiphoton microscopy (MPM). The angiogenic potential was evaluated by an ex vivo aortic ring assay, in which the endothelial cells were observed by histological staining after migration. The results show that the Fbg-blood gel prepared with 9 mg ml-1 fibrinogen and 50% blood of the Fbg solution volume has similar elasticity to human dental pulp and adequate degradation and swelling rates. It also allows cell survival and ECM secretion and enhances endothelial cell migration and formation of neovessel-like structures.