Articles

Photochemical and Molecular Dynamics Studies of Halide Binding in Flavoenzyme Glucose Oxidase
- Zhuang Bo
- Vos Marten
- Aleksandrov Alexey
ChemBioChem, Wiley-VCH Verlag , 2022, 23 (19), pp.e202200227 . Glucose oxidase (GOX), a characteristic flavoprotein oxidase with widespread industrial applications, binds fluoride (F−) and chloride (Cl−). We investigated binding properties of halide inhibitors of GOX through time-resolved spectral characterization of flavin-related photochemical processes and molecular dynamic simulations. Cl− and F− bind differently to the protein active site and have substantial but opposite effects on the population and decay of the flavin excited state. Cl− binds closer to the flavin, whose excited-state decays in <100 fs due to anion-π interactions. Such interactions appear absent in F− binding, which, however, significantly increases the active-site rigidity leading to more homogeneous, picosecond fluorescence decay kinetics. These findings are discussed in relation to the mechanism of halide inhibition of GOX by occupying the accommodation site of catalytic intermediates and increasing the active-site rigidity (10.1002/cbic.202200227)
DOI : 10.1002/cbic.202200227
Ultrafast photooxidation of protein-bound anionic flavin radicals
- Zhuang Bo
- Ramodiharilafy Rivo
- Liebl Ursula
- Aleksandrov Alexey
- Vos Marten H.
Proceedings of the National Academy of Sciences of the United States of America, National Academy of Sciences , 2022, 119 (8), pp.e2118924119 . The photophysical properties of anionic semireduced flavin radicals are largely unknown despite their importance in numerous biochemical reactions. Here, we studied the photoproducts of these intrinsically unstable species in five different flavoprotein oxidases where they can be stabilized, including the well-characterized glucose oxidase. Using ultrafast absorption and fluorescence spectroscopy, we unexpectedly found that photoexcitation systematically results in the oxidation of protein-bound anionic flavin radicals on a time scale of less than ∼100 fs. The thus generated photoproducts decay back in the remarkably narrow 10- to 20-ps time range. Based on molecular dynamics and quantum mechanics computations, positively charged active-site histidine and arginine residues are proposed to be the electron acceptor candidates. Altogether, we established that, in addition to the commonly known and extensively studied photoreduction of oxidized flavins in flavoproteins, the reverse process (i.e., the photooxidation of anionic flavin radicals) can also occur. We propose that this process may constitute an excited-state deactivation pathway for protein-bound anionic flavin radicals in general. This hitherto undocumented photochemical reaction in flavoproteins further extends the family of flavin photocycles. (10.1073/pnas.2118924119)
DOI : 10.1073/pnas.2118924119
Guidance by followers ensures long-range coordination of cell migration through α-Catenin mechanoperception
- Boutillon Arthur
- Escot Sophie
- Elouin Amélie
- Jahn Diego
- González-Tirado Sebastián
- Starruss Jörn
- Brusch Lutz
- David Nicolas B
Developmental Cell, Elsevier , 2022 . Morphogenesis, wound healing and some cancer metastases depend upon migration of cell collectives that need to be guided to their destination as well as coordinated with other cell movements. During zebrafish gastrulation, extension of the embryonic axis is led by the mesendodermal polster that migrates towards the animal pole, followed by axial mesoderm that is undergoing convergence and extension. We here investigate how polster cells are guided towards the animal pole. Using a combination of precise laser ablations, advanced transplantations and functional as well as silico approaches, we establish that the directional information guiding polster cells is mechanical, and is provided by the anteriorward migration of the following cells. This information is detected by cell-cell contact through E-Cadherin/α-Catenin mechanotransduction and propagates from cell to cell over the whole tissue. Such guidance of migrating cells by followers ensures long-range coordination of movements and developmental robustness. (10.1016/j.devcel.2022.05.001)
DOI : 10.1016/j.devcel.2022.05.001
Enhanced intrapulse difference frequency generation in the mid-infrared by a spectrally dependent polarization state
- Bournet Q.
- Guichard F.
- Natile M.
- Zaouter Y.
- Joffre M.
- Bonvalet A.
- Pupeza I.
- Hofer C.
- Druon F.
- Hanna Marc
- Georges Patrick
Optics Letters, Optical Society of America - OSA Publishing , 2022, 47 (2), pp.261 . We present a technique to optimize the intrapulse difference frequency generation efficiency for mid-IR generation. The approach employs a multi-order waveplate that is designed to selectively rotate the polarization state of the incoming spectral components on the relevant orthogonal axes for subsequent nonlinear interaction. We demonstrate a significant increase of the mid-IR average power generated by a factor ≥ 2.5 compared to the conventional scheme, due to an optimally distributed number of photons enrolled in the difference frequency generation process. (10.1364/OL.444908)
DOI : 10.1364/OL.444908
Flavoprotein Photochemistry: Fundamental Processes and Photocatalytic Perspectives
- Zhuang Bo
- Liebl Ursula
- Vos Marten H.
Journal of Physical Chemistry B, American Chemical Society , 2022, 126, pp.3199-3207 . Flavins are highly versatile redox-active and colored cofactors in a large variety of proteins. These do include photoenzymes and photoreceptors, although the vast majority performs non-light-driven physiological functions. Nevertheless, electron transfer between flavins and specific nearby amino acid residues (in particular tyrosine, tryptophan, and presumably histidine and arginine) takes place upon excitation of flavin in many flavoproteins. For oxidized flavoproteins these reactions potentially have a photoprotective role. In this Perspective, we outline work on the characterization of early reaction intermediates not only in the relatively well-studied resting oxidized forms but also in the fully reduced and the intrinsically unstable semireduced forms, where ultrafast photooxidation of flavin was recently demonstrated. Along different lines, flavoprotein-based novel photocatalysts for biotechnological applications are presently emerging, employing both substrate photooxidation and photoreduction strategies. Deep insight into the fundamental flavin photochemical reactions may help in guiding and optimizing their development and in the exploration of novel photocatalytic approaches. (10.1021/acs.jpcb.2c00969)
DOI : 10.1021/acs.jpcb.2c00969
G-quadruplexes in helminth parasites
- Cantara Alessio
- Luo Yu
- Dobrovolná Michaela
- Bohalova Natalia
- Fojta Miroslav
- Verga Daniela
- Guittat Lionel
- Cucchiarini Anne
- Savrimoutou Solène
- Häberli Cécile
- Guillon Jean
- Keiser Jennifer
- Brázda Václav
- Mergny Jean
Nucleic Acids Research, Oxford University Press , 2022, 50 (5), pp.2719–2735 . Parasitic helminths infecting humans are highly prevalent infecting ∼2 billion people worldwide, causing inflammatory responses, malnutrition and anemia that are the primary cause of morbidity. In addition, helminth infections of cattle have a significant economic impact on livestock production, milk yield and fertility. The etiological agents of helminth infections are mainly Nematodes (roundworms) and Platyhelminths (flatworms). G-quadruplexes (G4) are unusual nucleic acid structures formed by G-rich sequences that can be recognized by specific G4 ligands. Here we used the G4Hunter Web Tool to identify and compare potential G4 sequences (PQS) in the nuclear and mitochondrial genomes of various helminths to identify G4 ligand targets. PQS are nonrandomly distributed in these genomes and often located in the proximity of genes. Unexpectedly, a Nematode, Ascaris lumbricoides, was found to be highly enriched in stable PQS. This species can tolerate high-stability G4 structures, which are not counter selected at all, in stark contrast to most other species. We experimentally confirmed G4 formation for sequences found in four different parasitic helminths. Small molecules able to selectively recognize G4 were found to bind to Schistosoma mansoni G4 motifs. Two of these ligands demonstrated potent activity both against larval and adult stages of this parasite. (10.1093/nar/gkac129)
DOI : 10.1093/nar/gkac129
Early processes in heme-based CO-sensing proteins
- Vos Marten H
- Salman Mayla
- Liebl Ursula
Frontiers in Molecular Biosciences, Frontiers Media , 2022, 9, pp.1046412 . Carbon monoxide has been recognized relatively recently as signaling molecule, and only very few dedicated natural CO sensor proteins have been identified so far. These include in particular heme-based transcription factors: the bacterial sensor proteins CooA and RcoM. In these 6-coordinated systems, exchange between an internal protein residue and CO as a heme ligand in the sensor domain affects the properties of the DNA-binding domain. Using light to dissociate heme-ligand bonds can in principle initiate this switching process. We review the efforts to use this method to investigate early processes in ligand switching and signaling, with an emphasis on the CO-"trapping" properties of the heme cavity. These features are unusual for most heme proteins, but common for heme-based CO sensors. (10.3389/fmolb.2022.1046412)
DOI : 10.3389/fmolb.2022.1046412
Inline amplification of mid-infrared intrapulse difference frequency generation
- Bournet Q.
- Jonusas M.
- Zheng A.
- Guichard F.
- Natile M.
- Zaouter Y.
- Joffre M.
- Bonvalet A.
- Druon F.
- Hanna Marc
- Georges P.
Optics Letters, Optical Society of America - OSA Publishing , 2022, 47 (19), pp.4885 . We demonstrate an ultrafast mid-infrared source architecture that implements both intrapulse difference frequency generation (iDFG) and further optical parametric amplification (OPA), in an all-inline configuration. The source is driven by a nonlinearly compressed high-energy Yb-doped-fiber amplifier delivering 7.4 fs pulses at a central wavelength of 1030 nm, at a repetition rate of 250 kHz. It delivers 1 µJ, 73 fs pulses at a central wavelength of 8 µm, tunable over more than one octave. By enrolling all the pump photons in the iDFG process and recycling the long wavelength pump photons amplified in the iDFG in the subsequent OPA, we obtain an unprecedented overall optical efficiency of 2%. These performances, combining high energy and repetition rate in a very simple all-inline setup, make this technique ideally suited for a growing number of applications, such as high harmonic generation in solids or two-dimensional infrared spectroscopy experiments. (10.1364/OL.467792)
DOI : 10.1364/OL.467792
hnRNPA1/UP1 Unfolds KRAS G-Quadruplexes and Feeds a Regulatory Axis Controlling Gene Expression
- Ferino Annalisa
- Marquevielle Julien
- Choudhary Himanshi
- Cinque Giorgio
- Robert Coralie
- Bourdoncle Anne
- Picco Raffaella
- Mergny Jean-Louis
- Salgado Gilmar
- Xodo Luigi
ACS Omega, ACS Publications , 2021, 6 (49), pp.34092-34106 . (10.1021/acsomega.1c05538)
DOI : 10.1021/acsomega.1c05538
Exploring Sequence Space to Design Controllable G-Quadruplex Topology Switches
- Chen Jielin
- Cheng Mingpan
- Stadlbauer Petr
- Šponer Jiří
- Mergny Jean-Louis
- Ju Huangxian
- Zhou Jun
CCS Chemistry, Chinese Chemical Society , 2021, pp.3232 - 3246 . As nonclassical nucleic acid structures, G-quadruplexes (G4s) not only play important roles in gene regulation and stability maintenance, but are also widely used in nanotechnology. Structural diversity is one of the main factors explaining the popularity of G4s, but a comprehensive and integrated study of different factors determining G4 structural versatility is currently lacking. Herein, starting from a common G4 sequence, (G 3 T) 3 G 3 , as the parent chain, and then taking advantage of G4 versatility, we present a variety of strategies to control G4 structure, based on the regulation of loop length and flanking sequences, cation (type and concentration), and molecular crowding. These strategies allow us to convert the G4 topology from parallel to hybrid, to antiparallel, and then back to parallel. Such structural diversity reveals the coding regulation ability of G4 structures, with potential applications in nanotechnology. (10.31635/ccschem.021.202101357)
DOI : 10.31635/ccschem.021.202101357
The beginning and the end: flanking nucleotides induce a parallel G-quadruplex topology
- Chen Jielin
- Cheng Mingpan
- Salgado Gilmar F
- Stadlbauer Petr
- Zhang Xiaobo
- Amrane Samir
- Guédin Aurore
- He Fangni
- Šponer Jiří
- Ju Huangxian
- Mergny Jean-Louis
- Zhou Jun
Nucleic Acids Research, Oxford University Press , 2021, 49 (16), pp.9548-9559 . Abstract Genomic sequences susceptible to form G-quadruplexes (G4s) are always flanked by other nucleotides, but G4 formation in vitro is generally studied with short synthetic DNA or RNA oligonucleotides, for which bases adjacent to the G4 core are often omitted. Herein, we systematically studied the effects of flanking nucleotides on structural polymorphism of 371 different oligodeoxynucleotides that adopt intramolecular G4 structures. We found out that the addition of nucleotides favors the formation of a parallel fold, defined as the ‘flanking effect’ in this work. This ‘flanking effect’ was more pronounced when nucleotides were added at the 5′-end, and depended on loop arrangement. NMR experiments and molecular dynamics simulations revealed that flanking sequences at the 5′-end abolish a strong syn-specific hydrogen bond commonly found in non-parallel conformations, thus favoring a parallel topology. These analyses pave a new way for more accurate prediction of DNA G4 folding in a physiological context. (10.1093/nar/gkab681)
DOI : 10.1093/nar/gkab681
Phase-modulated rapid-scanning fluorescence-detected two-dimensional electronic spectroscopy
- Agathangelou Damianos
- Javed Ariba
- Sessa Francesco
- Solinas Xavier
- Joffre Manuel
- Ogilvie Jennifer
The Journal of Chemical Physics, American Institute of Physics , 2021, 155 (9), pp.094201 . (10.1063/5.0057649)
DOI : 10.1063/5.0057649
Novel G-quadruplex prone sequences emerge in the complete assembly of the human X chromosome
- Bohálová Natália
- Mergny Jean-Louis
- Brázda Václav
Biochimie, Elsevier , 2021, 191, pp.87-90 . (10.1016/j.biochi.2021.09.004)
DOI : 10.1016/j.biochi.2021.09.004
Multiscale conformational dynamics probed by time‐resolved circular dichroism from seconds to picoseconds
- Hache François
- Changenet Pascale
Chirality, Wiley , 2021, 33 (11), pp.747-757 . Time-resolved circular dichroism has been developed for a few decades to investigate rapid conformational changes in (bio)molecules. In our group, we have come up with several experimental set-ups allowing us to study pico-nanosecond local phenomena in molecular systems as well as much slower effects occurring in proteins and DNA in the folding processes. After an overview of the worldwide realizations in this domain, we present emblematic experiments that we have carried out, spanning time domain from picoseconds to seconds. (10.1002/chir.23359)
DOI : 10.1002/chir.23359
Luminescent lanthanide nanoparticle-based imaging enables ultra-sensitive, quantitative and multiplexed <i>in vitro</i> lateral flow immunoassays
- Mousseau F.
- Féraudet-Tarisse C.
- Simon S.
- Gacoin T.
- Alexandrou A.
- Bouzigues C I
Nanoscale, Royal Society of Chemistry , 2021, 13 (35), pp.14814 - 14824 . Lateral Flow Assays (LFAs) have been extensively used on-site to rapidly detect analytes, possibly in complex media. However, standard gold nanoparticle-based LFAs lack sensitivity and cannot provide quantitative measurements with high accuracy. To overcome these limitations, we image lanthanidedoped nanoparticles (YVO 4 :Eu 40%) as new luminescent LFA probes, using a homemade reader coupled to a smartphone and propose an original image analysis allowing strip quantification regardless of the shape of the test band signal. This method is demonstrated for the detection of staphylococcal enterotoxins SEA, SEG, SEH, and SEI. A systematic comparison to state-of-the-art gold nanoparticle-based LFA revealed an analytical sensitivity enhancement of at least one order of magnitude. We furthermore provided measurements of absolute toxin concentration over two orders of magnitude and demonstrated simultaneous quantitative detection of multiple toxins with unaltered sensitivity. In particular, we reached concentrations 100 times lower than the ones reported in the literature for on-site multiplexed LFA targeting enterotoxins. Altogether, these results highlight that our luminescent nanoparticle-based method provides a powerful and versatile on-site framework to detect multiple biomolecules with sensitivity approaching that obtained by ELISA. This paves the way to a change of paradigm in the field of analytical immunoassays by providing fast in situ quantitative high sensitivity detection of biomarkers or pathogens. (10.1039/d1nr03358a)
DOI : 10.1039/d1nr03358a
High precision dual-modulation differential terahertz ATR sensor for liquid measurements
- Zheng Xiujun
- Gevart Thomas
- Gallot Guilhem
Optics Letters, Optical Society of America - OSA Publishing , 2021, 46 (16), pp.4045-4048 . We describe a highly sensitive and stable quantum-cascade laser-based attenuated total reflection (ATR) terahertz sensor for the detection of very low concentration solutions, using a dual-modulation differential approach and ATR geometry. This sensor offers a very high dynamic range and a long-term stability of 40 dB, which extends the potential of terahertz radiation for the analysis of liquid and biological samples. The performance is illustrated by measurements on standard solutions of ions, sugars, and proteins, for concentrations down to 1 M.
Nanoaggregate-forming lipid-conjugated AS1411 aptamer as a promising tumor-targeted delivery system of anticancer agents in vitro
- Carvalho Josué
- Lopes-Nunes Jéssica
- Vialet Brune
- Rosado Tiago
- Gallardo Eugenia
- Vale João
- Eloy Catarina
- Ferreira Sofia
- Palmeira-De-Oliveira Rita
- Campello Maria Paula Cabral
- Paulo António
- Barthélémy Philippe
- Mergny Jean-Louis
- Salgado Gilmar
- Queiroz João
- Ellington Andrew
- Cruz Carla
Nanomedicine: Nanotechnology, Biology and Medicine, Elsevier , 2021, 36 (13), pp.102429 . Abstract The multidomain non-structural protein 3 (Nsp3) is the largest protein encoded by coronavirus (CoV) genomes and several regions of this protein are essential for viral replication. Of note, SARS-CoV Nsp3 contains a SARS-Unique Domain (SUD), which can bind Guanine-rich non-canonical nucleic acid structures called G-quadruplexes (G4) and is essential for SARS-CoV replication. We show herein that the SARS-CoV-2 Nsp3 protein also contains a SUD domain that interacts with G4s. Indeed, interactions between SUD proteins and both DNA and RNA G4s were evidenced by G4 pull-down, Surface Plasmon Resonance and Homogenous Time Resolved Fluorescence. These interactions can be disrupted by mutations that prevent oligonucleotides from folding into G4 structures and, interestingly, by molecules known as specific ligands of these G4s. Structural models for these interactions are proposed and reveal significant differences with the crystallographic and modeled 3D structures of the SARS-CoV SUD-NM/G4 interaction. Altogether, our results pave the way for further studies on the role of SUD/G4 interactions during SARS-CoV-2 replication and the use of inhibitors of these interactions as potential antiviral compounds. (10.1016/j.nano.2021.102429)
DOI : 10.1016/j.nano.2021.102429
Probing living cells permeabilization dynamics by terahertz attenuated total reflection
- Gallot Guilhem
Proceedings of SPIE, the International Society for Optical Engineering, SPIE, The International Society for Optical Engineering , 2021 . Using attenuated total reflection (ATR) in the terahertz domain, we demonstrate non-invasive, non-staining real time measurements of cytoplasm leakage during permeabilization of live epithelial cells by saponin detergent and after electropermeabilization. The origin of the contrast observed between cells and culture medium is addressed by both experimental and theoretical approaches, and gives access to permeabilization dynamics of live cells in real time. We show that terahertz modalities are more sensitive than fluorescence microscopy which is the reference optical technique for electropermeabilization. We propose analytical models for the influx and efflux of non-permeant molecules through permeabilized cell membranes. (10.1117/12.2596166)
DOI : 10.1117/12.2596166
The role of G-Quadruplex DNA in Paraspeckle formation in cancer
- Bhatt Uditi
- Kretzmann Amy
- Guédin Aurore
- Ou Arnold
- Kobelke Simon
- Bond Charles
- Evans Cameron
- Hurley Laurence
- Mergny Jean-Louis
- Iyer K. Swaminathan
- Fox Archa
- Smith Nicole
Biochimie, Elsevier , 2021 . (10.1016/j.biochi.2021.07.008)
DOI : 10.1016/j.biochi.2021.07.008
Noninvasive quantitative assessment of collagen degradation in parchments by polarization-resolved SHG microscopy
- Schmeltz Margaux
- Robinet Laurianne
- Heu-Thao Sylvie
- Sintès Jean-Marc
- Teulon Claire
- Ducourthial Guillaume
- Mahou Pierre
- Schanne-Klein Marie-Claire
- Latour Gaël
Science Advances, American Association for the Advancement of Science (AAAS) , 2021, 7 (29), pp.eabg1090 . Nondestructive and noninvasive investigation techniques are highly sought-after to establish the degradation state of historical parchments, which is up to now assessed by thermal techniques that are invasive and destructive. We show that advanced nonlinear optical (NLO) microscopy enables quantitative in situ mapping of parchment degradation at the micrometer scale. We introduce two parameters that are sensitive to different degradation stages: the ratio of two-photon excited fluorescence to second harmonic generation (SHG) signals probes severe degradation, while the anisotropy parameter extracted from polarization-resolved SHG measurements is sensitive to early degradation. This approach is first validated by comparing NLO quantitative parameters to thermal measurements on artificially altered contemporary parchments. We then analyze invaluable parchments from the Middle Ages and show that we can map their conservation state and assess the impact of a restoration process. NLO quantitative microscopy should therefore help to identify parchments most at risk and optimize restoration methods. (10.1126/sciadv.abg1090)
DOI : 10.1126/sciadv.abg1090
Deep and Spatially Controlled Volume Ablations using a Two-Photon Microscope in the Zebrafish Gastrula
- Boutillon Arthur
- Escot Sophie
- David Nicolas B.
Journal of visualized experiments : JoVE, JoVE , 2021 (173) . Morphogenesis involves many cell movements to organize cells into tissues and organs. For proper development, all these movements need to be tightly coordinated, and accumulating evidence suggests this is achieved, at least in part, through mechanical interactions. Testing this in the embryo requires direct physical perturbations. Laser ablations are an increasingly used option that allows relieving mechanical constraints or physically isolating two cell populations from each other. However, many ablations are performed with an ultraviolet (UV) laser, which offers limited axial resolution and tissue penetration. A method is described here to ablate deep, significant, and spatially well-defined volumes using a two-photon microscope. Ablations are demonstrated in a transgenic zebrafish line expressing the green fluorescent protein in the axial mesendoderm and used to sever the axial mesendoderm without affecting the overlying ectoderm or the underlying yolk cell. Cell behavior is monitored by live imaging before and after the ablation. The ablation protocol can be used at different developmental stages, on any cell type or tissue, at scales ranging from a few microns to more than a hundred microns. (10.3791/62815)
DOI : 10.3791/62815
Folding Dynamics of DNA G-Quadruplexes Probed by Millisecond Temperature Jump Circular Dichroism
- Laouer K
- Schmid M
- Wien F
- Changenet Pascale
- Hache François
Journal of Physical Chemistry B, American Chemical Society , 2021 . (10.1021/acs.jpcb.1c01993)
DOI : 10.1021/acs.jpcb.1c01993
Modeling nonlinear microscopy near index-mismatched interfaces
- Morizet Josephine
- Sartorello Giovanni
- Dray Nicolas
- Stringari Chiara
- Beaurepaire Emmanuel
- Olivier Nicolas
Optica, Optical Society of America - OSA Publishing , 2021, 8 (7), pp.944 . Nonlinear microscopy is widely used to characterize thick, optically heterogeneous biological samples. While quantitative image analysis requires accurately describing the contrast mechanisms at play, the majority of established numerical models neglect the influence of field distortion caused by sample heterogeneity near focus. In this work, we show experimentally and numerically that finite-difference time-domain (FDTD) methods are applicable to model focused fields interactions in the presence of heterogeneities, typical of nonlinear microscopy. We analyze the ubiquitous geometry of a vertical interface between index-mismatched media (water, glass, and lipids) and consider the cases of two-photon-excited fluorescence (2PEF), third-harmonic generation (THG) and polarized THG contrasts. We show that FDTD simulations can accurately reproduce experimental images obtained on model samples and in live adult zebrafish, in contrast with previous models neglecting field distortions caused by index mismatch at the micrometer scale. Accounting for these effects appears to be particularly critical when interpreting coherent and polarization-resolved microscopy data. (10.1364/OPTICA.421257)
DOI : 10.1364/OPTICA.421257
Quantitative structural imaging of keratoconic corneas using polarization-resolved SHG microscopy
- Raoux Clothilde
- Schmeltz Margaux
- Bied Marion
- Alnawaiseh Maged
- Hansen Uwe
- Latour Gaël
- Schanne-Klein Marie-Claire
Biomedical optics express, Optical Society of America - OSA Publishing , 2021, 12 . The human cornea is mainly composed of collagen fibrils aligned together within stacked lamellae. This lamellar structure can be affected in pathologies such as keratoconus, which is characterized by progressive corneal thinning and local steepening. In this study, we use polarization-resolved second harmonic generation (P-SHG) microscopy to characterize 8 control and 6 keratoconic human corneas. Automated processing of P-SHG images of transverse sections provides the collagen orientation in every pixel with sub-micrometer resolution. Series of P-SHG images recorded in the most anterior region of the stroma evidence sutural lamellae inclined at 22°± 5°to the corneal surface, but show no significant difference between control and keratoconic corneas. In contrast, series of P-SHG images acquired along the full thickness of the stroma show a loss of order in the lamellar structure of keratoconic corneas, in agreement with their defective mechanical properties. This structural difference is analyzed quantitatively by computing the entropy and the orientation index of the collagen orientation distribution and significant differences are obtained along the full thickness of the stroma. This study shows that P-SHG is an effective tool for automatic quantitative analysis of structural defects of human corneas and should be applied to other collagen-rich tissues. (10.1364/boe.426145)
DOI : 10.1364/boe.426145
Additive CHARMM36 Force Field for Nonstandard Amino Acids
- Croitoru Anastasia
- Park Sang-Jun
- Kumar Anmol
- Lee Jumin
- Im Wonpil
- Mackerell Alexander
- Aleksandrov Alexey
Journal of Chemical Theory and Computation, American Chemical Society , 2021, 17 (6), pp.3554-3570 . (10.1021/acs.jctc.1c00254)
DOI : 10.1021/acs.jctc.1c00254