Laboratoire d'optique et biosciences
Laboratoire d'optique et biosciences
Laboratoire d'optique et biosciences

Articles

  • Two-dimensional infrared spectroscopy using a fast-scanning interferometer and chirped pulse up-conversion at 100 kHz
    • Jonušas Mindaugas
    • Bournet Quentin
    • Bonvalet Adeline
    • Natile Michele
    • Ersen Andrei-Ovidiu
    • Guichard Florent
    • Zaouter Yoann
    • Georges Patrick
    • Druon Frédéric
    • Hanna Marc
    • Joffre Manuel
    The Journal of Chemical Physics, American Institute of Physics , 2025, 162 (17), pp.174201 . We report on a 100-kHz two-dimensional infrared (2DIR) spectrometer in the pump-probe geometry, which we apply to the measurement of the 2DIR spectrum of carboxy-hemoglobin. The probe pulses are spectrally resolved by chirped-pulse upconversion (CPU) using a fast 2048-pixel linescan CMOS camera. The two-pulse pump sequence is generated using a conventional interferometer with a fast-scanning mechanical delay line allowing to achieve a scanning frequency of 2 Hz. The resulting modulation frequency of 3.1 kHz is large enough to shift the relevant signal away from the low-frequency noise of the laser source. The combined use of an interferometer on the pump side and of CPU on the probe side opens the way to an improved spectral resolution in both pump and probe dimensions, as compared to currently-available 100-kHz 2DIR spectrometers based on pulse shapers and Mercury-Cadmium Telluride (MCT) detector arrays. (10.1063/5.0261494)
    DOI : 10.1063/5.0261494
  • Genome wide analyses reveal the role of mutator phenotypes in Mycobacterium tuberculosis drug resistance emergence
    • Zein-Eddine R.
    • Le Meur Adrien
    • Skouloubris S.
    • Jelsbak L.
    • Refrégier Guislaine
    • Myllykallio Hannu Hannu
    npj Antimicrobials and Resistance, Springer Nature , 2025, 3 (1), pp.35 . Antimicrobial combination therapy is widely used to combat Mycobacterium tuberculosis (Mtb), yet resistance rates continue to rise. Mutator strains, with defects in DNA repair genes, drive resistance in other bacterial infections, but their role in Mtb remains unclear. Here, we study the contribution of single nucleotide polymorphisms (SNPs) in DNA Repair, Replication, and Recombination (3 R) genes to Mtb resistance. Through large-scale bioinformatics analysis of 53,589 whole-genomes, we identified 18 novel SNPs in lineages 2 and 4 linked to genotypic drug resistance in 3 R genes, covering 12.5% of clinical isolates with available genome sequences. Notably, a number of the detected SNPs were positively selected during Mtb evolution. Experimental tests showed that mutM, fpgg2, xthA, and nucS mutants had increased the mutation frequency compared to the wild type. Our findings highlight the role of 3 R gene mutations in resistance, emphasizing the need for surveillance to improve early detection and control strategies. (10.1038/s44259-025-00107-1)
    DOI : 10.1038/s44259-025-00107-1
  • In vivo autofluorescence lifetime imaging of the Drosophila brain captures metabolic shifts associated with memory formation
    • Roussel Philémon
    • Zhou Mingyi
    • Stringari Chiara
    • Preat Thomas
    • Plaçais Pierre-Yves
    • Genovesio Auguste
    eLife, eLife Sciences Publication , 2025 . Abstract Neuronal energy regulation is increasingly recognized as a critical factor underlying brain functions and their pathological alterations, yet the metabolic dynamics that accompany cognitive processes remain poorly understood. As a label-free and minimally invasive technique, fluorescence lifetime imaging (FLIM) of coenzymes NADH and NADPH (collectively referred to as NAD(P)H) offers the possibility to resolve cellular metabolic profiles with high spatial precision. However, NAD(P)H FLIM’s capacity to detect subtle changes in neuronal metabolism associated with cognition has not been demonstrated. In this study, we applied NAD(P)H FLIM to map the metabolic profiles of Drosophila neurons in vivo across multiple scales, focusing on the primary centers for associative memory: the mushroom bodies (MBs). At a broad scale, we obtained an overview of the metabolic signatures of the main brain tissue and identified a marked difference between neuropil and cortex areas. At a finer scale, our findings revealed notable heterogeneity in the basal metabolic profiles of distinct MB neuron subtypes. Measurements performed after associative olfactory learning also uncovered a subtype-specific metabolic shift associated with memory formation, demonstrating the utility of NAD(P)H FLIM in detecting physiology-driven changes linked to brain function. These results establish a promising framework for studying cerebral energy dynamics in vivo. (10.7554/eLife.106040.1)
    DOI : 10.7554/eLife.106040.1
  • Ultra-Specific G-Quadruplex–Colistin Interaction for Efficient Transcriptome-Wide G4 Mapping
    • Wei Shijiong
    • Zhang Xiaobo
    • Feng Yilong
    • Tao Shentong
    • Qiu Dehui
    • Yan Xinrong
    • Li Guangming
    • Guittat Lionel
    • Zhang Wenli
    • Monchaud David
    • Mergny Jean-Louis
    • Ju Huangxian
    • Zhou Jun
    Journal of the American Chemical Society, American Chemical Society , 2025, 147, pp.9962 - 9971 . G-quadruplexes (G4s) are challenging targets for chemical biology interventions, notably because of their dynamic topological polymorphism. We found that the antibiotic smallmolecule colistin (COL) interacts specifically with a single subtype of G4 structures, the so-called parallel G4s. This interaction triggers the aggregation of the G4/COL complexes in a structure-specific manner, which can thus be separated from the bulk solution by centrifugation. This unprecedented mode of affinity-precipitation was exploited here to design the COL-induced RNA G4 precipitation and sequencing (CoRP-seq) protocol, which allows for the assessment of the prevalence of RNA G4s in the transcriptome of human cells in a straightforward manner. CoRP-seq shines by its ultraspecificity, simplicity, and practical convenience, which thus advances G4 mapping further and addresses unmet needs in the field of G4omics. (10.1021/jacs.5c01172)
    DOI : 10.1021/jacs.5c01172
  • Increasing the Accuracy and Robustness of the CHARMM General Force Field with an Expanded Training Set
    • Croitoru Anastasia
    • Kumar Anmol
    • Lambry Jean-Christophe
    • Lee Jihyeon
    • Sharif Suliman
    • Yu Wenbo
    • Mackerell Alexander
    • Aleksandrov Alexey
    Journal of Chemical Theory and Computation, American Chemical Society , 2025, 21 (6), pp.3044-3065 . <div><p>Small molecule empirical force fields (FFs), including the CHARMM General Force Field (CGenFF), are designed to have wide coverage of organic molecules and to rapidly assign parameters to molecules not explicitly included in the FF. Assignment of parameters to new molecules in CGenFF is based on a trained bond-angledihedral charge increment linear interpolation scheme for the partial atomic charges along with bonded parameters assigned based on analogy using a rules-based penalty score scheme associated with atom types and chemical connectivity. Accordingly, the accuracy of CGenFF is related to the extent of the training set of available parameters. In the present study that training set is extended by 1,390 molecules selected to represent connectivities new to CGenFF training compounds. Quantum mechanical (QM) data for optimized geometries, bond, valence angle, and dihedral angle potential energy scans, interactions with water, molecular dipole moments, and electrostatic potentials were used as target data. The resultant bonded parameters and partial atomic charges were used to train a new version of the CGenFF program, v5.0, which was used to generate parameters for a validation set of molecules, including drug-like molecules approved by the FDA, which were then benchmarked against both experimental and QM data. CGenFF v5.0 shows overall improvements with respect to QM intramolecular geometries, vibrations, dihedral potential energy scans, dipole moments and interactions with water. Tests of pure solvent properties of 216 molecules show small improvements versus the previous release of CGenFF v2.5.1 reflecting the high quality of the Lennard-Jones parameters that were explicitly optimized during the initial optimization of both the CGenFF and the CHARMM36 force field. CGenFF v5.0 represents an improvement that is anticipated to more accurately model intramolecular geometries and strain energies as well as non-covalent interactions of drug-like and other organic molecules.</p></div> (10.1021/acs.jctc.5c00046)
    DOI : 10.1021/acs.jctc.5c00046
  • Nance-Horan-Syndrome-like 1b controls mesodermal cell migration by regulating protrusion and actin dynamics during zebrafish gastrulation.
    • Escot Sophie
    • Hassanein Yara
    • Elouin Amélie
    • Torres-Paz Jorge
    • Mellottee Lucille
    • Ignace Amandine
    • David Nicolas B
    Communications Biology, Nature Publishing Group , 2025, 8 (1), pp.328 . Cell migrations are crucial for embryonic development, wound healing, the immune response, as well as for cancer progression. In most cells, the RAC1/Arp2/3/WAVE signalling pathway induces branched actin polymerisation, which protrudes the membrane and allows migration. Fine-tuning the activity of the RAC1/Arp2/3/WAVE complex modulates protrusion lifetime and migration persistence. Recently, NHSL1, a novel interactor in this complex has been identified as a negative regulator of cell migration in vitro. We here analysed its function in vivo, during zebrafish gastrulation, as nhsl1b is specifically expressed in migrating mesodermal cells. Loss and gain of function experiments revealed that nhsl1b is required for the proper migration of the mesoderm, controlling cell speed and migration persistence. Consistent with a role in regulating actin dynamics, Nhsl1b localises to the tip of actin-rich protrusions. However, in contrast to the in vitro situation, it appears to be a positive regulator of migration, with its loss of function reducing the length and lifetime of protrusions, whereas overexpression has the opposite effect. These results reveal that the effects of actin modulators depend on the cellular context, and highlight the importance of analysing their function in physiological contexts. (10.1101/2023.01.28.526006)
    DOI : 10.1101/2023.01.28.526006
  • Fast and Efficient Red‐absorbing Photoswitching Proteins Based on Flavin–Ligand Charge Transfer Complexes
    • Zhuang Bo
    • Liebl Ursula
    • Vos Marten
    • Sliwa Michel
    ChemPhotoChem, Wiley , 2025 . Recently a novel class of reversible protein photoswitches has been discovered that is based on a charge transfer (CT) complex composed of the flavin cofactor and a substrate‐analogue inhibitor molecule in the family of sarcosine oxidase flavoproteins. Here, excitation of the CT band results in barrierless dissociation of the CT complex on the femtosecond timescale followed by its thermally activated reformation, on the timescale of a few nanoseconds at ambient temperature. The photoreaction is thought to involve a well‐defined isomerization of the inhibitor without its dissociation from the protein. This reaction occurs with an unusually high quantum yield (~80%), is initiated by absorption in the red part of the visible absorption spectrum, and leads to a photoproduct absorbing in the blue spectral region (negative photochromism). Therefore, this class of photoswitches can be considered a promising template for developing a new class of fast negative photochromic compounds for Life Science applications provided the lifetime of the photoproducts (‘light state’) can be prolonged. Potential future developments will be discussed. (10.1002/cptc.202500012)
    DOI : 10.1002/cptc.202500012
  • Understanding the key challenges in tuberculosis drug discovery: what does the future hold?
    • Zein-Eddine Rima
    • Ramuz Masoud
    • Refrégier Guislaine
    • Lutzeyer Johannes F
    • Aleksandrov Alexey
    • Myllykallio Hannu
    Expert Opinion on Drug Discovery, Informa Healthcare , 2025, 20 (9), pp.1115-1130 . Introduction: Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a major global health concern. It spreads through airborne droplets and has a high mortality rate, particularly without treatment. Drug resistance is rising, with treatments against multidrug-resistant TB (MDR-TB) showing poor treatment success rates. The thick, lipid-rich wall of Mtb and its slow growth reduce antibiotic effectiveness, requiring long treatment courses of 4-6 months. Current therapies often fail against drugresistant strains, highlighting the urgent need for new, short-course treatment, affordable, and combination-friendly drugs. Areas covered: Within this perspective, the authors review and comment on the following topics regarding Mtb resistance emergence and treatment strategies: i) Existing treatment ii) Resistance evolution in Mtb; iii) Key challenges in drug discovery targeting Mtb; iv) emerging strategies and recent advances in Mtb drug discovery, and v) Next-generation approaches. Literature was identified through a search of PubMed, google scholar, and web of science, from January 2010 to March 2025. Expert opinion: AI is accelerating the discovery of bioavailable and safe preclinical drug candidates for TB, though data limitations and biological complexity remain challenging. Future progress requires multi-modal models, open-access datasets, and interdisciplinary collaboration. (10.1080/17460441.2025.2531229)
    DOI : 10.1080/17460441.2025.2531229
  • G-quadruplexes are promoter elements controlling nucleosome exclusion and RNA polymerase II pausing
    • Esnault Cyril
    • Zine El Aabidine Amal
    • Robert Marie-Cécile
    • Cucchiarini Anne
    • Magat Talha
    • Pigeot Alexia
    • Bouchouika Soumya
    • Garcia-Oliver Encar
    • Gawron Kevin
    • Basyuk Eugénia
    • Karpinska Magdalena A
    • Kozulic-Pirher Alja
    • Luo Yu
    • Verga Daniela
    • Mourad Raphael
    • Radulescu Ovidiu
    • Mergny Jean-Louis
    • Bertrand Edouard
    • Andrau Jean-Christophe
    Nature Genetics, Nature Publishing Group , 2025, 57 (8), pp.1981-1993 . Despite their central role in transcription, it has been difficult to define universal sequences associated to eukaryotic promoters. Within chromatin context, recruitment of the transcriptional machinery requires promoter opening but how DNA elements could contribute to this process is unclear. Here, we show that G-quadruplex (G4) secondary DNA structures are highly enriched at mammalian promoters. G4s are located at the deepest point of nucleosome exclusion at promoters and correlate with maximum promoter activity. We found that experimental G4s exclude nucleosomes in vivo and in vitro while favouring strong positioning. At model promoters, impairing G4s affected both transcriptional activity and chromatin opening. G4 destabilization also resulted in an inactive promoter state and affected transition to effective RNA production. Finally, G4 stabilization resulted in global reduction of proximal promoter pausing. Altogether, our data introduce G4s as bona fide promoter elements allowing nucleosome exclusion and facilitating pause release by the RNA Polymerase II. (10.1038/s41588-025-02263-6)
    DOI : 10.1038/s41588-025-02263-6
  • The Mappa mundi of Albi: Insight into the manufacturing, life and conservation state of an 8th century world map
    • Robinet Laurianne
    • Heu-Thao Sylvie
    • Galante Giulia
    • Latour Gaël
    • Tournié Aurélie
    • Daher Céline
    • Dan Anca
    • Schanne-Klein Marie-Claire
    • Michelin Anne
    • Deschaux Jocelyne
    Journal of Cultural Heritage, Elsevier , 2025, 74, pp.341-352 . The Mappa mundi of Albi is one of the oldest examples of spatial representation of the Western world. The small map conserved on the verso of folio 57 in manuscript 29 of the Médiathèque Pierre Amalric in Albi (France) was drawn on parchment, probably made in the second half of the 8th century, somewhere between south-western France and northern Spain, maybe in Albi itself. Because of its exceptional importance for the history of space representation, the map, together with the Index of seas and winds facing it, on the recto of folio 58, was recorded in the UNESCO Memory of the World Register in 2015. The detailed study published here has examined the manuscript’s structure and characterised the different constitutive materials. Observations and physicochemical analyses were performed on the map and the index, from the micro to the macroscale, combining optical microscopy, XRF, FORS, FTIR, and micro-Raman spectroscopy, hyperspectral imaging, proteomic analysis, and non-linear optical microscopy. Three manuscripts conserved at the same library or suspected to have been produced in the scriptorium of Albi have also been examined for comparisons. This material investigation complements the historical studies of the map by shedding new light on the manufacturing, life, and conservation state of this exceptional document. (10.1016/j.culher.2025.05.013)
    DOI : 10.1016/j.culher.2025.05.013
  • A Chimeric Photo-Controllable CRISPR/Cas12a System for Universal and Fast Diagnostics
    • Yan Xinrong
    • Liu Bin
    • Zhou Shuguang
    • Fan Yanjun
    • Wei Shijiong
    • Qiu Dehui
    • Xiang Henglong
    • Zhou Jiahang
    • Mergny Jean-Louis
    • Monchaud David
    • Ju Huangxian
    • Zhou Jun
    Analytical Chemistry, American Chemical Society , 2025, 97 (44), pp.24634 - 24642 . The potential of clustered regularly interspaced short palindromic repeats (CRISPR) and corresponding CRISPR-associated (Cas) protein systems (CRISPR/Cas) systems for biomedical applications is tremendous; however, precise control of their activity is essential to better harness this potential and, beyond this, to develop reliable diagnostic reagents. Herein, we report on such a strategy by controlling the CRISPR/Cas12a activity using a photo-controllable CRISPR RNA (crRNA). To this end, the 3′ end of crRNA was conjugated to a G-quadruplex (G4) block through a photocleavable linker: upon photo irradiation, the G4 trigger is removed, thus allowing for the DNA target to access and hybridize with the crRNA, and thus be processed by the CRISPR/Cas12a system. The efficiency of this approach was demonstrated by the detection of human papillomavirus 16 DNA in 50 clinical samples: our one-pot strategy was found to be as efficient as the routinely implemented method (qPCR), with 95.7% sensitivity and 100% specificity, in addition to be faster (25 versus 60 min) and both simpler and less expensive (being implementable as lateral flow test strips). Collectively, this new and fully controllable CRISPR/Cas system holds great potential for next-generation clinical diagnostics. (10.1021/acs.analchem.5c04782)
    DOI : 10.1021/acs.analchem.5c04782
  • Intrinsic variation of the polarization-resolved SHG from collagen: Multiscale analysis and application to parchments
    • Galante Giulia
    • Robinet Laurianne
    • Heu-Thao Sylvie
    • Caporal Clément
    • Latour Gaël
    • Schanne-Klein Marie-Claire
    APL Photonics, AIP Publishing LLC , 2025, 10 (5), pp.056106 . Second harmonic generation (SHG) microscopy is nowadays the gold standard technique for collagen structural imaging in intact tissues with sub-micrometer resolution. This multiphoton modality can be combined with polarimetry to provide key information about the 3D hierarchical organization of collagen. Notably, the so-called anisotropy parameter processed from polarization-resolved SHG (P-SHG) has been shown to vary with the orientational disorder of the fibrils within the focal volume and with their out-of-plane orientation. However, analytical equations describing both effects within the same formalism are still lacking. In this work, we present a unified multiscale theoretical approach of the intrinsic variations of the anisotropy parameter. We then measure these variations in the very same collagen samples to ensure reliable comparisons. To that end, we use parchments, which are materials made from animal skins and which contain almost exclusively collagen. These parchments are manufactured in different ways to obtain different collagen distributions. Our series of measurements exhibit a good agreement with our theoretical approach, which shows the relevance of P-SHG measurements to probe collagen multiscale organization in tissues. (10.1063/5.0250484)
    DOI : 10.1063/5.0250484
  • Synthesis of new 1,3-bis[(4-(substituted-aminomethyl)phenyl)methyl]benzene and 1,3-bis[(4-(substituted-aminomethyl)phenoxy)methyl]benzene derivatives, designed as novel potential G-quadruplex antimalarial ligands
    • Albenque-Rubio Sandra
    • Guillon Jean
    • Agnamey Patrice
    • Damiani Céline
    • Savrimoutou Solène
    • Ronga Luisa
    • Hanot Marie
    • Zangmo Tshering
    • Pinaud Noël
    • Moreau Stéphane
    • Mergny Jean-Louis
    • Marchivie Mathieu
    • Moukha Serge
    • Estela Fabienne
    • Dozolme Pascale
    • Cohen Anita
    • Sonnet Pascal
    Drugs and Drug Candidates, MDPI , 2025, 4 (3), pp.39 . Background: Based on our previously reported series of novel 1,3,5-tris[(4-(substituted-aminomethyl)phenyl)methyl]benzene and 1,3,5-tris[(4-(substituted-aminomethyl)phenoxy)methyl]benzene derivatives, we have now designed, synthesized, and tested a new series of novel restricted and simplified structural analogues of these compounds against Plasmodium falciparum in vitro; i.e., the 1,3-bis[(4-(substituted-aminomethyl)phenyl)methyl]benzene and 1,3-bis[(4-(substituted-aminomethyl)phenoxy)methyl]benzene compounds. Methods &amp; results: The pharmacological results revealed significant antimalarial activity, with IC50 values in the submicromolar to micromolar range. Additionally, the in vitro cytotoxicity of these new nitrogen-containing polyphenyl- or -phenoxymethylbenzene compounds was evaluated on human HepG2 cells. The compound 1f, the 1,3-bis[(4-(3-(morpholin-1-yl)propyl)aminomethyl)phenoxy)methyl]benzene derivative, emerged as one of the most potent and promising antimalarial candidates, demonstrating a cytotoxicity/antiprotozoal activity ratio of 594 against the chloroquine-sensitive Plasmodium falciparum 3D7 strain. Additionally, the 1,3-bis[((substituted aminomethyl)phenyl)methyl]benzene compound 1j and the 1,3-bis[((substituted aminomethyl)phenoxy)methyl]benzenes 2p and 2q also showed strong antimalarial potential, with selectivity indexes (SI) of over 303, 280, and 217, respectively, against the 3D7 strain, which has mefloquine-reduced sensitivity. Furthermore, the 1,3-bis[(4-(pyridin-2-ylethylaminomethyl)phenyl)methyl]benzene 2k was identified as the most noteworthy antimalarial compound, exhibiting a selectivity index (SI) that was superior to 178 against the chloroquine-resistant Plasmodium falciparum W2 strain. It has previously been suggested that the telomeres of P. falciparum may serve as potential targets for these polyaromatic compounds; thus, we assessed the ability of our novel derivatives to stabilize parasitic telomeric G-quadruplexes using a FRET melting assay. Conclusions: However, regarding the stabilization of the protozoan G-quadruplex, it was noted that the few substituted derivatives, which showed interesting stabilization profiles, were not necessarily the most effective antimalarial compounds against both Plasmodium strains. Moreover, these new compounds did not show promising stabilizing effects on the different G4 sequences. Therefore, no correlation arises between their antimalarial activity and the selectivity of their binding to G-quadruplexes. (10.3390/ddc4030039)
    DOI : 10.3390/ddc4030039
  • Spatial distribution and stability of Gd<sub>0.6</sub>Eu<sub>0.4</sub>VO<sub>4</sub> nanoparticles injected in mouse ear pinnae
    • Proiou Eleni
    • Pinakidou Fani
    • Paloura Eleni C
    • Pétri Nicolas
    • Gacoin Thierry
    • Laplace-Builhé Corinne
    • Schuck Götz
    • Alexandrou Antigoni
    • Katsikini Maria
    Trends in Analytical Chemistry, Elsevier , 2025, 182, pp.118049 . X-Ray Fluorescence (XRF) mapping is employed for the study of the spatial distribution of GdVO<sub>4</sub>:Eu nanoparticles (NPs) after their injection into mouse ear pinnae. The injected NP colloids were detectable in a concentration range from 5 to 30 mM in vanadate ions (90-530 nM in NP concentrations). The distribution maps were recorded separately for Gd, Eu, and V and reveal that the three elements are collocalized, indicating the NP stability after the injection. The distribution pattern of the NPs is not homogeneous; they follow bifurcated paths of easy flow demonstrating the complexity of the tissue-colloid interactions. The V-K, Gd-L 3 and Eu-L 3 -edge Xray Absorption Fine Structure (XAFS) spectra of the NPs recorded prior to and after their injection confirm that the integrity of the nanoparticles is preserved after injection. This combined XRF/XAFS analysis paves the way for studies on the long-term fate of injected Gd-containing NPs in tissues. (10.1016/j.trac.2024.118049)
    DOI : 10.1016/j.trac.2024.118049
  • Mechanism of Ultrafast Flavin Photoreduction in the Active Site of Flavoenzyme LSD1 Histone Demethylase
    • Zhuang Bo
    • Ramodiharilafy Rivo
    • Aleksandrov Alexey
    • Liebl Ursula
    • Vos Marten H
    Chemical Science, The Royal Society of Chemistry , 2025, 16, pp.338 - 344 . Photoreduction of oxidized flavins has a functional role in photocatalytic and photoreceptor flavoproteins. In flavoproteins without light-dependent physiological functions, ultrafast, reversible flavin photoreduction is supposedly photoprotective by nature, and holds potential for nonnatural photocatalytic applications. In this work, we combine protein mutagenesis, ultrafast spectroscopy, molecular dynamics simulations and quantum mechanics calculations to investigate the nonfunctional flavin photoreduction in a flavoenzyme, lysine-specific demethylase 1 (LSD1) which is pivotal in DNA transcription. LSD1 harbors an oxidized flavin adenine dinucleotide (FAD) cofactor and multiple electron-donating residues in the active site. Upon photoexcitation, the FAD cofactor is photoreduced in &lt; 200 fs by electron transfer (ET) from nearby residue(s), and the charge pairs recombine in ca. 2 ps. Site-directed mutagenesis pinpoints a specific tryptophan residue, W751, as the primary electron donor, whereas a tyrosine residue, Y761, despite being located closer to the flavin ring, does not effectively contribute to the process. Based on a hybrid quantum–classical computational approach, we characterize the W751–FAD and Y761–FAD charge-transfer states (CTW751 and CTY761, respectively), as well as the FAD locally excited state (LEFAD), and demonstrate that the coupling between LEFAD and CTW751 is larger than those involving CTY761 by an order of magnitude, rationalizing the experimental observations. More generally, this work highlights the role of the intrinsic protein environment and details of donor–acceptor molecular configurations on the dynamics of short-range ET involving a flavin cofactor and amino acid residue(s). (10.1039/D4SC06857B)
    DOI : 10.1039/D4SC06857B
  • Ultrafast dynamics of the UV-induced electronic relaxation in DNA guanine-thymine dinucleotides: from the Franck-Condon states to the minima of the potential energy surfaces
    • Petropoulos Vasilis
    • Martinez-Fernandez Lara
    • Uboldi Lorenzo
    • Maiuri Margherita
    • Cerullo Giulio
    • Balanikas Evangelos
    • Markovitsi Dimitra
    Physical Chemistry Chemical Physics, Royal Society of Chemistry , 2025 . We study the DNA dinucleotide 5’-dGpdT-3’ (abbreviated as GT) as a model system for the relaxation of the electronic excited states in stacked nucleobases. Quantum chemistry calculations determine the Franck-Condon... (10.1039/D5CP00788G)
    DOI : 10.1039/D5CP00788G
  • Factors Affecting the Population of Excited Charge Transfer States in Adenine/Guanine Dinucleotides: A Joint Computational and Transient Absorption Study
    • Petropoulos Vasilis
    • Martinez-Fernandez Lara
    • Uboldi Lorenzo
    • Maiuri Margherita
    • Cerullo Giulio
    • Balanikas Evangelos
    • Markovitsi Dimitra
    Biomolecules, MDPI , 2024, 14 (12), pp.1548 . There is compelling evidence that the absorption of low-energy UV radiation directly by DNA in solution generates guanine radicals with quantum yields that are strongly dependent on the secondary structure. Key players in this unexpected phenomenon are the photo-induced charge transfer (CT) states, in which an electric charge has been transferred from one nucleobase to another. The present work examines the factors affecting the population of these states during electronic relaxation. It focuses on two dinucleotides with opposite orientation: 5′-dApdG-3′ (AG) and 5′-dGpdA-3′ (GA). Quantum chemistry calculations determine their ground state geometry and the associated Franck–Condon states, map their relaxation pathways leading to excited state minima, and compute their absorption spectra. It has been shown that the most stable conformer is anti-syn for AG and anti-anti for GA. The ground state geometry governs both the excited states populated upon UV photon absorption and the type of excited state minima reached during their relaxation. Their fingerprints are detected in the transient absorption spectra recorded with excitation at 266 nm and a time resolution of 30 fs. Our measurements reveal that in the large majority of dinucleotides, chromophore coupling is already operative in the ground state and that the charge transfer process occurs within ~120 fs. The competition among various relaxation pathways affects the quantum yields of the CT state formation in each dinucleotide, which are estimated to be 0.18 and 0.32 for AG and GA, respectively. (10.3390/biom14121548)
    DOI : 10.3390/biom14121548
  • Archaerhodopsin 3 is an ideal template for the engineering of highly fluorescent optogenetic reporters
    • Herasymenko Krystyna
    • Walisinghe Danushka
    • Konno Masae
    • Barneschi Leonardo
    • de Waele Isabelle
    • Sliwa Michel
    • Inoue Keiichi
    • Olivucci Massimo
    • Haacke Stefan
    Chemical Science, The Royal Society of Chemistry , 2024, 16 (2), pp.761-774 . Archaerhodopsin-3 (AR-3) variants stand out among other rhodopsins in that they display a weak, but voltage-sensitive, near-infrared fluorescence emission. This has led to their application in optogenetics both in cell cultures and small animals. However, in the context of improving the fluorescence characteristics of the next generation of AR-3 reporters, an understanding of their ultrafast light-response in light-adapted conditions, is mandatory. To this end, we present a combined experimental and computational investigation of the excited state dynamics and quantum yields of AR-3 and its DETC and Arch-5 variants. The latter always display a mixture of all-trans/15-anti and 13-cis/15-syn isomers, which leads to a bi-exponential excited state decay. The isomerisation quantum yield is reduced more than 20 times as compared to WT AR-3 and proves that the steady-state fluorescence is induced by a single absorption photon event. In wild-type AR-3, we show that a 300 fs, barrier-less and vibrationally coherent isomerization is driven by an unusual covalent electronic character of its all-trans retinal chromophore leading to a metastable twisted diradical (TIDIR), in clear contrast to the standard charge-transfer scenario established for other microbial rhodopsins. We discuss how the presence of TIDIR makes AR-3 an ideal candidate for the design of variants with a one-photon induced fluorescence possibly reaching the emission quantum yield of the top natural emitter neorhodopsin (NeoR). (10.1039/d4sc05120c)
    DOI : 10.1039/d4sc05120c
  • Design, Synthesis, and In Vitro Antimalarial Evaluation of New 1,3,5-Tris[(4-(Substituted-Aminomethyl)Phenoxy)Methyl]Benzenes
    • Albenque-Rubio Sandra
    • Guillon Jean
    • Agnamey Patrice
    • Damiani Céline
    • Savrimoutou Solène
    • Mustière Romain
    • Pinaud Noël
    • Moreau Stéphane
    • Mergny Jean-Louis
    • Ronga Luisa
    • Kanavos Ioannis
    • Marchivie Mathieu
    • Moukha Serge
    • Dozolme Pascale
    • Sonnet Pascal
    • Cohen Anita
    Drugs and Drug Candidates, MDPI , 2024, 3 (3), pp.615-637 . By taking into account our previously described series of 1,3,5-tris[(4-(substituted-aminomethyl)phenyl)methyl]benzene compounds, we have now designed, prepared, and evaluated in vitro against Plasmodium falciparum a novel series of structural analogues of these molecules, i.e., the 1,3,5-tris[(4-(substituted-aminomethyl)phenoxy)methyl]benzene derivatives. The pharmacological data showed antimalarial activity with IC50 values in the sub and μM range. The in vitro cytotoxicity of these new nitrogen polyphenoxymethylbenzene compounds was also evaluated on human HepG2 cells. The 1,3,5-tris[(4-(substituted-aminomethyl)phenoxy)methyl]benzene derivative 1m was found as one of the most potent and promising antimalarial candidates with favorable cytotoxic to antiprotozoal properties in the P. falciparum strains W2 and 3D7. In conclusion, this 1,3,5-tris[(4-(pyridin-3-ylmethylaminomethyl)phenoxyl)methyl]benzene 1m (IC50 = 0.07 μM on W2, 0.06 μM on 3D7, and 62.11 μM on HepG2) was identified as the most promising antimalarial derivative with selectivity indexes (SI) of 887.29 on the W2 P. falciparum chloroquine-resistant strain, and of 1035.17 on the chloroquine-sensitive and mefloquine decreased sensitivity strain 3D7. It has been previously described that the telomeres of P. falciparum could represent potential targets for these types of polyaromatic compounds; therefore, the capacity of our novel derivatives to stabilize the parasitic telomeric G-quadruplexes was assessed using a FRET melting assay. However, with regard to the stabilization of the protozoal G-quadruplex, we observed that the best substituted derivatives 1, which exhibited some interesting stabilization profiles, were not the most active antimalarial compounds against the two Plasmodium strains. Thus, there were no correlations between their antimalarial activities and selectivities of their respective binding to G-quadruplexes. (10.3390/ddc3030035)
    DOI : 10.3390/ddc3030035
  • The Balance Between Shear Flow and Extracellular Matrix in Ovarian Cancer‐on‐Chip
    • Chen Changchong
    • Boché Alphonse
    • Wang Zixu
    • Lopez Elliot
    • Peng Juan
    • Carreiras Franck
    • Schanne-Klein Marie‐claire
    • Chen Yong
    • Lambert Ambroise
    • Aimé Carole
    Advanced Healthcare Materials, Wiley , 2024, 13 (23), pp.2400938 . Ovarian cancer is the most lethal gynecologic cancer in developed countries. In the tumor microenvironment, the extracellular matrix (ECM) and flow shear stress are key players in directing ovarian cancer cells invasion. Artificial ECM models based only on ECM proteins are used to build an ovarian tumor‐on‐chip to decipher the crosstalk between ECM and shear stress on the migratory behavior and cellular heterogeneity of ovarian tumor cells. This work shows that in the shear stress regime of the peritoneal cavity, the ECM plays a major role in driving individual or collective ovarian tumor cells migration. In the presence of basement membrane proteins, migration is more collective than on type I collagen regardless of shear stress. With increasing shear stress, individual cell migration is enhanced; while, no significant impact on collective migration is measured. This highlights the central position that ECM and flow shear stress should hold in in vitro ovarian cancer models to deepen understanding of cellular responses and improve development of ovarian cancer therapeutic platforms. In this frame, adding flow provides significant improvement in biological relevance over the authors’ previous work. Further steps for enhanced clinical relevance require not only multiple cell lines but also patient‐derived cells and sera. (10.1002/adhm.202400938)
    DOI : 10.1002/adhm.202400938
  • Quantitative Assessment of Collagen Remodeling during a Murine Pregnancy
    • Ramella-Roman Jessica C
    • Mahendroo Mala
    • Raoux Clothilde
    • Latour Gaël
    • Schanne-Klein Marie-Claire
    ACS photonics, American Chemical Society , 2024, 11 (9), pp.3536-3544 . <div><p>Uterine cervical remodeling is a fundamental feature of pregnancy, facilitating the delivery of the fetus through the cervical canal. Yet, we still know very little about this process due to the lack of methodologies that can quantitatively and unequivocally pinpoint the changes the cervix undergoes during pregnancy. We utilize polarization-resolved second harmonic generation to visualize the alterations the cervix extracellular matrix, specifically collagen, undergoes during pregnancy with exquisite resolution. This technique provides images of the collagen orientation at the pixel level (0.4 μm) over the entire murine cervical section. They show tight and ordered packing of collagen fibers around the os at the early stage of pregnancy and their disruption at the later stages. Furthermore, we utilize a straightforward statistical analysis to demonstrate the loss of order in the tissue, consistent with the loss of mechanical properties associated with this process. This work provides a deeper understanding of the parturition process and could support research into the cause of pathological or premature birth.</p></div> (10.1021/acsphotonics.4c00337)
    DOI : 10.1021/acsphotonics.4c00337
  • Confinement energy landscape classification reveals membrane receptor nano-organization mechanisms
    • Yu Chao
    • Richly Maximilian
    • Hoang Thi Thuy
    • El Beheiry Mohammed
    • Türkcan Silvan
    • Masson Jean-Baptiste
    • Alexandrou Antigoni
    • Bouzigues Cedric I
    Biophysical Journal, Biophysical Society , 2024, 123 (13), pp.1882-1895 . The cell membrane organization has an essential functional role through the control of membrane receptor confinement in micro- or nanodomains. Several mechanisms have been proposed to account for these properties, although some features have remained controversial, notably the nature, size, and stability of cholesterol- and sphingolipid-rich domains or lipid rafts. Here, we probed the effective energy landscape acting on single-nanoparticle-labeled membrane receptors confined in raft nanodomains— epidermal growth factor receptor (EGFR), Clostridium perfringens ε-toxin receptor (CPεTR), and Clostridium septicum α-toxin receptor (CSαTR)—and compared it with hop-diffusing transferrin receptors. By establishing a new analysis pipeline combining Bayesian inference, decision trees, and clustering approaches, we systematically classified single-protein trajectories according to the type of effective confining energy landscape. This revealed the existence of only two distinct organization modalities: confinement in a quadratic energy landscape for EGFR, CPεTR, and CSαTR (A), and free diffusion in confinement domains resulting from the steric hindrance due to F-actin barriers for transferrin receptor (B). The further characterization of effective confinement energy landscapes by Bayesian inference revealed the role of interactions with the domain environment in cholesterol- and sphingolipid-rich domains with (EGFR) or without (CPεTR and CSαTR) interactions with F-actin to regulate the confinement energy depth. These two distinct mechanisms result in the same organization type (A). We revealed that the apparent domain sizes for these receptor trajectories resulted from Brownian exploration of the energy landscape in a steady-state-like regime at a common effective temperature, independently of the underlying molecular mechanisms. These results highlight that confinement domains may be adequately described as interaction hotspots rather than rafts with abrupt domain boundaries. Altogether, these results support a new model for functional receptor confinement in membrane nanodomains and pave the way to the constitution of an atlas of membrane protein organization. (10.1016/j.bpj.2024.06.001)
    DOI : 10.1016/j.bpj.2024.06.001
  • Angle-Resolved Linear Dichroism to Probe the Organization of Highly Ordered Collagen Biomaterials
    • Krins Natacha
    • Wien Frank
    • Schmeltz Margaux
    • Pérez Javier
    • Dems Dounia
    • Debons Nicolas
    • Laberty-Robert Christel
    • Schanne-Klein Marie-Claire
    • Aimé Carole
    Biomacromolecules, American Chemical Society , 2024, 25 (9), pp.6181-6187 . Controlling the assembly of high-order structures is central to soft-matter and biomaterial engineering. Angle-resolved linear dichroism can probe the ordering of chiral collagen molecules in the dense state. Collagen triple helices were aligned by solvent evaporation. Their ordering gives a strong linear dichroism (LD) that changes sign and intensity with varying sample orientations with respect to the beam linear polarization. Being complementary to circular dichroism, which probes the structure of chiral (bio)molecules, LD can shift from the molecular to the supramolecular scale and from the investigation of the conformation to interactions. Supported by multiphoton microscopy and X-ray scattering, we show that LD provides a straightforward route to probe collagen alignment, determine the packing density, and monitor denaturation. This approach could be adapted to any assembly of chiral (bio)macromolecules, with key advantages in detecting large-scale assemblies with high specificity to aligned and chiral molecules and improved sensitivity compared to conventional techniques. (10.1021/acs.biomac.4c00860)
    DOI : 10.1021/acs.biomac.4c00860
  • Impact of trypsin on cell cytoplasm during detachment of cells studied by terahertz sensing
    • Lordon Blandine
    • Campion Tiffany
    • Gibot Laure
    • Gallot Guilhem
    Biophysical Journal, Biophysical Society , 2024, 123 (16), pp.2476-2483 . Trypsin is a very common enzyme used in cell culture to harvest cells by cleaving the proteins responsible for cell adhesion. However, trypsin also induces undesirable effects on cells, such as altering membrane proteins and the cytoskeleton, changing the composition of the cytoplasm and the cell volume, and even leading to cell death when used improperly. Using attenuated total reflection in the terahertz domain, confocal microscopy, and the propidium iodide test, we quantified in real time the change in cytoplasmic content induced by trypsin proteolysis on Madin-Darby canine kidney epithelial cells. We have observed a cytoplasmic modification from the very first seconds of trypsinization, following the change of cell volume due to mechanical re-equilibrium of the membrane. We found that the cytoplasmic alteration is associated with a transfer of small solutes: electrolytes and metabolites. We also found a very good nonlinear correlation between the side effects monitored by terahertz sensing and the cell height, regardless of the dependence of the cell height on trypsin concentration and exposure time. (10.1016/j.bpj.2024.06.011)
    DOI : 10.1016/j.bpj.2024.06.011
  • A Cost‐Effective Hemin‐Based Artificial Enzyme Allows for Practical Applications
    • Qiu Dehui
    • He Fangni
    • Liu Yuan
    • Zhou Zhaoxi
    • Yang Yuqin
    • Long Zhongwen
    • Chen Qianqian
    • Chen Desheng
    • Wei Shijiong
    • Mao Xuanxiang
    • Zhang Xiaobo
    • Mergny Jean‐louis
    • Monchaud David
    • Ju Huangxian
    • Zhou Jun
    Advanced Science, Wiley Open Access , 2024, 11 (32), pp.2402237 . Abstract Nanomaterials excel in mimicking the structure and function of natural enzymes while being far more interesting in terms of structural stability, functional versatility, recyclability, and large‐scale preparation. Herein, the story assembles hemin, histidine analogs, and G‐quadruplex DNA in a catalytically competent supramolecular assembly referred to as assembly‐activated hemin enzyme (AA‐heminzyme). The catalytic properties of AA‐heminzyme are investigated both in silico (by molecular docking and quantum chemical calculations) and in vitro (notably through a systematic comparison with its natural counterpart horseradish peroxidase, HRP). It is found that this artificial system is not only as efficient as HRP to oxidize various substrates (with a turnover number k cat of 115 s −1) but also more practically convenient (displaying better thermal stability, recoverability, and editability) and more economically viable, with a catalytic cost amounting to &lt;10% of that of HRP. The strategic interest of AA‐heminzyme is further demonstrated for both industrial wastewater remediation and biomarker detection (notably glutathione, for which the cost is decreased by 98% as compared to commercial kits). (10.1002/advs.202402237)
    DOI : 10.1002/advs.202402237