Articles

Strong and selective interactions of palmatine with G-rich sequences in TRF2 promoter; experimental and computational studies
- Fazelifar Pegah
- Cucchiarini Anne
- Khoshbin Zahra
- Mergny Jean-Louis
- Kazemi Noureini Sakineh
Journal of Biomolecular Structure and Dynamics, Taylor & Francis: STM, Behavioural Science and Public Health Titles , 2023, pp.in press . Background: G-rich sequences have the potential to fold into G-quadruplexes (GQs). G-quadruplexes, particularly those positioned in the regulatory regions of proto-oncogenes, have recently garnered attention in anti-cancer drug design. Methods: A thermal FRET assay was employed to conduct preliminary screening of various alkaloids, aiming to identify stronger interactions with a specific set of G-rich double-labeled oligonucleotides in both K+ and Na+ buffers. These oligonucleotides were derived from regions associated with Kit, Myc, Ceb, Bcl2, human telomeres, and potential G-quadruplex forming sequences found in the Nrf2 and Trf2 promoters. Palmatine generally increased the stability of different G-rich sequences into their folded GQ structures, more or less in a concentration dependent manner. The thermal stability and interaction of palmatine was further studied using transition FRET (t-FRET), CD and UV-visible spectroscopy and molecular dynamics simulation methods. Results: Palmatine showed the strongest interaction with TRF2 in both K+ and Na+ buffers even at equimolar concentration ratio. T-FRET studies revealed that palmatine has the potential to disrupt double-strand formation by the TRF2 sequence in the presence of its complementary strand. Palmatine exhibits a stronger interaction with G-rich strand DNA, promoting its folding into G-quadruplex structures. It is noteworthy that palmatine exhibits the strongest interaction with TRF2, which is the shortest sequence among the G-rich oligonucleotides studied, featuring only one nucleotide for two of its loops (10.1080/07391102.2023.2292793)
DOI : 10.1080/07391102.2023.2292793
Harnessing G-quadruplex ligands for lung cancer treatment: A comprehensive overview
- Figueiredo Joana
- Djavaheri-Mergny Mojgan
- Ferret Lucille
- Mergny Jean-Louis
- Cruz Carla
Drug Discovery Today, Elsevier , 2023, 28 (12), pp.103808 . Lung cancer (LC) remains a leading cause of mortality worldwide, and new therapeutic strategies are urgently needed. One such approach revolves around the utilization of four-stranded nucleic acid secondary structures, known as G-quadruplexes (G4), which are formed by G-rich sequences. G4 structures constitute enticing targets for therapeutic intervention. Ligands that bind selectively to G4 structures, present a promising strategy for regulating crucial cellular processes involved in the progression of LC, rendering them potent agents for lung cancer treatment. In this review, we offer a summary of the recent advancements in the development of ligands capable of targeting specific genes associated with the development and progression of lung cancer. (10.1016/j.drudis.2023.103808)
DOI : 10.1016/j.drudis.2023.103808
The future of CRISPR in Mycobacterium tuberculosis infection
- Zein Eddine Rima
- Refrégier Guislaine
- Cervantes Jorge
- Yokobori Noemí Kaoru
Journal of Biomedical Science, BioMed Central , 2023, 30 (1), pp.34 . Clustered Regularly Interspaced Short Palindromic repeats (CRISPR)-Cas systems rapidly raised from a bacterial genetic curiosity to the most popular tool for genetic modifications which revolutionized the study of microbial physiology. Due to the highly conserved nature of the CRISPR locus in Mycobacterium tuberculosis , the etiological agent of one of the deadliest infectious diseases globally, initially, little attention was paid to its CRISPR locus, other than as a phylogenetic marker. Recent research shows that M. tuberculosis has a partially functional Type III CRISPR, which provides a defense mechanism against foreign genetic elements mediated by the ancillary RNAse Csm6. With the advent of CRISPR-Cas based gene edition technologies, our possibilities to explore the biology of M. tuberculosis and its interaction with the host immune system are boosted. CRISPR-based diagnostic methods can lower the detection threshold to femtomolar levels, which could contribute to the diagnosis of the still elusive paucibacillary and extrapulmonary tuberculosis cases. In addition, one-pot and point-of-care tests are under development, and future challenges are discussed. We present in this literature review the potential and actual impact of CRISPR-Cas research on human tuberculosis understanding and management. Altogether, the CRISPR-revolution will revitalize the fight against tuberculosis with more research and technological developments. (10.1186/s12929-023-00932-4)
DOI : 10.1186/s12929-023-00932-4
Fluorescence to measure light intensity
- Lahlou Aliénor
- Tehrani Hessam Sepasi
- Coghill Ian
- Shpinov Yuriy
- Mandal Mrinal
- Plamont Marie-Aude
- Aujard Isabelle
- Niu Yuxi
- Nedbal Ladislav
- Lazár Dusan
- Mahou Pierre
- Supatto Willy
- Beaurepaire Emmanuel
- Eisenmann Isabelle
- Desprat Nicolas
- Croquette Vincent
- Jeanneret Raphaël
- Le Saux Thomas
- Jullien Ludovic
Nature Methods, Nature Publishing Group , 2023, 20, pp.1930–1938 . Abstract Despite the need for quantitative measurements of light intensity across many scientific disciplines, existing technologies for measuring light dose at the sample of a fluorescence microscope cannot simultaneously retrieve light intensity along with spatial distribution over a wide range of wavelengths and intensities. To address this limitation, we developed two rapid and straightforward protocols that use organic dyes and fluorescent proteins as actinometers. The first protocol relies on molecular systems whose fluorescence intensity decays and/or rises in a monoexponential fashion when constant light is applied. The second protocol relies on a broad-absorbing photochemically inert fluorophore to back-calculate the light intensity from one wavelength to another. As a demonstration of their use, the protocols are applied to quantitatively characterize the spatial distribution of light of various fluorescence imaging systems, and to calibrate illumination of commercially available instruments and light sources. (10.1038/s41592-023-02063-y)
DOI : 10.1038/s41592-023-02063-y
A Versatile G‐quadruplex (G4)‐coated Upconverted Metal‐Organic Framework for Hypoxic Tumor Therapy
- Mao Xuanxiang
- Zhang Xiaobo
- Chao Zhicong
- Qiu Dehui
- Wei Shijiong
- Luo Rengan
- Chen Desheng
- Zhang Yue
- Chen Yun
- Yang Yuanjiao
- Monchaud David
- Ju Huangxian
- Mergny Jean‐louis
- Lei Jianping
- Zhou Jun
Advanced Healthcare Materials, Wiley , 2023, 12 (28), pp.2300561 . Given the complexity of the tumor microenvironment, multiple strategies are being explored to tackle hypoxic tumors. One of the most efficient strategies combines several therapeutic modalities and typically requires the development of multifunctional nanocomposites through sophisticated synthetic procedures. Here, the G-quadruplex (G4)-forming sequence AS1411-A (d-(G2T)4TG(TG2)4A) was designed and used for its anti-tumor and biocatalytic properties, such as increasing the production of O2 ca. 2fold as compared to the parent AS1411 sequence. Subsequently, the AS1411-A/hemin complex (GH) was grafted on the surface and pores of a core-shell upconverted metalorganic framework (UMOF) to generate a UMGH nanoplatform. Compared with UMOF, UMGH exhibited enhanced colloidal stability, increased targeting of tumor cells and improved O2 production (8.5-fold) in situ. When irradiated with near-infrared (NIR) light, the UMGH antitumor properties were bolstered by photodynamic therapy (PDT), thanks to its ability to convert O2 into singlet oxygen (1 O2). Combined with the antiproliferative activity of AS1411-A, this novel approach herein lays the foundation for a new type of G4-based nanomedicine. (10.1002/adhm.202300561)
DOI : 10.1002/adhm.202300561
Chromatically Corrected Multicolor Multiphoton Microscopy
- Blanc Hugo
- Kaddour Gabriel
- David Nicolas B
- Supatto Willy
- Livet Jean
- Beaurepaire Emmanuel
- Mahou Pierre
ACS photonics, American Chemical Society , 2023 . Simultaneous imaging of multiple labels in tissues is key to studying complex biological processes. Although strategies for color multiphoton excitation have been established, chromatic aberration remains a major problem when multiple excitation wavelengths are used in a scanning microscope. Chromatic aberration introduces a spatial shift between the foci of beams of different wavelengths that varies across the field of view, severely degrading the performance of color imaging. In this work, we propose an adaptive correction strategy that solves this problem in two-beam microscopy techniques. Axial chromatic aberration is corrected by a refractive phase mask that introduces pure defocus into one beam, while lateral chromatic aberration is corrected by a piezoelectric mirror that dynamically compensates for lateral shifts during scanning. We show that this lightefficient approach allows seamless chromatic correction over the entire field of view of different multiphoton objectives without compromising spatial and temporal resolution and that the effective area for beam-mixing processes can be increased by more than 1 order of magnitude. We illustrate this approach with simultaneous three-color, two-photon imaging of developing zebrafish embryos and fixed Brainbow mouse brain slices over large areas. These results establish a robust and efficient method for chromatically corrected multiphoton imaging. (10.1021/acsphotonics.3c01104)
DOI : 10.1021/acsphotonics.3c01104
A new method for in vivo assessment of corneal transparency using spectral-domain OCT
- Vilbert Maëlle
- Bocheux Romain
- Georgeon Cristina
- Borderie Vincent
- Pernot Pascal
- Irsch Kristina
- Plamann Karsten
PLoS ONE, Public Library of Science , 2023, 18 (10), pp.e0291613 . Corneal transparency is essential to provide a clear view into and out of the eye, yet clinical means to assess such transparency are extremely limited and usually involve a subjective grading of visible opacities by means of slit-lamp biomicroscopy. Here, we describe an automated algorithm allowing extraction of quantitative corneal transparency parameters with standard clinical spectral-domain optical coherence tomography (SD-OCT). Our algorithm employs a novel pre-processing procedure to standardize SD-OCT image analysis and to numerically correct common instrumental artifacts before extracting mean intensity stromal-depth ( z ) profiles over a 6-mm-wide corneal area. The z -profiles are analyzed using our previously developed objective method that derives quantitative transparency parameters directly related to the physics of light propagation in tissues. Tissular heterogeneity is quantified by the Birge ratio B r and the photon mean-free path ( l s ) is determined for homogeneous tissues (i.e., B r ~1 ). SD-OCT images of 83 normal corneas (ages 22–50 years) from a standard SD-OCT device (RTVue-XR Avanti, Optovue Inc.) were processed to establish a normative dataset of transparency values. After confirming stromal homogeneity ( B r <10), we measured a median l s of 570 μm (interdecile range: 270–2400 μm). By also considering corneal thicknesses, this may be translated into a median fraction of transmitted (coherent) light T coh(stroma) of 51% (interdecile range: 22–83%). Excluding images with central saturation artifact raised our median T coh(stroma) to 73% (interdecile range: 34–84%). These transparency values are slightly lower than those previously reported, which we attribute to the detection configuration of SD-OCT with a relatively small and selective acceptance angle. No statistically significant correlation between transparency and age or thickness was found. In conclusion, our algorithm provides robust and quantitative measurements of corneal transparency from standard SD-OCT images with sufficient quality (such as ‘Line’ and ‘CrossLine’ B-scan modes without central saturation artifact) and addresses the demand for such an objective means in the clinical setting. (10.1371/journal.pone.0291613)
DOI : 10.1371/journal.pone.0291613
MiniBAR/GARRE1 is a dual Rac and Rab effector required for ciliogenesis
- Serres Murielle
- Shaughnessy Ronan
- Escot Sophie
- Hammich Hussein
- Cuvelier Frédérique
- Salles Audrey
- Rocancourt Murielle
- Verdon Quentin
- Gaffuri Anne-Lise
- Sourigues Yannick
- Malherbe Gilles
- Velikovsky Leonid
- Chardon Florian
- Sassoon Nathalie
- Tinevez Jean-Yves
- Callebaut Isabelle
- Formstecher Etienne
- Houdusse Anne
- David Nicolas
- Pylypenko Olena
- Echard Arnaud
Developmental Cell, Elsevier , 2023, 58, pp.1-18 . Cilia protrude from the cell surface and play critical roles in intracellular signaling, environmental sensing, and development. Reduced actin-dependent contractility and intracellular trafficking are both required for ciliogenesis, but little is known about how these processes are coordinated. Here, we identified a Rac1- and Rab35-binding protein with a truncated BAR (Bin/amphiphysin/Rvs) domain that we named MiniBAR (also known as KIAA0355/GARRE1), which plays a key role in ciliogenesis. MiniBAR colocalizes with Rac1 and Rab35 at the plasma membrane and on intracellular vesicles trafficking to the ciliary base and exhibits fast pulses at the ciliary membrane. MiniBAR depletion leads to short cilia, resulting from abnormal Rac-GTP/Rho-GTP levels and increased acto-myosin-II-dependent contractility together with defective trafficking of IFT88 and ARL13B into cilia. MiniBAR-depleted zebrafish embryos display dysfunctional short cilia and hallmarks of ciliopathies, including left-right asymmetry defects. Thus, MiniBAR is a dual Rac and Rab effector that controls both actin cytoskeleton and membrane trafficking for ciliogenesis. (10.1016/j.devcel.2023.09.010)
DOI : 10.1016/j.devcel.2023.09.010
Kidney Stone Classification Using Multimodal Multiphoton Microscopy
- Gleeson Matthew
- Morizet Joséphine
- Mahou Pierre
- Daudon Michel
- Bazin Dominique
- Stringari Chiara
- Schanne-Klein Marie-Claire
- Beaurepaire Emmanuel
ACS photonics, American Chemical Society , 2023, 10 (10), pp.3594–3604 . Kidney stones are a common form of nephrolithiasis, affecting up to 15% of the world’s population with a high probability of recurrence. These stones exhibit various chemical compositions and crystalline forms associated with different etiologies. Classification of the stones’ components is necessary to optimize treatment and suggest lifestyle changes to reduce the risk of recurrence. Current characterization methods usually require extensive sample preparation or are too detailed for the needs of a high-throughput laboratory. In this article, we present a kidney stone component classification scheme based on the multiphoton response of crushed samples that is label-free, requires minimal sample amounts, and simple preparation. We measure two-photon excited fluorescence, which is sensitive to protein content, second-harmonic generation, which is sensitive to crystalline symmetry, and polarization-resolved third-harmonic generation (pTHG), which is sensitive to crystal heterogeneity and birefringence. The combination of these three contrast modes can distinguish different materials, specifically calcium oxalate in monohydrate (COM), dihydrate (COD), or amorphous forms, cystine, and carbonate apatite. In addition, pTHG images have the potential to distinguish between COM and COD fragments and to provide information on the submicron organization of carbonate apatite fragments. (10.1021/acsphotonics.3c00651)
DOI : 10.1021/acsphotonics.3c00651
Real-time in vivo ROS monitoring with luminescent nanoparticles reveals skin inflammation dynamics
- Abdesselem M.
- Pétri N.
- Kuhner R.
- Mousseau F.
- Rouffiac V.
- Gacoin T.
- Laplace-Builhé C.
- Alexandrou A.
- Bouzigues C I
Biomedical optics express, Optical Society of America - OSA Publishing , 2023, 14 (10), pp.5392 . Reactive oxygen species (ROS) are key regulators in numerous pathological contexts, including cancer or inflammation. Their role is complex, which justifies the need for methods enabling their quantitative and time-resolved monitoring in vivo, in the perspective to profile tissues of individual patients. However, current ROS detection methods do not provide these features. Here, we propose a new method based on the imaging of lanthanide-ion nanoparticles (GdVO 4 :Eu), whose photoluminescence is modulated by the surrounding ROS concentration. We monitored their luminescence after intradermic injection in a mouse ear submitted to an inflammation-inducing topical stimulus. Based on this approach, we quantified the ROS concentration after inflammation induction and identified a two-step kinetics of ROS production, which may be attributed to the response of resident immune cells and their further recruitment at the inflammation locus. (10.1364/boe.501914)
DOI : 10.1364/boe.501914
Multititration: The New Method for Implementing Ultrasensitive and Quantitative Multiplexed In-Field Immunoassays Despite Cross-Reactivity?
- Mousseau Fanny
- Féraudet Tarisse Cécile
- Simon Stéphanie
- Gacoin Thierry
- Alexandrou Antigoni
- Bouzigues Cédric Ismael
Analytical Chemistry, American Chemical Society , 2023, 95 (36), pp.13509-13518 . The accurate in-field titration of multiple pathogens is essential to efficiently describe and monitor environmental or biological contamination, isolate, act, and treat adequately. This underscores the requirement of portable, fast, quantitative, and multiplexed detection technologies, which, however, have not been properly developed so far, notably because it has been hindered by the phenomenon of cross-reactivity. In this work, we proposed a new analytical method based on the imaging through a portable device of lanthanide-based nanoparticles (YVO 4 :Eu) for spatially multiplexed detection, relying on a multiparameter analysis, i.e., a simultaneous analysis of all of the luminescence signals through the comparison to a calibration surface built in the presence of multiple analytes of interest. We then demonstrated the possibility to simultaneously quantify by multiplexed lateral flow assay (xLFA) the three enterotoxins SEG, SEH, and SEI in unknown mixtures, over two concentration decades (from a dozen of pg•mL −1 to few ng•mL −1). Assays were performed in less than an hour (25 min of strip migration followed by 30 min of drying at room temperature), the time during which the presence of the operator was not required for more than 5 min, in order to dip the strip and have it imaged by the reader. The concepts of nominal concentration recovery, coefficient of variation (CV), limit of blank (LOB), and limit of detection (LOD) were discussed in detail in the context of multiplexed assays. With our new definitions, quantitative results demonstrated a high recovery of the nominal concentrations (115%), reliability (CV = 20%), and sensitivity (LOBs of 3, 27, and 6 pg•mL −1 for SEG, SEH, and SEI respectively, and LODs of 6, 48, and 11 pg•mL −1 for SEG, SEH, and SEI, respectively). Based on this method, we observed an increase in sensitivity of 100 compared to the other multiplexed LFA labeled with gold particles and we approached the sensitivity of the simplex enzyme-linked immunosorbent assay (ELISA) performed with the same capture and detection antibodies. To conclude, our results, which are applicable to virtually any kind of multiplexed test, pave the way to the next generation of in-field analytical immunoassays by providing fast, quantitative, and highly sensitive multiplexed detection of biomarkers or pathogens. (10.1021/acs.analchem.3c01846)
DOI : 10.1021/acs.analchem.3c01846
Apical size and deltaA expression predict adult neural stem cell decisions along lineage progression
- Mancini Laure
- Guirao Boris
- Ortica Sara
- Labusch Miriam
- Cheysson Felix
- Bonnet Valentin
- Phan Minh Son
- Herbert Sébastien
- Mahou Pierre
- Menant Emilie
- Bedu Sébastien
- Tinevez Jean-Yves
- Baroud Charles
- Beaurepaire Emmanuel
- Bellaiche Yohanns
- Bally-Cuif Laure
- Dray Nicolas
Science Advances, American Association for the Advancement of Science (AAAS) , 2023, 9 (35), pp.eadg7519 . The maintenance of neural stem cells (NSCs) in the adult brain depends on their activation frequency and division mode. Using long-term intravital imaging of NSCs in the zebrafish adult telencephalon, we reveal that apical surface area and expression of the Notch ligand DeltaA predict these NSC decisions. deltaA -negative NSCs constitute a bona fide self-renewing NSC pool and systematically engage in asymmetric divisions generating a self-renewing deltaA neg daughter, which regains the size and behavior of its mother, and a neurogenic deltaA pos daughter, eventually engaged in neuronal production following further quiescence-division phases. Pharmacological and genetic manipulations of Notch, DeltaA, and apical size further show that the prediction of activation frequency by apical size and the asymmetric divisions of deltaA neg NSCs are functionally independent of Notch. These results provide dynamic qualitative and quantitative readouts of NSC lineage progression in vivo and support a hierarchical organization of NSCs in differently fated subpopulations. (10.1126/sciadv.adg7519)
DOI : 10.1126/sciadv.adg7519
Last Year At Marienbad: Unusual Nucleic Acid structures
- Mergny Jean-Louis
- Trantírek Lukáš
- Capranico Giovanni
Biochimie, Elsevier , 2023, 214, pp.1-4 . (10.1016/j.biochi.2023.09.022)
DOI : 10.1016/j.biochi.2023.09.022
G-quadruplex forming sequences in the genes coding for cytochrome P450 enzymes and their potential roles in drug metabolism
- Saad Mona
- Zhang Rongxin
- Cucchiarini Anne
- Mehawej Cybel
- Mergny Jean-Louis
- Mroueh Mohamad
- Faour Wissam
Biochimie, Elsevier , 2023, 214, pp.45-56 . The majority of drugs are metabolized by cytochrome P450 (CYP) enzymes, primarily belonging to the CYP1, CYP2 and CYP3 families. Genetic variations are the main cause of inter-individual differences in drug response, which constitutes a major concern in pharmacotherapy. G-quadruplexes (G4s), are non-canonical DNA and RNA secondary structures formed by guanine-rich sequences. G4s have been implicated in cancer and gene regulation. In this study, we investigated putative G4-forming sequences (PQSs) in the CYP genes. Our findings reveal a high density of PQSs in the full genes of CYP family 2. Moreover, we observe an increased density of PQSs in the promoters of CYP family 1 genes compared to non-CYP450 genes. Importantly, stable PQSs were also identified in all studied CYP genes. Subsequently, we assessed the impact of the most frequently reported genetic mutations in the selected genes and the possible effect of these mutations on G4 formation as well as on the thermodynamic stability of predicted G4s. We found that 4 SNPs overlap G4 sequences and lead to mutated DNA and RNA G4 forming sequences in their context. Notably, the mutation in the CYP2C9 gene, which is associated with impaired (S)-warfarin metabolism in patients, alters a G4 sequence. We then demonstrated that at least 10 of the 13 chosen cytochrome P450 G4 candidates form G-quadruplex structures in vitro, using a combination of spectroscopic methods. In conclusion, our findings indicate the potential role of G-quadruplexes in cytochrome genes regulation, and emphasize the importance of G-quadruplexes in drug metabolism. (10.1016/j.biochi.2023.08.014)
DOI : 10.1016/j.biochi.2023.08.014
Unveiling the lamellar structure of the human cornea over its full thickness using polarization-resolved SHG microscopy
- Raoux Clothilde
- Chessel Anatole
- Mahou Pierre
- Latour Gaël
- Schanne-Klein Marie-Claire
Light: Science and Applications, Nature Publishing Group , 2023, 12 (1), pp.190 . A key property of the human cornea is to maintain its curvature and consequently its refraction capability despite daily changes in intraocular pressure. This is closely related to the multiscale structure of the corneal stroma, which consists of 1–3 µm-thick stacked lamellae made of thin collagen fibrils. Nevertheless, the distribution, size, and orientation of these lamellae along the depth of the cornea are poorly characterized up to now. In this study, we use second harmonic generation (SHG) microscopy to visualize the collagen distribution over the full depth of 10 intact and unstained human corneas (500–600 µm thick). We take advantage of the small coherence length in epi-detection to axially resolve the lamellae while maintaining the corneal physiological curvature. Moreover, as raw epi-detected SHG images are spatially homogenous because of the sub-wavelength size of stromal collagen fibrils, we use a polarimetric approach to measure the collagen orientation in every voxel. After a careful validation of this approach, we show that the collagen lamellae (i) are mostly oriented along the inferior–superior axis in the anterior stroma and along the nasal-temporal axis in the posterior stroma, with a gradual shift in between and (ii) exhibit more disorder in the anterior stroma. These results represent the first quantitative characterization of the lamellar structure of the human cornea continuously along its entire thickness with micrometric resolution. It also shows the unique potential of P-SHG microscopy for imaging of collagen distribution in thick dense tissues. (10.1038/s41377-023-01224-0)
DOI : 10.1038/s41377-023-01224-0
The H-NOX protein structure adapts to different mechanisms in sensors interacting with nitric oxide
- Yoo Byung-Kuk
- Kruglik Sergei
- Lambry Jean-Christophe
- Lamarre Isabelle
- Raman C.S.
- Nioche Pierre
- Négrerie Michel
Chemical Science, The Royal Society of Chemistry , 2023, 14 (31), pp.8408-8420 . Some classes of bacteria within phyla possess protein sensors identified as homologous to the heme domain of soluble guanylate cyclase, the mammalian NO-receptor. Named H-NOX domain (Heme-Nitric Oxide or OXygen-binding), their heme binds nitric oxide (NO) and O2 for some of them. The signaling pathways where these proteins act as NO or O2 sensors appear various and are fully established for only some species. Here, we investigated the reactivity of H-NOX from bacterial species toward NO with a mechanistic point of view using time-resolved spectroscopy. The present data show that H-NOXs modulate the dynamics of NO as a function of temperature, but in different ranges, changing its affinity by changing the probability of NO rebinding after dissociation in the picosecond time scale. This fundamental mechanism provides a means to adapt the heme structural response to the environment. In one particular H-NOX sensor the heme distortion induced by NO binding is relaxed in an ultrafast manner (∼15 ps) after NO dissociation, contrarily to other H-NOX proteins, providing another sensing mechanism through the H-NOX domain. Overall, our study links molecular dynamics with functional mechanism and adaptation. (10.1039/d3sc01685d)
DOI : 10.1039/d3sc01685d
G-quadruplexes in the evolution of hepatitis B virus
- Brázda Václav
- Dobrovolná Michaela
- Bohálová Natália
- Mergny Jean-Louis
Nucleic Acids Research, Oxford University Press , 2023, 51 . Abstract Hepatitis B virus (HBV) is one of the most dangerous human pathogenic viruses found in all corners of the world. Recent sequencing of ancient HBV viruses revealed that these viruses have accompanied humanity for several millenia. As G-quadruplexes are considered to be potential therapeutic targets in virology, we examined G-quadruplex-forming sequences (PQS) in modern and ancient HBV genomes. Our analyses showed the presence of PQS in all 232 tested HBV genomes, with a total number of 1258 motifs and an average frequency of 1.69 PQS per kbp. Notably, the PQS with the highest G4Hunter score in the reference genome is the most highly conserved. Interestingly, the density of PQS motifs is lower in ancient HBV genomes than in their modern counterparts (1.5 and 1.9/kb, respectively). This modern frequency of 1.90 is very close to the PQS frequency of the human genome (1.93) using identical parameters. This indicates that the PQS content in HBV increased over time to become closer to the PQS frequency in the human genome. No statistically significant differences were found between PQS densities in HBV lineages found in different continents. These results, which constitute the first paleogenomics analysis of G4 propensity, are in agreement with our hypothesis that, for viruses causing chronic infections, their PQS frequencies tend to converge evolutionarily with those of their hosts, as a kind of ‘genetic camouflage’ to both hijack host cell transcriptional regulatory systems and to avoid recognition as foreign material. (10.1093/nar/gkad556)
DOI : 10.1093/nar/gkad556
DNA topoisomerase 1 represses HIV-1 promoter activity through its interaction with a guanine quadruplex present in the LTR sequence
- Lista María José
- Jousset Anne-Caroline
- Cheng Mingpan
- Saint-André Violaine
- Perrot Elouan
- Rodrigues Melissa
- Di Primo Carmelo
- Gadelle Danielle
- Toccafondi Elenia
- Segeral Emmanuel
- Berlioz-Torrent Clarisse
- Emiliani Stéphane
- Mergny Jean-Louis
- Lavigne Marc
Retrovirology, BioMed Central , 2023, 20 (1), pp.10 . Background: Once integrated in the genome of infected cells, HIV-1 provirus is transcribed by the cellular transcription machinery. This process is regulated by both viral and cellular factors, which are necessary for an efficient viral replication as well as for the setting up of viral latency, leading to a repressed transcription of the integrated provirus. Results: In this study, we examined the role of two parameters in HIV-1 LTR promoter activity. We identified DNA topoisomerase1 (TOP1) to be a potent repressor of this promoter and linked this repression to its catalytic domain. Additionally, we confirmed the folding of a Guanine quadruplex (G4) structure in the HIV-1 promoter and its repressive effect. We demonstrated a direct interaction between TOP1 and this G4 structure, providing evidence of a functional relationship between the two repressive elements. Mutations abolishing G4 folding affected TOP1/G4 interaction and hindered G4-dependent inhibition of TOP1 catalytic activity in vitro. As a result, HIV-1 promoter activity was reactivated in a native chromatin environment. Lastly, we noticed an enrichment of predicted G4 sequences in the promoter of TOP1-repressed cellular genes. Conclusions: Our results demonstrate the formation of a TOP1/G4 complex on the HIV-1 LTR promoter and its repressive effect on the promoter activity. They reveal the existence of a new mechanism of TOP1/G4-dependent transcriptional repression conserved between viral and human genes. This mechanism contrasts with the known property of TOP1 as global transcriptional activator and offers new perspectives for anti-cancer and anti-viral strategies. (10.1186/s12977-023-00625-8)
DOI : 10.1186/s12977-023-00625-8
Third harmonic imaging contrast from tubular structures in the presence of index discontinuity
- Morizet Joséphine
- Olivier Nicolas
- Mahou Pierre
- Boutillon Arthur
- Stringari Chiara
- Beaurepaire Emmanuel
Scientific Reports, Nature Publishing Group , 2023, 13, pp.7850 . Accurate interpretation of third harmonic generation (THG) microscopy images in terms of sample optical properties and microstructure is generally hampered by the presence of excitation field distortions resulting from sample heterogeneity. Numerical methods that account for these artifacts need to be established. In this work, we experimentally and numerically analyze the THG contrast obtained from stretched hollow glass pipettes embedded in different liquids. We also characterize the nonlinear optical properties of 2,2 ′-thiodiethanol (TDE), a water-soluble index-matching medium. We find that index discontinuity not only changes the level and modulation amplitude of polarizationresolved THG signals, but can even change the polarization direction producing maximum THG near interfaces. We then show that a finite-difference time-domain (FDTD) modeling strategy can accurately account for contrast observed in optically heterogeneous samples, whereas reference Fourier-based numerical approaches are accurate only in the absence of index mismatch. This work opens perspectives for interpreting THG microscopy images of tubular objects and other geometries. (10.1038/s41598-023-34528-7)
DOI : 10.1038/s41598-023-34528-7
G-Quadruplex Structures Are Key Modulators of Somatic Structural Variants in Cancers
- Zhang Rongxin
- Shu Huiling
- Wang Yuqi
- Tao Tiantong
- Tu Jing
- Wang Cheng
- Mergny Jean-Louis
- Sun Xiao
Cancer Research, American Association for Cancer Research , 2023, 83 (8), pp.1234-1248 . Abstract G-quadruplexes (G4) are noncanonical secondary genome structures. Aberrant formation of G4s can impair genome integrity. Investigation of the relationship between G4s and somatic structural variants (SV) in cancers could provide a better understanding of the role of G4 formation in cancer development and progression. In this study, we combined bioinformatic approaches and multiomics data to investigate the connection between G4s and the somatic SVs. Somatic SV breakpoints were significantly enriched in G4 regions, regardless of SV subtypes. This enrichment was only observed in regions demonstrated to form G4s in cells (“active quadruplexes”), rather than in regions with a sequence compatible with G4 formation but without confirmed G4 formation (“potential quadruplexes”). Several genomic features affected the connection between G4s and SVs, with the enrichment being notably strengthened at the boundary of topologically associated domains. Somatic breakpoints were also preferentially associated with G4 regions with earlier replication timing and open chromatin status. In patients with cancer with homologous recombination repair defects, G4s and somatic breakpoints were substantially more strongly associated. Machine learning models were constructed that showed that G4 propensity is a potent feature for predicting the density of SV breakpoints. Altogether, these findings suggest that the G4 structures play a critical role in modulating the production of somatic SVs in cancers. Significance: G-quadruplex structure formation constitutes a critical step in the production of somatic structural variants in cancers, suggesting G-quadruplex structures as potential targets for future cancer prevention and treatment strategies. (10.1158/0008-5472.CAN-22-3089)
DOI : 10.1158/0008-5472.CAN-22-3089
DNA i-motif formation at neutral pH is driven by kinetic partitioning
- Školáková Petra
- Gajarský Martin
- Palacký Jan
- Šubert Denis
- Renčiuk Daniel
- Trantírek Lukáš
- Mergny Jean-Louis
- Vorlíčková Michaela
Nucleic Acids Research, Oxford University Press , 2023, 51 (6), pp.2950-2962 . Abstract Cytosine-rich DNA regions can form four-stranded structures based on hemi-protonated C.C+ pairs, called i-motifs (iMs). Using CD, UV absorption, NMR spectroscopy, and DSC calorimetry, we show that model (CnT3)3Cn (Cn) sequences adopt iM under neutral or slightly alkaline conditions for n > 3. However, the iMs are formed with long-lasting kinetics under these conditions and melt with significant hysteresis. Sequences with n > 6 melt in two or more separate steps, indicating the presence of different iM species, the proportion of which is dependent on temperature and incubation time. At ambient temperature, kinetically favored iMs of low stability are formed, most likely consisting of short C.C+ blocks. These species act as kinetic traps and prevent the assembly of thermodynamically favored, fully C.C+ paired iMs. A higher temperature is necessary to unfold the kinetic forms and enable their substitution by a slowly developing thermodynamic structure. This complicated kinetic partitioning process considerably slows down iM folding, making it much slower than the timeframes of biological reactions and, therefore, unlikely to have any biological relevance. Our data suggest kinetically driven iM species as more likely to be biologically relevant than thermodynamically most stable iM forms. (10.1093/nar/gkad119)
DOI : 10.1093/nar/gkad119
Autocatalytic effect boosts the production of medium-chain hydrocarbons by fatty acid photodecarboxylase
- Samire Poutoum
- Zhuang Bo
- Légeret Bertrand
- Baca-Porcel Ángel
- Peltier Gilles
- Sorigué Damien
- Aleksandrov Alexey
- Beisson Frédéric
- Müller Pavel
Science Advances, American Association for the Advancement of Science (AAAS) , 2023, 9 (13) . Ongoing climate change is driving the search for renewable and carbon-neutral alternatives to fossil fuels. Photocatalytic conversion of fatty acids to hydrocarbons by fatty acid photodecarboxylase (FAP) represents a promising route to green fuels. However, the alleged low activity of FAP on C2 to C12 fatty acids seemed to preclude the use for synthesis of gasoline-range hydrocarbons. Here, we reveal that Chlorella variabilis FAP ( Cv FAP) can convert n -octanoic acid in vitro four times faster than n -hexadecanoic acid, its best substrate reported to date. In vivo, this translates into a Cv FAP-based production rate over 10-fold higher for n -heptane than for n -pentadecane. Time-resolved spectroscopy and molecular modeling demonstrate that Cv FAP’s high catalytic activity on n -octanoic acid is, in part, due to an autocatalytic effect of its n -heptane product, which fills the rest of the binding pocket. These results represent an important step toward a bio-based and light-driven production of gasoline-like hydrocarbons. (10.1126/sciadv.adg3881)
DOI : 10.1126/sciadv.adg3881
Intensity noise in difference frequency generation-based tunable femtosecond MIR sources
- Bournet Q
- Natile M
- Jonusas M
- Guichard F
- Zaouter Y
- Joffre M
- Bonvalet A
- Druon F
- Hanna Marc
- Georges P
Optics Express, Optical Society of America - OSA Publishing , 2023, 31 (8), pp.12693-12702 . We characterize the intensity noise of two mid-infrared (MIR) ultrafast tunable (3.5-11 µm) sources based on difference frequency generation (DFG). While both sources are pumped by a high repetition rate Yb-doped amplifier delivering 200 µJ 300 fs at a central wavelength of 1030 nm, the first is based on intrapulse DFG (intraDFG), and the second on DFG at the output of an optical parametric amplifier (OPA). The noise properties are assessed through measurement of the relative intensity noise (RIN) power spectral density and pulse-to-pulse stability. The noise transfer mechanisms from the pump to the MIR beam is empirically demonstrated. As an example, improving the pump laser noise performance allows reduction of the integrated RIN (IRIN) of one of the MIR source from 2.7% RMS down to 0.4% RMS. The intensity noise is also measured at various stages and in several wavelength ranges in both laser system architectures, allowing us to identify the physical origin of their variation. This study presents numerical values for the pulse to pulse stability, and analyze the frequency content of the RINs of particular importance for the design of low-noise high repetition rate tunable MIR sources and future high performance time-resolved molecular spectroscopy experiments. (10.1364/oe.486509)
DOI : 10.1364/oe.486509
Chimeric Biocatalyst Combining Peptidic and Nucleic Acid Components Overcomes the Performance and Limitations of the Native Horseradish Peroxidase
- Zhang Xiaobo
- Qiu Dehui
- Chen Jielin
- Zhang Yue
- Wang Jiawei
- Chen Desheng
- Liu Yuan
- Cheng Mingpan
- Monchaud David
- Mergny Jean-Louis
- Ju Huangxian
- Zhou Jun
Journal of the American Chemical Society, American Chemical Society , 2023, 145 (8), pp.4517-4526 . Chimeric peptide-DNAzyme (CPDzyme) is a novel design of an artificial peroxidase that relies on the covalent assembly of DNA (quadruplex-DNA, or G4), peptides and an enzyme cofactor (hemin) in a single scaffold. An accurate control of the assembly of these different partners allows for the design of the efficient CPDzyme prototype G4-Hemin-KHRRH, found to be >2,000-fold more active (in terms of conversion number kcat) than the corresponding but non-covalent complex and, more importantly, >1.5-fold active than the corresponding native peroxidase (horseradish peroxidase, or HRP) when considering a single catalytic center. This unique performance originates in a series of improvements gradually made thanks to an accurate selection and arrangement of the different components of the CPDzyme, in order to benefit from synergistic interactions between them. The optimized prototype G4-Hemin-KHRRH is efficient and robust as it can be used under a wide range of non-physiologically relevant conditions (organic solvents, high temperature (95°C), in a wide range of pH (from 2 to 10)), thus compensating for the shortcomings of natural enzymes. Our approach thus opens broad prospects for the design of ever more efficient artificial enzymes. (10.1021/jacs.2c11318)
DOI : 10.1021/jacs.2c11318
Bacterial origins of thymidylate metabolism in Asgard archaea and Eukarya
- Filée Jonathan
- Becker Hubert F
- Mellottee Lucille
- Zein Eddine Rima
- Li Zhihui
- Yin Wenlu
- Lambry Jean-Christophe
- Liebl Ursula
- Myllykallio Hannu
Nature Communications, Nature Publishing Group , 2023 . Asgard archaea include the closest known archaeal relatives of eukaryotes. Here, we investigate the evolution and function of Asgard thymidylate synthases and other folate-dependent enzymes required for the biosynthesis of DNA, RNA, amino acids and vitamins, as well as syntrophic amino acid utilization. Phylogenies of Asgard folate-dependent enzymes are consistent with their horizontal transmission from various bacterial groups. We experimentally validate the functionality of thymidylate synthase ThyX of the cultured 'Candidatus Prometheoarchaeum syntrophicum'. The enzyme efficiently uses bacterial-like folates and is inhibited by mycobacterial ThyX inhibitors, even though the majority of experimentally tested archaea are known to use carbon carriers distinct from bacterial folates. Our phylogenetic analyses suggest that the eukaryotic thymidylate synthase, required for de novo DNA synthesis, is not closely related to archaeal enzymes and might have been transferred from bacteria to protoeukaryotes during eukaryogenesis. Altogether, our study suggests that the capacity of eukaryotic cells to duplicate their genetic material is a sum of archaeal (replisome) and bacterial (thymidylate synthase) characteristics. We also propose that recent prevalent lateral gene transfer from bacteria has markedly shaped the metabolism of Asgard archaea. (10.1038/s41467-023-36487-z)
DOI : 10.1038/s41467-023-36487-z