Articles

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
G-quadruplex ligands as potent regulators of lysosomes
- Ferret Lucille
- Alvarez-Valadez Karla
- Rivière Jennifer
- Muller Alexandra
- Bohálová Natalia
- Yu Luo
- Guittat Lionel
- Brázda Vaclav
- Kroemer Guido
- Mergny Jean-Louis
- Djavaheri-Mergny Mojgan
Autophagy, Taylor & Francis , 2023, 19 (7), pp.1901-1915 . Guanine-quadruplex structures (G4) are unusual nucleic acid conformations formed by guanine-rich DNA and RNA sequences and known to control gene expression mechanisms, from transcription to protein synthesis. So far, a number of molecules that recognize G4 have been developed for potential therapeutic applications in human pathologies, including cancer and infectious diseases. These molecules are called G4 ligands. When the biological effects of G4 ligands are studied, the analysis is often limited to nucleic acid targets. However, recent evidence indicates that G4 ligands may target other cellular components and compartments such as lysosomes and mitochondria. Here, we summarize our current knowledge of the regulation of lysosome by G4 ligands, underlying their potential functional impact on lysosome biology and autophagic flux, as well as on the transcrip- tional regulation of lysosomal genes. We outline the consequences of these effects on cell fate decisions and we systematically analyzed G4-prone sequences within the promoter of 435 lysosome- related genes. Finally, we propose some hypotheses about the mechanisms involved in the regula- tion of lysosomes by G4 ligands. (10.1080/15548627.2023.2170071)
DOI : 10.1080/15548627.2023.2170071
Label-free imaging of red blood cells and oxygenation with color third-order sum-frequency generation microscopy
- Ferrer Ortas Júlia
- Mahou Pierre
- Escot Sophie
- Stringari Chiara
- David Nicolas B
- Bally-Cuif Laure
- Dray Nicolas
- Négrerie Michel
- Supatto Willy
- Beaurepaire Emmanuel
Light: Science and Applications, Nature Publishing Group , 2023, 12 (1), pp.29 . Mapping red blood cells (RBCs) flow and oxygenation is of key importance for analyzing brain and tissue physiology. Current microscopy methods are limited either in sensitivity or in spatio-temporal resolution. In this work, we introduce a novel approach based on label-free third-order sum-frequency generation (TSFG) and third-harmonic generation (THG) contrasts. First, we propose a novel experimental scheme for color TSFG microscopy, which provides simultaneous measurements at several wavelengths encompassing the Soret absorption band of hemoglobin. We show that there is a strong three-photon (3P) resonance related to the Soret band of hemoglobin in THG and TSFG signals from zebrafish and human RBCs, and that this resonance is sensitive to RBC oxygenation state. We demonstrate that our color TSFG implementation enables specific detection of flowing RBCs in zebrafish embryos and is sensitive to RBC oxygenation dynamics with single-cell resolution and microsecond pixel times. Moreover, it can be implemented on a 3P microscope and provides label-free RBC-specific contrast at depths exceeding 600 µm in live adult zebrafish brain. Our results establish a new multiphoton contrast extending the palette of deep-tissue microscopy. (10.1038/s41377-022-01064-4)
DOI : 10.1038/s41377-022-01064-4
Collective cell migration due to guidance-by-followers is robust to multiple stimuli
- Müller Robert
- Boutillon Arthur
- Jahn Diego
- Starruß Jörn
- David Nicolas B
- Brusch Lutz
Frontiers in Applied Mathematics and Statistics, Frontiers Media S.A , 2023, 9, pp.1163583 . Collective cell migration is an important process during biological development and tissue repair but may turn malignant during tumor invasion. Mathematical and computational models are essential to unravel the mechanisms of self-organization that underlie the emergence of collective migration from the interactions among individual cells. Recently, guidance-by-followers was identified as one such underlying mechanism of collective cell migration in the embryo of the zebrafish. This poses the question of how the guidance stimuli are integrated when multiple cells interact simultaneously. In this study, we extend a recent individual-based model by an integration step of the vectorial guidance stimuli and compare model predictions obtained for different variants of the mechanism (arithmetic mean of stimuli, dominance of stimulus with largest transmission interface, and dominance of most head-on stimulus). Simulations are carried out and quantified within the modeling and simulation framework Morpheus. Collective cell migration is found to be robust and qualitatively identical for all considered variants of stimulus integration. Moreover, this study highlights the role of individual-based modeling approaches for understanding collective phenomena at the population scale that emerge from cell-cell interactions. (10.3389/fams.2023.1163583)
DOI : 10.3389/fams.2023.1163583
Evaluation of Slowfade Diamond as a buffer for STORM microscopy
- Boukhatem Hadjer
- Durel Beatrice
- Raimbault Manon
- Laurent Audrey
- Olivier Nicolas
Biomedical optics express, Optical Society of America - OSA Publishing , 2023, 14 (2), pp.550 . We study the potential of the commercial mounting medium Slowfade diamond as a buffer for STORM microscopy. We show that although it does not work with the popular far-red dyes typically used for STORM imaging, such as Alexa Fluor 647, it performs really well with a wide variety of green-excited dyes such as Alexa Fluor 532, Alexa Fluor 555 or CF 568. Moreover, imaging can be performed several months after the samples are mounted in this environment and kept in the fridge, providing a convenient way to preserve samples for STORM imaging, as well as to keep calibration samples, for example for metrology or teaching in particular in imaging facilities. (10.1364/BOE.473463)
DOI : 10.1364/BOE.473463
G4access identifies G-quadruplexes and their associations with open chromatin and imprinting control regions
- Esnault Cyril
- Magat Talha
- Aabidine Amal Zine El
- Garcia-Oliver Encar
- Cucchiarini Anne
- Bouchouika Soumya
- Llères David
- Goerke Lutz
- Luo Yu
- Verga Daniela
- Lacroix Laurent
- Feil Robert
- Spicuglia Salvatore
- Mergny Jean‐louis
- Andrau Jean Christophe
Nature Genetics, Nature Publishing Group , 2023, 55 (8), pp.1359–1369 . Metazoan promoters are enriched in secondary DNA structure-forming motifs, such as G-quadruplexes (G4s). Here we describe ‘G4access’, an approach to isolate and sequence G4s associated with open chromatin via nuclease digestion. G4access is antibody- and crosslinking-independent and enriches for computationally predicted G4s (pG4s), most of which are confirmed in vitro. Using G4access in human and mouse cells, we identify cell-type-specific G4 enrichment correlated with nucleosome exclusion and promoter transcription. G4access allows measurement of variations in G4 repertoire usage following G4 ligand treatment, HDAC and G4 helicases inhibitors. Applying G4access to cells from reciprocal hybrid mouse crosses suggests a role for G4s in the control of active imprinting regions. Consistently, we also observed that G4access peaks are unmethylated, while methylation at pG4s correlates with nucleosome repositioning on DNA. Overall, our study provides a new tool for studying G4s in cellular dynamics and highlights their association with open chromatin, transcription and their antagonism to DNA methylation. (10.1038/s41588-023-01437-4)
DOI : 10.1038/s41588-023-01437-4
Recent advances in the development of ultrafast electronic circular dichroism for probing the conformational dynamics of biomolecules in solution
- Changenet Pascale
- Hache François
The European Physical Journal. Special Topics, EDP Sciences / Springer Verlag , 2023, 232, pp.pages 2117–2129 . Conformational dynamics of biomolecules, which can span very large time scales ranging from seconds down to femtoseconds, play a key role in their function. In this regard, the combination of the high temporal resolution of ultrafast pump–probe spectroscopy and the structural sensitivity of electronic circular dichroism (CD) spectroscopy provides an extremely promising tool to follow these dynamics with a "virtually" unlimited temporal resolution. However, although CD spectroscopy is a widely used tool in structural biology to determine the secondary structure of biomolecules in solution, transposition of these measurements to the time domain (TRCD), on the sub-picosecond time scale, remains very challenging due to their weak signals prone to pump-induced polarization artifacts. Recent advances in laser technologies and non-linear optics, however, offer new perspectives for the development of femtosecond TRCD set-ups. In this review, we present recent developments in ultrafast TRCD spectroscopy. We discuss the advantages and drawbacks of the few existing functional experimental set-ups for their use to access the conformational dynamics of biomolecules at ultrashort time scales. (10.1140/epjs/s11734-022-00679-3)
DOI : 10.1140/epjs/s11734-022-00679-3
Elastic fiber alterations and calcifications in calcific uremic arteriolopathy
- Colboc Hester
- Moguelet Philippe
- Bazin Dominique
- Letavernier Emmanuel
- Sun Chenyu
- Chessel Anatole
- Carvalho Priscille
- Lok Catherine
- Dillies Anne-Sophie
- Chaby Guillaume
- Maillard Hervé
- Kottler Diane
- Goujon Elisa
- Jurus Christine
- Panaye Marine
- Tang Ellie
- Courville Philippe
- Boury Antoine
- Monfort Jean-Benoit
- Chasset François
- Senet Patricia
- Schanne-Klein Marie-Claire
Scientific Reports, Nature Publishing Group , 2023, 13, pp.15519 . Calcific uremic arteriolopathy (CUA) is a severely morbid disease, affecting mostly dialyzed end-stage renal disease (ESRD) patients, associated with calcium deposits in the skin. Calcifications have been identified in ESRD patients without CUA, indicating that their presence is not specific to the disease. The objective of this retrospective multicenter study was to compare elastic fiber structure and skin calcifications in ESRD patients with CUA to those without CUA using innovative structural techniques. Fourteen ESRD patients with CUA were compared to 12 ESRD patients without CUA. Analyses of elastic fiber structure and skin calcifications using multiphoton microscopy followed by machinelearning analysis and field-emission scanning electron microscopy coupled with energy dispersive X-ray were performed. Elastic fibers specifically appeared fragmented in CUA. Quantitative analyses of multiphoton images showed that they were significantly straighter in ESRD patients with CUA than without CUA. Interstitial and vascular calcifications were observed in both groups of ESRD patients, but vascular calcifications specifically appeared massive and circumferential in CUA. Unlike interstitial calcifications, massive circumferential vascular calcifications and elastic fibers straightening appeared specific to CUA. The origins of such specific elastic fiber's alteration are still to be explored and may involve relationships with ischemic vascular or inflammatory processes. (10.1038/s41598-023-42492-5)
DOI : 10.1038/s41598-023-42492-5
FLUTE: a Python GUI for interactive phasor analysis of FLIM data
- Gottlieb Dale
- Asadipour Bahar
- Kostina Polina
- Ung Thi Phuong Lien
- Stringari Chiara
Biological Imaging, Cambridge University Press , 2023, 3, pp.e21 . Fluorescence lifetime imaging microscopy (FLIM) is a powerful technique used to probe the local environment of fluorophores. The fit-free phasor approach to FLIM data is increasingly being used due to its ease of interpretation. To date, no open-source graphical user interface (GUI) for phasor analysis of FLIM data is available in Python, thus limiting the widespread use of phasor analysis in biomedical research. Here, we present Fluorescence Lifetime Ultimate Explorer (FLUTE), a Python GUI that is designed to fill this gap. FLUTE simplifies and automates many aspects of the analysis of FLIM data acquired in the time domain, such as calibrating the FLIM data, performing interactive exploration of the phasor plot, displaying phasor plots and FLIM images with different lifetime contrasts simultaneously, and calculating the distance from known molecular species. After applying desired filters and thresholds, the final edited datasets can be exported for further user-specific analysis. FLUTE has been tested using several FLIM datasets including autofluorescence of zebrafish embryos and in vitro cells. In summary, our user-friendly GUI extends the advantages of phasor plotting by making the data visualization and analysis easy and interactive, allows for analysis of large FLIM datasets, and accelerates FLIM analysis for non-specialized labs. (10.1017/s2633903x23000211)
DOI : 10.1017/s2633903x23000211
Excited-State Properties of Fully Reduced Flavins in Ferredoxin–NADP + Oxidoreductase
- Zhuang Bo
- Aleksandrov Alexey
- Seo Daisuke
- Vos Marten
Journal of Physical Chemistry Letters, American Chemical Society , 2023, 14 (4), pp.1096-1102 . The fully reduced flavin cofactor (FADred) in ferredoxin–NADP+ oxidoreductase (FNR) is a functional intermediate that displays different catalytic and steady-state spectral properties for enzymes from Bacillus subtilis (BsFNR), Chlorobaculum tepidum (CtFNR), and Rhodopseudomonas palustris (RpFNR). Using ultrafast spectroscopy, we reveal that at physiological pH, photoexcited FADred in BsFNR and RpFNR exhibits unprecedentedly fast decays (dominantly in 6 and 8 ps, respectively), whereas in CtFNR the decay is much slower (∼400 ps), as in other flavoproteins. Correlating these observations with the protonation states of FADred and the dynamic properties of the protein environment, we conclude that the excited state of neutral FADred can be intrinsically short-lived even in proteins, contrasting with the well-documented behavior of the anionic form that systematically displays markedly increased excited-state lifetime upon binding to proteins. This work provides new insight into the photochemistry of fully reduced flavins, which are emerging as functional initial states in bioengineered photocatalysts (10.1021/acs.jpclett.2c03741)
DOI : 10.1021/acs.jpclett.2c03741
Accurate calibration of optical tweezers close to a glass surface using interference rings in backscattered light
- Gillant Flavie
- Moreau Julien
- Richly Maximilian U
- Alexandrou Antigoni
- Perronet Karen
- Westbrook Nathalie
Journal of the European Optical Society : Rapid publications, European Optical Society , 2023, 19 . Mechanical forces play an important role in the behaviour of cells, from differentiation to migration and the development of diseases. Optical tweezers provide a quantitative tool to study these forces and must be combined with other tools, such as phase contrast and fluorescence microscopy. Detecting the retro-reflected trap beam is a convenient way to monitor the force applied by optical tweezers, while freeing top access to the sample. Accurate in situ calibration is required especially for single cells close to a surface where viscosity varies rapidly with height. Here, we take advantage of the well contrasted interference rings in the back focal plane of the objective to find the height of a trapped bead above a cover slip. We thus map the viscous drag dependence close to the surface and find agreement between four different measurement techniques for the trap stiffness down to 2 lm above the surface. Combining this detection scheme with phase contrast microscopy, we show that the phase ring in the back focal plane of the objective must be deported in a conjugate plane on the imaging path. This simplifies implementation of optical tweezers in combination with other techniques for biomechanical studies. (10.1051/jeos/2023026)
DOI : 10.1051/jeos/2023026
Second harmonic generation in the presence of walk-off and group velocity mismatch
- Hanna Marc
- Natile Michele
- Zaouter Yoann
- Joffre Manuel
- Georges Patrick
Journal of the Optical Society of America B, Optical Society of America , 2023, 40 (5), pp.930-938 . We study a second harmonic generation interaction geometry in the case where both group velocity mismatch and walk-off have significant impacts. This results in a frequency-converted beam exhibiting a pulse front tilt. Using the global response function of the crystal, we provide an analytical model that allows to predict the spatiotemporal structure of the second harmonic wave packet and verify its validity using numerical simulations and a simple experiment. Distinctive features of this geometry are the suppression of back-conversion and the ability to conserve the fundamental bandwidth in space and time domains. Subsequent compensation of the pulse front tilt should allow efficient generation of ultrashort pulses in the deep ultraviolet. (10.1364/josab.485597)
DOI : 10.1364/josab.485597
Photocycle alteration and increased enzymatic activity in genetically modified photoactivable adenylate cyclase OaPAC
- Raics Katalin
- Pirisi Katalin
- Zhuang Bo
- Fekete Zsuzsanna
- Kis-Bicskei Nikolett
- Pecsi Ildiko
- Ujfalusi Kinga Pozsonyi
- Telek Elek
- Li Yin
- Collado Jinnette Tolentino
- Tonge Peter
- Meech Stephen
- Vos Marten
- Bodis Emoke
- Lukacs Andras
Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology , 2023, 299 (8), pp.105056 . Photoactivated adenylate cyclases (PACs) are light activated enzymes that combine blue light sensing capacity with the ability to convert ATP to cAMP and pyrophosphate (PPi) in a light-dependent manner. In most of the known PACs blue light regulation is provided by a blue light sensing domain using flavin which undergoes a structural reorganization after blue-light absorption. This minor structural change then is translated toward the C-terminal of the protein, inducing a larger conformational change that results in the ATP conversion to cAMP. As cAMP is a key second messenger in numerous signal transduction pathways regulating various cellular functions, PACs are of great interest in optogenetic studies. The optimal optogenetic device must be “silent” in the dark and highly responsive upon light illumination. PAC from Oscillatoria acuminata is a very good candidate as its basal activity is very small in the dark and the conversion rates increase 20-fold upon light illumination. We studied the effect of replacing D67 to N, in the blue light using flavin domain. This mutation was found to accelerate the primary electron transfer process in the photosensing domain of the protein, as has been predicted. Furthermore, it resulted in a longer lived signaling state, which was formed with a lower quantum yield. Our studies show that the overall effects of the D67N mutation lead to a slightly higher conversion of ATP to cAMP, which points in the direction that by fine tuning the kinetic properties more responsive PACs and optogenetic devices can be generated (10.1016/j.jbc.2023.105056)
DOI : 10.1016/j.jbc.2023.105056