Articles

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
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
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
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
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
Individual heme a and heme a3 contributions to the Soret absorption spectrum of the reduced bovine cytochrome c oxidase
- Diuba Artem
- Vygodina Tatiana
- Azarkina Natalia
- Arutyunyan Alexander
- Soulimane Tewfik
- Vos Marten
- Konstantinov Alexander
Biochimica biophysica acta (BBA) - Bioenergetics, Elsevier , 2023, 1864, pp.148937 . Bovine cytochrome c oxidase (CcO) contains two hemes, a and a3, chemically identical but differing in coordination and spin state. The Soret absorption band of reduced aa3-type cytochrome c oxidase consists of overlapping bands of the hemes a2+ and a32+. It shows a peak at ∼444 nm and a distinct shoulder at ∼425 nm. However, attribution of individual spectral lineshapes to hemes a2+ and a32+ in the Soret is controversial. In the present work, we characterized spectral contributions of hemes a2+ and a32+ using two approaches. First, we reconstructed bovine CcO heme a2+ spectrum using a selective Ca2+-induced spectral shift of the heme a2+. Second, we investigated photobleaching of the reduced Thermus thermophilus ba3- and bovine aa3-oxidases in the Soret induced by femtosecond laser pulses in the Q-band. The resolved spectra show splitting of the electronic B0x-, B0y-transitions of both reduced hemes. The heme a2+ spectrum is shifted to the red relative to heme a32+ spectrum. The ∼425 nm shoulder is mostly attributed to heme a32+. (10.1016/j.bbabio.2022.148937)
DOI : 10.1016/j.bbabio.2022.148937
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
Impact of corpus callosum fiber tract crossing on polarimetric images of human brain histological sections: ex vivo studies in transmission configuration
- Ivanov Deyan
- Si Lu
- Felger Leonard
- Maragkou Theoni
- Schucht Philippe
- Schanne-Klein Marie-Claire
- Ma Hui
- Ossikovski Razvigor
- Novikova Tatiana
Journal of Biomedical Optics, Society of Photo-optical Instrumentation Engineers , 2023, 28 (10), pp.102908 . Significance: Imaging Mueller polarimetry is capable to trace in-plane orientation of brain fiber tracts by detecting the optical anisotropy of white matter of healthy brain. Brain tumor cells grow chaotically and destroy this anisotropy. Hence, the drop in scalar retardance values and randomization of the azimuth of the optical axis could serve as the optical marker for brain tumor zone delineation. Aim: The presence of underlying crossing fibers can also affect the values of scalar retardance and the azimuth of the optical axis. We studied and analyzed the impact of fiber crossing on the polarimetric images of thin histological sections of brain corpus callosum. Approach: We used the transmission Mueller microscope for imaging of two-layered stacks of thin sections of corpus callosum tissue to mimic the overlapping brain fiber tracts with different fiber orientations. The decomposition of the measured Mueller matrices was performed with differential and Lu–Chipman algorithms and completed by the statistical analysis of the maps of scalar retardance, azimuth of the optical axis, and depolarization. Results: Our results indicate the sensitivity of Mueller polarimetry to different spatial arrangement of brain fiber tracts as seen in the maps of scalar retardance and azimuth of optical axis of two-layered stacks of corpus callosum sections The depolarization varies slightly (<15 % ) with the orientation of the optical axes in both corpus callosum stripes, but its value increases by 2.5 to 3 times with the stack thickness. Conclusions: The crossing brain fiber tracts measured in transmission induce the drop in values of scalar retardance and randomization of the azimuth of the optical axis at optical path length of 15 μm. It suggests that the presence of nerve fibers crossing within the depth of few microns will be also detected in polarimetric maps of brain white matter measured in reflection configuration. (10.1117/1.JBO.28.10.102908)
DOI : 10.1117/1.JBO.28.10.102908
Topology of DNA G-Quadruplexes Can Be Harnessed in Holliday Junction-Based DNA Suprastructures to Control and Optimize Their Biocatalytic Properties
- Qiu Dehui
- Cheng Mingpan
- Stadlbauer Petr
- Chen Jielin
- Langer Michal
- Zhang Xiaobo
- Gao Qiang
- Ju Huangxian
- Šponer Jiří
- Mergny Jean-Louis
- Monchaud David
- Zhou Jun
ACS Catalysis, American Chemical Society , 2023, 12, pp.10722-10733 . The nature, composition, and topology of the active sites of both natural and artificial enzymes are key determinants of their catalytic performance. While interesting structural insights have been obtained for natural enzymes (e.g., horseradish peroxidase, HRP), the accurate catalytic microenvironment of HRP-mimicking DNA-based catalysts known as G-quadruplex (GQ)/hemin DNAzymes is still unclear. Herein, we report on a strategy allowing for fully controlling the nature of the active site of GQ DNAzyme, precisely manipulating the composition and topology of the hemin (Fe(III)protoporphyrin IX) cofactor binding site. This was achieved by introducing GQ within a Holliday junction (HJ) suprastructure that enables to seize control of both the GQ folding topology (parallel, antiparallel, hybrid) and the GQ strand directionality (clockwise, counterclockwise). By doing so, we demonstrate that the different GQ topologies are equivalent for both hemin binding and activation and that the flanking nucleotides (dA or dTC) modulate the activation of hemin in a GQ topology-dependent manner. Our experimental findings are supported by the most extensive molecular dynamics simulations ever been done on GQ DNAzyme, thus providing unique mechanistic insights into the biocatalytic activity of GQs. (10.1021/acscatal.3c02818)
DOI : 10.1021/acscatal.3c02818
Artifact-free balanced detection for the measurement of circular dichroism with a sub-picosecond time resolution
- Changenet Pascale
- Hache François
Optics Express, Optical Society of America - OSA Publishing , 2023, 31 (13), pp.21296 . Here we present the development of a subpicosecond spectropolarimeter enabling high sensitivity balanced detection of time-resolved circular dichroism (TRCD) signals from chiral sample in solution. The signals are measured with a conventional femtosecond pump-probe set-up using the combination of a quarter-waveplate and a Wollaston prism. This simple and robust method allows access to TRCD signals with improved signal-to-noise ratio and very short acquisition times. We provide a theoretical analysis of the artifacts of such detection geometry and the strategy to eliminate them. We illustrate the potential of this new detection with the study of the [Ru(phen) 3 ]·2PF 6 complexes in acetonitrile. (10.1364/OE.489468)
DOI : 10.1364/OE.489468
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
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
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
The paradoxes of Mycobacterium tuberculosis molecular evolution and consequences for the inference of tuberculosis emergence date
- Zein Eddine Rima
- Hak F.
- Le Meur A.
- Genestet C.
- Dumitrescu O.
- Guyeux C.
- Senelle G.
- Sola C.
- Refrégier G.
Tuberculosis, Elsevier , 2023, 143, pp.102378 . The date of Mycobacterium tuberculosis complex emergence has been the subject of long debates. New studies joining archaeological efforts with sequencing methods raise high hopes for solving whether this emergence is closer to 70,000 or to 6000 years before present. Inferring the date of emergence of this pathogen based on sequence data requires a molecular clock. Several clocks inferred from different types of loci and/or different samples, using both sound reasoning and reliable data, are actually very different, which we refer to as the paradoxes of M. tuberculosis molecular evolution. After having presented these paradoxes, we will remind the limits of the molecular clocks used in the different studies such as the assumption of homogeneous substitution rate. We will then review recent results that shed new light on the characteristics of M. tuberculosis molecular evolution: traces of diverse selection pressures, the impact of host dynamics, etc. We provide some ideas on what to do next to get nearer to a reliable dating of Mycobacterium tuberculosis complex emergence. Among them, the collection of additional remains from ancient tuberculosis seems still essential. (10.1016/j.tube.2023.102378)
DOI : 10.1016/j.tube.2023.102378
Quadruplexes and aging: G4-binding proteins regulate the presence of miRNA in small extracellular vesicles (sEVs)
- Brázda Václav
- Mergny Jean-Louis
Biochimie, Elsevier , 2023, pp.S0300-9084(23)00014-7 . The interaction between proteins and nucleic acids is a core element of life. Many proteins bind nucleic acids via a sequence-specific manner, but there are also many types of proteins that recognize various structural motifs. Researchers have recently found that proteins that can recognize DNA and RNA G- quadruplexes (G4s) are very important for basic cellular processes, particularly in eukaryotes. Some of these proteins are located outside the nucleus and interact with RNA, potentially affecting miRNA functions in intercellular communication, which is facilitated by small extracellular vesicles (sEVs). Im- balances in the production of sEVs are associated with various pathologies and senescence in humans. The distribution of miRNA into sEVs is regulated by two RNA-binding proteins, Alyref and FUS. Both proteins possess G-rich recognition motifs that are compatible with the formation of RNA parallel G4 structures. This lends credence to the new hypothesis that G4-formation in RNAs and their interaction with G4-binding proteins can affect the fate of miRNAs and control their distribution in sEVs that are associated with senescence and aging. (10.1016/j.biochi.2023.01.014)
DOI : 10.1016/j.biochi.2023.01.014
Terahertz Spectroscopy Sheds Light on Real‐Time Exchange Kinetics Occurring through Plasma Membrane during Photodynamic Therapy Treatment
- Zheng Xiujun
- Lordon Blandine
- Mingotaud Anne‐françoise
- Vicendo Patricia
- Brival Rachel
- Fourquaux Isabelle
- Gibot Laure
- Gallot Guilhem
Advanced Science, Wiley Open Access , 2023, 10 (18), pp.e2300589 . Methods to follow in real time complex processes occurring along living cell membranes such as cell permeabilization are rare. Here, the terahertz spectroscopy reveals early events in plasma membrane alteration generated during photodynamic therapy (PDT) protocol, events which are not observable in any other conventional biological techniques performed in parallel as comparison. Photodynamic process is examined in Madin-Darby canine kidney cells using Pheophorbide (Pheo) photosensitizer alone or alternatively encapsulated in poly(ethylene oxide)-block-poly(ε-caprolactone) micelles for drug delivery purpose. Terahertz spectroscopy (THz) reveals that plasma membrane permeabilization starts simultaneously with illumination and is stronger when photosensitizer is encapsulated. In parallel, the exchange of biological species is assessed. Over several hours, this conventional approach demonstrates significant differences between free and encapsulated Pheo, the latter leading to high penetration of propidium iodide, Na+ and Ca2+ ions, and a high level of leakage of K+, ATP, and lactate dehydrogenase. THz spectroscopy provides, in a single measurement, the relative number of defects per membrane surface created after PDT, which is not achieved by any other method, providing early, sensitive real-time information. THz spectroscopy is therefore a promising technique and can be applied to any biological topic requiring the examination of short-term plasma membrane permeabilization. (10.1002/advs.202300589)
DOI : 10.1002/advs.202300589
A sodium / potassium switch for G4-prone G / C-rich sequences
- Luo Yu
- Živković Martina Lenarčič
- Wang Jiawei
- Ryneš Jan
- Foldynová-Trantírková Silvie
- Trantírek Lukáš
- Verga Daniela
- Mergny Jean-Louis
Nucleic Acids Research, Oxford University Press , 2023, 52 (1), pp.448-461 . Metal ions are essential components for the survival of living organisms. For most species, intracellular and extracellular ionic conditions differ significantly. As G-quadruple x es (G4s) are ion-dependent structures, changes in the [Na+]/[K+] ratio may affect the folding of genomic G4s. More than 11000 putative G4 sequences in the human genome (hg19) contain at least two runs of three continuous cytosines, and these mixed G/C-rich sequences may form a quadruplex or a competing hairpin structure based on G-C base pairing. In this study, we examine how the [Na+]/[K+] ratio influences the structures of G/C-rich sequences. The natural G4 structure with a 9-nt long central loop, CEBwt, was chosen as a model sequence, and the loop bases were gradually replaced by cytosines. The series of CEB mutations revealed that the presence of cytosines in G4 loops does not prevent G4 f olding or decrease G4 stability but increases the probability of forming a competing structure, either a hairpin or an intermolecular duplex. Slow conversion to the quadruplex in vitro (in a potassium-rich buffer) and cells was demonstrated by NMR. 'Shape-shifting' sequences may respond to [Na+]/[K+] changes with delayed kinetics. (10.1093/nar/gkad1073)
DOI : 10.1093/nar/gkad1073
Simple Postsynthesis Thermal Treatment toward High Luminescence Performance of Rare Earth Vanadate Nanoparticles
- Perrella Rafael Vieira
- Mohammedi Rabei
- Kuhner Robin
- Cardone Christophe
- Larquet Eric
- Alexandrou Antigoni
- de Sousa Filho Paulo Cesar
- Gacoin Thierry
Crystal Growth & Design, American Chemical Society , 2023, 23 (8), pp.5389-5396 . Optical applications of colloidal oxide nanoparticles are often limited by low luminescence efficiencies caused by poor crystallinity and surface quenching. Bulk oxides prepared via conventional high-temperature annealing offer intense luminescence but commonly fail to yield stable colloidal dispersions. Coupling the best of these two situations to afford highly crystalline, dispersible nanoparticles with luminescence performance exceeding bulk solids is still challenging, thus requiring new safe, scalable, and reproducible methodologies. Herein we report a silicate-coating strategy followed by aggregate elimination to recover stable colloids of 40-150 nm single crystalline rare earth vanadates after unprotected annealing (800-1000 °C). Eu3+-doped nanoparticles showed enhanced photostability and ~50% emission quantum yields in water (λexc=280 nm), while Dy3+-, Tm3+-, and Yb3+/Er3+-doped vanadates provided remarkably intense multicolour emissions via downshift or upconversion luminescence. We correlated spectroscopic properties of pristine and annealed solids to microstructural characteristics to explain the superior emission features, opening new perspectives for sensing applications. (10.1021/acs.cgd.3c00308)
DOI : 10.1021/acs.cgd.3c00308
Label-free single-cell live imaging reveals fast metabolic switch in T lymphocytes
- Paillon Noémie
- Ung Thi Phuong Lien
- Dogniaux Stéphanie
- Stringari Chiara
- Hivroz Claire
Molecular Biology of the Cell, American Society for Cell Biology , 2023 . T cell activation induces a metabolic switch generating energy for proliferation, survival, and functions. We used non-invasive label-free two-photon fluorescence lifetime microscopy (2P-FLIM) to map the spatial and temporal dynamics of the metabolic NAD(P)H co-enzyme during T lymphocyte activation. This provides a readout of the OXPHOS and glycolysis rates at a single cell level. Analyzes were performed in the CD4+ leukemic T cell line Jurkat, and in human CD4+ primary T cells. Cells were activated on glass surfaces coated with activating antibodies mimicking immune synapse formation. Comparing the fraction of bound NAD(P)H between resting and activated T cells, we show that T cell activation induces a rapid switch toward glycolysis. This occurs after 10 minutes and remains stable for one hour. Three-dimensional analyzes revealed that the intracellular distribution of fraction of bound NAD(P)H increases at the immune synapse in activated cells. Finally, we show that fraction of bound NAD(P)H tends to negatively correlate with spreading of activated T cells, suggesting a link between actin remodeling and metabolic changes. This study highlights that 2P-FLIM measurement of fraction of bound NAD(P)H is well suited to follow a fast metabolic switch in 3D, in single T lymphocytes with subcellular resolution. (10.1091/mbc.E23-01-0009)
DOI : 10.1091/mbc.E23-01-0009