Articles

Archaerhodopsin 3 is an ideal template for the engineering of highly fluorescent optogenetic reporters
- Herasymenko Krystyna
- Walisinghe Danushka
- Konno Masae
- Barneschi Leonardo
- de Waele Isabelle
- Sliwa Michel
- Inoue Keiichi
- Olivucci Massimo
- Haacke Stefan
Chemical Science, The Royal Society of Chemistry , 2024, 16 (2), pp.761-774 . Archaerhodopsin-3 (AR-3) variants stand out among other rhodopsins in that they display a weak, but voltage-sensitive, near-infrared fluorescence emission. This has led to their application in optogenetics both in cell cultures and small animals. However, in the context of improving the fluorescence characteristics of the next generation of AR-3 reporters, an understanding of their ultrafast light-response in light-adapted conditions, is mandatory. To this end, we present a combined experimental and computational investigation of the excited state dynamics and quantum yields of AR-3 and its DETC and Arch-5 variants. The latter always display a mixture of all-trans/15-anti and 13-cis/15-syn isomers, which leads to a bi-exponential excited state decay. The isomerisation quantum yield is reduced more than 20 times as compared to WT AR-3 and proves that the steady-state fluorescence is induced by a single absorption photon event. In wild-type AR-3, we show that a 300 fs, barrier-less and vibrationally coherent isomerization is driven by an unusual covalent electronic character of its all-trans retinal chromophore leading to a metastable twisted diradical (TIDIR), in clear contrast to the standard charge-transfer scenario established for other microbial rhodopsins. We discuss how the presence of TIDIR makes AR-3 an ideal candidate for the design of variants with a one-photon induced fluorescence possibly reaching the emission quantum yield of the top natural emitter neorhodopsin (NeoR). (10.1039/d4sc05120c)
DOI : 10.1039/d4sc05120c
Design, Synthesis, and In Vitro Antimalarial Evaluation of New 1,3,5-Tris[(4-(Substituted-Aminomethyl)Phenoxy)Methyl]Benzenes
- Albenque-Rubio Sandra
- Guillon Jean
- Agnamey Patrice
- Damiani Céline
- Savrimoutou Solène
- Mustière Romain
- Pinaud Noël
- Moreau Stéphane
- Mergny Jean-Louis
- Ronga Luisa
- Kanavos Ioannis
- Marchivie Mathieu
- Moukha Serge
- Dozolme Pascale
- Sonnet Pascal
- Cohen Anita
Drugs and Drug Candidates, MDPI , 2024, 3 (3), pp.615-637 . By taking into account our previously described series of 1,3,5-tris[(4-(substituted-aminomethyl)phenyl)methyl]benzene compounds, we have now designed, prepared, and evaluated in vitro against Plasmodium falciparum a novel series of structural analogues of these molecules, i.e., the 1,3,5-tris[(4-(substituted-aminomethyl)phenoxy)methyl]benzene derivatives. The pharmacological data showed antimalarial activity with IC50 values in the sub and μM range. The in vitro cytotoxicity of these new nitrogen polyphenoxymethylbenzene compounds was also evaluated on human HepG2 cells. The 1,3,5-tris[(4-(substituted-aminomethyl)phenoxy)methyl]benzene derivative 1m was found as one of the most potent and promising antimalarial candidates with favorable cytotoxic to antiprotozoal properties in the P. falciparum strains W2 and 3D7. In conclusion, this 1,3,5-tris[(4-(pyridin-3-ylmethylaminomethyl)phenoxyl)methyl]benzene 1m (IC50 = 0.07 μM on W2, 0.06 μM on 3D7, and 62.11 μM on HepG2) was identified as the most promising antimalarial derivative with selectivity indexes (SI) of 887.29 on the W2 P. falciparum chloroquine-resistant strain, and of 1035.17 on the chloroquine-sensitive and mefloquine decreased sensitivity strain 3D7. It has been previously described that the telomeres of P. falciparum could represent potential targets for these types of polyaromatic compounds; therefore, the capacity of our novel derivatives to stabilize the parasitic telomeric G-quadruplexes was assessed using a FRET melting assay. However, with regard to the stabilization of the protozoal G-quadruplex, we observed that the best substituted derivatives 1, which exhibited some interesting stabilization profiles, were not the most active antimalarial compounds against the two Plasmodium strains. Thus, there were no correlations between their antimalarial activities and selectivities of their respective binding to G-quadruplexes. (10.3390/ddc3030035)
DOI : 10.3390/ddc3030035
The Balance Between Shear Flow and Extracellular Matrix in Ovarian Cancer‐on‐Chip
- Chen Changchong
- Boché Alphonse
- Wang Zixu
- Lopez Elliot
- Peng Juan
- Carreiras Franck
- Schanne-Klein Marie‐claire
- Chen Yong
- Lambert Ambroise
- Aimé Carole
Advanced Healthcare Materials, Wiley , 2024, 13 (23), pp.2400938 . Ovarian cancer is the most lethal gynecologic cancer in developed countries. In the tumor microenvironment, the extracellular matrix (ECM) and flow shear stress are key players in directing ovarian cancer cells invasion. Artificial ECM models based only on ECM proteins are used to build an ovarian tumor‐on‐chip to decipher the crosstalk between ECM and shear stress on the migratory behavior and cellular heterogeneity of ovarian tumor cells. This work shows that in the shear stress regime of the peritoneal cavity, the ECM plays a major role in driving individual or collective ovarian tumor cells migration. In the presence of basement membrane proteins, migration is more collective than on type I collagen regardless of shear stress. With increasing shear stress, individual cell migration is enhanced; while, no significant impact on collective migration is measured. This highlights the central position that ECM and flow shear stress should hold in in vitro ovarian cancer models to deepen understanding of cellular responses and improve development of ovarian cancer therapeutic platforms. In this frame, adding flow provides significant improvement in biological relevance over the authors’ previous work. Further steps for enhanced clinical relevance require not only multiple cell lines but also patient‐derived cells and sera. (10.1002/adhm.202400938)
DOI : 10.1002/adhm.202400938
Quantitative Assessment of Collagen Remodeling during a Murine Pregnancy
- Ramella-Roman Jessica C
- Mahendroo Mala
- Raoux Clothilde
- Latour Gaël
- Schanne-Klein Marie-Claire
ACS photonics, American Chemical Society , 2024, 11 (9), pp.3536-3544 . <div><p>Uterine cervical remodeling is a fundamental feature of pregnancy, facilitating the delivery of the fetus through the cervical canal. Yet, we still know very little about this process due to the lack of methodologies that can quantitatively and unequivocally pinpoint the changes the cervix undergoes during pregnancy. We utilize polarization-resolved second harmonic generation to visualize the alterations the cervix extracellular matrix, specifically collagen, undergoes during pregnancy with exquisite resolution. This technique provides images of the collagen orientation at the pixel level (0.4 μm) over the entire murine cervical section. They show tight and ordered packing of collagen fibers around the os at the early stage of pregnancy and their disruption at the later stages. Furthermore, we utilize a straightforward statistical analysis to demonstrate the loss of order in the tissue, consistent with the loss of mechanical properties associated with this process. This work provides a deeper understanding of the parturition process and could support research into the cause of pathological or premature birth.</p></div> (10.1021/acsphotonics.4c00337)
DOI : 10.1021/acsphotonics.4c00337
Confinement energy landscape classification reveals membrane receptor nano-organization mechanisms
- Yu Chao
- Richly Maximilian
- Hoang Thi Thuy
- El Beheiry Mohammed
- Türkcan Silvan
- Masson Jean-Baptiste
- Alexandrou Antigoni
- Bouzigues Cedric I
Biophysical Journal, Biophysical Society , 2024, 123 (13), pp.1882-1895 . The cell membrane organization has an essential functional role through the control of membrane receptor confinement in micro- or nanodomains. Several mechanisms have been proposed to account for these properties, although some features have remained controversial, notably the nature, size, and stability of cholesterol- and sphingolipid-rich domains or lipid rafts. Here, we probed the effective energy landscape acting on single-nanoparticle-labeled membrane receptors confined in raft nanodomains— epidermal growth factor receptor (EGFR), Clostridium perfringens ε-toxin receptor (CPεTR), and Clostridium septicum α-toxin receptor (CSαTR)—and compared it with hop-diffusing transferrin receptors. By establishing a new analysis pipeline combining Bayesian inference, decision trees, and clustering approaches, we systematically classified single-protein trajectories according to the type of effective confining energy landscape. This revealed the existence of only two distinct organization modalities: confinement in a quadratic energy landscape for EGFR, CPεTR, and CSαTR (A), and free diffusion in confinement domains resulting from the steric hindrance due to F-actin barriers for transferrin receptor (B). The further characterization of effective confinement energy landscapes by Bayesian inference revealed the role of interactions with the domain environment in cholesterol- and sphingolipid-rich domains with (EGFR) or without (CPεTR and CSαTR) interactions with F-actin to regulate the confinement energy depth. These two distinct mechanisms result in the same organization type (A). We revealed that the apparent domain sizes for these receptor trajectories resulted from Brownian exploration of the energy landscape in a steady-state-like regime at a common effective temperature, independently of the underlying molecular mechanisms. These results highlight that confinement domains may be adequately described as interaction hotspots rather than rafts with abrupt domain boundaries. Altogether, these results support a new model for functional receptor confinement in membrane nanodomains and pave the way to the constitution of an atlas of membrane protein organization. (10.1016/j.bpj.2024.06.001)
DOI : 10.1016/j.bpj.2024.06.001
Angle-Resolved Linear Dichroism to Probe the Organization of Highly Ordered Collagen Biomaterials
- Krins Natacha
- Wien Frank
- Schmeltz Margaux
- Pérez Javier
- Dems Dounia
- Debons Nicolas
- Laberty-Robert Christel
- Schanne-Klein Marie-Claire
- Aimé Carole
Biomacromolecules, American Chemical Society , 2024, 25 (9), pp.6181-6187 . Controlling the assembly of high-order structures is central to soft-matter and biomaterial engineering. Angle-resolved linear dichroism can probe the ordering of chiral collagen molecules in the dense state. Collagen triple helices were aligned by solvent evaporation. Their ordering gives a strong linear dichroism (LD) that changes sign and intensity with varying sample orientations with respect to the beam linear polarization. Being complementary to circular dichroism, which probes the structure of chiral (bio)molecules, LD can shift from the molecular to the supramolecular scale and from the investigation of the conformation to interactions. Supported by multiphoton microscopy and X-ray scattering, we show that LD provides a straightforward route to probe collagen alignment, determine the packing density, and monitor denaturation. This approach could be adapted to any assembly of chiral (bio)macromolecules, with key advantages in detecting large-scale assemblies with high specificity to aligned and chiral molecules and improved sensitivity compared to conventional techniques. (10.1021/acs.biomac.4c00860)
DOI : 10.1021/acs.biomac.4c00860
Impact of trypsin on cell cytoplasm during detachment of cells studied by terahertz sensing
- Lordon Blandine
- Campion Tiffany
- Gibot Laure
- Gallot Guilhem
Biophysical Journal, Biophysical Society , 2024, 123 (16), pp.2476-2483 . Trypsin is a very common enzyme used in cell culture to harvest cells by cleaving the proteins responsible for cell adhesion. However, trypsin also induces undesirable effects on cells, such as altering membrane proteins and the cytoskeleton, changing the composition of the cytoplasm and the cell volume, and even leading to cell death when used improperly. Using attenuated total reflection in the terahertz domain, confocal microscopy, and the propidium iodide test, we quantified in real time the change in cytoplasmic content induced by trypsin proteolysis on Madin-Darby canine kidney epithelial cells. We have observed a cytoplasmic modification from the very first seconds of trypsinization, following the change of cell volume due to mechanical re-equilibrium of the membrane. We found that the cytoplasmic alteration is associated with a transfer of small solutes: electrolytes and metabolites. We also found a very good nonlinear correlation between the side effects monitored by terahertz sensing and the cell height, regardless of the dependence of the cell height on trypsin concentration and exposure time. (10.1016/j.bpj.2024.06.011)
DOI : 10.1016/j.bpj.2024.06.011
A Cost‐Effective Hemin‐Based Artificial Enzyme Allows for Practical Applications
- Qiu Dehui
- He Fangni
- Liu Yuan
- Zhou Zhaoxi
- Yang Yuqin
- Long Zhongwen
- Chen Qianqian
- Chen Desheng
- Wei Shijiong
- Mao Xuanxiang
- Zhang Xiaobo
- Mergny Jean‐louis
- Monchaud David
- Ju Huangxian
- Zhou Jun
Advanced Science, Wiley Open Access , 2024, 11 (32), pp.2402237 . Abstract Nanomaterials excel in mimicking the structure and function of natural enzymes while being far more interesting in terms of structural stability, functional versatility, recyclability, and large‐scale preparation. Herein, the story assembles hemin, histidine analogs, and G‐quadruplex DNA in a catalytically competent supramolecular assembly referred to as assembly‐activated hemin enzyme (AA‐heminzyme). The catalytic properties of AA‐heminzyme are investigated both in silico (by molecular docking and quantum chemical calculations) and in vitro (notably through a systematic comparison with its natural counterpart horseradish peroxidase, HRP). It is found that this artificial system is not only as efficient as HRP to oxidize various substrates (with a turnover number k cat of 115 s −1) but also more practically convenient (displaying better thermal stability, recoverability, and editability) and more economically viable, with a catalytic cost amounting to <10% of that of HRP. The strategic interest of AA‐heminzyme is further demonstrated for both industrial wastewater remediation and biomarker detection (notably glutathione, for which the cost is decreased by 98% as compared to commercial kits). (10.1002/advs.202402237)
DOI : 10.1002/advs.202402237
G-quadruplex forming motifs in the promoter region of the B-MYB proto-oncogene
- Miranda André
- Cucchiarini Anne
- Esnault Cyril
- Andrau Jean-Christophe
- Oliveira Paula
- Mergny Jean‐louis
- Cruz Carla
International Journal of Biological Macromolecules, Elsevier , 2024, 270, pp.132244 . To combat cancer, a comprehensive understanding of the molecular mechanisms and behaviors involved in carcinogenesis is crucial, as tumorigenesis is a complex process influenced by various genetic events and disease hallmarks. The B-MYB gene encodes a transcription factor involved in cell cycle regulation, survival, and differentiation in normal cells. B-MYB can be transformed into an oncogene through mutations, and abnormal expression of B-MYB has been identified in various cancers, including lung cancer, and is associated with poor prognosis. Targeting this oncogene is a promising approach for anti-cancer drug design. B-MYB has been deemed undruggable in previous reports, necessitating the search for novel therapeutic options. In this study, we found that the B-MYB gene promoter contains several G/C rich motifs compatible with G-quadruplex (G4) formation. We investigated and validated the existence of G4 structures in the promoter region of B-MYB, first in vitro using a combination of bioinformatics, biophysical, and biochemical methods, then in cell with the recently developed G4access method. (10.1016/j.ijbiomac.2024.132244)
DOI : 10.1016/j.ijbiomac.2024.132244
Non-random spatial organization of telomeres varies during the cell cycle and requires LAP2 and BAF
- Keller Debora
- Stinus Sonia
- Umlauf David
- Gourbeyre Edith
- Biot Eric
- Olivier Nicolas
- Mahou Pierre
- Beaurepaire Emmanuel
- Andrey Philippe
- Crabbe Laure
iScience, Elsevier , 2024, 27 (4), pp.109343 . Spatial genome organization within the nucleus influences major biological processes and is impacted by the configuration of linear chromosomes. Here, we applied 3D spatial statistics and modeling on high-resolution telomere and centromere 3D-structured illumination microscopy images in cancer cells. We found a multi-scale organization of telomeres that dynamically evolved from a mixed clustered-and-regular distribution in early G1 to a purely regular distribution as cells progressed through the cell cycle. In parallel, our analysis revealed two pools of peripheral and internal telomeres, the proportions of which were inverted during the cell cycle. We then conducted a targeted screen using MadID to identify the molecular pathways driving or maintaining telomere anchoring to the nuclear envelope observed in early G1. Lamina-associated polypeptide (LAP) proteins were found transiently localized to telomeres in anaphase, a stage where LAP2α initiates the reformation of the nuclear envelope, and impacted telomere redistribution in the next interphase together with their partner barrier-to-autointegration factor (BAF). (10.1016/j.isci.2024.109343)
DOI : 10.1016/j.isci.2024.109343
Catalytic mechanism of fatty acid photodecarboxylase: on the detection and stability of the initial carbonyloxy radical intermediate
- Aleksandrov Alexey
- Bonvalet Adeline
- Müller Pavel
- Sorigué Damien
- Beisson Fred
- Antonucci Laura
- Solinas Xavier
- Joffre Manuel
- Vos Marten
Angewandte Chemie International Edition, Wiley-VCH Verlag , 2024, 63 (19), pp.e202401376 . In fatty acid photodecarboxylase (FAP), light‐induced formation of the primary radical product RCOO$^●$ from fatty acid RCOO– occurs in 300 ps, upon which CO2 is released quasi‐immediately. Based on the hypothesis that aliphatic RCOO$^●$ (spectroscopically uncharacterized because unstable) absorbs in the red similarly to aromatic carbonyloxy radicals such as 2,6‑dichlorobenzoyloxy radical (DCB$^●$), much longer‐lived linear RCOO$^●$ has been suggested recently. We performed quantum chemical reaction pathway and spectral calculations. These calculations are in line with the experimental DCB$^●$decarboxylation dynamics and spectral properties and show that in contrast to DCB$^●$, aliphatic RCOO$^●$ radicals a) decarboxylate with a very low energetic barrier and on the timescale of a few ps and b) exhibit little red absorption. A time‐resolved infrared spectroscopy experiment confirms very rapid, <<300 ps RCOO$^●$ decarboxylation in FAP. We argue that this property is required for the observed high quantum yield of hydrocarbons formation by FAP. (10.1002/anie.202401376)
DOI : 10.1002/anie.202401376
Modeling and Predicting Second-Harmonic Generation from Protein Molecular Structure
- Asadipour Bahar
- Beaurepaire Emmanuel
- Zhang Xingjian
- Chessel Anatole
- Mahou Pierre
- Supatto Willy
- Schanne-Klein Marie-Claire
- Stringari Chiara
Physical Review X, American Physical Society , 2024, 14 (1), pp.011038 . (10.1103/PhysRevX.14.011038)
DOI : 10.1103/PhysRevX.14.011038
Correction of non-random mutational biases along a linear bacterial chromosome by the mismatch repair endonuclease NucS
- Dagva Oyut
- Thibessard Annabelle
- Lorenzi Jean-Noël
- Labat Victor
- Piotrowski Emilie
- Rouhier Nicolas
- Myllykallio Hannu
- Leblond Pierre
- Bertrand Claire
Nucleic Acids Research, Oxford University Press , 2024, 52 (9), pp.5033-5047 . The linear chromosome of Streptomyces exhibits a highly compartmentalized structure with a conserved central region flanked by variable arms. As double strand break (DSB) repair mechanisms play a crucial role in shaping the genome plasticity of Streptomyces, we investigated the role of EndoMS/NucS, a recently characterized endonuclease involved in a non-canonical mismatch repair (MMR) mechanism in archaea and actinobacteria, that singularly corrects mismatches by creating a DSB. We showed that Streptomyces mutants lacking NucS display a marked colonial phenotype and a drastic increase in spontaneous mutation rate. In vitro biochemical assays revealed that NucS cooperates with the replication clamp to efficiently cleave G/T, G/G and T/T mismatched DNA by producing DSBs. These findings are consistent with the transition-shifted mutational spectrum observed in the mutant strains and reveal that NucS-dependent MMR specific task is to eliminate G/T mismatches generated by the DNA polymerase during replication. Interestingly, our data unveil a crescent-shaped distribution of the transition frequency from the replication origin towards the chromosomal ends, shedding light on a possible link between NucS-mediated DSBs and Streptomyces genome evolution. (10.1093/nar/gkae132)
DOI : 10.1093/nar/gkae132
G-quadruplex ligands in cancer therapy: Progress, challenges, and clinical perspectives
- Figueiredo Joana
- Mergny Jean-Louis
- Cruz Carla
Life Sciences, Elsevier , 2024, 340, pp.122481 . Guanine-rich sequences can form G-quadruplexes (G4) in living cells, making these structures promising anti- cancer targets. Compounds able to recognize these structures have been investigated as potential anticancer drugs; however, no G4 binder has yet been approved in the clinic. Here, we describe G4 ligands structure-activity relationships, in vivo effects as well as clinical trials. Addressing G4 ligand characteristics, targeting challenges, and structure-activity relationships, this review provides insights into the development of potent and selective G4-targeting molecules for therapeutic applications. (10.1016/j.lfs.2024.122481)
DOI : 10.1016/j.lfs.2024.122481
Chirped pulse upconversion for femtosecond mid-infrared spectroscopy at 100 kHz
- Jonušas Mindaugas
- Bournet Quentin
- Bonvalet Adeline
- Natile Michele
- Guichard Florent
- Zaouter Yoann
- Georges Patrick
- Druon Frédéric
- Hanna Marc
- Joffre Manuel
Optics Express, Optical Society of America - OSA Publishing , 2024, 32 (5), pp.8020 . We demonstrate that chirped pulse up-conversion (CPU), a method routinely used with systems based on 1-kHz Titanium:Sapphire lasers, can be extended to a repetition rate of 100 kHz with an Ytterbium diode-pumped femtosecond amplifier. Individual mid-infrared spectra can thus be measured directly in the near infrared using a fast CMOS linescan camera. After an appropriate Fourier processing, a spectral resolution of 1.1 cm-1 is reported, currently limited by our spectrometer. Additionally, we demonstrate the application of CPU to a pump-probe measurement of the vibrational relaxation in carboxy-hemoglobin, and we show that the combination of fast scanning and fast acquisition enables a straightforward removal of pump scattering interference. (10.1364/oe.515291)
DOI : 10.1364/oe.515291
Preferential formation of Z-RNA over intercalated motifs in long noncoding RNA
- Bhatt Uditi
- Cucchiarini Anne
- Luo Yu
- Evans Cameron
- Mergny Jean-Louis
- Iyer K. Swaminathan
- Smith Nicole
Genome Research, Cold Spring Harbor Laboratory Press , 2024, pp.gr.278236.123 . Secondary structure is a principal determinant of lncRNA function, predominantly regarding scaffold formation and interfaces with target molecules. Noncanonical secondary structures that form in nucleic acids have known roles in regulating gene expression and include G-quadruplexes (G4s), intercalated-motifs (iMs), and R-loops (RLs). In this paper, we utilized computational tools G4-iM Grinder and QmRLFS-finder to predict the formation of each of these structures throughout the lncRNA transcriptome in comparison to protein-coding transcripts. The importance of the predicted structures in lncRNA in biological contexts was assessed by combining our results with publicly available lncRNA tissue expression data followed by pathway analysis. The formation of predicted G4 (pG4) and iM (piM) structures in select lncRNA sequences was determined in vitro using biophysical experiments under near-physiological conditions. We found that the majority of the tested pG4s form highly stable G4 structures and identify many previously unreported G4s in biologically important lncRNAs. In contrast, none of the piM sequences were able to form iM structures, consistent with the idea that RNA is unable to form iMs. These C-rich sequences instead formed Z-RNA structures, which have not been previously observed in regions containing cytosine repeats and represent an interesting and under-explored target for protein-RNA interactions. Our results highlight the prevalence and potential structure-associated functions of noncanonical secondary structures in lncRNA and observe G4 and Z-RNA structure formation in many lncRNA sequences for the first time, furthering understanding of the structure-function relationship in lncRNAs. (10.1101/gr.278236.123)
DOI : 10.1101/gr.278236.123
Maximizing the efficiency of intrapulse difference frequency generation by pulse shaping and recycling
- Bournet Quentin
- Jonusas Mindaugas
- Guichard Florent
- Natile Michele
- Zaouter Yoann
- Joffre Manuel
- Bonvalet Adeline
- Druon Fréderic
- Hanna Marc
- Georges Patrick
Applied Physics B - Laser and Optics, Springer Verlag , 2024, 130 (2), pp.33 . <div><p>Intrapulse Difference Frequency Generation (iDFG) is an interesting technique for generating femtosecond pulses in the Mid-Infrared (MIR) range with unique properties such as robust Carrier-Envelope Phase (CEP) stability. However, its efficiency is low compared to other techniques. In this paper, we describe an iDFG system operating within the 4 to 10 µm range that features an original architecture to enhance efficiency. First, we introduce an interesting technique on the generation process. This approach involves polarization and spectral phase shaping techniques on the driving pulse to maximize the number of photons enrolled in the process. Second, we demonstrate that the polarization shaping allows further enhancement of efficiency by recycling the iDFG signal to pump a subsequent optical parametric amplification (OPA) stage. These two concepts and the associated parameters optimization are described into details, and supported by experimental results. Combined with a high-power Yb-fiber-based pump laser, these techniques allow to achieve record efficiencies, and generate µJ-level, fewcycle, tunable, CEP-stable pulses in the MIR at repetition rates above 100 kHz.</p></div> (10.1007/s00340-023-08162-0)
DOI : 10.1007/s00340-023-08162-0
Abundance of G-Quadruplex Forming Sequences in the Hepatitis Delta Virus Genomes
- Brázda Václav
- Valková Natália
- Dobrovolná Michaela
- Mergny Jean-Louis
ACS Omega, ACS Publications , 2024, 9, pp.4096-4101 . Hepatitis Delta virus (HDV) is a unique and highly unusual RNA satellite virus that depends on the presence of the hepatitis B virus (HBV) to be infectious. Its single-stranded RNA genome is very compact and variable, consisting of eight major genotypes distributed unequally on various continents. The significance of noncanonical secondary structures in DNA or RNA, such as G-quadruplexes (G4s), is becoming more evident especially for transcription, replication, and translation. G4s are formed from guanine-rich sequences and have been found in most eukaryotic and prokaryotic genomes, as well as viruses. In this study, we analyzed the G-quadruplex propensity of HDV genomes using G4Hunter. In contrast to the HBV virus, which has a G4 density similar to that of the human genome, the HDV virus possesses a significantly higher number of potential quadruplex-forming sequences (PQS) with a density more than four times higher than that of the human genome. This observation suggests a crucial role for G-quadruplexes in HDV, particularly because the tracks with the potential to form G-quadruplexes are well conserved. Furthermore, the high prevalence of the G-quadruplex-forming sequence could represent a promising therapeutic target to control HDV replication. (10.1021/acsomega.3c09288)
DOI : 10.1021/acsomega.3c09288
Arginine-Modified Hemin Enhances G-Quadruplex DNAzyme Peroxidase Activity for High Sensitivity Detection
- Liu Bin
- Wang Tian
- Qiu Dehui
- Yan Xinrong
- Liu Yuan
- Mergny Jean-Louis
- Zhang Xiaobo
- Monchaud David
- Ju Huangxian
- Zhou Jun
Analytical Chemistry, American Chemical Society , 2024, 96 (36), pp.14590-14597 . Hemin/G-quadruplex (hG4) complexes are frequently used as artificial peroxidase-like enzymatic systems (referred to as G4 DNAzymes) in many biosensing applications, in spite of a rather low efficiency, notably in terms of detection limits. To tackle this issue, we report herein on a strategy in which hemin is chemically modified with the amino acids found in the active site of parent horseradish peroxidase (HRP), in the aim of recreating an environment conducive to a high catalytic activity. When hemin conjugated with a single arginine, it associates with G4 to create an arginine-hemin/G4 (R-hG4) DNAzyme that exhibits improved catalytic performances, characterized by kinetic analysis and DFT calculations. The practical relevance of this system was demonstrated with the implementation of biosensing assays enabling the chemiluminescent detection of G4-containing DNA and colorimetry detection of FEN1 enzyme with a high efficiency and sensitivity. Our results thus provide a guide for future enzyme engineering campaigns to create ever more efficient peroxidase-mimicking DNA-based systems. (10.1021/acs.analchem.4c03013)
DOI : 10.1021/acs.analchem.4c03013
Broadband rapid-scanning phase-modulated Fourier transform electronic spectroscopy
- Javed Ariba
- Lüttig Julian
- Sanders Stephanie
- Sessa Francesco
- Gardiner Alastair
- Joffre Manuel
- Ogilvie Jennifer
Optics Express, Optical Society of America - OSA Publishing , 2024, 32 (16), pp.28035 . We present a phase-modulated approach for ultrabroadband Fourier transform electronic spectroscopy. To overcome the bandwidth limitations and spatial chirp introduced by acousto-optic modulators (AOMs), pulses from a 1 µm laser are modulated using AOMs prior to continuum generation. This phase modulation is transferred to the continuum generated in a yttrium aluminum garnet crystal. Separately generated phase-modulated continua in two arms of a Mach-Zehnder interferometer interfere with the difference of their modulation frequencies, enabling physical under-sampling of the signal and the suppression of low-frequency noise. By interferometrically tracking the relative time delay of the continua, we perform continuous, rapid-scanning Fourier transform electronic spectroscopy with a high signal-to-noise ratio and spectral resolution. As proof of principle, we measure the linear absorption and fluorescence excitation spectra of a laser dye and various biological samples. (10.1364/OE.530991)
DOI : 10.1364/OE.530991
Multiplexed Orthogonal Polarizations Harmonic Imaging for Probing Dynamic Biological Processes
- Gleeson Matthew
- Escot Sophie
- Solinas Xavier
- Asadipour Bahar
- Supatto Willy
- Mahou Pierre
- Stringari Chiara
- Beaurepaire Emmanuel
ACS photonics, American Chemical Society , 2024, 11 (8), pp.3259-3266 . Polarization-resolved nonlinear optical microscopy can provide label-free images of anisotropic biomaterials. Existing techniques are often limited by their complexity, chromaticity, or slowness. In this article, we propose a simple implementation based on the passive splitting of the excitation beam into slightly delayed orthogonally polarized pulse trains. Label-free second- and third-harmonic signals corresponding to each polarization state are separated by time-resolved photon counting, resulting in the acquisition of image pairs that are automatically registered with submicron spatial precision and nanosecond simultaneity. We demonstrate the utility of this small-footprint approach for live studies by presenting structural imaging of tubulin, myelinated axons, and blood vessels in zebrafish larvae, dynamic imaging of flowing microcrystals in the zebrafish larval otolith cavity, and mitotic spindles in dividing embryonic cells. (10.1021/acsphotonics.4c00660)
DOI : 10.1021/acsphotonics.4c00660
Inactivating negative regulators of cortical branched actin enhances persistence of single cell migration
- Fokin Artem I
- Boutillon Arthur
- James John
- Courtois Laura
- Vacher Sophie
- Simanov Gleb
- Wang Yanan
- Polesskaya Anna
- Bièche Ivan
- David Nicolas B
- Gautreau Alexis M
Journal of Cell Science, Company of Biologists , 2024, 137 (1), pp.jcs261332 . The Rac1-WAVE-Arp2/3 pathway pushes the plasma membrane by polymerizing branched actin, thereby powering membrane protrusions that mediate cell migration. Here, using knock-down (KD) or knock-out (KO), we combine the inactivation of the Arp2/3 inhibitory protein Arpin, the Arp2/3 subunit ARPC1A and the WAVE complex subunit, CYFIP2, that all enhance the polymerization of cortical branched actin (CBA). Inactivation of the 3 CBA negative regulators increases migration persistence of human breast MCF10A cells, and of endodermal cells in the zebrafish embryo, significantly more than any single or double inactivation. In the triple KO, but not triple KD cells, the “super-migrator” phenotype was associated with a heterogenous down-regulation of vimentin expression and a lack of coordination in collective behaviors, such as wound healing and acinus morphogenesis. Re-expression of vimentin in triple KO cells restored to a large extent normal persistence of single cell migration, suggesting that vimentin down-regulation contributes to the maintenance of the super-migrator phenotype in triple KO cells. Constant excessive production of branched actin at the cell cortex thus commits cells into a motile state through changes in gene expression. (10.1242/jcs.261332)
DOI : 10.1242/jcs.261332
Emerging Functional Connections Between Metabolism and Epigenetic Remodeling in Neural Differentiation
- Sánchez-Ramírez Edgar
- Ung Thi Phuong Lien
- Stringari Chiara
- Aguilar-Arnal Lorena
Molecular Neurobiology, Springer , 2024 . Stem cells possess extraordinary capacities for self-renewal and differentiation, making them highly valuable in regenerative medicine. Among these, neural stem cells (NSCs) play a fundamental role in neural development and repair processes. NSC characteristics and fate are intricately regulated by the microenvironment and intracellular signaling. Interestingly, metabolism plays a pivotal role in orchestrating the epigenome dynamics during neural differentiation, facilitating the transition from undifferentiated NSC to specialized neuronal and glial cell types. This intricate interplay between metabolism and the epigenome is essential for precisely regulating gene expression patterns and ensuring proper neural development. This review highlights the mechanisms behind metabolic regulation of NSC fate and their connections with epigenetic regulation to shape transcriptional programs of stemness and neural differentiation. A comprehensive understanding of these molecular gears appears fundamental for translational applications in regenerative medicine and personalized therapies for neurological conditions. (10.1007/s12035-024-04006-w)
DOI : 10.1007/s12035-024-04006-w
Synergistic effect of Gemcitabin-loaded metal organic frameworks nanoparticles with particle therapy
- Maury Pauline
- Hirayama Ryoichi
- Li Xue
- Mahou Pierre
- Porcel Erika
- Schanne-Klein Marie-Claire
- Lacombe Sandrine
- Gref Ruxandra
- Porcel Erika
International Journal of Pharmaceutics, Elsevier , 2024, 665, pp.124721 . Combination of nanoagents with radiations has opened up new perspectives in cancer treatment, improving both tumor diagnosis and therapeutic index. This work presents the first investigation of an innovative strategy that combines porous metal-organic frameworks (nanoMOFs) loaded with the anti-cancer drug Gemcitabine monophosphate (GemMP) and particle therapy-a globally emerging technique that offers more precise radiation targeting and enhanced biological efficacy compared to conventional radiotherapy. This radiochemotherapy has been confronted with two major obstacles limiting the efficacy of therapeutics when tested in vivo: (i) the presence of hypoxia, one of the most important causes for radiotherapy failure and (ii) the presence of a microenvironment, main biological barrier to the direct penetration of nanoparticles into cancer cells. On the one hand, this study explore the effects of hypoxia on drug delivery systems in combination with radiation, demonstrating that GemMP-loaded nanoMOFs significantly enhance the anticancer efficacy of particle therapy under both normoxic (pO<sub>2</sub> = 20 %) and hypoxic (pO<sub>2</sub> = 0.5 %) conditions. Notably, the presence of GemMP-loaded nanoMOFs allows the irradiation dose to be reduced by 1.4-fold in normoxia and at least 1.6-fold in hypoxia, achieving the same cytotoxic effect (SF=10 %) as carbon or helium ions alone. Synergistic effects between GemMP-loaded nanoMOFs and radiations have been observed and quantified. On the other hand, we also highlighted the ability of the nanoMOFs to diffuse through an extracellular matrix and accumulate in cells. An higher effect of the encapsulated GemMP than the free drug was observed, confirming the key role of the nanoMOFs in transporting the active substance to the cancer cells as a Trojan horse. This paves the way to the design of "all-in-one" nanodrugs where each component plays a role in the optimization of cancer therapy to maximize cytotoxic effects on hypoxic tumor cells while minimizing toxicity on healthy tissue. (10.1016/j.ijpharm.2024.124721)
DOI : 10.1016/j.ijpharm.2024.124721
In-cell NMR suggests that DNA i-motif levels are strongly depleted in living human cells
- Víšková Pavlína
- Ištvánková Eva
- Ryneš Jan
- Džatko Šimon
- Loja Tomáš
- Živković Martina Lenarčič
- Rigo Riccardo
- El-Khoury Roberto
- Serrano-Chacón Israel
- Damha Masad J
- González Carlos
- Mergny Jean-Louis
- Foldynová-Trantírková Silvie
- Trantírek Lukáš
Nature Communications, Nature Publishing Group , 2024, 15 (1), pp.1992 . I-Motifs (iM) are non-canonical DNA structures potentially forming in the accessible, single-stranded, cytosine-rich genomic regions with regulatory roles. Chromatin, protein interactions, and intracellular properties seem to govern iM formation at sites with i-motif formation propensity (iMFPS) in human cells, yet their specific contributions remain unclear. Using in-cell NMR with oligonucleotide iMFPS models, we monitor iM-associated structural equilibria in asynchronous and cell cycle-synchronized HeLa cells at 37 °C. Our findings show that iMFPS displaying pH<sub>T</sub> < 7 under reference in vitro conditions occur predominantly in unfolded states in cells, while those with pH<sub>T</sub> > 7 appear as a mix of folded and unfolded states depending on the cell cycle phase. Comparing these results with previous data obtained using an iM-specific antibody (iMab) reveals that cell cycle-dependent iM formation has a dual origin, and iM formation concerns only a tiny fraction (possibly 1%) of genomic sites with iM formation propensity. We propose a comprehensive model aligning observations from iMab and in-cell NMR and enabling the identification of iMFPS capable of adopting iM structures under physiological conditions in living human cells. Our results suggest that many iMFPS may have biological roles linked to their unfolded states (10.1038/s41467-024-46221-y)
DOI : 10.1038/s41467-024-46221-y