Articles

Daily life in the Open Biologist’s second job, as a Data Curator
- Scorza Livia C.T.
- Zieliński Tomasz
- Kalita Irina
- Lepore Alessia
- El Karoui Meriem
- Millar Andrew J
Wellcome Open Research, F1000Research , 2024, 9, pp.523 . Background: Data reusability is the driving force of the research data life cycle. However, implementing strategies to generate reusable data from the data creation to the sharing stages is still a significant challenge. Even when datasets supporting a study are publicly shared, the outputs are often incomplete and/or not reusable. The FAIR (Findable, Accessible, Interoperable, Reusable) principles were published as a general guidance to promote data reusability in research, but the practical implementation of FAIR principles in research groups is still falling behind. In biology, the lack of standard practices for a large diversity of data types, data storage and preservation issues, and the lack of familiarity among researchers are some of the main impeding factors to achieve FAIR data. Past literature describes biological curation from the perspective of data resources that aggregate data, often from publications. Methods: Our team works alongside data-generating, experimental researchers so our perspective aligns with publication authors rather than aggregators. We detail the processes for organizing datasets for publication, showcasing practical examples from data curation to data sharing. We also recommend strategies, tools and web resources to maximize data reusability, while maintaining research productivity. Conclusion: We propose a simple approach to address research data management challenges for experimentalists, designed to promote FAIR data sharing. This strategy not only simplifies data management, but also enhances data visibility, recognition and impact, ultimately benefiting the entire scientific community (10.12688/wellcomeopenres.22899.1)
DOI : 10.12688/wellcomeopenres.22899.1
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
The circularly permuted globin domain of Androglobin exhibits atypical heme stabilization and nitric oxide interaction
- Reeder Brandon J
- Deganutti Giuseppe
- Ukeri John
- Atanasio Silvia
- Svistunenko Dimitri A
- Ronchetti Christopher
- Mobarec Juan Carlos
- Welbourn Elizabeth
- Asaju Jeffrey
- Vos Marten H
- Wilson Michael T
- Reynolds Christopher A
Chemical Science, The Royal Society of Chemistry , 2024, 15 (18), pp.6738-6751 . In the decade since the discovery of androglobin, a multi-domain hemoglobin of metazoans associated with ciliogenesis and spermatogenesis, there has been little advance in the knowledge of the biochemical and structural properties of this unusual member of the hemoglobin superfamily. Using a method for aligning remote homologues, coupled with molecular modelling and molecular dynamics, we have identified a novel structural alignment to other hemoglobins. This has led to the first stable recombinant expression and characterization of the circularly permuted globin domain. Exceptional for eukaryotic globins is that a tyrosine takes the place of the highly conserved phenylalanine in the CD1 position, a critical point in stabilizing the heme. A disulfide bond, similar to that found in neuroglobin, forms a closed loop around the heme pocket, taking the place of androglobin’s missing CD loop and further supporting the heme pocket structure. Highly unusual in the globin superfamily is that the heme iron binds nitric oxide as a five-coordinate complex similar to other heme proteins that have nitric oxide storage functions. With rapid autoxidation and high nitrite reductase activity, the globin appears to be more tailored toward nitric oxide homeostasis or buffering. The use of our multi-template profile alignment method to yield the first biochemical characterisation of the circularly permuted globin domain of androglobin expands our knowledge of the fundamental functioning of this elusive protein and provides a pathway to better define the link between the biochemical traits of androglobin with proposed physiological functions. (10.1039/D4SC00953C)
DOI : 10.1039/D4SC00953C
Structure of a DNA G-quadruplex that Modulates SP1 Binding Sites Architecture in HIV-1 Promoter
- de Rache Aurore
- Marquevielle Julien
- Bouaziz Serge
- Vialet Brune
- Andreola Marie-Line
- Mergny Jean-Louis
- Amrane Samir
Journal of Molecular Biology, Elsevier , 2024, 436 (2), pp.168359 . Nucleic acid sequences containing guanine tracts are able to form non-canonical DNA or RNA structures known as G-quadruplexes (or G4s). These structures, based on the stacking of G-tetrads, are involved in various biological processes such as gene expression regulation. Here, we investigated a G4 forming sequence, HIVpro2, derived from the HIV-1 promoter. This motif is located 60 nucleotides upstream of the proviral Transcription Starting Site (TSS) and overlaps with two SP1 transcription factor binding sites. Using NMR spectroscopy, we determined that HIVpro2 forms a hybrid type G4 structure with a core that is interrupted by a single nucleotide bulge. An additional reverse-Hoogsteen AT base pair is stacked on top of the tetrad. SP1 transcription factor is known to regulate transcription activity of many genes through the recognition of Guanine-rich duplex motifs. Here, the formation of HIVpro2 G4 may modulate SP1 binding sites architecture by competing with the formation of the canonical duplex structure. Such DNA structural switch potentially participates to the regulation of viral transcription and may also interfere with HIV-1 reac- tivation or viral latency. (10.1016/j.jmb.2023.168359)
DOI : 10.1016/j.jmb.2023.168359
Mesure de la transparence cornéenne par l’analyse d’images oct
- Plamann Karsten
- Vilbert Maëlle
- Bocheux Romain
- Georgeon Cristina
- Borderie Vincent
- Pernot Pascal
- Irsch Kristina
Photoniques, EDP Sciences , 2024 (127), pp.46-51 . La cornée est la première des deux lentilles de l’oeil. La transparence de la cornée saine est due à sa structure très régulière qui peut être perturbée en présence de pathologies. Pour diagnostiquer la transparence cornéenne, nous avons développé des méthodes basées sur l’analyse d’images obtenues par tomographie par cohérence optique (OCT) qui permettent d’obtenir des valeurs physiques comme le libre parcours moyen des photons et le pourcentage de transmission cohérente de la lumière, qui impactent la vision. (10.1051/photon/202412746)
DOI : 10.1051/photon/202412746
The control of nitric oxide dynamics and interaction with substituted zinc-phthalocyanines
- Ben Brahim Nassim
- Touaiti Sarra
- Sellés Julien
- Lambry Jean-Christophe
- Negrerie Michel
Dalton Transactions, Royal Society of Chemistry , 2024, 53 (2), pp.772-780 . Phthalocyanines are artificial macrocycles that can harbour a central metal atom with four symmetric coordinations. Similar to metal-porphyrins, metal-phthalocyanines (M-PCs) may bind small molecules, especially diatomic gases such as NO and O2. Furthermore, various chemical chains can be grafted at the periphery of the M-PC macrocycle, which can change its properties, including the interaction with diatomic gases. In this study, we synthesized Zn-PCs with two different substituents and investigated their effects on the interaction and dynamics of nitric oxide (NO). Time-resolved absorption spectroscopy from picosecond to millisecond revealed that NO dynamics dramatically depends on the nature of the groups grafted to the Zn-PC macrocycle. These experimental results were rationalized by DFT calculations, which demonstrate that electrostatic interactions between NO and the quinoleinoxy substituent modify the potential energy surface and decrease the energy barrier for NO recombination, thus controlling its affinity. (10.1039/d3dt03356b)
DOI : 10.1039/d3dt03356b
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
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 . <p align="justify">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.</p> (10.3390/ddc3030035)
DOI : 10.3390/ddc3030035
DNA Quadruplex Structure with a Unique Cation Dependency
- Gajarsky Martin
- Stadlbauer Petr
- Sponer Jiri
- Cucchiarini Anne
- Dobrovolna Michaela
- Brazda Vaclav
- Mergny Jean-Louis
- Trantirek Lukas
- Lenarcic Zivkovic Martina
Angewandte Chemie International Edition, Wiley-VCH Verlag , 2023 . DNA quadruplex structures provide an additional layer of regulatory control in genome maintenance and gene expression and are widely used in nanotechnology. We report the discovery of a novel tetrastranded structure formed from a native G‐rich DNA sequence originating from the telomeric region of Caenorhabditis elegans. The structure is defined by multiple properties that distinguish it from all other known DNA quadruplexes. Most notably, the formation of a stable so‐called KNa‐quadruplex (KNaQ) requires concurrent coordination of K+ and Na+ ions at two distinct binding sites. This structure provides novel insight into G‐rich DNA folding under ionic conditions relevant to eukaryotic cell physiology and the structural evolution of telomeric DNA. It highlights the differences between the structural organization of human and nematode telomeric DNA, which should be considered when using C. elegans as a model in telomere biology, particularly in drug screening applications. Additionally, the absence/presence of KNaQ motifs in the host/parasite introduces an intriguing possibility of exploiting the KNaQ fold as a plausible antiparasitic drug target. The structure's unique shape and ion dependency and the possibility of controlling its folding by using low‐molecular‐weight ligands can be exploited for the design or discovery of novel recognition DNA elements and sensors. (10.1002/anie.202313226)
DOI : 10.1002/anie.202313226
Strong and selective interactions of palmatine with G-rich sequences in TRF2 promoter; experimental and computational studies
- Fazelifar Pegah
- Cucchiarini Anne
- Khoshbin Zahra
- Mergny Jean-Louis
- Kazemi Noureini Sakineh
Journal of Biomolecular Structure and Dynamics, Taylor & Francis: STM, Behavioural Science and Public Health Titles , 2023, pp.in press . Background: G-rich sequences have the potential to fold into G-quadruplexes (GQs). G-quadruplexes, particularly those positioned in the regulatory regions of proto-oncogenes, have recently garnered attention in anti-cancer drug design. Methods: A thermal FRET assay was employed to conduct preliminary screening of various alkaloids, aiming to identify stronger interactions with a specific set of G-rich double-labeled oligonucleotides in both K+ and Na+ buffers. These oligonucleotides were derived from regions associated with Kit, Myc, Ceb, Bcl2, human telomeres, and potential G-quadruplex forming sequences found in the Nrf2 and Trf2 promoters. Palmatine generally increased the stability of different G-rich sequences into their folded GQ structures, more or less in a concentration dependent manner. The thermal stability and interaction of palmatine was further studied using transition FRET (t-FRET), CD and UV-visible spectroscopy and molecular dynamics simulation methods. Results: Palmatine showed the strongest interaction with TRF2 in both K+ and Na+ buffers even at equimolar concentration ratio. T-FRET studies revealed that palmatine has the potential to disrupt double-strand formation by the TRF2 sequence in the presence of its complementary strand. Palmatine exhibits a stronger interaction with G-rich strand DNA, promoting its folding into G-quadruplex structures. It is noteworthy that palmatine exhibits the strongest interaction with TRF2, which is the shortest sequence among the G-rich oligonucleotides studied, featuring only one nucleotide for two of its loops (10.1080/07391102.2023.2292793)
DOI : 10.1080/07391102.2023.2292793
Harnessing G-quadruplex ligands for lung cancer treatment: A comprehensive overview
- Figueiredo Joana
- Djavaheri-Mergny Mojgan
- Ferret Lucille
- Mergny Jean-Louis
- Cruz Carla
Drug Discovery Today, Elsevier , 2023, 28 (12), pp.103808 . Lung cancer (LC) remains a leading cause of mortality worldwide, and new therapeutic strategies are urgently needed. One such approach revolves around the utilization of four-stranded nucleic acid secondary structures, known as G-quadruplexes (G4), which are formed by G-rich sequences. G4 structures constitute enticing targets for therapeutic intervention. Ligands that bind selectively to G4 structures, present a promising strategy for regulating crucial cellular processes involved in the progression of LC, rendering them potent agents for lung cancer treatment. In this review, we offer a summary of the recent advancements in the development of ligands capable of targeting specific genes associated with the development and progression of lung cancer. (10.1016/j.drudis.2023.103808)
DOI : 10.1016/j.drudis.2023.103808
The future of CRISPR in Mycobacterium tuberculosis infection
- Zein Eddine Rima
- Refrégier Guislaine
- Cervantes Jorge
- Yokobori Noemí Kaoru
Journal of Biomedical Science, BioMed Central , 2023, 30 (1), pp.34 . Clustered Regularly Interspaced Short Palindromic repeats (CRISPR)-Cas systems rapidly raised from a bacterial genetic curiosity to the most popular tool for genetic modifications which revolutionized the study of microbial physiology. Due to the highly conserved nature of the CRISPR locus in Mycobacterium tuberculosis , the etiological agent of one of the deadliest infectious diseases globally, initially, little attention was paid to its CRISPR locus, other than as a phylogenetic marker. Recent research shows that M. tuberculosis has a partially functional Type III CRISPR, which provides a defense mechanism against foreign genetic elements mediated by the ancillary RNAse Csm6. With the advent of CRISPR-Cas based gene edition technologies, our possibilities to explore the biology of M. tuberculosis and its interaction with the host immune system are boosted. CRISPR-based diagnostic methods can lower the detection threshold to femtomolar levels, which could contribute to the diagnosis of the still elusive paucibacillary and extrapulmonary tuberculosis cases. In addition, one-pot and point-of-care tests are under development, and future challenges are discussed. We present in this literature review the potential and actual impact of CRISPR-Cas research on human tuberculosis understanding and management. Altogether, the CRISPR-revolution will revitalize the fight against tuberculosis with more research and technological developments. (10.1186/s12929-023-00932-4)
DOI : 10.1186/s12929-023-00932-4
Fluorescence to measure light intensity
- Lahlou Aliénor
- Tehrani Hessam Sepasi
- Coghill Ian
- Shpinov Yuriy
- Mandal Mrinal
- Plamont Marie-Aude
- Aujard Isabelle
- Niu Yuxi
- Nedbal Ladislav
- Lazár Dusan
- Mahou Pierre
- Supatto Willy
- Beaurepaire Emmanuel
- Eisenmann Isabelle
- Desprat Nicolas
- Croquette Vincent
- Jeanneret Raphaël
- Le Saux Thomas
- Jullien Ludovic
Nature Methods, Nature Publishing Group , 2023, 20, pp.1930–1938 . Abstract Despite the need for quantitative measurements of light intensity across many scientific disciplines, existing technologies for measuring light dose at the sample of a fluorescence microscope cannot simultaneously retrieve light intensity along with spatial distribution over a wide range of wavelengths and intensities. To address this limitation, we developed two rapid and straightforward protocols that use organic dyes and fluorescent proteins as actinometers. The first protocol relies on molecular systems whose fluorescence intensity decays and/or rises in a monoexponential fashion when constant light is applied. The second protocol relies on a broad-absorbing photochemically inert fluorophore to back-calculate the light intensity from one wavelength to another. As a demonstration of their use, the protocols are applied to quantitatively characterize the spatial distribution of light of various fluorescence imaging systems, and to calibrate illumination of commercially available instruments and light sources. (10.1038/s41592-023-02063-y)
DOI : 10.1038/s41592-023-02063-y
A Versatile G‐quadruplex (G4)‐coated Upconverted Metal‐Organic Framework for Hypoxic Tumor Therapy
- Mao Xuanxiang
- Zhang Xiaobo
- Chao Zhicong
- Qiu Dehui
- Wei Shijiong
- Luo Rengan
- Chen Desheng
- Zhang Yue
- Chen Yun
- Yang Yuanjiao
- Monchaud David
- Ju Huangxian
- Mergny Jean‐louis
- Lei Jianping
- Zhou Jun
Advanced Healthcare Materials, Wiley , 2023, 12 (28), pp.2300561 . Given the complexity of the tumor microenvironment, multiple strategies are being explored to tackle hypoxic tumors. One of the most efficient strategies combines several therapeutic modalities and typically requires the development of multifunctional nanocomposites through sophisticated synthetic procedures. Here, the G-quadruplex (G4)-forming sequence AS1411-A (d-(G2T)4TG(TG2)4A) was designed and used for its anti-tumor and biocatalytic properties, such as increasing the production of O2 ca. 2fold as compared to the parent AS1411 sequence. Subsequently, the AS1411-A/hemin complex (GH) was grafted on the surface and pores of a core-shell upconverted metalorganic framework (UMOF) to generate a UMGH nanoplatform. Compared with UMOF, UMGH exhibited enhanced colloidal stability, increased targeting of tumor cells and improved O2 production (8.5-fold) in situ. When irradiated with near-infrared (NIR) light, the UMGH antitumor properties were bolstered by photodynamic therapy (PDT), thanks to its ability to convert O2 into singlet oxygen (1 O2). Combined with the antiproliferative activity of AS1411-A, this novel approach herein lays the foundation for a new type of G4-based nanomedicine. (10.1002/adhm.202300561)
DOI : 10.1002/adhm.202300561
Chromatically Corrected Multicolor Multiphoton Microscopy
- Blanc Hugo
- Kaddour Gabriel
- David Nicolas B
- Supatto Willy
- Livet Jean
- Beaurepaire Emmanuel
- Mahou Pierre
ACS photonics, American Chemical Society , 2023 . Simultaneous imaging of multiple labels in tissues is key to studying complex biological processes. Although strategies for color multiphoton excitation have been established, chromatic aberration remains a major problem when multiple excitation wavelengths are used in a scanning microscope. Chromatic aberration introduces a spatial shift between the foci of beams of different wavelengths that varies across the field of view, severely degrading the performance of color imaging. In this work, we propose an adaptive correction strategy that solves this problem in two-beam microscopy techniques. Axial chromatic aberration is corrected by a refractive phase mask that introduces pure defocus into one beam, while lateral chromatic aberration is corrected by a piezoelectric mirror that dynamically compensates for lateral shifts during scanning. We show that this lightefficient approach allows seamless chromatic correction over the entire field of view of different multiphoton objectives without compromising spatial and temporal resolution and that the effective area for beam-mixing processes can be increased by more than 1 order of magnitude. We illustrate this approach with simultaneous three-color, two-photon imaging of developing zebrafish embryos and fixed Brainbow mouse brain slices over large areas. These results establish a robust and efficient method for chromatically corrected multiphoton imaging. (10.1021/acsphotonics.3c01104)
DOI : 10.1021/acsphotonics.3c01104
A new method for in vivo assessment of corneal transparency using spectral-domain OCT
- Vilbert Maëlle
- Bocheux Romain
- Georgeon Cristina
- Borderie Vincent
- Pernot Pascal
- Irsch Kristina
- Plamann Karsten
PLoS ONE, Public Library of Science , 2023, 18 (10), pp.e0291613 . Corneal transparency is essential to provide a clear view into and out of the eye, yet clinical means to assess such transparency are extremely limited and usually involve a subjective grading of visible opacities by means of slit-lamp biomicroscopy. Here, we describe an automated algorithm allowing extraction of quantitative corneal transparency parameters with standard clinical spectral-domain optical coherence tomography (SD-OCT). Our algorithm employs a novel pre-processing procedure to standardize SD-OCT image analysis and to numerically correct common instrumental artifacts before extracting mean intensity stromal-depth ( z ) profiles over a 6-mm-wide corneal area. The z -profiles are analyzed using our previously developed objective method that derives quantitative transparency parameters directly related to the physics of light propagation in tissues. Tissular heterogeneity is quantified by the Birge ratio B r and the photon mean-free path ( l s ) is determined for homogeneous tissues (i.e., B r ~1 ). SD-OCT images of 83 normal corneas (ages 22–50 years) from a standard SD-OCT device (RTVue-XR Avanti, Optovue Inc.) were processed to establish a normative dataset of transparency values. After confirming stromal homogeneity ( B r <10), we measured a median l s of 570 μm (interdecile range: 270–2400 μm). By also considering corneal thicknesses, this may be translated into a median fraction of transmitted (coherent) light T coh(stroma) of 51% (interdecile range: 22–83%). Excluding images with central saturation artifact raised our median T coh(stroma) to 73% (interdecile range: 34–84%). These transparency values are slightly lower than those previously reported, which we attribute to the detection configuration of SD-OCT with a relatively small and selective acceptance angle. No statistically significant correlation between transparency and age or thickness was found. In conclusion, our algorithm provides robust and quantitative measurements of corneal transparency from standard SD-OCT images with sufficient quality (such as ‘Line’ and ‘CrossLine’ B-scan modes without central saturation artifact) and addresses the demand for such an objective means in the clinical setting. (10.1371/journal.pone.0291613)
DOI : 10.1371/journal.pone.0291613
MiniBAR/GARRE1 is a dual Rac and Rab effector required for ciliogenesis
- Serres Murielle
- Shaughnessy Ronan
- Escot Sophie
- Hammich Hussein
- Cuvelier Frédérique
- Salles Audrey
- Rocancourt Murielle
- Verdon Quentin
- Gaffuri Anne-Lise
- Sourigues Yannick
- Malherbe Gilles
- Velikovsky Leonid
- Chardon Florian
- Sassoon Nathalie
- Tinevez Jean-Yves
- Callebaut Isabelle
- Formstecher Etienne
- Houdusse Anne
- David Nicolas
- Pylypenko Olena
- Echard Arnaud
Developmental Cell, Elsevier , 2023, 58, pp.1-18 . Cilia protrude from the cell surface and play critical roles in intracellular signaling, environmental sensing, and development. Reduced actin-dependent contractility and intracellular trafficking are both required for ciliogenesis, but little is known about how these processes are coordinated. Here, we identified a Rac1- and Rab35-binding protein with a truncated BAR (Bin/amphiphysin/Rvs) domain that we named MiniBAR (also known as KIAA0355/GARRE1), which plays a key role in ciliogenesis. MiniBAR colocalizes with Rac1 and Rab35 at the plasma membrane and on intracellular vesicles trafficking to the ciliary base and exhibits fast pulses at the ciliary membrane. MiniBAR depletion leads to short cilia, resulting from abnormal Rac-GTP/Rho-GTP levels and increased acto-myosin-II-dependent contractility together with defective trafficking of IFT88 and ARL13B into cilia. MiniBAR-depleted zebrafish embryos display dysfunctional short cilia and hallmarks of ciliopathies, including left-right asymmetry defects. Thus, MiniBAR is a dual Rac and Rab effector that controls both actin cytoskeleton and membrane trafficking for ciliogenesis. (10.1016/j.devcel.2023.09.010)
DOI : 10.1016/j.devcel.2023.09.010
Kidney Stone Classification Using Multimodal Multiphoton Microscopy
- Gleeson Matthew
- Morizet Joséphine
- Mahou Pierre
- Daudon Michel
- Bazin Dominique
- Stringari Chiara
- Schanne-Klein Marie-Claire
- Beaurepaire Emmanuel
ACS photonics, American Chemical Society , 2023, 10 (10), pp.3594–3604 . Kidney stones are a common form of nephrolithiasis, affecting up to 15% of the world’s population with a high probability of recurrence. These stones exhibit various chemical compositions and crystalline forms associated with different etiologies. Classification of the stones’ components is necessary to optimize treatment and suggest lifestyle changes to reduce the risk of recurrence. Current characterization methods usually require extensive sample preparation or are too detailed for the needs of a high-throughput laboratory. In this article, we present a kidney stone component classification scheme based on the multiphoton response of crushed samples that is label-free, requires minimal sample amounts, and simple preparation. We measure two-photon excited fluorescence, which is sensitive to protein content, second-harmonic generation, which is sensitive to crystalline symmetry, and polarization-resolved third-harmonic generation (pTHG), which is sensitive to crystal heterogeneity and birefringence. The combination of these three contrast modes can distinguish different materials, specifically calcium oxalate in monohydrate (COM), dihydrate (COD), or amorphous forms, cystine, and carbonate apatite. In addition, pTHG images have the potential to distinguish between COM and COD fragments and to provide information on the submicron organization of carbonate apatite fragments. (10.1021/acsphotonics.3c00651)
DOI : 10.1021/acsphotonics.3c00651
Real-time in vivo ROS monitoring with luminescent nanoparticles reveals skin inflammation dynamics
- Abdesselem M.
- Pétri N.
- Kuhner R.
- Mousseau F.
- Rouffiac V.
- Gacoin T.
- Laplace-Builhé C.
- Alexandrou A.
- Bouzigues C I
Biomedical optics express, Optical Society of America - OSA Publishing , 2023, 14 (10), pp.5392 . Reactive oxygen species (ROS) are key regulators in numerous pathological contexts, including cancer or inflammation. Their role is complex, which justifies the need for methods enabling their quantitative and time-resolved monitoring in vivo, in the perspective to profile tissues of individual patients. However, current ROS detection methods do not provide these features. Here, we propose a new method based on the imaging of lanthanide-ion nanoparticles (GdVO 4 :Eu), whose photoluminescence is modulated by the surrounding ROS concentration. We monitored their luminescence after intradermic injection in a mouse ear submitted to an inflammation-inducing topical stimulus. Based on this approach, we quantified the ROS concentration after inflammation induction and identified a two-step kinetics of ROS production, which may be attributed to the response of resident immune cells and their further recruitment at the inflammation locus. (10.1364/boe.501914)
DOI : 10.1364/boe.501914
Multititration: The New Method for Implementing Ultrasensitive and Quantitative Multiplexed In-Field Immunoassays Despite Cross-Reactivity?
- Mousseau Fanny
- Féraudet Tarisse Cécile
- Simon Stéphanie
- Gacoin Thierry
- Alexandrou Antigoni
- Bouzigues Cédric Ismael
Analytical Chemistry, American Chemical Society , 2023, 95 (36), pp.13509-13518 . The accurate in-field titration of multiple pathogens is essential to efficiently describe and monitor environmental or biological contamination, isolate, act, and treat adequately. This underscores the requirement of portable, fast, quantitative, and multiplexed detection technologies, which, however, have not been properly developed so far, notably because it has been hindered by the phenomenon of cross-reactivity. In this work, we proposed a new analytical method based on the imaging through a portable device of lanthanide-based nanoparticles (YVO 4 :Eu) for spatially multiplexed detection, relying on a multiparameter analysis, i.e., a simultaneous analysis of all of the luminescence signals through the comparison to a calibration surface built in the presence of multiple analytes of interest. We then demonstrated the possibility to simultaneously quantify by multiplexed lateral flow assay (xLFA) the three enterotoxins SEG, SEH, and SEI in unknown mixtures, over two concentration decades (from a dozen of pg•mL −1 to few ng•mL −1). Assays were performed in less than an hour (25 min of strip migration followed by 30 min of drying at room temperature), the time during which the presence of the operator was not required for more than 5 min, in order to dip the strip and have it imaged by the reader. The concepts of nominal concentration recovery, coefficient of variation (CV), limit of blank (LOB), and limit of detection (LOD) were discussed in detail in the context of multiplexed assays. With our new definitions, quantitative results demonstrated a high recovery of the nominal concentrations (115%), reliability (CV = 20%), and sensitivity (LOBs of 3, 27, and 6 pg•mL −1 for SEG, SEH, and SEI respectively, and LODs of 6, 48, and 11 pg•mL −1 for SEG, SEH, and SEI, respectively). Based on this method, we observed an increase in sensitivity of 100 compared to the other multiplexed LFA labeled with gold particles and we approached the sensitivity of the simplex enzyme-linked immunosorbent assay (ELISA) performed with the same capture and detection antibodies. To conclude, our results, which are applicable to virtually any kind of multiplexed test, pave the way to the next generation of in-field analytical immunoassays by providing fast, quantitative, and highly sensitive multiplexed detection of biomarkers or pathogens. (10.1021/acs.analchem.3c01846)
DOI : 10.1021/acs.analchem.3c01846
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
Apical size and deltaA expression predict adult neural stem cell decisions along lineage progression
- Mancini Laure
- Guirao Boris
- Ortica Sara
- Labusch Miriam
- Cheysson Felix
- Bonnet Valentin
- Phan Minh Son
- Herbert Sébastien
- Mahou Pierre
- Menant Emilie
- Bedu Sébastien
- Tinevez Jean-Yves
- Baroud Charles
- Beaurepaire Emmanuel
- Bellaiche Yohanns
- Bally-Cuif Laure
- Dray Nicolas
Science Advances, American Association for the Advancement of Science (AAAS) , 2023, 9 (35), pp.eadg7519 . The maintenance of neural stem cells (NSCs) in the adult brain depends on their activation frequency and division mode. Using long-term intravital imaging of NSCs in the zebrafish adult telencephalon, we reveal that apical surface area and expression of the Notch ligand DeltaA predict these NSC decisions. deltaA -negative NSCs constitute a bona fide self-renewing NSC pool and systematically engage in asymmetric divisions generating a self-renewing deltaA neg daughter, which regains the size and behavior of its mother, and a neurogenic deltaA pos daughter, eventually engaged in neuronal production following further quiescence-division phases. Pharmacological and genetic manipulations of Notch, DeltaA, and apical size further show that the prediction of activation frequency by apical size and the asymmetric divisions of deltaA neg NSCs are functionally independent of Notch. These results provide dynamic qualitative and quantitative readouts of NSC lineage progression in vivo and support a hierarchical organization of NSCs in differently fated subpopulations. (10.1126/sciadv.adg7519)
DOI : 10.1126/sciadv.adg7519
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