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Article: Automated quantification of fluorescence and morphological changes in pretreated wood cells by fluorescence macroscopy.

Audibert, Edwige / Lebas, Berangère / Spriet, Corentin / Habrant, Anouck / Chabbert, Brigitte / Paës, Gabriel

2023 Volume 19, Issue 1, Page(s) 16

Abstract: Background: Lignocellulosic biomass is a complex network of polysaccharides and lignin that requires a pretreatment step to overcome recalcitrance and optimize valorisation into biobased products. Pretreatment of biomass induces chemical and ... ...

Abstract	Background: Lignocellulosic biomass is a complex network of polysaccharides and lignin that requires a pretreatment step to overcome recalcitrance and optimize valorisation into biobased products. Pretreatment of biomass induces chemical and morphological changes. Quantification of these changes is critical to understand biomass recalcitrance and to predict lignocellulose reactivity. In this study, we propose an automated method for the quantification of chemical and morphological parameters through fluorescence macroscopy, which was applied on wood samples (spruce, beechwood) pretreated with steam explosion. Results: Results in fluorescence macroscopy highlighted the impact of steam explosion on spruce and beechwood: fluorescence intensity of samples was highly altered, especially for the most severe conditions. Morphological changes were also revealed: shrinkage of cells and deformation of cell walls manifested as the loss of rectangularity or circular shape, for tracheids in spruce and vessels in beechwood respectively. Quantification of fluorescence intensity of cell walls and quantification of morphological parameters related to cell lumens were carried out accurately by applying the automated method onto the macroscopic images. The results showed that lumens area and circularity could be considered as complementary markers of cell deformation, and that fluorescence intensity of the cell walls could be related to morphological changes and to the conditions of pretreatment. Conclusions: The developed procedure allows simultaneous and effective quantification of morphological parameters and fluorescence intensity of the cell walls. This approach can be applied to fluorescence macroscopy as well as other imaging techniques and provides encouraging results towards the understanding of biomass architecture.
Language	English
Publishing date	2023-02-15
Publishing country	England
Document type	Journal Article
ZDB-ID	2203723-8
ISSN	1746-4811
ISSN	1746-4811
DOI	10.1186/s13007-023-00991-6
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Article ; Online: LIKE EARLY STARVATION 1 interacts with amylopectin during starch biosynthesis.

Osman, Rayan / Bossu, Mélanie / Dauvillée, David / Spriet, Corentin / Liu, Chun / Zeeman, Samuel C / D'Hulst, Christophe / Bompard, Coralie

Plant physiology

2024

Abstract: Starch is the major energy storage compound in plants. Both transient starch and long-lasting storage starch accumulate in the form of insoluble, partly crystalline granules. The structure of these granules is related to the structure of the branched ... ...

Abstract	Starch is the major energy storage compound in plants. Both transient starch and long-lasting storage starch accumulate in the form of insoluble, partly crystalline granules. The structure of these granules is related to the structure of the branched polymer amylopectin: linear chains of glucose units organized in double helices that align to form semi-crystalline lamellae, with branch points located in amorphous regions between them. EARLY STARVATION 1 (ESV1) and LIKE EARLY STARVATION 1 (LESV) proteins are involved in the maintenance of starch granule structure and in the phase transition of amylopectin, respectively, in Arabidopsis (Arabidopsis thaliana). These proteins contain a conserved tryptophan-rich C-terminal domain folded into an antiparallel β-sheet, likely responsible for binding of the proteins to starch, and different N-terminal domains whose structure and function are unknown. In this work, we combined biochemical and biophysical approaches to analyze the structures of LESV and ESV1 and their interactions with the different starch polyglucans. We determined that both proteins interact with amylopectin but not with amylose and that only LESV is capable of interacting with amylopectin during starch biosynthesis. While the C-terminal domain interacts with amylopectin in its semi-crystalline form, the N-terminal domain of LESV undergoes induced conformational changes that are probably involved in its specific function of mediating glucan phase transition. These results clarify the specific mechanism of action of these two proteins in the biosynthesis of starch granules.
Language	English
Publishing date	2024-04-04
Publishing country	United States
Document type	Journal Article
ZDB-ID	208914-2
ISSN	1532-2548 ; 0032-0889
ISSN (online)	1532-2548
ISSN	0032-0889
DOI	10.1093/plphys/kiae193
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Article ; Online: A Step-by-Step Guide for the Production of Recombinant Fluorescent TAT-HA-Tagged Proteins and their Transduction into Mammalian Cells.

Anny, Christer Abou / Nouaille, Sébastien / Fauré, Régis / Schulz, Céline / Spriet, Corentin / Huvent, Isabelle / Biot, Christophe / Lefebvre, Tony

Current protocols

2024 Volume 4, Issue 3, Page(s) e1016

Abstract: Investigating the function of target proteins for functional prospection or therapeutic applications typically requires the production and purification of recombinant proteins. The fusion of these proteins with tag peptides and fluorescently derived ... ...

Abstract	Investigating the function of target proteins for functional prospection or therapeutic applications typically requires the production and purification of recombinant proteins. The fusion of these proteins with tag peptides and fluorescently derived proteins allows the monitoring of candidate proteins using SDS-PAGE coupled with western blotting and fluorescent microscopy, respectively. However, protein engineering poses a significant challenge for many researchers. In this protocol, we describe step-by-step the engineering of a recombinant protein with various tags: TAT-HA (trans-activator of transduction-hemagglutinin), 6×His and EGFP (enhanced green fluorescent protein) or mCherry. Fusion proteins are produced in E. coli BL21(DE3) cells and purified by immobilized metal affinity chromatography (IMAC) using a Ni-nitrilotriacetic acid (NTA) column. Then, tagged recombinant proteins are introduced into cultured animal cells by using the penetrating peptide TAT-HA. Here, we present a thorough protocol providing a detailed guide encompassing every critical step from plasmid DNA molecular assembly to protein expression and subsequent purification and outlines the conditions necessary for protein transduction technology into animal cells in a comprehensive manner. We believe that this protocol will be a valuable resource for researchers seeking an exhaustive, step-by-step guide for the successful production and purification of recombinant proteins and their entry by transduction within living cells. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: DNA cloning, molecular assembly strategies, and protein production Basic Protocol 2: Protein purification Basic Protocol 3: Protein transduction in mammalian cells.
MeSH term(s)	Animals ; Escherichia coli/genetics ; Recombinant Proteins/genetics ; Recombinant Proteins/chemistry ; Peptides/genetics ; Peptides/metabolism ; Indicators and Reagents/metabolism ; Gene Products, tat/metabolism ; Coloring Agents/metabolism ; DNA/metabolism ; Mammals/genetics ; Mammals/metabolism
Chemical Substances	Recombinant Proteins ; Peptides ; Indicators and Reagents ; Gene Products, tat ; Coloring Agents ; DNA (9007-49-2)
Language	English
Publishing date	2024-03-21
Publishing country	United States
Document type	Journal Article
ISSN	2691-1299
ISSN (online)	2691-1299
DOI	10.1002/cpz1.1016
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Article ; Online: Further insight into the involvement of PII1 in starch granule initiation in Arabidopsis leaf chloroplasts

Vandromme, Camille / Spriet, Corentin / Putaux, Jean‐Luc / Dauvillée, David / Courseaux, Adeline / D'Hulst, Christophe / Wattebled, Fabrice

New Phytologist. 2023 July, v. 239, no. 1 p.132-145

2023

Abstract: The control of starch granule initiation in plant leaves is a complex process that requires active enzymes like Starch Synthase 4 and 3 (SS4 or SS3) and several noncatalytic proteins such as Protein Involved in starch Initiation 1 (PII1). In Arabidopsis ... ...

Abstract	The control of starch granule initiation in plant leaves is a complex process that requires active enzymes like Starch Synthase 4 and 3 (SS4 or SS3) and several noncatalytic proteins such as Protein Involved in starch Initiation 1 (PII1). In Arabidopsis leaves, SS4 is the main enzyme that control starch granule initiation, but in its absence, SS3 partly fulfills this function. How these proteins collectively act to control the initiation of starch granules remains elusive. PII1 and SS4 physically interact, and PII1 is required for SS4 to be fully active. However, Arabidopsis mutants lacking SS4 or PII1 still accumulate starch granules. Combining pii1 KO mutation with either ss3 or ss4 KO mutations provide new insights of how the remaining starch granules are synthesized. The ss3 pii1 line still accumulates starch, while the phenotype of ss4 pii1 is stronger than that of ss4. Our results indicate first that SS4 initiates starch granule synthesis in the absence of PII1 albeit being limited to one large lenticular granule per plastid. Second, that if in the absence of SS4, SS3 is able to initiate starch granules with low efficiency, this ability is further reduced with the additional absence of PII1.
Keywords	Arabidopsis ; chloroplasts ; leaves ; mutation ; phenotype ; starch granules ; starch synthase
Language	English
Dates of publication	2023-07
Size	p. 132-145.
Publishing place	John Wiley & Sons, Ltd
Document type	Article ; Online
Note	JOURNAL ARTICLE
ZDB-ID	208885-x
ISSN	1469-8137 ; 0028-646X
ISSN (online)	1469-8137
ISSN	0028-646X
DOI	10.1111/nph.18923
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: Measuring Interactions between Fluorescent Probes and Lignin in Plant Sections by sFLIM Based on Native Autofluorescence.

Terryn, Christine / Habrant, Anouck / Paës, Gabriel / Spriet, Corentin

Journal of visualized experiments : JoVE

2020 , Issue 155

Abstract: In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis process, which maintains enzymes far from their substrate. Characterization of these complex interactions ...

Abstract	In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis process, which maintains enzymes far from their substrate. Characterization of these complex interactions is thus a challenge in complex substrates such as LB. The method here measures molecular interactions between fluorophore-tagged molecules and native autofluorescent lignin, to be revealed by Förster resonance energy transfer (FRET). Contrary to FRET measurements in living cells using two exogenous fluorophores, FRET measurements in plants using lignin is not trivial due to its complex autofluorescence. We have developed an original acquisition and analysis pipeline with correlated observation of two complementary properties of fluorescence: fluorescence emission and lifetime. sFLIM (spectral and fluorescent lifetime imaging microscopy) provides the quantification of these interactions with high sensitivity, revealing different interaction levels between biomolecules and lignin.
MeSH term(s)	Fluorescence Resonance Energy Transfer ; Fluorescent Dyes/chemistry ; Fluorescent Dyes/metabolism ; Lignin/metabolism ; Microscopy, Fluorescence/methods ; Spectrometry, Fluorescence/methods ; Triticum/metabolism
Chemical Substances	Fluorescent Dyes ; Lignin (9005-53-2)
Language	English
Publishing date	2020-01-02
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Video-Audio Media
ZDB-ID	2259946-0
ISSN	1940-087X ; 1940-087X
ISSN (online)	1940-087X
ISSN	1940-087X
DOI	10.3791/59925
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Article: Measuring interactions between fluorescent probes and lignin in plant sections by sflim based on native autofluorescence

Terryn, Christine / Habrant, Anouck / Paës, Gabriel / Spriet, Corentin

Journal of visualized experiments. 2020 Jan. 02, , no. 155

2020

Abstract	In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis process, which maintains enzymes far from their substrate. Characterization of these complex interactions is thus a challenge in complex substrates such as LB. The method here measures molecular interactions between fluorophore-tagged molecules and native autofluorescent lignin, to be revealed by Förster resonance energy transfer (FRET). Contrary to FRET measurements in living cells using two exogenous fluorophores, FRET measurements in plants using lignin is not trivial due to its complex autofluorescence. We have developed an original acquisition and analysis pipeline with correlated observation of two complementary properties of fluorescence: fluorescence emission and lifetime. sFLIM (spectral and fluorescent lifetime imaging microscopy) provides the quantification of these interactions with high sensitivity, revealing different interaction levels between biomolecules and lignin.
Keywords	biomass ; energy transfer ; enzymatic hydrolysis ; enzyme activity ; enzymes ; fluorescence ; fluorescent dyes ; image analysis ; lignin ; lignocellulose ; microscopy
Language	English
Dates of publication	2020-0102
Size	p. e59925.
Publishing place	Journal of Visualized Experiments
Document type	Article
ZDB-ID	2259946-0
ISSN	1940-087X
ISSN	1940-087X
DOI	10.3791/59925
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: Further insight into the involvement of PII1 in starch granule initiation in Arabidopsis leaf chloroplasts.

Vandromme, Camille / Spriet, Corentin / Putaux, Jean-Luc / Dauvillée, David / Courseaux, Adeline / D'Hulst, Christophe / Wattebled, Fabrice

The New phytologist

2023 Volume 239, Issue 1, Page(s) 132–145

Abstract	The control of starch granule initiation in plant leaves is a complex process that requires active enzymes like Starch Synthase 4 and 3 (SS4 or SS3) and several noncatalytic proteins such as Protein Involved in starch Initiation 1 (PII1). In Arabidopsis leaves, SS4 is the main enzyme that control starch granule initiation, but in its absence, SS3 partly fulfills this function. How these proteins collectively act to control the initiation of starch granules remains elusive. PII1 and SS4 physically interact, and PII1 is required for SS4 to be fully active. However, Arabidopsis mutants lacking SS4 or PII1 still accumulate starch granules. Combining pii1 KO mutation with either ss3 or ss4 KO mutations provide new insights of how the remaining starch granules are synthesized. The ss3 pii1 line still accumulates starch, while the phenotype of ss4 pii1 is stronger than that of ss4. Our results indicate first that SS4 initiates starch granule synthesis in the absence of PII1 albeit being limited to one large lenticular granule per plastid. Second, that if in the absence of SS4, SS3 is able to initiate starch granules with low efficiency, this ability is further reduced with the additional absence of PII1.
MeSH term(s)	Arabidopsis/metabolism ; Starch/metabolism ; Arabidopsis Proteins/genetics ; Arabidopsis Proteins/metabolism ; Chloroplasts/metabolism ; Starch Synthase/genetics ; Plant Leaves/metabolism ; Mutation/genetics
Chemical Substances	Starch (9005-25-8) ; Arabidopsis Proteins ; Starch Synthase (EC 2.4.1.21)
Language	English
Publishing date	2023-04-19
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	208885-x
ISSN	1469-8137 ; 0028-646X
ISSN (online)	1469-8137
ISSN	0028-646X
DOI	10.1111/nph.18923
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article: TRPV6 Calcium Channel Targeting by Antibodies Raised against Extracellular Epitopes Induces Prostate Cancer Cell Apoptosis.

Haustrate, Aurélien / Shapovalov, George / Spriet, Corentin / Cordier, Clément / Kondratskyi, Artem / Noyer, Lucile / Foulquier, François / Prevarskaya, Natalia / Lehen'kyi, V'yacheslav

Cancers

2023 Volume 15, Issue 6

Abstract: The TRPV6 calcium channel is known to be up-regulated in various tumors. The efforts to target the TRPV6 channel in vivo are still ongoing to propose an effective therapy against cancer. Here, we report the generation of two antibodies raised against ... ...

Abstract	The TRPV6 calcium channel is known to be up-regulated in various tumors. The efforts to target the TRPV6 channel in vivo are still ongoing to propose an effective therapy against cancer. Here, we report the generation of two antibodies raised against extracellular epitopes corresponding to the extracellular loop between S1 and S2 (rb79) and the pore region (rb82). These antibodies generated a complex biphasic response with the transient activation of the TRPV6 channel. Store-operated calcium entry was consequently potentiated in the prostate cancer cell line LNCaP upon the treatment. Both rb79 and rb82 antibodies significantly decreased cell survival rate in a dose-dependent manner as compared to the control antibodies of the same isotype. This decrease was due to the enhanced cell death via apoptosis revealed using a sub-G1 peak in a cell cycle assay, TUNEL assay, and a Hoechst staining, having no effects in the PC3M
Language	English
Publishing date	2023-03-17
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2527080-1
ISSN	2072-6694
ISSN	2072-6694
DOI	10.3390/cancers15061825
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Article: Comparative Analysis of Hepatitis C Virus NS5A Dynamics and Localization in Assembly-Deficient Mutants.

Riva, Laura / Spriet, Corentin / Barois, Nicolas / Popescu, Costin-Ioan / Dubuisson, Jean / Rouillé, Yves

Pathogens (Basel, Switzerland)

2021 Volume 10, Issue 2

Abstract: The hepatitis C virus (HCV) life cycle is a tightly regulated process, during which structural and non-structural proteins cooperate. However, the interplay between HCV proteins during genomic RNA replication and progeny virion assembly is not completely ...

Abstract	The hepatitis C virus (HCV) life cycle is a tightly regulated process, during which structural and non-structural proteins cooperate. However, the interplay between HCV proteins during genomic RNA replication and progeny virion assembly is not completely understood. Here, we studied the dynamics and intracellular localization of non-structural 5A protein (NS5A), which is a protein involved both in genome replication and encapsidation. An NS5A-eGFP (enhanced green fluorescent protein) tagged version of the strain JFH-1-derived wild-type HCV was compared to the corresponding assembly-deficient viruses Δcore, NS5A basic cluster 352-533 mutant (BCM), and serine cluster 451 + 454 + 457 mutant (SC). These analyses highlighted an increase of NS5A motility when the viral protein core was lacking. Although to a lesser extent, NS5A motility was also increased in the BCM virus, which is characterized by a lack of interaction of NS5A with the viral RNA, impairing HCV genome encapsidation. This observation suggests that the more static NS5A population is mainly involved in viral assembly rather than in RNA replication. Finally, NS4B exhibited a reduced co-localization with NS5A and lipid droplets for both Δcore and SC mutants, which is characterized by the absence of interaction of NS5A with core. This observation strongly suggests that NS5A is involved in targeting NS4B to lipid droplets (LDs). In summary, this work contributes to a better understanding of the interplay between HCV proteins during the viral life cycle.
Language	English
Publishing date	2021-02-04
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2695572-6
ISSN	2076-0817
ISSN	2076-0817
DOI	10.3390/pathogens10020172
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Article: FRET-SLiM on native autofluorescence: a fast and reliable method to study interactions between fluorescent probes and lignin in plant cell wall.

Terryn, Christine / Paës, Gabriel / Spriet, Corentin

Plant methods

2018 Volume 14, Page(s) 74

Abstract: Background: Lignocellulosic biomass is a complex network of polymers making the cell walls of plants. It represents a feedstock of sustainable resources to be converted into fuels, chemicals and materials. Because of its complex architecture, ... ...

Abstract	Background: Lignocellulosic biomass is a complex network of polymers making the cell walls of plants. It represents a feedstock of sustainable resources to be converted into fuels, chemicals and materials. Because of its complex architecture, lignocellulose is a recalcitrant material that necessitates some pretreatments and several types of catalysts to be transformed efficiently. In particular, enzymes degrading lignocellulose can become inactivated due to their binding to lignin through non-specific interactions, leading to a loss in catalytic efficiency of industrial processes. Gaining more knowledge in the strength of interactions would allow optimizing enzymes and selecting appropriate pretreatments. Results: Measuring interactions directly in plant cell wall can theoretically be performed using confocal fluorescence techniques by evaluating fluorescence resonance energy transfer (FRET) between compatible fluorophores. In this study, autofluorescence of plant cell wall, mainly originating from lignin, was considered as a donor fluorophore while the acceptor was a common rhodamine-based fluorescent probe. To overcome complex plant cell wall fluorescence, which limits FRET analysis by standard techniques, we have developed an original approach, combining spectral and lifetime measurements. It consists in (1) dissecting autofluorescence signal in each spectral channel, (2) optimizing spectral channel choice for lifetime measurements and (3) achieving an unambiguous FRET signature with an autofluorescent donor fluorophore. Interactions between rhodamine-based probes of various sizes and untreated or pretreated wheat sample were evaluated, showing it was possible to discriminate interactions at the nano-scale, revealing some accessibility differences and the effect of pretreatment. Conclusions: SLiM measurement allows precise estimation of the optimal spectral range for FRET measurement. SLiM response allows for the first time doubtless FRET measurements between lignin as a donor, and an acceptor fluorophore with high accuracy and sensitivity related to lifetime decrease studies. As demonstrated, it thus becomes possible to measure interactions of fluorescent probes directly inside plant cell wall samples. This approach can thus be applied to various fields such as lignocellulose deconstruction to optimize the action of enzymes or plant cell wall development to assay in situ the biosynthesis of lignin.
Language	English
Publishing date	2018-08-27
Publishing country	England
Document type	Journal Article
ZDB-ID	2203723-8
ISSN	1746-4811
ISSN	1746-4811
DOI	10.1186/s13007-018-0342-3
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