Publications by authors named "Christine Terryn"

57 Publications

Exploring the Potential of β-Catenin -GlcNAcylation by Using Fluorescence-Based Engineering and Imaging.

Molecules 2020 Oct 1;25(19). Epub 2020 Oct 1.

Univ. Lille, CNRS, UMR 8576-UGSF-Unité de Glycobiologie Structurale et Fonctionnelle, F-59000 Lille, France.

Monitoring glycosylation changes within cells upon response to stimuli remains challenging because of the complexity of this large family of post-translational modifications (PTMs). We developed an original tool, enabling labeling and visualization of the cell cycle key-regulator β-catenin in its -GlcNAcylated form, based on intramolecular Förster resonance energy transfer (FRET) technology in cells. We opted for a bioorthogonal chemical reporter strategy based on the dual-labeling of β-catenin with a green fluorescent protein (GFP) for protein sequence combined with a chemically-clicked imaging probe for PTM, resulting in a fast and easy to monitor qualitative FRET assay. We validated this technology by imaging the -GlcNAcylation status of β-catenin in HeLa cells. The changes in -GlcNAcylation of β-catenin were varied by perturbing global cellular -GlcNAc levels with the inhibitors of -GlcNAc transferase (OGT) and -GlcNAcase (OGA). Finally, we provided a flowchart demonstrating how this technology is transposable to any kind of glycosylation.
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http://dx.doi.org/10.3390/molecules25194501DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7583010PMC
October 2020

Type I Collagen Aging Increases Expression and Activation of EGFR and Induces Resistance to Erlotinib in Lung Carcinoma in 3D Matrix Model.

Front Oncol 2020 10;10:1593. Epub 2020 Sep 10.

Université de Reims Champagne-Ardenne, Unité BioSpecT, EA7506, SFR CAP-Santé, UFR de Pharmacie, Reims, France.

Type I collagen is the major structural component of lung stroma. Because of its long half-life, type I collagen undergoes post-translational modifications such as glycation during aging process. These modifications have been shown to impact the structural organization of type I collagen fibers. In the present work we evaluated the impact of collagen aging on lung carcinoma cells response to erlotinib-induced cytotoxicity and apoptosis, and on Epidermal Growth Factor Receptor (EGFR) expression and phosphorylation. To this end, experiments were performed in 2D and 3D matrix models established from type I collagen extracted from adult (10 weeks-old) and old (100 weeks-old) rat's tail tendons. Our results show that old collagen induces a significant increase in EGFR expression and phosphorylation when compared to adult collagen in 3D matrix but not in 2D coating. Such modification was associated to an increase in the IC of erlotinib in the presence of old collagen and a lower sensitivity to drug-induced apoptosis. These data suggest that collagen aging confers resistance to the cytotoxic and apoptotic effects of therapies targeting EGFR kinase function in lung carcinoma. Moreover, our data underline the importance of the 3D matrix environment in this process.
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http://dx.doi.org/10.3389/fonc.2020.01593DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7511549PMC
September 2020

AgeR deletion decreases soluble fms-like tyrosine kinase 1 production and improves post-ischemic angiogenesis in uremic mice.

Angiogenesis 2021 02 23;24(1):47-55. Epub 2020 Sep 23.

Department of Nephrology, University Hospital of Limoges, Limoges, France.

Peripheral arterial disease occurs more frequently and has a worse prognosis in patients with chronic kidney disease (CKD). The receptor for advanced glycation end products (RAGE) is involved in multiple aspects of uremia-associated vasculopathy. Previous data suggest that the RAGE pathway may promote soluble fms-like tyrosine kinase 1 (sFlt1) production, an anti-angiogenic molecule. Thus, we tested the hypothesis that the deletion of AgeR would decrease sFlt1 production and improve post-ischemic revascularization in uremic condition. We used a well-established CKD model (5/6 nephrectomy) in WT and AgeR C57/Bl6 mice. Hindlimb ischemia was induced by femoral artery ligation. Revascularization was evaluated by complementary approaches: ischemic limb retraction, LASCA imagery, and capillary density. The production of sFlt1 was assessed at both RNA and protein levels. After hindlimb ischemia, uremic mice showed slower functional recovery (p < 0.01), decreased reperfusion (p < 0.01), lower capillary density (p = 0.02), and increased circulating sFlt1 levels (p = 0.03). AgeR deletion restored post-ischemic angiogenesis and was protective from sFlt1 increase in uremic mice. These findings show the main role of RAGE in post-ischemic angiogenesis impairment associated with CKD. RAGE may represent a key target for building new therapeutic approaches to improve the outcome of CKD patients with PAD.
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http://dx.doi.org/10.1007/s10456-020-09747-5DOI Listing
February 2021

Analysis of Hepatic Fibrosis Characteristics in Cirrhotic Patients with and without Hepatocellular Carcinoma by FTIR Spectral Imaging.

Molecules 2020 Sep 7;25(18). Epub 2020 Sep 7.

Université de Reims Champagne-Ardenne, BioSpecT-EA7506, UFR de Pharmacie, 51097 Reims, France.

The evolution of cirrhosis is marked by quantitative and qualitative modifications of the fibrosis tissue and an increasing risk of complications such as hepatocellular carcinoma (HCC). Our purpose was to identify by FTIR imaging the spectral characteristics of hepatic fibrosis in cirrhotic patients with and without HCC. FTIR images were collected at projected pixel sizes of 25 and 2.7 μm from paraffinized hepatic tissues of five patients with uncomplicated cirrhosis and five cirrhotic patients with HCC and analyzed by k-means clustering. When compared to the adjacent histological section, the spectral clusters corresponding to hepatic fibrosis and regeneration nodules were easily identified. The fibrosis area estimated by FTIR imaging was correlated to that evaluated by digital image analysis of histological sections and was higher in patients with HCC compared to those without complications. Qualitative differences were also observed when fibrosis areas were specifically targeted at higher resolution. The partition in two clusters of the fibrosis tissue highlighted subtle differences in the spectral characteristics of the two groups of patients. These data show that the quantitative and qualitative changes of fibrosis tissue occurring during the course of cirrhosis are detectable by FTIR imaging, suggesting the possibility of subclassifying cirrhosis into different steps of severity.
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http://dx.doi.org/10.3390/molecules25184092DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7570752PMC
September 2020

Receptor for Advanced Glycation End Products is Involved in Platelet Hyperactivation and Arterial Thrombosis during Chronic Kidney Disease.

Thromb Haemost 2020 Sep 29;120(9):1300-1312. Epub 2020 Jul 29.

UMR CNRS 7369 Matrice Extracellulaire et Dynamique Cellulaire (MEDyC), Team 2 "Matrix Aging and Vascular Remodelling," Université de Reims Champagne Ardenne, Reims, France.

Background:  Chronic kidney disease (CKD) is associated with a high cardiovascular mortality due to increased rates of vascular lesions and thrombotic events, as well as serum accumulation of uremic toxins. A subgroup of these toxins (advanced glycation end products [AGEs] and S100 proteins) can interact with the receptor for AGEs (RAGE). In this study, we analyzed the impact of CKD on platelet function and arterial thrombosis, and the potential role of RAGE in this process.

Methods:  Twelve weeks after induction of CKD in mice, platelet function and time to complete carotid artery occlusion were analyzed in four groups of animals (sham-operated, CKD, apolipoprotein E [Apoe], and Apoe/Ager mice).

Results:  Analysis of platelet function from whole blood and platelet-rich plasma showed hyperactivation of platelets only in CKD Apoe mice. There was no difference when experiments were done on washed platelets. However, preincubation of such platelets with AGEs or S100 proteins induced RAGE-mediated platelet hyperactivation. In vivo, CKD significantly reduced carotid occlusion times of Apoe mice (9.2 ± 1.1 vs. 11.1 ± 0.6 minutes for sham,  < 0.01). In contrast, CKD had no effect on occlusion times in Apoe/Ager mice. Moreover, carotid occlusion in Apoe CKD mice occurred significantly faster than in Apoe/Ager CKD mice ( < 0.0001).

Conclusion:  Our results show that CKD induces platelet hyperactivation, accelerates thrombus formation in a murine model of arterial thrombosis, and that RAGE deletion has a protective role. We propose that RAGE ligands binding to RAGE is involved in CKD-induced arterial thrombosis.
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http://dx.doi.org/10.1055/s-0040-1714101DOI Listing
September 2020

Neutrophil Extracellular Traps Generation Relates with Early Stage and Vascular Complications in Systemic Sclerosis.

J Clin Med 2020 Jul 7;9(7). Epub 2020 Jul 7.

EA7509 IRMAIC, University of Reims-Champagne-Ardenne, 51100 Reims, France.

Systemic sclerosis (SSc) is a systemic disease characterized by a great clinical and immunological heterogeneity whose pathophysiology is still being unraveled. Recently, innate immunity has been proposed to participate to the pathogenesis of SSc. In this study, we investigated the release of neutrophil extracellular traps (NETs) according to patient phenotype. Polymorphonuclear neutrophils (PMN) from 34 SSc patients and 26 healthy controls were stimulated by serum from SSc or healthy subject. NETs were visualized using epifluorescence microscope after DNA, myeloperoxidase, and Histone H3 tagging. Area of NETs were quantified using an original macro running in ImageJ software. PMN from SSc patients were significantly more prone to releasing NETs than control PMN after autologous stimulation. PMN from patients with severe vascular complications (pulmonary arterial hypertension, digital ulcers) produced more NETs than PMN from other SSc patients and their aberrant NET production appeared to be sustained over time. In patients with pulmonary interstitial disease or extensive cutaneous fibrosis, NET production was high at an early stage of the disease before progressively decreasing. Both serum factors and PMN activation status were involved in the enhanced production of NETs in SSc. Consequently, neutrophils and especially NETosis represent new physiopathological and therapeutic fields in SSc.
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http://dx.doi.org/10.3390/jcm9072136DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7408748PMC
July 2020

Mechanobiologically induced bone-like nodules: Matrix characterization from micro to nanoscale.

Nanomedicine 2020 10 29;29:102256. Epub 2020 Jun 29.

Université de Reims Champagne Ardenne, BIOS EA 4691, Reims, France; Université de Reims Champagne Ardenne, UFR d'Odontologie, Reims, France. Electronic address:

In bone tissue engineering, stem cells are known to form inhomogeneous bone-like nodules on a micrometric scale. Herein, micro- and nano-infrared (IR) micro-spectroscopies were used to decipher the chemical composition of the bone-like nodule. Histological and immunohistochemical analyses revealed a cohesive tissue with bone-markers positive cells surrounded by dense mineralized type-I collagen. Micro-IR gathered complementary information indicating a non-mature collagen at the top and periphery and a mature collagen within the nodule. Atomic force microscopy combined to IR (AFM-IR) analyses showed distinct spectra of "cell" and "collagen" rich areas. In contrast to the "cell" area, spectra of "collagen" area revealed the presence of carbohydrate moieties of collagen and/or the presence of glycoproteins. However, it was not possible to determine the collagen maturity, due to strong bands overlapping and/or possible protein orientation effects. Such findings could help developing protocols to allow a reliable characterization of in vitro generated complex bone tissues.
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http://dx.doi.org/10.1016/j.nano.2020.102256DOI Listing
October 2020

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

J Vis Exp 2020 01 2(155). Epub 2020 Jan 2.

Unité de Glycobiologie Structurale et Fonctionnelle, Université de Lille;

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.
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http://dx.doi.org/10.3791/59925DOI Listing
January 2020

Electron tomography reveals changes in spatial distribution of UBTF1 and UBTF2 isoforms within nucleolar components during rRNA synthesis inhibition.

J Struct Biol 2019 11 31;208(2):191-204. Epub 2019 Aug 31.

Unit of Cell and Tissue Biology, GIGA-Neurosciences, University of Liège, Liège, Belgium. Electronic address:

Upstream binding transcription factor (UBTF) is a co-regulator of RNA polymerase I by constituting an initiation complex on rRNA genes. UBTF plays a role in rDNA bending and its maintenance in "open" state. It exists as two splicing variants, UBTF1 and UBTF2, which cannot be discerned with antibodies raised against UBTF. We investigated the ultrastructural localization of each variant in cells synthesizing GFP-tagged UBTF1 or UBTF2 by using anti-GFP antibodies and pre-embedding nanogold strategy. Detailed 3D distribution of UBTF1 and 2 was also studied by electron tomography. In control cells, the two isoforms are very abundant within fibrillar centers, but their repartition strongly differs. Electron tomography shows that UBTF1 is disposed as fibrils that are folded in coils whereas UBTF2 is localized homogenously, preferentially at their cortical area. As UBTF is a useful marker to trace rDNA genes, we used these data to improve our previous model of 3D organization of active transcribing rDNA gene within fibrillar centers. Finally, when rRNA synthesis is inhibited during actinomycin D treatment or entry in mitosis, UBTF1 and UBTF2 show a similar distribution along extended 3D loop-like structures. Altogether these data suggest new roles for UBTF1 and UBTF2 isoforms in the organization of active and inactive rDNA genes.
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http://dx.doi.org/10.1016/j.jsb.2019.08.014DOI Listing
November 2019

Multimodal characterization of acid-pretreated poplar reveals spectral and structural parameters strongly correlate with saccharification.

Bioresour Technol 2019 Dec 16;293:122015. Epub 2019 Aug 16.

FARE Laboratory, INRA, Université de Reims Champagne Ardenne, Reims, France. Electronic address:

Lignocellulose biomass can be transformed into sustainable chemicals, materials and energy but its natural recalcitrance requires the use of pretreatment to enhance subsequent catalytic steps. Dilute acid pretreatment is one of the most common and efficient ones, however its impact has not yet been investigated simultaneously at nano- and cellular-scales. Poplar samples have been pretreated by dilute acid at different controlled severities, then characterized by combined structural and spectral techniques (scanning electron microscopy, confocal microscopy, autofluorescence, fluorescence lifetime, Raman). Results show that pretreatment favours lignin depolymerization until severity of 2.4-2.5 while at severity of 2.7 lignin seems to repolymerize as revealed by broadening of autofluorescence spectrum and strong decrease in fluorescence lifetime. Importantly, both nano-scale and cellular-scale markers can predict hydrolysis yield of pretreated samples, highlighting some connections in the multiscale recalcitrance of lignocellulose.
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http://dx.doi.org/10.1016/j.biortech.2019.122015DOI Listing
December 2019

In vivo efficacy of endothelial growth medium stimulated mesenchymal stem cells derived from patients with critical limb ischemia.

J Transl Med 2019 08 9;17(1):261. Epub 2019 Aug 9.

EA-3801, SFR CAP-santé, Université de Reims Champagne-Ardenne, 51092, Reims Cedex, France.

Background: Cell therapy has been proposed for patients with critical limb ischemia (CLI). Autologous bone marrow derived cells (BMCs) have been mostly used, mesenchymal stem cells (MSCs) being an alternative. The aim of this study was to characterize two types of MSCs and evaluate their efficacy.

Methods: MSCs were obtained from CLI-patients BMCs. Stimulated- (S-) MSCs were cultured in endothelial growth medium. Cells were characterized by the expression of cell surface markers, the relative expression of 6 genes, the secretion of 10 cytokines and the ability to form vessel-like structures. The cell proangiogenic properties was analysed in vivo, in a hindlimb ischemia model. Perfusion of lower limbs and functional tests were assessed for 28 days after cell infusion. Muscle histological analysis (neoangiogenesis, arteriogenesis and muscle repair) was performed.

Results: S-MSCs can be obtained from CLI-patients BMCs. They do not express endothelial specific markers but can be distinguished from MSCs by their secretome. S-MSCs have the ability to form tube-like structures and, in vivo, to induce blood flow recovery. No amputation was observed in S-MSCs treated mice. Functional tests showed improvement in treated groups with a superiority of MSCs and S-MSCs. In muscles, CD31+ and αSMA+ labelling were the highest in S-MSCs treated mice. S-MSCs induced the highest muscle repair.

Conclusions: S-MSCs exert angiogenic potential probably mediated by a paracrine mechanism. Their administration is associated with flow recovery, limb salvage and muscle repair. The secretome from S-MSCs or secretome-derived products may have a strong potential in vessel regeneration and muscle repair. Trial registration NCT00533104.
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http://dx.doi.org/10.1186/s12967-019-2003-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6688282PMC
August 2019

Low-diluted Phenacetinum disrupted the melanoma cancer cell migration.

Sci Rep 2019 06 24;9(1):9109. Epub 2019 Jun 24.

CNRS UMR7369 MEDyC, University of Reims Champagne-Ardenne, Reims, France.

Dynamic and reciprocal interactions generated by the communication between tumor cells and their matrix microenvironment, play a major role in the progression of a tumor. Indeed, the adhesion of specific sites to matrix components, associated with the repeated and coordinated formation of membrane protrusions, allow tumor cells to move along a determined pathway. Our study analyzed the mechanism of action of low-diluted Phenacetinum on murine cutaneous melanoma process in a fibronectin matrix environment. We demonstrated a reduction of dispersed cell migration, early and for as long as 24 h, by altering the formation of cell protrusions. Moreover, low-diluted Phenacetinum decreased cell stiffness highly on peripheral areas, due to a disruption of actin filaments located just under the plasma membrane. Finally, it modified the structure of the plasma membrane by accumulating large ordered lipid domains and disrupted B16 cell migration by a likely shift in the balance between ordered and disordered lipid phases. Whereas the correlation between the excess of lipid raft and cytoskeleton disrupting is not as yet established, it is clear that low-diluted Phenacetinum acts on the actin cytoskeleton organization, as confirmed by a decrease of cell stiffness affecting ultimately the establishment of an effective migration process.
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http://dx.doi.org/10.1038/s41598-019-45578-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6591484PMC
June 2019

Various Nucleolar Stress Inducers Result in Highly Distinct Changes in Water, Dry Mass and Elemental Content in Cancerous Cell Compartments: Investigation Using a Nano-Analytical Approach.

Nanotheranostics 2019 3;3(2):179-195. Epub 2019 May 3.

BioSpecT, EA 7506, Université de Reims Champagne Ardenne.

Numerous chemotherapeutic drugs that affect ribosome biogenesis in the nucleolus induce nucleolar stress. Improving our understanding of the effects of these drugs will require uncovering and comparing their impact on several biophysical parameters of the major cell compartments. Here, we quantified the water content and dry mass of cancerous cells treated with CX-5461, DRB or DAM to calculate macromolecular crowding and the volume occupied by free water, as well as elemental content. HeLa-H2B-GFP cells were treated with CX-5461, DRB or DAM. Water content and dry mass were measured in numerous regions of interest of ultrathin cryo-sections by quantitative scanning transmission electron microscope dark-field imaging and the elements quantified by energy dispersive X-ray spectrometry. The data were used to calculate macromolecular crowding and the volume occupied by free water in all cell compartments of control and treated cells. Hydrophobic and unfolded proteins were revealed by 8-Anilinonaphtalene-1-sulfonic acid (ANS) staining and imaging by two-photon microscopy. Immunolabeling of UBF, pNBS1 and pNF-κB was carried out and the images acquired with a confocal microscope for 3D imaging to address whether the localization of these proteins changes in treated cells. Treatment with CX-5461, DRB or DAM induced completely different changes in macromolecular crowding and elemental content. Macromolecular crowding and elemental content were much higher in CX-5461-treated, moderately higher in DRB-treated, and much lower in DAM-treated cells than control cells. None of the drugs alone induced nucleolar ANS staining but it was induced by heat-shock of control cells and cells previously treated with DAM. UBF and pNBS1 were systematically co-localized in the nucleolus of CX-5461- and DAM-treated cells. pNF-κB only localized to the nucleolar caps of pre-apoptotic DAM-treated cells. We directly quantified water and ion content in cell compartments using cryo-correlative electron microscopy. We show that different chemotherapeutic nucleolar stress inducers result in distinctive, thus far-unrecognized changes in macromolecular crowding and elemental content which are known to modify cell metabolism. Moreover we were able to correlate these changes to the sensitivity of treated cells to heat-shock and the behavior of nucleolar pNBS1 and pNF-κB.
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http://dx.doi.org/10.7150/ntno.31878DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6536780PMC
June 2020

Vincristine induces procoagulant activity of the human lymphoblastic leukemia cell line Jurkat through the release of extracellular vesicles.

J Thromb Thrombolysis 2019 Aug;48(2):195-202

EA3801-HERVI, Université de Reims Champagne-Ardenne, Reims, France.

Thromboembolic events are frequent and serious complications of acute lymphoblastic leukaemia treatment. The importance of chemotherapy in the pathogenesis of this increased risk is enhanced by the fact that thrombosis rarely occurs at diagnosis. Our study aims at investigating the effect of chemotherapy on pro-coagulant activity (PCA), phosphatidylserine (PS) exposure, tissue factor (TF) activity and derived extracellular vesicles (EV) of Jurkat cells. Jurkat cells were treated with two commonly used chemotherapeutics: Vincristine (VCR) or Daunorubicin (DNR), at relevant concentrations. PCA of cells and derived EV were evaluated using Thrombin generation Assay (TGA). Cells or EV were incubated with annexin V or anti TF antibodies to assess the respective contribution of TF and PS. PS exposure on cells was analysed by flow cytometry. Derived EV were evaluated in fluorescence microscopy and flow cytometry. Untreated Jurkat cells and EV support thrombin generation. Thrombin generation was abolished when PS activity was inhibited by annexin V. VCR treatment resulted in a time dependent increase of thrombin generation. After VCR exposure, TF activity increased as well as PS exposure increased on the cell surface. The increase in TF activity was abolished by annexin V indicating that PS was required. A spontaneous release of EV from Jurkat cells was observed and VCR treatment increased the number of generated EV. Our results indicate that VCR increased the PCA of Jurkat cells predominantly through PS exposure and increased EV generation. Lymphoid blasts derived EV could be biomarkers to determine high thrombotic risk ALL patients.
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http://dx.doi.org/10.1007/s11239-019-01894-xDOI Listing
August 2019

Fluorescence Lifetime Imaging of Plant Cell Walls.

Methods Mol Biol 2019 ;1992:77-82

Fractionation of AgroResources and Environment (FARE) Laboratory, INRA, University of Reims Champagne-Ardenne, Reims, France.

Fluorescence is a versatile property of many molecules called fluorophores. In plant cell walls, fluorescence is generally attributed to aromatic molecules such as lignin. In contrary to fluorescence intensity, fluorescence lifetime is independent from fluorophore concentration. So mapping fluorescence lifetime of plant cell walls represents a complementary approach to acquire chemical and structural information of cell wall components and interactions.
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http://dx.doi.org/10.1007/978-1-4939-9469-4_5DOI Listing
February 2020

NET Formation in Bullous Pemphigoid Patients With Relapse Is Modulated by IL-17 and IL-23 Interplay.

Front Immunol 2019 4;10:701. Epub 2019 Apr 4.

Laboratory of Dermatology, Faculty of Medicine of Reims, University of Champagne-Ardenne, Reims, France.

DNA extracellular traps (ETs), released by neutrophils (NETs), or eosinophils (EETs), play a pathogenic role in several autoimmune disorders. However, to date, NETs have never been investigated in bullous pemphigoid (BP) with respect to clinical and immunological activities, both at baseline and at time of relapse which have been characterized with specific IL-17 and IL-23 patterns. We sought to assess whether ETs were associated with BP as well as the relative contribution of IL-17 axis cytokines to NET induction. Skin biopsy specimens were obtained from 11 patients with BP. Immuno-detection of neutrophils and eosinophils combined to DNA staining allowed us to investigate the presence of NETs and EETs using confocal scanning microscopy. NETs release was evaluated by stimulating polymorphonuclear cells from BP patients with BP biological fluids in presence of IL-17A and IL-23 or of glucocorticoids. At baseline, ETs were observed in BP lesions at the site of dermal-epidermal cleavage. Despite an important infiltrate of eosinophils, ETs were essentially associated with neutrophils and were not related to BP clinical activity at diagnosis. observation of NETs was associated in 6 among 8 patients with serum capacity of NET induction. Notably both blister fluid and sera from BP patients at diagnosis and at time of relapse could induce NET formation . In contrast, a longitudinal investigation showed a decrease of NET formation with time of treatment in patients undergoing remission. Mimicking relapse, complementation of sera from BP patients with ongoing remission with either IL-17A or IL-23 increased NET formation. Conversely, IL-17A inhibited NET formation induced by serum from BP patients with relapse supplemented or not with IL-23. Finally, glucocorticoids also inhibited NET formation in BP. NET formation is an associated phenomenon with BP. Furthermore, we showed that IL-23 favored NET formation, whereas the effects of IL-17A are environment dependent. Indeed, IL-17A displayed a protective effect on NET formation when associated with IL-23, showing for the first-time differential effects of these two cytokines in BP.
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http://dx.doi.org/10.3389/fimmu.2019.00701DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6458298PMC
September 2020

Tumour cell blebbing and extracellular vesicle shedding: key role of matrikines and ribosomal protein SA.

Br J Cancer 2019 02 11;120(4):453-465. Epub 2019 Feb 11.

Université de Reims Champagne Ardenne, SFR CAP-Santé (FED 4231), Laboratoire de Biochimie Médicale et Biologie Moléculaire, Reims, France.

Background: Carcinogenesis occurs in elastin-rich tissues and leads to local inflammation and elastolytic proteinase release. This contributes to bioactive matrix fragment (Matrikine) accumulation like elastin degradation products (EDP) stimulating tumour cell invasive and metastatic properties. We previously demonstrate that EDPs exert protumoural activities through Hsp90 secretion to stabilised extracellular proteinases.

Methods: EDP influence on cancer cell blebbing and extracellular vesicle shedding were examined with a videomicroscope coupled with confocal Yokogawa spinning disk, by transmission electron microscopy, scanning electron microscopy and confocal microscopy. The ribosomal protein SA (RPSA) elastin receptor was identified after affinity chromatography by western blotting and cell immunolocalisation. mRNA expression was studied using real-time PCR. SiRNA were used to confirm the essential role of RPSA.

Results: We demonstrate that extracellular matrix degradation products like EDPs induce tumour amoeboid phenotype with cell membrane blebbing and shedding of extracellular vesicle containing Hsp90 and proteinases in the extracellular space. EDPs influence intracellular calcium influx and cytoskeleton reorganisation. Among matrikines, VGVAPG and AGVPGLGVG peptides reproduced EDP effects through RPSA binding.

Conclusions: Our data suggests that matrikines induce cancer cell blebbing and extracellular vesicle release through RPSA binding, favouring dissemination, cell-to-cell communication and growth of cancer cells in metastatic sites.
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http://dx.doi.org/10.1038/s41416-019-0382-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6461924PMC
February 2019

Identification of CD36 as a new interaction partner of membrane NEU1: potential implication in the pro-atherogenic effects of the elastin receptor complex.

Cell Mol Life Sci 2019 Feb 29;76(4):791-807. Epub 2018 Nov 29.

UMR CNRS 7369 Matrice Extracellulaire et Dynamique Cellulaire (MEDyC), Team 2 "Matrix Aging and Vascular Remodelling", Université de Reims Champagne Ardenne (URCA), UFR Sciences Exactes et Naturelles, Moulin de la Housse, BP1039, 51687, Reims Cedex 2, France.

In addition to its critical role in lysosomes for catabolism of sialoglycoconjugates, NEU1 is expressed at the plasma membrane and regulates a myriad of receptors by desialylation, playing a key role in many pathophysiological processes. Here, we developed a proteomic approach dedicated to the purification and identification by LC-MS/MS of plasma membrane NEU1 interaction partners in human macrophages. Already known interaction partners were identified as well as several new candidates such as the class B scavenger receptor CD36. Interaction between NEU1 and CD36 was confirmed by complementary approaches. We showed that elastin-derived peptides (EDP) desialylate CD36 and that this effect was blocked by the V14 peptide, which blocks the interaction between bioactive EDP and the elastin receptor complex (ERC). Importantly, EDP also increased the uptake of oxidized LDL by macrophages that is blocked by both the V14 peptide and the sialidase inhibitor 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (DANA). These results demonstrate, for the first time, that binding of EDP to the ERC indirectly modulates CD36 sialylation level and regulates oxidized LDL uptake through this sialidase. These effects could contribute to the previously reported proatherogenic role of EDP and add a new dimension in the regulation of biological processes through NEU1.
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http://dx.doi.org/10.1007/s00018-018-2978-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6514072PMC
February 2019

Contribution of Fluorescence Techniques in Determining the Efficiency of the Non-thermal Plasma Treatment.

Front Microbiol 2018 10;9:2171. Epub 2018 Sep 10.

Laboratoire de Biomatériaux et Inflammation en Site Osseux (EA 4691), SFR CAP-Santé, FED 4231, Université de Reims Champagne-Ardenne, Reims, France.

We have recently developed a non-thermal plasma (NTP) equipment intended to sterilize fragile medical devices and maintain the sterile state of items downstream the treatment. With traditional counts on agar plate a six log reduction of viability was obtained within 120 min of O, Ar, or N NTP treatments. However to determine the best NTP process, we studied the different physiological states of by flow cytometry (FC) and confocal laser scanning microscopy (CLSM) focusing on the esterasic activity and membrane integrity of the bacteria. Two fluorochromes, 5-(and-6)-carboxy-2,7-dichlorofluorescein diacetate and propidium iodide were used in order to distinguish three sub-populations: metabolically active, permeabilized, and damaged bacteria that can be in the viable but nonculturable state. FC and CLSM highlight that O and Ar NTP treatments were the most attractive processes. Indeed, a 5 min of Ar NTP generated a high destruction of the structure of bacteria and a 120 min of O NTP treatment led to the higher decrease of the total damaged bacteria population. SEM observations showed that in presence of clusters, bacteria of upper layers are easily altered compared to bacteria in the deeper layers. In conclusion, the plate counting method is not sufficient by itself to determine the best NTP treatment. FC and CLSM represent attractive indicator techniques to select the most efficient gas NTP treatment generating the lowest proportion of viable bacteria and the most debris.
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http://dx.doi.org/10.3389/fmicb.2018.02171DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6140754PMC
September 2018

FRET-SLiM on native autofluorescence: a fast and reliable method to study interactions between fluorescent probes and lignin in plant cell wall.

Plant Methods 2018 27;14:74. Epub 2018 Aug 27.

3TISBio, Unité de Glycobiologie Structurale et Fonctionnelle (UGSF), CNRS, UMR 8576, Université de Lille, 59000 Lille, France.

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.
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http://dx.doi.org/10.1186/s13007-018-0342-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6109981PMC
August 2018

Fluorescent Nano-Probes to Image Plant Cell Walls by Super-Resolution STED Microscopy.

Plants (Basel) 2018 Feb 6;7(1). Epub 2018 Feb 6.

Plateforme d'Imagerie Cellulaire et Tissulaire (PICT), Université de Reims Champagne-Ardenne, 51 rue Cognacq-Jay, 51100 Reims, France.

Lignocellulosic biomass is a complex network of polymers making up 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 requires some pretreatments and several types of catalysts to be transformed efficiently. Gaining more knowledge in the architecture of plant cell walls is therefore important to understand and optimize transformation processes. For the first time, super-resolution imaging of poplar wood samples has been performed using the Stimulated Emission Depletion (STED) technique. In comparison to standard confocal images, STED reveals new details in cell wall structure, allowing the identification of secondary walls and middle lamella with fine details, while keeping open the possibility to perform topochemistry by the use of relevant fluorescent nano-probes. In particular, the deconvolution of STED images increases the signal-to-noise ratio so that images become very well defined. The obtained results show that the STED super-resolution technique can be easily implemented by using cheap commercial fluorescent rhodamine-PEG nano-probes which outline the architecture of plant cell walls due to their interaction with lignin. Moreover, the sample preparation only requires easily-prepared plant sections of a few tens of micrometers, in addition to an easily-implemented post-treatment of images. Overall, the STED super-resolution technique in combination with a variety of nano-probes can provide a new vision of plant cell wall imaging by filling in the gap between classical photon microscopy and electron microscopy.
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http://dx.doi.org/10.3390/plants7010011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5874600PMC
February 2018

Nucleolar sub-compartments in motion during rRNA synthesis inhibition: Contraction of nucleolar condensed chromatin and gathering of fibrillar centers are concomitant.

PLoS One 2017 30;12(11):e0187977. Epub 2017 Nov 30.

CNRS UMR 7369, Université de Reims Champagne Ardenne, Reims, France.

The nucleolus produces the large polycistronic transcript (47S precursor) containing the 18S, 5.8S and 28S rRNA sequences and hosts most of the nuclear steps of pre-rRNA processing. Among numerous components it contains condensed chromatin and active rRNA genes which adopt a more accessible conformation. For this reason, it is a paradigm of chromosome territory organization. Active rRNA genes are clustered within several fibrillar centers (FCs), in which they are maintained in an open configuration by Upstream Binding Factor (UBF) molecules. Here, we used the reproducible reorganization of nucleolar components induced by the inhibition of rRNA synthesis by Actinomycin D (AMD) to address the steps of the spatiotemporal reorganization of FCs and nucleolar condensed chromatin. To reach that goal, we used two complementary approaches: i) time-lapse confocal imaging of cells expressing one or several GFP-tagged proteins (fibrillarin, UBF, histone H2B) and ii) ultrastructural identification of nucleolar components involved in the reorganization. Data obtained by time lapse confocal microscopy were analyzed through detailed 3D imaging. This allowed us to demonstrate that AMD treatment induces no fusion and no change in the relative position of the different nucleoli contained in one nucleus. In contrast, for each nucleolus, we observed step by step gathering and fusion of both FCs and nucleolar condensed chromatin. To analyze the reorganization of FCs and condensed chromatin at a higher resolution, we performed correlative light and electron microscopy electron microscopy (CLEM) imaging of the same cells. We demonstrated that threads of intranucleolar condensed chromatin are localized in a complex 3D network of vacuoles. Upon AMD treatment, these structures coalesce before migrating toward the perinucleolar condensed chromatin, to which they finally fuse. During their migration, FCs, which are all linked to ICC, are pulled by the latter to gather as caps disposed at the periphery of nucleoli.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0187977PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5708645PMC
December 2017

Seeing biomass recalcitrance through fluorescence.

Sci Rep 2017 08 18;7(1):8838. Epub 2017 Aug 18.

FARE laboratory, INRA, University of Reims Champagne-Ardenne, 2 esplanade Roland-Garros, 51100, Reims, France.

Lignocellulosic biomass is the only renewable carbon resource available in sufficient amount on Earth to go beyond the fossil-based carbon economy. Its transformation requires controlled breakdown of polymers into a set of molecules to make fuels, chemicals and materials. But biomass is a network of various inter-connected polymers which are very difficult to deconstruct optimally. In particular, saccharification potential of lignocellulosic biomass depends on several complex chemical and physical factors. For the first time, an easily measurable fluorescence properties of steam-exploded biomass samples from miscanthus, poplar and wheat straw was shown to be directly correlated to their saccharification potential. Fluorescence can thus be advantageously used as a predictive method of biomass saccharification. The loss in fluorescence occurring after the steam explosion pretreatment and increasing with pretreatment severity does not originate from the loss in lignin content, but rather from a decrease of the lignin β-aryl-ether linkage content. Fluorescence lifetime analysis demonstrates that monolignols making lignin become highly conjugated after steam explosion pretreatment. These results reveal that lignin chemical composition is a more important feature to consider than its content to understand and to predict biomass saccharification.
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http://dx.doi.org/10.1038/s41598-017-08740-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5562871PMC
August 2017

Lumican delays melanoma growth in mice and drives tumor molecular assembly as well as response to matrix-targeted TAX2 therapeutic peptide.

Sci Rep 2017 08 9;7(1):7700. Epub 2017 Aug 9.

Université de Reims Champagne-Ardenne, UFR Sciences Exactes et Naturelles, Campus Moulin de la Housse, 51100, Reims, France.

Lumican is a small leucine-rich proteoglycan (SLRP) being known as a key regulator of collagen fibrillogenesis. However, little attention has been given so far in studying its influence on tumor-associated matrix architecture. Here, we investigate the role of host lumican on tumor matrix organization as well as on disease progression considering an immunocompetent model of melanoma implanted in Lum vs. wild type syngeneic mice. Conjointly, lumican impact on tumor response to matrix-targeted therapy was evaluated considering a previously validated peptide, namely TAX2, that targets matricellular thrombospondin-1. Analysis of available genomics and proteomics databases for melanoma first established a correlation between lumican expression and patient outcome. In the B16 melanoma allograft model, endogenous lumican inhibits tumor growth and modulates response to TAX2 peptide. Indeed, IHC analyses revealed that lumican deficiency impacts intratumoral distribution of matricellular proteins, growth factor and stromal cells. Besides, innovative imaging approaches helped demonstrating that lumican host expression drives biochemical heterogeneity of s.c. tumors, while modulating intratumoral collagen deposition as well as organization. Altogether, the results obtained present lumican as a strong endogenous inhibitor of tumor growth, while identifying for the first time this proteoglycan as a major driver of tumor matrix coherent assembly.
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http://dx.doi.org/10.1038/s41598-017-07043-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5550434PMC
August 2017

Role of LRP-1 in cancer cell migration in 3-dimensional collagen matrix.

Cell Adh Migr 2017 07 27;11(4):316-326. Epub 2016 Jul 27.

a Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7369 Matrice Extracellulaire et Dynamique Cellulaire, Université de Reims Champagne-Ardenne, Unité de Formation et de Recherche Sciences Exactes et Naturelles , Reims , France.

The low-density lipoprotein receptor-related protein-1 (LRP-1) is a member of Low Density Lipoprotein Receptor (LDLR) family, which is ubiquitously expressed and which is described as a multifunctional endocytic receptor which mediates the clearance of various extracellular matrix molecules including serine proteinases, proteinase-inhibitor complexes, and matricellular proteins. Several studies showed that high LRP-1 expression promotes breast cancer cell invasiveness, and LRP-1 invalidation leads to cell motility abrogation in both tumor and non-tumor cells. Furthermore, our group has reported that LRP-1 silencing prevents the invasion of a follicular thyroid carcinoma despite increased pericellular proteolytic activities from MMP2 and uPA using a 2D-cell culture model. As the use of 3D culture systems is becoming more and more popular due to their promise as enhanced models of tissue physiology, the aim of the present work is to characterize for the first time how the 3D collagen type I matrix may impact the ability of LRP-1 to regulate the migratory properties of thyroid carcinoma using as a model FTC-133 cells. Our results show that inhibition of LRP-1 activity or expression leads to morphological changes affecting cell-matrix interactions, reorganizations of the actin-cytoskeleton especially by inhibiting FAK activation and increasing RhoA activity and MLC-2 phosphorylation, thus preventing cell migration. Taken together, our results suggest that LRP-1 silencing leads to a decrease of cell migratory capacity in a 3D configuration.
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http://dx.doi.org/10.1080/19336918.2016.1215788DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5569966PMC
July 2017

Tissue factor expressed by adherent cells contributes to hemodialysis-membrane thrombogenicity.

Thromb Res 2016 Aug 27;144:218-23. Epub 2016 May 27.

Equipe d'accueil 3801 Hémostase et Remodelage Vasculaire Post-Ischémie, Université de Reims, France. Electronic address:

End-stage renal patients present a high risk of thrombosis and bleeding. Consequently, it is challenging to prevent clotting during hemodialysis. If a contact system induces thrombin generation in the extra corporeal circuit, recent data suggest a role of tissue factor (TF) in hemodialysis-associated thrombosis. Using a method of elution, we collected adhering cells to an acrylonitrile membrane layered by polythyleneimine (AN69-ST). Using optic microscopy and flow cytometry, we observed that adherent cells were mainly constituted by activated polymorphonuclear neutrophils (PMNs). Using a sensitive fluorogenic method of thrombin generation, we found that adhering cells triggered thrombin generation in a TF-dependent manner. We next identified the presence of TF mRNA (Q-PCR) in adhering cells. Using immunofluorescence, we observed the presence of TF in PMNs and of TF-decorated neutrophil extracellular traps (NETs). As TF triggers thrombin generation after binding to serine protease FVIIa, we evaluated the effect of an inactivated human recombinant factor VIIa (hrFVIIai) in a sheep model of hemodialysis (HD). One single bolus of hrFVIIai maintained the full patency of the hemodialysis circuit without any measurable systemic anticoagulant effect. TF is a promising target for preventing thrombosis during HD.
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http://dx.doi.org/10.1016/j.thromres.2016.05.017DOI Listing
August 2016

Aging decreases collagen IV expression in vivo in the dermo-epidermal junction and in vitro in dermal fibroblasts: possible involvement of TGF-β1.

Eur J Dermatol 2016 Aug;26(4):350-60

Laboratoire de Biochimie Médicale et de Biologie Moléculaire, CNRS UMR 7369: Matrice Extracellulaire et Dynamique Cellulaire (MEDyC), UFR Médecine, Université de Reims Champagne-Ardenne, 51 rue Cognacq Jay, CS 30018, 51095.

Collagen IV is a major component of the dermo-epidermal junction (DEJ). To study expression of collagen IV upon aging in the DEJ and dermal fibroblasts isolated from the same patients. A model of senescent fibroblasts was developed in order to identify biological compounds that might restore the level of collagen IV. Skin fragments of women (30 to 70 years old) were collected. Localisation of collagen IV expression in the DEJ was studied by immunofluorescence. Fibroblast collagen IV expression was studied by real-time PCR, ELISA, and western blotting. Premature senescence was simulated by exposing fibroblasts to subcytotoxic H2O2 concentrations. Collagen IV decreased in the DEJ and fibroblasts relative to age. TGF-β1 treatment significantly increased collagen IV gene and protein expression in fibroblasts and restored expression in the model of senescence. Addition of TGF-β1-neutralizing antibody to fibroblast cultures decreased collagen IV expression. Taken together, the results suggest that the decrease in collagen IV in the DEJ, relative to age, could be due to a decrease in collagen IV expression by senescent dermal fibroblasts and may involve TGF-β1 signalling.
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http://dx.doi.org/10.1684/ejd.2016.2782DOI Listing
August 2016

Simultaneous detection of the protozoan parasites Toxoplasma, Cryptosporidium and Giardia in food matrices and their persistence on basil leaves.

Food Microbiol 2016 Aug 12;57:36-44. Epub 2016 Jan 12.

ACTALIA Food Safety Department, 310 rue Popielujko, 50000 Saint-Lô, France. Electronic address:

Toxoplasma gondii, Cryptosporidium spp. and Giardia intestinalis are emerging pathogen parasites in the food domain. However, without standardized methods for their detection in food matrices, parasitic foodborne outbreaks remain neglected. In this study, a new immunomagnetic separation assay (IMS Toxo) targeting the oocyst's wall of T. gondii was developed using a specific purified monoclonal antibody. Performance of this IMS Toxo coupled to microscopic and qPCR analyses was evaluated in terms of limit of detection (LOD) and recovery rate (RR) on: i) simple matrix (LOD = 5 oocysts; RR between 5 and 56%); ii) raspberries and basil (LOD = 33 oocysts/g; RR between 0.2 and 35%). Finally, to simultaneously extract the three protozoa from these food matrices, T. gondii oocysts were directly concentrated (without IMS Toxo) from the supernatant of the IMS of Cryptosporidium and Giardia (oo)cysts. This strategy associated to qPCR detection led to LOD <1 to 3 (oo)cysts/g and RR between 2 and 35%. This procedure was coupled to RT-qPCR analyses and showed that the three protozoa persisted on the leaves of basil and remained viable following storage at 4 °C for 8 days. These data strengthen the need to consider these protozoa in food safety.
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http://dx.doi.org/10.1016/j.fm.2016.01.002DOI Listing
August 2016

Highlighting the impact of aging on type I collagen: label-free investigation using confocal reflectance microscopy and diffuse reflectance spectroscopy in 3D matrix model.

Oncotarget 2016 Feb;7(8):8546-55

MéDIAN-Biophotonique et Technologies pour la Santé, Université de Reims Champagne-Ardenne, CNRS UMR 7369 MEDyC, UFR de Pharmacie, SFR CAP Santé, Reims, France.

During aging, alterations of extracellular matrix proteins contribute to various pathological phenotypes. Among these alterations, type I collagen cross-linking and associated glycation products accumulation over time detrimentally affects its physico-chemical properties, leading to alterations of tissue biomechanical stability. Here, different-age collagen 3D matrices using non-destructive and label-free biophotonic techniques were analysed to highlight the impact of collagen I aging on 3D constructs, at macroscopic and microscopic levels. Matrices were prepared with collagens extracted from tail tendons of rats (newborns, young and old adults) to be within the physiological aging process. The data of diffuse reflectance spectroscopy reveal that aging leads to an inhibition of fibril assembly and a resulting decrease of gel density. Investigations by confocal reflectance microscopy highlight poor-fibrillar structures in oldest collagen networks most likely related to the glycation products accumulation. Complementarily, an infrared analysis brings out marked spectral variations in the Amide I profile, specific of the peptidic bond conformation and for carbohydrates vibrations as function of collagen-age. Interestingly, we also highlight an unexpected behavior for newborn collagen, exhibiting poorly-organized networks and microscopic features close to the oldest collagen. These results demonstrate that changes in collagen optical properties are relevant for investigating the incidence of aging in 3D matrix models.
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http://dx.doi.org/10.18632/oncotarget.7385DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4890986PMC
February 2016

Stage-Specific Changes in the Water, Na+, Cl- and K+ Contents of Organelles during Apoptosis, Demonstrated by a Targeted Cryo Correlative Analytical Approach.

PLoS One 2016 11;11(2):e0148727. Epub 2016 Feb 11.

CNRS UMR 7369, Université de Reims Champagne Ardenne, Reims, France.

Many studies have demonstrated changes in the levels of several ions during apoptosis, but a few recent studies have reported conflicting results concerning the changes in water content in apoptotic cells. We used a correlative light and cryo-scanning transmission electron microscopy method to quantify water and ion/element contents simultaneously at a nanoscale resolution in the various compartments of cells, from the onset to the end of apoptosis. We used stably transfected HeLa cells producing H2B-GFP to identify the stages of apoptosis in cells and for a targeted elemental analysis within condensed chromatin, nucleoplasm, mitochondria and the cytosol. We found that the compartments of apoptotic cells contained, on average, 10% more water than control cells. During mitochondrial outer membrane permeabilization, we observed a strong increase in the Na+ and Cl- contents of the mitochondria and a strong decrease in mitochondrial K+ content. During the first step in apoptotic volume decrease (AVD), Na+ and Cl- levels decreased in all cell compartments, but remained higher than those in control cells. Conversely, during the second step of AVD, Na+ and Cl- levels increased considerably in the nucleus and mitochondria. During these two steps of AVD, K+ content decreased steadily in all cell compartments. We also determined in vivo ion status during caspase-3 activity and chromatin condensation. Finally, we found that actinomycin D-tolerant cells had water and K+ contents similar to those of cells entering apoptosis but lower Na+ and Cl- contents than both cells entering apoptosis and control cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0148727PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4807926PMC
July 2016