Identification of new potential molecular actors related to fiber quality in flax through Omics.

One of the biggest challenges for a more widespread utilization of plant fibers is to better understand the different molecular factors underlying the variability in fineness and mechanical properties of both elementary and scutched fibers. Accordingly, we analyzed genome-wide transcription profiling from bast fiber bearing tissues of seven different flax varieties (4 spring, 2 winter fiber varieties and 1 winter linseed) and identified 1041 differentially expressed genes between varieties, of which 97 were related to cell wall metabolism. KEGG analysis highlighted a number of different enriched pathways. Subsequent statistical analysis using Partial Least-Squares Discriminant Analysis showed that 73% of the total variance was explained by the first 3 X-variates corresponding to 56 differentially expressed genes. Calculation of Pearson correlations identified 5 genes showing a strong correlation between expression and morphometric data. Two-dimensional gel proteomic analysis on the two varieties showing the most discriminant and significant differences in morphometrics revealed 1490 protein spots of which 108 showed significant differential abundance. Mass spectrometry analysis successfully identified 46 proteins representing 32 non-redundant proteins. Statistical clusterization based on the expression level of genes corresponding to the 32 proteins showed clear discrimination into three separate clusters, reflecting the variety type (spring-/winter-fiber/oil). Four of the 32 proteins were also highly correlated with morphometric features. Examination of predicted functions for the 9 (5 + 4) identified genes highlighted lipid metabolism and senescence process. Calculation of Pearson correlation coefficients between expression data and retted fiber mechanical measurements (strength and maximum force) identified 3 significantly correlated genes. The genes were predicted to be connected to cell wall dynamics, either directly (Expansin-like protein), or indirectly (NAD(P)-binding Rossmann-fold superfamily protein). Taken together, our results have allowed the identification of molecular actors potentially associated with the determination of both in-planta fiber morphometrics, as well as ex-planta fiber mechanical properties, both of which are key parameters for elementary fiber and scutched fiber quality in flax.

Comparative Analysis of G-Layers in Bast Fiber and Xylem Cell Walls in Flax Using Raman Spectroscopy.

In a response to gravitropic stress, G-layers (gelatinous layers) were deposited in xylem cell walls of tilted flax plants. G-layers were produced in both tension wood (upper side) as expected but were also observed in opposite wood (lower side). Raman spectral profiles were acquired for xylem G-layers from the tension and opposite side as well as from the G-layer of bast fibers grown under non-tilted conditions. Statistical analysis by principal component analysis (PCA) and partial least square-discriminant analysis (PLS-DA) clearly distinguished bast fiber G-layers from xylem G-layers. Discriminating bands were observed for cellulose (380-1150-1376 cm-1), hemicelluloses (517-1094-1126-1452 cm-1) and aromatics (1270-1599-1658 cm-1). PCA did not allow separation of G-layers from tension/opposite-wood sides. In contrast, the two types of xylem G-layers could be incompletely discriminated through PLS-DA. Overall, the results suggested that while the architecture (polymer spatial distribution) of bast fibers G-layers and xylem G-layers are similar, they should be considered as belonging to a different cell wall layer category based upon ontogenetical and chemical composition parameters.

Ratiometric Fluorescent Safranin-O Staining Allows the Quantification of Lignin Contents In Muro.

In some specific vascular plant tissues, lignin can impregnate the entire cell wall to make it more rigid and hydrophobic. Different techniques have been developed in the past years to make possible the quantification of this polyphenolic polymer at the organ or tissue level, but difficulties of access to the cellular level remain. Here we describe an approach based on ratiometric emission measurements using safranin-O and the development of a macro adapted for the FIJI software, which makes it possible to quantify lignin in three different layers of the cell wall on images captured on a fluorescent confocal microscope.

Raman spectroscopy mapping of changes in the organization and relative quantities of cell wall polymers in bast fiber cell walls of flax plants exposed to gravitropic stress.

Flax is an important fiber crop that is subject to lodging. In order to gain more information about the potential role of the bast fiber cell wall in the return to the vertical position, 6-week-old flax plants were subjected to a long-term (6 week) gravitropic stress by stem tilting in an experimental set-up that excluded autotropism. Stress induced significant morphometric changes (lumen surface, lumen diameter, and cell wall thickness and lumen surface/total fiber surface ratio) in pulling- and opposite-side fibers compared to control fibers. Changes in the relative amounts and spatial distribution of cell wall polymers in flax bast fibers were determined by Raman vibrational spectroscopy. Following spectra acquisition, datasets (control, pulling- and opposite sides) were analyzed by principal component analysis, PC score imaging, and Raman chemical cartography of significant chemical bonds. Our results show that gravitropic stress induces discrete but significant changes in the composition and/or spatial organization of cellulose, hemicelluloses and lignin within the cell walls of both pulling side and opposite side fibers.

Development of a Once-Daily Modified-Release Formulation for the Short Half-Life RIPK1 Inhibitor GSK2982772 using DiffCORE Technology.

PURPOSE: GSK2982772 is a selective inhibitor of receptor-interacting protein kinase-1 (RIPK1) with a short 2- to 3-h half-life. In a previous modified-release (MR) study, a matrix monolithic formulation (80% GSK2982772 released over 12 h) provided a once-daily (QD) pharmacokinetic (PK) profile in the fasted state; however, it was susceptible to food effects. The current study evaluated the safety and PK of MR formulations using GSK proprietary DiffCORE™ technology. METHODS: Part A evaluated PK following single-dose (240 mg) fasted and fed (high-fat meal) administration of three DiffCORE MR formulations within pre-defined in vitro extremes of 80% GSK2982772 released over 12 h (MR-12 h) to 80% GSK2982772 released over 18 h (MR-18 h) versus an immediate-release formulation. Part B evaluated MR-16 h (120-960 mg) in different prandial states. RESULTS: Pharmacokinetic profiles for all MR formulations and doses tested in the fasted and fed states were consistent with QD dosing. CONCLUSIONS: The DiffCORE technology overcame the food effect vulnerability observed with the matrix monolithic formulation. The MR-16 h formulation was selected for further clinical development as a QD dosing regimen (NCT03649412 September 26, 2018).

REPRISAL: mapping lignification dynamics using chemistry, data segmentation, and ratiometric analysis.

This article describes a methodology for detailed mapping of the lignification capacity of plant cell walls that we have called "REPRISAL" for REPorter Ratiometrics Integrating Segmentation for Analyzing Lignification. REPRISAL consists of the combination of three separate approaches. In the first approach, H*, G*, and S* monolignol chemical reporters, corresponding to p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol, are used to label the growing lignin polymer in a fluorescent triple labeling strategy based on the sequential use of three main bioorthogonal chemical reactions. In the second step, an automatic parametric and/or artificial intelligence segmentation algorithm is developed that assigns fluorescent image pixels to three distinct cell wall zones corresponding to cell corners, compound middle lamella and secondary cell walls. The last step corresponds to the exploitation of a ratiometric approach enabling statistical analyses of differences in monolignol reporter distribution (ratiometric method [RM] 1) and proportions (RM 2) within the different cell wall zones. We first describe the use of this methodology to map developmentally related changes in the lignification capacity of wild-type Arabidopsis (Arabidopsis thaliana) interfascicular fiber cells. We then apply REPRISAL to analyze the Arabidopsis peroxidase (PRX) mutant prx64 and provide further evidence for the implication of the AtPRX64 protein in floral stem lignification. In addition, we also demonstrate the general applicability of REPRISAL by using it to map lignification capacity in poplar (Populus tremula × Populus alba), flax (Linum usitatissimum), and maize (Zea mays). Finally, we show that the methodology can be used to map the incorporation of a fucose reporter into noncellulosic cell wall polymers.

A randomised, placebo-controlled study of RIPK1 inhibitor GSK2982772 in patients with active ulcerative colitis.

OBJECTIVE: Tumour necrosis factor signalling via the receptor-interacting protein kinase 1 (RIPK1) pathway regulates colonic inflammation suggesting that RIPK1 inhibition may be a potential therapeutic target in ulcerative colitis (UC). This phase IIa, randomised, double-blind experimental medicine study investigated the safety, pharmacokinetics (PK), pharmacodynamics (PD) and preliminary efficacy of the RIPK1 inhibitor GSK2982772 in patients with active UC. DESIGN: In part A, prior to a protocol amendment, one patient was randomised to receive GSK2982772 60 mg twice daily for 42 days. After the amendment, patients were randomised 2:1 to receive GSK2982772 60 mg or placebo three times daily for 42 days. In part B, all patients switched to open-label GSK2982772 60 mg three times daily for 42 days. Safety, PK, PD biomarkers, histological disease activity, clinical efficacy and quality of life were assessed at days 43 and 85. RESULTS: Thirty-six patients were randomised (n=12, placebo/open-label GSK2982772; n=24, GSK2982772/open-label GSK2982772). Most adverse events were mild, with headache reported the most frequently across groups (placebo/open-label GSK2982772, n=2 (17%); GSK2982772/open-label GSK2982772, n=8 (33%)). GSK2982772 was well distributed into colonic tissue, with generally higher concentrations in colonic biopsy samples versus plasma. No apparent differences between treatment groups were observed for PD, histological disease activity, clinical disease activity or quality-of-life measures. At screening, all patients had Mayo endoscopic scores of 2 or 3. At day 43, no patients in the placebo/open-label GSK2982772 group achieved Mayo endoscopic scores of 0 or 1 vs 3/24 (13%) for GSK2982772/open-label GSK2982772. At day 85, 1/9 (11%) achieved scores of 0 or one for placebo/open-label GSK2982772 vs 3/22 (14%) for GSK2982772/open-label GSK2982772. CONCLUSION: GSK2982772 was generally well tolerated, with no treatment-related safety concerns identified. However, no significant differences in efficacy were observed between treatment groups, suggesting that GSK2982772 as monotherapy is not a promising treatment for patients with active UC. TRIAL REGISTRATION NUMBER: NCT02903966.

Development of a Prototype, Once-Daily, Modified-Release Formulation for the Short Half-Life RIPK1 Inhibitor GSK2982772.

PURPOSE: GSK2982772 is a selective inhibitor of receptor-interacting protein kinase-1, with a 2-3 h half-life. This study evaluated if a once-daily modified-release formulation of GSK2982772 could be developed with no significant food effect. METHODS: Part A evaluated the pharmacokinetics of GSK2982772 following fasted single-dose (120 mg) administration of two matrix minitab formulations (MT-8 h and MT-12 h) vs 120 mg immediate release (IR) and MT-12 h with a high-fat meal. Part B evaluated once-daily MT-12 h for 3 days at three dose levels. Part C evaluated a matrix monolithic (MM-12 h) formulation at two dose levels in different prandial states. RESULTS: All modified-release formulations dosed in the fasted state reduced maximum plasma concentration (Cmax), delayed time to Cmax, and decreased area under the curve (AUC) vs IR. When MT-12 h or MM-12 h were co-administered with a meal (standard or high-fat) Cmax and AUC increased. Dosing MM-12 h 1 h before a standard or high-fat meal had minimal impact on exposure vs fasted. CONCLUSIONS: MT-12 h and MM-12 h provided a QD pharmacokinetic profile in the fasted state, however when MT-12 h was dosed with a high-fat meal a QD profile was not maintained. ( ClinicalTrials.gov Identifier: NCT03266172).

Correction: EPR imaging of sinapyl alcohol and its application to the study of plant cell wall lignification.

Correction for 'EPR imaging of sinapyl alcohol and its application to the study of plant cell wall lignification' by Clémence Simon et al., Chem. Commun., 2021, DOI: .

EPR imaging of sinapyl alcohol and its application to the study of plant cell wall lignification.

In bioimaging, bioorthogonal chemistry is most often used to visualize chemical reporters by fluorescence in their native environment. Herein, we show that TEMPO-based probes can be ligated to monolignol reporters by Diels-Alder chemistry in plant cell walls, paving the way for the study of lignification by EPR spectroscopy and imaging.

Targeted Metagenomics of Retting in Flax: The Beginning of the Quest to Harness the Secret Powers of the Microbiota.

The mechanical and chemical properties of natural plant fibers are determined by many different factors, both intrinsic and extrinsic to the plant, during growth but also after harvest. A better understanding of how all these factors exert their effect and how they interact is necessary to be able to optimize fiber quality for use in different industries. One important factor is the post-harvest process known as retting, representing the first step in the extraction of bast fibers from the stem of species such as flax and hemp. During this process microorganisms colonize the stem and produce hydrolytic enzymes that target cell wall polymers thereby facilitating the progressive destruction of the stem and fiber bundles. Recent advances in sequencing technology have allowed researchers to implement targeted metagenomics leading to a much better characterization of the microbial communities involved in retting, as well as an improved understanding of microbial dynamics. In this paper we review how our current knowledge of the microbiology of retting has been improved by targeted metagenomics and discuss how related '-omics' approaches might be used to fully characterize the functional capability of the retting microbiome.

UDP-GLYCOSYLTRANSFERASE 72E3 Plays a Role in Lignification of Secondary Cell Walls in Arabidopsis.

Lignin is present in plant secondary cell walls and is among the most abundant biological polymers on Earth. In this work we investigated the potential role of the UGT72E gene family in regulating lignification in Arabidopsis. Chemical determination of floral stem lignin contents in ugt72e1, ugt72e2, and ugt72e3 mutants revealed no significant differences compared to WT plants. In contrast, the use of a novel safranin O ratiometric imaging technique indicated a significant increase in the cell wall lignin content of both interfascicular fibers and xylem from young regions of ugt72e3 mutant floral stems. These results were globally confirmed in interfascicular fibers by Raman microspectroscopy. Subsequent investigation using a bioorthogonal triple labelling strategy suggested that the augmentation in lignification was associated with an increased capacity of mutant cell walls to incorporate H-, G-, and S-monolignol reporters. Expression analysis showed that this increase was associated with an up-regulation of LAC17 and PRX71, which play a key role in lignin polymerization. Altogether, these results suggest that UGT72E3 can influence the kinetics of lignin deposition by regulating monolignol flow to the cell wall as well as the potential of this compartment to incorporate monomers into the growing lignin polymer.

Characterization of the UDP-glycosyltransferase UGT72 Family in Poplar and Identification of Genes Involved in the Glycosylation of Monolignols.

Monolignols are the building blocks for lignin polymerization in the apoplastic domain. Monolignol biosynthesis, transport, storage, glycosylation, and deglycosylation are the main biological processes partaking in their homeostasis. In Arabidopsis thaliana, members of the uridine diphosphate-dependent glucosyltransferases UGT72E and UGT72B subfamilies have been demonstrated to glycosylate monolignols. Here, the poplar UGT72 family, which is clustered into four groups, was characterized: Group 1 UGT72AZ1 and UGT72AZ2, homologs of Arabidopsis UGT72E1-3, as well as group 4 UGT72B37 and UGT72B39, homologs of Arabidopsis UGT72B1-3, glycosylate monolignols. In addition, promoter-GUS analyses indicated that poplar UGT72 members are expressed within vascular tissues. At the subcellular level, poplar UGT72s belonging to group 1 and group 4 were found to be associated with the nucleus and the endoplasmic reticulum. However, UGT72A2, belonging to group 2, was localized in bodies associated with chloroplasts, as well as possibly in chloroplasts. These results show a partial conservation of substrate recognition between Arabidopsis and poplar homologs, as well as divergent functions between different groups of the UGT72 family, for which the substrates remain unknown.

A rapid and quantitative safranin-based fluorescent microscopy method to evaluate cell wall lignification.

One of the main characteristics of plant cells is the presence of the cell wall located outside the plasma membrane. In particular cells, this wall can be reinforced by lignin, a polyphenolic polymer that plays a central role for vascular plants, conferring hydrophobicity to conducting tissues and mechanical support for upright growth. Lignin has been studied extensively by a range of different techniques, including anatomical and morphological analyses using dyes to characterize the polymer localization in situ. With the constant improvement of imaging techniques, it is now possible to revisit old qualitative techniques and adapt them to obtain efficient, highly resolutive, quantitative, fast and safe methodologies. In this study, we revisit and exploit the potential of fluorescent microscopy coupled to safranin-O staining to develop a quantitative approach for lignin content determination. The developed approach is based on ratiometric emission measurements and the development of an imagej macro. To demonstrate the potential of our methodology compared with other commonly used lignin reagents, we demonstrated the use of safranin-O staining to evaluate and compare lignin contents in previously characterized Arabidopsis thaliana lignin biosynthesis mutants. In addition, the analysis of lignin content and spatial distribution in the Arabidopsis laccase mutant also provided new biological insights into the effects of laccase gene downregulation in different cell types. Our safranin-O-based methodology, also validated for Linum usitatissimum (flax), Zea mays (maize) and Populus tremula x alba (poplar), significantly improves and speeds up anatomical and developmental investigations of lignin, which we hope will contribute to new discoveries in many areas of cell wall plant research.

One, Two, Three: A Bioorthogonal Triple Labelling Strategy for Studying the Dynamics of Plant Cell Wall Formation In Vivo.

Reported herein is an in vivo triple labelling strategy to monitor the formation of plant cell walls. Based on a combination of copper-catalysed alkyne-azide cycloaddition (CuAAC), strain-promoted azide-alkyne cycloaddition (SPAAC), and Diels-Alder reaction with inverse electronic demand (DARinv ), this methodology can be applied to various plant species of interest in research. It allowed detection of the differential incorporation of alkynyl-, azido-, and methylcyclopropenyl-tagged reporters of the three main monolignols into de novo biosynthesized lignin in different tissues, cell types, or cell wall layers. In addition, this triple labelling was implemented with different classes of chemical reporters, using two monolignol reporters in conjunction with alkynylfucose to simultaneously monitor the biosynthesis of lignin and non-cellulosic polysaccharides. This allowed observation of their deposition occurring contemporaneously in the same cell wall.

Nanoparticle-induced neuronal toxicity across placental barriers is mediated by autophagy and dependent on astrocytes.

The potential for maternal nanoparticle (NP) exposures to cause developmental toxicity in the fetus without the direct passage of NPs has previously been shown, but the mechanism remained elusive. We now demonstrate that exposure of cobalt and chromium NPs to BeWo cell barriers, an in vitro model of the human placenta, triggers impairment of the autophagic flux and release of interleukin-6. This contributes to the altered differentiation of human neural progenitor cells and DNA damage in the derived neurons and astrocytes. Crucially, neuronal DNA damage is mediated by astrocytes. Inhibiting the autophagic degradation in the BeWo barrier by overexpression of the dominant-negative human ATG4BC74A significantly reduces the levels of DNA damage in astrocytes. In vivo, indirect NP toxicity in mice results in neurodevelopmental abnormalities with reactive astrogliosis and increased DNA damage in the fetal hippocampus. Our results demonstrate the potential importance of autophagy to elicit NP toxicity and the risk of indirect developmental neurotoxicity after maternal NP exposure.

Visualizing Lignification Dynamics in Plants with Click Chemistry: Dual Labeling is BLISS!

Lignin is one of the most prevalent biopolymers on the planet and a major component of lignocellulosic biomass. This phenolic polymer plays a vital structural and protective role in the development and life of higher plants. Although the intricate mechanisms regulating lignification processes in vivo strongly impact the industrial valorization of many plant-derived products, the scientific community still has a long way to go to decipher them. In a simple three-step workflow, the dual labeling protocol presented herein enables bioimaging studies of actively lignifying zones of plant tissues. The first step consists in the metabolic incorporation of two independent chemical reporters, surrogates of the two native monolignols that give rise to lignin H- and G-units. After incorporation into growing lignin polymers, each reporter is then specifically labeled with its own fluorescent probe via a sequential combination of bioorthogonal SPAAC/CuAAC click reactions. Combined with lignin autofluorescence, this approach leads to the generation of three-color localization maps of lignin within plant cell walls by confocal fluorescence microscopy and provides precise spatial information on the presence or absence of active lignification machinery at the scale of plant tissues, cells and different cell wall layers.

Characterization of Bacterial and Fungal Community Dynamics by High-Throughput Sequencing (HTS) Metabarcoding during Flax Dew-Retting.

Flax dew-retting is a key step in the industrial extraction of fibers from flax stems and is dependent upon the production of a battery of hydrolytic enzymes produced by micro-organisms during this process. To explore the diversity and dynamics of bacterial and fungal communities involved in this process we applied a high-throughput sequencing (HTS) DNA metabarcoding approach (16S rRNA/ITS region, Illumina Miseq) on plant and soil samples obtained over a period of 7 weeks in July and August 2014. Twenty-three bacterial and six fungal phyla were identified in soil samples and 11 bacterial and four fungal phyla in plant samples. Dominant phyla were Proteobacteria, Bacteroidetes, Actinobacteria, and Firmicutes (bacteria) and Ascomycota, Basidiomycota, and Zygomycota (fungi) all of which have been previously associated with flax dew-retting except for Bacteroidetes and Basidiomycota that were identified for the first time. Rare phyla also identified for the first time in this process included Acidobacteria, CKC4, Chlorobi, Fibrobacteres, Gemmatimonadetes, Nitrospirae and TM6 (bacteria), and Chytridiomycota (fungi). No differences in microbial communities and colonization dynamics were observed between early and standard flax harvests. In contrast, the common agricultural practice of swath turning affects both bacterial and fungal community membership and structure in straw samples and may contribute to a more uniform retting. Prediction of community function using PICRUSt indicated the presence of a large collection of potential bacterial enzymes capable of hydrolyzing backbones and side-chains of cell wall polysaccharides. Assignment of functional guild (functional group) using FUNGuild software highlighted a change from parasitic to saprophytic trophic modes in fungi during retting. This work provides the first exhaustive description of the microbial communities involved in flax dew-retting and will provide a valuable benchmark in future studies aiming to evaluate the effects of other parameters (e.g., year-to year and site variability etc.) on this complex process.

BLISS: Shining a light on lignification in plants.

Lignin is a polyphenolic polymer of the plant cell wall formed by the oxidative polymerization of 3 main monomers called monolignols that give rise to the lignin H-, G- and S-units. Together with cellulose and hemicelluloses, lignin is a major component of plant biomass that is widely exploited by humans in numerous industrial processes. Despite recent advances in our understanding of monolignol biosynthesis, our current understanding of the spatio-temporal regulation of their transport and polymerization is more limited. In a recent publication, we have reported the development of an original Bioorthogonal Labeling Imaging Sequential Strategy (BLISS) that allows us to visualize the simultaneous incorporation dynamics of H and G monolignol reporters into lignifying cell walls of the flax stem. 11 Here, we extend the application of this strategy to other plant organs such as roots and rapidly discuss some of the contributions and perspectives of this new technique for improving our understanding of the lignification process in plants.

Spatial regulation of monolignol biosynthesis and laccase genes control developmental and stress-related lignin in flax.

BACKGROUND: Bast fibres are characterized by very thick secondary cell walls containing high amounts of cellulose and low lignin contents in contrast to the heavily lignified cell walls typically found in the xylem tissues. To improve the quality of the fiber-based products in the future, a thorough understanding of the main cell wall polymer biosynthetic pathways is required. In this study we have carried out a characterization of the genes involved in lignin biosynthesis in flax along with some of their regulation mechanisms. RESULTS: We have first identified the members of the phenylpropanoid gene families through a combination of in silico approaches. The more specific lignin genes were further characterized by high throughput transcriptomic approaches in different organs and physiological conditions and their cell/tissue expression was localized in the stems, roots and leaves. Laccases play an important role in the polymerization of monolignols. This multigenic family was determined and a miRNA was identified to play a role in the posttranscriptional regulation by cleaving the transcripts of some specific genes shown to be expressed in lignified tissues. In situ hybridization also showed that the miRNA precursor was expressed in the young xylem cells located near the vascular cambium. The results obtained in this work also allowed us to determine that most of the genes involved in lignin biosynthesis are included in a unique co-expression cluster and that MYB transcription factors are potentially good candidates for regulating these genes. CONCLUSIONS: Target engineering of cell walls to improve plant product quality requires good knowledge of the genes responsible for the production of the main polymers. For bast fiber plants such as flax, it is important to target the correct genes from the beginning since the difficulty to produce transgenic material does not make possible to test a large number of genes. Our work determined which of these genes could be potentially modified and showed that it was possible to target different regulatory pathways to modify lignification.