Impact of Heavy Metals on Cold Acclimation of Salix viminalis Roots.

In nature, plants are exposed to a range of climatic conditions. Those negatively impacting plant growth and survival are called abiotic stresses. Although abiotic stresses have been extensively studied separately, little is known about their interactions. Here, we investigate the impact of long-term mild metal exposure on the cold acclimation of Salix viminalis roots using physiological, transcriptomic, and proteomic approaches. We found that, while metal exposure significantly affected plant morphology and physiology, it did not impede cold acclimation. Cold acclimation alone increased glutathione content and glutathione reductase activity. It also resulted in the increase in transcripts and proteins belonging to the heat-shock proteins and related to the energy metabolism. Exposure to metals decreased antioxidant capacity but increased catalase and superoxide dismutase activity. It also resulted in the overexpression of transcripts and proteins related to metal homeostasis, protein folding, and the antioxidant machinery. The simultaneous exposure to both stressors resulted in effects that were not the simple addition of the effects of both stressors taken separately. At the antioxidant level, the response to both stressors was like the response to metals alone. While this should have led to a reduction of frost tolerance, this was not observed. The impact of the simultaneous exposure to metals and cold acclimation on the transcriptome was unique, while at the proteomic level the cold acclimation component seemed to be dominant. Some genes and proteins displayed positive interaction patterns. These genes and proteins were related to the mitigation and reparation of oxidative damage, sugar catabolism, and the production of lignans, trehalose, and raffinose. Interestingly, none of these genes and proteins belonged to the traditional ROS homeostasis system. These results highlight the importance of the under-studied role of lignans and the ROS damage repair and removal system in plants simultaneously exposed to multiple stressors.

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.

Proteomic responses of two spring wheat cultivars to the combined water deficit and aphid (Metopolophium dirhodum) treatments.

In the field, plants usually have to face the combined effects of abiotic and biotic stresses. In our study, two spring wheat cultivars-Septima and Quintus-were subjected to three water regimes [70%, 50%, and 40% soil water capacity (SWC)], aphid (Metopolophium dirhodum) infestation, or the combination of both stresses, i.e., water deficit (50%, 40% SWC) and aphids. The study has a 2 × 3 × 2 factorial design with three biological replicates. In the present study, the results of proteomic analysis using 2D-DIGE followed by MALDI-TOF/TOF protein identification are presented. Water deficit but also aphid infestation led to alterations in 113 protein spots including proteins assigned to a variety of biological processes ranging from signaling via energy metabolism, redox regulation, and stress and defense responses to secondary metabolism indicating a long-term adaptation to adverse conditions. The absence of specific proteins involved in plant response to herbivorous insects indicates a loss of resistance to aphids in modern wheat cultivars during the breeding process and is in accordance with the "plant vigor hypothesis." Septima revealed enhanced tolerance with respect to Quintus as indicated by higher values of morphophysiological characteristics (fresh aboveground biomass, leaf length, osmotic potential per full water saturation) and relative abundance of proteins involved in mitochondrial respiration and ATP biosynthesis.

Proteomics Evidence of a Systemic Response to Desiccation in the Resurrection Plant Haberlea rhodopensis.

Global warming and drought stress are expected to have a negative impact on agricultural productivity. Desiccation-tolerant species, which are able to tolerate the almost complete desiccation of their vegetative tissues, are appropriate models to study extreme drought tolerance and identify novel approaches to improve the resistance of crops to drought stress. In the present study, to better understand what makes resurrection plants extremely tolerant to drought, we performed transmission electron microscopy and integrative large-scale proteomics, including organellar and phosphorylation proteomics, and combined these investigations with previously published transcriptomic and metabolomics data from the resurrection plant Haberlea rhodopensis. The results revealed new evidence about organelle and cell preservation, posttranscriptional and posttranslational regulation, photosynthesis, primary metabolism, autophagy, and cell death in response to desiccation in H. rhodopensis. Different protective intrinsically disordered proteins, such as late embryogenesis abundant (LEA) proteins, thaumatin-like proteins (TLPs), and heat shock proteins (HSPs), were detected. We also found a constitutively abundant dehydrin in H. rhodopensis whose phosphorylation levels increased under stress in the chloroplast fraction. This integrative multi-omics analysis revealed a systemic response to desiccation in H. rhodopensis and certain targets for further genomic and evolutionary studies on DT mechanisms and genetic engineering towards the improvement of drought tolerance in crops.

Proteomic Studies of Roots in Hypoxia-Sensitive and -Tolerant Tomato Accessions Reveal Candidate Proteins Associated with Stress Priming.

Tomato (Solanum lycopersicum L.) is a vegetable frequently exposed to hypoxia stress induced either by being submerged, flooded or provided with limited oxygen in hydroponic cultivation systems. The purpose of the study was to establish the metabolic mechanisms responsible for overcoming hypoxia in two tomato accessions with different tolerance to this stress, selected based on morphological and physiological parameters. For this purpose, 3-week-old plants (plants at the juvenile stage) of waterlogging-tolerant (WL-T), i.e., POL 7/15, and waterlogging-sensitive (WL-S), i.e., PZ 215, accessions were exposed to hypoxia stress (waterlogging) for 7 days, then the plants were allowed to recover for 14 days, after which another 7 days of hypoxia treatment was applied. Root samples were collected at the end of each time-point and 2D-DIGE with MALDI TOF/TOF, and expression analyses of gene and protein-encoded alcohol dehydrogenase (ADH2) and immunolabelling of ADH were conducted. After collating the obtained results, the different responses to hypoxia stress in the selected tomato accessions were observed. Both the WL-S and WL-T tomato accessions revealed a high amount of ADH2, which indicates an intensive alcohol fermentation pathway during the first exposure to hypoxia. In comparison to the tolerant one, the expression of the adh2 gene was about two times higher for the sensitive tomato. Immunohistochemical analysis confirmed the presence of ADH in the parenchyma cells of the cortex and vascular tissue. During the second hypoxia stress, the sensitive accession showed a decreased accumulation of ADH protein and similar expression of the adh2 gene in comparison to the tolerant accession. Additionally, the proteome showed a greater protein abundance of glyceraldehyde-3-phosphate dehydrogenase in primed WL-S tomato. This could suggest that the sensitive tomato overcomes the oxygen limitation and adapts by reducing alcohol fermentation, which is toxic to plants because of the production of ethanol, and by enhancing glycolysis. Proteins detected in abundance in the sensitive accession are proposed as crucial factors for hypoxia stress priming and their function in hypoxia tolerance is discussed.

Effect of creatine and EDTA supplemented diets on European seabass (Dicentrarchus labrax) allergenicity, fish muscle quality and omics fingerprint.

The relatively easy access to fish worldwide, alongside the increase of aquaculture production contributes to increased fish consumption which result in higher prevalence of respective allergies. Allergies to fish constitute a significant concern worldwide. ß-parvalbumin is the main elicitor for IgE-mediated reactions. Creatine, involved in the muscle energy metabolism, and ethylenediamine tetraacetic acid (EDTA), a calcium chelator, are potential molecules to modulate parvalbumin. The purpose of this study was to test creatine (2, 5 and 8%) and EDTA (1.5, 3 and 4.5%) supplementation in fish diets to modulate ß-parvalbumin expression and structure and its allergenicity in farmed European seabass (Dicentrarchus labrax) while assessing its effects on the end-product quality. Fish welfare and muscle quality parameters were evaluated by plasma metabolites, rigor mortis, muscle pH and sensory and texture analysis. Proteomics was used to assess alterations in muscle proteome profile and metabolic fingerprinting by Fourier transform infrared spectroscopy was used to assess the liver metabolic profile. In addition, IgE-reactivity to parvalbumin was analysed using fish allergic patient sera. Metabolic fingerprinting of liver tissue revealed no major alterations in infrared spectra with creatine supplementation, while with EDTA, only absorption bands characteristic of lipids were altered. Comparative proteomics showed up regulation of (tropo) myosin and phosphoglycerate mutase 2 with Creatine supplementation. In the case of EDTA proteomics showed up regulation of proteins involved in cellular and ion homeostasis. Allergenicity seems not to be modulated with creatine or EDTA supplementation as no decreased expression levels were found and IgE-binding reactivity showed no quantitative differences.

Gene expression and metabolite analysis in barley inoculated with net blotch fungus and plant growth-promoting rhizobacteria.

Net blotch, caused by the ascomycete Drechslera teres, can compromise barley production. Beneficial bacteria strains are of substantial interest as biological agents for plant protection in agriculture. Belonging to the genus Paraburkholderia, a bacterium, referred to as strain B25, has been identified as protective for barley against net blotch. The strain Paraburkholderia phytofirmans (strain PsJN), which has no effect on the pathogen's growth, has been used as control. In this study, the expression of target genes involved in cell wall-related processes, defense responses, carbohydrate and phenylpropanoid pathways was studied under various conditions (with or without pathogen and/or with or without bacterial strains) at different time-points (0-6-12-48 h). The results show that specific genes were subjected to a circadian regulation and that the expression of most of them increased in barley infected with D. teres and/or bacterized with the strain PsJN. On the contrary, a decreased gene expression was observed in the presence of strain B25. To complement and enrich the gene expression analysis, untargeted metabolomics was carried out on the same samples. The data obtained show an increase in the production of lipid compounds in barley in the presence of the pathogen. In addition, the presence of strain B25 leads to a decrease in the production of defense compounds in this crop. The results contribute to advance the knowledge on the mechanisms occurring at the onset of D. teres infection and in the presence of a biocontrol agent limiting the severity of net blotch in barley.

The Cell Wall Proteome of Craterostigma plantagineum Cell Cultures Habituated to Dichlobenil and Isoxaben.

The remarkable desiccation tolerance of the vegetative tissues in the resurrection species Craterostigma plantagineum (Hochst.) is favored by its unique cell wall folding mechanism that allows the ordered and reversible shrinking of the cells without damaging neither the cell wall nor the underlying plasma membrane. The ability to withstand extreme drought is also maintained in abscisic acid pre-treated calli, which can be cultured both on solid and in liquid culture media. Cell wall research has greatly advanced, thanks to the use of inhibitors affecting the biosynthesis of e.g., cellulose, since they allowed the identification of the compensatory mechanisms underlying habituation. Considering the innate cell wall plasticity of C. plantagineum, the goal of this investigation was to understand whether habituation to the cellulose biosynthesis inhibitors dichlobenil and isoxaben entailed or not identical mechanisms as known for non-resurrection species and to decipher the cell wall proteome of habituated cells. The results showed that exposure of C. plantagineum calli/cells triggered abnormal phenotypes, as reported in non-resurrection species. Additionally, the data demonstrated that it was possible to habituate Craterostigma cells to dichlobenil and isoxaben and that gene expression and protein abundance did not follow the same trend. Shotgun and gel-based proteomics revealed a common set of proteins induced upon habituation, but also identified candidates solely induced by habituation to one of the two inhibitors. Finally, it is hypothesized that alterations in auxin levels are responsible for the increased abundance of cell wall-related proteins upon habituation.

The Resistance of Oilseed Rape Microspore-Derived Embryos to Osmotic Stress Is Associated With the Accumulation of Energy Metabolism Proteins, Redox Homeostasis, Higher Abscisic Acid, and Cytokinin Contents.

The present study aims to investigate the response of rapeseed microspore-derived embryos (MDE) to osmotic stress at the proteome level. The PEG-induced osmotic stress was studied in the cotyledonary stage of MDE of two genotypes: Cadeli (D) and Viking (V), previously reported to exhibit contrasting leaf proteome responses under drought. Two-dimensional difference gel electrophoresis (2D-DIGE) revealed 156 representative protein spots that have been selected for MALDI-TOF/TOF analysis. Sixty-three proteins have been successfully identified and divided into eight functional groups. Data are available via ProteomeXchange with identifier PXD024552. Eight selected protein accumulation trends were compared with real-time quantitative PCR (RT-qPCR). Biomass accumulation in treated D was significantly higher (3-fold) than in V, which indicates D is resistant to osmotic stress. Cultivar D displayed resistance strategy by the accumulation of proteins in energy metabolism, redox homeostasis, protein destination, and signaling functional groups, high ABA, and active cytokinins (CKs) contents. In contrast, the V protein profile displayed high requirements of energy and nutrients with a significant number of stress-related proteins and cell structure changes accompanied by quick downregulation of active CKs, as well as salicylic and jasmonic acids. Genes that were suitable for gene-targeting showed significantly higher expression in treated samples and were identified as phospholipase D alpha, peroxiredoxin antioxidant, and lactoylglutathione lyase. The MDE proteome profile has been compared with the leaf proteome evaluated in our previous study. Different mechanisms to cope with osmotic stress were revealed between the genotypes studied. This proteomic study is the first step to validate MDE as a suitable model for follow-up research on the characterization of new crossings and can be used for preselection of resistant genotypes.

Data on the in-vitro digestibility of acid gels prepared from brewers' spent grain protein isolates.

Brewers' Spent Grain (BSG) is the primary waste of the beer brewing process, which comprises a plethora of nutritionally appealing ingredients such as proteins, dietary fibres, essential lipids and micronutrients. In our previous study [1], the acid-induced gelation capacity of BSG protein isolate as influenced by the thermal pre-treatment severity was systematically investigated. In the present work, we aimed at providing a dataset outlining the gastrointestinal fate of the acid gels under simulating pre-absorptive digestion conditions adopting the INFOGEST static in-vitro digestion protocol. Protein hydrogel digestibility was assessed by quantification of the total soluble nitrogen content in the initial acid gels as well as the obtained gastric and small intestine chymes. The extent of proteolysis occurring in the oral, gastric and intestinal phases was investigated by SDS-PAGE and the molecular weight distribution of the proteins in the obtained gastric chymes and intestinal digesta was determined by image analysis. The dataset can be deployed to assist food scientists in the design and development of alternative protein-based food and food supplement products adopting the "waste-to-fork" concept.

Molecular investigation of Tuscan sweet cherries sampled over three years: gene expression analysis coupled to metabolomics and proteomics.

Sweet cherry (Prunus avium L.) is a stone fruit widely consumed and appreciated for its organoleptic properties, as well as its nutraceutical potential. We here investigated the characteristics of six non-commercial Tuscan varieties of sweet cherry maintained at the Regional Germplasm Bank of the CNR-IBE in Follonica (Italy) and sampled ca. 60 days post-anthesis over three consecutive years (2016-2017-2018). We adopted an approach merging genotyping and targeted gene expression profiling with metabolomics. To complement the data, a study of the soluble proteomes was also performed on two varieties showing the highest content of flavonoids. Metabolomics identified the presence of flavanols and proanthocyanidins in highest abundance in the varieties Morellona and Crognola, while gene expression revealed that some differences were present in genes involved in the phenylpropanoid pathway during the 3 years and among the varieties. Finally, proteomics on Morellona and Crognola showed variations in proteins involved in stress response, primary metabolism and cell wall expansion. To the best of our knowledge, this is the first multi-pronged study focused on Tuscan sweet cherry varieties providing insights into the differential abundance of genes, proteins and metabolites.

Protein changes as robust signatures of fish chronic stress: a proteomics approach to fish welfare research.

BACKGROUND: Aquaculture is a fast-growing industry and therefore welfare and environmental impact have become of utmost importance. Preventing stress associated to common aquaculture practices and optimizing the fish stress response by quantification of the stress level, are important steps towards the improvement of welfare standards. Stress is characterized by a cascade of physiological responses that, in-turn, induce further changes at the whole-animal level. These can either increase fitness or impair welfare. Nevertheless, monitorization of this dynamic process has, up until now, relied on indicators that are only a snapshot of the stress level experienced. Promising technological tools, such as proteomics, allow an unbiased approach for the discovery of potential biomarkers for stress monitoring. Within this scope, using Gilthead seabream (Sparus aurata) as a model, three chronic stress conditions, namely overcrowding, handling and hypoxia, were employed to evaluate the potential of the fish protein-based adaptations as reliable signatures of chronic stress, in contrast with the commonly used hormonal and metabolic indicators. RESULTS: A broad spectrum of biological variation regarding cortisol and glucose levels was observed, the values of which rose higher in net-handled fish. In this sense, a potential pattern of stressor-specificity was clear, as the level of response varied markedly between a persistent (crowding) and a repetitive stressor (handling). Gel-based proteomics analysis of the plasma proteome also revealed that net-handled fish had the highest number of differential proteins, compared to the other trials. Mass spectrometric analysis, followed by gene ontology enrichment and protein-protein interaction analyses, characterized those as humoral components of the innate immune system and key elements of the response to stimulus. CONCLUSIONS: Overall, this study represents the first screening of more reliable signatures of physiological adaptation to chronic stress in fish, allowing the future development of novel biomarker models to monitor fish welfare.

Primary Metabolism Is Distinctly Modulated by Plant Resistance Inducers in Coffea arabica Leaves Infected by Hemileia vastatrix.

Epidemics of coffee leaf rust (CLR) leads to great yield losses and huge depreciation of coffee marketing values, if no control measures are applied. Societal expectations of a more sustainable coffee production are increasingly imposing the replacement of fungicide treatments by alternative solutions. A protection strategy is to take advantage of the plant immune system by eliciting constitutive defenses. Based on such concept, plant resistance inducers (PRIs) have been developed. The Greenforce CuCa formulation, similarly to acibenzolar-S-methyl (ASM), shows promising results in the control of CLR (Hemileia vastatrix) in Coffea arabica cv. Mundo Novo. The molecular mechanisms of PRIs action are poorly understood. In order to contribute to its elucidation a proteomic, physiological (leaf gas-exchange) and biochemical (enzymatic) analyses were performed. Coffee leaves treated with Greenforce CuCa and ASM and inoculation with H. vastatrix were considered. Proteomics revealed that both PRIs lead to metabolic adjustments but, inducing distinct proteins. These proteins were related with photosynthesis, protein metabolism and stress responses. Greenforce CuCa increased photosynthesis and stomatal conductance, while ASM caused a decrease in these parameters. It was further observed that Greenforce CuCa reinforces the redox homeostasis of the leaf, while ASM seems to affect preferentially the secondary metabolism and the stress-related proteins. So, the PRIs prepare the plant to resist CLR but, inducing different defense mechanisms upon pathogen infection. The existence of a link between the primary metabolism and defense responses was evidenced. The identification of components of the plant primary metabolism, essential for plant growth and development that, simultaneously, participate in the plant defense responses can open new perspectives for plant breeding programs.

Physiological and proteomic response of Escherichia coli O157:H7 to a bioprotective lactic acid bacterium in a meat environment.

The enterohemorrhagic Escherichia (E.) coli (EHEC) is a pathogen of great concern for public health and the meat industry all over the world. The high economic losses in meat industry and the high costs of the illness highlight the necessity of additional efforts to control this pathogen. Previous studies have demonstrated the inhibitory activity of Enterococcus mundtii CRL35 towards EHEC, showing a specific proteomic response during the co-culture. In the present work, additional studies of the EHEC-Ent. mundtii interaction were carried out: i) differential protein expression of E. coli O157:H7 NCTC12900 growing in co-culture with Ent. mundtii in a meat environment, ii) the reciprocal influence between these two microorganisms in the adhesion to extracellular matrix (ECM) proteins and iii) the possible induction of the phage W933, coding for Shiga toxin (Stx1), by Ent. mundtii CRL35. Proteomic analysis showed a significant repression of a number of E. coli NCTC12900 proteins in co-culture respect to its single culture, these mostly related to the metabolism and transport of amino acids and nucleotides. On the other hand, statistically significant overexpression of EHEC proteins involved in stress, energy production, amino acid metabolism and transcription was observed at 30 h respect to 6 h when EHEC grew in co-culture. Data are available via ProteomeXchange with identifier PXD014588. Besides, EHEC showed a decreased adhesion capacity to ECM proteins in the presence of the bioprotective strain. Finally, Ent. mundtii CRL35 did not induce the lytic cycle of W933 bacteriophage, thus indicating its potential safe use for eliminating this pathogen. Overall, this study expands the knowledge of EHEC- Ent. mundtii CRL35 interaction in a meat environment, which will certainly contribute to find out effective biological strategies to eliminate this pathogen.

Phellem Cell-Wall Components Are Discriminants of Cork Quality in Quercus suber.

Cork is a renewable, non-wood high valued forest product, with relevant ecological and economic impact in the Mediterranean-type ecosystems. Currently, cork is ranked according to its commercial quality. The most valuable planks are chosen for cork stoppers production. Cork planks with adequate thickness and porosity are classified as stoppable quality cork (SQC). The chemical composition of cork is known, but the regulation of metabolic pathways responsible of cork production and composition, hence of cork quality, is largely unknown. Here, we tested the hypothesis that post-genomic events may be responsible for the development of SQC and N-SQC (non-stoppable quality cork). Here, we show that combined proteomics and targeted metabolomics (namely soluble and cell wall bound phenolics) analyzed on recently formed phellem allows discriminate cork planks of different quality. Phellem cells of SQC and N-SQC displayed different reducing capacity, with consequential impact on both enzymatic pathways (e.g., glycolysis) and other cellular functions, including cell wall assembly and suberization. Glycolysis and respiration related proteins were abundant in both cork quality groups, whereas the level of several proteins associated to mitochondrial metabolism was higher in N-SQC. The soluble and cell wall-bound phenolics in recently formed phellem clearly discriminated SQC from N-SCQ. In our study, SQC was characterized by a high incorporation of aromatic components of the phenylpropanoid pathway in the cell wall, together with a lower content of hydrolysable tannins. Here, we propose that the level of hydrolysable tannins may represent a valuable diagnostic tool for screening recently formed phellem, and used as a proxy for the quality grade of cork plank produced by each tree.

Distribution of cell-wall polysaccharides and proteins during growth of the hemp hypocotyl.

MAIN CONCLUSION: The immuno-ultrastructural investigation localized cell-wall polysaccharides of bast fibers during hemp hypocotyl growth. Moreover, for the first time, the localization of a peroxidase and laccase is provided in textile hemp. In the hypocotyl of textile hemp, elongation and girth increase are separated in time. This organ is therefore ideal for time-course analyses. Here, we follow the ultrastructural rearrangement of cell-wall components during the development of the hemp hypocotyl. An expression analysis of genes involved in the biosynthesis of cellulose, the chief polysaccharide of bast fiber cell walls and xylan, the main hemicellulose of secondary cell walls, is also provided. The analysis shows a higher expression of cellulose and xylan-related genes at 15 and 20 days after sowing, as compared to 9 days. In the young hypocotyl, the cell walls of bast fibers show cellulose microfibrils that are not yet compacted to form a mature G-layer. Crystalline cellulose is detected abundantly in the S1-layer, together with unsubstituted/low-substituted xylan and, to a lesser extent, in the G-layer. The LM5 galactan epitope is confined to the walls of parenchymatic cells. LM6-specific arabinans are detected at the interface between the cytoplasm and the gelatinous cell wall of bast fibers. The class III peroxidase antibody shows localization in the G-layer only at older developmental stages. The laccase antibody shows a distinctive labelling of the G-layer region closest to the S1-layer; the signal becomes more homogeneous as the hypocotyl matures. The data provide important insights on the cell wall distribution of polysaccharide and protein components in bast fibers during the hypocotyl growth of textile hemp.

Atmospheric Aerosol Assisted Pulsed Plasma Polymerization: An Environmentally Friendly Technique for Tunable Catechol-Bearing Thin Films.

In this work, an atmospheric aerosol assisted pulsed plasma process is reported as an environmentally friendly technique for the preparation of tunable catechol-bearing thin films under solvent and catalyst free conditions. The approach relies on the direct injection of dopamine acrylamide dissolved in 2-hydroxyethylmethacrylate as comonomer into the plasma zone. By adjusting the pulsing of the electrical discharge, the reactive plasma process can be alternatively switch ON (tON) and OFF (tOFF) during different periods of time, thus allowing a facile and fine tuning of the catechol density, morphology and deposition rate of the coating. An optimal tON/tOFF ratio is established, that permits maximizing the catechol content in the deposited film. Finally, a diagram, based on the average energy input into the process, is proposed allowing for easy custom synthesis of layers with specific chemical and physical properties, thus highlighting the utility of the developed dry plasma route.

DNA and Protein Analyses to Confirm the Absence of Cross-Contamination and Support the Clinical Reliability of Extensively Hydrolysed Diets for Adverse Food Reaction-Pets.

Adverse food reactions (AFR) are a common cause of skin diseases in cats and dogs. The correct diagnosis and management of AFR relies upon clinical nutrition. The reliability of commercial hypoallergenic diets commonly used in AFR has been questioned because studies have shown the presence of proteins not declared on the label ingredients. It is proposed that extensively hydrolysed protein-based diets constitute a reliable nutritional solution. Royal Canin Anallergenic™ Canine and Feline diets are formulated with very low molecular weight feather protein and purified corn starch. Protein gel electrophoresis and thin layer paper chromatography were used to characterize protein hydrolysis in these diets and their hydrolysed raw materials; protein species were identified by mass spectrometry. To detect cross-contaminating protein, species-specific DNA was measured and correlated with ancillary protein content using calibration curves. The only protein components detected in the extensively hydrolysed feather protein raw material were amino acids and small oligopeptides. GBSS-I (Granule-bound starch synthase 1) was detected in the finished diets; this has not been reported as a clinically apparent allergen in dogs or cats. The DNA threshold corresponding to the maximum acceptable level of ancillary protein was not exceeded in 99.9% of more than 2150 product batches tested and no products were released to the market with cross-contaminating proteins. These results demonstrate the extensive level of protein hydrolysis in Royal Canin Anallergenic™ Canine and Feline diets and the absence of cross-contaminating protein, both key requirements for a diet to be used during diagnosis and for management of pets with AFR.

Proteomic responses of carotenoid and retinol administration to Mongolian gerbils.

Various health benefits of carotenoids have been described. However, while human observational studies generally suggest positive health effects, supplementation with relatively high doses of individual carotenoids (supplements) have partly produced adverse effects. In the present study, we investigated the effect of several carotenoids on the proteomic response of male Mongolian gerbils (aged 6 weeks). Five groups of gerbils (n = 6 per group) received either retinol (vitamin A/53 mg per kg bw), all-trans ß-carotene (pro-vitamin A/100 mg kg-1), the non-pro vitamin A carotenoid lutein (100 mg kg-1), the acyclic carotenoid lycopene (100 mg kg-1) or vehicle (Cremophor EL), via oral single gavage. Gerbils were 12 h post-prandially sacrificed and blood plasma, liver, and white adipose tissue were collected. For liver and adipose tissue, a 2D-DIGE (difference gel electrophoresis) approach was conducted; for plasma, proteomic analyses were achieved by liquid chromatography-mass spectrometry. Compared to controls (vehicle), various proteins were showing significant abundance variations in plasma (66), liver (29) and adipose tissue (19), especially regarding structure (22), protein metabolism (15) and immune system/inflammation (19) functions, while proteins related to antioxidant effects were generally less abundant, suggesting no in vivo relevance. Surprisingly, a large overlap in protein regulation was found between lycopene and retinol exposure, while other carotenoids, including all-trans ß-carotene, did not show this overlap. Mainly retinoid acid receptor co-regulated proteins may mechanistically explain this overlapping regulation. This overlapping regulation may be related to common nuclear hormone receptor mediated signalling, though further studies using synthetic ligands of retinoid receptors targeting protein regulation are needed for confirmation.

Proteome response of dental pulp cells to exogenous FGF8.

FGF8 specifies early tooth development by directing the migration of the early tooth founder cells to the site of tooth emergence. To date the effect of the FGF8 in adult dental pulp has not been studied. We have assessed the regenerative potential of FGF8 by evaluating changes in the proteome landscape of dental pulp following short- and long-term exposure to recombinant FGF8 protein. In addition, we carried out qRT PCR analysis to determine extracellular/adhesion gene marker expression and assessed cell proliferation and mineralization in response to FGF8 treatment. 2D and mass spectrometry data showed differential expression of proteins implicated in cytoskeleton/ECM remodeling and migration, cell proliferation and odontogenic differentiation as evidenced by the upregulation of gelsolin, moesin, LMNA, WDR1, PLOD2, COPS5 and downregulation of P4HB. qRT PCR showed downregulation of proteins involved in cell-matrix adhesion such as ADAMTS8, LAMB3 and ANOS1 and increased expression of the angiogenesis marker PECAM1. We have observed that, FGF8 treatment was able to boost dental pulp cell proliferation and to enhance dental pulp mineralization. Collectively, our data suggest that, FGF8 treatment could promote endogenous healing of the dental pulp via recruitment of dental pulp progenitors as well as by promoting their angiogenic and odontogenic differentiation. SIGNIFICANCE: Dental pulp cells (DP) have been studied extensively for the purposes of mineralized tissue repair, particularly for the reconstruction of hard and soft tissue maxillofacial defects. Canonical FGF signaling has been implicated throughout multiple stages of tooth development by regulating cell proliferation, differentiation, survival as well as cellular migration. FGF8 expression is indispensible for normal tooth development and particularly for the migration of early tooth progenitors to the sites of tooth emergence. The present study provides proteome and qRT PCR data with regard to the future application and biological relevance of FGF8 in dental regenerative medicine. AUTHORS WITH ORCID: Rozaliya Tsikandelova - 0000-0003-0178-3767 Zornitsa Mihaylova - 0000-0003-1748-4489 Sébastien Planchon - 0000-0002-0455-0574 Nikolay Ishkitiev - 0000-0002-4351-5579.