Ketocarotenoid production in tomato triggers metabolic reprogramming and cellular adaptation: The quest for homeostasis.

Plants are sessile and therefore have developed an extraordinary capacity to adapt to external signals. Here, the focus is on the plasticity of the plant cell to respond to new intracellular cues. Ketocarotenoids are high-value natural red pigments with potent antioxidant activity. In the present study, system-level analyses have revealed that the heterologous biosynthesis of ketocarotenoids in tomato initiated a series of cellular and metabolic mechanisms to cope with the formation of metabolites that are non-endogenous to the plant. The broad multilevel changes were linked to, among others, (i) the remodelling of the plastidial membrane, where the synthesis and storage of ketocarotenoids occurs; (ii) the recruiting of core metabolic pathways for the generation of metabolite precursors and energy; and (iii) redox control. The involvement of the metabolites as regulators of cellular processes shown here reinforces their pivotal role suggested in the remodelled 'central dogma' concept. Furthermore, the role of metabolic reprogramming to ensure cellular homeostasis is proposed.

Diverting phenylpropanoid pathway flux from sinapine to produce industrially useful 4-vinyl derivatives of hydroxycinnamic acids in Brassicaceous oilseeds.

Sinapine (sinapoylcholine) is an antinutritive phenolic compound that can account for up to 2% of seed weight in brassicaceous oilseed crops and reduces the suitability of their protein-rich seed meal for use as animal feed. Sinapine biosynthesis draws on hydroxycinnamic acid precursors produced by the phenylpropanoid pathway. The 4-vinyl derivatives of several hydroxycinnamic acids have industrial applications. For example, 4-vinyl phenol (4-hydroxystyrene) is a building block for a range of synthetic polymers applied in resins, inks, elastomers, and coatings. Here we have expressed a modified bacterial phenolic acid decarboxylase (PAD) in developing seed of Camelina sativa to redirect phenylpropanoid pathway flux from sinapine biosynthesis to the production of 4-vinyl phenols. PAD expression led to a ∼95% reduction in sinapine content in seeds of both glasshouse and field grown C. sativa and to an accumulation of 4-vinyl derivatives of hydroxycinnamic acids, primarily as glycosides. The most prevalent aglycone was 4-vinyl phenol, but 4-vinyl guaiacol, 6-hydroxy-4-vinyl guaiacol and 4-vinylsyringol (Canolol) were also detected. The molar quantity of 4-vinyl phenol glycosides was more than twice that of sinapine in wild type seeds. PAD expression was not associated with an adverse effect on seed yield, harvest index, seed morphology, storage oil content or germination in either glasshouse or field experiments. Our data show that expression of PAD in brassicaceous oilseeds can supress sinapine accumulation, diverting phenylpropanoid pathway flux into 4-vinyl phenol derivatives, thereby also providing a non-petrochemical source of this class of industrial chemicals.

Laboratory phenomics predicts field performance and identifies superior indica haplotypes for early seedling vigour in dry direct-seeded rice.

Seedling vigour is an important agronomic trait and is gaining attention in Asian rice (Oryza sativa) as cultivation practices shift from transplanting to forms of direct seeding. To understand the genetic control of rice seedling vigour in dry direct seeded (aerobic) conditions we measured multiple seedling traits in 684 accessions from the 3000 Rice Genomes (3K-RG) population in both the laboratory and field at three planting depths. Our data show that phenotyping of mesocotyl length in laboratory conditions is a good predictor of field performance. By performing a genome wide association study, we found that the main QTL for mesocotyl length, percentage seedling emergence and shoot biomass are co-located on the short arm of chromosome 7. We show that haplotypes in the indica subgroup from this region can be used to predict the seedling vigour of 3K-RG accessions. The selected accessions may serve as potential donors in genomics-assisted breeding programs.

Thirty Years of sRNA-Mediated Regulation in Staphylococcus aureus: From Initial Discoveries to In Vivo Biological Implications.

Staphylococcus aureus is a widespread livestock and human pathogen that colonizes diverse microenvironments within its host. Its adaptation to the environmental conditions encountered within humans relies on coordinated gene expression. This requires a sophisticated regulatory network, among which regulatory RNAs (usually called sRNAs) have emerged as key players over the last 30 years. In S. aureus, sRNAs regulate target genes at the post-transcriptional level through base-pair interactions. The functional characterization of a subset revealed that they participate in all biological processes, including virulence, metabolic adaptation, and antibiotic resistance. In this review, we report 30 years of S. aureus sRNA studies, from their discovery to the in-depth characterizations of some of them. We also discuss their actual in vivo contribution, which is still lagging behind, and their place within the complex regulatory network. These shall be key aspects to consider in order to clearly uncover their in vivo biological functions.

Engineering of tomato for the sustainable production of ketocarotenoids and its evaluation in aquaculture feed.

Ketocarotenoids are high-value pigments used commercially across multiple industrial sectors as colorants and supplements. Chemical synthesis using petrochemical-derived precursors remains the production method of choice. Aquaculture is an example where ketocarotenoid supplementation of feed is necessary to achieve product viability. The biosynthesis of ketocarotenoids, such as canthaxanthin, phoenicoxanthin, or astaxanthin in plants is rare. In the present study, complex engineering of the carotenoid pathway has been performed to produce high-value ketocarotenoids in tomato fruit (3.0 mg/g dry weight). The strategy adopted involved pathway extension beyond ß-carotene through the expression of the ß-carotene hydroxylase (CrtZ) and oxyxgenase (CrtW) from Brevundimonas sp. in tomato fruit, followed by ß-carotene enhancement through the introgression of a lycopene ß-cyclase (ß-Cyc) allele from a Solanum galapagense background. Detailed biochemical analysis, carried out using chromatographic, UV/VIS, and MS approaches, identified the predominant carotenoid as fatty acid (C14:0 and C16:0) esters of phoenicoxanthin, present in the S stereoisomer configuration. Under a field-like environment with low resource input, scalability was shown with the potential to deliver 23 kg of ketocarotenoid/hectare. To illustrate the potential of this "generally recognized as safe" material with minimal, low-energy bioprocessing, two independent aquaculture trials were performed. The plant-based feeds developed were more efficient than the synthetic feed to color trout flesh (up to twofold increase in the retention of the main ketocarotenoids in the fish fillets). This achievement has the potential to create a new paradigm in the renewable production of economically competitive feed additives for the aquaculture industry and beyond.

Genome Wide Analysis of Fatty Acid Desaturation and Its Response to Temperature.

Plants modify the polyunsaturated fatty acid content of their membrane and storage lipids in order to adapt to changes in temperature. In developing seeds, this response is largely controlled by the activities of the microsomal ω-6 and ω-3 fatty acid desaturases, FAD2 and FAD3. Although temperature regulation of desaturation has been studied at the molecular and biochemical levels, the genetic control of this trait is poorly understood. Here, we have characterized the response of Arabidopsis (Arabidopsis thaliana) seed lipids to variation in ambient temperature and found that heat inhibits both ω-6 and ω-3 desaturation in phosphatidylcholine, leading to a proportional change in triacylglycerol composition. Analysis of the 19 parental accessions of the multiparent advanced generation intercross (MAGIC) population showed that significant natural variation exists in the temperature responsiveness of ω-6 desaturation. A combination of quantitative trait locus (QTL) analysis and genome-wide association studies (GWAS) using the MAGIC population suggests that ω-6 desaturation is largely controlled by cis-acting sequence variants in the FAD2 5' untranslated region intron that determine the expression level of the gene. However, the temperature responsiveness of ω-6 desaturation is controlled by a separate QTL on chromosome 2. The identity of this locus is unknown, but genome-wide association studies identified potentially causal sequence variants within ∼40 genes in an ∼450-kb region of the QTL.

Awake1, an ABC-Type Transporter, Reveals an Essential Role for Suberin in the Control of Seed Dormancy.

The mother plant plays an important dynamic role in the control of dormancy of her progeny seed in response to environmental signals. In order to further understand the mechanisms by which this dormancy control takes place in Arabidopsis (Arabidopsis thaliana), we conducted a forward genetic screen to isolate mutants that fail to enter dormancy in response to variation in temperature during seed set. We show that, for the first of these mutants, designated awake1, the maternal allele is required for entry into strongly dormant states and that awake1 mutants show seed phenotypes shown previously to be associated with the loss of suberin in the seed. We identify awake1 as an allele of ABCG20, an ATP-binding cassette transporter-encoding gene required for the transport of fatty acids during suberin deposition, and show that further suberin-deficient mutants have seed dormancy defects. Seed coat suberin composition is affected by temperature during seed maturation, but this response appears to be independent of ABCG20. We conclude that seed coat suberin is essential for seed dormancy imposition by low temperature and that the exclusion of oxygen and water from the seed by the suberin and tannin layers is important for dormancy imposition.

Multigene engineering of triacylglycerol metabolism boosts seed oil content in Arabidopsis.

Increasing the yield of oilseed crops is an important objective for biotechnologists. A number of individual genes involved in triacylglycerol metabolism have previously been reported to enhance the oil content of seeds when their expression is altered. However, it has yet to be established whether specific combinations of these genes can be used to achieve an additive effect and whether this leads to enhanced yield. Using Arabidopsis (Arabidopsis thaliana) as an experimental system, we show that seed-specific overexpression of WRINKLED1 (a transcriptional regulator of glycolysis and fatty acid synthesis) and DIACYLGLYCEROL ACYLTRANSFERASE1 (a triacylglycerol biosynthetic enzyme) combined with suppression of the triacylglycerol lipase SUGAR-DEPENDENT1 results in a higher percentage seed oil content and greater seed mass than manipulation of each gene individually. Analysis of total seed yield per plant suggests that, despite a reduction in seed number, the total yield of oil is also increased.

Remarkable reproducibility of enzyme activity profiles in tomato fruits grown under contrasting environments provides a roadmap for studies of fruit metabolism.

To assess the influence of the environment on fruit metabolism, tomato (Solanum lycopersicum 'Moneymaker') plants were grown under contrasting conditions (optimal for commercial, water limited, or shaded production) and locations. Samples were harvested at nine stages of development, and 36 enzyme activities of central metabolism were measured as well as protein, starch, and major metabolites, such as hexoses, sucrose, organic acids, and amino acids. The most remarkable result was the high reproducibility of enzyme activities throughout development, irrespective of conditions or location. Hierarchical clustering of enzyme activities also revealed tight relationships between metabolic pathways and phases of development. Thus, cell division was characterized by high activities of fructokinase, glucokinase, pyruvate kinase, and tricarboxylic acid cycle enzymes, indicating ATP production as a priority, whereas cell expansion was characterized by enzymes involved in the lower part of glycolysis, suggesting a metabolic reprogramming to anaplerosis. As expected, enzymes involved in the accumulation of sugars, citrate, and glutamate were strongly increased during ripening. However, a group of enzymes involved in ATP production, which is probably fueled by starch degradation, was also increased. Metabolites levels seemed more sensitive than enzymes to the environment, although such differences tended to decrease at ripening. The integration of enzyme and metabolite data obtained under contrasting growth conditions using principal component analysis suggests that, with the exceptions of alanine amino transferase and glutamate and malate dehydrogenase and malate, there are no links between single enzyme activities and metabolite time courses or levels.

The sugar-dependent1 lipase limits triacylglycerol accumulation in vegetative tissues of Arabidopsis.

There has been considerable interest recently in the prospect of engineering crops to produce triacylglycerol (TAG) in their vegetative tissues as a means to achieve a step change in oil yield. Here, we show that disruption of TAG hydrolysis in the Arabidopsis (Arabidopsis thaliana) lipase mutant sugar-dependent1 (sdp1) leads to a substantial accumulation of TAG in roots and stems but comparatively much lower TAG accumulation in leaves. TAG content in sdp1 roots increases with the age of the plant and can reach more than 1% of dry weight at maturity, a 50-fold increase over the wild type. TAG accumulation in sdp1 roots requires both ACYL-COENZYME A:DIACYLGLYCEROL ACYLTRANSFERASE1 (DGAT1) and PHOSPHATIDYLCHOLINE:DIACYLGLYCEROL ACYLTRANSFERASE1 and can also be strongly stimulated by the provision of exogenous sugar. In transgenic plants constitutively coexpressing WRINKLED1 and DGAT1, sdp1 also doubles the accumulation of TAG in roots, stems, and leaves, with levels ranging from 5% to 8% of dry weight. Finally, provision of 3% (w/v) exogenous Suc can further boost root TAG content in these transgenic plants to 17% of dry weight. This level of TAG is similar to seed tissues in many plant species and establishes the efficacy of an engineering strategy to produce oil in vegetative tissues that involves simultaneous manipulation of carbohydrate supply, fatty acid synthesis, TAG synthesis, and also TAG breakdown.

A diminution in ascorbate oxidase activity affects carbon allocation and improves yield in tomato under water deficit.

The regulation of carbon allocation between photosynthetic source leaves and sink tissues in response to stress is an important factor controlling plant yield. Ascorbate oxidase is an apoplastic enzyme, which controls the redox state of the apoplastic ascorbate pool. RNA interference was used to decrease ascorbate oxidase activity in tomato (Solanum lycopersicum L.). Fruit yield was increased in these lines under three conditions where assimilate became limiting for wild-type plants: when fruit trusses were left unpruned, when leaves were removed or when water supply was limited. Several alterations in the transgenic lines could contribute to the improved yield and favour transport of assimilate from leaves to fruits in the ascorbate oxidase lines. Ascorbate oxidase plants showed increases in stomatal conductance and leaf and fruit sugar content, as well as an altered apoplastic hexose:sucrose ratio. Modifications in gene expression, enzyme activity and the fruit metabolome were coherent with the notion of the ascorbate oxidase RNAi lines showing altered sink strength. Ascorbate oxidase may therefore be a target for strategies aimed at improving water productivity in crop species.

Ex vivo activity of the ACT new components pyronaridine and piperaquine in comparison with conventional ACT drugs against isolates of Plasmodium falciparum.

BACKGROUND: The aim of the present work was to assess i) ex vivo activity of pyronaridine (PND) and piperaquine (PPQ), as new components of artemisinin-based combination therapy (ACT), to define susceptibility baseline, ii) their activities compared to other partner drugs, namely monodesethylamodiaquine (MDAQ), lumefantrine (LMF), mefloquine (MQ), artesunate (AS) and dihydroartemisinin (DHA) against 181 Plasmodium falciparum isolates from African countries, India and Thailand, and iii) in vitro cross-resistance with other quinoline drugs, chloroquine (CQ) or quinine (QN). METHODS: The susceptibility of the 181 P. falciparum isolates to the nine anti-malarial drugs was assessed using the standard 42-hours 3H-hypoxanthine uptake inhibition method. RESULTS: The IC50 values for PND ranged from 0.55 to 80.0 nM (geometric mean = 19.9 nM) and from 11.8 to 217.3 nM for PPQ (geometric mean = 66.8 nM). A significant positive correlation was shown between responses to PPQ and PND responses (rho = 0.46) and between PPQ and MDAQ (rho = 0.30). No significant correlation was shown between PPQ IC50 and responses to other anti-malarial drugs. A significant positive correlation was shown between responses to PND and MDAQ (rho = 0.37), PND and LMF (rho = 0.28), PND and QN (rho = 0.24), PND and AS (rho = 0.19), PND and DHA (rho = 0.18) and PND and CQ (rho = 0.16). All these coefficients of correlation are too low to suggest cross-resistance between PPQ or PND and the other drugs. CONCLUSIONS: In this study, the excellent anti-malarial activity of PPQ and PND was confirmed. The absence of cross-resistance with quinolines and artemisinin derivatives is consistent with the efficacy of the combinations of PPQ and DHA or PND and AS in areas where parasites are resistant to conventional anti-malarial drugs.

A native promoter-gene fusion created by CRISPR/Cas9-mediated genomic deletion offers a transgene-free method to drive oil accumulation in leaves.

Achieving gain-of-function phenotypes without inserting foreign DNA is an important challenge for plant biotechnologists. Here, we show that a gene can be brought under the control of a promoter from an upstream gene by deleting the intervening genomic sequence using dual-guide CRISPR/Cas9. We fuse the promoter of a nonessential photosynthesis-related gene to DIACYLGLYCEROL ACYLTRANSFERASE 2 (DGAT2) in the lipase-deficient sugar-dependent 1 mutant of Arabidopsis thaliana to drive ectopic oil accumulation in leaves. DGAT2 expression is enhanced more than 20-fold and the triacylglycerol content increases by around 30-fold. This deletion strategy offers a transgene-free route to engineering traits that rely on transcriptional gain-of-function, such as producing high lipid forage to increase the productivity and sustainability of ruminant farming.

Production of human milk fat substitute by engineered strains of Yarrowia lipolytica.

Human milk fat has a distinctive stereoisomeric structure where palmitic acid is esterified to the middle (sn-2) position on the glycerol backbone of the triacylglycerol and unsaturated fatty acids to the outer (sn-1/3) positions. This configuration allows for more efficient nutrient absorption in the infant gut. However, the fat used in most infant formulas originates from plants, which exclude palmitic acid from the sn-2 position. Oleaginous yeasts provide an alternative source of lipids for human nutrition. However, these yeasts also exclude palmitic acid from the sn-2 position of their triacylglycerol. Here we show that Yarrowia lipolytica can be engineered to produce triacylglycerol with more than 60% of the palmitic acid in the sn-2 position, by expression of lysophosphatidic acid acyltransferases with palmitoyl-Coenzyme A specificity. The engineered Y. lipolytica strains can be cultured on glycerol, glucose, palm oil or a mixture of substrates, under nitrogen limited condition, to produce triacylglycerol with a fatty acid composition that resembles human milk fat, in terms of the major molecular species (palmitic, oleic and linoleic acids). Culture on palm oil or a mixture of glucose and palm oil produced the highest lipid titre and a triacylglycerol composition that is most similar with human milk fat. Our data show that an oleaginous yeast can be engineered to produce a human milk fat substitute (ß-palmitate), that could be used as an ingredient in infant formulas.

Cisatracurium pharmacokinetics and pharmacodynamics during hypothermic cardiopulmonary bypass in infants and children.

BACKGROUND: Hypothermia potentiates neuromuscular blockade in adults during cardiopulmonary bypass (CPB) but the pediatric literature is sparse. Temperature-dependent Hoffman degradation of cisatracurium may allow reduction in infusion rate (IR) during hypothermia. The effect of hypothermic CPB on the pharmacokinetics (PK) and pharmacodynamics (PD) of cisatracurium has not been described in children. METHODS AND MATERIALS: Using neuromuscular monitoring with a Datex Relaxograph, cisatracurium IR was adjusted to obtain a pseudo-steady state during each phase of surgery (pre-CPB, CPB, post-CPB). Paired samples were taken at each phase. Cisatracurium plasma concentrations (Cpss) were determined by HPLC. Core and skin temperatures were recorded. RESULTS: Data from ten infants were analyzed: Group 1: mean 33.6°C; Group 2: mean 21.9°C. To maintain T1% between 5% and 10% in Group 2, the IR was decreased by a mean of 89% (P < 0.001). IR was not significantly different in Group 1. Post-CPB IR approximated pre-CPB rates in both groups. During CPB, Cpss fell by 27% in Group 1 and by 50% in Group 2 (P = 0.039). Post-CPB Cpss was not significantly different to pre-CPB in either group. Clearance did not change significantly in Group 1 but fell significantly in Group 2 during CPB (P = 0.002). Clearance post-CPB was unchanged from pre-CPB. CONCLUSIONS: Cisatracurium IR may be decreased by around 60% during CPB with moderate hypothermia but can be maintained at baseline during mild hypothermia.

Galleria mellonella as a Suitable Model of Bacterial Infection: Past, Present and Future.

The increasing interest for Galleria mellonella larvae as an infection model is evidenced by the number of papers reporting its use, which increases exponentially since the early 2010s. This popularity was initially linked to limitation of conventional animal models due to financial, technical and ethical aspects. In comparison, alternative models (e.g. models using Caenorhabditis elegans, Drosophila melanogaster or G. mellonella) were cheap, simple to use and not limited by ethical regulation. Since then, similar results have been established with G. mellonella model comparatively to vertebrates, and it is more and more often used as a robust model per se, not only as an alternative to the murine model. This review attempts to summarize the current knowledge supporting the development of this model, both on immunological and microbiological aspects. For that, we focus on investigation of virulence and new therapies for the most important pathogenic bacteria. We also discuss points out directions for standardization, as well as recent advances and new perspectives for monitoring host-pathogen interactions.

Galleria mellonella Larvae as an Infection Model to Investigate sRNA-Mediated Pathogenesis in Staphylococcus aureus.

Small regulatory RNAs (sRNAs) are key players in bacterial regulatory networks. Monitoring their expression inside living colonized or infected organisms is essential for identifying sRNA functions, but few studies have looked at sRNA expression during host infection with bacterial pathogens. Insufficient in vivo studies monitoring sRNA expression attest to the difficulties in collecting such data, we therefore developed a non-mammalian infection model using larval Galleria mellonella to analyze the roles of Staphylococcus aureus sRNAs during larval infection and to quickly determine possible sRNA involvement in staphylococcal virulence before proceeding to more complicated animal testing. We began by using the model to test infected larvae for immunohistochemical evidence of infection as well as host inflammatory responses over time. To monitor sRNA expression during infection, total RNAs were extracted from the larvae and invading bacteria at different time points. The expression profiles of the tested sRNAs were distinct and they fluctuated over time, with expression of both sprD and sprC increased during infection and associated with mortality, while rnaIII expression remained barely detectable over time. A strong correlation was observed between sprD expression and the mortality. To confirm these results, we used sRNA-knockout mutants to investigate sRNA involvement in Staphylococcus aureus pathogenesis, finding that the decrease in death rates is delayed when either sprD or sprC was lacking. These results demonstrate the relevance of this G. mellonella model for investigating the role of sRNAs as transcriptional regulators involved in staphylococcal virulence. This insect model provides a fast and easy method for monitoring sRNA (and mRNA) participation in S. aureus pathogenesis, and can also be used for other human bacterial pathogens.

Medical Infrared Thermography in Odontogenic Facial Cellulitis as a Clinical Decision Support Tool. A Technical Note.

BACKGROUND: Odontogenic cellulitis are frequent infections of the head and neck fascial spaces that can sometimes spread and be life-threatening, requiring urgent hospitalization. Early diagnosis of facial cellulitis with diffuse inflammatory process is crucial in patient management but not always obvious in the field. Medical infrared thermography (MIT) is a noninvasive tool increasingly used to evaluate skin temperature maps and delineate inflammatory lesions. OBJECTIVE: The aim of this work was to evaluate the use of MIT to improve the clinical examination of patients with facial cellulitis. METHODS: Image processing work was carried out to highlight the thermal gradient resulting from inflammation linked to infection, in 2 patients with facial cellulitis. RESULTS: In real-time, MIT allowed to precisely locate the inflammatory focus linked to cellulitis with no propagation to danger areas such as infraorbital space or around pharyngeal axis. CONCLUSION: Here, we show the first cases using MIT as a powerful complementary tool in the clinical evaluation of patients with facial cellulitis. SIGNIFICANCE: This technology could help optimize the hospitalization decision through a facilitated assessment of infection spread in head and neck tissues and helping to incision for drainage.

Adhesion of Staphylococcus aureus to epithelial cells: an in vitro approach to study interactions within the nasal microbiota.

Introduction. Staphylococcus aureus is a skin and mucous commensal bacterium of warm-blooded animals. In humans, the nose is the main ecological niche of S. aureus, and nasal carriage is a risk factor for developing an endogenous infection. S. aureus nasal colonization is a multifactorial process, involving inter-species interactions among the nasal microbiota.Aims. The objectives of this study were to characterize the microbiota of carriers and non-carriers of S. aureus and to demonstrate the importance of inter-species relationships in the adhesion of S. aureus, a key step in nasal colonization.Methodology. First, we characterized the nasal microbiota from 30 S. aureus carriers and non-carriers by a culturomic approach. We then evaluated the adhesion of S. aureus, first alone and then along with other bacteria of the nasal microbiota. To do that, we used an in vitro model to measure the interactions among bacteria in the presence of epithelial cells.Results. Analysis of the nasal microbiota of the carriers and non-carriers of S. aureus made it possible to observe that each microbiota has specific features in terms of composition. However, this composition differs significantly between carriers and non-carriers mainly through two bacterial groups: coagulase-negative staphylococci and corynebacteria. In a second part, adhesion of S. aureus to epithelial cells showed competition between S. aureus and these bacteria, suggesting a limitation of nasal colonization by S. aureus.Conclusion. These findings demonstrate the existence of a negative correlation between S. aureus and other species which inhibits adhesion and could limit nasal colonization.