Impact of Carbon Fixation, Distribution and Storage on the Production of Farnesene and Limonene in Synechocystis PCC 6803 and Synechococcus PCC 7002.

Terpenes are high-value chemicals which can be produced by engineered cyanobacteria from sustainable resources, solar energy, water and CO2. We previously reported that the euryhaline unicellular cyanobacteria Synechocystis sp. PCC 6803 (S.6803) and Synechococcus sp. PCC 7002 (S.7002) produce farnesene and limonene, respectively, more efficiently than other terpenes. In the present study, we attempted to enhance farnesene production in S.6803 and limonene production in S.7002. Practically, we tested the influence of key cyanobacterial enzymes acting in carbon fixation (RubisCO, PRK, CcmK3 and CcmK4), utilization (CrtE, CrtR and CruF) and storage (PhaA and PhaB) on terpene production in S.6803, and we compared some of the findings with the data obtained in S.7002. We report that the overproduction of RubisCO from S.7002 and PRK from Cyanothece sp. PCC 7425 increased farnesene production in S.6803, but not limonene production in S.7002. The overexpression of the crtE genes (synthesis of terpene precursors) from S.6803 or S.7002 did not increase farnesene production in S.6803. In contrast, the overexpression of the crtE gene from S.6803, but not S.7002, increased farnesene production in S.7002, emphasizing the physiological difference between these two model cyanobacteria. Furthermore, the deletion of the crtR and cruF genes (carotenoid synthesis) and phaAB genes (carbon storage) did not increase the production of farnesene in S.6803. Finally, as a containment strategy of genetically modified strains of S.6803, we report that the deletion of the ccmK3K4 genes (carboxysome for CO2 fixation) did not affect the production of limonene, but decreased the production of farnesene in S.6803.

Assessing Behavioral Disorders with SDQ in Very Preterm Children at 5 Years of Age in LIFT Cohort.

BACKGROUND: Preterm-born children are at risk of behavioral disorders and the systematic assessment of these disorders remains a challenge. Questions remain about the accuracy of self-reported parent questionnaires and the real everyday life behavior of the child. AIM: To evaluate the association between SDQ reported by parents in the preterm and behavioral difficulties in the everyday school life environment reported by teacher. METHODS: All children born before 33 weeks and who followed-up in the LIFT (Loire Infant Follow-up team) network were included. The Strengths and Difficulties Parental Questionnaire (SDQ), completed at 5 years, was used to check for behavioral difficulties and identified three groups: "normal", "borderline" and "abnormal". Then, the SDQ results were compared to the Global School Adaptation Score (GSA) at 5 years. RESULTS: Out of the 1825 children followed in the cohort at the age of 5, 1397 questionnaires were analyzed. A total of 11.1% of children had an abnormal score, and 9.7% had a borderline score. Male gender and a lower birth weight z-score were significantly associated with the "abnormal SDQ" group. There is a significant relationship between the probability of being in the "abnormal SDQ" group at 5 years and with difficulty in global school adaptation at 5 years, as well as an SDQ borderline score in the preterm (p < 0.001). CONCLUSIONS: SDQ abnormal and borderline scores are associated with behavioral difficulties in the classroom and everyday life behavior. In preterm children, one should be alerted even by a borderline SDQ score.

Exploring the Potential of the Model Cyanobacteria Synechococcus PCC 7002 and PCC 7942 for the Photoproduction of High-Value Terpenes: A Comparison with Synechocystis PCC 6803.

We have performed the first comparative analysis of the potential of two physiologically-diverse model cyanobacteria, Synechococcus PCC 7002 (S.7002) and Synechococcus elongatus PCC 7942 (S.7942), for the photosynthetic production of four chemically-different high-value terpenes: two monoterpenes limonene and pinene, and two sesquiterpenes bisabolene and farnesene. We showed, for the first time, that S.7002 and S.7942 can produce farnesene and bisabolene, respectively. Both cyanobacteria produced farnesene (S.7942 produced more efficiently than S.7002) more efficiently than the other tested terpenes (especially pinene, the weakest produced terpene). S.7002 produced limonene more efficiently than bisabolene, whereas S.7942 produced bisabolene more efficiently than limonene. These findings suggest that S.7942 is better suited to produce sesquiterpenes than monoterpenes. Interestingly, higher levels of terpenes were produced by S.7942 and S.7002 expressing a terpene-synthase gene from both an RSF1010-derived replicating plasmid and a neutral chromosomal site, as compared to either the plasmid alone or the chromosome alone. These results suggest that in both cyanobacteria, the production of terpenes is more limited by the activity of terpene synthases than the abundance of terpene precursors. Finally, higher levels of terpenes were produced by S.7002 growing on urea (a frequent pollutant) as compared to nitrate or ammonium, the standard nitrogen sources for cyanobacteria.

Exploring the potential of the model cyanobacterium Synechocystis PCC 6803 for the photosynthetic production of various high-value terpenes.

BACKGROUND: The robust model cyanobacterium Synechocystis PCC 6803 is increasingly explored for its potential to use solar energy, water and atmospheric CO2 for the carbon-neutral production of terpenes, the high-value chemicals that can be used for the production of drugs, flavors, fragrances and biofuels. However, as terpenes are chemically diverse, it is extremely difficult to predict whether Synechocystis is a suitable chassis for the photosynthetic production of various terpenes or only a few of them. RESULTS: We have performed the first-time engineering and comparative analysis of the best-studied cyanobacterium Synechocystis PCC 6803 for the photosynthetic production of five chemically diverse high-value terpenes: two monoterpenes (C10H16) limonene (cyclic molecule) and pinene (bicyclic), and three sesquiterpenes (C15H24) bisabolene (cyclic), farnesene (linear) and santalene (cyclic). All terpene producers appeared to grow well and to be genetically stable, as shown by the absence of changes in their production levels during the 5-9-month periods of their sub-cultivation under photoautotrophic conditions). We also found that Synechocystis PCC 6803 can efficiently and stably produce farnesene and santalene, which had never been produced before by this model organism or any other cyanobacteria, respectively. Similar production levels were observed for cells growing on nitrate (the standard nitrogen source for cyanobacteria) or urea (cheaper than nitrate). Furthermore, higher levels of farnesene were produced by cloning the heterologous farnesene synthase gene in a RSF1010-derived replicating plasmid as compared to the well-used slr0168 neutral cloning site of the chromosome. CONCLUSIONS:  Altogether, the present results indicate that Synechocystis PCC 6803 is better suited to produce sesquiterpenes (particularly farnesene, the most highly produced terpene of this study) than monoterpenes (especially pinene).

Unraveling the Microbiome of Necrotizing Enterocolitis: Insights in Novel Microbial and Metabolomic Biomarkers.

Necrotizing enterocolitis (NEC) is among the most relevant gastrointestinal diseases affecting mostly prematurely born infants with low birth weight. While intestinal dysbiosis has been proposed as one of the possible factors involved in NEC pathogenesis, the role of the gut microbiota remains poorly understood. In this study, the gut microbiota of preterm infants was explored to highlight differences in the composition between infants affected by NEC and infants prior to NEC development. A large-scale gut microbiome analysis was performed, including 47 shotgun sequencing data sets generated in the framework of this study, along with 124 retrieved from publicly available repositories. Meta-analysis led to the identification of preterm community state types (PT-CSTs), which recur in healthy controls and NEC infants. Such analyses revealed an overgrowth of a range of opportunistic microbial species accompanying the loss of gut microbial biodiversity in NEC subjects. Moreover, longitudinal insights into preterm infants prior to NEC development indicated Clostridium neonatale and Clostridium perfringens species as potential biomarkers for predictive early diagnosis of this disease. Furthermore, functional investigation of the enzymatic reaction profiles associated with pre-NEC condition suggested DL-lactate as a putative metabolic biomarker for early detection of NEC onset. IMPORTANCE Necrotizing enterocolitis (NEC) is a severe gastrointestinal disease occurring predominantly in premature infants whose etiology is still not fully understood. In this study, the analysis of infant fecal samples through shotgun metagenomics approaches revealed a marked reduction of the intestinal (bio)diversity and an overgrowth of (opportunistic) pathogens associated with the NEC development. In particular, dissection of the infant's gut microbiome before NEC diagnosis highlighted the potential involvement of Clostridium genus members in the progression of NEC. Remarkably, our analyses highlighted a gastrointestinal DL-lactate accumulation among NEC patients that might represent a novel potential functional biomarker for the early diagnosis of NEC.

Human milk pasteurisation reduces pre-lipolysis but not digestive lipolysis and moderately decreases intestinal lipid uptake in a combination of preterm infant in vitro models.

Donor human milk, pasteurised for safety reasons, is the first alternative for feeding preterm infants when mothers' own milk is unavailable. Breastmilk pasteurisation impact on lipid digestion and absorption was evaluated by a static in vitro digestion model for preterm infants coupled with intestinal absorption using Caco-2/TC7 cells. Lipid absorption was quantified by digital image analysis of lipid droplets, by measurement of basolateral triglyceride concentration and by analysing the expression of major genes involved. After in vitro digestion, lipolysis extent was 13% lower in pasteurised human milk (PHM) than in raw human milk (RHM). In Caco-2/TC7 cells, the number of lipid droplets was identical for both milk types, while the mean droplet area was 17% smaller with PHM. Altogether, pasteurisation decreased the pre-lipolysis of human milk. This initial difference in free fatty acid amount was only partially buffered by the subsequent processes of in vitro digestion and cellular lipid absorption.

Multimodal gadolinium oxysulfide nanoparticles for bioimaging: A comprehensive biodistribution, elimination and toxicological study.

For some years now, gadolinium oxysulfide nanoparticles (NPs) appear as strong candidates for very efficient multimodal in vivo imaging by: 1) Magnetic Resonance (MRI), 2) X-ray Computed Tomography (CT) and 3) photoluminescence imaging. In this paper, we present a selection of results centered on the evaluation of physico-chemical stability, toxicity, bio-distribution and excretion mechanisms of Gd2O2S:Ln3+ nanoparticles intravenously injected in rats. Two formulations are here tested with a common matrix and different dopants: Gd2O2S:Eu3+5% and Gd2O2S:Yb3+4%/Tm3+0.1%. The NPs appear to be almost insoluble in pure water and human plasma but corrosion/degradation phenomenon appears in acidic conditions classically encountered in cell lysosomes. Whole body in vivo distribution, excretion and toxicity evaluation revealed a high tolerance of nanoparticles with a long-lasting imaging signal associated with a slow hepatobiliary clearance and very weak urinary excretion. The results show that the majority of the injected product (>60%) has been excreted through the feces after five months. Experiments have evidenced that the NPs mainly accumulate in macrophage-rich organs, that is mainly liver and spleen and to a lesser extent lungs and bones (mainly marrow). No significant amounts have been detected in other organs such as heart, kidneys, brain, intestine and skin. Gd2O2S:Ln3+ NPs appeared to be very well tolerated up to 400 mg/kg when administered intravenously. STATEMENT OF SIGNIFICANCE: Since 2011, we have focused our work on Gd2O2S nanoparticles (NPs) for multimodal bioimaging using fluorescence, Magnetic Resonance Imaging (MRI) and Computed Tomography with very efficient results already published. However, since the European Medicines Agency has concluded its review of gadolinium contrast agents, confirming recommendations to restrict the use of some linear gadolinium agents used in MRI, a particular attention must be paid to any new contrast media containing gadolinium. Therefore, we present in this paper a compilation of studies about toxicity, bio-distribution and excretion mechanisms of Gd2O2S:Ln3+ NPs intravenously injected into rats. We also present an in vitro kinetic study of NPs degradation in aqueous and biological media to provide some information on chemical and biological stability.

Multimodal gadolinium oxysulfide nanoparticles: a versatile contrast agent for mesenchymal stem cell labeling.

Despite a clear development of innovative therapies based on stem cell manipulation, the availability of new tools to better understand and follow stem cell behavior and improve their biomedical applications is not adequate. Indeed, an ideal tracking device must have good ability to label stem cells as well as complete neutrality relative to their biology. Furthermore, preclinical studies imply in vitro and in vivo approaches that often require several kinds of labeling and/or detection procedures. Consequently, the multimodality concept presented in this work may present a solution to this problem as it has the potential to combine complementary imaging techniques. Spherical europium-doped gadolinium oxysulfide (Gd2O2S:Eu3+) nanoparticles are presented as a candidate as they are detectable by (1) magnetic resonance (MRI), (2) X-ray and (3) photoluminescence imaging. Whole body in vivo distribution, elimination and toxicity evaluation revealed a high tolerance of nanoparticles with a long-lasting MRI signal and slow hepatobiliary and renal clearance. In vitro labeling of a wide variety of cells unveils the nanoparticle potential for efficient and universal cell tracking. Emphasis on mesenchymal stromal cells (MSCs) leads to the definition of optimal conditions for labeling and tracking in the context of cell therapy: concentrations below 50 µg mL-1 and diameters between 170 and 300 nm. Viability, proliferation, migration and differentiation towards mesodermal lineages are preserved under these conditions, and cell labeling appears to be persistent and without any leakage. Ex vivo detection of as few as five thousand Gd2O2S:Eu3+-labeled MSCs by MRI combined with in vitro examination with fluorescence microscopy highlights the feasibility of cell tracking in cell therapy using this new nanoplatform.