The modification of metabolic profile in plant cell cultures as a strategy to investigate the biological role of secondary metabolites

I metaboliti secondari sono considerati necessari per l\u2019interazione della pianta con l\u2019ambiente; tuttavia la precisa funzione di specifici metaboliti \ue8 nota solo in pochi casi. Un possibile approccio per studiarne i ruoli nella pianta \ue8 quello di modificare il profilo metabolico di colture cellulari in vitro e indagare sull\u2019impatto di tali modifiche in relazione al fenotipo. L\u2019approccio sperimentale proposto nel presente progetto di ricerca pu\uf2 essere riassunto in quattro passaggi principali: 1) analisi dettagliata del profilo metabolico, in termini di metaboliti secondari, delle linee cellulari; 2) il profilo metabolico delle cellule viene modificato attraverso la somministrazione di precursori e inibitori e l\u2019effetto dei trattamenti sul metaboloma viene monitorato attraverso HPLC-DAD e HPLC-ESI-MS; 3) le linee cellulari vengono sottoposte a brevi stress a gli effetti vengono caratterizzati da un punto di vista citologico al fine di identificare specifici tratti fenotipici causati dall\u2019applicazione di specifici stress; 4) vengono valutati gli effetti della modifica del metaboloma in relazione alla risposta cellulare nei confronti dello stress. Tre linee cellulari, appartenenti a differenti specie vegetali, sono state scelte per la loro abilit\ue0 di accumulare un diverso insieme di metaboliti secondari: T2b (Ocimum basilicum), Sw4i (Petunia hybrida) e R3M (Daucus carota). La linea cellulare di basilico, T2b, \ue8 stata oggetto di una strategia che ha previsto l\u2019uso dell\u2019inibitore della biosintesi di coumaroyl CoA (3,4-(metilenediossi) acido cinnamico, MDCA) accoppiato con la somministrazione di un precursore degli antociani (diidroquercetina, DHQ), al fine di variare il rapporto tra acido rosmarinico (RA) e antocianine (AC). Per la linea cellulare di petunia, Sw4i, sono state messe a punto due diverse strategie per modularne il profilo metabolico. Attraverso la somministrazione di diidroquercetina, precursore di AC tipo cianidina e delfinidina, \ue8 stata ottenuta una modifica del rapporto tra antocianine metilate e non metilate. Nella seconda strategia, l\u2019uso dell\u2019acido piperonilico (PIP), inibitore della biosintesi dell\u2019acido p-cumarico, ha permesso di modificare l\u2019accumulo di AC acilate nei confronti AC non acilate, con una significativa riduzione dei livelli di AC acilate. Le cellule di carota R3M sono state trattate con PIP al fine di diminuire l\u2019accumulo di AC acilate. Diversamente dai risultati ottenuti con petunia, dopo il trattamento, le AC non acilate hanno subito una forte riduzione. Questo risultato inaspettato potrebbe dipendere dalla non-specificit\ue0 degli enzimi aciltransferasi in R3M. Infatti, PIP e acido cinnamico (substrato della reazione inibita dal PIP) potrebbero essere usati per generare AC acilate a scapito dell\u2019accumulo di AC non acilate. Nella linea cellulare R3M, \ue8 stato possibile indagare il ruolo protettivo di specifiche classi di metaboliti secondari dopo l\u2019applicazione dello stress, attraverso la somministrazione di precursori e inibitori. Il trattamento termico di 1 h a 44\ub0C induceva la comparsa di placche citoplasmatiche circondate da reticolo endoplasmico; \ue8 stato inoltre dimostrato che le cellule di carota che mostravano questa morfologia erano destinate ad una morte cellulare di tipo lento. Esperimenti precedenti, mediante somministrazione di acidi idrossicinnamici (HCA) prima del trattamento termico, hanno causato un aumento delle AC acilate e dei derivati degli HCA e una riduzione del numero di cellule che presentava le placche. In seguito al trattamento con PIP, \ue8 stata ottenuta una diminuzione dei livelli di AC non acilate e di derivati degli HCA e un aumento del numero di cellule che mostrava un fenotipo stressato. Confrontando i due risultati ottenuti mediante i due approcci complementari, non \ue8 possibile valutare il ruolo degli AC. Nonostante ci\uf2, entrambe le strategie hanno suggerito un ruolo degli HCA nella prevenzione dei danni cellulari indotti dallo stress al calore in cellule di carota R3M.Secondary metabolites are considered necessary for plant adaptation to the variable environment; however, only for few secondary metabolites the specific functions are known in detail. One possible approach to elucidate their roles in plant is to modify the secondary metabolite profile of in vitro cell cultures and investigate the impact of such modifications on the phenotype. The general experimental approach followed in this investigation can be summarized by these four different steps: 1) detailed analysis of the secondary metabolite profile of the chosen cell lines; 2) the metabolite profile of the cells is modified by precursor and inhibitor administration, i.e. the main object of this research project, and the effects of any treatment on the metabolome are monitored though HPLC-DAD and HPLC-ESI-MS; 3) short stresses are applied to cells and the effects of stress are characterized from a cytological point of view in order to identify specific phenotypic traits caused by stress application; 4) the effects of the metabolome modification on cellular response against the stress are evaluated. Three in vitro cell lines derived from different plant species, T2b (Ocimum basilicum), Sw4i (Petunia hybrida), R3M (Daucus carota), have been chosen for their ability to accumulate different set of secondary metabolites. In the basil cell line, T2b, a strategy using the inhibitor of the biosynthesis of coumaroyl CoA (3,4- (methylenedioxy) cinnamic acid, MDCA) coupled with the supplementation of an AC precursor (dihydroquercetin, DHQ) was performed, in order to change the ratio between rosmarinic acid (RA) and anthocyanins (ACs). In the petunia cell line, Sw4i, two different strategies were designed in order to modulate the metabolic profile. A modification of the ratio between methylated ACs (petunidin, malvidin, peonidin) and non-methylated ACs (delphinidin, cyanidin) was obtained feeding cells with DHQ, a precursor of cyanidin and delphinidin based-ACs in petunia species. In the second strategy, the use of piperonylic acid (PIP), an inhibitor of the biosynthesis of p-coumaric acid, allowed to modify the accumulation of acylated ACs versus non-acylated ACs, with a significant decrease of acylated ACs. In the carrot cell line, R3M, cells were treated with PIP in order to decrease the accumulation of acylated ACs, as obtained in petunia. Surprisingly, after the treatment, non-acylated ACs underwent a strong reduction. This unexpected result might be due to the non-specificity of acyltransferase enzymes in carrot cell line. In fact, PIP supplied to cells and cinnamic acid accumulated as substrate of the inhibited reaction, might be used to generate acylated ACs at the expense of non-acylated ACs. In R3M cell line, the protective role of specific classes of secondary metabolites after stress application could be investigated both through precursor and inhibitor administration. The heat treatment at 44\ub0C for 1 h induced the appearance of cytoplasmic patches surrounded by endoplasmic reticulum; it was also demonstrated that the carrot cells showing this morphology were committed to a slow cell death fate. Previous experiments of feeding with hydroxycinnamic acids (HCAs) before the heat treatment caused an increase in acylated ACs and HCAs derivatives and the reduction of the number of cells with patches. By supplying PIP to R3M cells we obtained the decrease of the level of non-acylated ACs and HCA derivatives and an increase of the number of cells with stressed phenotype. Comparing the results obtained by the two complementary approaches, it was not possible to assess the role of ACs. However both strategies suggested a role of HCAs in the prevention of cellular damages induced by heat stress in R3M carrot cells

Impact of phenylpropanoid compounds on heat stress tolerance in carrot cell cultures

The phenylpropanoid and flavonoid families include thousands of specialized metabolites that influence a wide range of processes in plants, including seed dispersal, auxin transport, photoprotection, mechanical support and protection against insect herbivory. Such metabolites play a key role in the protection of plants against abiotic stress, in many cases through their well-known ability to inhibit the formation of reactive oxygen species (ROS). However, the precise role of specific phenylpropanoid and flavonoid molecules is unclear. We therefore investigated the role of specific anthocyanins (ACs) and other phenylpropanoids that accumulate in carrot cells cultivated in vitro, focusing on their supposed ability to protect cells from heat stress. First we characterized the effects of heat stress to identify quantifiable morphological traits as markers of heat stress susceptibility. We then fed the cultures with precursors to induce the targeted accumulation of specific compounds, and compared the impact of heat stress in these cultures and unfed controls. Data modeling based on Projection to Latent Structures (PLS) regression revealed that metabolites containing coumaric or caffeic acid, including ACs, correlate with less heat damage. Further experiments suggested that one of the cellular targets damaged by heat stress and protected by these metabolites is the actin microfilament cytoskeleton

Proceedings of the third French-Ukrainian workshop on the instrumentation developments for HEP

The reports collected in these proceedings have been presented in the third French-Ukrainian workshop on the instrumentation developments for high-energy physics held at LAL, Orsay on October 15-16. The workshop was conducted in the scope of the IDEATE International Associated Laboratory (LIA). Joint developments between French and Ukrainian laboratories and universities as well as new proposals have been discussed. The main topics of the papers presented in the Proceedings are developments for accelerator and beam monitoring, detector developments, joint developments for large-scale high-energy and astroparticle physics projects, medical applications.Comment: 3rd French-Ukrainian workshop on the instrumentation developments for High Energy Physics, October 15-16, 2015, LAL, Orsay, France, 94 page

Late Post-traumatic Epilepsy in Children and Young Adults : Impropriety of Long-Term Antiepileptic Prophylaxis and Risks in Tapering

Background: After traumatic brain injury, epilepsy affects up to 20\uc2 % of children. It is a risk factor, for both clinical recovery and cognitive performance; therefore pharmacological therapy is advisable. Current guidelines recommend prophylaxis to be initiated as soon as possible and tapered 1\uc2 week after trauma. However, no guideline exists for paediatric patients and the clinical practice is heterogeneous. Objective: In our institute, prophylaxis was routinely tapered 6\uc2 months after trauma. Therefore we investigated whether this prophylaxis or its tapering influenced the development of post-traumatic epilepsy, together with several clinical-demographic factors. Methods: The study population comprised all patients with post-traumatic brain injury referred to this institute between 2002 and 2009 who consented to participate. Clinical, epileptological and pharmacological data were collected. The role of prophylaxis and several other predictors on occurrence of post-traumatic epilepsy was analysed through logistic regressions. Results: Two hundred and three patients (145 paediatric) were followed for 57\uc2 months on average. Risk factors for epilepsy were past neurosurgery [odds ratio (OR)\uc2 =\uc2 2.61, 95\uc2 % confidence interval (CI) 1.15\u20135.96], presence of epileptiform anomalies (OR\uc2 =\uc2 6.92, 95\uc2 % CI 3.02\u201315.86) and the presence of prophylaxis (OR\uc2 =\uc2 2.49, 95\uc2 % CI 1.12\u20135.52), while higher intelligence quotient (IQ) was protective (OR\uc2 =\uc2 0.96, 95\uc2 % CI 0.95\u20130.98). While evaluating possible different effects within and after 6\uc2 months (tapering, for those under prophylaxis), we found that epileptiform anomalies (OR\uc2 =\uc2 7.61, 95\uc2 % CI 2.33\u201324.93, and OR\uc2 =\uc2 8.21, 95\uc2 % CI 3.00\u201322.44) and IQ (OR\uc2 =\uc2 0.96, 95\uc2 % CI 0.94\u20130.98, and OR\uc2 =\uc2 0.97, 95\uc2 % CI 0.95\u20130.98) were always significant predictors of epilepsy, while neurosurgery (OR\uc2 =\uc2 4.38, 95\uc2 % CI 1.10\u201317.45) was significant only within 6\uc2 months from trauma, and prophylaxis (OR\uc2 =\uc2 3.98, 95\uc2 % CI 1.62\u20139.75) only afterwards. Conclusions: These results suggest that prophylaxis was irrelevant when present; furthermore its tapering increased the risk of epilepsy. Since the presence of epileptiform anomalies was the main predictor of post-traumatic epilepsy, such anomalies may be useful to better direct the choice of prophylaxis

Purification of molybdenum oxide, growth and characterization of medium size zinc molybdate crystals for the LUMINEU program

The LUMINEU program aims at performing a pilot experiment on neutrinoless double beta decay of 100Mo using radiopure ZnMoO4 crystals operated as scintillating bolometers. Growth of high quality radiopure crystals is a complex task, since there are no commercially available molybdenum compounds with the required levels of purity and radioactive contamination. This paper discusses approaches to purify molybdenum and synthesize compound for high quality radiopure ZnMoO4 crystal growth. A combination of a double sublimation (with addition of zinc molybdate) with subsequent recrystallization in aqueous solutions (using zinc molybdate as a collector) was used. Zinc molybdate crystals up to 1.5 kg were grown by the low-thermal-gradient Czochralski technique, their optical, luminescent, diamagnetic, thermal and bolometric properties were tested.Comment: Contribution to Proc. of Int. Workshop on Radiopure Scintillators RPSCINT 2013, 17-20 September 2013, Kyiv, Ukraine; to be published in EPJ Web of Conferences; expected to be online in January 2014; 6 pages, 6 figures, and 3 table

Scintillating bolometers based on ZnMoO4 and Zn100MoO4 crystals to search for 0ν2β decay of 100Mo (LUMINEU project): first tests at the Modane Underground Laboratory

The technology of scintillating bolometers based on zinc molybdate (ZnMoO4) crystals is under development within the LUMINEU project to search for decay of 100Mo with the goal to set the basis for large scale experiments capable to explore the inverted hierarchy region of the neutrino mass pattern. Advanced ZnMoO4 crystal scintillators with mass of ∼0.3 kg were developed and Zn100MoO4 crystal from enriched 100Mo was produced for the first time by using the low-thermal-gradient Czochralski technique. One ZnMoO4 scintillator and two samples (59 g and 63 g) cut from the enriched boule were tested aboveground at milli-Kelvin temperature as scintillating bolometers showing a high detection performance. The first results of the low background measurements with three ZnMoO4 and two enriched detectors installed in the EDELWEISS set-up at the Modane Underground Laboratory (France) are presented

Development of 100^{100}Mo-containing scintillating bolometers for a high-sensitivity neutrinoless double-beta decay search

We report recent achievements in the development of scintillating bolometers to search for neutrinoless double-beta decay of $^{100}$Mo. The presented results have been obtained in the framework of the LUMINEU, LUCIFER and EDELWEISS collaborations, and are now part of the R\&D activities towards CUPID (CUORE Update with Particle IDentification), a proposed next-generation double-beta decay experiment based on the CUORE experience. We have developed a technology for the production of large mass ($\sim$1 kg), high optical quality, radiopure zinc and lithium molybdate crystal scintillators (ZnMoO$_4$ and Li$_2$MoO$_4$, respectively) from deeply purified natural and $^{100}$Mo-enriched molybdenum. The procedure is applied for a routine production of enriched crystals. Furthermore, the technology of a single detector module consisting of a large-volume ($\sim 100$~cm$^3$) Zn$^{100}$MoO$_4$ and Li$_2$$^{100}$MoO$_4$ scintillating bolometer has been established, demonstrating performance and radiopurity that are close to satisfy the demands of CUPID. In particular, the FWHM energy resolution of the detectors at 2615 keV --- near the $Q$-value of the double-beta transition of $^{100}$Mo (3034~keV) --- is $\approx$ 4--10~keV. The achieved rejection of $\alpha$-induced dominant background above 2.6~MeV is at the level of more than 99.9\%. The bulk activity of $^{232}$Th ($^{228}$Th) and $^{226}$Ra in the crystals is below 10 $\mu$Bq/kg. Both crystallization and detector technologies favor Li$_2$MoO$_4$, which was selected as a main element for the realization of a CUPID demonstrator (CUPID-0/Mo) with $\sim$7 kg of $^{100}$Mo

Correlated accumulation of anthocyanins and rosmarinic acid in mechanically stressed red cell suspensions of basil (Ocimum basilicum)

A red basil cell line (T2b) rich in rosmarinic acid (RA) was selected for the stable production of anthocyanins (ACs) in the dark. Cell suspension cultures were subjected to mechanical stress through increased agitation (switch from 90 to 150 rpm) to determine the relationship between AC and RA accumulation. Cell extracts were analyzed by HPLC and LC\u2013MS, and the resulting data were processed with multivariate statistical analysis. MS and MS/MS spectra facilitated the putative annotation of several complex cyanidin-based ACs, which were esterified with coumaric acid and, in some cases, also with malonic acid. It was also possible to identify various RA-related molecules, some caffeic and coumaric acid derivatives and some flavanones. Mechanical stress increased the total AC and RA contents, but reduced biomass accumulation. Many metabolites were induced by mechanical stress, including RA and some of its derivatives, most ACs, hydroxycinnamic acids and flavonoids, whereas the abundance of some RA dimers was reduced. Although AC and RA share a common early biosynthetic pathway (from phenylalanine to 4-coumaroyl-CoA) and could have similar or overlapping functions providing antioxidant activity against stress-generated reactive oxygen species, there appeared to be no competition between their individual pathways

The modification of phenylpropanoid profile affects sensitivity to heat stress in cell cultures of Daucus carota

Precursors and inhibitors of the phenylpropanoid pathway were used for modifying the level of specific secondary metabolites in R3M carrot cell culture, a red pigmented line characterised by the production of cyanidin and hydroxycinnamic acid (HCA) derivatives, in order to evaluate the effects of profile modification on heat stress response. The heat treatment (1h at 44 \ub0C) caused the appearance of cytoplasmic patches surrounded by endoplasmic reticulum, the arrest of endocellular movements, and a strong reduction of the viable cell number, since the patched cells were committed to slow cell death. Cytochalasin D, an anti-microfilament agent, caused the formation of structures similar to the heat-induced patches at specific concentrations. These results suggest that the heat stress-determined microfilament damages caused the formation of cytoplasmic patches, structures which anticipate programmed cell death. Feeding R3M cells with dihydroquercetin (a precursor of cyanidin) and HCAs before the heat treatment caused an increase in acylated anthocyanins and HCA derivatives and a reduction in the number of patched and dead cells. The supply of piperonilic acid, an inhibitor of the biosynthesis of p-coumaric acid (an intermediate of anthocyanin biosynthesis and substrate of their acylation) caused a decrease in the level of specific phenylpropanoids and an increase in the number of cells with stressed phenotype. These data suggest that specific phenylpropanoids can play a protective role against heat stress even though the detailed analysis of the correlation between the level of single metabolites and stress response is in progress