Artificial growing system of sweet basil (Ocimum basilicum L.) for agro-industrial production of secondary metabolites

This work aimed at developing artificial cultivation systems of sweet basil for the extraction of RA in consideration of its commercial value as ingredient of medicinal, herbal and plant protection products. A number of experiments were conducted with sweet basil seedlings of different cultivars (green-leaved “Genovese” and “Superbo”, and purple-leaved “Dark Opal”) grown in both in vitro and in vivo culture. The latter system was based on greenhouse hydroponic system, in particular on floating system. In all experiments, several CADs of interest (caffeic acid, caftaric acid, chlorogenic acid, cicoric acid, cynarin, ferulic acid, t-cinnamic acid, p-coumaric acid, rosmarinic acid) were quantified using HPLC and HPLC-MS. Apart from rosmarinic acid (RA) and a methylated form of this substance, the levels of other selected CADs were close or below the detection limit (0.05 mg g-1 DW) in all the analyzed samples, irrespective of genotype, growing conditions or plant tissue. Work is in progress to elucidate the nature and the origin (genuine in vivo biosynthesis or artefact during the methanol extraction) of the methylated form of RA detected in our samples. All the determinations were conducted on fresh samples as in preliminary experiments it was found that desiccation at 70°C reduced markedly (up to 40%) RA content in basil tissues. Desiccation is generally used for post-harvest preservation of medicinal plant materials; dried materials is easy to handle and process and less prone to microbial degradation. If fresh material has to be used for the extraction of the metabolites of interest, greenhouse cultivations must be located very close to the processing facilities and cold rooms may be necessary for short-term storage, following a production-delivery scheme similar to the one used by fresh-cuts products industry. Of course, detailed economical analysis is necessary to compare the overall costs of RA production from fresh or dry sweet basil material. In general, the RA concentrations found in sweet basil grown either in vitro or in vivo were very high in comparison with those reported in the literature for this species. The highest RA content were detected in young leaves of hydroponically-grown plants fertilized with 0.5 mol m3 NO3- (up to 10% DW) and in micro-propagated plantlets in RITA™ vessels (up to more than 15% DW). Among the three cultivars of sweet basil under investigation, the purple-leaved cultivar contained less RA than green-leaved genotypes. The former cultivar is very rich in anthocyanins pigments and a competition between RA and anthocyanins synthesis, which have a common precursor (4-coumaroyl-CoA), can be hypothesized. In general, floating raft system provided a suitable growing method of sweet basil for agro-industrial production of RA, as a large amount of biomass with high concentration of this compound can be produced in few weeks. The highest RA content was detected in root and leaf tissues at full bloom; RA content was up to 10% DW in the youngest leaves. Even if the root biomass production is limited, in comparison with the shoot (above or less 10% of total dry mass), the utilization of the roots system for the extraction of RA may be hypothesized, as the floating system facilitates the harvesting of those tissues, clean and easy to processed. Our experiments allowed the optimization of N nutrition of hydroponically-grown plants. It was found that the standard N concentration used in hydroponic culture (10.0 mol m-3 or higher) could be reduced considerably, with important implications from the environmental point of view. The use of a total NO3- concentration of 5.0 mol m-3 resulted in optimal plant growth, while the supply of much lower concentration (0.5 mol m-3 NO3- ) induced a strong increase of RA accumulation (up to 97 mg g-1 DW in the young leaves), but a simultaneous reduction of biomass production. In contrast, the addition of NH4+ to the nutrient solution was detrimental to both biomass accumulation and RA synthesis and its use should be avoided in hydroponic culture of sweet basil. Plant growth and RA accumulation were scarcely affected by hypoxia condition and mild NaCl salinity. These results have some implications from the operational point of view, as they suggest that poor quality irrigation water can be used in water culture of sweet basil and that the aeration of nutrient solution is not a crucial factor for optimal plant growth and RA production of this species. As regard in vitro culture, high RA accumulation levels were detected in micropropagated plantlets. The content of this metabolite varied during the micropropagation phases and in dependence of BA concentration in the growing medium, which however affected differently purple-leaved and green-leaved plants. In fact, increasing BA concentration from 0.1 to 1 mg L-1 resulted in higher RA accumulation in green-leaved genotypes whereas an opposite effect was observed in the purple-leaved Dark Opal. Plant tissue content of RA was also affected by the type of cultural vessel adopted for explants growth. The highest RA accumulation was found in RITA™-cultured shoots and this was associated with completely-heterotrophic growth and large accumulation of ethylene in the internal atmosphere, as compared to other vessel types. The levels of RA determined in micropropagated plants exceeded by far the concentrations reported in the literature for in vitro-grown sweet basil tissues or cells. On the other hand, the concentrations were similar to those reported for the plants grown in vivo, whereas the biomass production was dramatically lower. Due to the high cost and low biomass production of in vitro growing systems, greenhouse hydroponic culture seems much more cost-effective and probably easier from the operational point of view. Hydroponic production of sweet basil and other herbs for fresh or minimally-processed vegetables market is a well-developed technology that can be easily applied to the production of biomass to be extracted for active principles. Production efficiency could be improved by the cultivation of selected varieties, as sweet basil RA content may vary markedly in dependence of the genotype. Moreover, future work may be done at the aim to elucidate the biochemical and molecular mechanisms underlying the shift from primary and secondary metabolism and the influence of growing factors on RA synthesis. For that, in vitro culture could represent a suitable model. Tyrosine aminotransferase (TAT), hydroxycinnamoyl-CoA: hydroxyphenyllactate hydroxycinnamoyl transferase (RAS) and PAL are key-enzymes of RA biosynthesis. Genes sequences encoding for these enzymes were isolated in leaf tissues of sweet basil plants (cv. Genovese) grown both in vivo and in vitro. Real time PCR experiments are in progress to investigate the RNA expression levels of these genes in plants grown under conditions that have been found to affect markedly RA accumulation, for instance in N-deficient or NH4+-fed plants

Aspetti fisiologici legati alla conservazione di pomodoro e kiwi di IV gamma

ASPETTI FISIOLOGICI LEGATI ALLA CONSERVAZIONE DI POMODORO E KIWI DI IV GAMMA Gli ortaggi e la frutta di IV gamma (minimally processed) sono una categoria di alimenti vegetali freschi ad alto contenuto di servizio in quanto vengono sottoposti a minime lavorazioni atte a renderli pronti al consumo. Le lavorazioni minime in genere consistono in lavaggi, taglio e confezionamento in contenitori di vario tipo in genere di plastica, talvolta a permeabilità selettiva per i gas, eventualmente associati alla modificazione della atmosfera interna. Attualmente, i prodotto di IV gamma stanno riscuotendo un particolare successo in considerazione dei cambiamenti degli stili di vita e delle abitudini alimentari della popolazione, soprattutto di quella urbana. Tali mutamenti si sono tradotti in una richiesta di cibi ad elevata praticità d’impiego, fra cui compaiono gli alimenti “pronti all’uso”, in primis i prodotti di IV gamma. Gli ortaggi e la frutta per essere commercializzati in vaschette necessitano di un insieme di lavorazioni che provvedono a garantire la freschezza insieme alla sicurezza igienico-sanitaria. La serbevolezza di questi prodotti può essere limitata da numerosi processi degenerativi quali l’ossidazione e il rammollimento dei tessuti. Questi fenomeni sono provocati soprattutto dal taglio, l’operazione tipica del “lavorazione minima”. Tutta la tecnologia post-raccolta, in particolare per laIV gamma, è finalizzata al contenimento delle ossidazioni, ricorrendo fondamentalmente ai metodi della refrigerazione, sin dalla raccolta in campo (pre-refrigerazione). Lo studio sperimentale riportato in questa tesi è stata condotto allo scopo di analizzare il comportamento di due specie vegetali diverse, pomodoro e kiwi, sottoposte a sei diverse condizioni di conservazione: frutti integri o tagliati conservati a temperatura ambiente o a 4°C in vaschette virtualmente ermetiche o microforate (dello stesso volume, comunque), per tempi fino a 15 giorni, più frequentemente per una settimana. Per valutare la perdita di consistenza dei tessuti è stato utilizzato la determinazione della perdita di elettroliti, mentre in fase di conservazione sono state misurate per via gas-cromatografica le concentrazioni e di etilene e CO2 ed è stato quindi calcolato, con un apposito algoritmo di calcolo, il tasso di evoluzione endogena dei due gas, legata alla biosintesi dell’ormone e all’attività respiratoria. Come previsto, i frutti di pomodoro e kiwi hanno mostrato una diversa attività respiratoria e una diversa capacità di sintesi dell’etilene, entrambe diminuite dalla bassa temperatura (Figure ); il pomodoro respira maggiormente e produce più etilene del kiwi. Il taglio ha aumentato l’evoluzione della CO2 e dell’etilene dai campioni; questo effetto è stato più evidente nel kiwi, probabilmente perché nella fetta del kiwi si ha una maggiore superficie esposta all’azione del taglio rispetto a quanto avviene nel pomodoro. Nelle vaschette virtualmente ermetiche sono stati rilevati livelli di etilene (Figura ) e CO2 (Figura ) nettamente superiori rispetto a quelli delle vaschette microforate, indipendentemente dalla temperatura di conservazione. Le maggiori concentrazioni di etilene all’interno delle vaschette chiuse hanno indotto, probabilmente, una sintesi autocatalitica dell’ormone; in effetti, il tasso orario di produzione dell’etilene è risultato maggiore nel caso delle vaschette chiuse. A temperatura ambiente, i contenitori chiusi sembrano determinare una condizione di stress per i campioni, che reagiscono con un incremento della respirazione. A bassa temperatura, invece, la respirazione è maggiore nei campioni conservati nelle vaschette microforate e questo risultato sembra riconducibile alla minore concentrazione di CO2. L’effetto del taglio sull’integrità cellulare membrane, valutata attraverso la misura della perdita di elettroliti, si è osservato solo nel caso di una frigo-conservazione relativamente lunga e non sembra strettamente correlato al tasso respiratorio e alla sintesi di etilene dei campioni. Le differenze riscontrate tra i due tipi di frutto studiate suggeriscono una stretta relazione, seppur indiretta, fra biosintesi di etilene e integrità cellulare. D’altra parte, l’uso di due diverse tipologie di contenitore ha determinato una differente concentrazione dei due gas che ne ha marcatamente condizionato la sintesi da parte dei campioni dei frutti tagliati, senza però influenzare in modo significativo la perdita di elettroliti. Pertanto, considerando le due specie separatamente, il danno cellulare provocato dall’operazione del taglio e dalla frigo-conservazione non sembra condizionato dall’etilene e dalla respirazione. La conclusione generale, quindi, è che sono necessarie ulteriori indagini per una migliore comprensione dei complessi rapporti tra sintesi di etilene, respirazione, composizione della atmosfera di conservazione e resistenza allo stress indotto dal taglio e, soprattutto nel caso di frutti come il pomodoro, della bassa temperatura

Iodine biofortification of crops: agronomic biofortification, metabolic engineering and iodine bioavailability

Iodine deficiency is a widespread micronutrient malnutrition problem, and the addition of iodine to table salt represents the most common prophylaxis tool. The biofortification of crops with iodine is a recent strategy to further enrich the human diet with a potentially cost-effective, well accepted and bioavailable iodine source. Understanding how iodine functions in higher plants is key to establishing suitable biofortification approaches. This review describes the current knowledge regarding iodine physiology in higher plants, and provides updates on recent agronomic and metabolic engineering strategies of biofortification. Whereas the direct administration of iodine is effective to increase the iodine content in many plant species, a more sophisticated genetic engineering approach seems to be necessary for the iodine biofortification of some important staple crops

Tomato fruits: a good target for iodine biofortification

Iodine is a trace element that is fundamental for human health: its deficiency affects about two billion people worldwide. Fruits and vegetables are usually poor sources of iodine; however, plants can accumulate iodine if it is either present or exogenously administered to the soil. The biofortification of crops with iodine has therefore been proposed as a strategy for improving human nutrition. A greenhouse pot experiment was carried out to evaluate the possibility of biofortifying tomato fruits with iodine. Increasing concentrations of iodine supplied as KI or KIO3 were administered to plants as root treatments and the iodine accumulation in fruits was measured. The influences of the soil organic matter content or the nitrate level in the nutritive solution were analyzed. Finally, yield and qualitative properties of the biofortified tomatoes were considered, as well as the possible influence of fruit storage and processing on the iodine content. Results showed that the use of both the iodized salts induced a significant increase in the fruit’s iodine content in doses that did not affect plant growth and development. The final levels ranged from a few mg up to 10 mg iodine kg-1 fruit fresh weight and are more than adequate for a biofortification program, since 150 μg iodine per day is the recommended dietary allowance for adults. In general, the iodine treatments scarcely affected fruit appearance and quality, even with the highest concentrations applied. In contrast, the use of KI in plants fertilized with low doses of nitrate induced moderate phytotoxicity symptoms. Organic matter-rich soils improved the plant’s health and production, with only mild reductions in iodine stored in the fruits. Finally, a short period of storage at room temperature or a 30-min boiling treatment did not reduce the iodine content in the fruits, if the peel was maintained. All these results suggest that tomato is a particularly suitable crop for iodine biofortification programs

Atypical antipsychotic therapy in Parkinson's disease psychosis: A retrospective study.

ObjectiveParkinson's disease psychosis (PDP) is a frequent complication of idiopathic Parkinson's disease (iPD) with significant impact on quality of life and association with poorer outcomes. Atypical antipsychotic drugs (APDs) are often used for the treatment of PDP; however, their use is often complicated by adverse drug reactions (ADRs). In this study, we present patients with PDP who were treated with the most commonly used atypical antipsychotic agents and review their respective ADRs.MethodsA retrospective study was carried out to include a total of 45 patients with iPD who visited a movement disorders clinic between 2006 and 2015. All PDP patients treated with atypical APDs were included in the analysis for their specific ADRs.ResultsForty-five iPD patients (mean age of onset: 62.67 ± 9.86 years) were included, of those 10 patients had psychosis (mean age of onset: 76.80 ± 4.61 years). Of the 45 patients, 22.2% were found to have psychotic symptoms, of whom 70% had hallucinations, 20% had delusions, and 10% illusions. Seventy percent of psychotic symptoms occurred after ten or more years from diagnosis of iPD. PDP patients were treated with quetiapine, olanzapine, and risperidone separately or in combination, all of which were found to have certain ADRs.LimitationsThis study was limited by its retrospective study design and small sample size and with likely selection bias.ConclusionsThe prevalence of PDP is relatively high in older patients with iPD. The uses of the currently available atypical APDs in this patient population are often complicated by ADRs. The selective 5-HT 2A inverse agonist, pimavanserin, could be a better alternative in the treatment of PDP

Brain-Derived Neurotrophic Factor (BDNF) and Serotonin Transporter (SERT) in Platelets of Patients with Mild Huntington’s Disease: Relationships with Social Cognition Symptoms

Peripheral biological correlates of early-stage Huntington’s disease (HD) are currently attracting much interest given their possible use as prognostic predictors of later neurodegeneration. Since deficits in social-cognition processing are present among the initial disease symptoms, aim of this work was to appraise, in blood platelets, Brain-Derived Neurotrophic Factor (BDNF) and serotonin (5-HT) transporter (SERT), two proteins involved in human adaptive behavior as potential biochemical correlates of such disabilities in mild-HD. Thirteen gene positive and symptomatic patients (9M/4W, HD-stage II, age> 40y) together 11 gender/age matched controls without a concurrent diagnosis of psychiatric disorders, underwent a blood test to determine BDNF storage and membrane-bound SERT in platelets by ELISA immune-enzyme and [3H]-paroxetine ([3H]-PAR) binding assays, respectively. Concomitantly, all subjects were examined through a battery of socio-cognitive and emotion recognition questionnaires. Results showed moderately increased intra-platelet BDNF amounts (+20-22%) in patients versus controls, whereas [3H]-PAR binding parameters, maximum density (Bmax) and dissociation constant (KD), did not appreciably vary between the two groups. While patients displaying significantly reduced cognitive/emotion abilities, biochemical parameters and clinical features or psychosocial scores did not correlate each other, except for platelet BDNF and the illness duration, positively correlated, or for SERT KDs and angry voice recognition ability, negatively correlated in both controls and patients. Therefore, in this pilot investigation, platelet BDNF and SERT did not specifically underlie psychosocial deficits in stage II-HD. Higher platelet BDNF storage in patients showing lasting-mild symptoms would derive from compensatory mechanisms. Thus, supplementary investigations are warranted by also comparing patients in other illness’s phases

Time-dependent recovery of brain hypometabolism in neuro-COVID-19 patients

Purpose We evaluated brain metabolic dysfunctions and associations with neurological and biological parameters in acute, subacute and chronic COVID-19 phases to provide deeper insights into the pathophysiology of the disease.Methods Twenty-six patients with neurological symptoms (neuro-COVID-19) and [F-18]FDG-PET were included. Seven patients were acute (< 1 month (m) after onset), 12 subacute (4 >= 1-m, 4 >= 2-m and 4 >= 3-m) and 7 with neuro-post-COVID-19 (3 >= 5-m and 4 >= 7-9-m). One patient was evaluated longitudinally (acute and 5-m). Brain hypo- and hypermetabolism were analysed at single-subject and group levels. Correlations between severity/extent of brain hypo- and hypermetabolism and biological (oxygen saturation and C-reactive protein) and clinical variables (global cognition and Body Mass Index) were assessed.Results The "fronto-insular cortex" emerged as the hypometabolic hallmark of neuro-COVID-19. Acute patients showed the most severe hypometabolism affecting several cortical regions. Three-m and 5-m patients showed a progressive reduction of hypometabolism, with limited frontal clusters. After 7-9 months, no brain hypometabolism was detected. The patient evaluated longitudinally showed a diffuse brain hypometabolism in the acute phase, almost recovered after 5 months. Brain hypometabolism correlated with cognitive dysfunction, low blood saturation and high inflammatory status. Hypermetabolism in the brainstem, cerebellum, hippocampus and amygdala persisted over time and correlated with inflammation status.Conclusion Synergistic effects of systemic virus-mediated inflammation and transient hypoxia yield a dysfunction of the fronto-insular cortex, a signature of CNS involvement in neuro-COVID-19. This brain dysfunction is likely to be transient and almost reversible. The long-lasting brain hypermetabolism seems to reflect persistent inflammation processes

Accumulation of anthocyanins in tomato skin extends shelf life

Shelf life is one of the most important traits for the tomato (Solanum lycopersicum) industry. Two key factors, post-harvest over-ripening and susceptibility to post-harvest pathogen infection, determine tomato shelf life. Anthocyanins accumulate in the skin of Aft/Aft atv/atv tomatoes, the result of introgressing alleles affecting anthocyanin biosynthesis in fruit from two wild relatives of tomato, which results in extended fruit shelf life. Compared with ordinary, anthocyanin-less tomatoes, the fruits of Aft/Aft atv/atv keep longer during storage and are less susceptible to Botrytis cinerea, a major tomato pathogen, post-harvest. Using genetically modified tomatoes over-producing anthocyanins, we confirmed that skin-specific accumulation of anthocyanins in tomato is sufficient to reduce the susceptibility of fruit to Botrytis cinerea. Our data indicate that accumulation of anthocyanins in tomato fruit, achieved either by traditional breeding or genetic engineering can be an effective way to extend tomato shelf life

Sustained seizure freedom with adjunctive brivaracetam in patients with focal onset seizures

The maintenance of seizure control over time is a clinical priority in patients with epilepsy. The aim of this study was to assess the sustained seizure frequency reduction with adjunctive brivaracetam (BRV) in real-world practice. Patients with focal epilepsy prescribed add-on BRV were identified. Study outcomes included sustained seizure freedom and sustained seizure response, defined as a 100% and a ≥50% reduction in baseline seizure frequency that continued without interruption and without BRV withdrawal through the 12-month follow-up. Nine hundred ninety-four patients with a median age of 45 (interquartile range = 32–56) years were included. During the 1-year study period, sustained seizure freedom was achieved by 142 (14.3%) patients, of whom 72 (50.7%) were seizure-free from Day 1 of BRV treatment. Sustained seizure freedom was maintained for ≥6, ≥9, and 12 months by 14.3%, 11.9%, and 7.2% of patients from the study cohort. Sustained seizure response was reached by 383 (38.5%) patients; 236 of 383 (61.6%) achieved sustained ≥50% reduction in seizure frequency by Day 1, 94 of 383 (24.5%) by Month 4, and 53 of 383 (13.8%) by Month 7 up to Month 12. Adjunctive BRV was associated with sustained seizure frequency reduction from the first day of treatment in a subset of patients with uncontrolled focal epilepsy