Profile and potential bioactivity of the miRNome and metabolome expressed in Malva sylvestris L. leaf and flower.

Malva sylvestris L. (common mallow) is a plant species widely used in phytotherapy and ethnobotanical practices since time immemorial. Characterizing the components of this herb might promote a better comprehension of its biological effects on the human body but also favour the identification of the molecular processes that occur in the plant tissues. Thus, in the present contribution, the scientific knowledge about the metabolomic profile of the common mallow was expanded. In particular, the phytocomplex of leaves and flowers from this botanical species and the extraction capacity of different concentrations of ethanol (i.e., 95%, 70%, 50%, and 0%; v/v in ddH2O) for it were investigated by spectrophotometric and chromatographic approaches. In detail, 95% ethanol extracts showed the worst capacity in isolating total phenols and flavonoids, while all the hydroalcoholic samples revealed a specific ability in purifying the anthocyanins. HPLC-DAD system detected and quantified 20 phenolic secondary metabolites, whose concentration in the several extracts depended on their own chemical nature and the percentage of ethanol used in the preparation. In addition, the stability of the purified phytochemicals after resuspension in pure ddH2O was also proved, considering a potential employment of them in biological/medical studies which include in vitro and in vivo experiments on mammalian models. Here, for the first time, the expressed miRNome in M. sylvestris was also defined by Next Generation Sequencing, revealing the presence of 33 microRNAs (miRNAs), 10 typical for leaves and 2 for flowers. Then, both plant and human putative mRNA targets for the detected miRNAs were predicted by bioinformatics analyses, with the aim to clarify the possible role of these small nucleic acids in the common mallow plant tissues and to try to understand if they could exert a potential cross-kingdom regulatory activity on the human health. Surprisingly, our investigations revealed that 19 miRNAs out of 33 were putatively able to modulate, in the plant cells, the expression of various chromosome scaffold proteins. In parallel, we found, in the human transcriptome, a total of 383 mRNAs involved in 5 fundamental mammalian cellular processes (i.e., apoptosis, senescence, cell-cycle, oxidative stress, and invasiveness) that theoretically could be bound and regulated by M. sylvestris miRNAs. The evidence collected in this work would suggest that the beneficial properties of the use of M. sylvestris, documented by the folk medicine, are probably linked to their content of miRNAs and not only to the action of phytochemicals (e.g., anthocyanins). This would open new perspectives about the possibility to develop gene therapies based on miRNAs isolated from medicinal plants, including M. sylvestris.

Looking for Plant microRNAs in Human Blood Samples: Bioinformatics Evidence and Perspectives.

Literature has proposed the existence of a cross kingdom regulation (CRK) between human and plants. In this context, microRNAs present in edible plants would be acquired through diet by the consumer's organism and transported via bloodstream to tissues, where they would modulate gene expression. However, the validity of this phenomenon is strongly debated; indeed, some scholars have discussed both the methodologies and the results obtained in previous works. To date, only one study has performed a bioinformatics analysis on small RNA-sequencing data for checking the presence of plant miRNAs (pmiRNAs) in human plasma. For that investigation, the lack of reliable controls, which led to the misidentification of human RNAs as pmiRNAs, has been deeply criticized. Thus, in the present contribution, we aim to demonstrate the existence of pmiRNAs in human blood, adopting a bioinformatics approach characterized by more stringent conditions and filtering. The information obtained from 380 experiments produced in 5 different next generation sequencing (NGS) projects was examined, revealing the presence of 350 circulating pmiRNAs across the analysed data set. Although one of the NGS projects shows results likely to be attributed to sample contamination, the others appear to provide reliable support for the acquisition of pmiRNAs through diet. To infer the potential biological activity of the identified pmiRNAs, we predicted their putative human mRNA targets, finding with great surprise that they appear to be mainly involved in neurogenesis and nervous system development. Unfortunately, no consensus was identified within the sequences of detected pmiRNAs, in order to justify their stability or capability to be preserved in human plasma. We believe that the issue regarding CKR still needs further clarifications, even if the present findings would offer a solid support that this hypothesis is not impossible.

Leaf-buds of Pistacia atlantica: a novel source of bioactive molecules with high anti-inflammatory, antioxidant, anti-tyrosinase and antimicrobial properties.

In this study, for the first time, the anti-inflammatory, antioxidant, anti-tyrosinase and antimicrobial property of P. atlantica Desf. subsp. atlantica leaf-bud extract have been investigated. The anti-inflammatory activity was determined in vivo by reducing carrageenan-induced hind paw edema in mice, while the antiradical function was evaluated using DPPH, total antioxidant capacity (TAC) and reduction power assays. The extract induced a significant reduction of the edema, from 1 to 6 h in a dose-dependent manner (150, 200 and 300 mg/kg). Histological observations of the inflamed tissues also confirmed this. An effective antioxidant activity of the plant samples was demonstrated, showing an EC50 = 0.183 ± 0.005 mg/mL for the DPPH test, a value of 28.776 ± 2.541 mg AAE/g for the TAC and an EC50 = 0.136 ± 0.003 mg/mL for reducing power. The leaf-bud extract also revealed a good antimicrobial activity against S. aureus and L. monocytogenes (mean diameter of inhibition zones of 13.2 and 17.0 mm, respectively), while a slight antifungal effect was observed. The plant preparation was then documented to inhibit tyrosinase activity, with an EC50 value of 0.098 ± 0.00 mg/mL in a dose-dependent manner. HPLC-DAD analysis revealed that dimethyl-allyl caffeic acid and rutin were the most abundant molecules. The current data documented that P. atlantica leaf-bud extract has strong biological properties and constitutes a potential source of pharmacological molecules.

Phytochemicals and quality level of food plants grown in an aquaponics system.

BACKGROUND: Beyond nutrition, fruits and vegetables can be considered as natural sources of bioactive molecules, for which beneficial effects on human health are widely recognised. To improve food quality, soilless growing systems could represent a good strategy for promoting a sustainable food production chain, although the nutritional and nutraceutical properties of their products should be investigated in depth. The main quality traits and the volatile and non-volatile secondary metabolites of Solanum lycopersicum L., Petroselinum crispum (Mill.) Fuss and Ocimun basilicum L. grown in an aquaponics system and in organic farming were quantified and compared. RESULTS: On a fresh basis, soil-grown P. crispum and O. basilicum showed significantly higher total phenolics and antioxidant activity compared to aquaponic crops, whereas, on a dry basis, both plants showed opposite results. Soil-grown S. lycopersicum was significantly richer in total phenolics, whereas the aquaponic type showed a higher antioxidant activity. Aquaponics induced the accumulation of resveratrol in P. crispum, rosmarinic acid and myricetin in O. basilicum, and lycopene in S. lycopersicum. Among the volatile compounds, in O. basilicum, linalool was the main constituent in both treatments, whereas τ-cadinol represented the second constituent in aquaponic crops. The volatile profiles of P. crispum did not differ significantly between the two cultivation methods. CONCLUSION: The overall quality of organic and aquaponics cultures appeared to be comparable. The results showed that aquaponic farming method can be an innovative, rapid and sustainable way of producing quality food. © 2021 Society of Chemical Industry.

Plant miR171 modulates mTOR pathway in HEK293 cells by targeting GNA12.

Plant microRNAs have shown the capacity to regulate mammalian systems. The potential bioactivity of miR171vr, an isoform of the plant miR171, on human embryonic kidney 293 (HEK293) cells was investigated. Bioinformatics simulations revealed that human G protein subunit alpha 12 (GNA12) transcript could represent an excellent target for miR171vr. To confirm this prediction, in vitro experiments were performed using a synthetic microRNA designed on miR171vr sequence. MiR-treated cells showed a significant decrease of GNA12 mRNA and protein levels, confirming the putative cross-kingdom interaction. In addition, miR171vr determined the modulation of GNA12 downstream signaling factors, including mTOR, as expected. Finally, the effect of the plant miRNA on HEK293 cell growth and its stability in presence of several stressors, such as those miming digestive processes and procedures for preparing food, were evaluated. All this preliminary evidence would suggest that miR171vr, introduced by diet or as supplement in gene therapies, could potentially influence human gene expression, especially for treating disorders where GNA12 is over-expressed (i.e. oral cancer, breast and prostate adenocarcinoma) or mTOR kinase is down-regulated (e.g. obesity, type 2 diabetes, neurodegeneration).

Induction of Antioxidant Metabolites in Moringa oleifera Callus by Abiotic Stresses.

Moringa oleifeira has recently been subjected to numerous scientific studies pursuing its biological properties. However, biotechnological approaches promoting the synthesis of pharmacological compounds in this species are still scarce, despite the fact that moringa metabolites have shown significant nutraceutical effects. For this reason, in vitro cultures of moringa callus, obtained from leaf explantation, were subjected to various abiotic stresses such as temperature, salicylic acid, and NaCl, to identify the best growth conditions for the production of high levels of antioxidant molecules. Temperature stresses (exposure to 4 and 45 °C) led to no significant variation in moringa callus, in terms of antiradical metabolites, whereas salicylic acid (200 µM) and NaCl (50-100 µM) affected an increase of total phenolic compounds, after 15 and 30 days of treatment. Overall, the treatment with 100 µM NaCl for 30 days showed the highest free radical scavenging activity, comparable to that measured in moringa leaf. In addition, high doses of NaCl (200 µM) inhibited callus growth and reduced the amount and bioactivity of the secondary metabolites of callus. This study provides useful information to standardize growth conditions for the production of secondary metabolites in moringa in vitro cultures, a biotechnological system that could be employed for a rapid, controlled, and guaranteed production of antioxidant molecules for pharmaceutical purposes.

Plant defense factors involved in Olea europaea resistance against Xylella fastidiosa infection.

Olive quick decline syndrome (OQDS) is a dangerous plant disease, caused by the bacterium Xylella fastidiosa, which targets olive (Olea europaea). Since field observations suggested that some olive cultivars (i.e. Leccino) were more resistant to OQDS than others (i.e. Cellina di Nardò), the plant defense strategies adopted by olive to contrast X. fastidiosa infection were investigated. In the present study, ELISA and genetic approaches were used to confirm plant infection, while microbial colonization mechanism and distribution in host plant tissues and reactive oxygen species (ROS) levels were examined by light, scanning electron and confocal microscopy analyses. Spectrophotometric and chromatographic techniques were performed to measure secondary metabolites content and qPCR assay was carried out for monitoring plant gene expression variation. Our analysis showed that X. fastidiosa caused accumulation of ROS in Leccino samples compared to Cellina di Nardò. Moreover, the infection induced the up-regulation of defense-related genes, such as NADPH oxidase, some protein kinases, pathogen plant response factors and metabolic enzymes. We also found that Leccino plants enhanced the production of specific antioxidant and antimicrobial molecules, to fight the pathogen and avoid its spreading into xylem vessels. We provided new information on OQDS resistance mechanism applied by Leccino cultivar. In particular, we evidenced that high concentrations of ROS, switching on plant defence signalling pathways, may represent a key factor in fighting X. fastidiosa infection.

Antibacterial Activity of Different Blossom Honeys: New Findings.

Antibacterial activity is the most investigated biological property of honey. The goal of this study was to evaluate the antibacterial activity of 57 Slovak blossom honeys against Staphylococcus aureus and Pseudomonas aeruginosa and investigate the role of several bioactive substances in antibacterial action of honeys. Inhibitory and bactericidal activities of honeys were studied to determine the minimum inhibitory and bactericidal concentrations. The contents of glucose oxidase (GOX) enzyme, hydrogen peroxide (H2O2), and total polyphenols (TP) were determined in honeys. We found that honey samples showed different antibacterial efficacy against the tested bacteria as follows: wildflower honeys > acacia honeys > rapeseed honeys. Overall antibacterial activity of the honeys was statistically-significantly correlated with the contents of H2O2 and TP in honeys. A strong correlation was found between the H2O2 and TP content. On the other hand, no correlation was found between the content of GOX and level of H2O2. Antibacterial activity of 12 selected honeys was markedly reduced by treatment with catalase, but it remained relatively stable after inactivation of GOX with proteinase-K digestion. Obtained results suggest that the antibacterial activity of blossom honeys is mainly mediated by H2O2 levels present in honeys which are affected mainly by polyphenolic substances and not directly by GOX content.

Growth of Pseudomonas aeruginosa in zinc poor environments is promoted by a nicotianamine-related metallophore.

Previous studies have suggested that P. aeruginosa possesses redundant zinc uptake systems. To identify uncharacterized zinc transporters, we analyzed the genome-wide transcriptional responses of P. aeruginosa PA14 to zinc restriction. This approach led to the identification of an operon (zrmABCD) regulated by the zinc uptake regulator Zur, that encodes for a metallophore-mediated zinc import system. This operon includes the genes for an uncharacterized TonB-dependent Outer Membrane Protein (ZrmA) and for a putative nicotianamine synthase (ZrmB). The simultaneous inactivation of the ZnuABC transporter and of one of these two genes markedly decreases the ability of P. aeruginosa to grow in zinc-poor media and compromises intracellular zinc accumulation. Our data demonstrate that ZrmB is involved in the synthesis of a metallophore which is released outside the cell and mediates zinc uptake through the ZrmA receptor. We also show that alterations in zinc homeostasis severely affect the ability of P. aeruginosa to cause acute lung and systemic infections in C57BL/6 mice, likely due to the involvement of zinc in the expression of several virulence traits. These findings disclose a hitherto unappreciated role of zinc in P. aeruginosa pathogenicity and reveal that this microorganism can obtain zinc through a strategy resembling siderophore-mediated iron uptake.

From Robinia pseudoacacia L. nectar to Acacia monofloral honey: biochemical changes and variation of biological properties.

BACKGROUND: Robinia pseudoacacia L. nectar and its derivative monofloral honey were systematically compared in this study, to understand how much the starting solution reflected the final product, after re-elaboration by Apis mellifera ligustica Spinola. RESULTS: Subjected to dehydration in the hive, nectar changed in its water and sugar content when transformed into honey, as physicochemical and gas chromatographic-mass spectrometric analyses revealed. Spectrophotometric measurements and characterization by high-performance liquid chromatography-diode array detection of 18 plant molecules demonstrated honey to be richer than nectar in secondary metabolites. For the first time, the hypothesis of the existence of a nectar redox cycle in R. pseudoacacia was reported, as previously described for Nicotiana sp., based on 1D-protein profiles, western blot analysis and detection of H2 O2 and ascorbate. The bioactivity of both matrices was also investigated. Antiradical in vitro tests showed that Acacia honey was more antioxidant than nectar, which was even able to induce oxidative stress directly in a eukaryotic cell system. Antimicrobial assays demonstrated that nectar was bacteriostatic, due to H2 O2 activity, whereas honey was even bactericidal. CONCLUSION: All these data support the ecological role of nectar and honey in nature: protection of the gynoecium from pathogens and preservation from degradative processes, respectively. © 2018 Society of Chemical Industry.

Botanical influence on phenolic profile and antioxidant level of Italian honeys.

Honeybees directly transfer plant compounds from nectar into honey. Each plant species possesses a specific metabolic profile, the amount and the typology of plant molecules that may be detected in honey vary according to their botanical origin. Aim of the present work was the spectrophotometrical determination of concentration ranges of simple phenols and flavonoids in 460 several Italian monofloral honeys, in order to individuate specific intervals of plant metabolites for each typology of honey. Moreover, an LC-MS analysis was performed to determine amount of various secondary metabolites in the samples, with the purpose to use them as potential molecular markers in support to honey melissopalynological classification. As plant molecules have a strong reducing power, the antioxidant activity of the honeys was evaluated by two antiradical assays, DPPH and FRAP. The free radical scavenging effect of each monofloral group was correlated to the concentration of simple phenols and flavonoids, with the aim to deduce the existence of possible relationships between these parameters. In conclusion, dark honeys (Castanea sativa, honeydew, Erica sp. and Eucalyptus sp.) appeared to be the richest in secondary metabolites and, consequently, showed higher antioxidant activity. However, all analyzed monofloral honeys showed to be good sources of antioxidants.

OeFAD8, OeLIP and OeOSM expression and activity in cold-acclimation of Olea europaea, a perennial dicot without winter-dormancy.

MAIN CONCLUSION: Cold-acclimation genes in woody dicots without winter-dormancy, e.g., olive-tree, need investigation. Positive relationships between OeFAD8, OeOSM , and OeLIP19 and olive-tree cold-acclimation exist, and couple with increased lipid unsaturation and cutinisation. Olive-tree is a woody species with no winter-dormancy and low frost-tolerance. However, cold-tolerant genotypes were empirically selected, highlighting that cold-acclimation might be acquired. Proteins needed for olive-tree cold-acclimation are unknown, even if roles for osmotin (OeOSM) as leaf cryoprotectant, and seed lipid-transfer protein for endosperm cutinisation under cold, were demonstrated. In other species, FAD8, coding a desaturase producing α-linolenic acid, is activated by temperature-lowering, concomitantly with bZIP-LIP19 genes. The research was focussed on finding OeLIP19 gene(s) in olive-tree genome, and analyze it/their expression, and that of OeFAD8 and OeOSM, in drupes and leaves under different cold-conditions/developmental stages/genotypes, in comparison with changes in unsaturated lipids and cell wall cutinisation. Cold-induced cytosolic calcium transients always occurred in leaves/drupes of some genotypes, e.g., Moraiolo, but ceased in others, e.g., Canino, at specific drupe stages/cold-treatments, suggesting cold-acclimation acquisition only in the latter genotypes. Canino and Moraiolo were selected for further analyses. Cold-acclimation in Canino was confirmed by an electrolyte leakage from leaf/drupe membranes highly reduced in comparison with Moraiolo. Strong increases in fruit-epicarp/leaf-epidermis cutinisation characterized cold-acclimated Canino, and positively coupled with OeOSM expression, and immunolocalization of the coded protein. OeFAD8 expression increased with cold-acclimation, as the production of α-linolenic acid, and related compounds. An OeLIP19 gene was isolated. Its levels changed with a trend similar to OeFAD8. All together, results sustain a positive relationship between OeFAD8, OeOSM and OeLIP19 expression in olive-tree cold-acclimation. The parallel changes in unsaturated lipids and cutinisation concur to suggest orchestrated roles of the coded proteins in the process.

Ribosomal stress activates eEF2K-eEF2 pathway causing translation elongation inhibition and recruitment of terminal oligopyrimidine (TOP) mRNAs on polysomes.

The synthesis of adequate amounts of ribosomes is an essential task for the cell. It is therefore not surprising that regulatory circuits exist to organize the synthesis of ribosomal components. It has been shown that defect in ribosome biogenesis (ribosomal stress) induces apoptosis or cell cycle arrest through activation of the tumor suppressor p53. This mechanism is thought to be implicated in the pathophysiology of a group of genetic diseases such as Diamond Blackfan Anemia which are called ribosomopathies. We have identified an additional response to ribosomal stress that includes the activation of eukaryotic translation elongation factor 2 kinase with a consequent inhibition of translation elongation. This leads to a translational reprogramming in the cell that involves the structurally defined group of messengers called terminal oligopyrimidine (TOP) mRNAs which encode ribosomal proteins and translation factors. In fact, while general protein synthesis is decreased by the impairment of elongation, TOP mRNAs are recruited on polysomes causing a relative increase in the synthesis of TOP mRNA-encoded proteins compared to other proteins. Therefore, in response to ribosomal stress, there is a change in the translation pattern of the cell which may help restore a sufficient level of ribosomes.

Ultrastructure and development of the floral nectary from Borago officinalis L. and phytochemical changes in its secretion.

Although Boraginaceae have been classified as good sources of nectar for many insects, little is still known about their nectar and nectaries. Thus, in the present contribution, we investigated the nectar production dynamics and chemistry in Borago officinalis L. (borage or starflower), together with its potential interaction capacity with pollinators. A peak of nectar secretion (∼5.1 µL per flower) was recorded at anthesis, to decrease linearly during the following 9 days. In addition, TEM and SEM analyses were performed to understand ultrastructure and morphological changes occurring in borage nectary before and after anthesis, but also after its secretory phase. Evidence suggested that nectar was transported by the apoplastic route (mainly from parenchyma to epidermis) and then released essentially by exocytotic processes, that is a granulocrine secretion. This theory was corroborated by monitoring the signal of complex polysaccharides and calcium, respectively, via Thiéry staining and ESI/EELS technique. After the secretory phase, nectary underwent degeneration, probably through autophagic events and/or senescence induction. Furthermore, nectar (Nec) and other flower structures (i.e., sepals, gynoecia with nectaries, and petals) from borage were characterized by spectrophotometry and HPLC-DAD, in terms of plant secondary metabolites, both at early (E-) and late (L-) phase from anthesis. The content of phytochemicals was quantified and discussed for all samples, highlighting potential biological roles of these compounds in the borage flower (e.g., antimicrobial, antioxidant, staining effects). Surprisingly, a high significant accumulation of flavonoids was registered in L-Nec, with respect to E-Nec, indicating that this phenomenon might be functional and able to hide molecular (e.g., defence against pathogens) and/or ecological (e.g., last call for pollinators) purposes. Indeed, it is known that these plant metabolites influence nectar palatability, encouraging the approach of specialist pollinators, deterring nectar robbers, and altering the behaviour of insects.

Nutraceutical Content and Biological Properties of Lipophilic and Hydrophilic Fractions of the Phytocomplex from Pistacia atlantica Desf. Buds, Roots, and Fruits.

The aim of the present investigation was to obtain 12 aqueous extracts and 1 oil from Pistacia atlantica Desf. subsp. atlantica specimens. The samples differed for processed plant organs (i.e., roots, buds, and fruits), gender and geographical station of the collected trees. Total phenols, flavonoids, and condensed tannins were determined, revealing that bud extracts exhibited the highest phenolic content (386.785 ± 16.227 mg GAE/g DM), followed by fruit and root preparations. Similar results were detected for flavonoids and tannins, whose quantitation ranged from 0.014 ± 0.005 to 74.780 ± 9.724 mg CE/g DM and from 0.037 ± 0.003 to 14.793 ± 0.821 mg CE/g DM, respectively. The biochemical profile of the extracts was further characterized by HPLC-DAD, in terms of specific phenolics. This analysis identified gallic acid as a typical metabolite for ripe fruit, while hydroxytyrosol for female roots and male buds. In parallel, P. atlantica fruit oil was profiled by GC-MS analysis, which detected 37 lipophilic components, including palmitic acid (the major component, ~55%), anacardol, tetradecanol, arachidic acid, squalene, and some terpenes. The samples revealed interesting antioxidant activity, with EC50 values ranging from 0.073 ± 0.001 to 193.594 ± 28.942 mg/mL and from 0.029 ± 0.001 to 103.086 ± 20.540 mg/mL, in that order, for DPPH and reducing power assays. Concerning the total antioxidant capacity, the results ranged from 0.053 ± 0.008 to 51.648 ± 1.659 mg AAE/g DM. Finally, the antimicrobial potential of the plant extracts was estimated against 7 bacterial species and 2 fungal strains, known to be human pathogens, demonstrating a good antibiotic effect for the bud extracts. All these findings strongly suggest that P. atlantica would represent a natural reservoir for novel additives to be used in therapeutic, food, and cosmetic products.

Microparticles from dental calculus disclose paleoenvironmental and palaeoecological records.

Plants have always represented a key element in landscape delineation. Indeed, plant diversity, whose distribution is influenced by geographic/climatic variability, has affected both environmental and human ecology. The present contribution represents a multi-proxy study focused on the detection of starch, pollen and non-pollen palynomorphs in ancient dental calculus collected from pre-historical individuals buried at La Sassa and Pila archaeological sites (Central Italy). The collected record suggested the potential use of plant taxa by the people living in Central Italy during the Copper-Middle Bronze Age and expanded the body of evidence reported by previous palynological and palaeoecological studies. The application of a microscopic approach provided information about domesticated crops and/or gathered wild plants and inferred considerations on ancient environments, water sources, and past health and diseases. Moreover, the research supplied data to define the natural resources (e.g., C4-plant intake) and the social use of the space during that period. Another important aspect was the finding of plant clues referable to woody habitats, characterised by broad-leaved deciduous taxa and generally indicative of a warm-temperate climate and grassy vegetation. Other unusual records (e.g., diatoms, brachysclereids) participated in defining the prehistoric ecological framework. Thus, this work provides an overview on the potential of the human dental calculus analysis to delineate some features of the ancient plant ecology and biodiversity.

Influence of environmental conditions on the production of nutraceuticals in Italian edible plant landraces.

Autochthonous plant varieties, also referred to as landraces, represent an important genetic resource, being well-adapted to the environment in which they have been selected. Landraces usually show profiles rich in nutraceuticals, making them an effective and valuable alternative to commercial agri-products, as well as potential candidates for crop improvement programs. Basilicata region is recognized as an Italian hotspot for agrobiodiversity, due to its complex orography. Thus, this work aimed to characterize and monitor, for two successive years, the content of secondary metabolites and related antioxidant properties of seven different species, four officinal (i.e., wild fennel - Feoniculum vulgare Mill.; oregano - Origanum vulgare L.; thyme - Thymus vulgaris L.; valerian - Valeriana officinalis L.) and three fruit species (i.e., fig - Ficus carica L. cv. Dottato; sweet cherry Prunus avium L. cv. Majatica; plum - Prunus domestica L. cv. Cascavella Gialla), collected in three different sites of this region. In detail, spectrophotometric tests were performed to assess the concentration of phenolic compounds, flavonoids, and - for officinal plants - also terpenoids, together with the antiradical activity (FRAP assays). In addition, to better typify the phytocomplexes of these landraces, HPLC-DAD and GC-MS analyses were carried out. In general, officinal plants showed higher values of nutraceutical compounds and related bioactivity with respect to fruit species. The data showed how different accessions of the same species had different phytochemical profiles, according to the sampling area and the year of collection, suggesting a role for both genetic and environmental factors in determining the observed results. Therefore, the final goal of this research was also to find a possible correlation between environmental factors and nutraceutics. The greatest correlation was found in valerian, where a lower water intake seemed to lead to a higher accumulation of antioxidants, and in plum, where the flavonoid content correlated positively with high temperatures. All these outcomes contribute at valorising Basilicata landraces for their aptitude to be high-quality foods and, at the same time, promoting the preservation of the agrobiodiversity for this region.

Sustainability in Aquaponics: Industrial Spirulina Waste as a Biofertilizer for Lactuca sativa L. Plants.

Aquaponics represents an alternative to traditional soil cultivation. To solve the problem of nutrient depletion that occurs in this biotechnological system, the application of a spirulina-based biofertilizer was assessed. The microalgal waste used in this study came from industrial processing. Four different dilutions of the supernatant portion of this waste were sprayed on lettuce plants cultivated in an aquaponics system installed at the Botanical Gardens of the Tor Vergata University of Rome. The biofertilizer was characterized to evaluate its amount of macro- and micronutrients. The analysis conducted on the plants involved both morpho-biometric aspects and qualitative-quantitative measurements. The experiments showed that the spirulina extract had a positive effect on the growth and nutraceutical content of the lettuce plants; the obtained results highlighted that a dilution of 75% was the best for treatment. The use of the proposed organic and recycled fertilizer could increase the sustainability of crop cultivation and promote the functioning of aquaponics systems.

Pollen variability in Quercus L. species and relative systematic implications.

This study aims to address one of the challenges related to the complexity of the Quercus L. genus, that is the identification of structural elements favouring the systematic identification of the oak pollen. Thus, in this contribution, we explored the variation of morphometric and chemical parameters in pollen samples collected from 47 different Quercus species and hybrids. Several qualitative (e.g., outline in polar view, class, aperture structures) and quantitative (e.g., diameter, exine and sporoderm thickness, autofluorescence, content in proteins and plant metabolites) features were evaluated by optic microscopy and spectrophotometric assays. Statistical analyses were also carried out to assess significant correlations and clustering effects among the studied taxa, based on phenotypical and biochemical data, to identify the parameters which could be useful for taxonomic discrimination at inter- and intra-specific level. Only few morphological traits showed the potentiality to be diagnostic, such as pollen diameter and outline in polar view. The intensity of pollen autofluorescence varied among the samples but it did not seem to correlate with protein, carotenoid, phenolic and flavonoid content. However, differences in protein and carotenoid levels were detected, suggesting them as possible taxonomic discriminants for oak pollen. Thus, our work represents a step forward in understanding morphology and biochemistry of oak pollen, constitutes an experimental set-up applicable in future systematic studies on other genera, and opens new perspectives for further molecular investigations on Quercus species.

AKT-driven epithelial-mesenchymal transition is affected by copper bioavailability in HER2 negative breast cancer cells via a LOXL2-independent mechanism.

BACKGROUND: The main mechanism underlying cancer dissemination is the epithelial to mesenchymal transition (EMT). This process is orchestrated by cytokines like TGFß, involving "non-canonical" AKT- or STAT3-driven pathways. Recently, the alteration of copper homeostasis seems involved in the onset and progression of cancer. METHODS: We expose different breast cancer cell lines, including two triple negative (TNBC) ones, an HER2 enriched and one cell line representative of the Luminal A molecular subtype, to short- or long-term copper-chelation by triethylenetetramine (TRIEN). We analyse changes in the expression of EMT markers (E-cadherin, fibronectin, vimentin and αSMA), in the levels and activity of extracellular matrix components (LOXL2, fibronectin and MMP2/9) and of copper homeostasis markers by Western blot analyses, immunofluorescence, enzyme activity assays and RT-qPCR. Boyden Chamber and wound healing assays revealed the impact of copper chelation on cell migration. Additionally, we explored whether perturbation of copper homeostasis affects EMT prompted by TGFß. Metabolomic and lipidomic analyses were applied to search the effects of copper chelation on the metabolism of breast cancer cells. Finally, bioinformatics analysis of data on breast cancer patients obtained from different databases was employed to correlate changes in kinases and copper markers with patients' survival. RESULTS: Remarkably, only HER2 negative breast cancer cells differently responded to short- or long-term exposure to TRIEN, initially becoming more aggressive but, upon prolonged exposure, retrieving epithelial features, reducing their invasiveness. This phenomenon may be related to the different impact of the short and prolonged activation of the AKT kinase and to the repression of STAT3 signalling. Bioinformatics analyses confirmed the positive correlation of breast cancer patients' survival with AKT activation and up-regulation of CCS. Eventually, metabolomics studies demonstrate a prevalence of glycolysis over mitochondrial energetic metabolism and of lipidome changes in TNBC cells upon TRIEN treatment. CONCLUSIONS: We provide evidence of a pivotal role of copper in AKT-driven EMT activation, acting independently of HER2 in TNBC cells and via a profound change in their metabolism. Our results support the use of copper-chelators as an adjuvant therapeutic strategy for TNBC.