Biological Activity and Metabolomics of Griffonia simplicifolia Seeds Extracted with Different Methodologies.

Griffonia simplicifolia, a tropical plant endemic to West Africa, is highly regarded for its significant pharmacological potential. The objective of this study was to evaluate the metabolomic profile and to explore the antioxidant properties, antiproliferative activity, and antimicrobial potential of G. simplicifolia seed extracts obtained through either maceration, microwave-assisted extraction (MAE), or Soxhlet extraction using water, acetone, methanol and ethanol as solvents. Overall, methanol possessed superior total extraction efficiency. HPLC analyses confirmed the efficacy of acetone and ethanol as optimal solvents for the extraction of flavonoids and flavan-3-ols, whereas MAE exhibited enhanced effectiveness in extracting N-containing compounds, including 5-hydroxytryptophan (5-HTP). HPLC-MS analyses identified forty-three compounds, including thirty-four phenolic compounds and nine N-containing molecules. Isomyricitrin, taxifolin and a flavonol glucuronide were the main polyphenols, whereas 5-HTP was the main N-containing compound. Hydroalcoholic G. simplicifolia extracts showed the highest radical scavenging and metal-reducing antioxidant power, suggesting that most of the contribution to antioxidant activity depends on the more polar bioactive compounds. G. simplicifolia extracts showed dose-dependent antiproliferative activity against three distinct cancer cell lines (HeLa, HepG2, and MCF-7), with notable variations observed among both the different extracts and cell lines and divergent GI50 values, emphasizing substantial discrepancies in cell sensitivity to the various extracts. Furthermore, G. simplicifolia extracts revealed antibiotic activity against Staphylococcus aureus. Our results highlight the potential of G. simplicifolia phytochemicals in the development of functional foods, nutraceuticals, and dietary supplements.

14-3-3 Proteins and the Plasma Membrane H+-ATPase Are Involved in Maize (Zea mays) Magnetic Induction.

The geomagnetic field (GMF) is a natural component of the biosphere, and, during evolution, all organisms experienced its presence while some evolved the ability to perceive magnetic fields (MF). We studied the response of 14-3-3 proteins and the plasma membrane (PM) proton pump H+-ATPase to reduced GMF values by lowering the GMF intensity to a near-null magnetic field (NNMF). Seedling morphology, H+-ATPase activity and content, 14-3-3 protein content, binding to PM and phosphorylation, gene expression, and ROS quantification were assessed in maize (Zea mays) dark-grown seedlings. Phytohormone and melatonin quantification were also assessed by LG-MS/MS. Our results suggest that the GMF regulates the PM H+-ATPase, and that NNMF conditions alter the proton pump activity by reducing the binding of 14-3-3 proteins. This effect was associated with both a reduction in H2O2 and downregulation of genes coding for enzymes involved in ROS production and scavenging, as well as calcium homeostasis. These early events were followed by the downregulation of IAA synthesis and gene expression and the increase in both cytokinin and ABA, which were associated with a reduction in root growth. The expression of the homolog of the MagR gene, ZmISCA2, paralleled that of CRY1, suggesting a possible role of ISCA in maize magnetic induction. Interestingly, melatonin, a widespread molecule present in many kingdoms, was increased by the GMF reduction, suggesting a still unknown role of this molecule in magnetoreception.

Recent Advances in Plant-Insect Interactions.

Plant-insect interaction is a fast-developing research field that continues to increase the interest of numerous scientists, many of whom come from heterogeneous backgrounds [...].

Discrimination of Green Coffee (Coffea arabica and Coffea canephora) of Different Geographical Origin Based on Antioxidant Activity, High-Throughput Metabolomics, and DNA RFLP Fingerprinting.

The genus Coffea is known for the two species C. arabica (CA) and C. canephora (CC), which are used to prepare the beverage coffee. Proper identification of green beans of coffee varieties is based on phenotypic and phytochemical/molecular characteristics. In this work, a combination of chemical (UV/Vis, HPLC-DAD-MS/MS, GC-MS, and GC-FID) and molecular (PCR-RFLP) fingerprinting was used to discriminate commercial green coffee accessions from different geographical origin. The highest content of polyphenols and flavonoids was always found in CC accessions, whereas CA showed lower values. ABTS and FRAP assays showed a significant correlation between phenolic content and antioxidant activity in most CC accessions. We identified 32 different compounds, including 28 flavonoids and four N-containing compounds. The highest contents of caffeine and melatonin were detected in CC accessions, whereas the highest levels of quercetin and kaempferol derivatives were found in CA accessions. Fatty acids of CC accessions were characterized by low levels of linoleic and cis octadecenoic acid and high amounts of elaidic acid and myristic acid. Discrimination of species according to their geographical origin was achieved using high-throughput data analysis, combining all measured parameters. Lastly, PCR-RFLP analysis was instrumental for the identification of recognition markers for the majority of accessions. Using the restriction enzyme AluI on the trnL-trnF region, we clearly discriminated C. canephora from C. arabica, whereas the cleavage performed by the restriction enzymes MseI and XholI on the 5S-rRNA-NTS region produced specific discrimination patterns useful for the correct identification of the different coffee accessions. This work extends our previous studies and provides new information on the complete flavonoid profile, combining high-throughput data with DNA fingerprinting to assess the geographical discrimination of green coffee.

Beta-Caryophyllene Modifies Intracellular Lipid Composition in a Cell Model of Hepatic Steatosis by Acting through CB2 and PPAR Receptors.

Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease; however, no specific pharmacological therapy has yet been approved for this condition. Plant-derived extracts can be an important source for the development of new drugs. The aim of this study was to investigate the effects of (E)-ß-caryophyllene (BCP), a phytocannabinoid recently found to be beneficial against metabolic diseases, on HepG2 steatotic hepatocytes. Using a fluorescence-based lipid quantification assay and GC-MS analysis, we show that BCP is able to decrease lipid accumulation in steatotic conditions and to change the typical steatotic lipid profile by primarily reducing saturated fatty acids. By employing specific antagonists, we demonstrate that BCP action is mediated by multiple receptors: CB2 cannabinoid receptor, peroxisome proliferator-activated receptor α (PPARα) and γ (PPARγ). Interestingly, BCP was able to counteract the increase in CB2 and the reduction in PPARα receptor expression observed in steatotic conditions. Moreover, through immunofluorescence and confocal microscopy, we demonstrate that CB2 receptors are mainly intracellularly localized and that BCP is internalized in HepG2 cells with a maximum peak at 2 h, suggesting a direct interaction with intracellular receptors. The results obtained with BCP in normal and steatotic hepatocytes encourage future applications in the treatment of NAFLD.

Coinheritance of PIEZO1 variants and multi-locus red blood cell defects account for the symptomatic phenotype in beta-thalassemia carriers.

Workflow of the study with some examples of the achieved results.

Bacterial Volatiles (mVOC) Emitted by the Phytopathogen Erwinia amylovora Promote Arabidopsis thaliana Growth and Oxidative Stress.

Phytopathogens are well known for their devastating activity that causes worldwide significant crop losses. However, their exploitation for crop welfare is relatively unknown. Here, we show that the microbial volatile organic compound (mVOC) profile of the bacterial phytopathogen, Erwinia amylovora, enhances Arabidopsis thaliana shoot and root growth. GC-MS head-space analyses revealed the presence of typical microbial volatiles, including 1-nonanol and 1-dodecanol. E. amylovora mVOCs triggered early signaling events including plasma transmembrane potential Vm depolarization, cytosolic Ca2+ fluctuation, K+-gated channel activity, and reactive oxygen species (ROS) and nitric oxide (NO) burst from few minutes to 16 h upon exposure. These early events were followed by the modulation of the expression of genes involved in plant growth and defense responses and responsive to phytohormones, including abscisic acid, gibberellin, and auxin (including the efflux carriers PIN1 and PIN3). When tested, synthetic 1-nonanol and 1-dodecanol induced root growth and modulated genes coding for ROS. Our results show that E. amylovora mVOCs affect A. thaliana growth through a cascade of early and late signaling events that involve phytohormones and ROS.

The Geomagnetic Field (GMF) Is Necessary for Black Garden Ant (Lasius niger L.) Foraging and Modulates Orientation Potentially through Aminergic Regulation and MagR Expression.

The geomagnetic field (GMF) can affect a wide range of animal behaviors in various habitats, primarily providing orientation cues for homing or migratory events. Foraging patterns, such as those implemented by Lasius niger, are excellent models to delve into the effects of GMF on orientation abilities. In this work, we assessed the role of GMF by comparing the L. niger foraging and orientation performance, brain biogenic amine (BA) contents, and the expression of genes related to the magnetosensory complex and reactive oxygen species (ROS) of workers exposed to near-null magnetic fields (NNMF, ~40 nT) and GMF (~42 µT). NNMF affected workers' orientation by increasing the time needed to find the food source and return to the nest. Moreover, under NNMF conditions, a general drop in BAs, but not melatonin, suggested that the lower foraging performance might be correlated to a decrease in locomotory and chemical perception abilities, potentially driven by dopaminergic and serotoninergic regulations, respectively. The variation in the regulation of genes related to the magnetosensory complex in NNMF shed light on the mechanism of ant GMF perception. Overall, our work provides evidence that the GMF, along with chemical and visual cues, is necessary for the L. niger orientation process.

The Geomagnetic Field (GMF) Is Required for Lima Bean Photosynthesis and Reactive Oxygen Species Production.

Plants evolved in the presence of the Earth's magnetic field (or geomagnetic field, GMF). Variations in MF intensity and inclination are perceived by plants as an abiotic stress condition with responses at the genomic and metabolic level, with changes in growth and developmental processes. The reduction of GMF to near null magnetic field (NNMF) values by the use of a triaxial Helmholtz coils system was used to evaluate the requirement of the GMF for Lima bean (Phaseolus lunatus L.) photosynthesis and reactive oxygen species (ROS) production. The leaf area, stomatal density, chloroplast ultrastructure and some biochemical parameters including leaf carbohydrate, total carbon, protein content and δ13C were affected by NNMF conditions, as were the chlorophyll and carotenoid levels. RubisCO activity and content were also reduced in NNMF. The GMF was required for the reaction center's efficiency and for the reduction of quinones. NNMF conditions downregulated the expression of the MagR homologs PlIScA2 and PlcpIScA, implying a connection between magnetoreception and photosynthetic efficiency. Finally, we showed that the GMF induced a higher expression of genes involved in ROS production, with increased contents of both H2O2 and other peroxides. Our results show that, in Lima bean, the GMF is required for photosynthesis and that PlIScA2 and PlcpIScA may play a role in the modulation of MF-dependent responses of photosynthesis and plant oxidative stress.

Biology of Two-Spotted Spider Mite (Tetranychus urticae): Ultrastructure, Photosynthesis, Guanine Transcriptomics, Carotenoids and Chlorophylls Metabolism, and Decoyinine as a Potential Acaricide.

Two-Spotted Spider Mites (TSSMs, Tetranychus urticae Koch 1836 (Acari: Tetranychidae)) is one of the most important pests in many crop plants, and their feeding activity is based on sucking leaf cell contents. The purpose of this study was to evaluate the interaction between TSSMs and their host Lima bean (Phaseolus lunatus) by analyzing the metabolomics of leaf pigments and the transcriptomics of TSSM guanine production. We also used epifluorescence, confocal laser scanning, and transmission electron microscopies to study the morphology and structure of TSSMs and their excreta. Finally, we evaluated the potential photosynthetic ability of TSSMs and the activity and content of Ribulose-1,5-bisphosphate Carboxylase/Oxigenase (RubisCO). We found that TSSMs express several genes involved in guanine production, including Guanosine Monophosphate Synthetase (GMPS) and decoyinine (DCY), a potential inhibitor of GMPS, was found to reduce TSSMs proliferation in infested Lima bean leaves. Despite the presence of intact chloroplasts and chlorophyll in TSSMs, we demonstrate that TSSMs do not retain any photosynthetic activity. Our results show for the first time the transcriptomics of guanine production in TSSMs and provide new insight into the catabolic activity of TSSMs on leaf chlorophyll and carotenoids. Finally, we preliminary demonstrate that DCY has an acaricidal potential against TSSMs.

Transcriptomics and Metabolomics of Reactive Oxygen Species Modulation in Near-Null Magnetic Field-Induced Arabidopsis thaliana.

The geomagnetic field (GMF) is a natural component of Earth's biosphere. GMF reduction to near-null values (NNMF) induces gene expression modulation that generates biomolecular, morphological, and developmental changes. Here, we evaluate the effect of NNMF on gene expression and reactive oxygen species (ROS) production in time-course experiments on Arabidopsis thaliana. Plants exposed to NNMF in a triaxial Helmholtz coils system were sampled from 10 min to 96 h to evaluate differentially expressed genes (DEGs) of oxidative stress responses by gene microarray. In 24-96 h developing stages, H2O2 and polyphenols were also analyzed from roots and shoots. A total of 194 DEGs involved in oxidative reactions were selected, many of which showed a fold change ≥±2 in at least one timing point. Heatmap clustering showed DEGs both between roots/shoots and among the different time points. NNMF induced a lower H2O2 than GMF, in agreement with the expression of ROS-related genes. Forty-four polyphenols were identified, the content of which progressively decreased during NNMF exposition time. The comparison between polyphenols content and DEGs showed overlapping patterns. These results indicate that GMF reduction induces metabolomic and transcriptomic modulation of ROS-scavenging enzymes and H2O2 production in A. thaliana, which is paralleled by the regulation of antioxidant polyphenols.

Tackling the Future Pandemics: Broad-Spectrum Antiviral Agents (BSAAs) Based on A-Type Proanthocyanidins.

A-type proanthocyanidins (PAC-As) are plant-derived natural polyphenols that occur as oligomers or polymers of flavan-3-ol monomers, such as (+)-catechin and (-)-epicatechin, connected through an unusual double A linkage. PAC-As are present in leaves, seeds, flowers, bark, and fruits of many plants, and are thought to exert protective natural roles against microbial pathogens, insects, and herbivores. Consequently, when tested in isolation, PAC-As have shown several biological effects, through antioxidant, antibacterial, immunomodulatory, and antiviral activities. PAC-As have been observed in fact to inhibit replication of many different human viruses, and both enveloped and non-enveloped DNA and RNA viruses proved sensible to their inhibitory effect. Mechanistic studies revealed that PAC-As cause reduction of infectivity of viral particles they come in contact with, as a result of their propensity to interact with virion surface capsid proteins or envelope glycoproteins essential for viral attachment and entry. As viral infections and new virus outbreaks are a major public health concern, development of effective Broad-Spectrum Antiviral Agents (BSAAs) that can be rapidly deployable even against future emerging viruses is an urgent priority. This review summarizes the antiviral activities and mechanism of action of PAC-As, and their potential to be deployed as BSAAs against present and future viral infections.

Calcium Signaling in Plant-Insect Interactions.

In plant-insect interactions, calcium (Ca2+) variations are among the earliest events associated with the plant perception of biotic stress. Upon herbivory, Ca2+ waves travel long distances to transmit and convert the local signal to a systemic defense program. Reactive oxygen species (ROS), Ca2+ and electrical signaling are interlinked to form a network supporting rapid signal transmission, whereas the Ca2+ message is decoded and relayed by Ca2+-binding proteins (including calmodulin, Ca2+-dependent protein kinases, annexins and calcineurin B-like proteins). Monitoring the generation of Ca2+ signals at the whole plant or cell level and their long-distance propagation during biotic interactions requires innovative imaging techniques based on sensitive sensors and using genetically encoded indicators. This review summarizes the recent advances in Ca2+ signaling upon herbivory and reviews the most recent Ca2+ imaging techniques and methods.

Iron-sulfur complex assembly: Potential players of magnetic induction in plants.

Iron-sulfur (Fe-S) clusters are involved in fundamental biological reactions and represent a highly regulated process involving a complex sequence of mitochondrial, cytosolic and nuclear-catalyzed protein-protein interactions. Iron-sulfur complex assembly (ISCA) scaffold proteins are involved in Fe-S cluster biosynthesis, nitrogen and sulfur metabolism. ISCA proteins are involved in abiotic stress responses and in the pigeon they act as a magnetic sensor by forming a magnetosensor (MagS) complex with cryptochrome (Cry). MagR gene exists in the genomes of humans, plants, and microorganisms and the interaction between Cry and MagR is highly conserved. Owing to the extensive presence of ISCA proteins in plants and the occurrence of homology between animal and human MagR with at least four Arabidopsis ISCAs and several ISCAs from different plant species, we believe that a mechanism similar to pigeon magnetoperception might be present in plants. We suggest that plant ISCA proteins, homologous of the animal MagR, are good candidates and could contribute to a better understanding of plant magnetic induction. We thus urge more studies in this regard to fully uncover the plant molecular mechanisms underlying MagR/Cry mediated magnetic induction and the possible coupling between light and magnetic induction.

Hb Alessandria [ß37(C3)Trp→Leu; HBB: c.113G>T]: a Novel Variant on the ß-Globin Chain with Slightly Increased Affinity for Oxygen Detected by Capillary Electrophoresis.

We report a novel mutation on the ß-globin gene in a 68-year-old woman of Sicilian origin living in Alessandria, Italy. This mutation produces a hemoglobin (Hb) variant of Hb A that was detected by the capillary electrophoresis (CE) method during measurement of Hb A1c. The variant Hb did not separate from Hb A using different high performance liquid chromatography (HPLC) instruments. Direct DNA sequencing revealed a G>T transversion at codon 37 and subsequent substitution of a tryptophan residue for a leucine residue. The new Hb variant was named Hb Alessandria [ß37(C3)Trp→Leu; HBB: c.113G>T]. The p50 value was slightly decreased while the stability test at 37 °C in isopropyl alcohol and the main erythrocyte parameters were normal. Overall, the patient appeared clinically normal.

An increase in the membrane lipids recycling by PDAT overexpression stimulates the accumulation of triacylglycerol in Nannochloropsis gaditana.

Oleaginous microalgae represent potential feedstocks for the sustainable production of lipids thanks to their ability to accumulate triacylglycerols (TAGs). TAG accumulation in several algal species is strongly induced under specific conditions such as nutrient deprivation and high light which, however, also negatively impact growth. Genetic modification of lipogenic pathways can potentially enhance TAG accumulation without negatively affecting growth, avoiding the trade-off between biomass and lipid productivity. In this study, the phospholipid: diacylglycerol acyltransferase (PDAT), an enzyme involved in membrane lipid recycling, was overexpressed in the seawater alga Nannochloropsis gaditana. PDAT overexpression induced increased TAG content in actively growing algae cultures while no effects were observed in conditions naturally stimulating strong lipid accumulation such as high light and nitrogen starvation. The increase of TAG content was confirmed also in a strain cultivated in industrially relevant conditions even though PDAT overexpression, if too strong, the gene overexpression becomes detrimental for growth in the longer term. Results overall suggest that genetic modulation of the PDAT gene represents a promising strategy to increase microalgae lipid content by minimizing negative effects on biomass productivity.

Metabolomics-Based Profiling, Antioxidant Power, and Uropathogenic Bacterial Anti-Adhesion Activity of SP4TM, a Formulation with a High Content of Type-A Proanthocyanidins.

Flavonoids and proanthocyanidins (PACs) have been the subject of intense scientific investigations, both for their antioxidant properties and anti-adhesion activity against uropathogenic bacteria. We investigated the metabolomics and antioxidant capacity of SP4TM, a patent-pending formulation based on a mixture of plant extracts with a high content of bioactive PACs and other polyphenols. The total content of polyphenols (885.51 ± 14.19 mg/g), flavonoids (135.52 ± 8.98 mg/g), anthocyanins (54.84 ± 2.97 mg/g), and PACs (379.43 ± 12.44 mg/g) was quantified using UV-Vis assays. Use of HPLC-ESI-MS/MS revealed the presence of 5 flavanols (100.77 ± 3.90 mg g-1 d.wt), 11 flavonols (59.96 ± 1.83 mg g-1 d.wt), and 8 anthocyanins (46.96 ± 1.59 mg g-1 d.wt), whereas MALDI-TOF MS showed that SP4TM contains PACs with one or more type-A interflavan bonds at each degree of polymerization. Regarding antioxidant properties, LUCS technology on HepG2 cells evidenced the ability of SP4TM to neutralize intracellular free radicals, inhibit membrane lipid peroxidation, quench H2O2, and reduce free radicals mainly through chelating mechanism, as demonstrated by a higher FRAP value (2643.28 ± 39.86 mmol/g) compared with ABTS (139.92 ± 6.16 mmol/g) and DPPH (89.51 ± 3.91 mmol/g). Finally, the SP4TM type-A PAC content strongly prevented bacterial adhesion of P-fimbriated uropathogenic Escherichia coli (0.23 mg/mL). In conclusion, SP4TM has a strong antioxidant capacity involving multitarget mechanisms and is a potential supplement to fight urinary tract infections due to its ability to inhibit uropathogenic E. coli adhesion.

Astaxanthin and eicosapentaenoic acid production by S4, a new mutant strain of Nannochloropsis gaditana.

BACKGROUND: Astaxanthin is a ketocarotenoid with high antioxidant power used in different fields as healthcare, food/feed supplementation and as pigmenting agent in aquaculture. Primary producers of astaxanthin are some species of microalgae, unicellular photosynthetic organisms, as Haematococcus lacustris. Astaxanthin production by cultivation of Haematococcus lacustris is costly due to low biomass productivity, high risk of contamination and the requirement of downstream extraction processes, causing an extremely high price on the market. Some microalgae species are also primary producers of omega-3 fatty acids, essential nutrients for humans, being related to cardiovascular wellness, and required for visual and cognitive development. One of the main well-known producers of omega-3 fatty eicosapentaenoic acid (EPA) is the marine microalga Nannochloropsis gaditana (named also Microchloropsis gaditana): this species has been already approved by the Food and Drug Administration (FDA) for human consumption and it is characterized by a fast grow phenotype. RESULTS: Here we obtained by chemical mutagenesis a Nannochloropsis gaditana mutant strain, called S4, characterized by increased carotenoid to chlorophyll ratio. S4 strain showed improved photosynthetic activity, increased lipid productivity and increased ketocarotenoids accumulation, producing not only canthaxanthin but also astaxanthin, usually found only in traces in the WT strain. Ketocarotenoids produced in S4 strain were extractible in different organic solvents, with the highest efficiency observed upon microwaves pre-treatment followed by methanol extraction. By cultivation of S4 strain at different irradiances it was possible to produce up to 1.3 and 5.2 mgL-1 day-1 of ketocarotenoids and EPA respectively, in a single cultivation phase, even in absence of stressing conditions. Genome sequencing of S4 strain allowed to identify 199 single nucleotide polymorphisms (SNP): among the mutated genes, mutations in a carotenoid oxygenase gene and in a glutamate synthase gene could explain the different carotenoids content and the lower chlorophylls content, respectively. CONCLUSIONS: By chemical mutagenesis and selection of strain with increased carotenoids to chlorophyll ratio it was possible to isolate a new Nannochloropsis gaditana strain, called S4 strain, characterized by increased lipids and ketocarotenoids accumulation. S4 strain can thus be considered as novel platform for ketocarotenoids and EPA production for different industrial applications.