Class Of Metabolites Research Articles

Advances in the Study of Halogenated Natural Products.

Halogenated natural products are an important class of secondary metabolites that are widely distributed in nature. The presence of halogen atoms usually enhances the pharmacological activity of the compounds. As a result, halogenated natural products have shown promising pharmacological activities in antibacterial, antitumour, anti-inflammatory and antiplasmodial properties, providing a rich resource for the development of new drugs. To date, more than 62% of halogenated compounds are produced by marine organisms, mainly including marine sponges, algae, corals, fungi and other organisms. In addition, terrestrial microorganisms, including bacteria and fungi, also produce halogenated metabolites, which are equally important sources of halogenated natural products. The biosynthesis of halogenated natural products involves the synergistic action of multiple enzymes that efficiently and selectively bind halogen atoms to organic molecules, a process that enhances the biological activity of the compounds. Halogenated natural products have a wide range of uses as important raw materials in the agricultural, pharmaceutical and chemical industries. This paper reviews the progress of research on halogenated compounds and their biosynthesis in recent years, laying the foundation for further utilisation and development of halogenated compounds.

Metabolic Blockade-Based Genome Mining of Malbranchea circinata SDU050: Discovery of Diverse Secondary Metabolites.

Malbranchea circinata SDU050, a fungus derived from deep-sea sediment, is a prolific producer of diverse secondary metabolites. Genome sequencing revealed the presence of at least 69 biosynthetic gene clusters (BGCs), including 30 encoding type I polyketide synthases (PKSs). This study reports the isolation and identification of four classes of secondary metabolites from wild-type M. circinata SDU050, alongside five additional metabolite classes, including three novel cytochalasins (7-9), obtained from a mutant strain through the metabolic blockade strategy. Furthermore, bioinformatic analysis of the BGC associated with the isocoumarin sclerin (1) enabled the deduction of its biosynthetic pathway based on gene function predictions. Bioactivity assays demonstrated that sclerin (1) and (-)-mycousnine (10) exhibited weak antibacterial activity against Gram-positive bacteria such as Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), and Bacillus subtilis. These findings underscore the chemical diversity and biosynthetic potential of M. circinata SDU050 and highlight an effective strategy for exploring marine fungal metabolites.

Effects of Echinacea purpurea and Alkylamides on Respiratory Virus Replication and IL-8 Expression In Vitro.

Echinacea purpurea is a perennial medicinal herb with important immunomodulatory and anti-inflammatory properties, especially purported for the alleviation of cold and flu symptoms. Different classes of secondary metabolites of the plant, such as alkylamides, caffeic acid derivatives, polysaccharides, flavonoids, and glycoproteins, are believed to be biologically and pharmacologically active. Although previous research suggests that the alkylamides present in Echinacea may be responsible for reducing the symptoms associated with the common cold or flu through their immunomodulatory activity, the roles of specific alkylamides and their targets (i.e., immune and/or antiviral) have not been well-elucidated or established. This study tested the antiviral and cytokine regulatory activity of various specific alkylamides that are present predominantly in Echinacea root extracts and found that one specific alkylamide, Dodeca-2E,4E-Dienoic acid isobutylamide, had potent antiviral activity against rhinovirus (the causative agent of most common colds) and influenza virus, as well as potent inhibition of IL-8 cytokine production. IL-8 is responsible for many of the symptoms associated with the common cold and is upregulated in other common respiratory infections. The broad activity and low cytotoxicity of this specific alkylamide support its potential use for treating rhinovirus and influenza virus infections.

Strategic Acyl Carrier Protein Engineering Enables Functional Type II Polyketide Synthase Reconstitution In Vitro.

Microbial polyketides represent a structurally diverse class of secondary metabolites with medicinally relevant properties. Aromatic polyketides are produced by type II polyketide synthase (PKS) systems, each minimally composed of a ketosynthase-chain length factor (KS-CLF) and a phosphopantetheinylated acyl carrier protein (holo-ACP). Although type II PKSs are found throughout the bacterial kingdom, and despite their importance to strategic bioengineering, type II PKSs have not been well-studied in vitro. In cases where the KS-CLF can be accessed via E. coli heterologous expression, often the cognate ACPs are not activatable by the broad specificity Bacillus subtilis surfactin-producing phosphopantetheinyl transferase (PPTase) Sfp and, conversely, in systems where the ACP can be activated by Sfp, the corresponding KS-CLF is typically not readily obtained. Here, we report the high-yield heterologous expression of both cyanobacterial Gloeocapsa sp. PCC 7428 minimal type II PKS (gloPKS) components in E. coli, which allowed us to study this minimal type II PKS in vitro. Initially, neither the cognate PPTase nor Sfp converted gloACP to its active holo state. However, by examining sequence differences between Sfp-compatible and -incompatible ACPs, we identified two conserved residues in gloACP that, when mutated, enabled high-yield phosphopantetheinylation of gloACP by Sfp. Using analogous mutations, other previously Sfp-incompatible type II PKS ACPs from different bacterial phyla were also rendered activatable by Sfp. This demonstrates the generalizability of our approach and breaks down a longstanding barrier to type II PKS studies and the exploration of complex biosynthetic pathways.

The Entry of Pollinating Fig Wasps Plays a Pivotal Role in the Developmental Phase and Metabolic Expression Changes in Ficus hookeriana Figs

The fig (the syconium of the Ficus tree) and its pollinating fig wasp represent exceptional examples for researching plant–insect interactions due to their remarkable specificity in species interaction and mutually beneficial symbiotic relationship. However, the mechanisms underlying the developmental process of monoecious figs in response to the entry of pollinating fig wasps (pollinators) and the metabolic changes occurring during this process remain elusive. Our study employed a combination of controlled experiments in the field and LC-MS methods to investigate the impact of pollinating fig wasp entry on the developmental phase of figs, as well as the metabolic alterations occurring during this process. A total of 381 metabolites and 155 differential metabolites were identified, with the predominant classes of metabolites being organic acids, lipids, and benzene aromatic compounds. The results suggest that in the absence of wasp entry, the receptive phase of fig would exhibit an extended duration. However, upon the entry of fig wasps, the receptive phase of figs would terminate within a span of 1 to 2 days, concomitant with substantial fluctuations in the composition and proportions of metabolites within the fig. Our research focuses on the analysis of linoleic acid metabolism, phenylpropanoid biosynthesis, and flavonoid biosynthesis pathways. Our findings suggest that the entry of wasps triggers alterations in the metabolic regulatory mechanisms of figs. Prior to wasp entry, metabolites primarily regulate fig growth and development. However, after wasp entry, metabolites predominantly govern lipid accumulation and the establishment of defense mechanisms, indicating a transition in fig development. This metabolic perspective explains why figs promptly enter an interflower phase that is not attractive to pollinating fig wasps after their entry, and how figs achieve reproductive balance through the regulation of different metabolic pathways. This study provides scientific evidence for elucidating the stability mechanism of the fig wasp mutualistic system.

Hemical Profile And Allelopathic Potential of Asparagus setaceus (KUNTH) Jessop (ASPARAGACEAE)

Objective: This work aimed to elucidate the chemical profile and the allelopathic potential of the ethanolic extract of Asparagus setaceus leaves collected in the morning (8 A.M) and afternoon (2 P.M). Method: The extract was obtained using cold ethanol and the secondary metabolites classes were identified with specific reagents. For allelopathic activity, the test solution was obtained from 100 mg of ethanolic extract, dissolved in 100 mL of 66% ethanol, obtaining concentrations from 6.25% to 100%. Two control groups were used: distilled water and 66% ethanol alcohol in the evaluation of the following parameters: germination percentage (GP), germination speed index (GSI) and stem and radicle length. Results and Discussion: The tabulated results were analyzed by two-way ANOVA, through analysis of variance and Tukey's test. The pH values of Asparagus setaceus ethanolic extract in the morning ranged from 9 to 12, for Asparagus setaceus ethanolic extract in the afternoon ranged from 8 to 12. In the phytochemical analysis, the morning extract showed a greater number of metabolites when compared to the same extract prepared in the afternoon, causing greater negative interference observed in the germination speed index and in the development of seedlings. Research Implications: Therefore, the combined action of these compounds and factors such as humidity, temperature and soil pattern should be considered when evaluating the different results found, and further studies are needed to investigate the specific constituents of allelopathic activity.

Untargeted Characterization and Biological Activity of Amazonian Aqueous Stem Bark Extracts by Liquid and Gas Chromatography-Mass Spectrometry.

Aqueous stem bark extracts of Aspidosperma rigidum Rusby, Couroupita guianensis Aubl., Monteverdia laevis (Reissek) Biral, and Protium sagotianum Marchand have been reported as traditional remedies in several countries of the Amazonian region. Despite previous research, further investigation to characterize secondary metabolites and the biological activity of extracts is needed to derive potential applications. Metabolic profiling was carried out using liquid and gas chromatography coupled with mass spectrometry (UHPLC-MS/MS and GC-MS). The chemical composition of the studied plants was further compared by principal component analysis (PCA). Additionally, chemical profiles were correlated with antimicrobial and toxicity activities, which suggested potential metabolites for future research. We identified 16 and 32 compounds by UHPLC-MS/MS and GC-MS analysis, respectively. Antimicrobial activity was detected in three stem bark extracts. C. guianensis showed inhibition of all tested microorganisms, including antibiotic-resistant strains. Molecular networking approaches, in silico tools, and Pearson's correlation showed that antifungal compounds could be a terpene glycoside (r = 0.918) and/or a phenolic (r = 0.882) metabolite class. This study highlights the use of the established procedure in exploring the metabolomes of these species, which could be a novel source of antimicrobial drug discovery. Coupling the observed biological potential with UHPLC-MS/MS data has also accelerated the tracing of their bioactive compounds. These findings update the state of the art regarding the chemical composition and biological activity of the plant extracts, defining potential new applications for the pharmaceutical applications.

ITASO: A Novel Photosensitive Probe for High-Throughput and Selective Submetabolomic Analysis via Flow Injection-Mass Spectrometry.

Flow injection mass spectrometry (FI-MS) is widely employed for high-throughput metabolome analysis, yet the absence of prior separation leads to significant matrix effects, thereby limiting the metabolome coverage. In this study, we introduce a novel photosensitive MS probe, iTASO-ONH2, integrated with FI-MS to establish a high-throughput strategy for submetabolome analyses. The iTASO probe features a conjugated-imino sulfonate moiety for efficient photolysis under 365 nm irradiation and a reactive group for selective metabolite labeling. The iTASO-ONH2 probe effectively and selectively labels carbonyl compounds, forming highly stable labeled products. Upon UV exposure, the labeled products rapidly release sulfonic acid-containing photolysis products, detectable with high sensitivity in ESI-negative mode and low matrix effect, offering femtomole-level detection sensitivity. The iTASO-ONH2-based FI-MS strategy was applied to fecal samples from chronic sleep-deprived and control mice, revealing 192 potential carbonyl compounds of which 37 exhibited significant alterations. Additionally, three other photosensitive probes─iTASO-NH2, iTASO-NHS, and iTASO-MAL─were synthesized to selectively label carboxyl, amino, and thiol metabolites, respectively, underscoring the versatility of the iTASO-based FI-MS strategy for submetabolomic analysis across diverse metabolite classes.

Metabolic profiling of Perilla leaves of different colors: insights into metabolite variation and bioactive compound distribution

BackgroundPerilla frutescens (L.) Britt. (Lamiaceae) leaves are essential culinary and medicinal herbs, native to East Asian countries. Three types of Perilla leaves, including green, green/purple and purple, are mainly found, and the leaf color is a critical attribute for consumer preferences. However, the extent of diversity and variability of metabolites in Perilla leaves of different colors remain elusive. Thus, in this study, we applied LC-QqQ-MS (liquid chromatography triple quadrupole tandem mass spectrometry)-based widely targeted metabolic profiling to investigate variation in the metabolite profiles of green (PF1), green/purple (PF2) and purple (PF3) Perilla leaves.ResultsWe qualitatively and quantitatively identified a total of 1239 metabolites in Perilla leaves of different colors. Amino acids and derivatives (15.50%), flavonoids (14.77%), phenolic acids (12.19%), lipids (9.60%), carbohydrates and derivatives (8.47%), organic acids and derivatives (7.99%), nucleotides and derivatives (7.99%), and terpenoids (5.25%) were the major classes of metabolites in perilla leaves. In contrast to green leaves, the principal component analysis and correlation analysis revealed a considerable influence of genotypes for the variation in purple leaves’ metabolite profiles. Differentially accumulated metabolites (DAMs) analysis revealed that flavonoids, phenolic acids, and amino acids and derivatives were the major DAMs, and the phenylpropanoid pathway was the most differentially regulated. All DAMs, including four potential metabolic discriminative biomarkers, were screened out. In addition, we revealed the accumulation patterns of bioactive compounds in the leaves of different colors. It was noteworthy that most of the differentially accumulated flavonoids showed a higher relative content in PF3, followed by PF2 and PF1. Glycosylated cyanidins and delphinidins were identified as the key compounds responsible for the purple coloration of leaves. Finally, we found that the variation in glutamate metabolism may be one of the main contributors for variation in metabolite profiles and biological attributes of Perilla leaves of different colors.ConclusionOur results showed that the metabolic processes, mainly phenylpropanoid biosynthesis and amino acid metabolism, are differently regulated in green, green/purple and purple Perilla leaves. Furthermore, they offer valuable data for a comprehensive use of Perilla leaves and exploring gene-metabolites interactions in Perilla leaves.

Ajwa date extract (Phoenix dactylifera L.): Phytochemical analysis, antiviral activity against herpes simplex virus-I and coxsackie B4 virus, and in silico study.

To investigate the phytochemical composition of Ajwa date extract and evaluate its antiviral activity and mechanism of action. High perfomance liquid chromatography, gas chromatography-mass spectrometry, and liquid chromatography-mass spectrometry were used to analyze the phytochemical profile of Ajwa date extract. The antiviral activity was assessed using the MTT colorimetric assay against herpes simplex virus type I (HSV-I) and coxsackievirus B4 (CVB-4). Assessment of the mechanism of action against HSV-I was carried out using 3 protocols. Molecular docking and quantum chemical calculations were carried out to predict the binding affinities of the identified compounds to viral glycoprotein D. A total of 17 metabolites belonging to different classes of metabolites, mainly flavonoids, phenolic acid derivatives, fatty acids, and sugar derivatives. Ajwa extract exhibited antiviral activity against HSV-I with an IC: 50 of 113.99±4.67 μg/mL, whereas it showed limited activity against CVB-4. The antiviral activity of Ajwa extract was mainly attributed to its cell protectant activity by preventing adherence of viral to host cell with an IC: 50 equal to 57.82±1.37μg/mL. Molecular docking studies indicated that chlorogenic acid had the strongest binding affinity to viral glycoprotein D, which suggests its potential role in inhibiting viral entry into host cells. The Ajwa date extract demonstrated promising antiviral activity, especially against HSV-I. Integrating in vitro and in silico analyses provided valuable insights into the mechanisms of action.

Antiproliferative Activity of New Bis-Thionaphthoquinones in Cervical Cancer

Over the past decades, natural products have been a primary source of anticancer agents. Naphthoquinones, a large class of secondary metabolites, exhibit known anticancer activity. Thionaphthoquinones, which are naphthoquinones with added thiol groups, have demonstrated similar potential to their precursors. Cervical cancer is the third most common and fourth most lethal type of cancer among women worldwide. This study aimed to investigate the antiproliferative activity of new bis-thionaphthoquinones in cervical cancer cells. Five new molecules (D1-D5) were synthesized via multicomponent reaction and characterized by mass spectrometry, infrared spectroscopy, and nuclear magnetic resonance. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to evaluate antiproliferative activity in HeLa and NIH3T3 cells. Flow cytometry was employed to analyze deoxyribonucleic acid (DNA) fragmentation. PharmMapper was utilized for target fishing. Among the tested molecules, bis-thionaphthoquinone D4 exhibited the highest potency and selectivity, with a half maximal inhibitory concentration (IC50) of 42.7 μM and a selectivity index of 2.05. Additionally, it also induced DNA fragmentation, suggesting its potential to induce cell death. Carbonic anhydrase II was identified as the most likely target for D4. Therefore, bis-thionaphthoquinone D4 demonstrated both potency and selectivity, making it a promising drug candidate for the treatment of cervical cancer.

A novel approach for the synthesis of the cyclic lipopeptide globomycin.

Cyclic lipopeptides (CLiPs) are a highly diverse class of secondary metabolites produced by bacteria and fungi. Examples of CLiPs have been found that possess potent antimicrobial activity against multidrug-resistant Gram-negative bacteria. Globomycin is a 19-membered CLiP that kills both Gram-positive and Gram-negative bacteria through inhibition of lipoprotein signal peptidase II (Lsp). It can only be obtained in small quantities from its Streptomyces producer strain, so there has been much interest in development of synthetic methods to access globomycin and analogues. Globomycin contains an N-terminal anti-α-methyl-β-hydroxy nonanoyl lipid tail, whose hydroxyl group forms an ester with the C-terminal carboxylate. Constructing the anti-arrangement between the α-methyl and β-hydroxy is synthetically challenging and previous globomycin syntheses are not compatible with diversification of the lipid tail after the stereocenters have been installed. Herein, we describe a new approach for the synthesis of globomycin that allows for facile lipid diversification. Using an anti-Evans Aldol condensation, a common intermediate is obtained that allows different "lipid swapping" through Grubbs-catalyzed cross-metathesis. Upon auxiliary cleavage, the resulting lipid can then be utilized in solid-phase peptide synthesis. Given the plethora of lipopeptides that contain β-hydroxy lipids, this method offers a convenient approach for convergent generation of lipopeptide analogues.

Solvent Selection in the Quality Analysis of White Tea by Nuclear Magnetic Resonance Spectroscopy

The study aimed to assess the influence of diverse solvent schemes on the qualitative analysis of white tea and to establish a more comprehensive quality evaluation. A systematic study of solvent optimization was carried out using the quality analysis of white tea as an exemplar, highlighting the critical role of selecting appropriate solvents in NMR-based sample preparation process. Six sample dissolution protocols, encompassing extraction and reconstitution solvents, were evaluated. The aqueous methanol solution proved effective as an extraction solvent, offering optimal metabolite coverage and spectral resolution. For reconstitution solvents, D2O facilitated identification of specific metabolites, such as amino acids, organic acids and carbohydrates, while DMSO-d6 exhibited better performance in detecting catechins, alkaloids and lipid resonances. Additionally, DMSO-d6 enabled better discrimination among various white tea varieties, whereas D2O system demonstrated better ability for estimating the storage duration of white tea. The characteristic metabolites were exactly those that each of the two solvents is suited to solubilize. Therefore, it can be concluded that solvents suitable for the detection of a class of metabolites are more likely to highlight differences in that type of metabolites and multi-solvent combination captures more comprehensive information, revealing the necessity of optimizing solvent protocols for different assay targets.

HILIC-MS/MS Multi-targeted Method for Metabolomics Applications.

Metabolomics aims at identification and quantitation of key end point metabolites, basically polar, in order to study changes in biochemical activities in response to pathophysiological stimuli or genetic modifications. Targeted profiling assays enjoying a growing popularity over the last years with LC-MS/MS as a powerful tool for development of such (semi-)quantitative methods for a large number of metabolites. Here we describe a method for absolute quantitation of ca. 100 metabolites belonging to key metabolite classes such as sugars, amino acids, nucleotides, organic acids, and amines with a hydrophilic interaction liquid chromatography (HILIC) system comprised with ultra (high) performance liquid chromatography (UHPLC) with detection on a triple quadrupole mass spectrometer operating in both positive and negative modes.

Bio-guided isolation and bioinformatic studies of cytotoxic phytosterols from Acanthospermum hispidum DC against breast (MCF7) and colorectal (HT29) cancer cells

Acanthospermum hispidum DC (Asteraceae), a traditional medicinal plant, plays a role as an alternative remedy for various diseases, such as bacterial and viral infections, jaundice, malaria, fever, gastrointestinal disorders, headache, convulsions, and snake bites. Flavonoids, alkaloids, saponins, tannins, terpenes, steroids, and cardioactive glycosides are the distinct classes of metabolites in the plant. Although A. hispidum was suggested as a promising antitumor phytomedicine, no studies identified its potential cytotoxic components. In this study, the cytotoxic compounds of A. hispidum were isolated using chromatographic techniques guided by in vitro MTT cytotoxicity assay against selected cancer cell lines; breast cancer (MCF7), colorectal adenocarcinoma (HT29), and hepatoblastoma (HepG2). The selective index (SI) was assessed on MRC5 (Normal human fetal lung fibroblast) cell line. The dichloromethane fraction (DCM) showed remarkable cytotoxic activity against MCF7 and HT29 (%Cell viability; 46.15 and 60.5, respectively). Hence, the main bioactive fraction from DCM was purified to afford two phytosterols; stigmasterol (1) and β-sitosterol (2), which were identified by 1-D and 2-D NMR spectroscopy. Cytotoxic evaluation of 1 and 2 revealed that β-sitosterol showed better selective cytotoxicity against MCF7 and HT29 (IC50 4.07 μg/mL, SI 2.63; IC50 4.52 μg/mL, SI 2.37) compared to stigmasterol (IC50 5.43 μg/mL, SI 1.38; IC50 4.21 μg/mL, SI 1.78), respectively. Bioinformatic assessments of drug-likeness and ADMET properties demonstrated that most criteria were obeyed by the investigated compounds except for their poor solubility, which recommended the preparation of special dosage forms, such as nanoformulation to enhance their oral bioavailability. Swiss Target prediction indicated that nuclear receptors represent the main target class (40%). Whereas caspase-3 stimulant activity (a key enzyme in apoptosis) was predicted by the PASS prediction tool as a potential anticancer mechanism. Our study suggests A. hispidum as a potential source of bioactive phytosterols and as a chemopreventive medicinal plant.

Localization of Secondary Metabolites in Relict Gymnosperms of the Genus Sequoia In Vivo and in Cell Cultures In Vitro, and the Biological Activity of Their Extracts.

In order to scientifically search for new sources of secondary metabolites with valuable qualities for phytopharmacognosy, tasks requiring a step-by-step solution were set. The primary task is the development of technologies for obtaining in vitro highly productive biomass of cells of relict gymnosperms of the genus Sequoia, capable of accumulating various classes of secondary metabolites. The study of the accumulation and localization of secondary metabolites allowed us to evaluate the biological activity and cytotoxicity of in vitro Sequoia cultures. In our study, histochemical methods were used to determine the localization of secondary compounds (phenolic and terpenoid in nature) in plant tissues. Secondary metabolites-polyphenols, catechins, and terpenoids-are mainly localized in the epidermal, parenchymal, and conductive tissues of Sequoia leaves and stems. In callus and suspension cultures of Sequoia, secondary metabolites were localized in cell walls and vacuoles. The mineral composition of the nutrient medium (MS and WPM), the light source (photoperiod), and the endogenous content of polyphenols in the primary explant influenced the initiation and growth characteristics of the in vitro culture of Sequoia plants. Inhibition of growth in suspension cultures on the WPM nutrient medium was noted. The cultivation of Sequoia cell lines at a 16 h photoperiod stimulated the formation of polyphenols but had a negative effect on the growth of callus cultures. Extractive substances obtained from intact and callus tissues of evergreen Sequoia demonstrate high biological (fungicidal) activity and cytotoxicity. The inhibitory effect on Fusarium oxisporum was noted when 200 mg/L of Sequoia extract was added to the nutrient medium. Extracts of redwood callus cultures were low in toxicity to normal FetMSC cells but inhibited the growth of lines of "immortal" cervical HeLa cancer cells and human glioblastoma A172. Intact tissues of Sequoia plants and cell cultures initiated from them in vitro are producers of secondary metabolites with high biological activity.

DNEA: an R package for fast and versatile data-driven network analysis of metabolomics data

BackgroundMetabolomics is a high-throughput technology that measures small molecule metabolites in cells, tissues or biofluids. Analysis of metabolomics data is a multi-step process that involves data processing, quality control and normalization, followed by statistical and bioinformatics analysis. The latter step often involves pathway analysis to aid biological interpretation of the data. This approach is limited to endogenous metabolites that can be readily mapped to metabolic pathways. An alternative to pathway analysis that can be used for any classes of metabolites, including unknown compounds that are ubiquitous in untargeted metabolomics data, involves defining metabolite-metabolite interactions using experimental data. Our group has developed several network-based methods that use partial correlations of experimentally determined metabolite measurements. These were implemented in CorrelationCalculator and Filigree, two software tools for the analysis of metabolomics data we developed previously. The latter tool implements the Differential Network Enrichment Analysis (DNEA) algorithm. This analysis is useful for building differential networks from metabolomics data containing two experimental groups and identifying differentially enriched metabolic modules. While Filigree is a user-friendly tool, it has certain limitations when used for the analysis of large-scale metabolomics datasets.ResultsWe developed the DNEA R package for the data-driven network analysis of metabolomics data. We present the DNEA workflow and functionality, algorithm enhancements implemented with respect to the package’s predecessor, Filigree, and discuss best practices for analyses. We tested the performance of the DNEA R package and illustrated its features using publicly available metabolomics data from the environmental determinants of diabetes in the young. To our knowledge, this package is the only publicly available tool designed for the construction of biological networks and subsequent enrichment testing for datasets containing exogenous, secondary, and unknown compounds. This greatly expands the scope of traditional enrichment analysis tools that can be used to analyze a relatively small set of well-annotated metabolites.ConclusionsThe DNEA R package is a more flexible and powerful implementation of our previously published software tool, Filigree. The modular structure of the package, along with the parallel processing framework built into the most computationally extensive steps of the algorithm, make it a powerful tool for the analysis of large and complex metabolomics datasets.

The phospho-ferrozine assay: a tool to study bacterial redox-active metabolites produced at the plant root.

Soil microbial communities are pivotal to plant health and nutrient acquisition. It is becoming increasingly clear that many interactions, both among and between microbes and plants, are governed by small bioactive molecules or "secondary metabolites" that can aid in communication, competition, and nutrient uptake. Yet, secondary metabolite biogeography - who makes what, where, and why-is in its infancy. Further, secondary metabolite biosynthesis genes are often silent or weakly expressed under standard laboratory conditions, making it incredibly difficult to study these small molecules. To begin to address these dual challenges, we focused on redox-active metabolites (RAMs), a specific class of small molecules, and took advantage of recent findings that many RAMs aid in acquiring phosphorus and that their production is frequently stimulated by stress for this macronutrient. We developed a screen for RAM-producing bacteria that leverages phosphorus limitation to stimulate metabolite biosynthesis and uses a colorimetric (ferrozine) iron-reduction assay to identify redox activity. We isolated 557 root-associated bacteria from grasses collected at sites across the United States (Santa Rita Experimental Range [AZ], Konza Prairie Biological Station [KS], and Harvard Forest [MA]) and from commercial tomato plants and screened them for RAM production. We identified 128 soil isolates of at least 19 genera across Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes that produced RAMs under phosphorus stress. Our work reveals that the production of RAMs under phosphorus stress is common across diverse soil bacteria and provides an approach to screen for these small molecules rapidly.IMPORTANCEBy secreting secondary metabolites, bacteria at the plant root can defend against diseases and help acquire essential nutrients. However, the genes that synthesize secondary metabolites are typically inactive or are weakly expressed under standard laboratory conditions. This fact makes it difficult to study these small molecules and hinders the discovery of novel small molecules that may play crucial roles in agricultural and biomedical settings. Here, we focus on redox-active metabolites (RAMs), a class of secondary metabolites that can help bacteria solubilize phosphorus and are often produced when phosphorus is limited. We developed a screen that rapidly identifies RAM-producing bacteria by utilizing a colorimetric iron-reduction assay in combination with phosphorus limitation to stimulate biosynthesis. The screen reveals that RAM-producing bacteria are far more prevalent in soil than previously appreciated and that this approach can be used to identify RAM producers.

Integrated Transcriptomic and Metabolomic Analysis Revealed Regulatory Mechanisms on Flavonoids Biosynthesis in the Skin of Passion Fruit (Passiflora spp.).

Passion fruit is one of the most famous fruit crops in tropical and subtropical regions due to its high edible, medicinal, and ornamental value. Flavonoids, a class of plant secondary metabolites, have important health-related roles. In this study, a total of 151 flavonoid metabolites were identified, of which 25 key metabolites may be the main contributors to the purple phenotype. Using RNA sequencing, 11,180 differentially expressed genes (DEGs) were identified. Among these, 48 flavonoid biosynthesis genes (PAL, 4CL, C4H, CHS, CHI, F3H, DFR, ANS, and UFGT) and 123 transcription factors were identified. Furthermore, 12 distinct modules were identified through weighted gene coexpression network analysis, of which the brown module displays a robust positive correlation with numerous flavonoid metabolites. Overexpression of PeMYB114 significantly promoted flavonoids accumulation in tobacco leaves. Our study provided a key candidate gene for molecular breeding to improve color traits in passion fruit.

Functional and Genomic Insights into the Biotechnological Potential of Vibrio spp. Isolated from Deeply Polluted and Pristine Environments.

Vibrio spp. are remarkably diverse bacteria, being worthy of investigation not only for their antibiotic resistance and virulence, but also for their biotechnological potential. Indeed, there is increasing evidence that these bacteria display industrially relevant traits, particularly as producers of antimicrobial substances, tensioactive/emulsifying compounds, and enzymes. Here, our aim was to investigate the potential of Vibrio strains isolated from two different marine sources to produce such biotechnologically applicable substances. From the eighteen analyzed strains, five were isolated from plastic particles from a heavily polluted urban estuary and 13 from calcareous sponges inhabiting submarine caves in an isolated volcanic archipelago in the Atlantic Ocean. Enzymatic screening revealed that most strains were agarolytic and cellulolytic. Overall, six strains showed antimicrobial activity against Staphylococcus aureus ATCC 29,213, with four of them active towards Escherichia coli ATCC 25,922 as well. Additionally, eight strains were positive for the production of bioemulsifiers. Genomic analyses of four strains further revealed insights regarding the enzymatic arsenal, as shown by the detection of several key gene clusters pertaining to the chitin degradation pathway, and also encoding diverse classes of antimicrobial-active metabolites. Our findings highlight the biotechnological potential of Vibrio spp., evidencing their functional diversity and the need for continued and sustained prospecting of this bacterial genus to uncover its potential high-value-added bioproducts.