Citrus fruits are valued not only for their nutritional benefits but also for their rich phytochemical content. Metabolomics has emerged as a comprehensive technique for assessing the chemical composition of fruits. The botanical connection between leaves, flowers, and fruits is reflected in both their structure and chemical composition, particularly in the flow of nutrients between plant organs. We introduced a new logarithm ratio-based approach to compare metabolite profiles between fruits and leaves. We hypothesize that this method allows for the analysis of multiple citrus metabolomic profiles to reveal known and novel correlation patterns, reflecting the dynamic connections between metabolic sources. To test this hypothesis, we leveraged comprehensive leaf metabolomic profiles from over 200 accessions in the Givaudan Citrus Variety Collection and reviewed published metabolomics data for fruits and juices of matching citrus types. By employing logarithm-transformed metabolic ratios within each dataset, we accounted for systematic differences across metabolomic platforms, achieving an unbiased analysis.

Long bean (Vigna unguiculata subsp. sesquipedalis, asparagus bean, Asian yardlong bean), the vegetable type of cowpea [Vigna unguiculata (L.) Walp], is a climate-resilient and nutritious food legume grown by Southeast Asian farmers in the Central Valley of California and marketed to Asian immigrant communities across the United States. Insect pests are major threats, reducing yield and quality of all current lines. Modern plant breeding protocols and extension activities were implemented to develop resistant lines using sources of natural resistance found in African cowpea germplasm. Three aphid-resistant long bean lines, Dark Green 1994 (Reg no. GP-320, PI 702995), Light Green 2055 (Reg. no. GP-321, PI 702996), and Purple 2056 (Reg. no. GP-322, PI 702997), were developed by introgression of two known quantitative trait loci (QTL) for aphid resistance into three local elite lines through marker-assisted backcrossing (MABC). One bush-type long bean line, Bush 2074 (Reg. no. GP-319, PI 702994), carrying two known QTL for root-knot nematode resistance, was also developed to enable scaling up production and to improve nematode management in large-scale commercial farming. These improved lines were evaluated in controlled experiments which also served in outreach activities to enable adoption. Each of these advanced lines, when forming a near-isogenic pair with its recurrent parent, can provide useful genetic materials for resistance gene discovery. The aphid-resistant long bean lines can be substituted for current local lines to minimize insecticide use. The nematode-resistant long bean line can serve as climate-smart green beans used in mechanical harvest systems. The long bean near-isogenic lines and founder parents could be useful materials for resistance gene discovery.

Cholesterol is an essential membrane structural component and steroid hormone precursor, and is involved in numerous signaling processes. Astrocytes regulate brain cholesterol homeostasis and they supply cholesterol to the needs of neurons. ATP-binding cassette transporter A1 (ABCA1) is the main cholesterol efflux transporter in astrocytes. Here we show dysregulated cholesterol homeostasis in astrocytes generated from human induced pluripotent stem cells (iPSCs) derived from males with fragile X syndrome (FXS), which is the most common cause of inherited intellectual disability. ABCA1 levels are reduced in FXS human and mouse astrocytes when compared with controls. Accumulation of cholesterol associates with increased desmosterol and polyunsaturated phospholipids in the lipidome of FXS mouse astrocytes. Abnormal astrocytic responses to cytokine exposure together with altered anti-inflammatory and cytokine profiles of human FXS astrocyte secretome suggest contribution of inflammatory factors to altered cholesterol homeostasis. Our results demonstrate changes of astrocytic lipid metabolism, which can critically regulate membrane properties and affect cholesterol transport in FXS astrocytes, providing target for therapy in FXS.

A key aspect of parasitic nematode infection is the nematodes' ability to evade and/or suppress host immunity. This immunomodulatory ability is likely driven by the release of hundreds of excretory/secretory proteins (ESPs) during infection. While ESPs have been shown to display immunosuppressive effects on various hosts, our understanding of the molecular interactions between individual proteins released and host immunity requires further study. We have recently identified a secreted phospholipase A2 (sPLA2) released from the entomopathogenic nematode (EPN) Steinernema carpocapsae we have named Sc-sPLA2. We report that Sc-sPLA2 increased mortality of Drosophila melanogaster infected with Streptococcus pneumoniae and promoted increased bacterial growth. Furthermore, our data showed that Sc-sPLA2 was able to downregulate both Toll and Imd pathway-associated antimicrobial peptides (AMPs) including drosomycin and defensin, in addition to suppressing phagocytosis in the hemolymph. Sc-sPLA2 was also found to be toxic to D. melanogaster with the severity being both dose- and time-dependent. Collectively, our data highlighted that Sc-sPLA2 possessed both toxic and immunosuppressive capabilities.

The RAP (RNA-binding domain abundant in Apicomplexans) protein family has been identified in various organisms. Despite expansion of this protein family in apicomplexan parasites, their main biological functions remain unknown. In this study, we use inducible knockdown studies in the human malaria parasite, Plasmodium falciparum, to show that two RAP proteins, PF3D7_0105200 (PfRAP01) and PF3D7_1470600 (PfRAP21), are essential for parasite survival and localize to the mitochondrion. Using transcriptomics, metabolomics, and proteomics profiling experiments, we further demonstrate that these RAP proteins are involved in mitochondrial RNA metabolism. Using high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (eCLIP-seq), we validate that PfRAP01 and PfRAP21 are true RNA-binding proteins and interact specifically with mitochondrial rRNAs. Finally, mitochondrial enrichment experiments followed by deep sequencing of small RNAs demonstrate that PfRAP21 controls mitochondrial rRNA expression. Collectively, our results establish the role of these RAP proteins in mitoribosome activity and contribute to further understanding this protein family in malaria parasites.

Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor that, upon activation by ligands, heterodimerizes with retinoid X receptor (RXR), binds to PPAR response elements (PPREs), and activates transcription of downstream genes. As PPARγ plays a central role in adipogenesis, fatty acid storage, and glucose metabolism, PPARγ-specific pharmaceuticals (e.g., thiazolidinediones) have been developed to treat Type II diabetes and obesity within human populations. However, to our knowledge, no prior studies have concurrently assessed the effects of PPARγ ligand exposure on genome-wide PPARγ binding as well as effects on the transcriptome and lipidome within human cells at biologically active, non-cytotoxic concentrations. In addition to quantifying concentration-dependent effects of ciglitazone (a reference PPARγ agonist) and GW 9662 (a reference PPARγ antagonist) on human hepatocarcinoma (HepG2) cell viability, PPARγ abundance in situ, and neutral lipids, HepG2 cells were exposed to either vehicle (0.1% DMSO), ciglitazone, or GW 9662 for up to 24 h, and then harvested for 1) chromatin immunoprecipitation-sequencing (ChIP-seq) to identify PPARγ-bound regions across the entire genome, 2) mRNA-sequencing (mRNA-seq) to identify potential impacts on the transcriptome, and 3) lipidomics to identify potential alterations in lipid profiles. Following exposure to ciglitazone and GW 9662, we found that PPARγ levels were not significantly different after 2–8 h of exposure. While ciglitazone and GW 9662 resulted in a concentration-dependent increase in neutral lipids, the magnitude and localization of PPARγ-bound regions across the genome (as identified by ChIP-seq) did not vary by treatment. However, mRNA-seq and lipidomics revealed that exposure of HepG2 cells to ciglitazone and GW 9662 resulted in significant, treatment-specific effects on the transcriptome and lipidome. Overall, our findings suggest that exposure of human cells to PPARγ ligands at biologically active, non-cytotoxic concentrations results in toxicity that may be driven by a combination of both PPARγ-dependent and PPARγ-independent mechanisms.

Commiphora wightii (Arn.) Bhandari, known as guggul, produces a medicinally important gum resin which is used extensively by Ayurvedic physicians to treat various ailments. However, most of the studies on C. wightii have been limited to its gum resin. Comprehensive metabolic profiling of leaves, stem and gum resin samples was undertaken to analyse aqueous and non-aqueous metabolites from three distinct chemotypes (NBRI-101, NBRI-102 and NBRI-103) shortlisted from different agro-climatic zones. GC-MS, HPLC and NMR spectroscopy were used for comprehensive metabolomics. Multivariate analysis showed characteristic variation in quinic and citric acids, myo-inositol and glycine (aqueous metabolites) and 2,6-di-tert-butyl-phenol, trans-farnesol and guggulsterones (non-aqueous metabolites) amongst the three chemotypes. Quinic acid, citric acid and myo-ionositol were detected in substantial quantities from leaves and stem samples which provide opportunities for novel nutraceutical and pharmaceutical formulations. Quinic acid, from the leaves, was identified as a marker metabolite for early selection of high guggulsterones-yielding cultivars.

Metabolomics represents a powerful, complementary approach for studying biological system responses to various biotic and abiotic stimuli. A major challenge in metabolomics is the lack of reliable annotations for all metabolites detected in complex MS and/or NMR data. To meet this challenge, we have developed an integrated UHPLC-QTOF-MS/MS-SPE-NMR system for higher-throughput metabolite identifications, which provides advanced biological context and enhances the scientific value of metabolomics data for understanding systems biology. This integrated instrumental method is less labor-intensive and more cost-effective than conventional individual methods (LC; MS; SPE; NMR). It enables the simultaneous purification and identification of primary and secondary metabolites present in biological samples. In this chapter, we describe the configuration and use of UHPLC-MS/MS-SPE-NMR in metabolite analyses ranging from sample extraction to higher-throughput metabolite annotation. With the integrated UHPLC-QTOF-MS/MS-SPE-NMR method, we have purified and confidently identified more than 100 previously known as well as unknown triterpene and flavonoid glycosides while noting that most of the identified compounds are not commercially available.

Lignin provides essential mechanical support for plant cell walls but decreases the digestibility of forage crops and increases the recalcitrance of biofuel crops. Attempts to modify lignin content and/or composition by genetic modification often result in negative growth effects. Although several studies have attempted to address the basis for such effects in individual transgenic lines, no common mechanism linking lignin modification with perturbations in plant growth and development has yet been identified. To address whether a common mechanism exists, we have analyzed transposon insertion mutants resulting in independent loss of function of five enzymes of the monolignol pathway, as well as one double mutant, in the model legume Medicago truncatula These plants exhibit growth phenotypes from essentially wild type to severely retarded. Extensive phenotypic, transcriptomic, and metabolomics analyses, including structural characterization of differentially expressed compounds, revealed diverse phenotypic consequences of lignin pathway perturbation that were perceived early in plant development but were not predicted by lignin content or composition alone. Notable phenotypes among the mutants with severe growth impairment were increased trichome numbers, accumulation of a variety of triterpene saponins, and extensive but differential ectopic expression of defense response genes. No currently proposed model explains the observed phenotypes across all lines. We propose that reallocation of resources into defense pathways is linked to the severity of the final growth phenotype in monolignol pathway mutants of M. truncatula, although it remains unclear whether this is a cause or an effect of the growth impairment.

Aim of the study was to undertake comprehensive metabolic profiling of plant parts of Commiphora wightii during two contrasting seasons i.e. summer and winter; compared seasonal metabolic variations; and assess antioxidant activity of fractions for commercial applications. Leaves, young stems and gum-resin extracts from summer and winter seasons were analyzed using GC-MS, HPLC and NMR spectroscopy. The antioxidant activity on each set was determined by DPPH free radical scavenging assay. Complete metabolic profiling from two contrasting seasons identified one hundred and four major known and unknown metabolites. Also, two alkylated phenols, 2,4-di- tert-butyl phenol and 3-(3,5-di- tert-butyl-4-hydroxyphenyl) propanoic acid not reported earlier from this taxon were isolated from the vegetative part. Comparative analysis of seasonal metabolic profiles of leaves, young stems and gum-resin revealed significant variations in concentrations of several metabolites. Multivariate principal component analysis (PCA) showed significant qualitative and quantitative variations in the polar (glycine, quinic acid and myo-inositol) and non-polar metabolites (alkylated phenols, guggulsterones and α-tocopherol) between the two seasons. Variation amongst metabolites such as myo-inositol, quinic acid α- tocopherol and alkylated phenols that are important for nutraceutical industry in the two contrasting seasons is a useful finding. These metabolites are of medicinal and nutraceutical importance and are commonly used in nutraceuticals and dietary supplement industry. DPPH radical scavenging activity (IC50 values) of polar and non-polar extracts varied significantly between summer and winter seasons. The antioxidant activity can be attributed to major polar metabolite, quinic acid biosynthesized in excess during winter, and to non-polar metabolites like alkylated phenols and α-tocopherol present during the summer season. The study shall be useful for medicinal, nutraceutical and dietary supplement industry for selection of polar or non-polar extracts from a particular season for obtaining targeted products with optimized functionality.

Aim of the study was to undertake comprehensive metabolic profiling of plant parts of Commiphora wightii during two contrasting seasons i.e. summer and winter; compared seasonal metabolic variations; and assess antioxidant activity of fractions for commercial applications. Leaves, young stems and gum-resin extracts from summer and winter seasons were analyzed using GC-MS, HPLC and NMR spectroscopy. The antioxidant activity on each set was determined by DPPH free radical scavenging assay. Complete metabolic profiling from two contrasting seasons identified one hundred and four major known and unknown metabolites. Also, two alkylated phenols, 2,4-di- tert -butyl phenol and 3-(3,5-di- tert -butyl-4-hydroxyphenyl) propanoic acid not reported earlier from this taxon were isolated from the vegetative part. Comparative analysis of seasonal metabolic profiles of leaves, young stems and gum-resin revealed significant variations in concentrations of several metabolites. Multivariate principal component analysis (PCA) showed significant qualitative and quantitative variations in the polar (glycine, quinic acid and myo -inositol) and non-polar metabolites (alkylated phenols, guggulsterones and α-tocopherol) between the two seasons. Variation amongst metabolites such as myo -inositol, quinic acid α- tocopherol and alkylated phenols that are important for nutraceutical industry in the two contrasting seasons is a useful finding. These metabolites are of medicinal and nutraceutical importance and are commonly used in nutraceuticals and dietary supplement industry. DPPH radical scavenging activity (IC 50 values) of polar and non-polar extracts varied significantly between summer and winter seasons. The antioxidant activity can be attributed to major polar metabolite, quinic acid biosynthesized in excess during winter, and to non-polar metabolites like alkylated phenols and α-tocopherol present during the summer season. The study shall be useful for medicinal, nutraceutical and dietary supplement industry for selection of polar or non-polar extracts from a particular season for obtaining targeted products with optimized functionality.

Flavonoids are a class of specialized metabolites found in many different plant species. They protect against UV radiation, scavenge reactive oxygen species, are involved in plant defense responses, and are associated with plant-microorganism interactions. They have also been reported to possess health-promoting effects including anti-inflammatory, antioxidant, anticancer activity, and antihypertensive effects. Flavonoids encompass >10,000 structures where the types and amounts depend on the plant species, developmental stage, organ, and growth conditions. The diversity of flavonoid structures represents a significant challenge in the analysis of plant flavonoids. Many analytical techniques have been developed to detect and quantify flavonoids, and the most productive of these techniques use liquid chromatography (LC) coupled to mass spectrometry (MS) to analyze flavonoids due to the excellent combination of selectivity and sensitivity of MS. In addition, mass spectral libraries have been constructed to further aid flavonoid identification. Here, the use of ultra-high pressure liquid chromatography coupled to mass spectrometry (UHPLC-MS) in plant flavonoid analyses, with an emphasis on sample extraction, flavonoid separation, and MS detection, is described. © 2018 by John Wiley & Sons, Inc.

Biogenic synthesis of silver nanoparticles for enhanced antimicrobial activity has gained a lot of momentum making it an urgent need to search for a suitable biocandidate which could be utilized for efficient capping and shaping of silver nanoparticles with enhanced bactericidal activity utilizing its secondary metabolites. Current work illustrates the enhancement of antimicrobial efficacy of silver nanoparticles by reducing and modifying their surface with antimicrobial metabolites of cell free filtrate of Trichoderma viride (MTCC 5661) in comparison to citrate stabilized silver nanoparticles. Nanoparticles were characterized by visual observations, UV-visible spectroscopy, zetasizer, and transmission electron microscopy (TEM). Synthesized particles were monodispersed, spherical in shape and 10-20 nm in size. Presence of metabolites on surface of biosynthesized silver nanoparticles was observed by gas chromatography-mass spectroscopy (GC-MS), energy dispersive X-ray analysis (EDAX), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR). The antimicrobial activity of both silver nanoparticles was tested against Shigella sonnei, Pseudomonas aeruginosa (Gram-negative) and Staphylococcus aureus (Gram-positive) by growth inhibition curve analysis and colony formation unit assay. Further, it was noted that internalization of biosynthesized nanoparticles inside the bacterial cell was much higher as compared to citrate stabilized particles which in turn lead to higher production of reactive oxygen species. Increase in oxidative stress caused severe damage to bacterial membrane enhancing further uptake of particles and revoking other pathways for bacterial disintegration resulting in complete and rapid death of pathogens as evidenced by fluorescein diacetate/propidium iodide dual staining and TEM. Thus, study reveals that biologically synthesized silver nanoarchitecture coated with antimicrobial metabolites of T. viride was more potent than their chemical counterpart in killing of pathogenic bacteria.

A rapid and reliable method was developed and validated for determining betulin and betulinic acid in bark in Betula utilis by high-resolution magic angle spinning H-1 nuclear magnetic resonance (HR-MAS NMR) spectroscopy. HR-MAS NMR spectroscopy clearly distinguished the resonances of betulin and betulinic acid in the bark of all accessions of B. utilis. The concentrations of betulin and betulinic acid were calculated and added to the spectra. The determination of the targeted metabolites in chloroform extract of bark of each accession of B. utilis was performed by high-performance liquid chromatography (HPLC). Quantitatively, betulin was present at higher concentrations than betulinic acid in all accessions. The HR-MAS NMR and HPLC results showed that betulin and betulinic acid varied significantly among accessions of B. utilis. Principal component analysis of the NMR and HPLC results provided classification into three metabolic groups in which the betulin concentration was high, moderate, or low. The results show that HR-MAS NMR is rapid for fingerprinting of betulin and betulinic acid in the bark of B. utilis, while minimizing the drawbacks associated with solvent extraction.

Ethnopharmacological relevance: Terminalia bellerica Roxb. fruits are rich in a variety of biologically active ingredients. Tharu and Buksa tribes of Udham Singh Nagar, Uttarakhand, India use grilled fruits of Terminalia bellerica as an effective cure for diarrheaAim of the study: We validated the ethnobotanical claim by comparing the antidiarrheal effect of grilled fruits (GF) with dried fruits (DF).Materials and methods: The 50% ethanolic extracts of GF and DF were successively fractionated; the antioxidant and bacterial inhibition activity were studied using DPPH free radical scavenging, anti-lipid peroxidation and broth dilution method respectively. Difference in metabolites of ethyl acetate fractions of GF and DF was analyzed using GC-MS, gallic acid content was determined through HPTLC. Further the in-vivo antidiarrheal effect of ethyl acetate fractions of DF and GF was studied on castor oil induced diarrhea model.Results: The ethyl acetate fractions showed potential DPPH free radical scavenging (IC50 11.13 mu g/ml in DF and 8.56 mu g/ml in GF), anti-lipid peroxidation and antibacterial activity. The non-targeted metabolic profiling showed higher content of tartaric acid, valeric acid, gallic acid, succinic acid, oxalic acid, malonic acid, malic acid, 1,2,3 trisbenzene, uridine and 11-eicosenoic acid in GF. The HPTLC results indicated that gallic acid content was 2.8 (+/- 0.14) and 4.92 (+/- 0.28) mg/g while ellagic acid content was 4.7 (+/- 0.32) and 4.45 (+/- 0.45) mg/g thy powder in DF and GF respectively. According to in vivo antidiarrheal activity DF and GF (100 mg/kg oral) inhibited diarrhea by 41.87% and 71.72% respectively.Conclusion: Grilling significantly altered the levels of metabolites in T. bellerica fruits which could be responsible for its increased therapeutic potential.

Guggul gum resin from Commiphora wightii (syn. Commiphora mukul) has been used for centuries in Ayurveda to treat a variety of ailments. The NMR and GC–MS based non-targeted metabolite profiling identified 118 chemically diverse metabolites including amino acids, fatty acids, organic acids, phenolic acids, pregnane-derivatives, steroids, sterols, sugars, sugar alcohol, terpenoids, and tocopherol from aqueous and non-aqueous extracts of leaves, stem, roots, latex and fruits of C. wightii. Out of 118, 51 structurally diverse aqueous metabolites were characterized by NMR spectroscopy. For the first time quinic acid and myo-inositol were identified as the major metabolites in C. wightii. Very high concentration of quinic acid was found in fruits (553.5±39.38mgg−1 dry wt.) and leaves (212.9±10.37mgg−1 dry wt.). Similarly, high concentration of myo-inositol (168.8±13.84mgg−1 dry wt.) was observed from fruits. The other metabolites of cosmeceutical, medicinal, nutraceutical and industrial significance such as α-tocopherol, n-methylpyrrolidone (NMP), trans-farnesol, prostaglandin F2, protocatechuic, gallic and cinnamic acids were identified from non-aqueous extracts using GC–MS. These important metabolites have thus far not been reported from this plant. Isolation of a fungal endophyte, (Nigrospora sps.) from this plant is the first report. The fungal endophyte produced a substantial quantity of bostrycin and deoxybostrycin known for their antitumor properties. Very high concentrations of quinic acid and myo-inositol in leaves and fruits; a substantial quantity of α-tocopherol and NMP in leaves, trans-farnesol in fruits, bostrycin and deoxybostrycin from its endophyte makes the taxa distinct, since these metabolites with medicinal properties find immense applications as dietary supplements and nutraceuticals.