Morphological, Molecular and Metabolic Characterization of the Pigmented Fungus Subramaniula asteroids.

Chaetomiaceae fungi are ascosporulating fungi whose importance as human pathogens has been frequently ignored. In the current study, a new isolate of the genus Subramaniula was described. The fungus was isolated from the soil of Wadi Om Nefa'a, Hurghada in the Red Sea Governorate, Egypt. Previously, Subramaniula were misidentified as Papulaspora spp. According to molecular analysis, the fungus was identified as Subramaniula asteroids OP484336. Remarkably, this species has been found among other fungi responsible for keratitis in humans and has been recorded for the first time in Egypt. Analysing the Subramaniula asteroids' metabolic profile was one of the objectives of the current study because little is known about this family's metabolome. The fungal extract's untargeted metabolic profiling was carried out by gas chromatography-mass spectroscopy (GC/MS), 1H and 1H-HSQC nuclear magnetic resonance (NMR) data, and their corresponding databases. In total, fifty-nine metabolites have been reported in the polar and non-polar extracts. The majority of polar metabolites are amino acids and carbohydrates. The non-polar extract's main components were 1-dodecanamine, N,N-dimethyl-, 1-tetradecanamine, N,N-dimethyl-, and 9-octadecenoic acid ethyl ester. The current study is the first to provide a metabolic profile of Subramaniula asteroids, which can be used in chemotaxonomical classification, antifungal drug development, and biological activity investigation of the studied species.

Isolation, Characterization, and Metabolic Profiling of Ceratorhiza hydrophila from the Aquatic Plant Myriophyllum spicatum.

The goal of the present study was to investigate the antibacterial properties, enzyme production, and metabolic profiling of a new Ceratorhiza hydrophila strain isolated from the submerged aquatic plant Myriophyllum spicatum. Furthermore, the fungus' morphological characterization and DNA sequencing have been described. The fungus has been identified and submitted to the GenBank as Ceratorhiza hydrophila isolate EG19 and the fungus ID is MK387081. The enzyme analyses showed its ability to produce protease and cellulase enzymes. According to the CSLI standard, the ethyl acetate extract of C. hydrophila showed intermediate antibacterial activity against Streptococcus pneumonia, Micrococcus luteus, and Staphylococcus aureus. Metabolic profiling has been carried out using 700 MHz NMR spectroscopy. Based on the 1H and 1H-13C heteronuclear single quantum coherence (HSQC) NMR data and NMR databases, 23 compounds have been identified. The identified metabolites include 31% amino acids, 9% sugars, 9% amines, 4% sugar alcohols, and 4% alkaloids. This is the first report for the metabolic characterization of C. hydrophila, which gave preliminary information about the fungus. It is expected that our findings not only will pave the way to other perspectives in enormous applications using C. hydrophila as a new promising source of antimicrobial agents and essential metabolites, but also it will be valuable in the classification and chemotaxonomy of the species.

Metabolic profiling, in vitro propagation, and genetic assessment of the endangered rare plant Anarrhinum pubescens.

BACKGROUND: Anarrhinum pubescens Fresen. (Plantaginaceae) is a rare plant, endemic to the Saint Catherine area, of South Sinai, Egypt. Earlier studies have reported the isolation of cytotoxic and anti-cholinesterase iridoid glucosides from the aerial parts of the plant. The present study aimed to investigate the chemical profiling of the wild plant shoots as well as establish efficient protocols for in vitro plant regeneration and proliferation with further assessment of the genetic stability of the in vitro regenerated plants. RESULTS: Twenty-seven metabolites have been identified in wild plant shoots using the Nuclear Magnetic Resonance (NMR) spectroscopy. The metabolites include alkaloids, amino acids, carbohydrates, organic acids, vitamins, and a phenol. In vitro propagation of the plant was carried out through nodal cutting-micropropagation and leaf segment-direct organogenesis. The best results were obtained when nodal cutting explants were cultured on Murashige and Skoog medium with Gamborg B5 vitamins supplemented with 6-benzylaminopurine (BAP) (1.0 mg/L) and naphthaleneacetic acid (NAA) (0.05 mg/L), which gave a shoot formation capacity of 100% and a mean number of shoots of 27.67 ± 1.4/explant. These shoots were successfully rooted and transferred to the greenhouse and the survival rate was 75%. Genetic fidelity evaluation of the micropropagated clones was carried out using random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) molecular markers. Jaccard's similarity coefficient indicated a similarity as high as 98% and 95% from RAPD and ISSR markers, respectively. CONCLUSIONS: This study provides the chemical profiling of the aerial part of Anarrhinum pubescens. Moreover, in vitro regeneration through different tissue culture techniques has been established for mass propagation of the plant, and the genetic fidelity of the in vitro regenerated plants was confirmed as well. Our work on the in vitro propagation of A. pubescens will be helpful in ex situ conservation and identification of bioactive metabolites.

Comparative Analysis of Au and Au@SiO2 Nanoparticle-Protein Interactions for Evaluation as Platforms in Theranostic Applications.

Gold nanoparticles are utilized in a variety of sensing and detection technologies because of their unique physiochemical properties. Their tunable size, shape, and surface charge enable them to be used in an array of platforms. The purpose of this study is to conduct a thorough spectroscopic characterization of Au and functionalized hybrid Au@SiO2 nanoparticles under physiological conditions and in the presence of two proteins known to be abundant in serum, bovine serum albumin and human ubiquitin. The information obtained from this study will enable us to develop design principles to synthesize an array of surface-enhanced Raman spectroscopy-based nanoparticles as platforms for theranostic applications. We are particularly interested in tailoring the surface chemistry of the Au@SiO2 nanoparticles for applications in theranostic technologies. We employ common spectroscopic techniques, with particular emphasis on circular dichroism and heteronuclear single quantum correlation nuclear magnetic resonance (HSQC NMR) spectroscopy, as combinatorial tools to understand protein conformational dynamics, binding site interactions, and protein corona for the design of nanoparticles capable of reaching their intended target in vivo. Our results conclude that protein adsorption onto the nanoparticle surface prevents nanoparticle aggregation. We observed that varying the ionic strength and type of ion influences the aggregation and aggregation rate of each respective nanoparticle. The conformation of proteins and the absorption of proteins on the surface of Au nanoparticles are also influenced by ionic strength. Using two-dimensional [15N-1H]-HSQC NMR experiments to compare the interactions of Au and Au@SiO2 nanoparticles with 15N-ubiquitin, we observed small chemical shift perturbations in some amino acid peaks and differences in binding site interactions with ubiquitin and respective nanoparticles.

New iridoid glycosides from Anarrhinum pubescens.

Six metabolites (1-6) were isolated from the aerial parts of Anarrhinum pubescens Fresen. (Plantaginaceae) growing in Saint Catherine region in Egypt; two of them (1 and 4) are here reported to be newly identified naturally occurring iridoids. The isolated metabolites were identified as 6-O-foliamenthoyl-(6'-O-cinnamoyl)-antirrhinoside (1), 6'-O-cinnamoyl-antirrhinoside (2), the iridoid dimer, pubescensoside (4), antirrhinoside (5), 10-hydroxy-antirrhinoside (6), and the flavonoid, diosmin (3). Identification of the new metabolites was based on analysis of their collected spectroscopic data (NMR and HR-ESI-MS). Furthermore, compounds (1, 4, and 5) were subjected to cytotoxic testing against the human lung carcinoma cell line (A-549); compound 4 showed better cytotoxic activity as indicated by the obtained (IC50).

NMR Analysis Reveals a Wealth of Metabolites in Root-Knot Nematode Resistant Roots of Citrullus amarus Watermelon Plants.

Citrullus amarus ( CA ) (previously known as Citrullus lanatus var. citroides ) accessions collected in southern Africa are known to have resistance to root-knot nematodes (RKN) and are suitable rootstocks for grafted watermelon. The objective of this study was to conduct a comparative metabolomics analysis and identify unique metabolites in roots of CA accessions versus roots of watermelon cultivars ( Citrullus lanatus (Thunb.) Matsum. and Nakai var. lanatus; CL ). Nuclear magnetic resonance (NMR) technology and principal component analysis (PCA) were used to analyze and compare metabolic profiles of seven CA accessions resistant to RKN along with two RKN-susceptible watermelon cultivars (Charleston Gray and Crimson Sweet). Calculation of the Mahalanobis distance revealed that the CA United States Plant Introduction (PI) 189225 (Line number 1832) and PI 482324 (1849) have the most distinct metabolic profiles compared with the watermelon cultivars Charleston Gray and Crimson Sweet, respectively. Several amino acids identified in the CA accessions were reported in previous studies to have a nematicidal effect. The results in this study indicate that roots of watermelon accessions collected in the wild are rich in metabolic compounds. These metabolic compounds may have been diminished in watermelon cultivars as a consequence of many years of cultivation and selection for desirable fruit qualities.

Differential roles of melatonin in plant-host resistance and pathogen suppression in cucurbits.

Since the 1950s, research on the animal neurohormone, melatonin, has focused on its multiregulatory effect on patients suffering from insomnia, cancer, and Alzheimer's disease. In plants, melatonin plays major role in plant growth and development, and is inducible in response to diverse biotic and abiotic stresses. However, studies on the direct role of melatonin in disease suppression and as a signaling molecule in host-pathogen defense mechanism are lacking. This study provides insight on the predicted biosynthetic pathway of melatonin in watermelon (Citrullus lanatus), and how application of melatonin, an environmental-friendly immune inducer, can boost plant immunity and suppress pathogen growth where fungicide resistance and lack of genetic resistance are major problems. We evaluated the effect of spray-applied melatonin and also transformed watermelon plants with the melatonin biosynthetic gene SNAT (serotonin N-acetyltransferase) to determine the role of melatonin in plant defense. Increased melatonin levels in plants were found to boost resistance against the foliar pathogen Podosphaera xanthii (powdery mildew), and the soil-borne oomycete Phytophthora capsici in watermelon and other cucurbits. Further, transcriptomic data on melatonin-sprayed (1 mmol/L) watermelon leaves suggest that melatonin alters the expression of genes involved in both PAMP-mediated (pathogen-associated molecular pattern) and ETI-mediated (effector-triggered immunity) defenses. Twenty-seven upregulated genes were associated with constitutive defense as well as initial priming of the melatonin-induced plant resistance response. Our results indicate that developing strategies to increase melatonin levels in specialty crops such as watermelon can lead to resistance against diverse filamentous pathogens.

NMR-based metabolomic analysis of wild, greenhouse, and in vitro regenerated shoots of Cymbopogon schoenanthus subsp. proximus with GC-MS assessment of proximadiol.

Cymbopogon schoenanthus subsp. proximus is a wild plant distributed in subtropical and east Africa extending from the north to the southern parts of Egypt. Widely used in folk medicine, it is the source of the diuretic sesquiterpene proximadiol. Nuclear magnetic resonance metabolomic analysis of polar extracts of shoots from wild, greenhouse, somatic embryos, and direct and indirect organogenic in vitro cultures was carried out. Metabolic profiling yielded 39 compounds, of which common metabolites were 15 (38.4%). Unique metabolites were trehalose (2.5%) in the wild plants, 2-hydroxylisobutyrate, galactarate and tyrosine (7.6%) in indirect organogenic shoots. Tartrate was found only in direct regenerated shoots (2.5%). Metabolites identified in greenhouse and embryogenic shoots showed no unique compounds. Multivariate analysis revealed significant differences between all tested shoots. 4-aminobutyrate, alanine, glutamine, glucose, fructose, and sucrose were the most significantly different metabolites. Proximadiol was identified and quantitatively measured from the non-polar extract of different types of shoots using gas chromatography and mass spectrometry (GC-MS). Concentrations ranged from 3.6 ± 0.03 to 198.6 ± 7.2 µg/100 mg dry weight in regenerated shoots from somatic embryogenesis and in wild plant shoots, respectively. Direct organogenesis yielded the highest in vitro concentration (20.3 ± 0.5 µg/100 mg dry weight). This study reported the metabolic profiling of C. schoenanthus polar extract and identified primary metabolites that are unique to the wild type and shoots regenerated from different in vitro cultures. Proximadiol was quantified and the in vitro culture system yielding the highest concentration relative to the wild plant was identified.

NMR Spectroscopy Identifies Metabolites Translocated from Powdery Mildew Resistant Rootstocks to Susceptible Watermelon Scions.

Powdery mildew (PM) disease causes significant loss in watermelon. Due to the unavailability of a commercial watermelon variety that is resistant to PM, grafting susceptible cultivars on wild resistant rootstocks is being explored as a short-term management strategy to combat this disease. Nuclear magnetic resonance-based metabolic profiles of susceptible and resistant rootstocks of watermelon and their corresponding susceptible scions (Mickey Lee) were compared to screen for potential metabolites related to PM resistance using multivariate principal component analysis. Significant score plot differences between the susceptible and resistant groups were revealed through Mahalanobis distance analysis. Significantly different spectral buckets and their corresponding metabolites (including choline, fumarate, 5-hydroxyindole-3-acetate, and melatonin) have been identified quantitatively using multivariate loading plots and verified by volcano plot analyses. The data suggest that these metabolites were translocated from the powdery mildew resistant rootstocks to their corresponding powdery mildew susceptible scions and can be related to PM disease resistance.

Cold adaptation shapes the robustness of metabolic networks in Drosophila melanogaster.

When ectotherms are exposed to low temperatures, they enter a cold-induced coma (chill coma) that prevents resource acquisition, mating, oviposition, and escape from predation. There is substantial variation in time taken to recover from chill coma both within and among species, and this variation is correlated with habitat temperatures such that insects from cold environments recover more quickly. This suggests an adaptive response, but the mechanisms underlying variation in recovery times are unknown, making it difficult to decisively test adaptive hypotheses. We use replicated lines of Drosophila melanogaster selected in the laboratory for fast (hardy) or slow (susceptible) chill-coma recovery times to investigate modifications to metabolic profiles associated with cold adaptation. We measured metabolite concentrations of flies before, during, and after cold exposure using nuclear magnetic resonance (NMR) spectroscopy to test the hypotheses that hardy flies maintain metabolic homeostasis better during cold exposure and recovery, and that their metabolic networks are more robust to cold-induced perturbations. The metabolites of cold-hardy flies were less cold responsive and their metabolic networks during cold exposure were more robust, supporting our hypotheses. Metabolites involved in membrane lipid synthesis, tryptophan metabolism, oxidative stress, energy balance, and proline metabolism were altered by selection on cold tolerance. We discuss the potential significance of these alterations.

NMR-based metabolomics study of the biochemical relationship between sugarcane callus tissues and their respective nutrient culture media.

The culture of sugarcane leaf explant onto culture induction medium triggers the stimulation of cell metabolism into both embryogenic and non-embryogenic callus tissues. Previous analyses demonstrated that embryogenic and non-embryogenic callus tissues have distinct metabolic profiles. This study is the follow-up to understand the biochemical relationship between the nutrient media and callus tissues using one-dimensional (1D (1)H) and two-dimensional (2D (1)H-(13)C) NMR spectroscopy followed by principal component analysis (PCA). 1D (1)H spectral comparisons of fresh unspent media (FM), embryogenic callus media (ECM), non-embryogenic callus media (NECM), embryogenic callus (EC), and non-embryogenic callus (NEC), showed different metabolic relationships between callus tissues and media. Based on metabolite fold change analysis, significantly changing sugar compounds such as glucose, fructose, sucrose, and maltose were maintained in large quantities by EC only. Significantly different amino acid compounds such as valine, leucine, alanine, threonine, asparagine, and glutamine and different organic acid derivatives such as lactate, 2-hydroxyisobutyrate, 4-aminobutyrate, malonate, and choline were present in EC, NEC, and NECM, which indicates that EC maintained these nutrients, while NEC either maintained or secreted the metabolites. These media and callus-specific results suggest that EC and NEC utilize and/or secrete media nutrients differently.

Tissue-Specific Metabolic Profile Study of Moringa oleifera L. Using Nuclear Magnetic Resonance Spectroscopy.

Moringa oleifera, an important multipurpose crop, is rich in various phytochemicals: flavonoids, antioxidants, vitamins, minerals and carotenes. The purpose of this study was to profile the groups of metabolites in leaf and stem tissues of M. oleifera. Various sugars, amino acids, and organic acid derivatives were found in all of the M. oleifera tissues with different profiles/peak intensities depending on the tissue. 1D proton nuclear magnetic resonance (NMR) was applied for collecting metabolite spectra. Approximately 30 metabolites with 2 unknown peaks were identified with Chenomx and verified with MMCD databases using carbon data. Among these metabolites, 22 metabolites were identified as common in both leaf and stem tissues. Of the remaining 8 metabolites, 4-aminobutyrate, adenosine, guanosine, tyrosine, and p-cresol were found only in leaf tissues; however, glutamate, glutamine, and tryptophan were found only in stem tissues. Biochemical pathway analysis revealed that 28 identified metabolites were interconnected with 36 different pathways as well as related to different fatty acids and secondary metabolites synthesis biochemical pathways. It is well known that different tissues of M. oleifera have nutritional, medicinal, and therapeutic values; therefore, our main objective is to provide a publicly available M. oliefera tissue specific metabolite database.

Identification of isethionic acid and other small molecule metabolites of Fragilariopsis cylindrus with nuclear magnetic resonance.

Nuclear magnetic resonance (NMR) spectroscopy has been used to obtain metabolic profiles of the polar diatom Fragilariopsis cylindrus, leading to the identification of a novel metabolite in this organism. Initial results from an ongoing metabolomics study have led to the discovery of isethionic acid (2-hydroxyethanesulfonic acid, CAS: 107-36-8) as a major metabolite in F. cylindrus. This compound is being produced by the organism under normal culture conditions. This finding is the first report of a diatom producing isethionic acid. In addition to isethionic acid, four other metabolites, dimethylsulfoniopropionate (DMSP), betaine, homarine, and proline were present and may serve as osmoprotectants in F. cylindrus. NMR-based metabolite profiles of F. cylindrus were obtained along a growth curve of the organism. The relative concentration levels of the five metabolites were monitored over a growth period of F. cylindrus from 18 to 25 days. All showed an increase in relative concentration with time, except for proline, which began to decrease after day 21.

NMR-based microbial metabolomics and the temperature-dependent coral pathogen Vibrio coralliilyticus.

Coral bleaching occurs when the symbioses between coral animals and their zooxanthellae is disrupted, either as part of a natural cycle or as the result of unusual events. The bacterium Vibrio coralliilyticus (type strain ATCC BAA-450) has been linked to coral disease globally (for example in the Mediterranean, Red Sea, Indian Ocean, and Great Barrier Reef) and like many other Vibrio species exhibits a temperature-dependent pathogenicity. The temperature-dependence of V. corallillyticus in regard to its metabolome was investigated. Nuclear magnetic resonance (NMR) spectra were obtained of methanol-water extracts of intracellula rmetabolites (endometabolome) from multiple samples of the bacteria cultured into late stationary phase at 27 degrees C (virulent form) and 24 degrees C (avirulent form). The spectra were subjected to principal components analysis (PCA), and significant temperature-based separations in PC1, PC2, and PC3 dimensions were observed. Betaine, succinate, and glutamate were identified as metabolites that caused the greatest temperature-based separations in the PC scores plots. With increasing temperature, betaine was shown to be down regulated, while succinate and glutamate were up regulated.

NMR-derived folate-bound structure of dihydrofolate reductase 1 from the halophile Haloferax volcanii.

Folate binds to dihydrofolate reductase (DHFR) to form a binary complex whose structure maintains the overall configuration of the enzyme; however, some significant changes are evident when a comparison is made to the enzyme. The structure of DHFR1 from the halophilic Halopherax volcanii was solved in its folate-bound form using nuclear magnetic resonance spectroscopy. NOE data obtained from the (15)N-NOESY-HSQC and (13)C-NOESY-HSQC experiments of the triply labeled ((1)H, (13)C, and (15)N) binary complex were used as input for the structure calculation with the Crystallography and Nuclear Magnetic Resonance System program. The resulting family of structures was compared with the enzyme solved by both nuclear magnetic resonance and X-ray crystallography and also to the mesophilic folate-bound enzyme from Escherichia coli. The binary complex exhibited less convergence of structure in the alpha2-helix and differences in the hinge residues D39 and A94. In comparison to the previously reported mesophilic binary complex solved by X-ray crystallography, the halophilic binary complex reported here does not agree with the convergence of the M20 loop to a single structure. The corresponding L21 loop of the halophilic binary complex family of structures solved by nuclear magnetic resonance indicates variability in this region.

Structure in an extreme environment: NMR at high salt.

Proteins from halophiles have adapted to challenging environmental conditions and require salt for their structure and function. How halophilic proteins adapted to a hypersaline environment is still an intriguing question. It is important to mimic the physiological conditions of the archae extreme halophiles when characterizing their enzymes, including structural characterization. The NMR derived structure of Haloferax volcanii dihydrofolate reductase in 3.5 M NaCl is presented, and represents the first high salt structure calculated using NMR data. Structure calculations show that this protein has a solution structure which is similar to the previously determined crystal structure with a difference at the N terminus of beta3 and the type of beta-turn connection beta7 and beta8.

NMR assignments of the binary hvDHFR1:folate complex.

A better understanding of how salt affects enzyme activity can be gained via NMR studies of binary hvDHFR1:folate complex. Chemical shift assignments of the 17.9 kDa enzyme with bound substrate prepare the way for ongoing research of the effects of salt on enzyme flexibility through relaxation studies.