HR-MAS NMR Research Articles

High-Resolution Magic-Angle Spinning Nuclear Magnetic Resonance Identifies Impairment of Metabolism by T-2 Toxin, in Relation to Toxicity, in Zebrafish Embryo Model.

Among the widespread trichothecene mycotoxins, T-2 toxin is considered the most toxic congener. In the present study, we utilized high-resolution magic-angle spinning nuclear magnetic resonance (HRMAS NMR), coupled to the zebrafish (Danio rerio) embryo model, as a toxicometabolomics approach to elucidate the cellular, molecular and biochemical pathways associated with T-2 toxicity. Aligned with previous studies in the zebrafish embryo model, exposure to T-2 toxin was lethal in the high parts-per-billion (ppb) range, with a median lethal concentration (LC50) of 105 ppb. Exposure to the toxins was, furthermore, associated with system-specific alterations in the production of reactive oxygen species (ROS), including decreased ROS production in the liver and increased ROS in the brain region, in the exposed embryos. Moreover, metabolic profiling based on HRMAS NMR revealed the modulation of numerous, interrelated metabolites, specifically including those associated with (1) phase I and II detoxification, and antioxidant pathways; (2) disruption of the phosphocholine lipids of cell membranes; (3) mitochondrial energy metabolism, including apparent disruption of the tricarboxylic acid (TCA) cycle, and the electron transport chain of oxidative phosphorylation, as well as "upstream" effects on carbohydrate, i.e., glucose metabolism; and (4) several compensatory catabolic pathways. Taken together, these observations enabled development of an integrated, system-level model of T-2 toxicity in relation to human and animal health.

MRI and HR-MAS NMR spectroscopy to correlate structural characteristics and the metabolome of Fiano and Pallagrello grapes with the action of field spray preparation 500 and the soil spatial microvariability

BackgroundA number of Italian grape berry varieties, such as Fiano (F) and Pallagrello Nero (P), represent National strategic products. Therefore, it is important to identify soil conditions emphasizing their peculiar characteristics as well as find innovative and sustainable treatments improving their compositional and nutraceutical quality. The field spray preparation 500 is a biodynamic product that is presumed to serve as biostimulant on the vine. However, so far, the scientific results probing its effectiveness are still lacking. Moreover, it is necessary to establish a reliable relationship between the grape quality and the spatial microvariability of the vineyard’s soil. On this basis, the main objective of this work consisted in correlating structural and morphological characteristics (via MRI), the primary metabolome (via semi-solid state HRMAS NMR) and important nutraceutical parameters (total phenols and antioxidants via DPPH assay) of F and P grapes with both the action of preparation 500 biostimulant and the vineyard soil microvariability, based on soil apparent electrical conductivity.ResultsHRMAS enabled the identification of the primary metabolome of F and P. The elaboration of 1H NMR spectra through chemometrics revealed significant changes in F and P grapes, accounting for both soil microvariability and the application of field spray (the latter also confirmed by PLS-DA and Heat-map clustering). Interestingly, for both F and P it was observed a significantly lower content of carbohydrates after biostimulant treatment while MRI revealed diagnostic structural and internal details of intact grapes. The combined use of proton parametric indices, such as relaxation times and diffusion coefficients, indicated alterations induced in grapes by both the spatial microvariability of the soil and the effects of investigated biostimulant. Interestingly, a tight correlation was found between MRI transverse relaxation time and the contents in total phenols and antioxidants.ConclusionsOur results have proven that both soil spatial microvariability and the application of field spray preparation 500 significantly affect the structural, metabolomic and nutraceutical characteristics of grapes. Moreover, the preparation 500 treatment has increased the nutraceutical value of grapes. Importantly, these data may be potentially used to promote and protect biodynamic grape and predict the quality of the resulting wines.Graphical

Morpho- and Chemotyping of Holopelagic Sargassum Species Causing Massive Strandings in the Caribbean Region

The specific identification of three major morphotypes of the tropical holopelagic Sargassum species causing massive strandings on the African and Caribbean coastlines was attempted by morphological characterisation as well as quantitative and qualitative analyses of several metabolites. Of the 25 morphological variables studied on 208 samples from the North Atlantic Ocean, 22 were used to establish a dichotomous identification key, allowing without any doubt the identification of each morphotype based on their morphological criteria alone. We also attempted to differentiate morphotypes using chemical fingerprintings (HR-MAS NMR) and markers by analysing pigment level and composition using High Pressure Liquid Chromatography, terpene profiles by Thin Layer Chromatography, phenolic compound levels by the Folin-Ciocalteu assay and structures by 2D Nuclear Magnetic Resonance spectroscopy, and fatty acid composition by Gas Chromatography. While pigment level and composition, terpene profiles, and phenolic contents were not discriminating, quantification of eight fatty acids enabled the differentiation of the three morphotypes. Furthermore, phlorotannin purification permitted their structural characterisation allowing discrimination between the three morphotypes. Our study highlights the potential of the free fatty acid profile and phlorotannin structure as good chemomarkers in order to discriminate between the three morphotypes of holopelagic Sargassum.

Amphiphilic polymer Co-networks based on cross-linked tetra-PEG-b-PCL star block copolymers

A new class of AB4-type amphiphilic polymer co-networks (ACN) was prepared by hetero-complementary 2-(4-nitrophenyl)-benzoxazinone/amine end group cross-linking chemistry in dimethyl sulfoxide (DMSO). For this purpose, a well-defined star block copolymer (tetra-PEG-b-PCL) consisting of a hydrophilic tetra-arm PEG core (Mn ∼ 5 kg mol−1) and hydrophobic PCL wings with a total molar mass of Mn ∼ 10 kg mol−1 was synthesized by ring-opening polymerization (ROP). This tetra-PEG-b-PCL star block copolymer was functionalized either with 2-(4-nitrophenyl)-benzoxazinone or with amino terminal groups. The ACN synthesis was carried out by conversion of these two star block copolymers. The overlap concentration of c* ∼76 g L−1 determined by viscosity measurements of both stars showed no significant temperature dependence. The network syntheses carried out at different concentrations (0.5 c* to 2.5 c*) resulted in conversion degrees in the range of p ≈ 95 % or higher as detected by HR MAS NMR spectroscopy. As in previous work on the synthesis of A4B4 networks using the same cross-linking chemistry, deviations from the expected network structure (increased proportion of double links between two stars) were detected also here by multi quantum NMR spectroscopy. The expected environmentally sensitive behavior of the ACNs was confirmed in terms of their equilibrium swelling in solvents of different quality to PEG and PCL. The scaling of the swelling and viscoelasticity data as a function of preparation concentration showed deviations from the expectations of a perfectly cross-linked star polymer network. Finally, a two-step gelation process was observed by rheology at 30 °C in DMSO, which is related to associations between the 2-(4-nitrophenyl)-benzoxazinone groups surrounded by PCL blocks that become apparent on the time scale of rheology when the molar mass is increased as a result of cross-linking.

Preparation of Single-Helical Curdlan Hydrogel and Its Activation with Coagulation Factor G.

β-1,3-glucans are a kind of natural polysaccharide with immunomodulatory, antitumor, and anti-inflammatory properties. Curdlan, as the simplest linear β-1,3-glucan, possesses a variety of biological activities and thermogelation properties. However, due to the complexity and variability of the conformations of curdlan, the exact structure-activity relationship remains unclear. We prepare a chemically crosslinked curdlan hydrogel with the unique single-helical skeleton (named S gel) in 0.4 wt% NaOH at 40 °C, confirmed by diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS). X-ray diffractometry (XRD) data show that S gel maintains the single-helical crystal structure, and the degree of crystallinity of the S gel is ~24%, which is slightly lower than that of the raw powder (~31%). Scanning electron microscopy (SEM) reveals that S gel has a continuous network structure, with large pores measuring 50-200 μm, which is consistent with its high swelling property. Using the 13C high-resolution magic angle spinning nuclear magnetic resonance (HRMAS NMR) method, we determine that most of the single-helical skeleton carbon signals in the swollen S gel are visible, suggesting that the single-helical skeleton of S gel exhibits fascinating mobility at room temperature. Finally, we reveal that the binding of S gel to coagulation Factor G from tachypleus amebocyte lysate increases and saturates at 20 μL tachypleus amebocyte lysate per mg of S gel. Our prepared S gel can avoid the transformation of curdlan conformations and retain the bioactivity of binding to coagulation Factor G, making it a valuable material for use in the food industry and the pharmaceutical field. This work deepens the understanding of the relationship between the single-helical structure and the activity of curdlan, promoting the development and application of β-1,3-glucans.

Functional implications of NHR-210 enrichment in C. elegans cephalic sheath glia: insights into metabolic and mitochondrial disruptions in Parkinson's disease models.

Glial cells constitute nearly half of the mammalian nervous system's cellular composition. The glia in C. elegans perform majority of tasks comparable to those conducted by their mammalian equivalents. The cephalic sheath (CEPsh)glia, which are known to be the counterparts of mammalian astrocytes, are enriched with two nuclear hormone receptors (NHRs)-NHR-210 and NHR-231. This unique enrichment makes the CEPsh glia and these NHRs intriguing subjects of study concerning neuronal health. We endeavored to assess the role of these NHRs in neurodegenerative diseases and related functional processes, using transgenic C. elegans expressing human alpha-synuclein. We employed RNAi-mediated silencing, followed by behavioural, functional, and metabolic profiling in relation to suppression of NHR-210 and 231. Our findings revealed that depleting nhr-210 changes dopamine-associated behaviour and mitochondrial function in human alpha synuclein-expressing strains NL5901 and UA44, through a putative target, pgp-9, a transmembrane transporter. Considering the alteration in mitochondrial function and the involvement of a transmembrane transporter, we performed metabolomics study via HR-MAS NMR spectroscopy. Remarkably, substantial modifications in ATP, betaine, lactate, and glycine levels were seen upon the absence of nhr-210. We also detected considerable changes in metabolic pathways such as phenylalanine, tyrosine, and tryptophan biosynthesis metabolism; glycine, serine, and threonine metabolism; as well as glyoxalate and dicarboxylate metabolism. In conclusion, the deficiency of the nuclear hormone receptor nhr-210 in alpha-synucleinexpressing strain of C. elegans, results in altered mitochondrial function, coupled with alterations in vital metabolite levels. These findings underline the functional and physiological importance of nhr-210 enrichment in CEPsh glia.

A Metabolomics Study by 1H HRMAS NMR: From Sheep Milk to a Pressed-Curd Cheese: A Proof of Concept

For the first time, High-Resolution Magic Angle Nuclear Magnetic Resonance spectroscopy (NMR-HRMAS) was applied to directly identify specific metabolites from a Spanish raw ewe’s milk and enzymatic coagulation pressed-curd cheese (Protected Geographical Indication: Castellano) manufactured by two procedures (traditional/artisanal vs. industrial) and including the ewe’s raw milk. The NMR parameters were optimized to study the complex matrixes of this type of cheese. In addition, conventional overcrowded 1H-NMR-HRMAS spectra were selectively simplified by a Carr–Purcell–Meiboom–Gill (CPMG) sequence or a stimulated echo pulse sequence by bipolar gradients (DIFF), thus modulating spin–spin relaxation times and diffusion of molecular components, respectively. 1H-NMR-HRMAS spectroscopy displayed important information about cheese metabolites, which can be associated with different manufacturing processes (industrial vs. traditional) and ripening times (from 2 to 90 days). These results support that this spectroscopy is a useful technique to monitor the ripening process, from raw milk to commercial ripened cheese, using a minimum intact sample, implying the absence of time-consuming sample pretreatments.

Integrated Metabolomics and Proteomics of Symptomatic and Early Presymptomatic States of Colitis.

Colitis has a multifactorial pathogenesis with a strong cross-talk among microbiota, hypoxia, and tissue metabolism. Here, we aimed to characterize the molecular signature of the disease in symptomatic and presymptomatic stages of the inflammatory process at the tissue and fecal level. The study is based on two different murine models for colitis, and HR-MAS NMR on "intact" colon tissues and LC-MS/MS on colon tissue extracts were used to derive untargeted metabolomics and proteomics information, respectively. Solution NMR was used to derive metabolomic profiles of the fecal extracts. By combining metabolomic and proteomic analyses of the tissues, we found increased anaerobic glycolysis, accompanied by an altered citric acid cycle and oxidative phosphorylation in inflamed colons; these changes associate with inflammation-induced hypoxia taking place in colon tissues. Different colitis states were also characterized by significantly different metabolomic profiles of fecal extracts, attributable to both the dysbiosis characteristic of colitis as well as the dysregulated tissue metabolism. Strong and distinctive tissue and fecal metabolomic signatures can be detected before the onset of symptoms. Therefore, untargeted metabolomics of tissues and fecal extracts provides a comprehensive picture of the changes accompanying the disease onset already at preclinical stages, highlighting the diagnostic potential of global metabolomics for inflammatory diseases.

Metabolic characterization of ovarian cancer using j-resolved 1h hr mas NMR spectroscopy

Metabolic characterization of ovarian cancer using j-resolved 1h hr mas NMR spectroscopy

HR-MAS NMR Metabolomics Profile of Vero Cells under the Influence of Virus Infection and nsP2 Inhibitor: A Chikungunya Case Study.

The arbovirus Chikungunya (CHIKV) is transmitted by Aedes mosquitoes in urban environments, and in humans, it triggers debilitating symptoms involving long-term complications, including arthritis and Guillain-Barré syndrome. The development of antiviral therapies is relevant, as no efficacious vaccine or drug has yet been approved for clinical application. As a detailed map of molecules underlying the viral infection can be obtained from the metabolome, we validated the metabolic signatures of Vero E6 cells prior to infection (CC), following CHIKV infection (CV) and also upon the inclusion of the nsP2 protease inhibitor wedelolactone (CWV), a coumestan which inhibits viral replication processes. The metabolome groups evidenced significant changes in the levels of lactate, myo-inositol, phosphocholine, glucose, betaine and a few specific amino acids. This study forms a preliminary basis for identifying metabolites through HR-MAS NMR (High Resolution Magic Angle Spinning Nuclear Magnetic Ressonance Spectroscopy) and proposing the affected metabolic pathways of cells following viral infection and upon incorporation of putative antiviral molecules.

Complex I, V, and MDH2 deficient human skin fibroblasts reveal distinct metabolic signatures by 1 H HR-MAS NMR.

In this study, we investigated the metabolic signatures of different mitochondrial defects (two different complex I and complex V, and the one MDH2 defect) in human skin fibroblasts (HSF). We hypothesized that using a selective culture medium would cause defect specific adaptation of the metabolome and further our understanding of the biochemical implications for the studied defects. All cells were cultivated under galactose stress condition and compared to glucose-based cell culture condition. We investigated the bioenergetic profile using Seahorse XFe96 cell analyzer and assessed the extracellular metabolic footprints and the intracellular metabolic fingerprints using NMR. The galactose-based culture condition forced a bioenergetic switch from a glycolytic to an oxidative state in all cell lines which improved overall separation of controls from the different defect groups. The extracellular metabolome was discriminative for separating controls from defects but not the specific defects, whereas the intracellular metabolome suggests CI and CV changes and revealed clear MDH2 defect-specific changes in metabolites associated with the TCA cycle, malate aspartate shuttle, and the choline metabolism, which are pronounced under galactose condition.

Metabolomic profiles of intact tissues reflect clinically relevant prostate cancer subtypes

BackgroundProstate cancer (PC) is a heterogenous multifocal disease ranging from indolent to lethal states. For improved treatment-stratification, reliable approaches are needed to faithfully differentiate between high- and low-risk tumors and to predict therapy response at diagnosis.MethodsA metabolomic approach based on high resolution magic angle spinning nuclear magnetic resonance (HR MAS NMR) analysis was applied on intact biopsies samples (n = 111) obtained from patients (n = 31) treated by prostatectomy, and combined with advanced multi- and univariate statistical analysis methods to identify metabolomic profiles reflecting tumor differentiation (Gleason scores and the International Society of Urological Pathology (ISUP) grade) and subtypes based on tumor immunoreactivity for Ki67 (cell proliferation) and prostate specific antigen (PSA, marker for androgen receptor activity).ResultsValidated metabolic profiles were obtained that clearly distinguished cancer tissues from benign prostate tissues. Subsequently, metabolic signatures were identified that further divided cancer tissues into two clinically relevant groups, namely ISUP Grade 2 (n = 29) and ISUP Grade 3 (n = 17) tumors. Furthermore, metabolic profiles associated with different tumor subtypes were identified. Tumors with low Ki67 and high PSA (subtype A, n = 21) displayed metabolite patterns significantly different from tumors with high Ki67 and low PSA (subtype B, n = 28). In total, seven metabolites; choline, peak for combined phosphocholine/glycerophosphocholine metabolites (PC + GPC), glycine, creatine, combined signal of glutamate/glutamine (Glx), taurine and lactate, showed significant alterations between PC subtypes A and B.ConclusionsThe metabolic profiles of intact biopsies obtained by our non-invasive HR MAS NMR approach together with advanced chemometric tools reliably identified PC and specifically differentiated highly aggressive tumors from less aggressive ones. Thus, this approach has proven the potential of exploiting cancer-specific metabolites in clinical settings for obtaining personalized treatment strategies in PC.

The OL101 O antigen locus specifies a novel Klebsiella pneumoniae serotype O13 structure

Klebsiella pneumoniae is one of the priority objects for the development of new therapies against infections. The species has been perceived as of limited variety of O antigens (11 O serotypes identified to date). That trait makes lipopolysaccharide an attractive target for protective antibodies. Nowadays, K. pneumoniae O antigens encoding genes are often analysed by bioinformatic tools, such as Kaptive, indicating higher actual diversity of the O antigen loci. One of the novel K. pneumoniae O loci for which the antigen structure has not been elucidated so far is OL101. In this study, four clinical isolates predicted as OL101 were characterized and found to have the O antigen structure composed of β-Kdop-[→3)-α-l-Rhap-(1 → 4)-α-d-Glcp]n, representing a novel serotype O13. Identification of the β-Kdop terminus was based on the analysis of the complete LPS molecule by the HR-MAS NMR spectroscopy. The bioinformatic analysis of 71,377 K. pneumoniae genomes from public databases (July 2023) revealed a notable OL101 prevalence of 6.55 %.

HR-MAS DREAMTIME NMR for Slow Spinning ex Vivo and in Vivo Samples.

HR-MAS NMR is a powerful tool, capable of monitoring molecular changes in intact heterogeneous samples. However, one of the biggest limitations of 1H NMR is its narrow spectral width which leads to considerable overlap in complex natural samples. DREAMTIME NMR is a highly selective technique that allows users to isolate suites of metabolites from congested spectra. This permits targeted metabolomics by NMR and is ideal for monitoring specific processes. To date, DREAMTIME has only been employed in solution-state NMR, here it is adapted for HR-MAS applications. At high spinning speeds (>5 kHz), DREAMTIME works with minimal modifications. However, spinning over 3-4 kHz leads to cell lysis, and if maintaining sample integrity is necessary, slower spinning (<2.5 kHz) is required. Very slow spinning (≤500 Hz) is advantageous for in vivo analysis to increase organism survival; however, sidebands from water pose a problem. To address this, a version of DREAMTIME, termed DREAMTIME-SLOWMAS, is introduced. Both techniques are compared at 2500, 500, and 50 Hz, using ex vivo worm tissue. Following this, DREAMTIME-SLOWMAS is applied to monitor key metabolites of anoxic stress in living shrimp at 500 Hz. Thus, standard DREAMTIME works well under MAS conditions and is recommended for samples reswollen in D2O or spun >2500 Hz. For slow spinning in vivo or intact tissue samples, DREAMTIME-SLOWMAS provides an excellent way to target process-specific metabolites while maintaining sample integrity. Overall, DREAMTIME should find widespread application wherever targeted molecular information is required from complex samples with a high degree of spectral overlap.

The Mechanical and Biological Performance of Photopolymerized Gelatin-Based Hydrogels as a Function of the Reaction Media.

From the first experiments with biomaterials to mimic tissue properties, the mechanical and biochemical characterization have evolved extensively. A number of properties can be described, however, what should be essential is to conduct a proper and physiologically relevant characterization. Herein, we describe the influence of the reaction media and buffer media -phosphate buffer saline (PBS) and Dulbecco's modified Eagle's medium (DMEM) with two different glucose concentrations- in GelMA hydrogel mechanics and in the biological behavior of two tumoral cell lines (Caco-2 and HCT-116). All scaffolds were photocrosslinked using UV light under identical conditions and evaluated for mass swelling ratio and stiffness. Our results indicate that stiffness is highly susceptible to the reaction media, but not to the swelling media. In addition, PBS-prepared hydrogels exhibited a higher photopolymerization degree as confirmed by HR-MAS NMR. These findings correlate with the biological response of Caco-2 and HCT-116 cells seeded on the substrates, which demonstrated flatter morphologies on stiffer hydrogels. Overall, cell viability and proliferation were excellent for both cell lines, and Caco-2 cells displayed a characteristic apical-basal polarization as observed in F-actin/Nuclei fluorescence images. These characterization experiments highlight the importance of conducting mechanical testing of biomaterials in the same medium as cell culture. This article is protected by copyright. All rights reserved.

High-Resolution Magic Angle Spinning (HRMAS) NMR Identifies Oxidative Stress and Impairment of Energy Metabolism by Zearalenone in Embryonic Stages of Zebrafish (Danio rerio), Olive Flounder (Paralichthys olivaceus) and Yellowtail Snapper (Ocyurus chrysurus).

Zearalenone (ZEA) is a mycotoxin, commonly found in agricultural products, linked to adverse health impacts in humans and livestock. However, less is known regarding effects on fish as both ecological receptors and economically relevant "receptors" through contamination of aquaculture feeds. In the present study, a metabolomics approach utilizing high-resolution magic angle spinning nuclear magnetic resonance (HRMAS NMR) was applied to intact embryos of zebrafish (Danio rerio), and two marine fish species, olive flounder (Paralichthys olivaceus) and yellowtail snapper (Ocyurus chrysurus), to investigate the biochemical pathways altered by ZEA exposure. Following the assessment of embryotoxicity, metabolic profiling of embryos exposed to sub-lethal concentrations showed significant overlap between the three species and, specifically, identified metabolites linked to hepatocytes, oxidative stress, membrane disruption, mitochondrial dysfunction, and impaired energy metabolism. These findings were further supported by analyses of tissue-specific production of reactive oxygen species (ROS) and lipidomics profiling and enabled an integrated model of ZEA toxicity in the early life stages of marine and freshwater fish species. The metabolic pathways and targets identified may, furthermore, serve as potential biomarkers for monitoring ZEA exposure and effects in fish in relation to ecotoxicology and aquaculture.

Linear PEI‐based responsive hydrogels: Synthesis and characterization

AbstractTwo responsive PEI hydrogels (P1.5E and P2E) were synthesized through a click reaction using linear PEI polymers with different cross‐linker contents (ethylene glycol diglycidyl ether—EGDE) and avoiding organic solvents. Through 13C HRMAS NMR and DSC studies the presence of residual epoxy groups and different protonation states of the linear PEI segments in the hydrogel were detected. The swelling and rheological behavior was dependent on the cross‐linking degree. The hydrogel with the lower cross‐linking degree (P1.5E) presented higher swelling values, but it was less resistant to the deformation. Besides, the swelling capacity and the resistance to deformation were found to be dependent on the interchain repulsions and charge of the amine groups, which endowed the hydrogels with stimuli‐responsive behavior to pH or ionic strength changes. For both hydrogels, the swelling capacity and rheological properties were inversely proportional and directly proportional to the pH increase, respectively. Both materials presented adsorption capacity for the azo dye methyl orange (MO) and Cu2+ ions. The interaction of the materials with the pollutants was also dependent on the cross‐linking degree, being P1.5E the one that presented higher sorption capacities. Furthermore, the hydrogels did not release toxic molecules, as assessed by lettuce seeds germination experiments.

Dynamics of diffusion, evaporation, and retention of organic solvents in paints by unilateral NMR and HR-MAS NMR spectroscopy

Transport of organic solvents in polymeric matrices plays a central role in many processes of interest to the coatings industry. Previous studies have focused on individual processes taking place in the solvent-paint system. In the present study, we report results on the molecular dynamics in a solvent/paint system during the entire sequence of absorption, diffusion, swelling, and evaporation processes, obtained non-invasively, in situ, and in real time using unilateral NMR relaxometry. The associated chemical structures were investigated using HR-MAS NMR spectroscopy. Four distinct ranges of molecular motion in the solvent/paint film were identified based on proton NMR transverse relaxation time (T2) data. The fast motion of the freely moving solvent molecules was discriminated from the slower motion of the so-called bound phase of the solvent molecules physically and chemically interacting with the cross-linked polymer network. In addition, two distinct domains of the cross-linked and mobile phases of the polymerized oil were observed, allowing the study of both the dynamics of the swelling-contraction process of the polymer and of the solubilization process. Such non-invasive analyses provide significant information on transport phenomena of the solvent in the paint film, as well as on the effects of various solvents applied to oil paintings.

Metabolic effects of diclofenac on the aquatic food chain - 1H-NMR study of water flea-zebrafish system.

In the environment, aquatic organisms are not only directly exposed to pollutants, but the effects can be exacerbated along the food chain. In this study, we investigated the effect of the food (water flea) on the secondary consumer (zebrafish) with the exposure diclofenac (DCF) Both organisms were exposed to an environmentally relevant concentrations (15µg/L) of diclofenac for five days, and zebrafish were fed exposed and non-exposed water fleas, respectively. Metabolites of the water fleas were directly analyzed using HRMAS NMR, and for zebrafish, polar metabolite were extracted and analyzed using liquid NMR. Metabolic profiling was performed and statistically significant metabolites which affected by DCF exposure were identified. There were more than 20 metabolites with variable importance (VIP) score greater than 1.0 in comparisons in fish groups, and identified metabolites differed depending on the effect of exposure and the effect of food. Specifically, exposure to DCF significantly increased alanine and decreased NAD + in zebrafish, which means energy demand was increased. Additionally, the effects of exposed food decreased in guanosine, a neuroprotective metabolite, which explained that the neurometabolic pathway was perturbated by the feeding of exposed food. Our results which short-term exposed primary consumers to pollutants indirectly affected the metabolism of secondary consumers suggest that the long-term exposure further study remains to be investigated.

Compost-derived thermophilic microorganisms producing glycoside hydrolase activities as new potential biocatalysts for sustainable processes

BackgroundThe management of the organic waste recycling process determines the interest in the thermophiles microorganisms involved in composting. Although many microbial enzymes have been isolated and studied for their industrial and commercial uses, there is still a continuous search for microorganisms which could synthesize industrially feasible enzymes, especially when the microbial diversity of cow dung itself makes a potential source of biotechnological enzymes.ResultsThe composting process studied at the Experimental Station of the University of Naples Federico II (Castel Volturno, Caserta, Italy) was characterized by fresh saw dust 40%, bovine manure 58%, and 2% mature compost as raw organic substrates, and its thermophilic phase exceeded a temperature of 55 °C for at least 5 days, thus achieving sanitation. Six microbial strains were isolated and designated as follow: CV1-1, CV1-2, CV2-1, CV2-2, CV2-3 and CV2-4. Based on 16S rRNA gene sequence, HRMAS–NMR spectroscopy, and biochemical investigations, they were ascribed to the genera Geobacillus and Bacillus. All the microbial isolates were qualitatively screened on plates for the presence of hydrolytic activities, and they were quantitatively screened in liquid for glycoside hydrolase enzymes in the extracellular, cell-bound, and cytosolic fractions. Based on these results, strains CV2-1 and CV2-3 were also quantitatively screened for the presence of cellulase and pectinase activities, and pH and temperature optimum plus thermostability of cellulase from CV2-1 were analyzed.ConclusionsThe isolation and the identification of these thermophilic microorganisms such as Geobacillus toebii, Geobacillus galactosidasius, Bacillus composti, Bacillus thermophilus and Aeribacillus composti have allowed the study of the biodiversity of compost, with emphasis on their primary metabolome through an innovative and underutilized technique, that is HRMAS–NMR, also highlighting it as a novel approach to bacterial cell analysis. Subsequently, this study has permitted the identification of enzymatic activities able to degrade cellulose and other polymeric substrates, such as the one investigated from strain CV2-1, which could be interesting from an industrial and a biotechnological point of view, furthermore, increasing the knowledge for potential applicability in different industrial fields as an efficient and environmentally friendly technique.Graphical