Micronized Powder of Raspberry Pomace as a Source of Bioactive Compounds.

Red raspberries, which contain a variety of nutrients and phytochemicals that are beneficial for human health, can be utilized as a raw material in the creation of several supplements. This research suggests micronized powder of raspberry pomace production. The molecular characteristics (FTIR), sugar, and biological potential (phenolic compounds and antioxidant activity) of micronized raspberry powders were investigated. FTIR spectroscopy results revealed spectral changes in the ranges with maxima at ~1720, 1635, and 1326, as well as intensity changes in practically the entire spectral range analyzed. The discrepancies clearly indicate that the micronization of the raspberry byproduct samples cleaved the intramolecular hydrogen bonds in the polysaccharides present in the samples, thus increasing the respective content of simple saccharides. In comparison to the control powders, more glucose and fructose were recovered from the micronized samples of the raspberry powders. The study's micronized powders were found to contain nine different types of phenolic compounds, including rutin, elagic acid derivatives, cyanidin-3-sophoroside, cyanidin-3-(2-glucosylrutinoside), cyanidin-3-rutinoside, pelargonidin-3-rutinoside, and elagic acid derivatives. Significantly higher concentrations of ellagic acid and ellagic acid derivatives and rutin were found in the micronized samples than in the control sample. The antioxidant potential assessed by ABTS and FRAP significantly increased following the micronization procedure.

Experimental-theoretical study of laccase as a detoxifier of aflatoxins.

We investigate laccase-mediated detoxification of aflatoxins, fungal carcinogenic food contaminants. Our experimental comparison between two aflatoxins with similar structures (AFB1 and AFG2) shows significant differences in laccase-mediated detoxification. A multi-scale modeling approach (Docking, Molecular Dynamics, and Density Functional Theory) identifies the highly substrate-specific changes required to improve laccase detoxifying performance. We employ a large-scale density functional theory-based approach, involving more than 7000 atoms, to identify the amino acid residues that determine the affinity of laccase for aflatoxins. From this study we conclude: (1) AFB1 is more challenging to degrade, to the point of complete degradation stalling; (2) AFG2 is easier to degrade by laccase due to its lack of side products and favorable binding dynamics; and (3) ample opportunities to optimize laccase for aflatoxin degradation exist, especially via mutations leading to π-π stacking. This study identifies a way to optimize laccase for aflatoxin bioremediation and, more generally, contributes to the research efforts aimed at rational enzyme optimization.

Metabolically-incorporated deuterium in myelin localized by neutron diffraction and identified by mass spectrometry.

Myelin is a natural and dynamic multilamellar membrane structure that continues to be of significant biological and neurological interest, especially with respect to its biosynthesis and assembly during its normal formation, maintenance, and pathological breakdown. To explore the usefulness of neutron diffraction in the structural analysis of myelin, we investigated the use of in vivo labeling by metabolically incorporating non-toxic levels of deuterium (2H; D) via drinking water into a pregnant dam (D-dam) and her developing embryos. All of the mice were sacrificed when the pups (D-pups) were 55 days old. Myelinated sciatic nerves were dissected, fixed in glutaraldehyde and examined by neutron diffraction. Parallel samples that were unfixed (trigeminal nerves) were frozen for mass spectrometry (MS). The diffraction patterns of the nerves from deuterium-fed mice (D-mice) vs. the controls (H-mice) had major differences in the intensities of the Bragg peaks but no appreciable differences in myelin periodicity. Neutron scattering density profiles showed an appreciable increase in density at the center of the lipid-rich membrane bilayer. This increase was greater in D-pups than in D-dam, and its localization was consistent with deuteration of lipid hydrocarbon, which predominates over transmembrane protein in myelin. MS analysis of the lipids isolated from the trigeminal nerves demonstrated that in the pups the percentage of lipids that had one or more deuterium atoms was uniformly high across lipid species (97.6% â€‹± â€‹2.0%), whereas in the mother the lipids were substantially less deuterated (60.6% â€‹± â€‹26.4%) with levels varying among lipid species and subspecies. The mass distribution pattern of deuterium-containing isotopologues indicated the fraction (in %) of each lipid (sub-)species having one or more deuteriums incorporated: in the D-pups, the pattern was always bell-shaped, and the average number of D atoms ranged from a low of ∼4 in fatty acid to a high of ∼9 in cerebroside. By contrast, in D-dam most lipids had more complex, overlapping distributions that were weighted toward a lower average number of deuteriums, which ranged from a low of ∼3-4 in fatty acid and in one species of sulfatide to a high of 6-7 in cerebroside and sphingomyelin. The consistently high level of deuteration in D-pups can be attributed to their de novo lipogenesis during gestation and rapid, postnatal myelination. The widely varying levels of deuteration in D-dam, by contrast, likely depends on the relative metabolic stability of the particular lipid species during myelin maintenance. Our current findings demonstrate that stably-incorporated D label can be detected and localized using neutron diffraction in a complex tissue such as myelin; and moreover, that MS can be used to screen a broad range of deuterated lipid species to monitor differential rates of lipid turnover. In addition to helping to develop a comprehensive understanding of the de novo synthesis and turnover of specific lipids in normal and abnormal myelin, our results also suggest application to studies on myelin proteins (which constitute only 20-30% by dry mass of the myelin, vs. 70-80% for lipid), as well as more broadly to the molecular constituents of other biological tissues.

Methods to Assess Phosphodiesterase and/or Adenylyl Cyclase Activity Via Heterologous Expression in Fission Yeast.

Heterologous expression of cyclic nucleotide phosphodiesterases (PDEs) and adenylyl cyclases (ACs) in the fission yeast Schizosaccharomyces pombe can be used in combination with PKA-repressed reporters to either carry out high throughput screens for small molecule inhibitors of these target enzymes or to assess hit compounds and their analogs from such screens. Here, we describe two methods for testing panels of such compounds. The first uses a growth assay for which growth in medium containing the pyrimidine analog 5-fluoro orotic acid (5FOA) occurs in response to inhibiting PDE activity to activate PKA. The second uses mass spectrometry to directly measure the impact of compound treatment to study compounds that modulate either PDE or AC activity.

Functional Analysis of the Differences in the Dimensions of Two Types of Boiled and Steamed Rice Grains.

The study tested how the cooking process can change the dimensions of rice grains. The impact of set times of cooking or steaming process on the characteristics such as length, width, and height of two varieties of rice, namely, long-grain white and parboiled, was investigated. The measurements of the dimension characteristics obtained at different times of the cooking process were converted to functional data. Different methods of multivariate functional data analysis, namely, functional multivariate analysis of variance, functional discriminant coordinates, and cluster analysis, were applied to discover the differences between the two varieties and the two heat treatment methods.

Luminescence of Lanthanide Complexes with Perfluorinated Alkoxide Ligands.

Four groups of rare-earth complexes, comprising 11 new compounds, with fluorinated O-donor ligands ([K(THF)6][Ln(OC4F9)4(THF)2] (1-Ln; Ln = Ce, Nd), [K](THF)x[Ln(OC4F9)4(THF)y] (2-Ln; Ln = Eu, Gd, Dy), [K(THF)2][Ln(pinF)2(THF)3] (3-Ln; Ln = Ce, Nd), and [K(THF)2][Ln(pinF)2(THF)2] (4-Ln; Ln = Eu, Gd, Dy, Y) have been synthesized and characterized. Single-crystal X-ray diffraction data were collected for all compounds except 2-Ln. Species 1-Ln, 3-Ln, and 4-Ln are uncommon examples of six-coordinate (Eu, Gd, Dy, and Y) and seven-coordinate (Ce and Nd) LnIII centers in all-O-donor environments. Species 1-Ln, 2-Ln, 3-Ln, and 4-Ln are all luminescent (except where Ln = Gd and Y), with the solid-state emission of 1-Ce being exceptionally blue-shifted for a Ce complex. The emission spectra of the six Nd, Eu, and Dy complexes do not show large differences based on the ligand and are generally consistent with the well-known free-ion spectra. Time-dependent density functional theory results show that 1-Ce and 3-Ce undergo allowed 5f → 4d excitations, consistent with luminescence lifetime measurements in the nanosecond range. Eu-containing 2-Eu and 4-Eu, however, were found to have luminescence lifetimes in the millisecond range, indicating phosphorescence rather than fluorescence. The performance of a pair of multireference models for prediction of the Ln = Nd, Eu, and Dy absorption spectra was assessed. It was found that spectroscopy-oriented configuration interaction as applied to a simplified model in which the free-ion lanthanide was embedded in ligand-centered Löwdin point charges performed as well (Nd) or better (Eu and Dy) than canonical NEVPT2 calculations, when the ligand orbitals were included in the treatment.

Therapeutic benefit of combining calorie-restricted ketogenic diet and glutamine targeting in late-stage experimental glioblastoma.

Glioblastoma (GBM) is an aggressive primary human brain tumour that has resisted effective therapy for decades. Although glucose and glutamine are the major fuels that drive GBM growth and invasion, few studies have targeted these fuels for therapeutic management. The glutamine antagonist, 6-diazo-5-oxo-L-norleucine (DON), was administered together with a calorically restricted ketogenic diet (KD-R) to treat late-stage orthotopic growth in two syngeneic GBM mouse models: VM-M3 and CT-2A. DON targets glutaminolysis, while the KD-R reduces glucose and, simultaneously, elevates neuroprotective and non-fermentable ketone bodies. The diet/drug therapeutic strategy killed tumour cells while reversing disease symptoms, and improving overall mouse survival. The therapeutic strategy also reduces edema, hemorrhage, and inflammation. Moreover, the KD-R diet facilitated DON delivery to the brain and allowed a lower dosage to achieve therapeutic effect. The findings support the importance of glucose and glutamine in driving GBM growth and provide a therapeutic strategy for non-toxic metabolic management.

DART-MS in-source collision induced dissociation and high mass accuracy for new psychoactive substance determinations.

The influx of new psychoactive substances is a problem that is challenging the analytical capabilities of enforcement agencies. Cathinone designer drugs are less likely to be included in routine drug screens and typical drug formulations are commonly mixtures with continually shifting components. Ambient ionization mass spectrometry employs relatively mild conditions to desorb and ionize solid samples, imparting much less energy than that associated with conventional mass spectrometry methods. Direct analysis in real time mass spectrometry (DART-MS) is an ambient ionization method that was employed to rapidly screen cathinones, alone and in mixtures, readily enabling differentiation of the active drug(s) from various cutting agents. Accurate mass determinations provided preliminary identification of the various components of drug mixtures. The data generated in forensic mass spectrometry can be used for both elemental composition formulations and isotope abundance calculations for determination of unknown psychoactive substances, and we demonstrate how this data could be applied to the presence of new drugs as the active components shift in response to regulations. Isotope abundance calculations were used to develop a candidate pool of possible molecular formulas associated with cathinones as a specific class of designer drugs. Together, the combination of a time-of-flight (TOF) mass analyzer along with in-source collision-induced dissociation (CID) spectra were used to drastically narrow the pool of candidates to a single molecular formula. The [M+H](+) and product ion peaks provided data for presumptive analysis of various substituted synthetic cathinones in a manner that is complementary to conventional GC-MS analysis of new psychoactive substances.

DART-MS for rapid, preliminary screening of urine for DMAA.

Dimethylamylamine (DMAA) is a sympathomimetic amine found in weight-loss/workout supplements or used as an appetite suppressant. DMAA is a stimulant that is banned by the World Anti-Doping Agency (WADA). Adverse health effects as well as fatalities have been implicated with its use. Direct analysis in real time mass spectrometry (DART-MS) is an ambient ionization method that was employed to rapidly identify the presence of DMAA in various samples without any extraction or preparations whatsoever. DMAA was first identified in supplements, sampled directly in their solid forms. Furthermore, DMAA was detected directly in urine over 48 h as a means of indicating recent abuse of the substance. DART-MS analysis is instantaneous, and coupled with the high mass accuracy associated with the time-of-flight mass analyzer, results in unequivocal identification of the presence of DMAA. These features demonstrate DART-MS as an attractive potential alternative screening method for the presence of drugs and medications or for toxicological investigations.

DART-MS as a preliminary screening method for "herbal incense": chemical analysis of synthetic cannabinoids.

Direct analysis in real time mass spectrometry (DART-MS) served as a method for rapid high-throughput screening of six commercially available "Spice" products, detecting various combinations of five synthetic cannabinoids. Direct analysis in real time is an ambient ionization process that, along with high mass accuracy time-of-flight (TOF)-MS to 0.0001 Da, was employed to establish the presence of cannabinoids. Mass spectra were acquired by simply suspending a small portion of sample between the ion source and the mass spectrometer inlet. The ability to test minute amounts of sample is a major advantage when very limited amounts of evidentiary material are available. In addition, reports are widespread regarding the testing backlogs that now exist because of the large influx of designer drugs. This method circumvents time-consuming sample extraction, derivatization, chromatographic, and other sample preparative steps required for analysis by more conventional mass spectrometric methods. Accordingly, the synthetic cannabinoids AM-2201, JWH-122, JWH-203, JWH-210, and RCS-4 were identified in commercially available herbal Spice products, singly and in tandem, at concentrations within the range of 4-141 mg/g of material. Direct analysis in real time mass spectrometry decreases the time necessary to triage analytical evidence, and therefore, it has the potential to contribute to backlog reduction and more timely criminal prosecution.

Direct analysis in real time mass spectrometry (DART-MS) of "bath salt" cathinone drug mixtures.

Rapid and versatile direct analysis in real time mass spectrometry (DART-MS) methods were developed for detection and characterization of synthetic cathinone designer drugs, also known as "bath salts". The speed and efficiency associated with DART-MS testing of such highly unpredictable samples demonstrate the technique as an attractive alternative to conventional GC-MS and LC-MS methods. A series of isobaric and closely related synthetic cathinones, alone and in mixtures, were differentiated using high mass accuracy and in-source collision induced dissociation (CID). Crime laboratories have observed a dramatic rise in the use of these substances, which has caused sample testing backlogs, particularly since the myriad of structurally related compounds are challenging to efficiently differentiate. This challenge is compounded by the perpetual emergence of new structural variants as soon as older generation derivatives become scheduled. Because of the numerous chemical substances that fall into these categories, along with the varying composition and complexity of mixtures of these drugs, DART-MS CID has the potential to dramatically streamline sample analysis, minimize the number of sample preparation steps, and enable rapid characterization of emerging structural analogs.

Direct analysis in real time mass spectrometry with collision-induced dissociation for structural analysis of synthetic cannabinoids.

RATIONALE: The emergence of numerous cannabinoid designer drugs has been tied to large spikes in emergency room visits and overdoses. Identifying these substances is difficult for the following reasons: (1) the compounds are novel, closely structurally related, and do not usually test positive in drug screens; (2) novel analogs rapidly appear on the market; (3) no standard protocols exist for their identification; and (4) customized and extensive sample preparation/extraction and analysis procedures are required to demonstrate their presence. METHODS: Direct analysis in real time mass spectrometry (DART-MS) employing collision-induced dissociation (CID) provided confirmatory structural information that was useful in characterizing the various cannabinoid analogs, including those contained in mixtures. CID analysis illustrated that, although closely related compounds fragment in a similar fashion, their structural differences still resulted in multiple diagnostic peaks that provided additional confidence towards structural identification. RESULTS: DART-MS spectra were acquired under CID conditions to rapidly differentiate among five synthetic cannabinoids contained within 'herbal' products purchased locally in New York State (USA). The spectra exhibited [M+H](+) ions and product ions unique to each cannabinoid that corresponded to major structural features. Five different cannabinoid analogs, alone and as mixtures of at least two cannabinoids, were identified in six herbal products and differentiated by their CID product ion patterns. CONCLUSIONS: Illicit synthetic cannabinoid products continue to be readily available despite national and international restrictions. These products contain a wide range of active components, and, in many cases, multiple active ingredients. DART-MS allows rapid analyses of these synthetic cannabinoids based on the exact masses of their [M+H](+) ions and product ion peaks generated using CID.

Rapid identification of synthetic cannabinoids in herbal samples via direct analysis in real time mass spectrometry.

RATIONALE: Dozens of synthetic cannabinoid analogs purposefully meant to circumvent legal restrictions associated with controlled substances continue to be manufactured and promoted as producing 'legal highs'. These designer drugs are difficult to identify in conventional drug screens not only because routine protocols have not been developed for their detection, but also because their association with complex plant matrices during manufacture generally requires labor-intensive extraction and sample preparation for analysis. To address this new and important challenge in forensic chemistry, Direct Analysis in Real Time Mass Spectrometry (DART-MS) is applied to the analysis of these designer drugs. METHODS: DART-MS was employed to sample synthetic cannabinoids directly on botanical matrices. The ambient ionization method associated with DART-MS permitted the analysis of solid herbal samples directly, without the need for extraction or sample preparation. The high mass resolution time-of-flight analyzer allowed identification of these substances despite their presence within a complex matrix and enabled differentiation of closely related analogs. RESULTS: DART-MS was performed to rapidly identify the synthetic cannabinoids AM-251 and JWH-015. For each cannabinoid, three hundred micrograms (300 µg) of material was easily detected within an excess of background matrix by mass. CONCLUSIONS: New variations of herbal blends containing a wide range of base components and laced with synthetic cannabinoids are being produced, making their presence difficult to track by conventional methods. DART-MS permits rapid identification of trace synthetic cannabinoids within complex biological matrices, with excellent sensitivity and specificity compared with standard methods.

Routine analysis and characterization of highly insoluble polycyclic aromatic compounds by direct analysis in real time mass spectrometry (DART).

Direct Analysis in Real Time (DART), is used for the first time for the routine rapid analysis of highly insoluble polycyclic aromatic compounds. Direct analysis of such compounds as solid samples under solvent-free conditions shows that DART is a powerful analytical platform capable of providing high-throughput analysis for these complex samples, requiring no special sample pre-treatment or instrument setup.

HRMS directly from TLC slides. A powerful tool for rapid analysis of organic mixtures.

High-resolution mass spectra (HRMS) of individual spots on thin-layer chromatography (TLC) slides can now be obtained quickly and easily at atmospheric pressure, with zero sample preparation, using commercially available instrumentation. The method is complementary to GC-mass spectrometry but is not limited to compounds of high volatility and high thermal stability. TLC-HRMS can be used to monitor chemical reactions in real time and has the capacity thereby to accelerate significantly the pace of synthetic organic chemistry.