Utilization of bis-MPA Dendrimers for the Calibration of Ion Mobility Collision Cross Section Calculations.

Ion mobility-mass spectrometry (IM-MS) has become increasingly popular with the rapid expansion of available techniques and instrumentation. To enable accuracy, standardization, and repeatability of IM-MS measurements, the community requires reliable and well-defined reference materials for calibration and tuning of the equipment. To address this need, synthetic dendrimers of high chemical and structural purity were tested on three ion mobility platforms as potential calibrants. First, synthesized dendrimers were characterized by drift tube ion mobility (DTIMS), using an Agilent 6560 IM-qTOF-MS to assess their drift tube collision cross section (DTCCS) values. Then, assessment of obtained CCS values on trapped ion mobility (TIMS) and traveling wave ion mobility (TWIMS) ion mobility platforms were compared to those found by DTIMS. Across all three systems, dendrimers were found to have high potential for m/z and ion mobility calibration in the CCS range of 160-1700 Å2. To further validate their use as calibrants, drift tube calculated CCS values for dendrimers were utilized to calibrate calculations of CCS for known standards including Agilent Tuning mix, the CCS Major mix from Waters, and SPLASH LIPIDOMIX. Additionally, structures of sodiated dendrimers were computated along with theoretical CCS values which showed good agreement with the experimental CCS values. On the basis of the results presented, we recommend the use of dendrimers as alternatives and/or complementary compounds to commonly used calibrants for ion mobility platforms.

Increased renal elimination of endogenous and synthetic pyrimidine nucleosides in concentrative nucleoside transporter 1 deficient mice.

Concentrative nucleoside transporters (CNTs) are active nucleoside influx systems, but their in vivo roles are poorly defined. By generating CNT1 knockout (KO) mice, here we identify a role of CNT1 in the renal reabsorption of nucleosides. Deletion of CNT1 in mice increases the urinary excretion of endogenous pyrimidine nucleosides with compensatory alterations in purine nucleoside metabolism. In addition, CNT1 KO mice exhibits high urinary excretion of the nucleoside analog gemcitabine (dFdC), which results in poor tumor growth control in CNT1 KO mice harboring syngeneic pancreatic tumors. Interestingly, increasing the dFdC dose to attain an area under the concentration-time curve level equivalent to that achieved by wild-type (WT) mice rescues antitumor efficacy. The findings provide new insights into how CNT1 regulates reabsorption of endogenous and synthetic nucleosides in murine kidneys and suggest that the functional status of CNTs may account for the optimal action of pyrimidine nucleoside analog therapeutics in humans.

Simulation of Energy-Resolved Mass Spectrometry Distributions from Surface-Induced Dissociation.

Understanding the relationship between protein structure and experimental data is crucial for utilizing experiments to solve biochemical problems and optimizing the use of sparse experimental data for structural interpretation. Tandem mass spectrometry (MS/MS) can be used with a variety of methods to collect structural data for proteins. One example is surface-induced dissociation (SID), which is used to break apart protein complexes (via a surface collision) into intact subcomplexes and can be performed at multiple laboratory frame SID collision energies. These energy-resolved MS/MS experiments have shown that the profile of the breakages depends on the acceleration energy of the collision. It is possible to extract an appearance energy (AE) from energy-resolved mass spectrometry (ERMS) data, which shows the relative intensity of each type of subcomplex as a function of SID acceleration energy. We previously determined that these AE values for specific interfaces correlated with structural features related to interface strength. In this study, we further examined the structural relationships by developing a method to predict the full ERMS plot from the structure, rather than extracting a single value. First, we noted that for proteins with multiple interface types, we could reproduce the correct shapes of breakdown curves, further confirming previous structural hypotheses. Next, we demonstrated that interface size and energy density (measured using Rosetta) correlated with data derived from the ERMS plot (R2 = 0.71). Furthermore, based on this trend, we used native crystal structures to predict ERMS. The majority of predictions resulted in good agreement, and the average root-mean-square error was 0.20 for the 20 complexes in our data set. We also show that if additional information on cleavage as a function of collision energy could be obtained, the accuracy of predictions improved further. Finally, we demonstrated that ERMS prediction results were better for the native than for inaccurate models in 17/20 cases. An application to run this simulation has been developed in Rosetta, which is freely available for use.

Detection and characterization of a rare two-component lantibiotic, amyloliquecidin GF610 produced by Bacillus velezensis, using a combination of culture, molecular and bioinformatic analyses.

AIM: To detect and characterize novel lantibiotics from a collection of Bacillus spp. using a multifaceted analytical approach. METHODS AND RESULTS: A previously completed microassay identified 45 Bacillus isolates with anti-Listeria activity. The isolates were PCR screened using degenerate primers targeting conserved sequences in lanM-type lantibiotics. B. velezensis GF610 produced a PCR product whose sequence, along with genome mining and bioinformatics, guided the liquid chromatographic analysis of strain's cell-free extracts and the mass spectrometry of purified fractions. Results revealed a new amyloliquecidin variant (designated GF610) produced by the strain. Amyloliquecidin GF610 is a two-component lantibiotic with α and ß peptides having monoisotopic masses of 3026 and 2451 Da, and molecular formulae C130 H191 N35 O39 S5 and C110 H158 N26 O30 S4 , respectively. Amyloliquecidin GF610 is active against Listeria monocytogenes, Clostridium sporogenes, Clostridioides difficile, Staphylococcus aureus and Alicyclobacillus acidoterrestris with minimum inhibitory concentrations (MICs) in the range of 0.5-7.0 µmol l-1 . CONCLUSIONS: The proposed multifaceted analytical approach was valuable to provide a deep and proper characterization of a novel bacteriocin, amyloliquecidin GF610, with high antimicrobial activity against Gram-positive bacteria. SIGNIFICANCE AND IMPACT: The discovered Amyloliquecidin GF610 is potentially useful in food, agricultural or medical applications. The analytical approach followed may facilitate future discoveries of two-component lantibiotics, which are challenging compounds to detect and characterize.

Destruction and reconstruction of UO22+ using gas-phase reactions.

While the strong axial U[double bond, length as m-dash]O bonds confer high stability and inertness to UO22+, it has been shown that the axial oxo ligands can be eliminated or replaced in the gas-phase using collision-induced dissociation (CID) reactions. We report here tandem mass spectrometry experiments initiated with a gas-phase complex that includes UO22+ coordinated by a 2,6-difluorobenzoate ligand. After decarboxylation to form a difluorophenide coordinated uranyl ion, [UO2(C6F2H3)]+, CID causes elimination of CO, and then CO and C2H2 in sequential dissociation steps, to leave a reactive uranium fluoride ion, [UF2(C2H)]+. Reaction of [UF2(C2H)]+ with CH3OH creates [UF2(OCH3)]+, [UF(OCH3)2]+ and [UF(OCH3)2(CH3OH)]+. Cleavage of C-O bonds within these species results in the elimination of methyl cation (CH3+). Subsequent CID steps convert [UF(OCH3)2]+ to [UO2(F)]+ and similarly, [U(OCH3)3]+ to [UO2(OCH3)]+. Our experiments show removal of both uranyl oxo ligands in "top-down" CID reactions and replacement in "bottom-up" ion-molecule and dissociation steps.

Tandem surface-induced dissociation of protein complexes on an ultrahigh resolution platform.

We describe instrumentation for conducting tandem surface-induced dissociation (tSID) of native protein complexes on an ultrahigh resolution Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. The two stages of SID are accomplished with split lenses replacing the entrance lenses of the quadrupole mass filter (stage 1, referred to herein as SID-Q) and the collision cell (stage 2, Q-SID). After SID-Q, the scattered projectile ions and subcomplexes formed in transit traverse the 20 mm pre-filter prior to the mass-selecting quadrupole, providing preliminary insights into the SID fragmentation kinetics of noncovalent protein complexes. The isolated SID fragments (subcomplexes) are then fragmented by SID in the collision cell entrance lens (Q-SID), generating subcomplexes of subcomplexes. We show that the ultrahigh resolution of the FT-ICR can be used for deconvolving species overlapping in m/z, which are particularly prominent in tandem SID spectra due to the combination of symmetric charge partitioning and narrow product ion charge state distributions. Various protein complex topologies are explored, including homotetramers, homopentamers, a homohexamer, and a heterohexamer.

Surface-Induced Dissociation of Protein Complexes Selected by Trapped Ion Mobility Spectrometry.

Native mass spectrometry (nMS), particularly in conjunction with gas-phase ion mobility spectrometry measurements, has proven useful as a structural biology tool for evaluating the stoichiometry, conformation, and topology of protein complexes. Here, we demonstrate the combination of trapped ion mobility spectrometry (TIMS) and surface-induced dissociation (SID) on a Bruker SolariX XR 15 T FT-ICR mass spectrometer for the structural analysis of protein complexes. We successfully performed SID on mobility-selected protein complexes, including the streptavidin tetramer and cholera toxin B with bound ligands. Additionally, TIMS-SID was employed on a mixture of the peptides desArg1 and desArg9 bradykinin to mobility-separate and identify the individual peptides. Importantly, results show that native-like conformations can be maintained throughout the TIMS analysis. The TIMS-SID spectra are analogous to SID spectra acquired using quadrupole mass selection, indicating little measurable, if any, structural rearrangement during mobility selection. Mobility parking was used on the ion or mobility of interest and 50-200 SID mass spectra were averaged. High-quality TIMS-SID spectra were acquired over a period of 2-10 min, comparable to or slightly longer than SID coupled with ion mobility on various instrument platforms in our laboratory. The ultrahigh resolving power of the 15 T FT-ICR allowed for the identification and relative quantification of overlapping SID fragments with the same nominal m/z based on isotope patterns, and it shows promise as a platform to probe small mass differences, such as protein/ligand binding or post-translational modifications. These results represent the potential of TIMS-SID-MS for the analysis of both protein complexes and peptides.

Proanthocyanidin Structural Details Revealed by Ultrahigh Resolution FT-ICR MALDI-Mass Spectrometry, 1H-13C HSQC NMR, and Thiolysis-HPLC-DAD.

Proanthocyanidins (condensed tannins) are important in food chemistry, agriculture, and health, driving demand for improvements in structure determination. We used ultrahigh resolution Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR MS) methods to determine the exact composition of individual species in heterogeneous mixtures of proanthocyanidin polymers from Sorghum bicolor grain and Neptunia lutea leaves. Fragmentation patterns obtained with FT-ICR ESI MS-MS (electrospray ionization) confirmed structural details from thiolysis-high-performance liquid chromatography (HPLC)-diode array detection (DAD) and 1H-13C heteronuclear single quantum coherence (HSQC) NMR. We found that A-type linkages were characteristic of shorter polymers in predominantly B-linked proanthocyanidin. We suggest that supramolecular complex formation between proanthocyanidins and matrix components such as 2,5-dihydroxybenzoic acid was responsible for anomalous 152 dalton peaks, incorrectly assigned as 3-O-galloylation, when using FT-ICR matrix-assisted laser desorption ionization (MALDI-MS). Our data illustrate the power of the ultrahigh resolution FT-ICR methods but include the caveat that MALDI-MS must be paired with complementary analytical tools to avoid artifacts.

Genome-Guided Mass Spectrometry Expedited the Discovery of Paraplantaricin TC318, a Lantibiotic Produced by Lactobacillus paraplantarum Strain Isolated From Cheese.

The quest for potent alternatives to the currently used antimicrobials is urged by health professionals, considering the rapid rise in resistance to preservatives and antibiotics among pathogens. The current study was initiated to search for novel and effective bacteriocins from food microbes, preferably lactic acid bacteria (LAB), for potential use as preservatives. Advances in genome-guided mass spectrometry (MS) were implemented to expedite identifying and elucidating the structure of the recovered antimicrobial agent. A LAB strain, OSY-TC318, was isolated from a Turkish cheese, and the crude extract of the cultured strain inhibited the growth of various pathogenic and spoilage bacteria such as Bacillus cereus, Clostridium sporogenes, Enterococcus faecalis, Listeria monocytogenes, Salmonella enterica ser. Typhimurium, and Staphylococcus aureus. The antimicrobial producer was identified as Lactobacillus paraplantarum using MS biotyping and genomic analysis. Additionally, L. paraplantarum OSY-TC318 was distinguished from closely related strains using comparative genomic analysis. Based on in silico analysis, the genome of the new strain contained a complete lantibiotic biosynthetic gene cluster, encoding a novel lantibiotic that was designated as paraplantaricin TC318. The bioinformatic analysis of the gene cluster led to the prediction of the biosynthetic pathway, amino acid sequence, and theoretical molecular mass of paraplantaricin TC318. To verify the genomic analysis predictions, paraplantaricin TC318 was purified from the producer cellular crude extract using liquid chromatography, followed by structural elucidation using Fourier transform ion cyclotron resonance MS analysis. This genome-guided MS analysis revealed that the molecular mass of paraplantaricin TC318 is 2,263.900 Da, its chemical formula is C106H133N27O22S4, and its primary sequence is F-K-S-W-S-L-C-T-F-G-C-G-H-T-G-S-F-N-S-F-C-C. This lantibiotic, which differs from mutacin 1140 at positions 9, 12, 13, and 20, is considered a new member of the epidermin group in class I lantibiotics. In conclusion, the study revealed a new L. paraplantarum strain producing a novel lantibiotic that is potentially useful in food and medical applications.

Obesogenic Memory Maintains Adipose Tissue Inflammation and Insulin Resistance.

BACKGROUND: Obesity is characterized by visceral adipose tissue (AT) inflammation. Immunosuppressive regulatory T cells (Tregs), phagocytic M2-like macrophages, and innate lymphoid cells type 2 (ILC2) control lean AT inflammation to maintain systemic insulin sensitivity, while the loss of these cells in obesity leads to AT inflammation and insulin resistance (IR). OBJECTIVE: The objective of this study was to determine if weight loss following obesity would correct AT inflammation and systemic metabolism. RESULTS: After six months of high fat diet (HFD) in male C57/Bl6 mice, flow analyses of epidydimal AT stromal vascular fraction (SVF) revealed depleted Tregs by 50%, doubling of CD8+ T cells, tripling of pro-inflammatory M1-like macrophages, and an 80% drop in ILC2 cells associated with changes in pro-inflammatory adipocyte and macrophage gene expression. Despite normalization of body weight, fat, and adipocyte size, mice ingesting 3 months of high-fat diet (HFD) followed by 3 months of chow-diet remained more insulin resistant and glucose intolerant than chow-fed animals. Adipocytes, AT Tregs, CD8+ T cells, ILC2 cells, and M1-like macrophages all failed to normalize with weight loss. CONCLUSIONS: Persistent AT inflammation contributes to the maintenance of IR despite body weight and fat normalization in previously obese mice. These findings highlight the importance of obesity prevention to avoid the consequences of "obesogenic memory."

Simple and Minimally Invasive SID Devices for Native Mass Spectrometry.

We describe a set of simple devices for surface-induced dissociation of proteins and protein complexes on three instrument platforms. All of the devices use a novel yet simple split lens geometry that is minimally invasive (requiring a few millimeters along the ion path axis) and is easier to operate than prior generations of devices. The split lens is designed to be small enough to replace the entrance lens of a Bruker FT-ICR collision cell, the dynamic range enhancement (DRE) lens of a Waters Q-IM-TOF, or the exit lens of a transfer multipole of a Thermo Scientific Extended Mass Range (EMR) Orbitrap. Despite the decrease in size and reduction in number of electrodes to 3 (from 10 to 12 in Gen 1 and ∼6 in Gen 2), we show sensitivity improvement in a variety of cases across all platforms while also maintaining SID capabilities across a wide mass and energy range. The coupling of SID, high resolution, and ion mobility is demonstrated for a variety of protein complexes of varying topologies.

Stimulation of retrotrapezoid nucleus Phox2b-expressing neurons rescues breathing dysfunction in an experimental Parkinson's disease rat model.

Emerging evidence from multiple studies indicates that Parkinson's disease (PD) patients suffer from a spectrum of autonomic and respiratory motor deficiencies in addition to the classical motor symptoms attributed to substantia nigra degeneration of dopaminergic neurons. Animal models of PD show a decrease in the resting respiratory rate as well as a decrease in the number of Phox2b-expressing retrotrapezoid nucleus (RTN) neurons. The aim of this study was to determine the extent to which substantia nigra pars compact (SNc) degeneration induced RTN biomolecular changes and to identify the extent to which RTN pharmacological or optogenetic stimulations rescue respiratory function following PD-induction. SNc degeneration was achieved in adult male Wistar rats by bilateral striatal 6-hydroxydopamine injection. For proteomic analysis, laser capture microdissection and pressure catapulting were used to isolate the RTN for subsequent comparative proteomic analysis and Ingenuity Pathway Analysis (IPA). The respiratory parameters were evaluated by whole-body plethysmography and electromyographic analysis of respiratory muscles. The results confirmed reduction in the number of dopaminergic neurons of SNc and respiratory rate in the PD-animals. Our proteomic data suggested extensive RTN remodeling, and that pharmacological or optogenetic stimulations of the diseased RTN neurons promoted rescued the respiratory deficiency. Our data indicate that despite neuroanatomical and biomolecular RTN pathologies, that RTN-directed interventions can rescue respiratory control dysfunction.

Chemical Alterations of Dissolved Organic Matter by Permanganate Oxidation.

Dissolved organic matter (DOM) is ubiquitous in raw drinking water and can efficiently scavenge oxidants, such as permanganate. Here, changes to DOM induced by permanganate oxidation under typical drinking water treatment conditions (6 µM, 1 h) to bulk DOM properties, DOM functional groups, and DOM chemical formulae were examined for two DOM isolate types (terrestrial and microbial). Permanganate oxidation did not mineralize DOM, rather changes were compositional in nature. Optical properties suggest that permanganate oxidation decreased DOM aromaticity (decreased SUVA-254), decreased DOM electron-donating capacity, and decreased DOM average molecular weight (increased E2/E3 ratios). Fourier-transform-infrared spectroscopy second derivative analyses revealed that permanganate does not oxidize DOM alkene groups, suggesting permanganate access to functional groups may be important. Four ionization techniques were used with ultrahigh-resolution mass spectrometry: negative and positive ion mode electrospray ionization and negative and positive ion mode laser/desorption ionization. The results from all four techniques were combined to understand changes in DOM chemical formulae. It was concluded that nitrogen-containing aromatic compounds and alkylbenzenes were oxidized by permanganate to form nitrogen-containing aliphatic compounds and benzoic acid-containing compounds. This work highlights how multiple ionization techniques coupled with UHR-MS can enable a more detailed characterization of DOM.

Application of UAV in Topographic Modelling and Structural Geological Mapping of Quarries and Their Surroundings-Delineation of Fault-Bordered Raw Material Reserves.

A 3D surface model of an active limestone quarry and a vegetation-covered plateau was created using unmanned aerial vehicle (UAV) technique in combination with terrestrial laser scanning (TLS). The aim of the research was to identify major fault zones that dissect the inaccessible quarry faces and to prepare a model that shows the location of these fault zones at the entire study area. An additional purpose was to calculate reserves of the four identified lithological units. It was only possible to measure faults at the lowermost two meters of the quarry faces. At the upper parts of the quarry and on the vegetation-covered plateau where no field geological information was available, remote sensing was used. Former logs of core drillings were obtained for the modelling of the spatial distribution of four lithological units representing cover beds and various quality of limestone reserves. With the comparison of core data, field measurements and remote sensing, it was possible to depict major faults. Waste material volumes and limestone reserves were calculated for five blocks that are surrounded by these faults. The paper demonstrates that, with remote sensing and with localised control field measurements, it is possible: (a) to provide all geometric data of faults and (b) to create a 3D model with fault planes even at no exposure or at hardly accessible areas. The surface model with detected faults serves as a basis for calculating geological reserves.

Trimethylamine N-oxide and incident atherosclerotic events in high-risk individuals with diabetes: an ACCORD trial post hoc analysis.

Introduction: Type 2 diabetes mellitus (T2D) confers high atherosclerotic cardiovascular disease (ASCVD) risk. The metabolite trimethylamine N-oxide (TMAO) derived via gut flora has been linked to excess ASCVD. Research design and methods: We analyzed data, biospecimens, and major adverse cardiovascular events (MACEs) from the prospective multicenter randomized Action to Control Cardiovascular Risk in Diabetes (ACCORD) trial to assess its value in 330 high-risk individuals with T2D without evident atherosclerotic disease at enrollment. Results: Incident cardiovascular events occurred in 165 cases; 165 controls matched by age, sex, and treatment arm experienced no incident events during follow-up. Cases and controls (mean age 64.5 years) had similar mean glycated hemoglobin (HbA1c) (8.2%) and mean 10-year ASCVD risk (23.5%); groups also had similar use of statins and antihypertensive medications at baseline and follow-up. Baseline plasma TMAO levels did not differ between groups after adjusting for ASCVD risk score, HbA1c, and estimated glomerular filtration rate, nor did TMAO distinguish patients suffering incident MACE from those who remained event-free. Conclusions: TMAO's prognostic value for incident ASCVD events may be blunted when applied to individuals with T2D with poor glycemic control and high baseline ASCVD risk. These results behoove further translational investigations of unique mechanisms of ASCVD risk in T2D.

Accurate and Efficient Determination of Unknown Metabolites in Metabolomics by NMR-Based Molecular Motif Identification.

Knowledge of the chemical identity of metabolite molecules is critical for the understanding of the complex biological systems to which they belong. Since metabolite identities and their concentrations are often directly linked to the phenotype, such information can be used to map biochemical pathways and understand their role in health and disease. A very large number of metabolites however are still unknown; i.e., their spectroscopic signatures do not match those in existing databases, suggesting unknown molecule identification is both imperative and challenging. Although metabolites are structurally highly diverse, the majority shares a rather limited number of structural motifs, which are defined by sets of 1H and 13C chemical shifts of the same spin system. This allows one to characterize unknown metabolites by a divide-and-conquer strategy that identifies their structural motifs first. Here, we present the structural motif-based approach "SUMMIT Motif" for the de novo identification of unknown molecular structures in complex mixtures, without the need for extensive purification, using NMR in tandem with two newly curated NMR molecular structural motif metabolomics databases (MSMMDBs). For the identification of structural motif(s), first, the 1H and 13C chemical shifts of all the individual spin systems are extracted from 2D and 3D NMR spectra of the complex mixture. Next, the molecular structural motifs are identified by querying these chemical shifts against the new MSMMDBs. One database, COLMAR MSMMDB, was derived from experimental NMR chemical shifts of known metabolites taken from the COLMAR metabolomics database, while the other MSMMDB, pNMR MSMMDB, is based on predicted chemical shifts of metabolites of several existing large metabolomics databases. For molecules consisting of multiple spin systems, spin systems are connected via long-range scalar J-couplings. When this motif-based identification method was applied to the hydrophilic extract of mouse bile fluid, two unknown metabolites could be successfully identified. This approach is both accurate and efficient for the identification of unknown metabolites and hence enables the discovery of new biochemical processes and potential biomarkers.

Design and Performance of a Second-Generation Surface-Induced Dissociation Cell for Fourier Transform Ion Cyclotron Resonance Mass Spectrometry of Native Protein Complexes.

A second-generation ("Gen 2") device capable of surface-induced dissociation (SID) and collision-induced dissociation (CID) for Fourier transform ion cyclotron resonance mass spectrometry of protein complexes has been designed, simulated, fabricated, and experimentally compared to a first-generation device ("Gen 1"). The primary goals of the redesign were to (1) simplify SID by reducing the number of electrodes, (2) increase CID and SID sensitivity by lengthening the collision cell, and (3) increase the mass range of the device for analysis of larger multimeric proteins, all while maintaining the normal instrument configuration and operation. Compared to Gen 1, Gen 2 exhibits an approximately 10× increase in sensitivity in flythrough mode, 7× increase in CID sensitivity for protonated leucine enkephalin (m/z 556), and 14× increase of CID sensitivity of 53 kDa streptavidin tetramer. It also approximately doubles the useful mass range (from m/z 8000 to m/z 15 000) using a rectilinear ion trap with a smaller inscribed radius or triples it (to m/z 22 000) using a hexapole collision cell and yields a 3-10× increase in SID sensitivity. We demonstrate the increased mass range and sensitivity on a variety of model molecules spanning nearly 3 orders of magnitude in absolute mass and present examples where the high resolution of the FT-ICR is advantageous for deconvoluting overlapping SID fragments.

Metabolic Regulation of Glycolysis and AMP Activated Protein Kinase Pathways during Black Raspberry-Mediated Oral Cancer Chemoprevention.

Oral cancer is a public health problem with an incidence of almost 50,000 and a mortality of 10,000 each year in the USA alone. Black raspberries (BRBs) have been shown to inhibit oral carcinogenesis in several preclinical models, but our understanding of how BRB phytochemicals affect the metabolic pathways during oral carcinogenesis remains incomplete. We used a well-established rat oral cancer model to determine potential metabolic pathways impacted by BRBs during oral carcinogenesis. F344 rats were exposed to the oral carcinogen 4-nitroquinoline-1-oxide in drinking water for 14 weeks, then regular drinking water for six weeks. Carcinogen exposed rats were fed a 5% or 10% BRB supplemented diet or control diet for six weeks after carcinogen exposure. RNA-Seq transcriptome analysis on rat tongue, and mass spectrometry and NMR metabolomics analysis on rat urine were performed. We tentatively identified 57 differentially or uniquely expressed metabolites and over 662 modulated genes in rats being fed with BRB. Glycolysis and AMPK pathways were modulated during BRB-mediated oral cancer chemoprevention. Glycolytic enzymes Aldoa, Hk2, Tpi1, Pgam2, Pfkl, and Pkm2 as well as the PKA-AMPK pathway genes Prkaa2, Pde4a, Pde10a, Ywhag, and Crebbp were downregulated by BRBs during oral cancer chemoprevention. Furthermore, the glycolysis metabolite glucose-6-phosphate decreased in BRB-administered rats. Our data reveal the novel metabolic pathways modulated by BRB phytochemicals that can be targeted during the chemoprevention of oral cancer.

Spectroscopic Evidence for Lactam Formation in Terminal Ornithine b2+ and b3+ Fragment Ions.

Infrared multiple photon dissociation action spectroscopy was performed on the AlaOrn b2+ and AlaAlaOrn b3+ fragment ions from ornithine-containing tetrapeptides. Infrared spectra were obtained in the fingerprint region (1000-2000 cm-1) using the infrared free electron lasers at the Centre Laser Infrarouge d'Orsay (CLIO) facility in Orsay, France, and the free electron lasers for infrared experiments (FELIX) facility in Nijmegen, the Netherlands. A novel terminal ornithine lactam AO+ b2+ structure was synthesized for experimental comparison and spectroscopy confirms that the b2+ fragment ion from AOAA forms a lactam structure. Comparison of experimental spectra with scaled harmonic frequencies at the B3LYP/6-31+G(d,p) level of theory shows that AO+ b2+ forms a terminal lactam protonated either on the lactam carbonyl oxygen or the N-terminal nitrogen atom. Several low-lying conformers of these isomers are likely populated following IRMPD dissociation. Similarly, a comparison of the experimental IRMPD spectrum with calculated spectra shows that AAO+ b3+-ions also adopt a lactam structure, again with multiple different protonation sites, during fragmentation. This study provides spectroscopic confirmation for the lactam cyclization proposed for the "ornithine effect" and represents an alternative bn+ structure to the oxazolone and diketopiperazine/macrocycle structures most often formed.