Pinoresinol rescues developmental phenotypes of Arabidopsis phenylpropanoid mutants overexpressing FERULATE 5-HYDROXYLASE.

Most phenylpropanoid pathway flux is directed toward the production of monolignols, but this pathway also generates multiple bioactive metabolites. The monolignols coniferyl and sinapyl alcohol polymerize to form guaiacyl (G) and syringyl (S) units in lignin, components that are characteristic of plant secondary cell walls. Lignin negatively impacts the saccharification potential of lignocellulosic biomass. Although manipulation of its content and composition through genetic engineering has reduced biomass recalcitrance, in some cases, these genetic manipulations lead to impaired growth. The reduced-growth phenotype is often attributed to poor water transport due to xylem collapse in low-lignin mutants, but alternative models suggest that it could be caused by the hyper- or hypoaccumulation of phenylpropanoid intermediates. In Arabidopsis thaliana, overexpression of FERULATE 5-HYDROXYLASE (F5H) shifts the normal G/S lignin ratio to nearly pure S lignin and does not result in substantial changes to plant growth. In contrast, when we overexpressed F5H in the low-lignin mutants cinnamyl dehydrogenase c and d (cadc cadd), cinnamoyl-CoA reductase 1, and reduced epidermal fluorescence 3, plant growth was severely compromised. In addition, cadc cadd plants overexpressing F5H exhibited defects in lateral root development. Exogenous coniferyl alcohol (CA) and its dimeric coupling product, pinoresinol, rescue these phenotypes. These data suggest that mutations in the phenylpropanoid pathway limit the biosynthesis of pinoresinol, and this effect is exacerbated by overexpression of F5H, which further draws down cellular pools of its precursor, CA. Overall, these genetic manipulations appear to restrict the synthesis of pinoresinol or a downstream metabolite that is necessary for plant growth.

Quantitative Proteomic and Metabolomic Profiling Reveals Altered Mitochondrial Metabolism and Folate Biosynthesis Pathways in the Aging Drosophila Eye.

Aging is associated with increased risk of ocular disease, suggesting that age-associated molecular changes in the eye increase its vulnerability to damage. Although there are common pathways involved in aging at an organismal level, different tissues and cell types exhibit specific changes in gene expression with advanced age. Drosophila melanogaster is an established model system for studying aging and neurodegenerative disease that also provides a valuable model for studying age-associated ocular disease. Flies, like humans, exhibit decreased visual function and increased risk of retinal degeneration with age. Here, we profiled the aging proteome and metabolome of the Drosophila eye and compared these data with age-associated transcriptomic changes from both eyes and photoreceptors to identify alterations in pathways that could lead to age-related phenotypes in the eye. Of note, the proteomic and metabolomic changes observed in the aging eye are distinct from those observed in the head or whole fly, suggesting that tissue-specific changes in protein abundance and metabolism occur in the aging fly. Our integration of the proteomic, metabolomic, and transcriptomic data reveals that changes in metabolism, potentially due to decreases in availability of B vitamins, together with chronic activation of the immune response, may underpin many of the events observed in the aging Drosophila eye. We propose that targeting these pathways in the genetically tractable Drosophila system may help to identify potential neuroprotective approaches for neurodegenerative and age-related ocular diseases. Data are available via ProteomeXchange with identifier PXD027090.

Metabolomic signature of spermatozoa established during holding time is responsible for differences in boar sperm freezability†.

Holding at room temperature is the first step in most boar semen cryopreservation protocols. It is well accepted that a holding time (HT) of 24 h increases sperm cryotolerance. However, the effect of HT on ejaculates with different freezability is not entirely clear. The aim of this study was to understand how HT influences spermatic and seminal plasma metabolite profiles of boar ejaculates and how these possible changes affect freezability. A total of 27 ejaculates were collected and extended to 1:1 (v: v) with BTS and split into two aliquots. The first aliquot was cryopreserved without HT (0 h), and the second was held at 17°C for 24 h before cryopreservation. Spermatozoa and seminal plasma were collected by centrifugation at two times, before HT (0 h) and after HT (24 h), and subsequently frozen until metabolite extraction and UPLC-MS analysis. After thawing, the semen samples were evaluated for kinetics, membrane integrity, mitochondrial potential, membrane lipid peroxidation, and fluidity. The ejaculates were then allocated into two phenotypes (good ejaculate freezers [GEF] and poor ejaculate freezers [PEF]) based on the percent reduction in sperm quality (%RSQ) as determined by the difference in total motility and membrane integrity between raw and post-thaw samples cryopreserved after 24 h of HT. The metabolic profile of the seminal plasma did not seem to influence ejaculate freezability, but that of the spermatozoa were markedly different between GEF and PEF. We identified a number of metabolic markers in the sperm cells (including inosine, hypoxanthine, creatine, ADP, niacinamide, spermine, and 2-methylbutyrylcarnitine) that were directly related to the improvement of ejaculate freezability during HT; these were components of metabolic pathways associated with energy production. Furthermore, PEF showed an upregulation in the arginine and proline as well as the glutathione metabolism pathways. These findings help to better understand the effect of HT on boar sperm freezability and propose prospective metabolic markers that may predict freezability; this has implications in both basic and applied sciences.

Repurposing FDA-approved sulphonamide carbonic anhydrase inhibitors for treatment of Neisseria gonorrhoeae.

Neisseria gonorrhoeae is a high-priority pathogen of concern due to the growing prevalence of resistance development against approved antibiotics. Herein, we report the anti-gonococcal activity of ethoxzolamide, the FDA-approved human carbonic anhydrase inhibitor. Ethoxzolamide displayed an MIC50, against a panel of N. gonorrhoeae isolates, of 0.125 µg/mL, 16-fold more potent than acetazolamide, although both molecules exhibited almost similar potency against the gonococcal carbonic anhydrase enzyme (NgCA) in vitro. Acetazolamide displayed an inhibition constant (Ki) versus NgCA of 74 nM, while Ethoxzolamide's Ki was estimated to 94 nM. Therefore, the increased anti-gonococcal potency of ethoxzolamide was attributed to its increased permeability in N. gonorrhoeae as compared to that of acetazolamide. Both drugs demonstrated bacteriostatic activity against N. gonorrhoeae, exhibited post-antibiotic effects up to 10 hours, and resistance was not observed against both. Taken together, these results indicate that acetazolamide and ethoxzolamide warrant further investigation for translation into effective anti-N. gonorrhoeae agents.

Repeatability of in vitro carboplatin elution from carboplatin-impregnated calcium sulfate hemihydrate beads made in a clinic setting.

OBJECTIVE: To evaluate the intra-lot and inter-lot consistency and total carboplatin elution over 25 days from carboplatin-impregnated calcium sulfate hemihydrate (C-I CSH) beads manufactured in a clinic setting. STUDY DESIGN: In vitro elution study. METHODS: Two volumes of carboplatin were mixed with CSH to yield 4 mg and 8 mg C-I CSH doses. Two lots of beads were made for each concentration and split into five doses (n = 10 per concentration). Beads hardened in molds and were placed in a covered six-well plate, submerged in phosphate-buffered saline, and incubated with samples collected at 12 time points (0, 6, 12, and 24 hours and 2, 3, 5, 7, 10, 14, 18, and 25 days). The amount of carboplatin in each sample was evaluated by high-performance liquid chromatography/mass spectrometry. Correction for carboplatin degradation and dilution was applied, and eluted carboplatin was calculated. Intra-lot and inter-lot coefficient of variation (CV) was calculated for each concentration. RESULTS: The intra-lot CV ranged between 7.9% and 23.1%, and the inter-lot CV ranged from 3.5% to 10.3%, with improvement noted in each successive lot of beads. Mean peak eluted carboplatin was 2.45 ± 0.43 mg (61%) and 3.68 ± 0.41 mg (45.9%) for the 4-mg and 8-mg C-I CSH beads, respectively, with both occurring at the 12-hour timepoint. CONCLUSION: Progressive improvement in variability with successive lots of beads indicated a learning curve with bead manufacturing with a low variation both within and between lots of C-I CSH beads. CLINICAL SIGNIFICANCE: On-site mixing of carboplatin with commercial CSH bead powder leads to a low variation of carboplatin per bead dose.

The Cell Wall Arabinose-Deficient Arabidopsis thaliana Mutant murus5 Encodes a Defective Allele of REVERSIBLY GLYCOSYLATED POLYPEPTIDE2.

Traditional marker-based mapping and next-generation sequencing was used to determine that the Arabidopsis (Arabidopsis thaliana) low cell wall arabinose mutant murus5 (mur5) encodes a defective allele of REVERSIBLY GLYCOSYLATED POLYPEPTIDE2 (RGP2). Marker analysis of 13 F2 confirmed mutant progeny from a recombinant mapping population gave a rough map position on the upper arm of chromosome 5, and deep sequencing of DNA from these 13 lines gave five candidate genes with G→A (C→T) transitions predicted to result in amino acid changes. Of these five, only insertional mutant alleles of RGP2, a gene that encodes a UDP-arabinose mutase that interconverts UDP-arabinopyranose and UDP-arabinofuranose, exhibited the low cell wall arabinose phenotype. The identities of mur5 and two SALK insertional alleles were confirmed by allelism tests and overexpression of wild-type RGP2 complementary DNA placed under the control of the 35S promoter in the three alleles. The mur5 mutation results in the conversion of cysteine-257 to tyrosine-257 within a conserved hydrophobic cluster predicted to be distal to the active site and essential for protein stability and possible heterodimerization with other isoforms of RGP.

HPLC-MS Analysis of Lichen-Derived Metabolites in the Life Stages of Crambidia cephalica (Grote & Robinson).

Tiger moths (Lepidoptera: Erebidae: Arctiinae: Arctiini) are notable for their specialized associations with hosts that produce toxic secondary compounds, and are thus an ideal study system for understanding insect-plant interactions and the evolution of antipredatory defense. Likewise, their sister lineage (Arctiinae: Lithosiini) has been documented feeding on algae and lichens, and is known to sequester lichen-derived secondary compounds from the larval to adult stages. Prevalence of lichenivory in this early radiation (ca. 3000 species) may provide clues to the phylogenetic basis for storied chemical sequestration within all tiger moths. Despite the evolutionary significance of this trait, we lack a basic understanding of the extent of lichenivory among lithosiines, and the distribution of sequestered chemicals among life stages. The dynamics of chemical sequestration throughout the lifecycle for the lichen moth Crambidia cephalica were investigated by testing the hypothesis that lichen-derived metabolites are unequally distributed among life stages, and that laboratory-reared C. cephalica have less metabolite diversity than wild-caught individuals. Crambidia cephalica was reared on Physcia, and analyzed using high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS). Several putative lichen-derived metabolites were detected across three life stages, i.e., larval, pupal, and adult, and differences among life stages and lichen host were observed. These results provide evidence that multiple lichen-derived metabolites are sequestered by C. cephalica; some metabolites are retained through adulthood, and others are lost or modified in earlier life stages. The presence of differing lichen-derived metabolites across life stages may indicate functional properties of the metabolites for C. cephalica with regards to chemical protection from antagonists, and other physiological processes.

Chemical characterization of α-pinene secondary organic aerosol constituents using gas chromatography, liquid chromatography, and paper spray-based mass spectrometry techniques.

RATIONALE: Despite ample research into the atmospheric oxidation of α-pinene, an important precursor to biogenic secondary organic aerosol formation, the identification of its reaction products, specifically organic nitrates, which impact atmospheric NOx concentrations, is still incomplete. This negatively impacts our understanding of α-pinene oxidation chemistry and its relation to air quality. METHODS: Photochemical chamber experiments were conducted in conjunction with mass spectrometric techniques, including gas chromatography/mass spectrometry (GC/MS), high-performance liquid chromatography/time-of-flight (HPLC/TOF), and paper spray ionization MS, to investigate products from the OH radical initiated oxidation of α-pinene under high NOx conditions. RESULTS: Over 30 compounds were tentatively identified, including those newly detected from photochemical chamber studies of α-pinene oxidation, pinocamphenol, fencholenic aldehyde, and α-pinene-derived nitrate isomers. α-Pinene-derived hydroxynitrate isomers were successfully detected using chromatographic methods, demonstrating, for the first time, the identification of individual first-generation organic nitrate products derived from α-pinene. The application of paper spray ionization to particle-phase compounds collected on filters represents a novel method for the direct analysis of filter samples at ambient pressure and temperature. CONCLUSIONS: The use of HPLC/TOF and paper spray ionization methods to identify previously unobserved α-pinene-derived products helps lower the uncertainty in α-pinene oxidation chemistry and provides new platforms that can be used to identify and quantify important atmospheric compounds that relate to air quality in a complex sample matrix, such as ambient aerosol particles. Additionally, the use of paper spray ionization for direct filter analysis is a fast, relatively inexpensive sample preparation technique that can be used to reduce sample manipulation from solvent-induced reactions. Copyright © 2016 John Wiley & Sons, Ltd.

Contribution of CoA ligases to benzenoid biosynthesis in petunia flowers.

Biosynthesis of benzoic acid from Phe requires shortening of the side chain by two carbons, which can occur via the ß-oxidative or nonoxidative pathways. The first step in the ß-oxidative pathway is cinnamoyl-CoA formation, likely catalyzed by a member of the 4-coumarate:CoA ligase (4CL) family that converts a range of trans-cinnamic acid derivatives into the corresponding CoA thioesters. Using a functional genomics approach, we identified two potential CoA-ligases from petunia (Petunia hybrida) petal-specific cDNA libraries. The cognate proteins share only 25% amino acid identity and are highly expressed in petunia corollas. Biochemical characterization of the recombinant proteins revealed that one of these proteins (Ph-4CL1) has broad substrate specificity and represents a bona fide 4CL, whereas the other is a cinnamate:CoA ligase (Ph-CNL). RNA interference suppression of Ph-4CL1 did not affect the petunia benzenoid scent profile, whereas downregulation of Ph-CNL resulted in a decrease in emission of benzylbenzoate, phenylethylbenzoate, and methylbenzoate. Green fluorescent protein localization studies revealed that the Ph-4CL1 protein is localized in the cytosol, whereas Ph-CNL is in peroxisomes. Our results indicate that subcellular compartmentalization of enzymes affects their involvement in the benzenoid network and provide evidence that cinnamoyl-CoA formation by Ph-CNL in the peroxisomes is the committed step in the ß-oxidative pathway.

Implementation of multiomic mass spectrometry approaches for the evaluation of human health following environmental exposure.

Omics analyses collectively refer to the possibility of profiling genetic variants, RNA, epigenetic markers, proteins, lipids, and metabolites. The most common analytical approaches used for detecting molecules present within biofluids related to metabolism are vibrational spectroscopy techniques, represented by infrared, Raman, and nuclear magnetic resonance (NMR) spectroscopies and mass spectrometry (MS). Omics-based assessments utilizing MS are rapidly expanding and being applied to various scientific disciplines and clinical settings. Most of the omics instruments are operated by specialists in dedicated laboratories; however, the development of miniature portable omics has made the technology more available to users for field applications. Variations in molecular information gained from omics approaches are useful for evaluating human health following environmental exposure and the development and progression of numerous diseases. As MS technology develops so do statistical and machine learning methods for the detection of molecular deviations from personalized metabolism, which are correlated to altered health conditions, and they are intended to provide a multi-disciplinary overview for researchers interested in adding multiomic analysis to their current efforts. This includes an introduction to mass spectrometry-based omics technologies, current state-of-the-art capabilities and their respective strengths and limitations for surveying molecular information. Furthermore, we describe how knowledge gained from these assessments can be applied to personalized medicine and diagnostic strategies.

Dietary Phosphorus Levels Influence Protein-Derived Uremic Toxin Production in Nephrectomized Male Rats.

Gut microbiota-derived uremic toxins (UT) accumulate in patients with chronic kidney disease (CKD). Dietary phosphorus and protein restriction are common in CKD treatment, but the relationship between dietary phosphorus, a key nutrient for the gut microbiota, and protein-derived UT is poorly studied. Thus, we explored the relationship between dietary phosphorus and serum UT in CKD rats. For this exploratory study, we used serum samples from a larger study on the effects of dietary phosphorus on intestinal phosphorus absorption in nephrectomized (Nx, n = 22) or sham-operated (sham, n = 18) male Sprague Dawley rats. Rats were randomized to diet treatment groups of low or high phosphorus (0.1% or 1.2% w/w, respectively) for 1 week, with serum trimethylamine oxide (TMAO), indoxyl sulfate (IS), and p-cresol sulfate (pCS) analyzed by LC-MS. Nx rats had significantly higher levels of serum TMAO, IS, and pCS compared to sham rats (all p < 0.0001). IS showed a significant interaction between diet and CKD status, where serum IS was higher with the high-phosphorus diet in both Nx and sham rats, but to a greater extent in the Nx rats. Serum TMAO (p = 0.24) and pCS (p = 0.34) were not affected by dietary phosphorus levels. High dietary phosphorus intake for 1 week results in higher serum IS in both Nx and sham rats. The results of this exploratory study indicate that reducing dietary phosphorus intake in CKD may have beneficial effects on UT accumulation.

RNAi suppression of Arogenate Dehydratase1 reveals that phenylalanine is synthesized predominantly via the arogenate pathway in petunia petals.

l-Phe, a protein building block and precursor of numerous phenolic compounds, is synthesized from prephenate via an arogenate and/or phenylpyruvate route in which arogenate dehydratase (ADT) or prephenate dehydratase, respectively, plays a key role. Here, we used Petunia hybrida flowers, which are rich in Phe-derived volatiles, to determine the biosynthetic routes involved in Phe formation in planta. Of the three identified petunia ADTs, expression of ADT1 was the highest in petunia petals and positively correlated with endogenous Phe levels throughout flower development. ADT1 showed strict substrate specificity toward arogenate, although with the lowest catalytic efficiency among the three ADTs. ADT1 suppression via RNA interference in petunia petals significantly reduced ADT activity, levels of Phe, and downstream phenylpropanoid/benzenoid volatiles. Unexpectedly, arogenate levels were unaltered, while shikimate and Trp levels were decreased in transgenic petals. Stable isotope labeling experiments showed that ADT1 suppression led to downregulation of carbon flux toward shikimic acid. However, an exogenous supply of shikimate bypassed this negative regulation and resulted in elevated arogenate accumulation. Feeding with shikimate also led to prephenate and phenylpyruvate accumulation and a partial recovery of the reduced Phe level in transgenic petals, suggesting that the phenylpyruvate route can also operate in planta. These results provide genetic evidence that Phe is synthesized predominantly via arogenate in petunia petals and uncover a novel posttranscriptional regulation of the shikimate pathway.

Quantitative LC-MS/MS analysis of arachidonoyl amino acids in mouse brain with treatment of FAAH inhibitor.

An additional class of endogenous lipid amides, N-arachidonoyl amino acids (Ara-AAs), is growing in significance in the field of endocannabinoids. The development, validation, and application of a sensitive and selective method to simultaneously monitor and quantify the level of Ara-AAs along with anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) in mouse brain has been established. The linearity of the method over the concentration ranges of 0.2-120 pg/µl for the standards of N-arachidonoyl amino acids, N-arachidonoyl alanine (NAAla), serine (NASer), γ-aminobutyric acid (NAGABA), and glycine (NAGly); 0.7-90 pg/µl for AEA-d(0)/d(8); and 7.5-950 pg/µl for 2-AG was determined with R(2) values of 0.99. Also the effects of the FAAH inhibitor URB 597 on the endogenous levels of these analytes were investigated. AEA and NASer brain levels exhibit a dose-dependent increase after systemic administration of URB 597, whereas NAGly and NAGABA were significantly decreased after treatment. NAAla and 2-AG were not altered after URB 597 treatment. The potential benefit of establishing this assay extends beyond the quantification of the Ara-AAs along with AEA and 2-AG in mouse brain, to reveal a variety of pharmacological effects and physiological roles of these analytes.

Step-by-Step Approach to Build Multiple Reaction Monitoring (MRM) Profiling Instrument Acquisition Methods for Class-based Lipid Exploratory Analysis by Mass Spectrometry.

Multiple reaction monitoring (MRM) profiling is a strategy for the exploratory analysis of small molecules and lipids by direct sample injection, ie, without the use of chromatographic separation. It is based on instrument methods that comprise a list of ion transitions (MRMs), in which the precursor ion is the expected ionized m/z of the lipid at its species level, ie, the description of lipid class and number of carbon and double bonds in the fatty acid chain(s), and the product ion is a fragment expected for the lipid class or for the fatty acid neutral loss. The Lipid Maps database is expanding constantly, and therefore the MRM-profiling methods associated with this database need to be continuously updated. Here, we provide a comprehensive overview and the key references for the MRM-profiling methodology and workflow, followed by a step-by-step approach to build MRM-profiling instrument acquisition methods for class-based lipid exploratory analysis based on the Lipid Maps database. The detailed workflow includes (1) importing the list of lipids from the database; (2) for a given class, combining isomeric lipids described at full structural level into 1 entry to obtain the neutral mass at species level; (3) attributing the standard Lipid Maps abbreviated nomenclature for the lipid at its species level; (4) predicting the ionized precursor ions; and (5) adding the expected product ion. We also describe how to simulate the precursor ion for the suspect screening of modified lipids using lipid oxidation and their expected product ions as an example. After determining the MRMs, information about collision energy, dwell time, and other instrument parameters are added to finalize the acquisition method. As an example of final method output, we describe the format for Agilent MassHunter v.B.06 and provide the parameters in which optimization can be performed by lipid class using one or more lipid standards.

Proteomics and metabolomics profiling of meat exudate to determine the impact of postmortem aging on oxidative stability of beef muscles.

The objective of this study was to characterize the major proteomes and metabolites in beef exudate and determine their relationship to color and oxidative quality of beef muscles. Beef loin (LD) and tenderloin (PM) muscles were cut into sections, individually vacuum-packaged, and aged for 9, 16 and 23 days at 2 °C. Following aging, beef exudates were collected and analyzed for both proteomics and metabolomics profiles. Proteome analysis indicated clustering by muscle types, while metabolomics profiling further clustered the samples based on the aging periods. The PM exudate had a greater concentration of oxidative enzymes, while the LD exudate contained more glycolytic enzymes. Greater lipid, nucleotide, carnitine and glucoside metabolites were observed in LD and 23d exudates. HSP70 and laminin proteins, together with glucosides metabolites, were correlated to muscle oxidative stability. The results indicated that meat exudate could be a viable analytical matrix to determine changes in quality attributes of meat with aging.

Profiling hydroxycinnamoyl-coenzyme A thioesters: unlocking the back door of phenylpropanoid metabolism.

In plants, 20 to 30% of photosynthetically fixed carbon is directed toward lignin and other phenylpropanoid compounds for which hydroxycinnamoyl-coenzyme A (CoA) esters are key intermediates. CoA thioesters, ubiquitous metabolites found in all living cells (often at trace levels), have traditionally been challenging to measure. Here we report a hydrophilic interaction liquid chromatography (HILIC) method, coupled with tandem mass spectrometry (MS/MS), that allows simultaneous sensitive quantification of previously undetectable hydroxycinnamoyl-CoA esters and an extended range of acyl-CoAs from plant tissues. This method provides rapid liquid chromatography (LC) analysis (10 min/sample) and the ability for qualitative assessment of acyl-CoAs by MS/MS precursor ion scanning.

Evaluation of a tandem gas chromatography/time-of-flight mass spectrometry metabolomics platform as a single method to investigate the effect of starvation on whole-animal metabolism in rainbow trout (Oncorhynchus mykiss).

This study was conducted to evaluate the use of a two-dimensional gas chromatography/time-of-flight mass spectrometry (GC×GC/TOF-MS) metabolomic platform to comprehensively analyze the effect of starvation on whole-animal metabolism in rainbow trout (Oncorhynchus mykiss). Trout were either fed a commercial diet at 2% body mass twice daily or starved for 4 weeks. Metabolomic analysis was conducted on serum, liver and muscle tissue from each fish. Database searching and statistical analysis revealed that concentrations of more than 50 positively identified molecules changed significantly (P<0.05) as a result of starvation. Our results indicate that starving rainbow trout for 4 weeks promotes increased utilization of select tissue fatty acids in liver and muscle. However, starvation did not significantly affect protein catabolism in peripheral tissues, as indicated by reductions in the level of serum amino acids in starved fish. In contrast, starvation appears to promote protein catabolism in liver as the level of methionine, proline and lysine metabolite 2-piperidine carboxylic acid increased significantly. Also, starvation resulted in significant changes in the level of numerous xenobiotics that could indicate the origin of particular feed ingredients and selective retention of these molecules in tissues. We suggest that metabolomic analysis using GC×GC/TOF-MS is an effective tool in studying whole-animal metabolism and the fate of important xenobiotic compounds in rainbow trout as numerous polar and non-polar metabolites were rapidly and accurately profiled using a single method.

Changes in lipid profile and SOX-2 expression in RM-1 cells after co-culture with preimplantation embryos or with deproteinated blastocyst extracts.

The embryonic environment can modify cancer cell metabolism, and it is reported to induce the loss of tumorigenic properties and even affect the differentiation of cancer cells into normal tissues. The cellular mechanisms related to this remarkable phenomenon, which is likely mediated by cell-to-cell communication, have been previously investigated with particular focus on the proteins and genes involved. In this study we report the optimization and results of a straightforward in vitro system where mouse prostate carcinoma (RM-1) cells were co-cultured for three days with preimplantation mouse embryos or spiked with deproteinated extracts from mouse blastocysts. Compared to controls, both treatments induced RM-1 cells to increase the expression of the SOX-2 gene, which is related to cellular stemness, as well as altered their lipid composition. Specific acyl-carnitines, diacylglycerols, phosphatidylglycerols, phosphatidylinositols, phosphatidylserines and cardiolipins selected using an elastic net model discriminated the treated RM-1 cells from controls. Note that the tumorigenic properties of the treated RM-1 cells were not evaluated in this research. Due to the nature of the lipids impacted in the treated RM-1 cells, we hypothesize that mitochondrial metabolism has been altered, and that small molecules both secreted from and present within the embryos might be involved in the induction of metabolic changes observed in the RM-1 cells. These molecules, which could influence cancer cell metabolism, may still be unknown (i.e. structure, role).

Characterizing the Flavor Precursors and Liberation Mechanisms of Various Dry-Aging Methods in Cull Beef Loins Using Metabolomics and Microbiome Approaches.

The objective of this study was to characterize and compare the dry-aging flavor precursors and their liberation mechanisms in beef aged with different methods. Thirteen paired loins were collected at 5 days postmortem, divided into four sections, and randomly assigned into four aging methods (wet-aging (WA), conventional dry-aging (DA), dry-aging in a water-permeable bag (DWA), and UV-light dry-aging (UDA)). All sections were aged for 28 days at 2 °C, 65% RH, and a 0.8 m/s airflow before trimming and sample collection for chemical, metabolomics, and microbiome analyses. Higher concentrations of free amino acids and reducing sugars were observed in all dry-aging samples (p < 0.05). Similarly, metabolomics revealed greater short-chain peptides in the dry-aged beef (p < 0.05). The DWA samples had an increase in polyunsaturated free fatty acids (C18:2trans, C18:3n3, C20:2, and C20:5; p < 0.05) along with higher volatile compound concentrations compared to other aging methods (aldehyde, nonanal, octanal, octanol, and carbon disulfide; p < 0.05). Microbiome profiling identified a clear separation in beta diversity between dry and wet aging methods. The Pseudomonas spp. are the most prominent bacterial species in dry-aged meat, potentially contributing to the greater accumulation of flavor precursor concentrations in addition to the dehydration process during the dry-aging. Minor microbial species involvement, such as Bacillus spp., could potentially liberate unique and potent flavor precursors.

Identification of a naturally-occurring canine model for early detection and intervention research in high grade urothelial carcinoma.

Background: Early detection and intervention research is expected to improve the outcomes for patients with high grade muscle invasive urothelial carcinoma (InvUC). With limited patients in suitable high-risk study cohorts, relevant animal model research is critical. Experimental animal models often fail to adequately represent human cancer. The purpose of this study was to determine the suitability of dogs with high breed-associated risk for naturally-occurring InvUC to serve as relevant models for early detection and intervention research. The feasibility of screening and early intervention, and similarities and differences between canine and human tumors, and early and later canine tumors were determined. Methods: STs (n=120) ≥ 6 years old with no outward evidence of urinary disease were screened at 6-month intervals for 3 years with physical exam, ultrasonography, and urinalysis with sediment exam. Cystoscopic biopsy was performed in dogs with positive screening tests. The pathological, clinical, and molecular characteristics of the "early" cancer detected by screening were determined. Transcriptomic signatures were compared between the early tumors and published findings in human InvUC, and to more advanced "later" canine tumors from STs who had the typical presentation of hematuria and urinary dysfunction. An early intervention trial of an oral cyclooxygenase inhibitor, deracoxib, was conducted in dogs with cancer detected through screening. Results: Biopsy-confirmed bladder cancer was detected in 32 (27%) of 120 STs including InvUC (n=29, three starting as dysplasia), grade 1 noninvasive cancer (n=2), and carcinoma in situ (n=1). Transcriptomic signatures including druggable targets such as EGFR and the PI3K-AKT-mTOR pathway, were very similar between canine and human InvUC, especially within luminal and basal molecular subtypes. Marked transcriptomic differences were noted between early and later canine tumors, particularly within luminal subtype tumors. The deracoxib remission rate (42% CR+PR) compared very favorably to that with single-agent cyclooxygenase inhibitors in more advanced canine InvUC (17-25%), supporting the value of early intervention. Conclusions: The study defined a novel naturally-occurring animal model to complement experimental models for early detection and intervention research in InvUC. Research incorporating the canine model is expected to lead to improved outcomes for humans, as well as pet dogs, facing bladder cancer.