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.

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.

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.

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.

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.

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.

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).

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.

Effect of Dry-Aging on Quality and Palatability Attributes and Flavor-Related Metabolites of Pork Loins.

This study evaluated the effect of dry-aging on quality, palatability, and flavor-related compounds of pork loins. Ten pork loins were obtained at 7 days postmortem, divided into three equal portions, randomly assigned into three different aging methods (wet-aging (W), conventional dry-aging (DA), and UV-light dry-aging (UDA)), and aged for 21 days at 2 °C, 70% RH, and 0.8 m/s airflow. The results showed similar instrumental tenderness values across all treatments (p > 0.05), while DA and UDA had a greater water-holding capacity than WA (p < 0.05). Both DA and UDA were observed to have comparable color stability to WA up to 5 days of retail display (p > 0.05). Greater lipid oxidation was measured in both DA and UDA at the end of display compared to WA (p < 0.05). The UV light minimized microorganisms concentration on both surface and lean portions of UDA compared to other treatments (p < 0.05). The consumer panel was not able to differentiate any sensory traits and overall likeness between the treatments (p > 0.05). Metabolomics analysis, however, identified more flavor-related compounds in dry-aged meat. These findings suggested that dry-aging can be used for pork loins for value-seeking consumers, as it has a potential to generate unique dry-aged flavor in meat with no adverse impacts on meat quality and microbiological attributes.

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.

Developmental atrazine exposure in zebrafish produces the same major metabolites as mammals along with altered behavioral outcomes.

Atrazine (ATZ) is the second most commonly applied agricultural herbicide in the United States. Due to contamination concerns, the U.S. EPA has set the maximum contaminant level in potable water sources at 3 parts per billion (ppb; µg/l). Depending on the time of year and sampling location, water sources often exceed this limit. ATZ is an endocrine disrupting chemical in multiple species observed to target the neuroendocrine system. In this study the zebrafish vertebrate model was used to test the hypothesis that a developmental ATZ exposure generates metabolites similar to those found in mammals and alters morphology and behavior in developing larvae. Adult AB zebrafish were bred, embryos were collected, and exposed to 0, 0.3, 3, or 30 ppb ATZ from 1 to 120 h post fertilization (hpf). Targeted metabolomic analysis found that zebrafish produce the same major ATZ metabolites as mammals: desethyl atrazine (DEA), deisopropyl atrazine (DIA), and diaminochloroatrazine (DACT). The visual motor response test at 120 hpf detected hyperactivity in larvae in the 0.3 ppb treatment group and hypoactivity in the 30 ppb treatment group (p < 0.05). Further analysis into behavior during the dark and light phases showed zebrafish larvae exposed to 0.3 ppb ATZ had an increase in total distance moved in the first light phase and time spent moving in the first dark and light phases (p < 0.05). Alternatively, a decrease in total distance moved was observed in the second and third dark phases in zebrafish exposed to 30 ppb ATZ (p < 0.05). No significant differences were observed for any of the morphological measurements following ATZ exposure from 1 to 120 hpf (p > 0.05). These findings suggest that a ATZ exposure during early development generates metabolite profiles similar to mammals and leads to behavioral alterations supporting ATZ as a neurodevelopmental toxicant.

Elucidating mechanisms involved in flavor generation of dry-aged beef loins using metabolomics approach.

The present study was conducted to identify flavor-related chemical compounds and to elucidate beef flavor development in response to dry-aging. Paired grass-fed beef loins (n = 18) were obtained at 7 d postmortem, cut into two sections and assigned to 3 aging methods: conventional dry-aging (DA), vacuum packaged wet-aging (WA) and dry-aging in a bag (DW) for 28 days. Following aging, samples were analyzed for UPLC-MS metabolomics, volatile, fatty acid profiling, and consumer sensory comment analysis. Greater number of proteins and nucleotides derived metabolites were liberated in dry-aged samples compared to WA (P < 0.05). In particular, the liberation of gammaglutmayl peptides and glutamine metabolites through the glutathione metabolism were identified. While fatty acid profile was not affected by treatments (P > 0.05), higher concentrations of volatile compounds were found in the dry-aged (P < 0.05). Dry-aging process decreased the presence of terpenoid and steroid lipid group, which could possibly result in reducing undesirable flavor of grass-fed beef.

Effects of administration of ascorbic acid and low-dose hydrocortisone after infusion of sublethal doses of lipopolysaccharide to horses.

BACKGROUND: Sepsis is associated with ascorbic acid (AA) depletion and critical illness-related corticosteroid insufficiency (CIRCI) in humans. HYPOTHESES: Intravenous infusion of lipopolysaccharide (LPS) would (a) decrease endogneous AA concentrations, (b) induce CIRCI and (c) administration of a combination of AA and hydrocortisone (HC) would have decreased indices of inflammation compared to either drug alone. ANIMALS: Thirty-two healthy horses. METHODS: Randomized placebo-controlled experimental trial. Horses were assigned to 1 of 4 groups (saline, AA and HC, AA only, or HC only). Treatments were administered 1 hour after completion of LPS infusion. Clinical signs, clinicopathological variables, pro-inflammatory cytokine gene expression and production, and plasma AA concentrations were assessed at various time points. Serum cortisol concentrations and ACTH stimulation tests were used to detect CIRCI. RESULTS: There was no effect of drug on clinical signs or pro-inflammatory cytokine gene expression or production compared to controls at any time point. Administration of AA was associated with higher blood neutrophil counts 6 hours after LPS infusion (11.01 ± 1.02 K/µl) compared to other groups (8.99 ± 0.94 K/µL; P < .009). Adminstration of HC was associated with higher blood neutrophil counts 12 hours after LPS infusion (10.40 ± 0.75 K/µl) compared to other groups (6.88 ± 0.68 K/µl; P < .001). Serum cortisol increased from 5.11 ± 1.48 µg/dL before LPS administration to 9.59 ± 1.83 µg/dL 1 h after completion of LPS infusion (T1) without an effect of treatment (P = 0.59). CONCLUSIONS AND CLINICAL IMPORTANCE: Ascorbic acid and HC appeared to protect against LPS-induced neutrophil depletion and could be considered as adjunctive therapy in horses with endotoxemia.

Ketorolac Is Not More Effective Than Flunixin Meglumine or Phenylbutazone in Reducing Foot Pain in Horses.

The objective was to compare the analgesic efficacy of ketorolac tromethamine (KT) and two other nonsteroidal anti-inflammatory drugs (NSAIDs), including flunixin meglumine (FM) and phenylbutazone (PB), using a heart bar shoe (HBS) model of reversible foot lameness in horses. Nine adult horses were used in a blinded, randomized, placebo-controlled crossover study. After induction of left front limb lameness using a modified HBS model, one of three NSAIDs (KT, 2.0 mg/kg IV; FM, 1.1 mg/kg IV; PB, 4.4 mg/kg IV) or saline (placebo) was administered IV as a single dose. Lameness was assessed every 30 minutes for 2 hours, then every hour up to 12 hours using both a lameness grading scale (lameness score; LS) and a body-mounted inertial sensor system (lameness locator; LL). High-performance liquid chromatography and mass spectrometry were used to measure plasma drug concentration at various time points. There was no difference in percent reduction of LS or LL value between KT and any other group, or between FM and placebo. The PB group showed a significantly higher percentage in LS reduction than the placebo and FM groups. The mean percent reduction in LL value was greater for the PB group than that for the placebo and FM groups. Plasma drug concentration was similar among horses for each drug at each time point, with drug concentrations decreasing over time. Thus, variation in plasma drug concentration did not influence lameness reduction for any drug. Ketorolac tromethamine was not superior to FM or PB in reducing lameness using a HBS model.

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.

Proteomic and metabolomic profiling reveals the involvement of apoptosis in meat quality characteristics of ovine M. longissimus from different callipyge genotypes.

Proteome and metabolome changes in muscles from callipyge mutation (+/C) and non-callipyge phenotype (+/+, C/+, and C/C) lambs were profiled to provide insight into the biochemical changes affecting meat quality attributes. M. longissimus thoracis from lambs with all four possible callipyge genotype (n = 4, C/+, C/C, +/C, and +/+) were collected after 3d aging and analyzed using mass-spectrometry based platforms. Among identified proteomes, cytochrome c (pro-apoptotic protein) was detected with significantly lower abundances in +/C. Anti-apoptotic HSP70, BAG3, and PARK7 were over-abundant in +/C, which could result in delayed apoptosis and possibly attributed to tougher meat in callipyge lambs. Eight glycolysis enzymes were overabundant in +/C lambs, whereas 3 enzymes involved in TCA cycle were overabundant in non-callipyge ones (C/C and/or C/+). Twenty-five metabolites were affected by genotypes (P < .05), including metabolic co-factors, polyphenols, and AA/short peptides. Our omics results provided insightful information for revealing the differences in biochemical attributes caused by callipyge mutation.

Overcoming cellulose recalcitrance in woody biomass for the lignin-first biorefinery.

BACKGROUND: Low-temperature swelling of cotton linter cellulose and subsequent gelatinization in trifluoroacetic acid (TFA) greatly enhance rates of enzymatic digestion or maleic acid-AlCl3 catalyzed conversion to hydroxymethylfurfural (HMF) and levulinic acid (LA). However, lignin inhibits low-temperature swelling of TFA-treated intact wood particles from hybrid poplar (Populus tremula × P. alba) and results in greatly reduced yields of glucose or catalytic conversion compared to lignin-free cellulose. Previous studies have established that wood particles from transgenic lines of hybrid poplar with high syringyl (S) lignin content give greater glucose yields following enzymatic digestion. RESULTS: Low-temperature (- 20 °C) treatment of S-lignin-rich poplar wood particles in TFA slightly increased yields of glucose from enzymatic digestions and HMF and LA from maleic acid-AlCl3 catalysis. Subsequent gelatinization at 55 °C resulted in over 80% digestion of cellulose in only 3 to 6 h with high-S-lignin wood, compared to 20-60% digestion in the wild-type poplar hybrid and transgenic lines high in guaiacyl lignin or 5-hydroxy-G lignin. Disassembly of lignin in woody particles by Ni/C catalytic systems improved yields of glucose by enzymatic digestion or catalytic conversion to HMF and LA. Although lignin was completely removed by Ni/C-catalyzed delignification (CDL) treatment, recalcitrance to enzymatic digestion of cellulose from the high-S lines was reduced compared to other lignin variants. However, cellulose still exhibited considerable recalcitrance to complete enzymatic digestion or catalytic conversion after complete delignification. Low-temperature swelling of the CDL-treated wood particles in TFA resulted in nearly complete enzymatic hydrolysis, regardless of original lignin composition. CONCLUSIONS: Genetic modification of lignin composition can enhance the portfolio of aromatic products obtained from lignocellulosic biomass while promoting disassembly into biofuel and bioproduct substrates. CDL enhances rates of enzymatic digestion and chemical conversion, but cellulose remains intrinsically recalcitrant. Cold TFA is sufficient to overcome this recalcitrance after CDL treatment. Our results inform a 'no carbon left behind' strategy to convert total woody biomass into lignin, cellulose, and hemicellulose value streams for the future biorefinery.

Absorption of Transdermal Fluoxetine Compounded in a Lipoderm Base Compared to Oral Fluoxetine in Client-owned Cats.

The objective of this study was to compare serum concentrations of transdermal fluoxetine compounded in Lipoderm base versus commercially available oral fluoxetine tablets. Sixteen clinically healthy, client-owned cats that were at least one year of age were enrolled. Cats weighed between three and seven kilograms, had no comorbidities, and were behavior medication naïve. Cats were recruited from January 2016 through April 2016. Eight cats were assigned to each medication group based on owner preference. The cats received either oral (1 mg/kg) or transdermal (5 mg/kg; maximum 25 mg daily) fluoxetine compounded in a transdermal base (PCCA Lipoderm), administered daily for 60 days. Serum levels of fluoxetine and norfluoxetine were assessed as a surrogate for relative efficacy. Serum was collected and analyzed by high-performance liquid chromatography-mass spectrometry/mass spectrometry at baseline and days 5, 10, 30, 45, and 60 post-drug start. Adverse effects were monitored during physical exams, speaking with owners, and laboratory analysis of liver function tests at baseline and days 5, 30, and 60 post-drug start. Serum fluoxetine concentrations significantly differed between the treatment groups at days 45 and 60 post-drug start. Norfluoxetine concentrations significantly differed at days 30, 45, and 60 post-drug start. Blood concentrations of fluoxetine and norfluoxetine significantly differed between oral and transdermal routes after 30 days of treatment. Oral fluoxetine concentrations were consistently higher. Transdermal fluoxetine appeared to be well-tolerated, but a lack of knowledge regarding effective blood levels makes it unclear if a clinical effective response would be obtained at the blood concentrations achieved.

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.