Olive- and Coconut-Oil-Enriched Diets Decreased Secondary Bile Acids and Regulated Metabolic and Transcriptomic Markers of Brain Injury in the Frontal Cortexes of NAFLD Pigs.

The objective of this study was to investigate the effect of dietary fatty acid (FA) saturation and carbon chain length on brain bile acid (BA) metabolism and neuronal number in a pig model of pediatric NAFLD. Thirty 20-day-old Iberian pigs, pair-housed in pens, were randomly assigned to receive one of three hypercaloric diets for 10 weeks: (1) lard-enriched (LAR; n = 5 pens), (2) olive-oil-enriched (OLI, n = 5), and (3) coconut-oil-enriched (COC; n = 5). Pig behavior and activity were analyzed throughout the study. All animals were euthanized on week 10 and frontal cortex (FC) samples were collected for immunohistochemistry, metabolomic, and transcriptomic analyses. Data were analyzed by multivariate and univariate statistics. No differences were observed in relative brain weight, neuronal number, or cognitive functioning between diets. Pig activity and FC levels of neuroprotective secondary BAs and betaine decreased in the COC and OLI groups compared with LAR, and paralleled the severity of NAFLD. In addition, OLI-fed pigs showed downregulation of genes involved in neurotransmission, synaptic transmission, and nervous tissue development. Similarly, COC-fed pigs showed upregulation of neurogenesis and myelin repair genes, which caused the accumulation of medium-chain acylcarnitines in brain tissue. In conclusion, our results indicate that secondary BA levels in the FCs of NAFLD pigs are affected by dietary FA composition and are associated with metabolic and transcriptomic markers of brain injury. Dietary interventions that aim to replace saturated FAs by medium-chain or monounsaturated FAs in high-fat hypercaloric diets may have a negative effect on brain health in NAFLD patients.

Dietary fat composition shapes bile acid metabolism and severity of liver injury in a pig model of pediatric NAFLD.

The objective of this study was to investigate the effect of dietary fatty acid (FA) composition on bile acid (BA) metabolism in a pig model of NAFLD, by using a multiomics approach combined with histology and serum biochemistry. Thirty 20-day-old Iberian pigs pair-housed in pens were randomly assigned to receive 1 of 3 hypercaloric diets for 10 wk: 1) lard-enriched (LAR; n = 5 pens), 2) olive oil-enriched (OLI; n = 5), and 3) coconut oil-enriched (COC; n = 5). Animals were euthanized on week 10 after blood sampling, and liver, colon, and distal ileum (DI) were collected for histology, metabolomics, and transcriptomics. Data were analyzed by multivariate and univariate statistics. Compared with OLI and LAR, COC increased primary and secondary BAs in liver, plasma, and colon. In addition, both COC and OLI reduced circulating fibroblast growth factor 19, increased hepatic necrosis, composite lesion score, and liver enzymes in serum, and upregulated genes involved in hepatocyte proliferation and DNA repair. The severity of liver disease in COC and OLI pigs was associated with increased levels of phosphatidylcholines, medium-chain triacylglycerides, trimethylamine-N-oxide, and long-chain acylcarnitines in the liver, and the expression of profibrotic markers in DI, but not with changes in the composition or size of BA pool. In conclusion, our results indicate a role of dietary FAs in the regulation of BA metabolism and progression of NAFLD. Interventions that aim to modify the composition of dietary FAs, rather than to regulate BA metabolism or signaling, may be more effective in the treatment of NAFLD.NEW & NOTEWORTHY Bile acid homeostasis and signaling is disrupted in NAFLD and may play a central role in the development of the disease. However, there are no studies addressing the impact of diet on bile acid metabolism in patients with NAFLD. In juvenile Iberian pigs, we show that fatty acid composition in high-fat high-fructose diets affects BA levels in liver, plasma, and colon but these changes were not associated with the severity of the disease.

Consumption of High-Fructose Corn Syrup Compared with Sucrose Promotes Adiposity and Increased Triglyceridemia but Comparable NAFLD Severity in Juvenile Iberian Pigs.

BACKGROUND: Fructose consumption has been linked to nonalcoholic fatty liver disease (NAFLD) in children. However, the effect of high-fructose corn syrup (HFCS) compared with sucrose in pediatric NAFLD has not been investigated. OBJECTIVES: We tested whether the isocaloric substitution of dietary sucrose by HFCS would increase the severity of NAFLD in juvenile pigs, and whether this effect would be associated with changes in gut histology, SCFA production, and microbial diversity. METHODS: Iberian pigs, 53-d-old and pair-housed in pens balanced for weight and sex, were randomly assigned to receive a mash diet top-dressed with increasing amounts of sucrose (SUC; n = 3 pens; 281.6-486.8 g/kg diet) or HFCS (n = 4; 444.3-724.8 g/kg diet) during 16 wk. Diets exceeded the animal's energy requirements by providing sugars in excess, but met the requirements for all other nutrients. Animals were killed at 165 d of age after blood sampling, and liver, muscle, and gut were collected for histology, metabolome, and microbiome analyses. Data were analyzed by multivariate and univariate statistics. RESULTS: Compared with SUC, HFCS increased subcutaneous fat, triacylglycerides in plasma, and butyrate in colon (P ≤ 0.05). In addition, HFCS decreased UMP and short-chain acyl carnitines in liver, and urea nitrogen and creatinine in serum (P ≤ 0.05). Microbiome analysis showed a 24.8% average dissimilarity between HFCS and SUC associated with changes in SCFA-producing bacteria. Body weight gain, intramuscular fat, histological and serum markers of liver injury, and circulating hormones, glucose, and proinflammatory cytokines did not differ between diets. CONCLUSIONS: Fructose consumption derived from HFCS promoted butyrate synthesis, triglyceridemia, and subcutaneous lipid deposition in juvenile Iberian pigs, but did not increase serum and histological markers of NAFLD compared with a sucrose-enriched diet. Longer studies could be needed to observe differences in liver injury among sugar types.

Dysregulated FXR-FGF19 signaling and choline metabolism are associated with gut dysbiosis and hyperplasia in a novel pig model of pediatric NASH.

To investigate the role of bile acids (BAs) in the pathogenesis of diet-induced nonalcoholic steatohepatitis (NASH), we fed a "Western-style diet" [high fructose, high fat (HFF)] enriched with fructose, cholesterol, and saturated fat for 10 wk to juvenile Iberian pigs. We also supplemented probiotics with in vitro BA deconjugating activity to evaluate their potential therapeutic effect in NASH. Liver lipid and function, cytokines, and hormones were analyzed using commercially available kits. Metabolites, BAs, and fatty acids were measured by liquid chromatography-mass spectrometry. Histology and gene and protein expression analyses were performed using standard protocols. HFF-fed pigs developed NASH, cholestasis, and impaired enterohepatic Farnesoid-X receptor (FXR)-fibroblast growth factor 19 (FGF19) signaling in the absence of obesity and insulin resistance. Choline depletion in HFF livers was associated with decreased lipoprotein and cholesterol in serum and an increase of choline-containing phospholipids in colon contents and trimethylamine-N-oxide in the liver. Additionally, gut dysbiosis and hyperplasia increased with the severity of NASH, and were correlated with increased colonic levels of choline metabolites and secondary BAs. Supplementation of probiotics in the HFF diet enhanced NASH, inhibited hepatic autophagy, increased excretion of taurine and choline, and decreased gut microbial diversity. In conclusion, dysregulation of BA homeostasis was associated with injury and choline depletion in the liver, as well as increased biliary secretion, gut metabolism and excretion of choline-based phospholipids. Choline depletion limited lipoprotein synthesis, resulting in hepatic steatosis, whereas secondary BAs and choline-containing phospholipids in colon may have promoted dysbiosis, hyperplasia, and trimethylamine synthesis, causing further damage to the liver.NEW & NOTEWORTHY Impaired Farnesoid-X receptor (FXR)-fibroblast growth factor 19 (FGF19) signaling and cholestasis has been described in nonalcoholic fatty liver disease (NAFLD) patients. However, therapeutic interventions with FXR agonists have produced contradictory results. In a swine model of pediatric nonalcoholic steatohepatitis (NASH), we show that the uncoupling of intestinal FXR-FGF19 signaling and a decrease in FGF19 levels are associated with a choline-deficient phenotype of NASH and increased choline excretion in the gut, with the subsequent dysbiosis, colonic hyperplasia, and accumulation of trimethylamine-N-oxide in the liver.

Synthesis and Electronic Properties of Fluoreno[2,1-a]fluorenedione and Fluoreno[1,2-a]fluorenedione.

The [2,1-a]- and [1,2-a]-isomers of fluorenofluorenedione have been synthesized via intramolecular Friedel-Crafts acylations. DFT calculations indicate that the [1,2-a]-isomer adopts a twisted, helical C2-symmetric structure and that its protonated form is the thermodynamic product of the Friedel-Crafts acylation in hot sulfuric acid. Absorption spectroscopy and cyclic voltammetry measurements provide experimental estimations of frontier molecular orbital energy levels, which are reported and discussed.

Characterization of Reagent Pencils for Deposition of Reagents onto Paper-Based Microfluidic Devices.

Reagent pencils allow for solvent-free deposition of reagents onto paper-based microfluidic devices. The pencils are portable, easy to use, extend the shelf-life of reagents, and offer a platform for customizing diagnostic devices at the point of care. In this work, reagent pencils were characterized by measuring the wear resistance of pencil cores made from polyethylene glycols (PEGs) with different molecular weights and incorporating various concentrations of three different reagents using a standard pin abrasion test, as well as by measuring the efficiency of reagent delivery from the pencils to the test zones of paper-based microfluidic devices using absorption spectroscopy and digital image colorimetry. The molecular weight of the PEG, concentration of the reagent, and the molecular weight of the reagent were all found to have an inverse correlation with the wear of the pencil cores, but the amount of reagent delivered to the test zone of a device correlated most strongly with the concentration of the reagent in the pencil core. Up to 49% of the total reagent deposited on a device with a pencil was released into the test zone, compared to 58% for reagents deposited from a solution. The results suggest that reagent pencils can be prepared for a variety of reagents using PEGs with molecular weights in the range of 2000 to 6000 g/mol.

Reagent pencils: a new technique for solvent-free deposition of reagents onto paper-based microfluidic devices.

Custom-made pencils containing reagents dispersed in a solid matrix were developed to enable rapid and solvent-free deposition of reagents onto membrane-based fluidic devices. The technique is as simple as drawing with the reagent pencils on a device. When aqueous samples are added to the device, the reagents dissolve from the pencil matrix and become available to react with analytes in the sample. Colorimetric glucose assays conducted on devices prepared using reagent pencils had comparable accuracy and precision to assays conducted on conventional devices prepared with reagents deposited from solution. Most importantly, sensitive reagents, such as enzymes, are stable in the pencils under ambient conditions, and no significant decrease in the activity of the enzyme horseradish peroxidase stored in a pencil was observed after 63 days. Reagent pencils offer a new option for preparing and customizing diagnostic tests at the point of care without the need for specialized equipment.

Supramolecular assemblies of DNA with neutral nucleoside amphiphiles.

A neutral uridine-based amphiphile is described which condenses plasmid DNA. AFM studies show that the three distinct structural components of the amphiphile (i.e, nucleobase, alkyl chains, and poly(ethylene glycol)) are required for the formation of DNA-amphiphile supramolecular assemblies on a mica surface.

Bonding in HNO-myoglobin as characterized by X-ray absorption and resonance raman spectroscopies.

The EXAFS and resonance Raman spectra on the HNO-myoglobin adduct, 1, are consistent with the presence of HNO bound to a heme center. The three-dimensional structure about the heme center of 1 obtained from multiple-scattering (MS) analysis of the EXAFS of the heme protein yielded an Fe-N-O bond angle of 131 degrees and an Fe-N bond length of 1.82 A, which compare well with published values for model complexes containing RNO ligands. Resonance Raman spectra identified the nu(N=O) stretch at 1385 cm-1 (confirmed by 15N labeling), which corresponds well with those reported for small molecule HNO complexes. The wavelength of the nu(Fe-N) at 636 cm-1 of 1 is significantly higher than those of MbIINO and MbIIINO (554 and 595 cm-1, respectively). The XAFS, XANES, and resonance Raman data are all consistent with the structure deduced from the NMR experiments, providing more detail on the bonding between HNO and the metal center.

DNA-PEG-DNA triblock macromolecules for reagentless DNA detection.

The sandwich assay is the most common design for electrochemical DNA sensors. This assay consists of three individual DNA components: an immobilized capture strand, a target strand, and a probe strand containing a redox-active reporter group. We report a simplified DNA assay where two strands of ssDNA, the capture and probe strands, are linked together via a flexible poly(ethylene glycol) (PEG) spacer forming an ABA triblock macromolecule. We have developed an electrochemical assay where the detection signal arises as a consequence of a large structural change induced upon hybridization with target DNA. In this system, the DNA-PEG-DNA macromolecule folds or wraps around the target DNA, bringing the ferrocene probe in close proximity to the electrode, affording an electrochemical response.

Conformationally gated electrochemical gene detection.

The synthesis and characterization of a 26-base DNA hairpin containing both a redox-active reporter (ferrocene) and terminal thiol functionality for electrochemical gene detection is described. This electrochemical DNA sensor exploits electron-transfer dynamics that alter as a consequence of a large structural rearrangement (hairpin-to-duplex) induced by hybridization of the target DNA sequence. Melting temperature and circular dichroism studies confirm that the 26-mer DNA forms a hairpin structure in the absence of target DNA. The loop region of the DNA hairpin is shown to form a stable duplex in the presence of complementary single-stranded DNA. Atomic force microscopy and ellipsometry experiments of immobilized self-assembled DNA monolayers suggest that hybridization with complementary DNA affords a conformational change that alters the electrochemical response.

Electron-transfer chemistry of Ru-linker-(heme)-modified myoglobin: rapid intraprotein reduction of a photogenerated porphyrin cation radical.

We report the synthesis and characterization of RuC7, a complex in which a heme is covalently attached to a [Ru(bpy)(3)](2+) complex through a -(CH(2))(7)- linker. Insertion of RuC7 into horse heart apomyoglobin gives RuC7Mb, a Ru(heme)-protein conjugate in which [Ru(bpy)(3)](2+) emission is highly quenched. The rate of photoinduced electron transfer (ET) from the resting (Ru(2+)/Fe(3+)) to the transient (Ru(3+)/Fe(2+)) state of RuC7Mb is >10(8) s(-1); the back ET rate (to regenerate Ru(2+)/Fe(3+)) is 1.4 x 10(7) s(-1). Irreversible oxidative quenching by [Co(NH(3))(5)Cl](2+) generates Ru(3+)/Fe(3+): the Ru(3+) complex then oxidizes the porphyrin to a cation radical (P*+); in a subsequent step, P*+ oxidizes both Fe(3+) (to give Fe(IV)=O) and an amino acid residue. The rate of intramolecular reduction of P*+ is 9.8 x 10(3) s(-1); the rate of ferryl formation is 2.9 x 10(3) s(-1). Strong EPR signals attributable to tyrosine and tryptophan radicals were recorded after RuC7MbM(3+) (M = Fe, Mn) was flash-quenched/frozen.

Electrocatalytic reductions of nitrite, nitric oxide, and nitrous oxide by thermophilic cytochrome P450 CYP119 in film-modified electrodes and an analytical comparison of its catalytic activities with myoglobin.

Previous investigations of nitrite and nitric oxide reduction by myoglobin in surfactant film modified electrodes characterized several distinct steps in the denitrification pathway, including isolation of a nitroxyl adduct similar to that proposed in the P450nor catalytic cycle. To investigate the effect of the axial ligand on these biomimetic reductions, we report here a comparison of the electrocatalytic activity of myoglobin (Mb) with a thermophilic cytochrome P450 CYP119. Electrocatalytic nitrite reduction by CYP119 is very similar to that by Mb: two catalytic waves at analogous potentials are observed, the first corresponding to the reduction of nitric oxide, the second to the production of ammonia. CYP119 is a much more selective catalyst, giving almost exclusively ammonia during the initial half-hour of reductive electrolysis of nitrite. More careful investigations of specific steps in the catalytic cycle show comparable rates of nitrite dehydration and almost identical potentials and lifetimes for ferrous nitroxyl intermediate (Fe(II)-NO(-)) in CYP119 and Mb. The catalytic efficiency of nitric oxide reduction is reduced for CYP119 as compared to Mb, attributable to both a lower affinity of the protein for NO and a decreased rate of N-N coupling. Isotopic labeling studies show ammonia incorporation into nitrous oxide produced during nitrite reduction, as has been termed co-denitrification for certain bacterial and fungal nitrite reductases. Mb has a much higher co-denitrification activity than CYP119. Conversely, CYP119 is shown to be slightly more efficient at the two-electron reduction of N(2)O to N(2). These results suggest that thiolate ligation does not significantly alter the catalytic reactivity, but the dramatic difference in product distribution may suggest an important role for protein stability in the selectivity of biocatalysts.

Efficient trapping of HNO by deoxymyoglobin.

Nitrosyl hydride, HNO, also commonly termed nitroxyl, is a transient species that has been implicated in the biological activity of nitric oxide, NO. Herein, we report the first generation of a stable HNO-metal complex by direct trapping of free HNO. Deoxymyoglobin (Mb-Fe(II)) rapidly reacts with HNO produced from the decomposition of methylsulfonylhydroxylamine (MSHA) or Angeli's salt (AS) in aqueous solutions from pH 7 to pH 10, forming an adduct, Mb-HNO. The unique 1H NMR signal of the Fe-bound HNO at 14.8 ppm allows definitive proof of its formation. The generation of Mb-HNO and quantification of various myoglobin byproducts were accomplished by correlation of 1H NMR, UV-vis, and EPR spectroscopies. Typically, the maximum Mb-HNO yield obtained is 60-80%; competitive side reactions with byproducts as well as the further reactivity of the Mb-HNO decrease the overall yield. At pH 10, the observed rate of Mb-HNO generation by trapping HNO from MSHA is close to that for MSHA decomposition; kinetic simulations give a lower limit to the bimolecular rate of trapping as 1.4 x 10(4) M(-1) s(-1). The binding of HNO to deoxymyoglobin is rapid and essentially irreversible, which suggests that the biological activity of nitroxyl may be mediated by its reactivity with ferrous heme proteins such as myoglobin and hemoglobin.

Dendritic molecular capsules for hydrophobic compounds.

Reichardt's dye, a highly solvatochromic dye, was encapsulated within poly (glycerol succinic acid) ([Gn]-PGLSA-OH) dendrimers to investigate the interior environment of these dendritic macromolecules. The absorption maximum for the encapsulated Reichardt's dye in water was indicative of a relatively high dielectric constant present within the dye/dendrimer complex. (1)H NMR of the encapsulated complex showed the presence of aromatic protons from Reichardt's dye along with the aliphatic protons of the dendrimer. Additionally, there were substantial changes in T(1) and T(2) times of the encapsulated dye when compared with the free dye, and (1)H NOESY spectra for the complex showed a significant number of intermolecular NOE cross-peaks. These data reveal the close through-space proximity of the dye to the dendrimer and the restricted motion of the encapsulated dye. To demonstrate the potential use of these macromolecules as drug delivery vehicles, the poorly water-soluble anticancer drug 10-hydroxycamptothecin (10HCPT) was encapsulated within a carboxylated PGLSA dendrimer ([G4]-PGLSA-COONa). Cytotoxicity assays with human breast cancer cells showed a significant reduction of cell viability, demonstrating that 10HCPT retains activity upon encapsulation.

A novel heme and peroxide-dependent tryptophan-tyrosine cross-link in a mutant of cytochrome c peroxidase.

The crystal structure of a cytochrome c peroxidase mutant where the distal catalytic His52 is converted to Tyr reveals that the tyrosine side-chain forms a covalent bond with the indole ring nitrogen atom of Trp51. We hypothesize that this novel bond results from peroxide activation by the heme iron followed by oxidation of Trp51 and Tyr52. This hypothesis has been tested by incorporation of a redox-inactive Zn-protoporphyrin into the protein, and the resulting crystal structure shows the absence of a Trp51-Tyr52 cross-link. Instead, the Tyr52 side-chain orients away from the heme active-site pocket, which requires a substantial rearrangement of residues 72-80 and 134-144. Additional experiments where heme-containing crystals of the mutant were treated with peroxide support our hypothesis that this novel Trp-Tyr cross-link is a peroxide-dependent process mediated by the heme iron.

Synthesis and characterization of pi-stacked phenothiazine-labeled oligodeoxynucleotides.

[reaction: see text] A facile procedure for the incorporation of N-methyl phenothiazine as the terminal nucleoside in oligodeoxynucleotides is reported. The phenothiazine nucleoside analogue is synthesized and then incorporated into DNA using an automated DNA solid-phase synthesizer. Phenothiazine-labeled oligodeoxynucleotides form stable B-form duplexes with higher melting temperatures compared to unlabeled DNA duplexes.

Mesopone cytochrome c peroxidase: functional model of heme oxygenated oxidases.

The effect of heme ring oxygenation on enzyme structure and function has been examined in a reconstituted cytochrome c peroxidase. Oxochlorin derivatives were formed by OsO(4) treatment of mesoporphyrin followed by acid-catalyzed pinacol rearrangement. The northern oxochlorin isomers were isolated by chromatography, and the regio-isomers assignments determined by 2D COSY and NOE 1H NMR. The major isomer, 4-mesoporphyrinone (Mp), was metallated with FeCl(2) and reconstituted into cytochrome c peroxidase (CcP) forming a hybrid green protein, MpCcP. The heme-altered enzyme has 99% wild-type peroxidase activity with cytochrome c. EPR spectroscopy of MpCcP intermediate compound I verifies the formation of the Trp(191) radical similar to wild-type CcP in the reaction cycle. Peroxidase activity with small molecules is varied: guaiacol turnover increases approximately five-fold while that with ferrocyanide is approximately 85% of native. The electron-withdrawing oxo-substitutents on the cofactor cause a approximately 60-mV increase in Fe(III)/Fe(II) reduction potential. The present investigation represents the first structural characterization of an oxochlorin protein with X-ray intensity data collected to 1.70 A. Although a mixture of R- and S-mesopone isomers of the FeMP cofactor was used during heme incorporation into the apo-protein, only the S-isomer is found in the crystallized protein.

Enhanced electron transfer and lauric acid hydroxylation by site-directed mutagenesis of CYP119.

CYP119, a cytochrome P450 from a thermophilic organism for which a crystal structure is available, is shown here to hydroxylate lauric acid in a reaction supported by putidaredoxin and putidaredoxin reductase. This fatty acid hydroxylation activity is increased 15-fold by T214V and D77R mutations. The T214V mutation increases the rate by facilitating substrate binding and enhancing the associated spin state change, whereas the D77R mutation improves binding of the heterologous redox partner putidaredoxin to CYP119 and the rate of electron transfer from it to the heme group. A sequence alignment with P450(cam) can, therefore, be used to identify a part of the binding site for putidaredoxin on an unrelated P450 enzyme. This information can be used to engineer by mutagenesis an improved complementarity of the protein-protein interface that results in improved electron transfer from putidaredoxin to the P450 enzyme. As a result, the catalytic activity of the thermo- and barostable CYP119 has been incorporated into a catalytic system that hydroxylates fatty acids.