Metabolic transitions regulate global protein fatty acylation.

Intermediary metabolites and flux through various pathways have emerged as key determinants of post-translational modifications. Independently, dynamic fluctuations in their concentrations are known to drive cellular energetics in a bi-directional manner. Notably, intracellular fatty acid pools that drastically change during fed and fasted states act as precursors for both ATP production and fatty acylation of proteins. Protein fatty acylation is well regarded for its role in regulating structure and functions of diverse proteins; however, the effect of intracellular concentrations of fatty acids on protein modification is less understood. In this regard, we unequivocally demonstrate that metabolic contexts, viz. fed and fasted states, dictate the extent of global fatty acylation. Moreover, we show that presence or absence of glucose that influences cellular and mitochondrial uptake/utilization of fatty acids and affects palmitoylation and oleoylation, which is consistent with their intracellular abundance in fed and fasted states. Employing complementary approaches including click-chemistry, lipidomics, and imaging, we show the top-down control of cellular metabolic state. Importantly, our results establish the crucial role of mitochondria and retrograde signaling components like SIRT4, AMPK, and mTOR in orchestrating protein fatty acylation at a whole cell level. Specifically, pharmacogenetic perturbations that alter either mitochondrial functions and/or retrograde signaling affect protein fatty acylation. Besides illustrating the cross-talk between carbohydrate and lipid metabolism in mediating bulk post-translational modification, our findings also highlight the involvement of mitochondrial energetics.

The carboxy terminus causes interfacial assembly of oleate hydratase on a membrane bilayer.

The soluble flavoprotein oleate hydratase (OhyA) hydrates the 9-cis double bond of unsaturated fatty acids. OhyA substrates are embedded in membrane bilayers; OhyA must remove the fatty acid from the bilayer and enclose it in the active site. Here, we show that the positively charged helix-turn-helix motif in the carboxy terminus (CTD) is responsible for interacting with the negatively charged phosphatidylglycerol (PG) bilayer. Super-resolution microscopy of Staphylococcus aureus cells expressing green fluorescent protein fused to OhyA or the CTD sequence shows subcellular localization along the cellular boundary, indicating OhyA is membrane-associated and the CTD sequence is sufficient for membrane recruitment. Using cryo-electron microscopy, we solved the OhyA dimer structure and conducted 3D variability analysis of the reconstructions to assess CTD flexibility. Our surface plasmon resonance experiments corroborated that OhyA binds the PG bilayer with nanomolar affinity and we found the CTD sequence has intrinsic PG binding properties. We determined that the nuclear magnetic resonance structure of a peptide containing the CTD sequence resembles the OhyA crystal structure. We observed intermolecular NOE from PG liposome protons next to the phosphate group to the CTD peptide. The addition of paramagnetic MnCl2 indicated the CTD peptide binds the PG surface but does not insert into the bilayer. Molecular dynamics simulations, supported by site-directed mutagenesis experiments, identify key residues in the helix-turn-helix that drive membrane association. The data show that the OhyA CTD binds the phosphate layer of the PG surface to obtain bilayer-embedded unsaturated fatty acids.

Ceramide analog C2-cer induces a loss in insulin sensitivity in muscle cells through the salvage/recycling pathway.

Ceramides have been shown to play a major role in the onset of skeletal muscle insulin resistance and therefore in the prevalence of type 2 diabetes. However, many of the studies involved in the discovery of deleterious ceramide actions used a nonphysiological, cell-permeable, short-chain ceramide analog, the C2-ceramide (C2-cer). In the present study, we determined how C2-cer promotes insulin resistance in muscle cells. We demonstrate that C2-cer enters the salvage/recycling pathway and becomes deacylated, yielding sphingosine, re-acylation of which depends on the availability of long chain fatty acids provided by the lipogenesis pathway in muscle cells. Importantly, we show these salvaged ceramides are actually responsible for the inhibition of insulin signaling induced by C2-cer. Interestingly, we also show that the exogenous and endogenous monounsaturated fatty acid oleate prevents C2-cer to be recycled into endogenous ceramide species in a diacylglycerol O-acyltransferase 1-dependent mechanism, which forces free fatty acid metabolism towards triacylglyceride production. Altogether, the study highlights for the first time that C2-cer induces a loss in insulin sensitivity through the salvage/recycling pathway in muscle cells. This study also validates C2-cer as a convenient tool to decipher mechanisms by which long-chain ceramides mediate insulin resistance in muscle cells and suggests that in addition to the de novo ceramide synthesis, recycling of ceramide could contribute to muscle insulin resistance observed in obesity and type 2 diabetes.

Ultralong Nanowires of Cadmium Phosphate Hydroxide Synthesized Using a Cadmium Oleate Precursor Hydrothermal Method and Sulfidation Conversion to Ultralong CdS Nanowires.

Ultralong nanowires with ultrahigh aspect ratios exhibit high flexibility, and they are promising for applications in various fields. Herein, a cadmium oleate precursor hydrothermal method is developed for the synthesis of ultralong nanowires of cadmium phosphate hydroxide. In this method, water-soluble cadmium salt is used as the cadmium source, water-soluble phosphate is used as the phosphorus source, and sodium oleate is adopted as a reactant to form cadmium oleate precursor and as a structure-directing agent. By using this method, ultralong nanowires of cadmium phosphate hydroxide are successfully synthesized using CdCl2, sodium oleate, and NaH2PO4 as reactants in an aqueous solution by hydrothermal treatment at 180 °C for 24 h. In addition, a new type of flexible fire-resistant inorganic paper with good electrical insulation performance is fabricated using ultralong nanowires of cadmium phosphate hydroxide. As an example of the extended application of this synthetic method, ultralong nanowires of cadmium phosphate hydroxide can be converted to ultralong CdS nanowires through a convenient sulfidation reaction. In this way, ultralong CdS nanowires are successfully synthesized by simple sulfidation of ultralong nanowires of cadmium phosphate hydroxide under mild conditions. The as-prepared ultralong nanowires of cadmium phosphate hydroxide are promising for applications as the precursors and templates for synthesizing other inorganic ultralong nanowires and have wide applications in various fields.

Long-term prevention of bladder cancer progression by alpha1-oleate alone or in combination with chemotherapy.

Bladder cancer is common and one of the most costly cancer forms, due to a lack of curative therapies. Recently, clinical safety and efficacy of the alpha1-oleate complex was demonstrated in a placebo-controlled study of nonmuscle invasive bladder cancer. Our study investigated if long-term therapeutic efficacy is improved by repeated treatment cycles and by combining alpha1-oleate with low-dose chemotherapy. Rapidly growing bladder tumors were treated by intravesical instillation of alpha1-oleate, Epirubicin or Mitomycin C alone or in combination. One treatment cycle arrested tumor growth, with a protective effect lasting at least 4 weeks in mice receiving 8.5 mM of alpha1-oleate alone or 1.7 mM of alpha-oleate combined with Epirubicin or Mitomycin C. Repeated treatment cycles extended protection, defined by a lack of bladder pathology and a virtual absence of bladder cancer-specific gene expression. Synergy with Epirubicin was detected at the lower alpha1-oleate concentration and in vitro, alpha1-oleate was shown to enhance the uptake and nuclear translocation of Epirubicin, by tumor cells. Effects at the chromatin level affecting cell proliferation were further suggested by reduced BrdU incorporation. In addition, alpha1-oleate triggered DNA fragmentation, defined by the TUNEL assay. The results suggest that bladder cancer development may be prevented long-term in the murine model, by alpha1-oleate alone or in combination with low-dose Epirubicin. In addition, the combination of alpha1-oleate and Epirubicin reduced the size of established tumors. Exploring these potent preventive and therapeutic effects will be of immediate interest in patients with bladder cancer.

Hepatic oleate regulates one-carbon metabolism during high carbohydrate feeding.

Obesity is a major risk factor for type 2 diabetes, coronary heart disease, and strok. These diseases are associated with profound alterations in gene expression in metabolic tissues. Epigenetic-mediated regulation of gene expression is one mechanism through which environmental factors, such as diet, modify gene expression and disease predisposition. However, epigenetic control of gene expression in obesity and insulin resistance is not fully characterized. We discovered that liver-specific stearoyl-CoA desaturase-1 (Scd1) knockout mice (LKO) fed a high-carbohydrate low-fat diet exhibit dramatic changes in hepatic gene expression and metabolites of the folate cycle and one-carbon metabolism respectively for the synthesis of S-adenosylmethionine (SAM). LKO mice show an increased ratio of S-adenosylmethionine to S-adenosylhomocysteine, a marker for increased cellular methylation capacity. Furthermore, expression of DNA and histone methyltransferase genes is up-regulated while the mRNA and protein levels of the non-DNA methyltransferases including phosphatidylethanolamine methyltransferase (PEMT), Betaine homocysteine methyltransferase (Bhmt), and the SAM-utilizing enzymes such as glycine-N-methyltransferase (Gnmt) and guanidinoacetate methyltransferase (Gamt) are generally down-regulated. Feeding LKO mice a high carbohydrate diet supplemented with triolein, but not tristearin, and increased endogenous hepatic synthesis of oleate but not palmitoleate in Scd1 global knockout mice normalized one carbon gene expression and metabolite levels. Additionally, changes in one carbon gene expression are independent of the PGC-1α-mediated ER stress response previously reported in the LKO mice. Together, these results highlight the important role of oleate in maintaining one-carbon cycle homeostasis and point to observed changes in one-carbon metabolism as a novel mediator of the Scd1 deficiency-induced liver phenotype.

Efficacy of minocycline hydrochloride aspiration sclerotherapy with additional monoethanolamine oleate aspiration sclerotherapy for symptomatic hepatic cysts.

AIM: Minocycline hydrochloride (MINO) aspiration sclerotherapy (AS) has been widely used for treating hepatic cysts (HC). However, cyst recurrence remains problematic. Information on monoethanolamine oleate (EO) AS, another effective HC treatment, is currently limited. We investigated the efficacy of EO on ineffective MINO treatments, and the relationship between MINO AS and cyst fluid pH. METHODS: A total of 22 cases with symptomatic HC underwent AS with 500 mg of MINO from January 2016 to June 2021. Cyst fluid pH was measured before and after MINO injection. Cyst volume ratio (CVR, %) after 2 weeks was calculated as follows:cyst volume 2 weeks after MINO injection / pre-treatment cyst volume × 100. Treatment was completed if CVR after 2 weeks was ≤35% (MINO-group). For patients with CVR >35%, 2 g of EO was added (MINO/EO-group). Cyst volume ratio was measured every 12 months thereafter. RESULTS: There were no recurrence symptoms in any of the patients during follow-up. Of the 22 cases, 21 had CVR ≤20% after 12 months. The MINO/EO-group (n = 8) tended to have smaller CVRs after 12 months than the MINO-group (n = 14). Cyst volume ratio after 2 weeks was correlated to pH change (p = 0.012) and was larger in patients whose pH decreased by <1.5 (p = 0.015). All adverse events were mild, including in elderly patients. CONCLUSION: Adding EO is an effective and safe treatment for symptomatic HC when MINO AS alone is insufficient. Patients with pH decreases of <1.5 should be considered for additional EO treatment.

The presence of high-risk varices after sclerotherapy in biliary atresia.

BACKGROUND: Esophagogastric varices (EGVs) may develop as a result of portal hypertension in children with biliary atresia (BA). Although endoscopic injection sclerotherapy (EIS) with ethanolamine oleate (EO) is reported useful for children, risk factors associated with the presence of high-risk EGVs after treatment remain unknown. METHODS: The subjects were BA patients under 15 years of age who underwent EO-EIS. We retrospectively reviewed a total of 28 treatment sessions of EGVs with red signs and those larger than F2, which were considered to be at high risk of bleeding. Survival analysis was performed for the presence of high-risk EGVs at the time of follow-up endoscopy as the occurrence of an event. RESULTS: Univariate analysis showed a significantly increased risk of the presence of high-risk EGVs post-EO-EIS in patients with increased liver stiffness (LS) and Mac-2 binding protein glycan isomer (M2BPGi), with hazard ratios of 1.48 and 1.15, respectively. The median presence-free period was significantly shorter in the LS ≥ 2.8 m/s patients than in those with LS <2.8 m/s (189 vs. 266 days). Similarly, the median presence-free period was significantly shorter in patients with M2BPGi ≥ 4.0 than in those with M2BPGi < 4.0 (182 vs. 203 days). The results of multivariate analysis revealed that the risk of the presence of high-risk EGVs was significantly higher only in the high-LS group, with a hazard ratio of 2.76. CONCLUSIONS: Increased LS is associated with risk of the presence of high-risk EGVs following EO-EIS in children with BA.

Investigation of the enhancement for Echinocandin B fermentation with methyl oleate from transcription level.

Echinocandin B (ECB) is the key precursor compound of the antifungal drug Anidulafungin. The effects of the five precursor amino acids on ECB biosynthesis were firstly investigated. It showed that although L-threonine was a main compound of the hexapeptide scaffold of ECB, exogenous addition of L-threonine had no significant effect on the increase of ECB fermentation titer. Meanwhile, the ECB fermentation titer with methyl oleate showed two times higher than that of the other carbon sources. Transcription level analysis of the key genes for ECB biosynthesis indicated that the gene an655543 related to L-threonine biosynthesis showed higher value during the fermentation process, therefore, the exogenous addition of L-threonine had no obvious affection. Furthermore, it indicated that the transcription level of gene ecdA might be the main restriction factor for the ECB biosynthesis. The study provided the research foundation for the modification of the ECB producing strains in the following work.

Diode laser versus sclerotherapy: bloodless approaches in the treatment of oral pyogenic granuloma (randomised controlled clinical trial).

Oral pyogenic granuloma (PG) is traditionally treated by surgical excision which is associated with bleeding, pain and a high rate of recurrence. Our research aimed to clinically assess the effectiveness of diode laser versus sclerotherapy, as bloodless approach, in the treatment of oral PG. We randomly divided 20 patients with oral PG into two groups, with those in the test group being managed via diode laser application and those in the control group via injections of ethanolamine oleate as a sclerosing agent. All patients were evaluated intraoperatively for bleeding severity and postoperatively for pain. The quality of healing was also assessed using Landry healing index after the 1st, 2nd and 4th weeks. Additionally, the patients were recalled after 3, 6 and 9 months from the end of treatment for recurrence evaluation. Our results revealed that intraoperative bleeding did not differ significantly between both groups while postoperative pain decreased significantly in the sclerotherapy group compared to the laser group. For different intervals, the sclerotherapy group had a higher healing quality index than the laser group, although the difference was not statistically significant. However, recurrence occurred in the laser group, there were no cases of recurrence in the sclerotherapy group in all intervals. In conclusion, diode laser treatment of PG is a reliable, less invasive, and sensitive procedure that requires an experienced operator and specialised equipment. However, ethanolamine oleate sclerotherapy is an inexpensive, simple technique besides being less prone to recurrence problems, especially when treatment duration is not a concern.

Efficient removal of NaOl from mineral processing wastewater using Al-electrocoagulation.

Sodium oleate (NaOl) is widely used as collector for oxidised ore flotation, and residual NaOl in mineral processing wastewater is a serious threat to mine environment. In this work, the feasibility of electrocoagulation (EC) as an alternative for chemical oxygen demand (COD) removal from NaOl-containing wastewater was demonstrated. Major variables were evaluated to optimise EC, and related mechanisms were proposed to interpret the observations in EC experiments. The initial pH of the wastewater greatly affected the COD removal efficiency, which was likely to be related to the variation of predominant species. When the pH was below 8.93 (original pH), liquid HOl(l) was the predominant specie, which could be rapidly removed by EC thought charge neutralisation and adsorption. At original pH or higher, Ol- could react with dissolved Al3+ to form insoluble Al(Ol)3, which was subsequently removed through charge neutralisation and adsorption. The presence of fine mineral particles could reduce repulsion force of the suspended solids and promote flocculation, whereas the presence of water glass had an opposite effect. These results demonstrated that EC can be employed as an effective process to purify NaOl-containing wastewater. This study will contribute to deepening our understanding of EC technology for NaOl removal and provide useful information to researchers in mineral processing industry.

Transcriptome analysis revealed detoxification gene expression changes in Tetranychus cinnabarinus challenged with ethyl oleate.

Natural acaricides are potential biorational mite control alternatives to conventional chemical acaricides. However, little is known about the molecular mechanism of defense response to natural acaricides in mites. We previously reported significant acaricidal properties of ethyl oleate (EO) against Tetranychus cinnabarinus (here referred to as a sibling species of two-spotted spider mite, Tetranychus urticae), a highly polyphagous pest devastating crops in fields and greenhouses worldwide. In this study, we explored the molecular responses of T. cinnabarinus exposed to EO using RNA-Seq and differentially expressed gene (DEG) analysis. A total of 131, 185, and 154 DEGs were identified in T. cinnabarinus after 1, 6, and 24 h of EO treatment. In addition, 36 putative detoxification-related DEGs, including 10 cytochrome P450s (P450s), three glutathione S-transferases (GSTs), nine UDP-glycosyltransferases (UGTs), eight esterases (ESTs), and six ATP-binding cassette transporters (ABC transporters), were identified. Interestingly, the upregulation of these detoxification-related genes might be the main defense response of T. cinnabarinus exposed to EO. A quantitative real-time PCR analysis indicated that the expression profiles of 19 random DEGs were consistent with the RNA-Seq results. These findings serve as valuable information for a better understanding of the acaricide-mite interaction and molecular mechanisms involved in the defense response of T. cinnabarinus against EO.

A pH-Controlled Solid Inhibitor Based on PAM Hydrogel for Steel Corrosion Protection in Wide Range pH NaCl Medium.

To provide carbon steel a long-term corrosion protection effect in NaCl solutions with different pH values, based on poly-acrylamide (PAM) and oleate imidazoline (OIM), a solid corrosion inhibitor with the properties of pH-controlled release was synthesized. SEM, FTIR and TGA results indicated that the OIM inhibitors were successfully loaded into PAM hydrogel with a high OIM encapsulation content (39.64 wt.%). The OIM release behavior from the hydrogel structure has two stages, quick release and sustained release. The pH of solutions could affect the initial release kinetics of OIM inhibitors and the diffusion path in the hydrogel structure. Weight loss measurement of L80 steel in different pH solutions with OIM@PAM proved the inhibitor responsive release mechanism and anticorrosion performance. The inhibition efficiency of OIM@PAM can maintain over 80% after long-term immersion in a harsh corrosive environment (pH 3), which is much higher than the inhibition efficiency of OIM@PAM in a moderate corrosive solution.

Effects of Hydrogen Bonds between Ethoxylated Alcohols and Sodium Oleate on Collecting Performance in Flotation of Quartz.

Hydrogen bonds play an important role in the interaction between surfactants. In this study, the effect of three different ethoxylated alcohols (OP-10, NP-10, AEO-9) on the collecting behavior of sodium oleate (NaOL) in the flotation of quartz was investigated. To explore the mechanism, the hydrogen bond between ethoxylated alcohols and NaOL was analyzed using molecular dynamics (MD) simulation. The results showed that ethoxylated alcohols promoted the collecting performance of NaOL and reduced the dosage of the activator CaO and the collector NaOL in the flotation of quartz. The Zeta potential measurement illustrated that ethoxylated alcohols promoted the adsorption of OL- on the activated quartz surface and the degree of promotion was in the order of OP-10 > NP-10 > AEO-9. The MD simulation results showed that a hydrogen bond presented between ethoxylated alcohols and OL-. Due to the hydrogen bond between the ethoxylated alcohols and OL-, the attraction force between OL- and the quartz surface increased with the addition of ethoxylated alcohols in the order of OP-10 > NP-10 > AEO-9 based on the MD simulation results. As the result, the addition of ethoxylated alcohols increased the adsorption density of OL- on the activated quartz surface, which explained the promotion of the collecting performance of OL- in the flotation of quartz.

Structure Identification of Adsorbed Anionic-Nonionic Binary Surfactant Layers Based on Interfacial Shear Rheology Studies and Surface Tension Isotherms.

Mixtures of anionic sodium oleate (NaOl) and nonionic ethoxylated or alkoxylated surfactants improve the selective separation of magnesite particles from mineral ores during the process of flotation. Apart from triggering the hydrophobicity of magnesite particles, these surfactant molecules adsorb to the air-liquid interface of flotation bubbles, changing the interfacial properties and thus affecting the flotation efficiency. The structure of adsorbed surfactants layers at the air-liquid interface depends on the adsorption kinetics of each surfactant and the reformation of intermolecular forces upon mixing. Up to now, researchers use surface tension measurements to understand the nature of intermolecular interactions in such binary surfactant mixtures. Aiming to adapt better to the dynamic character of flotation, the present work explores the interfacial rheology of NaOl mixtures with different nonionic surfactants to study the interfacial arrangement and viscoelastic properties of adsorbed surfactants under the application of shear forces. Interfacial shear viscosity results reveal the tendency on nonionic molecules to displace NaOl molecules from the interface. The critical nonionic surfactant concentration needed to complete NaOl displacement at the interface depends on the length of its hydrophilic part and on the geometry of its hydrophobic chain. The above indications are supported by surface tension isotherms.

Application of small angle X-ray scattering in exploring the effect of edible oils with different unsaturation FAs on bioaccessibility of stigmasterol oleate.

BACKGROUND: Phytosterol can improve its lipid solubility, lipophilic/hydrophilic balance and bioaccessibility by esterification with fatty acids, which increases its practical application range in the food industry. In the present study, small angle X-ray scattering combined with the pH-stat in vitro digestion model was applied to continuously monitor the molecular structure evolution of mixed micelles during digestion and investigate the effect of three edible oils (olive oil with 72.41 ± 0.57% oleic, sunflower seed oil with 63.45 ± 0.78% linoleic, refined linseed oil with 51.74 ± 0.34% linolenic) on bioaccessibility of stigmasterol oleate in vitro. RESULTS: The release degree and rate of fatty acids in the three edible oil systems (kOO+ST-OA = 0.0501, kSO+ ST-OA = 0.0357, kLO+ST-OA = 0.0323) was compared. The three different edible oils had similar impact on the formation of dietary mixed micelles during the simulatedin vitro digestion of stigmasterol oleate, although there were significant differences in molecular morphology and composition of mixed micelles. The results showed that the vesicles formed by linoleic oil (SO system) or linolenic oil (LO system) were easy to dissociate. The largest average number and diameter of vesicles (5.55 × 1016 cm-3 and 2230.75 Å), the most stable vesicle structure and the fastest fatty acid release rate were observed in the OO system. CONCLUSION: Compared to linoleic (SO system) or linolenic (LO system), the oleic (OO system) could facilitate the transformation of micelles to vesicles and maintain the stability of its membrane, significantly promotin the dissolution of stigmasterol and improving bioaccessibility. © 2023 Society of Chemical Industry.

Improved antioxidant activity of rutin via lipase-mediated esterification with oleic acid.

BACKGROUND: Oxidation is a major problem for oils and fats, which can be mitigated by antioxidants. Rutin has excellent antioxidant activity, but its poor lipid solubility greatly limits its practical application. In this study, an efficient enzymatic synthesis route of lipophilic rutin ester was established using oleic acid as an acyl donor, and the antioxidant potential of rutin oleate was evaluated for the first time by proton (1 H) nuclear magnetic resonance (NMR) spectroscopy. RESULTS: The synthesized product was finally identified as rutin oleate by Fourier transform infrared, high-performance liquid chromatography-mass spectrometry, and 1 H, carbon-13, and DEPT-135 NMR analyses, and the acylation site was the 4‴-OH of the rhamnose group in the rutin molecule. The maximum conversion was over 93% after 48 h of reaction using Novozym 435 as catalyst under the best conditions among these tests. The conversion of rutin ester decreased with the increase of carbon chain length and the number of carbon-carbon double bonds of the fatty acid molecule. Most importantly, rutin oleate exhibited antioxidant capacity comparable to butylated hydroxytoluene and its counterparts (rutin and oleic acid) at low temperatures (60° C), but had a significant advantage at high temperatures (120° C). CONCLUSION: The antioxidant activity of rutin was significantly enhanced by lipase-mediated esterification with oleic acid. Therefore, rutin oleate could be further developed as a novel antioxidant for use in oil- and fat-based foods. © 2023 Society of Chemical Industry.

Antioxidant monoammonium glycyrrhizinate alleviates damage from oxidative stress in perinatal cows.

This study was conducted to evaluate the antioxidant capability of dietary supplementation with monoammonium glycyrrhizinate (MAG) in perinatal cows. Glycyrrhizic acid has been shown to have strong antioxidant activity and we hypothesised that the aglycone of glycyrrhizin and MAG, could reduce damage from oxidative stress in perinatal cows by enhancing antioxidant capacity. Blood and milk samples were collected from three groups of healthy perinatal cows that were similar in body weight, parity, milk yield in the last milk cycle, etc., receiving dietary MAG supplementation ([Day 0 = parturition]: 0 g/day, [n = 13)] 3 g/day [n = 13] or 6 g/day [n = 11]) from -28 to 56 day (0 day = parturition). Compared with 0 g/day controls (CON), milk fat was significantly decreased in cows fed with MAG, and 3 g/day had the greatest effect. A diet containing 3 g/day MAG decreased the serum alanine aminotransferase (ALT) level compared with CON at -7 day post-partum. ALT was also lower at 5 day post-partum in cows fed with 3 g/day MAG compared to 6 g/day. The administration of 3 g/day and 6 g/day MAG decreased serum aspartate transaminase (AST) at 3 day post-partum. Supplementation of MAG in cows increased total antioxidant capacity (T-AOC) in serum, and cows given 3 g MAG per day had higher T-AOC than controls on post-partum 7 day. At the end of the experiment, we isolated and cultured primary hepatocytes to determine the effect of MAG on oxidative stress caused by incubation with the sodium oleate (SO). SO increased lipid synthesis, but pre-treatment with MAG prevented the fatty buildup. SO treatment increased AST and ALT levels and malondialdehyde concentration, but decreased T-AOC and superoxide dismutase (SOD). Incubation with MAG increased antioxidant capacity and inhibited oxidant damage in bovine hepatocytes. SO stimulated expression of the antioxidant genes, NAD(P)H quinone dehydrogenase 1 (NQO1) and SOD1, in the nuclear factor erythroid 2-related factor 2 (NRF2) pathway, and catalase 1 (CAT1); this increase was accentuated by MAG pre-treatment. The results suggest that MAG can alleviate the damage caused by oxidative stress in perinatal cows by enhancing antioxidant activity.

Structure and mechanism of Staphylococcus aureus oleate hydratase (OhyA).

Flavin adenine dinucleotide (FAD)-dependent bacterial oleate hydratases (OhyAs) catalyze the addition of water to isolated fatty acid carbon-carbon double bonds. Staphylococcus aureus uses OhyA to counteract the host innate immune response by inactivating antimicrobial unsaturated fatty acids. Mechanistic information explaining how OhyAs catalyze regiospecific and stereospecific hydration is required to understand their biological functions and the potential for engineering new products. In this study, we deduced the catalytic mechanism of OhyA from multiple structures of S. aureus OhyA in binary and ternary complexes with combinations of ligands along with biochemical analyses of relevant mutants. The substrate-free state shows Arg81 is the gatekeeper that controls fatty acid entrance to the active site. FAD binding engages the catalytic loop to simultaneously rotate Glu82 into its active conformation and Arg81 out of the hydrophobic substrate tunnel, allowing the fatty acid to rotate into the active site. FAD binding also dehydrates the active site, leaving a single water molecule connected to Glu82. This active site water is a hydronium ion based on the analysis of its hydrogen bond network in the OhyA•PEG400•FAD complex. We conclude that OhyA accelerates acid-catalyzed alkene hydration by positioning the fatty acid double bond to attack the active site hydronium ion, followed by the addition of water to the transient carbocation intermediate. Structural transitions within S. aureus OhyA channel oleate to the active site, curl oleate around the substrate water, and stabilize the hydroxylated product to inactivate antimicrobial fatty acids.

Paenibacillus oleatilyticus sp. nov., isolated from soil.

A gram-stain-negative, endo-spore forming, facultatively anaerobic, motile, rod-shaped bacterial strain SM69T, isolated from soil samples of Rohtak, Haryana, India was characterized using polyphasic approach. White colonies were 2-3 mm, in diameter and growth occurred between 20 and 55 °C, pH 6.0-10.0 with 0-2.0% (w/v) NaCl. Based on 16S rRNA gene sequence similarity the strain is placed in the genus Paenibacillus as it is closely related to 'Paenibacillus tyrfis MSt1T' (99.7%) and P. elgii SD17T (99.6%). The cell wall peptidoglycan contained meso-diaminopimelic acid. The dominant fatty acids included anteiso-C15: 0 (50%), C16: 0 (12%) and anteiso-C17: 0 (10%). Major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. The size of the draft genome was 7,848,017 bp, with 53.1% G+C content. dDDH (51.6%) and ANI (93.5%) of strain SM69T with its close relatives indicates that it represents a novel species, for which the name Paenibacillus oleatilyticus sp. nov. (Type strain SM69T = MCC 3064T = JCM 33981T = KACC 21649T) is proposed.