Free Acid Form Research Articles

Effects of methionine as free amino acid and dipeptide on productive efficiency and meat quality of broilers under acute and chronic heat stress

Context Methionine in the form of free amino acid has been widely studied in broilers challenged by heat stress (HS). However, the effects of methionine dipeptide in broilers subjected to HS are not known. Aims To evaluate the effects of methionine as free amino acid and dipeptide on the performance, oxidative status, plasma parameters and meat quality of broilers subjected to acute and chronic HS. Methods Broilers were evaluated at the following three experimental periods: 24 h of evaluation (21–22 days of age); 10 days of evaluation (22–32 days of age); and 20 days of evaluation (22–42 days of age). Broilers were divided into two groups; one group was raised in thermal comfort, and the other group was raised in continuous HS of 30°C. In both groups, animals received a diet without methionine supplementation (MD), with supplementation of methionine as free amino acid (dl-M), and with supplementation of methionine as dipeptide (dl-MM). Key results HS reduced body weight gain after 10 and 20 days of evaluation. Broilers under HS condition fed the MD diet had the highest concentrations of thiobarbituric acid reactive substances (TBARS) at 22 days of age and carbonylated proteins (CP) at 32 days of age. At 42 days of age, broilers fed the MD diet had higher concentrations of TBARS and CP. At 32 days of age, broilers under HS had lower high-density lipoprotein and higher low-density lipoprotein concentrations. In breast meat, broilers in thermal comfort fed the dl-M or dl-MM diets had a lower cooking loss. Broilers in HS fed the dl-M diet had the lowest cooking loss. HS reduced the pH of the meat of legs. Conclusions The acute and chronic HS affect the broiler performance in different ways. Methionine supplementation contributes to reduce the effects of HS. There were no notable differences between the supplementation of dl-M or dl-MM. Implications Productivity and the quality of the chicken meat are the most important attributes of the production. The ambient temperature can influence these parameters. The methionine in its most varied forms, due to its direct and indirect antioxidant function, has been shown to be an effective source of protection for the animal in adverse conditions such as during HS.

Release of Anti-Inflammatory Palmitoleic Acid and Its Positional Isomers by Mouse Peritoneal Macrophages.

Positional isomers of hexadecenoic acid are considered as fatty acids with anti-inflammatory properties. The best known of them, palmitoleic acid (cis-9-hexadecenoic acid, 16:1n-7), has been identified as a lipokine with important beneficial actions in metabolic diseases. Hypogeic acid (cis-7-hexadecenoic acid, 16:1n-9) has been regarded as a possible biomarker of foamy cell formation during atherosclerosis. Notwithstanding the importance of these isomers as possible regulators of inflammatory responses, very little is known about the regulation of their levels and distribution and mobilization among the different lipid pools within the cell. In this work, we describe that the bulk of hexadecenoic fatty acids found in mouse peritoneal macrophages is esterified in a unique phosphatidylcholine species, which contains palmitic acid at the sn-1 position, and hexadecenoic acid at the sn-2 position. This species markedly decreases when the macrophages are activated with inflammatory stimuli, in parallel with net mobilization of free hexadecenoic acid. Using pharmacological inhibitors and specific gene-silencing approaches, we demonstrate that hexadecenoic acids are selectively released by calcium-independent group VIA phospholipase A2 under activation conditions. While most of the released hexadecenoic acid accumulates in free fatty acid form, a significant part is also transferred to other phospholipids to form hexadecenoate-containing inositol phospholipids, which are known to possess growth-factor-like-properties, and are also used to form fatty acid esters of hydroxy fatty acids, compounds with known anti-diabetic and anti-inflammatory properties. Collectively, these data unveil new pathways and mechanisms for the utilization of palmitoleic acid and its isomers during inflammatory conditions, and raise the intriguing possibility that part of the anti-inflammatory activity of these fatty acids may be due to conversion to other lipid mediators.

Acute dietary saturated fat intake suppresses human monocyte subset inflammatory and chemokine responses

Abstract Background Dietary saturated fat (SF) intake is implicated in increasing cardiovascular risk. Studies suggest SF may incite an inflammatory immune response yet the mechanisms for this remain unclear. Circulating blood monocyte subsets (classical “CD16low” and non-classical/intermediate “CD16high”) are thought to differentially contribute to the macrophage foam cell burden in atherosclerotic plaque, yet they are heterogenous and much is still unknown about their response to dietary lipid. Purpose This dietary intervention study hypothesised that a subset-specific monocyte response occurs following acute SF intake in healthy individuals.Top of Form Methods 10 healthy participants were fed a coconut oil, lauric acid-rich meal. Monocyte subsets were purified from fasting and 4h postprandial blood by fluorescence activated cytometry (FACS) into CD16low and CD16high subsets. Surface marker expression was recorded by FACS, gene expression by qPCR, lipid loading by fluorescence microscopy, cytokine responses by ELISA and mass spectrometry was utilised to trace dietary fat into circulating monocytes. Triglyceride rich lipoprotein (TGRL) was obtained through ultracentrifugation of postprandial blood. TLR agonists LPS (TLR4) and R848 (TLR7/8) were used to replicate infectious stimuli to provoke a cytokine response. Results SF meal intake resulted in a 4h postprandial rise in serum triglycerides (TG). Other than a small rise in HLA-DR in postprandial CD16low monocytes, no changes in CD14, CD16 or CD11c levels were noted. Inflammatory gene expression was downregulated although a rise in lipid handling gene ABCA1 was noted in both subsets. Postprandial monocytes accumulated cytoplasmic neutral lipid droplets (larger and more numerate droplets in CD16low). Mass-spectrometry confirmed entry of dietary lipid into circulating monocytes. Postprandial monocyte functional responses were impaired; lipid-loaded postprandial monocytes were less able to migrate towards a chemokine gradient. Following TLR challenge, the inflammatory response was abrogated in postprandial monocytes, further suppressed by in vitro postprandial TGRL addition. Lipidomic analyses confirmed postprandial monocyte lauric acid accumulation in the form of free fatty acid and TG trilaurin, and 9(S)-, 13(S)-HODE and 17(S)-HDoHE, that are metabolites of docosahexanoic acid (DHA) and possess anti-inflammatory properties. The attached graphical abstract summarises the pertinent findings. Conclusions Acute dietary SF intake of a lauric acid-rich meal results in postprandial monocyte suppression of inflammatory TLR responses, a reduction in the ability to migrate towards a chemokine gradient whilst accumulating cytoplasmic neutral lipid droplets. The immunosuppression may be caused by accumulation of anti-inflammatory metabolites of DHA. Graphical Abstract Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): British Heart Foundation, UK

DGLA from the Microalga Lobosphaera Incsa P127 Modulates Inflammatory Response, Inhibits iNOS Expression and Alleviates NO Secretion in RAW264.7 Murine Macrophages.

Microalgae have been considered as a renewable source of nutritional, cosmetic and pharmaceutical compounds. The ability to produce health-beneficial long-chain polyunsaturated fatty acids (LC-PUFA) is of high interest. LC-PUFA and their metabolic lipid mediators, modulate key inflammatory pathways in numerous models. In particular, the metabolism of arachidonic acid under inflammatory challenge influences the immune reactivity of macrophages. However, less is known about another omega-6 LC-PUFA, dihomo-γ-linolenic acid (DGLA), which exhibits potent anti-inflammatory activities, which contrast with its delta-5 desaturase product, arachidonic acid (ARA). In this work, we examined whether administrating DGLA would modulate the inflammatory response in the RAW264.7 murine macrophage cell line. DGLA was applied for 24 h in the forms of carboxylic (free) acid, ethyl ester, and ethyl esters obtained from the DGLA-accumulating delta-5 desaturase mutant strain P127 of the green microalga Lobosphaera incisa. DGLA induced a dose-dependent increase in the RAW264.7 cells’ basal secretion of the prostaglandin PGE1. Upon bacterial lipopolysaccharide (LPS) stimuli, the enhanced production of pro-inflammatory cytokines, tumor necrosis factor alpha (TNFα) and interleukin 1β (IL-1β), was affected little by DGLA, while interleukin 6 (IL-6), nitric oxide, and total reactive oxygen species (ROS) decreased significantly. DGLA administered at 100 µM in all forms attenuated the LPS-induced expression of the key inflammatory genes in a concerted manner, in particular iNOS, IL-6, and LxR, in the form of free acid. PGE1 was the major prostaglandin detected in DGLA-supplemented culture supernatants, whose production prevailed over ARA-derived PGE2 and PGD2, which were less affected by LPS-stimulation compared with the vehicle control. An overall pattern of change indicated DGLA’s induced alleviation of the inflammatory state. Finally, our results indicate that microalgae-derived, DGLA-enriched ethyl esters (30%) exhibited similar activities to DGLA ethyl esters, strengthening the potential of this microalga as a potent source of this rare anti-inflammatory fatty acid.

Optimization of pH, time, temperature, variety and concentration of the added fatty acid and the initial count of added lactic acid Bacteria strains to improve microbial conjugated linoleic acid production in fermented ground beef

The aim of the present study was to determine the optimum pH, time, temperature, variety and concentration of the added fatty acid and the initial count of added Lactobacillus plantarum AB20–961 and Lactobacillus plantarum DSM2601 for high conjugated linoleic acid (CLA) production in ground beef. The highest CLA production with using safflower fatty acids by L. plantarum AB20–961 and L. plantarum DSM2601 was 7.91 and 38.31 mg CLA/g fat, respectively (P < 0.05). Optimum conditions for both strains were 37 °C fermentation temperature, 5% added fatty acid in free form and 8 log CFU/g initial count. Additionally, the optimum pH and fermentation time were 7.94 pH and 78.78 h for L. plantarum AB20–961 and 7.68 and 72.57 h for L. plantarum DSM2601. The results indicated that both L. plantarum strains with optimum conditions determined in the present study may be applied in order to enrich CLA content in ground beef and satisfy consumer demands for the fermented meat products with functional components.

Monosulfonated Azo Dyes: A Crystallographic Study of the Molecular Structures of the Free Acid, Anionic and Dianionic Forms

Crystallographic studies of monosulfonated azo dyes have concentrated on the salt forms that contain the azo anion. Here we present a study that compares the structures of these anions with protonated free acid forms and with doubly deprotonated dianion forms. To this end, the new single crystal diffraction structures of 13 systematically related free acid forms of monosulfonated azo dyes are presented, together with three new structures of doubly deprotonated forms and two new structures of Na salt forms of azo anions. No structures of dideprotonated monosulfonated azo dyes have previously been reported and this paper also reports the first crystal structure of an azo dye with a hydronium cation. The geometries of the free acid, anion and dianion forms are compared to literature equivalents. It is shown that protonation of the azo bond gives predictable bond lengthening and shortening, which is of a greater magnitude than similar effects caused by azo-hydrazone tautomerisation, or the smaller again effects caused by the resonance electron donation from O or N based substituents. The dianion containing structures have twisted dianion geometries that can be understood based on the resonance effects of the phenoxide groups and upon the needs to bond to a relatively high number of metal cations.

Determination of Cyclopropenoid Fatty Acids in Ewe Milk Fat by GC-MS after Intravenous Administration of Sterculic Acid.

Cyclopropenoid fatty acids (CPEFA), found in oilseeds from Malvaceae and Sterculiaceae, have been shown to interfere with the endogenous synthesis of several bioactive lipids of dairy fat, such as cis-9, trans-11 18:2 and cis-9 18:1, by inhibiting Δ9-desaturase. No previous study has reported the presence of sterculic acid in animal fat and its incorporation in tissues after its administration, due to the lack of a proper methodology. In the present research, a GC-MS method based on cold base derivatization to fatty acids methylesters was developed to determine CPEFA in ewe milk triglycerides, after infusing sterculic acid (0.5 g/day) to six lactating ewes. An alternative derivatization based on silanyzation followed by GC-MS analysis was also tested, showing its possible applicability when CPEFA are present in the form of free fatty acids. Sterculic acid was detected in ewe milk triglycerides, demonstrating its incorporation from the bloodstream into milk by the mammary gland. The mean transfer rate represented 8.0 ± 1.0% of the daily dose. This study provides, for the first time, the presence of sterculic acid in milk fat, supporting the importance of understanding its occurrence in vivo and encouraging further research to determine whether it can be present in foods, such as dairy products, obtained under practical farming conditions.

β-hydroxy-β-methylbutyrate (HMB) Does Not Improve Resistance Exercise-Induced Changes In Body Composition: A Systematic-review And Meta-analysis

β-hydroxy-β-methylbutyrate (HMB) is a leucine metabolite used as a nutritional supplement purported to increase lean body mass and performance in response to resistance exercise training (RET). However, literature definitive evidence-based answer to the question of the efficacy of HMB is lacking. PURPOSE: The aim of this systematic-review and meta-analysis was to determine the efficacy of HMB supplementation, in the calcium (HMB-Ca) or free acid (HMB-FA) form, to augment lean body mass and strength gains during RET. METHODS: A systematic search on Medline, Embase, CINAHL and SportDiscus, from 1996-Oct 2019 was conducted. Inclusion criteria for studies were: randomized controlled trial (RCT), RET ≥ 3 weeks (training sessions at least 2 x/week), male subjects <50 y, and ingesting 3g/d of HMB-Ca or HMB-FA with or without protein or amino acids. Random-effects meta-analysis was performed in Review Manager V.5.3. Cochrane risk-of-bias tool for randomized trials (RoB 2) was applied. Studies with 3 domains judged as unknown risk or at least 1 domain judged as high risk of bias were excluded from the analysis. Industry-related sponsorship or authorship were considered high risk of bias. The following outcomes were investigated: total body mass (TBM), lean body mass (LBM), fat mass (FM), total 1 repetition maximum (RM), bench press (BP) 1RM, and lower body (LwB) 1RM. RESULTS: Fourteen studies fit the inclusion criteria. However, after removing studies according to RoB2 scoring, the number of analysed studies dropped to seven. A total of 291 male participants (18-45y) were included, and the mean study duration was 8 ± 3 weeks with a training frequency of 2-5 d/week. No significant effects were found on TBM (0.34kg [-0.09, 0.77], p=0.12), body composition (LBM: -0.06kg [-0.55, 0.42], p=0.80; FM: 0.11kg [-0.12, 0.34], p=0.34) or strength (total 1RM: 1.30kg [-3.12, 5.72], p=0.56; BP 1RM: 1.49kg [-1.33, 4.30], p=0.30 and LwB 1RM: 3.96kg [-1.09, 9.02], p=0.12). CONCLUSION: This meta-analysis showed that HMB does not improve changes in body mass and composition caused by RET. In addition, effects on strength were not significant. Therefore, the claims for HMB consumption to optimize RET effects seem to be based on studies with considerable risk of bias. When such studies are not considered, there is no support for HMB ingestion.

Machine-Vision-Enabled Salt Dissolution Analysis.

Salt formation is a well-established method to increase the solubility of ionizable drug candidates. However, possible conversion of salt to its original form of free acid or base—disproportionation—can have a drastic effect on the solubility and consequently the bioavailability of a drug. Therefore, during the salt selection process, the salt dissolution behavior should be well understood. Improved understanding could be achieved by a method that enables simultaneous screening of small sample amounts and detailed dissolution process analysis. Here, we use a machine-vision-based single-particle analysis (SPA) method to successfully determine the pH-solubility profile, intrinsic solubility, common-ion effect, pKa, pHmax, and Ksp values of three model compounds in a fast and low sample consumption (<1 mg) manner. Moreover, the SPA method enables, with a particle-scale resolution, in situ observation of the disproportionation process and its immediate effect on the morphology and solubility of dissolving species. In this study, a potentially higher energy thermodynamic solid-state form of diclofenac free acid and an intriguing conversion to liquid verapamil free base were observed upon disproportionation of the respective salts. As such, the SPA method offers a low sample consumption platform for fast yet elaborate characterization of the salt dissolution behavior.

Preparation of phosphatidylcholine containing capric acid at the sn-1 position via Novozym® 435-catalyzed acidolysis

Abstract PC from soybean and C10:0 were subjected to lipase-mediated acidolysis/interesterification for the preparation of PC containing C10:0 (C10:0-PC). Free fatty acid form of C10:0 was selected as the best acyl donor in comparison to triacylglycerol from of C10:0 and ethyl ester from of C10:0 because higher C10:0-PC yield (46.2 ± 1.8 mol%) was obtained when free fatty acid form of C10:0 was used as acyl donor while that of triacylglycerol and ethyl ester were 24.9 ± 3.1 mol% and N.D., respectively in a preliminary reaction condition. In addition, Novozym® 435 was selected as the best catalyst compared to the commercial lipases Lipozyme® TL IM, Lipozyme® RM IM and Lipase OF® because the highest C10:0-PC yield (46.2 ± 1.8 mol%) was obtained when Novozym® 435 was used as a catalyst in a preliminary reaction condition. The reaction was then optimized based on the evaluation of acyl ratio (C10:0/PC), solvents, and reaction time, with mainly pursing C10:0-PC yield. It was demonstrated that high levels of acyl ratio decrease C10:0-incorporation, that solvents having low polarity are more effective in this reaction, and that the hydrolysis reaction was superior to the acidolysis reaction at the early stage of the process. Finally, C10:0-PC yield of 61.8 ± 3.0 mol% was obtained in the optimum reaction conditions, In this reaction system, Novozym® 435 showed regiospecificity at the sn-1 position of the PC molecule.

Synthesis of DHA/EPA Ethyl Esters via Lipase-Catalyzed Acidolysis Using Novozym® 435: A Kinetic Study

DHA/EPA ethyl ester is mainly used in the treatment of arteriosclerosis and hyperlipidemia. In this study, DHA+EPA ethyl ester was synthesized via lipase-catalyzed acidolysis of ethyl acetate (EA) with DHA+EPA concentrate in n-hexane using Novozym® 435. The DHA+EPA concentrate (in free fatty acid form), contained 54.4% DHA and 16.8% EPA, was used as raw material. A central composite design combined with response surface methodology (RSM) was used to evaluate the relationship between substrate concentrations and initial rate of DHA+EPA ethyl ester production. The results indicated that the reaction followed the ordered mechanism and as such, the ordered mechanism model was used to estimate the maximum reaction rate (Vmax) and kinetic constants. The ordered mechanism model was also combined with the batch reaction equation to simulate and predict the conversion of DHA+EPA ethyl ester in lipase-catalyzed acidolysis. The integral equation showed a good predictive relationship between the simulated and experimental results. 88–94% conversion yields were obtained from 100–400 mM DHA+EPA concentrate at a constant enzyme activity of 200 U, substrate ratio of 1:1 (DHA+EPA: EA), and reaction time of 300 min.

A Novel Non‐Prostaglandin EP2‐Receptor Agonist for Glaucoma Treatment: Omidenepag Isopropyl (DE‐117)

PurposeElevated intraocular pressure (IOP) is often associated with Primary Open‐Angle Glaucoma (POAG), an optic neuropathy that afflicts millions of people and which can cause blindness if left untreated. Omidenepag Isopropyl (OMDI; DE‐117) is a novel pro‐drug non‐prostaglandin (PG) which hydrolyzes to release its active free acid form (Omidenepag; OMD) into the aqueous humor (AQH) after topical ocular (t.o.) instillation. The purpose of the current studies was to characterize OMDI and OMD in several in vitro and in vivo model systems in order to determine its IOP‐lowering and other pharmacological properties.MethodsWell‐accepted in vitro cell‐based assays and in vivo IOP measurement techniques were utilized in the current studies.ResultsOMD was found to be a highly selective EP2 receptor (EP2R) agonist that bound to the EP2R with a high affinity (Ki = 3.6 nM) and which potently increased intracellular cAMP (EC50 = 8.3 nM). Once daily (q.d.), single t.o. dosing with OMDI (0.0001–0.01%) potently and efficaciously lowered IOP in ocular normotensive rabbits, dogs and monkeys, and also in ocular hypertensive (OHT) monkeys. Repeated t.o. dosing with OMDI in dogs did not cause tachyphylaxis of IOP reduction.Additive IOP‐lowering to OMDI was observed with Timolol, Brimonidine and Brinzolamide in rabbits and monkeys. OMDI 0.002% t.o. lowered IOP by increasing AQH efflux via trabecular meshwork (TM) and uveoscleral (UVSC) outflow pathways in OHT monkey eyes. In a phase‐3 clinical study, 0.002% OMDI (q.d.; t.o.), an optimal dose, lowered IOP by 5–7 mmHg in OHT/POAG patients (baseline IOPs of 22–34 mmHg) that was maintained over 12‐months with equivalent efficacy to Xalatan (0.005%, q.d.; t.o.). OHT/POAG patients that were low‐ or non‐responders to Xalatan (0.005%, q.d.; t.o.) exhibited significant IOP‐lowering (additional 3 mmHg) when switched to OMDI 0.002% (q.d.; t.o.). OMDI was well tolerated and produced transient conjunctival hyperemia, and mild iritis and macular edema (ME) in a few patients. However, no iris color changes, DUES, or abnormal eyelash growth were noted after 12‐month q.d. t.o. dosing with 0.002% OMDI, unlike FP‐receptor PG agonists (Bimatoprost and Latanoprost). Additive IOP‐lowering was also observed in OHT/POAG patients when OMDI (0.002%; q.d.) and Timolol (a known ocular hypotensive beta‐antagonist; 0.05%, b.i.d.) were t.o. instilled.ConclusionIn conclusion, the non‐prostaglandin organic molecule, OMD, is a highly selective and potent EP2R agonist whose isopropyl ester (OMDI) was shown to be a potent and efficacious ocular hypotensive agent in several animal species and in the OHT/POAG patient population studied. OMDI promoted AQH outflow via both the TM and UVSC pathways to lower IOP, and it achieved a comparable ocular hypotensive efficacy to Latanoprost (Xalatan 0.005% q.d.; t.o.). Additional IOP reduction was observed in Xalatan‐non/low responders when switched over to 0.002% OMDI. Additive IOP‐lowering efficacy was also observed when OMDI was t.o. dosed along with other ocular hypotensive drugs. These pharmacological and clinical efficacy features of OMDI support further development of this drug candidate for treating OHT and glaucoma.

In vitro metabolism of sunscreen compounds by liquid chromatography/high-resolution tandem mass spectrometry.

Exposure to UV light can induce adverse effects on human health, such as photo-aging, immunosuppression, and cancer. Sunscreens are used to prevent the absorption of UV rays, but certain UV-filtering compounds have been shown to disrupt endocrine systems or act as carcinogens. To assess the effects of the exposure to such compounds, it is important to study the pathways by which they are biotransformed in the body. Liquid chromatography coupled to high-resolution tandem mass spectrometry (LC/HRMS/MS) was employed to evaluate the oxidative metabolism and, specifically, the formation of reactive metabolites of six active ingredients commonly used in sunscreen formulations: oxybenzone, avobenzone, homosalate, octisalate, octocrylene, and octinoxate. In vitro incubations were performed with human and rat liver microsomes in the presence of β-nicotinamide adenine dinucleotide phosphate and glutathione. An LC/HRMS/MS method was developed to identify metabolites employing a biphenyl reversed-phase column for separating parent molecules, metabolites, and glutathione (GSH) adducts. Each tested compound resulted in the formation of several metabolites, including at least one GSH adduct. Compounds containing ester groups were hydrolyzed, and some metabolites of the free acid forms were also detected. High-resolution MS/MS data was crucial for the structural elucidation of metabolites and GSH adducts. Fragmentation pathways were proposed for all parent compounds, as well as each described metabolite and adduct. The results of this study will help better understand the metabolism and detoxification pathways of these xenobiotics.

Simultaneous detection of plant growth regulators jasmonic acid and methyl jasmonate in plant samples by a monoclonal antibody-based ELISA.

Methyl jasmonate (MeJA) and its free-acid form, jasmonic acid (JA), collectively referred to as jasmonates (JAs), are natural plant growth regulators that are widely present in higher plants. Simultaneous detection of JA and MeJA in plant samples is of significance and is a great challenging issue. In this study, coupling with two extraction methods, a sensitive monoclonal antibody (mAb) based enzyme-linked immunosorbent assay (ELISA) for simultaneous detection of JA and MeJA in plant samples was developed. The JA-bovine serum albumin (BSA) conjugate was used as an immunogen for the production of mAb. As the produced mAb exhibited higher recognition ability towards MeJA than towards JA, ELISA was established using MeJA as the standard. Under optimal experimental conditions, the IC50 and LOD values of ELISA for MeJA were 2.02 ng mL-1 and 0.20 ng mL-1, respectively. In the first extraction method, MeJA in plant samples was evaporated and only JA was extracted. In the second extraction method, both JA and MeJA were extracted. After methylation, JA in the extracts was converted into MeJA, and the whole MeJA in the extracts was measured by ELISA. Plant samples including the leaves of Salvia splendens, the flowers of Salvia splendens and the fruit of grapes were collected. JA and MeJA in these samples were detected by the proposed ELISA. It was found that the concentrations of JA in these three plant samples were about 3-5 times higher than those of MeJA in those samples. ELISA was also confirmed by HPLC. There was a good correlation between ELISA and HPLC.

Integrated Production of Biodiesel and Concentration of Polyunsaturated Fatty Acid in Glycerides Through Effective Enzymatic Catalysis.

DHA (docosahexaenoic acid) and EPA (eicosapentaenoic acid) contained in glycerides have been reported to be more advantageous for their intake than their counterpart in the form of free fatty acid or fatty acid esters. This work attempts to achieve the flexible concentration of DHA and EPA in glycerides as well as biodiesel production via a two-step process catalyzed by lipases. In the first step, several commercial lipases were investigated and Novozym ET2.0 demonstrated the highest potential in selective concentration of DHA and EPA. Over 85% of EPA and other fatty acids were converted to its corresponding FAEEs (fatty acid ethyl esters), while over 80% of DHA remained in glycerides under the optimized conditions. After the first step ethanolysis, the oil phase was subject to molecular distillation and a 97.5% biodiesel (FAEE) content could be obtained. Further flexible enrichment of DHA and EPA in glycerides was realized by immobilized lipase Novozym 435-mediated transesterification of glycerides (remaining in the heavy phase after molecular distillation) with DHA- or EPA-rich EE, and glycerides with 67.1% DHA and 13.1% EPA, or glycerides with 41.1% EPA and 38.0% DHA could be obtained flexibly. This work demonstrated an effective approach for DHA and EPA enrichment combined with biodiesel production through enzymatic catalysis.

SWATH Differential Abundance Proteomics and Cellular Assays Show In Vitro Anticancer Activity of Arachidonic Acid- and Docosahexaenoic Acid-Based Monoacylglycerols in HT-29 Colorectal Cancer Cells.

Colorectal cancer (CRC) is one of the most common and mortal types of cancer. There is increasing evidence that some polyunsaturated fatty acids (PUFAs) exercise specific inhibitory actions on cancer cells through different mechanisms, as a previous study on CRC cells demonstrated for two very long-chain PUFA. These were docosahexaenoic acid (DHA, 22:6n3) and arachidonic acid (ARA, 20:4n6) in the free fatty acid (FFA) form. In this work, similar design and technology have been used to investigate the actions of both DHA and ARA as monoacylglycerol (MAG) molecules, and results have been compared with those obtained using the corresponding FFA. Cell assays revealed that ARA- and DHA-MAG exercised dose- and time-dependent antiproliferative actions, with DHA-MAG acting on cancer cells more efficiently than ARA-MAG. Sequential window acquisition of all theoretical mass spectra (SWATH)—mass spectrometry massive quantitative proteomics, validated by parallel reaction monitoring and followed by pathway analysis, revealed that DHA-MAG had a massive effect in the proteasome complex, while the ARA-MAG main effect was related to DNA replication. Prostaglandin synthesis also resulted as inhibited by DHA-MAG. Results clearly demonstrated the ability of both ARA- and DHA-MAG to induce cell death in colon cancer cells, which suggests a direct relationship between chemical structure and antitumoral actions.

Enhancing the incorporation of lysine derivatives into proteins with methylester forms of unnatural amino acids

We have improved the incorporation of l- and d-forms of unnatural amino acid (UAA) Nε-thiaprolyl-l-lysine (ThzK) into ubiquitin (UB) and green fluorescent protein (GFP) by 2–6 folds with the use of the methylester forms of the UAAs in E coli cell culture. We also improved the yields of UAA-incorporated UB and GFP with the methylester forms of Nε-Boc-l-Lysine (BocK) and Nε-propargyl-l-Lysine (PrK) by 2–5 folds compared to their free acid forms. Our work demonstrated that using methylester-capped UAAs for protein expression is a useful strategy to enhance the yields of UAA-incorporated proteins.

Enantioselective chemoenzymatic synthesis of (R)-γ-valerolactone from levulinic acid

A number of chemicals with high industrial value can be synthesized from levulinic acid, a feasible building block readily available from cellulosic biomass. Among them, γ-valerolactone is a versatile chemical precursor for the synthesis of value-added products including bio-active molecules, bio-fuels, and carbon-based chemicals. In this study, a novel two-step chemoenzymatic conversion of levulinic acid to (R)-γ-valerolactone via 4-hydroxyvaleric acid was investigated. For that purpose, an engineered 3-hydroxybutyrate dehydrogenase (e3HBDH) with improved catalytic activity toward levulinic acid was employed in the first-step reaction, and dehydration with 1 % (v/v) sulfuric acid was applied for the lactonization of 4-hydroxyvaleric acid to γ-valerolactone in the second step. As a result, enantiomerically pure (R)-γ-valerolactone (>99 % ee) was successfully produced from the free acid form of levulinic acid with the maximum yield of approximately 100 %.

Ester coupling of ibuprofen in hydrogel matrix: A facile one-step strategy for controlled anti-inflammatory drug release

Ibuprofen (IBU) is a non-steroidal anti-inflammatory drug (NSAID) commonly used in the treatment of pain, fever and inflammation. However, the administration of IBU in its free carboxylic acid form is strongly dependent on its limited solubility in aqueous solution. This mandates for an increased drug concentration to reach the therapeutic window, and promotes the alternative use of IBU sodium salt, even if this latter form poses significant constraints in terms of tunable release due to its uncontrolled and rapid diffusion. A potential solution is represented by oral administration through physical encapsulation of ibuprofen in designed carriers, despite this route limits the application of this therapeutic agent. In this work, we propose the covalent tethering of ibuprofen to a hydrogel matrix via esterification reaction. Exploiting the cleavability of the ester bond under physiological conditions, we propose a controlled drug delivery system where the whole drug payload can be released, thus overcoming the questioned aspects of over-dosage and solubility-dependent administration. In particular, we tested the biological activity of cleaved ibuprofen in terms of cyclooxygenase inhibition, reporting that chemical tethering did not alter the efficiency of the NSAID. Moreover, due to the sol-gel transition of the hydrogel matrix, these ibuprofen-functionalized hydrogels could be used as injectable tools in several clinical scenarios, performing a localized drug release and opening advanced avenues for in situ treatments.

ПРИМЕНЕНИЕ СОЛЕЙ d-МЕТАЛЛОВ 4-го ПЕРИОДА В КАЧЕСТВЕ КОАГУЛЯНТОВ В ТЕХНОЛОГИИ ПРОИЗВОДСТВА ЭМУЛЬСИОННЫХ КАУЧУКОВ

Currently, much attention is paid to improving the technology of rubbers obtained by emulsion polymerization. The stage of isolation of rubber from latex is one of the problematic stages of this production. The development of more advanced coagulating systems remains relevant. For the first time, the possibility of using d-metal salts of the fourth period of the periodic table of Mendeleev’s salts as coagulants in rubber isolation is considered: chloride of zinc, nickel and cobalt. The influence of the consumption of these salts on the completeness of rubber isolation is investigated, the properties of rubbers are estimated. It is found that zinc chloride has the best coagulating ability. The formation of insoluble complex compounds between the coagulating agent and surfactants of the latex system is established. It is revealed that the acidification of the coagulating system leads to the release of higher carboxylic acids in free form and the formation of sulfuric salts of zinc, cobalt and nickel, which remain in the aqueous phase (serum). The presence of these salts in the serum allows its usage in the technological process of emulsion rubbers production for the preparation of aqueous solutions of chlorides of these salts, as well as acidifying agent (sulfuric acid). The positive outcome of the considered issues contributes to the solution of a number of environmental problems. The studied rubber mixtures and vulcanizates prepared on the basis of experimental rubbers meet the regulatory requirements.