Medium-chain Free Fatty Acids Research Articles

Molecular Mechanisms Underlying Medium-Chain Free Fatty Acid-Regulated Activity of the Phospholipase PlaF from Pseudomonas aeruginosa.

PlaF is a membrane-bound phospholipase A1 from Pseudomonas aeruginosa that is involved in remodeling membrane glycerophospholipids (GPLs) and modulating virulence-associated signaling and metabolic pathways. Previously, we identified the role of medium-chain free fatty acids (FFAs) in inhibiting PlaF activity and promoting homodimerization, yet the underlying molecular mechanism remained elusive. Here, we used unbiased and biased molecular dynamics simulations and free energy computations to assess how PlaF interacts with FFAs localized in the water milieu surrounding the bilayer or within the bilayer and how these interactions regulate PlaF activity. Medium-chain FFAs localized in the upper bilayer leaflet can stabilize inactive dimeric PlaF, likely through interactions with charged surface residues, as has been experimentally validated. Potential of mean force (PMF) computations indicate that membrane-bound FFAs may facilitate the activation of monomeric PlaF by lowering the activation barrier for changing into a tilted, active configuration. We estimated that the coupled equilibria of PlaF monomerization-dimerization and tilting at the physiological concentration of PlaF lead to the majority of PlaF forming inactive dimers when in a cell membrane loaded with decanoic acid (C10). This is in agreement with a suggested in vivo product feedback loop and gas chromatography-mass spectrometry profiling results, indicating that PlaF catalyzes the release of C10 from P. aeruginosa membranes. Additionally, we found that C10 in the water milieu can access the catalytic site of active monomeric PlaF, contributing to the competitive component of C10-mediated PlaF inhibition. Our study provides mechanistic insights into how medium-chain FFAs may regulate the activity of PlaF, a potential bacterial drug target.

Evaluation of strategies to narrow the product chain-length distribution of microbially synthesized free fatty acids

The dominant strategy for tailoring the chain-length distribution of free fatty acids (FFA) synthesized by heterologous hosts is expression of a selective acyl-acyl carrier protein (ACP) thioesterase. However, few of these enzymes can generate a precise (greater than 90% of a desired chain-length) product distribution when expressed in a microbial or plant host. The presence of alternative chain-lengths can complicate purification in situations where blends of fatty acids are not desired. We report the assessment of several strategies for improving the dodecanoyl-ACP thioesterase from the California bay laurel to exhibit more selective production of medium-chain free fatty acids to near exclusivity. We demonstrated that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) was an effective library screening technique for identification of thioesterase variants with favorable shifts in chain-length specificity. This strategy proved to be a more effective screening technique than several rational approaches discussed herein. With this data, we isolated four thioesterase variants which exhibited a more selective FFA distribution over wildtype when expressed in the fatty acid accumulating E. coli strain, RL08. We then combined mutations from the MALDI isolates to generate BTE-MMD19, a thioesterase variant capable of producing free fatty acids consisting of 90% of C12 products. Of the four mutations which conferred a specificity shift, we noted that three affected the shape of the binding pocket, while one occurred on the positively charged acyl carrier protein landing pad. Finally, we fused the maltose binding protein (MBP) from E. coli to the N – terminus of BTE-MMD19 to improve enzyme solubility and achieve a titer of 1.9 g per L of twelve-carbon fatty acids in a shake flask.

Untargeted Metabolome Analysis of Alcohol-Exposed Pregnancies Reveals Metabolite Differences That Are Associated with Infant Birth Outcomes.

Prenatal alcohol exposure can produce offspring growth deficits and is a leading cause of neurodevelopmental disability. We used untargeted metabolomics to generate mechanistic insight into how alcohol impairs fetal development. In the Western Cape Province of South Africa, 52 women between gestational weeks 5-36 (mean 18.5 ± 6.5) were recruited, and they provided a finger-prick fasting bloodspot that underwent mass spectrometry. Metabolomic data were analyzed using partial least squares-discriminant analyses (PLS-DA) to identify metabolites that correlated with alcohol exposure and infant birth outcomes. Women who consumed alcohol in the past seven days were distinguished by a metabolite profile that included reduced sphingomyelins, cholesterol, and pregnenolones, and elevated fatty acids, acyl and amino acyl carnitines, and androsterones. Using PLS-DA, 25 of the top 30 metabolites differentiating maternal groups were reduced by alcohol with medium-chain free fatty acids and oxidized sugar derivatives having the greatest influence. A separate ortho-PLS-DA analysis identified a common set of 13 metabolites that were associated with infant length, weight, and head circumference. These included monoacylglycerols, glycerol-3-phosphate, and unidentified metabolites, and most of their associations were negative, implying they represent processes having adverse consequences for fetal development.

Engineering Pseudomonas putida KT2440 for chain length tailored free fatty acid and oleochemical production

Despite advances in understanding the metabolism of Pseudomonas putida KT2440, a promising bacterial host for producing valuable chemicals from plant-derived feedstocks, a strain capable of producing free fatty acid-derived chemicals has not been developed. Guided by functional genomics, we engineered P. putida to produce medium- and long-chain free fatty acids (FFAs) to titers of up to 670 mg/L. Additionally, by taking advantage of the varying substrate preferences of paralogous native fatty acyl-CoA ligases, we employed a strategy to control FFA chain length that resulted in a P. putida strain specialized in producing medium-chain FFAs. Finally, we demonstrate the production of oleochemicals in these strains by synthesizing medium-chain fatty acid methyl esters, compounds useful as biodiesel blending agents, in various media including sorghum hydrolysate at titers greater than 300 mg/L. This work paves the road to produce high-value oleochemicals and biofuels from cheap feedstocks, such as plant biomass, using this host.

Development and Validation of a New GC-FID Method for the Determination of Short and Medium Chain Free Fatty Acids in Wine

A new analytical method for the determination of six volatile short and medium-chain fatty acids (acetic, propionic, isobutyric, isovaleric, hexanoic, and octanoic acids) through liquid-liquid extraction with diethyl ether, followed by GC-FID analysis, was developed and validated. The extraction conditions were optimized by evaluating the effect of the number of extractions (1 to 3) and the effect of the addition of salts (NaH2PO4, (NH4)2SO4, NaCl, (NH4)2SO4/NaH2PO4) to increase the concentration of the analytes in the ethyl ether phase. Results showed that a single extraction allows obtaining the highest sensitivity (due to the impossibility of evaporating the solvent to avoid losses of the analytes). The use of salting out agents, in particular, NaH2PO4, showed an important increase in the extraction extent, on average, 1.5 times higher as compared to the extraction performed without salt. The proposed method is rapid, requiring a total of 30 min for preparation and analysis, and it makes use of small amounts of sample (500 µL) and solvent (400 µL). The method was then applied to quantify the analytes in 5 white wines and 5 red wines, allowing to highlight some clear differences between red and white wines, with the red ones having a significantly higher amount of acetic acid (715.7 ± 142.3 mg/L in red wines and 351.5 ± 21.2 mg/L in white wines) and the white wines having a significantly higher amount of hexanoic and octanoic acid (6.1 ± 3.0 mg/L and 2.6 ± 0.8 mg/L, respectively, are the mean concentrations in white wines, and 4.7 ± 0.8 and 2.4 ± 0.4 mg/L, respectively, are the mean concentrations in red wines).

Fatty acids composition and lipolysis of Parmigiano Reggiano PDO cheese: effect of the milk cooling temperature at the farm.

The aim was to study the influence of cooling milk at 9°C at the farm versus keeping it at 20°C on Parmigiano Reggiano cheese lipolysis. A total of six cheesemaking trials (3 in winter and 3 in summer) were performed. In each trial, milk was divided continuously into two identical aliquots, one of which was kept at 9°C (MC9) and the other at 20°C (MC20). For each trial and milk temperature, vat milk (V-milk) and the resulting 21 month ripened cheese were analysed. Fat and dry matter and fat/casein ratio were lower in MC9 V-milk (p≤0.05) than in MC20. Total bacteria, mesophilic lactic acid and psychrotrophic and lipolytic bacteria showed significant differences (p≤0.05) between the two V-milks. Regarding cheese, fat content resulted lower and crude protein higher (p≤0.05) both in outer (OZ) and in inner zone (IZ) of the MC9 cheese wheels. Concerning total fatty acids, the MC9 OZ had a lower concentration of butyric, capric (p≤0.05) and medium chain fatty acids (p≤0.05), while the MC9 IZ had lower content of butyric (p≤0.05), caproic (p≤0.01) and short chain fatty acids (p≤0.05). The levels of short chain and medium chain free fatty acids (p≤0.05) were lower and that of long chain fatty acids (p≤0.05) was higher in MC9 OZ cheese. The principal component analysis of total and free fatty acids resulted in a clear separation among samples by seasons, whereas slight differences were observed between the two different milk temperatures. Storing milk at 9°C at the herd affects the chemical composition of Parmigiano Reggiano, with repercussion on lipolysis. However, the changes are not very relevant, and since the cheese can present a high variability among the different cheese factories, such changes should be considered within the "normal variations" of Parmigiano Reggiano chemical characteristics.

Lipidomics Analysis of Free Fatty Acids in Human Plasma of Healthy and Diabetic Subjects by Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS).

Targeted analytical methods for the determination of free fatty acids (FFAs) in human plasma are of high interest because they may help in identifying biomarkers for diseases and in monitoring the progress of a disease. The determination of FFAs is of particular importance in the case of metabolic disorders because FFAs have been associated with diabetes. We present a liquid chromatography-high resolution mass spectrometry (LC-HRMS) method, which allows the simultaneous determination of 74 FFAs in human plasma. The method is fast (10-min run) and straightforward, avoiding any derivatization step and tedious sample preparation. A total of 35 standard saturated and unsaturated FFAs, as well as 39 oxygenated (either hydroxy or oxo) saturated FFAs, were simultaneously detected and quantified in plasma samples from 29 subjects with type 2 diabetes mellitus (T2D), 14 with type 1 diabetes mellitus (T1D), and 28 healthy subjects. Alterations in the levels of medium-chain FFAs (C6:0 to C10:0) were observed between the control group and T2D and T1D patients.

Metabolic engineering strategies to produce medium-chain oleochemicals via acyl-ACP:CoA transacylase activity

Microbial lipid metabolism is an attractive route for producing oleochemicals. The predominant strategy centers on heterologous thioesterases to synthesize desired chain-length fatty acids. To convert acids to oleochemicals (e.g., fatty alcohols, ketones), the narrowed fatty acid pool needs to be reactivated as coenzyme A thioesters at cost of one ATP per reactivation - an expense that could be saved if the acyl-chain was directly transferred from ACP- to CoA-thioester. Here, we demonstrate such an alternative acyl-transferase strategy by heterologous expression of PhaG, an enzyme first identified in Pseudomonads, that transfers 3-hydroxy acyl-chains between acyl-carrier protein and coenzyme A thioester forms for creating polyhydroxyalkanoate monomers. We use it to create a pool of acyl-CoA’s that can be redirected to oleochemical products. Through bioprospecting, mutagenesis, and metabolic engineering, we develop three strains of Escherichia coli capable of producing over 1 g/L of medium-chain free fatty acids, fatty alcohols, and methyl ketones.

Short-Chain Fatty Acid Receptors and Cardiovascular Function.

Increasing experimental and clinical evidence points toward a very important role for the gut microbiome and its associated metabolism in human health and disease, including in cardiovascular disorders. Free fatty acids (FFAs) are metabolically produced and utilized as energy substrates during almost every biological process in the human body. Contrary to long- and medium-chain FFAs, which are mainly synthesized from dietary triglycerides, short-chain FFAs (SCFAs) derive from the gut microbiota-mediated fermentation of indigestible dietary fiber. Originally thought to serve only as energy sources, FFAs are now known to act as ligands for a specific group of cell surface receptors called FFA receptors (FFARs), thereby inducing intracellular signaling to exert a variety of cellular and tissue effects. All FFARs are G protein-coupled receptors (GPCRs) that play integral roles in the regulation of metabolism, immunity, inflammation, hormone/neurotransmitter secretion, etc. Four different FFAR types are known to date, with FFAR1 (formerly known as GPR40) and FFAR4 (formerly known as GPR120) mediating long- and medium-chain FFA actions, while FFAR3 (formerly GPR41) and FFAR2 (formerly GPR43) are essentially the SCFA receptors (SCFARs), responding to all SCFAs, including acetic acid, propionic acid, and butyric acid. As with various other organ systems/tissues, the important roles the SCFARs (FFAR2 and FFAR3) play in physiology and in various disorders of the cardiovascular system have been revealed over the last fifteen years. In this review, we discuss the cardiovascular implications of some key (patho)physiological functions of SCFAR signaling pathways, particularly those regulating the neurohormonal control of circulation and adipose tissue homeostasis. Wherever appropriate, we also highlight the potential of these receptors as therapeutic targets for cardiovascular disorders.

Metabolomic and inflammatory signatures of symptom dimensions in major depression

BackgroundMajor depressive disorder (MDD) is a highly heterogenous disease, both in terms of clinical profiles and pathobiological alterations. Recently, immunometabolic dysregulations were shown to be correlated with atypical, energy-related symptoms but less so with the Melancholic or Anxious distress symptom dimensions of depression in The Netherlands Study of Depression and Anxiety (NESDA) study. In this study, we aimed to replicate these immunometabolic associations and to characterize the metabolomic correlates of each of the three MDD dimensions. MethodsUsing three clinical rating scales, Melancholic, and Anxious distress, and Immunometabolic (IMD) dimensions were characterized in 158 patients who participated in the Predictors of Remission to Individual and Combined Treatments (PReDICT) study and from whom plasma and serum samples were available. The NESDA-defined inflammatory index, a composite measure of interleukin-6 and C-reactive protein, was measured from pre-treatment plasma samples and a metabolomic profile was defined using serum samples analyzed on three metabolomics platforms targeting fatty acids and complex lipids, amino acids, acylcarnitines, and gut microbiome-derived metabolites among other metabolites of central metabolism. ResultsThe IMD clinical dimension and the inflammatory index were positively correlated (r = 0.19, p = 0.019) after controlling for age, sex, and body mass index, whereas the Melancholic and Anxious distress dimensions were not, replicating the previous NESDA findings. The three symptom dimensions had distinct metabolomic signatures using both univariate and set enrichment statistics. IMD severity correlated mainly with gut-derived metabolites and a few acylcarnitines and long chain saturated free fatty acids. Melancholia severity was significantly correlated with several phosphatidylcholines, primarily the ether-linked variety, lysophosphatidylcholines, as well as several amino acids. Anxious distress severity correlated with several medium and long chain free fatty acids, both saturated and polyunsaturated ones, sphingomyelins, as well as several amino acids and bile acids. ConclusionThe IMD dimension of depression appears reliably associated with markers of inflammation. Metabolomics provides powerful tools to inform about depression heterogeneity and molecular mechanisms related to clinical dimensions in MDD, which include a link to gut microbiome and lipids implicated in membrane structure and function.

GPR84 signaling promotes intestinal mucosal inflammation via enhancing NLRP3 inflammasome activation in macrophages

The putative medium-chain free fatty acid receptor GPR84 is a G protein-coupled receptor primarily expressed in myeloid cells that constitute the innate immune system, including neutrophils, monocytes, and macrophages in the periphery and microglia in the brain. The fact that GPR84 expression in leukocytes is remarkably increased under acute inflammatory stimuli such as lipopolysaccharide (LPS) and TNFα suggests that it may play a role in the development of inflammatory and fibrotic diseases. Here we demonstrate that GPR84 is highly upregulated in inflamed colon tissues of active ulcerative colitis (UC) patients and dextran sulfate sodium (DSS)-induced colitis mice. Infiltrating GPR84+ macrophages are significantly increased in the colonic mucosa of both the UC patients and the mice with colitis. Consistently, GPR84−/− mice are resistant to the development of colitis induced by DSS. GPR84 activation imposes pro-inflammatory properties in colonic macrophages through enhancing NLRP3 inflammasome activation, while the loss of GPR84 prevents the M1 polarization and properties of proinflammatory macrophages. CLH536, a novel GPR84 antagonist discovered by us, suppresses colitis by reducing the polarization and function of pro-inflammatory macrophages. These results define a unique role of GPR84 in innate immune cells and intestinal inflammation, and suggest that GPR84 may serve as a potential drug target for the treatment of UC.

Medium and long chain free fatty acid receptors in the pathophysiology of respiratory diseases

Chronic inflammatory diseases of the respiratory tract, including asthma and chronic obstructive pulmonary disease, are a global problem of our time due to the widespread prevalence and difficulty of controlling the course. The mechanism of chronic inflammation in the bronchopulmonary system is closely related to metabolic disorders of lipids and their derivatives. Lipids and their mediators play both a pro-inflammatory and anti-inflammatory role in chronic inflammatory bronchopulmonary pathology. In particular, free fatty acids (FFAs) perform important signaling and regu latory functions in the body, coordinating metabolic and immune relationships. The mechanism that potentially binds FFAs and inflammatory reactions involves the activation of their receptors (FFAR – free fatty acid receptor), which are expressed on the cells of the respiratory tract, as well as on nerve and immune cells. Currently, FFARs are considered attractive targets in the treatment of chronic bronchopulmonary pathology, since modulation of their activity through the use of alimentary polyunsaturated fatty acids (PUFA) can affect the activity and resolution of neuroimmune inflammation in the bronchopulmonary system. However, controversial issues regarding their effectiveness and dose standardization of PUFA continue to limit their widespread use. This review summarizes the literature data on the role of medium- and longchain FFAs in the body’s immunoregulation in normal conditions and in chronic bronchopulmonary pathology. Data on medium and long chain FFA receptors – FFAR1 and FFAR4, FFAR-mediated signaling pathways in the regulation of metabolism and immune responses are systematized. The perspective and complex issues of the use of fatty acids in the treatment of chronic bronchopulmonary pathology are discussed.

Effects of medium-chain free fatty acids on performance, some biochemical parameters and meat fatty acids profile of broiler chickens

Effects of medium-chain free fatty acids on performance, some biochemical parameters and meat fatty acids profile of broiler chickens

Fatty acid sensing GPCR (GPR84) signaling safeguards cartilage homeostasis and protects against osteoarthritis

It is well known that free fatty acids (FFAs) have beneficial effects on the skeletal system, however, which fatty acid sensing GPCR(s) and how the GPCR(s) regulating cartilage development and osteoarthritis (OA) pathogenesis is largely unknown. In this study, we found Gpr84, a receptor for medium-chain FFAs (MCFA), was the only FFA-sensing GPCR in human and mouse chondrocytes that exhibited elevated expression when stimulated by interleukin (IL)-1β. Gpr84-deficiency upregulated cartilage catabolic regulator expression and downregulated anabolic factor expression in the IL-1β-induced cell model and the destabilization of the medial meniscus (DMM)-induced OA mouse model. Gpr84−/− mice exhibited an aggravated OA phenotype characterized by severe cartilage degradation, osteophyte formation and subchondral bone sclerosis. Moreover, activating Gpr84 directly enhanced cartilage extracellular matrix (ECM) generation while knockout of Gpr84 suppressed ECM-related gene expression. Especially, the agonists of GPR84 protected human OA cartilage explants against degeneration by inducing cartilage anabolic factor expression. At the molecular level, GPR84 activation inhibited IL-1β-induced NF-κB signaling pathway. Furthermore, deletion of Gpr84 had little effect on articular and spine cartilaginous tissues during skeletal growth. Together, all of our results demonstrated that fatty acid sensing GPCR (Gpr84) signaling played a critical role in OA pathogenesis, and activation of GPR84 or MCFA supplementation has potential in preventing the pathogenesis and progression of OA without severe cartilaginous side effect.

Modulation of cerebral ketone metabolism following traumatic brain injury in humans

Adaptive metabolic response to injury includes the utilization of alternative energy substrates – such as ketone bodies (KB) – to protect the brain against further damage. Here, we examined cerebral ketone metabolism in patients with traumatic brain injury (TBI; n = 34 subjects) monitored with cerebral microdialysis to measure total brain interstitial tissue KB levels (acetoacetate and β-hydroxybutyrate). Nutrition – from fasting vs. stable nutrition state – was associated with a significant decrease of brain KB (34.7 [10th–90th percentiles 10.7–189] µmol/L vs. 13.1 [6.5–64.3] µmol/L, p < 0.001) and blood KB (668 [168.4–3824.9] vs. 129.4 [82.6–1033.8] µmol/L, p < 0.01). Blood KB correlated with brain KB (Spearman’s rho 0.56, p = 0.0013). Continuous feeding with medium-chain triglycerides-enriched enteral nutrition did not increase blood KB, and provided a modest increase in blood and brain free medium chain fatty acids. Higher brain KB at the acute TBI phase correlated with age and brain lactate, pyruvate and glutamate, but not brain glucose. These novel findings suggest that nutritional ketosis was the main determinant of cerebral KB metabolism following TBI. Age and cerebral metabolic distress contributed to brain KB supporting the hypothesis that ketones might act as alternative energy substrates to glucose. Further studies testing KB supplementation after TBI are warranted.

Induction of GPR40 positively regulates cell motile and growth activities in breast cancer MCF-7 cells

Free fatty acid (FFA) receptors belong to a member of G-protein-coupled receptors. GPCR 120 (GPR120) and GPR40 are identified as FFA receptors and activated via the binding of long- and medium-chain FFAs. The aim of this study was to assess the effects of GPR120 and GPR40 on cell motility and growth in breast cancer cells treated with tamoxifen (TAM). MCF-7 cells were continuously treated with TAM for approximately 6 months. The expression level of GPR40 gene was markedly higher in the long-term TAM treated (MCF-TAM) cells than in MCF-7 cells. In cell motility assay, MCF-TAM cells indicated the high cell motile activity, compared with MCF-7 cells. The cell motile activity of MCF-TAM cells was suppressed by a selective GPR40 antagonist, GW1100. To evaluate the effects of GPR40 on cell growth activity under estrogen-free conditions, cells were maintained in serum-free DMEM without phenol red for 2 days. In estrogen-free conditioned medium, the cell growth rate of MCF-TAM cells was significantly higher than that of MCF-7 cells. In addition, treatment of GW1100 reduced the cell growth rate of MCF-TAM cells. These results suggest that the cell motile and growth activities may be positively regulated through the induction of GPR40 by the long-term TAM treatment in MCF-7 cells.

Medium chain triglyceride diet reduces anxiety-like behaviors and enhances social competitiveness in rats

Medium-chain triglycerides (MCT) are emerging as unique dietary supplements that are potentially relevant for the amelioration of brain dysfunctions. MCT are converted into ketones and free medium chain fatty acids that, in the brain, are highly effective energy sources to mitochondria and potentially less harmful than glucose metabolism to neurons. Given the recently established link between mitochondrial dysfunction and high anxiety and depression, we performed this study to investigate the effectiveness of an MCT-enriched diet to ameliorate anxiety- and depression-related behaviors in rats. Male rats were distributed into groups, according to their anxiety-like behaviors in the elevated plus maze. Each group was given either MCT-supplemented diet or an isocaloric control diet for fifteen days. Starting from the eighth day of diet, rats were exposed to different behavioral tests. MCT-fed rats exhibited reduced anxiety-like behaviors and enhanced social competitiveness, while their coping responses in the forced swim test were not affected by the treatment. When evaluated at the end of the two-week MCT diet, mitochondrial respiration was reduced in the medial prefrontal cortex (mPFC) while unchanged in the nucleus accumbens. In the mPFC, enzymes related to glycolysis and oxidative phosphorylation were also decreased by MCT diet, while proteins controlling glucose and glutamate transport were increased. Altogether, our findings strongly suggest the effectiveness of MCT diet to exert anxiolytic effects. In the brain, our results point to the mPFC as a brain region in which MCT supplementation improves transport and control of energy substrates.

503 Defining the Minimum Inhibitory Concentration of Synthetic and Commercial Medium Chain Fatty Acid Based Products Against Salmonella Typhimurium.

Research has confirmed that a 2% inclusion rate of a blend of C6:0, C8:0, and C10:0 in swine diets and ingredients can reduce Salmonella enterica serotype Typhimurium. However, it is unclear how the chain length and concentration of medium chain fatty acids (MCFA) impacts bacteriostatic properties. This can be tested through a minimum inhibitory concentration (MIC) benchtop assay, which identifies the lowest concentration of a chemical that prevents visible growth of a bacterium. The objective of this set of experiments was to utilize MIC as a mechanism to screen commercial or developmental feed additives containing MCFA for potential to mitigate Salmonella Typhimurium. First, the MIC of four synthetic MCFA treatments (C6:0, C8:0, C10:0, and 1:1:1 ratio of C:6:C8:C10 blend) was determined for Salmonella Typhimurium. The MIC of each treatment was conducted in a modified microbroth dilution and had three replicates. The MIC of C6:0 and C8:0 (0.4 and 0.5%, respectively) were similar (P > 0.05) to one another, but lower (P < 0.05) than the MIC of C10:0 or the blend of C6:C8:C10 (0.6% and >1.0%, respectively). It was therefore hypothesized that products containing high concentrations of C6:0 or C8:0 would have the greatest potential to mitigate Salmonella Typhimurium. Next, the fatty acid profile of 24 feed additive products that were commercially developed or in the development process and containing MCFA was determined via gas chromatography. As a result, four products in the development phase from varying companies plus coconut oil were selected as having a high potential for mitigating Salmonella Typhimurium based on their C6:0 and C8:0 levels. Development products 1, 2, 3, 4, and coconut oil contained 29.5, 43.1, 27.4, 0.98, and 6.82 mg/g of C6:0 and 123.20, 610.3, 248.7, 227.1, and 72.0 mg/g of C8:0, respectively. The MIC of these products vs. Salmonella Typhimurium was then determined. The MIC of Product 1 and Product 2 (0.5% and 1.3%, respectively) were similar (P > 0.05) to one another, but lower (P < 0.05) than the MIC of Products 3 or 4 (3.8% and 4.3%, respectively). All four products had a lower (P < 0.05) MIC than coconut oil (> 5.0%). In summary, current or future feed additives containing high concentrations of C6:0 and C8:0 appear to be more effective at mitigating Salmonella Typhimurium than those containing C10:0. Furthermore, it is hypothesized that free MCFA have greater antibacterial potential than those in triglyceride form.

Effect of substituting barley with glycerol as energy feed on feed intake, milk production and milk quality in dairy cows in mid or late lactation

The experiment reported in this research paper aimed to determine the level at which glycerol can substitute barley in grass-clover silage-based ration for dairy cows in mid or late lactation, without affecting milk production, milk composition, milk free fatty acid (FFA) profile, and milk sensory quality. Forty Holstein cows were used in a 4 × 4 Latin square experimental design. Crude glycerol substituted barley in the partially mixed ration (PMR) of the cows at inclusion levels of 0% (Gly0), 6% (Gly6), 12% (Gly12), and 18% (Gly18) of dietary dry matter (DM). Individual milk production, feed intake, and milking frequency were recorded daily, while milk composition and milk FA daily were analyzed weekly. Milk sensory analysis was performed on fresh and 7 d stored samples for the four diets. The PMR intake increased almost 1kg from Gly0 to Gly12, and decreased by approximately 1kg from Gly12 to Gly18. Concentrate intake at the automated milking unit tended to decrease by half a kg from Gly0 to Gly6. Milk yield decreased linearly with increasing proportions of glycerol in the diet while fat and protein contents in milk increased. This resulted in a quadratic effect of dietary glycerol level on ECM yield, with maximum yield with Gly6 and the lowest yield with Gly18. Protein and lactose yields decreased linearly with increasing proportions of glycerol in the diet. With increasing glycerol proportion in the diet, palmitic acid, oleic acid, linoleic acid, linolenic acid, and conjugated linoleic acid proportions in milk decreased linearly, while most of the short and medium chain FFA proportions in milk increased. To conclude, glycerol, as an energy feed, can substitute barley up to 18% of dietary DM for dairy cows in mid or late lactation without affecting milk quality. However, including more than 12% dietary glycerol might decrease ECM yield.

A subset of sweet-sensing neurons identified by IR56d are necessary and sufficient for fatty acid taste.

Fat represents a calorically potent food source that yields approximately twice the amount of energy as carbohydrates or proteins per unit of mass. The highly palatable taste of free fatty acids (FAs), one of the building blocks of fat, promotes food consumption, activates reward circuitry, and is thought to contribute to hedonic feeding underlying many metabolism-related disorders. Despite a role in the etiology of metabolic diseases, little is known about how dietary fats are detected by the gustatory system to promote feeding. Previously, we showed that a broad population of sugar-sensing taste neurons expressing Gustatory Receptor 64f (Gr64f) is required for reflexive feeding responses to both FAs and sugars. Here, we report a genetic silencing screen to identify specific populations of taste neurons that mediate fatty acid (FA) taste. We find neurons identified by expression of Ionotropic Receptor 56d (IR56d) are necessary and sufficient for reflexive feeding response to FAs. Functional imaging reveals that IR56d-expressing neurons are responsive to short- and medium-chain FAs. Silencing IR56d neurons selectively abolishes FA taste, and their activation is sufficient to drive feeding responses. Analysis of co-expression with Gr64f identifies two subpopulations of IR56d-expressing neurons. While physiological imaging reveals that both populations are responsive to FAs, IR56d/Gr64f neurons are activated by medium-chain FAs and are sufficient for reflexive feeding response to FAs. Moreover, flies can discriminate between sugar and FAs in an aversive taste memory assay, indicating that FA taste is a unique modality in Drosophila. Taken together, these findings localize FA taste within the Drosophila gustatory center and provide an opportunity to investigate discrimination between different categories of appetitive tastants.