Changes in the nutritional quality of decaying leaf litter in a stream based on fatty acid content

We examined the nutritional quality of decaying leaf litter in a third-order forested stream, using measurements of fatty acid (FA) composition over time. We measured changes in concentrations of total, polyunsaturated, microalgal, and microbial marker FAs in mixed-species leaf packs in spring and autumn and effects of including/excluding macroinvertebrates. Initial concentrations of total FAs in litter were significantly less in spring (5.2 mg/g) than in autumn (6.9 mg/g; F = 6.3; P = 0.03), but total FA concentrations in litter placed in the stream declined significantly over 120 days in both spring (62%; F = 10.9; P < 0.001) and autumn (56%; F = 19.4; P = 0.0001). Quantities of most FAs declined at a greater rate than that of bulk leaf matter. The presence or absence of macroinvertebrates (5 mm vs. 250 μm mesh) had no effect on FA concentration or composition of decomposing litter. Omega-3 polyunsaturated FAs were either nearly absent (20:5ω3) or depleted preferentially over other FAs (18:3ω3). During decomposition the polyunsaturated FA linoleic acid (18:2ω6, common in fungi), declined in concentration more rapidly than other FAs in the spring, but in autumn declined at slower rates, perhaps suggesting greater fungal activity in autumn. Quantities of bacterial (e.g., 16:1ω7) and fungal (e.g., 18:1ω9) FA markers increased over time in autumn (and 16:1ω7 also in spring). Our data provide no evidence for increasing nutritional FA quality of litter during decay and microbial colonization, based on total and polyunsaturated FAs, despite measured increases in bacterial and fungal FA over time. Routine measurements of FA composition of litter could provide insights into the nutrition of allochthonous matter and the importance of fungi and bacteria during decomposition.     

Proteomic Analysis of Differentially Expressed Proteins in Resistant Soybean Leaves after Phakopsora pachyrhizi Infection

Soybean rust caused by Phakopsora pachyrhizi is a destructive foliar disease in nearly all soybean‐producing countries. Understanding the host responses at the molecular level is certainly essential for effective control of the disease. To identify proteins involved in the resistance to soybean rust, differential proteomic analysis was conducted in soybean leaves of a resistant genotype after P. pachyrhizi infection. A total of 41 protein spots exhibiting a fold change >1.5 between the non‐inoculated and P. pachyrhizi‐inoculated soybean leaves at 12 and 24 h postinoculation (hpi) were unambiguously identified and functionally grouped into seven categories. Twenty proteins were up‐regulated and four proteins were down‐regulated at 12 hpi, whereas 18 proteins were up‐regulated and eight proteins were down‐regulated at 24 hpi. Generally, proteins involved in photosynthesis were down‐regulated, whereas proteins associated with disease and defense response, protein folding and assembly, carbohydrate metabolism and energy production were up‐regulated. Results are discussed in terms of the functional implications of the proteins identified, with special emphasis on their putative roles in defense. Abundance changes of these proteins, together with their putative functions reveal a comprehensive picture of the host response in rust‐resistant soybean leaves and provide a useful platform for better understanding of the molecular basis of soybean rust resistance.     

Age‐associated changes in long‐chain fatty acid profile during healthy aging promote pro‐inflammatory monocyte polarization via PPARγ

Differences in lipid metabolism associate with age‐related disease development and lifespan. Inflammation is a common link between metabolic dysregulation and aging. Saturated fatty acids (FAs) initiate pro‐inflammatory signalling from many cells including monocytes; however, no existing studies have quantified age‐associated changes in individual FAs in relation to inflammatory phenotype. Therefore, we have determined the plasma concentrations of distinct FAs by gas chromatography in 26 healthy younger individuals (age < 30 years) and 21 healthy FA individuals (age > 50 years). Linear mixed models were used to explore the association between circulating FAs, age and cytokines. We showed that plasma saturated, poly‐ and mono‐unsaturated FAs increase with age. Circulating TNF‐α and IL‐6 concentrations increased with age, whereas IL‐10 and TGF‐β1 concentrations decreased. Oxidation of MitoSOX Red was higher in leucocytes from FA adults, and plasma oxidized glutathione concentrations were higher. There was significant colinearity between plasma saturated FAs, indicative of their metabolic relationships. Higher levels of the saturated FAs C18:0 and C24:0 were associated with lower TGF‐β1 concentrations, and higher C16:0 were associated with higher TNF‐α concentrations. We further examined effects of the aging FA profile on monocyte polarization and metabolism in THP1 monocytes. Monocytes preincubated with C16:0 increased secretion of pro‐inflammatory cytokines in response to phorbol myristate acetate‐induced differentiation through ceramide‐dependent inhibition of PPARγ activity. Conversely, C18:1 primed a pro‐resolving macrophage which was PPARγ dependent and ceramide dependent and which required oxidative phosphorylation. These data suggest that a midlife adult FA profile impairs the switch from proinflammatory to lower energy, requiring anti‐inflammatory macrophages through metabolic reprogramming.     

The size and phospholipid composition of lipid droplets can influence their proteome

The proteomic makeup of lipid droplets (LDs) is believed to regulate the function of LDs, which are now recognized as important cellular organelles that are associated with many human metabolic disorders. However, factors that help determine LD proteome remain to be identified and characterized. Here we analyzed the phospholipid and protein composition of LDs isolated from wild type (WT) yeast cells, and also from fld1Δ, cds1, and ino2Δ mutant cells which produce ‘supersized’ LDs. LDs of fld1Δ and WT cells exhibited similar phospholipid profiles, whereas LDs of cds1 and ino2Δ strains had a higher (cds1) or lower (ino2Δ) percentage of phosphatidylcholine than those of WT, respectively. Unexpectedly, the presence of most known LD resident proteins was greatly reduced in the LD fraction isolated from cds1 and ino2Δ, including neutral lipid hydrolases. Consistent with this result, mobilization of neutral lipids was seriously impaired in these two strains. Contrary to the reduction of LD resident proteins, the Hsp90 family molecular chaperones, Hsc82 and Hsp82, were greatly increased in the LD fractions of cds1 and ino2Δ strains without changes at the level of expression. These data demonstrate the impact of LD phopholipids and size on the makeup of LD proteome.     

Lipid droplet–mediated ER homeostasis regulates autophagy and cell survival during starvation

Lipid droplets (LDs) are conserved organelles for intracellular neutral lipid storage. Recent studies suggest that LDs function as direct lipid sources for autophagy, a central catabolic process in homeostasis and stress response. Here, we demonstrate that LDs are dispensable as a membrane source for autophagy, but fulfill critical functions for endoplasmic reticulum (ER) homeostasis linked to autophagy regulation. In the absence of LDs, yeast cells display alterations in their phospholipid composition and fail to buffer de novo fatty acid (FA) synthesis causing chronic stress and morphologic changes in the ER. These defects compromise regulation of autophagy, including formation of multiple aberrant Atg8 puncta and drastically impaired autophagosome biogenesis, leading to severe defects in nutrient stress survival. Importantly, metabolically corrected phospholipid composition and improved FA resistance of LD-deficient cells cure autophagy and cell survival. Together, our findings provide novel insight into the complex interrelation between LD-mediated lipid homeostasis and the regulation of autophagy potentially relevant for neurodegenerative and metabolic diseases.     

Use of RNA Sequencing to Perform Comprehensive Analysis of Long Noncoding RNA Expression Profiles in Macrophages Infected with Trichosporon asahii

Trichosporon asahii (T. asahii) is a clinically important opportunistic pathogenic fungus capable of causing systemic lethal infection in immunosuppressive and immunodeficient hosts. However, the mechanism of the host immune response upon T. asahii infection has not been elucidated. Recent evidence has shown that long noncoding RNAs (lncRNAs) play key roles in regulating the immune response to resist microbial infections. In this study, we analyzed the expression profiles of lncRNAs at 12 and 24 h post-infection (hpi) in THP-1 cells infected with T. asahii using RNA sequencing (RNA-Seq). A total of 64 and 160 lncRNAs displayed significant differentially expressed (DE) at 12 h and 24 hpi, respectively. Among these lncRNAs, 18 lncRNAs were continuous DE at two time points. The DE of eight candidate lncRNAs were verified by real time quantitative polymerase chain reaction (RT-qPCR). Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses were performed to analyze the cis-target genes of 18 DE lncRNAs. The results showed that they were enriched in signaling pathways related to the host immune response, indicating that these lncRNAs might play important roles in fungi–host interactions. Finally, we explored the function of lncRNA NEAT1 and found that the expression of TNF-α and IL-1β declined after NEAT1 knockdown in T. asahii-infected THP-1 cells. To our knowledge, this is the first report of a expression analysis of lncRNAs in macrophages infected with T. asahii. Our study helps to elucidate the role of lncRNAs in the host immune response to early infection by T. asahii.

     

Quantitative live-cell PALM reveals nanoscopic Faa4 redistributions and dynamics on lipid droplets during metabolic transitions of yeast

Lipid droplets (LDs) are dynamic organelles for lipid storage and homeostasis. Cells respond to metabolic changes by regulating the spatial distribution of LDs and enzymes required for LD growth and turnover. The small size of LDs precludes the observation of their associated enzyme densities and dynamics with conventional fluorescence microscopy. Here we employ quantitative photo-activated localization microscopy to study the density of the fatty acid (FA) activating enzyme Faa4 on LDs in live yeast cells with single-molecule sensitivity and 30 nm resolution. During the log phase LDs colocalize with the endoplasmic reticulum (ER) where their emergence and expansion are mediated by the highest observed Faa4 densities. During transition to the stationary phase, LDs with a ∼2-fold increased surface area translocate to the vacuolar surface and lumen and exhibit a ∼2.5-fold increase in Faa4 density. The increased Faa4 density on LDs further suggests its role in LD expansion, is caused by its ∼5-fold increased expression level, and is specific to exogenous FA chain-lengths. When lipolysis is induced by refreshed medium, Faa4 shuttles through ER- and lipophagy to the vacuole, where it may activate FAs for membrane expansion and degrade Faa4 to reset its cellular abundance to levels in the log phase.     

Chlamydia trachomatis Infection Leads to Defined Alterations to the Lipid Droplet Proteome in Epithelial Cells

The obligate intracellular bacterium Chlamydia trachomatis is a major human pathogen and a main cause of genital and ocular diseases. During its intracellular cycle, C. trachomatis replicates inside a membrane-bound vacuole termed an “inclusion”. Acquisition of lipids (and other nutrients) from the host cell is a critical step in chlamydial replication. Lipid droplets (LD) are ubiquitous, ER-derived neutral lipid-rich storage organelles surrounded by a phospholipids monolayer and associated proteins. Previous studies have shown that LDs accumulate at the periphery of, and eventually translocate into, the chlamydial inclusion. These observations point out to Chlamydia-mediated manipulation of LDs in infected cells, which may impact the function and thereby the protein composition of these organelles. By means of a label-free quantitative mass spectrometry approach we found that the LD proteome is modified in the context of C. trachomatis infection. We determined that LDs isolated from C. trachomatis-infected cells were enriched in proteins related to lipid metabolism, biosynthesis and LD-specific functions. Interestingly, consistent with the observation that LDs intimately associate with the inclusion, a subset of inclusion membrane proteins co-purified with LD protein extracts. Finally, genetic ablation of LDs negatively affected generation of C. trachomatis infectious progeny, consistent with a role for LD biogenesis in optimal chlamydial growth.     

Fatty Acid Patterns of Seeds of Some Salvia Species from Iran – A Chemotaxonomic Approach

In this study, the seed oil content and fatty acid (FA) profile of 21 populations from 16 wild Salvia species of Iran were analyzed by GC. Patterns of chemical variations of the oils among species were identified via numerical analyses and also the taxonomic status of the infrageneric grouping was outlined in the genus. Salvia species were scored based on the contents of main FAs using principal coordinate analysis (PCO). The results showed that the total oil content in the seeds varied significantly, and ranged from 6.68 to 38.53% dry weight. α‐Linolenic (18:3ω3, 1.69 – 53.56%), linoleic (18:2ω6, 13.04 – 60.64%), oleic (18:1ω9, 6.15 – 27.06%), palmitic (16:0, 3.77 – 9.27%), and stearic (18:0, 1.78 – 3.05%) acid were identified as five major FAs in the oils. The amount of ω‐3 and ω‐6 FAs ranged between 1.90 – 53.80% and 13.46 – 60.83% of total FAs in the seed oils, respectively. The results confirmed that FA profiles were distinctive among the species and that they can be used as chemotaxonomic markers. The discrimination of Salvia species according to their botanical classification at intersectional level was supported. In general, seed oils of Salvia species were rich sources of polyunsaturated FAs, except in linoleic and α‐linolenic acid, and may be valuable for food and pharmaceutical industries.     

Lipophagy maintains energy homeostasis in the kidney proximal tubule during prolonged starvation

Macroautophagy/autophagy is a self-degradation process that combats starvation. Lipids are the main energy source in kidney proximal tubular cells (PTCs). During starvation, PTCs increase fatty acid (FA) uptake, form intracellular lipid droplets (LDs), and hydrolyze them for use. The involvement of autophagy in lipid metabolism in the kidney remains largely unknown. Here, we investigated the autophagy-mediated regulation of renal lipid metabolism during prolonged starvation using PTC-specific Atg5-deficient (atg5-TSKO) mice and an in vitro serum starvation model. Twenty-four h of starvation comparably induced LD formation in the PTCs of control and atg5-TSKO mice; however, additional 24 h of starvation reduced the number of LDs in control mice, whereas increases were observed in atg5-TSKO mice. Autophagic degradation of LDs (lipophagy) in PTCs was demonstrated by electron microscopic observation and biochemical analysis. In vitro pulse-chase assays demonstrated that lipophagy mobilizes FAs from LDs to mitochondria during starvation, whereas impaired LD degradation in autophagy-deficient PTCs led to decreased ATP production and subsequent cell death. In contrast to the in vitro assay, despite impaired LD degradation, kidney ATP content was preserved in 48-h starved atg5-TSKO mice, probably due to increased utilization of ketone bodies. This compensatory mechanism was accompanied by a higher plasma FGF21 (fibroblast growth factor 21) level and its expression in the PTCs; however, this was not essential for the production of ketone bodies in the liver during prolonged starvation. In conclusion, lipophagy combats prolonged starvation in PTCs to avoid cellular energy depletion.     

Revealing the three dimensional architecture of focal adhesion components to explain Ca2 +-mediated turnover of focal adhesions

BackgroundFocal adhesions (FAs) are large, dynamic protein complexes located close to the plasma membrane, which serve as the mechanical linkages and a biochemical signaling hub of cells. The coordinated and dynamic regulation of focal adhesion is required for cell migration. Degradation, or turnover, of FAs is a major event at the trailing edge of a migratory cell, and is mediated by Ca^2 +/calpain-dependent proteolysis and disassembly. Here, we investigated how Ca^2 + influx induces cascades of FA turnover in living cells.MethodsImages obtained with a total internal reflection fluorescence microscope (TIRFM) showed that Ca^2 + ions induce different processes in the FA molecules focal adhesion kinase (FAK), paxillin, vinculin, and talin. Three mutated calpain-resistant FA molecules, FAK-V744G, paxillin-S95G, and talin-L432G, were used to clarify the role of each FA molecule in FA turnover.ResultsVinculin was resistant to degradation and was not significantly affected by the presence of mutated calpain-resistant FA molecules. In contrast, talin was more sensitive to calpain-mediated turnover than the other molecules. Three-dimensional (3D) fluorescence imaging and immunoblotting demonstrated that outer FA molecules were more sensitive to calpain-mediated proteolysis than internal FA molecules. Furthermore, cell contraction is not involved in degradation of FA.ConclusionsThese results suggest that Ca^2 +-mediated degradation of FAs was mediated by both proteolysis and disassembly. The 3D architecture of FAs is related to the different dynamics of FA molecule degradation during Ca^2 +-mediated FA turnover.General significanceThis study will help us to clearly understand the underlying mechanism of focal adhesion turnover by Ca^2 +.     

Fatty acid composition of lipids from mushrooms belonging to the family Boletaceae

The non-polar lipid content and fatty acid (FA) composition of 11 mushroom species of the family Boletaceae were determined. The non-polar lipid content ranged from 2.0 (Leccinum aurantiacum and Boletus erythropus) to 5.4 % (w/w) d.w. (Suillus grevillei) with an average value of 2.9 %. More than 25 different FAs were found in the mushroom lipids. Unsaturated FAs, mainly linoleic and oleic acids, accounted for about 83 % of the total FAs, while palmitic acid was the main saturated FA. Some FAs are identified for the first time in Boletaceae and in higher Basidiomycetes (cis-11,12-methyleneoctadecanoic acid, 7-cis,10-cis hexadecadienoic) or in fungi (cis-11,12-methyleneoctadecanoic acid). There were significant differences (P < 0.05) in the contents of specific FAs between mushroom species.     

Comparative proteomics reveals that lipid droplet-anchored mitochondria are more sensitive to cold in brown adipocytes

Brown adipose tissue (BAT) is specialized for uncoupled heat production through mitochondrion fueled majorly from fatty acids (FAs) of lipid droplets (LDs). How the interaction between the two organelles contributes the generation of heat remains elusive. Here, we report that LD-anchored mitochondria (LDAM) were observed in the BAT of mice raised at three different temperatures, 30 °C, 23 °C, and 6 °C. The biochemical analyses including Western blotting of electron transport chain subunits showed that LDAM were functional. Comparative proteomics analysis was conducted, which revealed differential expressions of proteins between LDAM and cytoplasmic mitochondria (CM) at different temperatures. Higher expressions of proteins at low temperature were observed for i) FA β-oxidation in LDAM including FA synthesis and uncoupling, ii) pseudo-futile cycle in CM, and iii) two shuttle systems: glycerol 3-phosphate in both CM and LDAM and citrate malate in CM. Together, these results suggest that LDs and LDAM form a preorganized and functional organelle complex that permits the rapid response to cold.     

Changes in the content and composition of lipid fatty acids in tobacco leaves and roots at low-temperature hardening

Changes in the fatty acid (FA) composition of leaf and root lipids of heat-loving tobacco (Nicotiana tabacum L., cv. Samsun) plants during low-temperature hardening (8°C for 6 days) were studied. Hardening could improve leaf but not root cold tolerance. As this took place, the relative content of polyunsaturated (18:2n-6 and 18:3n-3) FAs increased and the proportion of saturated and monounsaturated FAs decreased. In contrast, in the roots hardening slightly increased the concentration of saturated FAs (16:0 and 18:0) and reduced the level of unsaturated FAs (18:1n-9, 18:2n-6, and 18:3n-3). At the same time, root lipids contained much C_20–24 FAs, and their content increased during hardening. It was suggested that an increased FA saturation and elevated proportion of C_20–24 FAs in the root lipids resulting in the lower membrane fluidity could be a reason for incapability of heat-loving tobacco plant roots of hardening and plant death at the lowtemperature stress.     

The cellular ceramide transport protein CERT promotes Chlamydia psittaci infection and controls bacterial sphingolipid uptake

Chlamydiaceae are bacterial pathogens that cause diverse diseases in humans and animals. Despite their broad host and tissue tropism, all Chlamydia species share an obligate intracellular cycle of development and have evolved sophisticated mechanisms to interact with their eukaryotic host cells. Here, we have analysed interactions of the zoonotic pathogen Chlamydia psittaci with a human epithelial cell line. We found that C. psittaci recruits the ceramide transport protein (CERT) to its inclusion. Chemical inhibition and CRISPR/Cas9‐mediated knockout of CERT showed that CERT is a crucial factor for C. psittaci infections thereby affecting different stages of the infection including inclusion growth and infectious progeny formation. Interestingly, the uptake of fluorescently labelled sphingolipids in bacteria inside the inclusion was accelerated in CERT‐knockout cells indicating that C. psittaci can exploit CERT‐independent sphingolipid uptake pathways. Moreover, the CERT‐specific inhibitor HPA‐12 strongly diminished sphingolipid transport to inclusions of infected CERT‐knockout cells, suggesting that other HPA‐12‐sensitive factors are involved in sphingolipid trafficking to C. psittaci. Further analysis is required to decipher these interactions and to understand their contributions to bacterial development, host range, tissue tropism, and disease outcome.     

Effects of reduced diameter of bag cultures on content of essential fatty acids and cell density in a continuous algal production system

Cell density and fatty acid (FA) content of Pavlova lutheri and Chaetoceros muelleri were analysed in a continuous algal production system (250-L bags) with reduced diameter. The cell density and FA content and composition in the algal production system were determined in replicate bags over a period of 5 weeks. The results showed that the cell density and essential FAs increased during the experiment for both species. After 5 weeks the mean cell numbers had increased to 6.0 ± 0.3 × 10⁶ cells mL⁻¹ in the P. lutheri bags and 6.0 ± 0.4 × 10⁶ cells mL⁻¹ in the C. muelleri bags. The content of total FAs increased significantly (p < 0.05) in all of the bags during the experiment. At the end of the experiment the mean total FA content were 2.7 ± 0.3 pg cell⁻¹ in the P. lutheri bags and 1.8 ± 0.1 pg cell⁻¹ in the C. muelleri bags. Maximum total FA content registered was 3.0 pg cell⁻¹ in one of the P. lutheri bags. The content of the essential FAs (ARA, EPA, DHA) increased over time in both of the species. At the end of the experiment the content of EPA (0.6 ± 0.1 pg cell⁻¹) and DHA (0.3 ± 0.0 pg cell⁻¹) were highest in the P. lutheri bags, while ARA (0.1 ± 0.0 pg cell⁻¹) was highest in C. muelleri. EPA and DHA constituted 22% and 11%, respectively, of total FA content in P. lutheri, while ARA constituted 6% of total FA content in C. muelleri. The results from this experiment indicate that flagellates such as P. lutheri perform better in narrow bags with improved light conditions, while diatoms like C. muelleri perform better in wider bags under light limitation. Implications for bivalve hatcheries are discussed.     

N‐6 From Different Sources Protect From Metabolic Alterations to Obese Patients: A Factor Analysis

First, to analyze the interactions among fatty acids (FAs) from diet, plasma and subcutaneous and visceral adipose tissue (AT), and second, the relationship among FAs from these different sources and obesity‐related alterations in extreme obesity. We studied 20 extreme obese subjects. A food‐frequency questionnaire was used to determine the FA intakes. Serum and AT (subcutaneous and visceral) FA concentrations were determined by gas chromatography. Cardiometabolic risk parameters were assessed. Principal factor analysis was performed to define specific FA factors in the metabolic alterations. We found important associations among diet, plasma, and AT FA and cardiometabolic parameters. In this regard, it is interesting to highlight the negative associations between plasma cholesterol and dietary n‐3 FA. In the subcutaneous depot, as occurred in plasma, n‐6 and polyunsaturated FAs (PUFA) were negatively associated with triacylglycerols (TGs). Factor analysis revealed TGs as the unique cardiovascular risk parameter appearing in the first factor (F1), together with n‐6 (load factor = 0.94) and PUFA (0.91). Besides, n‐3 from diet and plasma appeared in the third factor inversely related to cholesterol, low‐density lipoprotein cholesterol (LDL‐c), and insulin. In an opposite way, dietary and AT trans FAs and saturated FA (SFA) were associated to an increase of the metabolic risk. We have shown, for the first time, the importance of n‐6 and PUFAs composition as protective factors against metabolic alterations in extreme obese subjects. These findings support current dietary recommendations to increase PUFA intakes and restrict saturated and trans FA intakes even in extreme obesity.