Allergic inflammation triggers dyslipidemia via IgG signalling.

BACKGROUND: Allergic diseases begin early in life and are often chronic, thus creating an inflammatory environment that may precede or exacerbate other pathologies. In this regard, allergy has been associated to metabolic disorders and with a higher risk of cardiovascular disease, but the underlying mechanisms remain incompletely understood. METHODS: We used a murine model of allergy and atherosclerosis, different diets and sensitization methods, and cell-depleting strategies to ascertain the contribution of acute and late phase inflammation to dyslipidemia. Untargeted lipidomic analyses were applied to define the lipid fingerprint of allergic inflammation at different phases of allergic pathology. Expression of genes related to lipid metabolism was assessed in liver and adipose tissue at different times post-allergen challenge. Also, changes in serum triglycerides (TGs) were evaluated in a group of 59 patients ≥14 days after the onset of an allergic reaction. RESULTS: We found that allergic inflammation induces a unique lipid signature that is characterized by increased serum TGs and changes in the expression of genes related to lipid metabolism in liver and adipose tissue. Alterations in blood TGs following an allergic reaction are independent of T-cell-driven late phase inflammation. On the contrary, the IgG-mediated alternative pathway of anaphylaxis is sufficient to induce a TG increase and a unique lipid profile. Lastly, we demonstrated an increase in serum TGs in 59 patients after undergoing an allergic reaction. CONCLUSION: Overall, this study reveals that IgG-mediated allergic inflammation regulates lipid metabolism.

Toward Sustainable Wax Extraction from the Saccharum officinarum L. Filter Cake Byproduct: Process Optimization, Physicochemical Characterization, and Antioxidant Performance.

Saccharum officinarum L. exploitation and processing result in different byproducts, such as filter cake (FC). This study aimed to establish the most suitable experimental conditions to obtain lipophilic bioactive compounds from FC industrial residues, considering their high efficiency, cost-effectiveness, extraction yield, composition, and physicochemical properties. Results indicated that the most appropriate methodology consisted of the pretreatment of the FC sample with H2SO4, followed by ethanolic extraction (B6 method), avoiding energy-consumption FC drying steps and providing ethanol recovery (approx. 90%). The obtained B6 extract yield was 9.59 ± 0.27 g/100 g of FC dry weight, and this methodology proved to be more efficient in obtaining fatty alcohols (20.28 ± 1.48 g/kg extract) and phytosterols (31.56 ± 0.18 g/kg extract) while maintaining lower total monosaccharide concentration (26.19 ± 1.82 mg/g extract). Furthermore, the geographically related multivariate analysis in wax composition and antioxidant activity was evaluated by comparing B6 waxes from Guariba (G) and Univalem (U), both provided by Brazil and collected in June 2020. Overall, the wax composition is affected, but the antioxidant activity is uncompromised, which indicates that the optimized wax extraction method can be applied to FC.

Impaired hepatic lipid metabolism and biomarkers in fatty liver disease.

The liver plays a crucial role in lipid metabolism and metabolic homeostasis. Non-Alcoholic Fatty Liver Disease (NAFLD) is the most common chronic liver disease worldwide and currently has no specific treatments. Lifestyle modifications such as weight loss, exercise, and dietary changes are recommended to reduce the risk factors associated with the disease. Oxidized cholesterol products, some phospholipids and diacylglycerols can activate inflammatory pathways and contribute to the progression to Non-Alcoholic Steatohepatitis. Monitoring the whole plasma and liver lipidome may provide insights into the onset, development, and prevention of inflammatory-related diseases. As Lipid Droplets (LDs) represent augmented lipid reservoirs in NAFLD, new developments are being made on different therapies focused on LD associated proteins modulation (seipin, PLIN-2), as well as LD lipophagy mechanisms. The information covered in this publication provides an overview of the available research on lipid biomarkers linked to NAFLD and can be used to guide the development of future pharmacological therapies.

Novel Lipids to Regulate Obesity and Brain Function: Comparing Available Evidence and Insights from QSAR In Silico Models.

Lipid molecules, such as policosanol, ergosterol, sphingomyelin, omega 3 rich phosphatidylcholine, α-tocopherol, and sodium butyrate, have emerged as novel additions to the portfolio of bioactive lipids. In this state-of-the-art review, we discuss these lipids, and their activity against obesity and mental or neurological disorders, with a focus on their proposed cellular targets and the ways in which they produce their beneficial effects. Furthermore, this available information is compared with that provided by in silico Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) models in order to understand the usefulness of these tools for the discovery of new bioactive compounds. Accordingly, it was possible to highlight how these lipids interact with various cellular targets related to the molecule transportation and absorption (e.g., α-tocopherol transfer protein for α-Tocopherol, ATP-binding cassette ABC transporters or Apolipoprotein E for sphingomyelins and phospholipids) or other processes, such as the regulation of gene expression (involving Sterol Regulatory Element-Binding Proteins for ergosterol or Peroxisome Proliferator-Activated Receptors in the case of policosanol) and inflammation (the regulation of interleukins by sodium butyrate). When comparing the literature with in silico Quantitative Structure-Activity Relationship (QSAR) models, it was observed that although they are useful for selecting bioactive molecules when compared in batch, the information they provide does not coincide when assessed individually. Our review highlights the importance of considering a broad range of lipids as potential bioactives and the need for accurate prediction of ADMET parameters in the discovery of new biomolecules. The information presented here provides a useful resource for researchers interested in developing new strategies for the treatment of obesity and mental or neurological disorders.

Differential Lipid Accumulation on HepG2 Cells Triggered by Palmitic and Linoleic Fatty Acids Exposure.

Lipid metabolism pathways such as ß-oxidation, lipolysis and, lipogenesis, are mainly associated with normal liver function. However, steatosis is a growing pathology caused by the accumulation of lipids in hepatic cells due to increased lipogenesis, dysregulated lipid metabolism, and/or reduced lipolysis. Accordingly, this investigation hypothesizes a selective in vitro accumulation of palmitic and linoleic fatty acids on hepatocytes. After assessing the metabolic inhibition, apoptotic effect, and reactive oxygen species (ROS) generation by linoleic (LA) and palmitic (PA) fatty acids, HepG2 cells were exposed to different ratios of LA and PA to study the lipid accumulation using the lipophilic dye Oil Red O. Lipidomic studies were also carried out after lipid isolation. Results revealed that LA was highly accumulated and induced ROS production when compared to PA. Lipid profile modifications were observed after LA:PA 1:1 (v/v) exposure, which led to a four-fold increase in triglycerides (TGs) (mainly in linoleic acid-containing species), as well as a increase in cholesterol and polyunsaturated fatty acids (PUFA) content when compared to the control cells. The present work highlights the importance of balancing both PA and LA fatty acids concentrations in HepG2 cells to maintain normal levels of free fatty acids (FFAs), cholesterol, and TGs and to minimize some of the observed in vitro effects (i.e., apoptosis, ROS generation and lipid accumulation) caused by these fatty acids.

Cannabidiol and Cannabigerol Exert Antimicrobial Activity without Compromising Skin Microbiota.

Cannabidiol (CBD) and cannabigerol (CBG) are two pharmacologically active phytocannabinoids of Cannabis sativa L. Their antimicrobial activity needs further elucidation, particularly for CBG, as reports on this cannabinoid are scarce. We investigated CBD and CBG's antimicrobial potential, including their ability to inhibit the formation and cause the removal of biofilms. Our results demonstrate that both molecules present activity against planktonic bacteria and biofilms, with both cannabinoids removing mature biofilms at concentrations below the determined minimum inhibitory concentrations. We report for the first time minimum inhibitory and lethal concentrations for Pseudomonas aeruginosa and Escherichia coli (ranging from 400 to 3180 µM), as well as the ability of cannabinoids to inhibit Staphylococci adhesion to keratinocytes, with CBG demonstrating higher activity than CBD. The value of these molecules as preservative ingredients for cosmetics was also assayed, with CBG meeting the USP 51 challenge test criteria for antimicrobial effectiveness. Further, the exact formulation showed no negative impact on skin microbiota. Our results suggest that phytocannabinoids can be promising topical antimicrobial agents when searching for novel therapeutic candidates for different skin conditions. Additional research is needed to clarify phytocannabinoids' mechanisms of action, aiming to develop practical applications in dermatological use.

Microbial Conjugated Linolenic Acid-Enriched Fermented Milk Using Lipase-Hydrolyzed Flaxseed Oil: Biochemical, Organoleptic and Storage Traits.

The bioactive conjugated linolenic acid (CLNA) can be microbiologically produced by different probiotic strains when in the presence of α-linolenic acid (α-LNA). Food matrices are a good vector, such as has been previously demonstrated with fermented milk enriched with microbial CLNA by Bifidobacterium breve DSM 20091 from lipase-hydrolyzed flaxseed oil. The aim of the present work was to further assess the nutritional, biochemical and organoleptic properties of the developed dairy product, as well as its storage stability throughout 28 days at 4 °C, proving its suitability for consumption. Milk lactose hydrolyzed into glucose (0.89 g/100 g) and galactose (0.88 g/100 g), which were further metabolized into lactic (0.42 g/100 g), acetic (0.44 g/100 g) and propionic (0.85 g/100 g) acids. Titratable acidity reached 0.69% and pH 4.93. Compared with the control (no CLNA), fat content was slightly higher (2.0 g/100 g). Acetic acid was the major volatile (83.32%), lacking important dairy flavor contributors, like acetaldehyde. Sensory analysis revealed predominant astringency and bitterness. No microbial concerns arose during storage, but the CLNA content increased, and some saturated fatty acids seemed to oxidize. In conclusion, the CLNA-enriched fermented milk revealed reasonable compositional properties, yet further improvements are needed for optimal consumer acceptance and a prolonged shelf-life.

Suitability of Solvent-Assisted Extraction for Recovery of Lipophilic Phytochemicals in Sugarcane Straw and Bagasse.

Sugarcane is primarily harvested to meet up to 80% of global sugar demand. Recently, lipids recovered from their biomass (straw and bagasse) have attracted much attention due to their possible utilisation in biofuel production but also by the presence of health-promoting compounds as phytosterols (i.e., improvement of cardiovascular function) or 1-octacosanol (i.e., anti-obesity). Although this fraction is commonly obtained through solid-liquid isolation, there is scarce information about how different solvents affect the composition of the extracts. This research work aimed to study whether, in sugarcane straw and bagasse samples, Soxtec extraction with widely used dichloromethane (DCM) would be suitable to recover most of the lipid classes when compared to other available solvents such as food grade ethanol (EtOH) or solvents without regulation restrictions for food and drug applications (i.e., acetone and ethyl acetate). The obtained results allow concluding that sugarcane waxes from straw and bagasse are complex lipid mixtures of polar and non-polar compounds. According to the extraction yield, the best results were obtained with ethanol (5.12 ± 0.30% and 1.97 ± 0.31%) for both straw and bagasse, respectively. The extractant greatly influenced the lipid composition of the obtained product. Thus, DCM enriched the isolates in glycerolipids (mono-, di- and triglycerides), free fatty acids, fatty alcohols, fatty aldehydes, phytosterols and hydrocarbons. On the other hand, EtOH resulted in polar isolates rich in glycolipids. Therefore, depending on the application and objectives of future research studies, the solvent to recover such lipids needs to be carefully selected.

Phytosterols and Novel Triterpenes Recovered from Industrial Fermentation Coproducts Exert In Vitro Anti-Inflammatory Activity in Macrophages.

The unstoppable growth of human population that occurs in parallel with all manufacturing activities leads to a relentless increase in the demand for resources, cultivation land, and energy. In response, currently, there is significant interest in developing strategies to optimize any available resources and their biowaste. While solutions initially focused on recovering biomolecules with applications in food, energy, or materials, the feasibility of synthetic biology in this field has been demonstrated in recent years. For instance, it is possible to genetically modify Saccharomyces cerevisiae to produce terpenes for commercial applications (i.e., against malaria or as biodiesel). But the production process, similar to any industrial activity, generates biowastes containing promising biomolecules (from fermentation) that if recovered may have applications in different areas. To test this hypothesis, in the present study, the lipid composition of by-products from the industrial production of ß-farnesene by genetically modified Saccharomyces cerevisiae are studied to identify potentially bioactive compounds, their recovery, and finally, their stability and in vitro bioactivity. The assayed biowaste showed the presence of triterpenes, phytosterols, and 1-octacosanol which were recovered through molecular distillation into a single fraction. During the assayed stability test, compositional modifications were observed, mainly for the phytosterols and 1-octacosanol, probably due to oxidative reactions. However, such changes did not affect the in vitro bioactivity in macrophages, where it was found that the obtained fraction decreased the production of TNF-α and IL-6 in lipopolysaccharide (LPS)-induced inflammation.

Bioactive Sugarcane Lipids in a Circular Economy Context.

Most of the global sugar and ethanol supply trade comes from the harvesting of Saccharum officinarum (i.e., sugarcane). Its industrial processing results in numerous by-products and waste streams, such as tops, straw, filter cake, molasses and bagasse. The recovery of lipids (i.e., octacosanol, phytosterols, long-chain aldehydes and triterpenoids) from these residues is an excellent starting point for the development of new products for various application fields, such as health and well-being, representing an important feature of the circular economy. By selecting green scalable extraction procedures, industry can reduce its environmental impact. Refluxed ethanol extraction methods have been demonstrated to meet these characteristics. On the other hand, effective non-solvent methodologies such as molecular distillation and supercritical CO2 extraction can fractionate lipids based on high temperature and pressure application with similar yields. Sugarcane lipophilic extracts are usually analyzed through gas chromatography (GC) and liquid chromatography (LC) techniques. In many cases, the identification of such compounds involves the development of high-temperature GC-MS/FID techniques. On the other hand, for the identification and quantification of thermolabile lipids, LC-MS techniques are suitable for the separation and identification of major lipid classes. Generically, its composition includes terpenes, phytosterols, tocopherol, free fatty acids, fatty alcohols, wax esters, triglycerides, diglycerides and monoglycerides. These compounds are already known for their interesting application in various fields such as pharma and cosmetics due to their anti-hypercholesterolemic, anti-hyperglycemic, antioxidant and anti-inflammatory properties.

Lactobacillus mulieris sp. nov., a new species of Lactobacillus delbrueckii group.

One Gram-stain-positive, non-motile, non-spore-forming, catalase-negative, and coccobacilli-shaped strain, designated c10Ua161MT, was isolated from a urine sample from a reproductive-age healthy woman. Comparative 16S rRNA gene sequence analysis indicated that strain c10Ua161MT belonged to the genus Lactobacillus. Phylogenetic analysis based on pheS and rpoA gene sequences strongly supported a clade encompassing strains c10Ua161MT and eight other strains from public databases, distinct from currently recognized species of the genus Lactobacillus. In silico Average Nucleotide Identity (ANI) and Genome-to-Genome Distance Calculator (GGDC), showed 87.9 and 34.3 % identity to the closest relative Lactobacillus jensenii, respectively. The major fatty acids of strain c10Ua161MT were C18 : 1ω9c (65.0%), C16 : 0 (17.8%), and summed feature 8 (10.2 %; comprising C18 : 1ω7c, and/or C18 : 1ω6c). The DNA G+C content of the strains is 34.2 mol%. On the basis of data presented here, strain c10Ua161MT represents a novel species of the genus Lactobacillus, for which the name Lactobacillus mulieris sp. nov. is proposed. The type strain is c10Ua161MT (=CECT 9755T=DSM 108704T).

Microbial Production of Conjugated Linoleic Acid and Conjugated Linolenic Acid Relies on a Multienzymatic System.

Conjugated linoleic acids (CLAs) and conjugated linolenic acids (CLNAs) have gained significant attention due to their anticarcinogenic and lipid/energy metabolism-modulatory effects. However, their concentration in foodstuffs is insufficient for any therapeutic application to be implemented. From a biotechnological standpoint, microbial production of these conjugated fatty acids (CFAs) has been explored as an alternative, and strains of the genera Propionibacterium, Lactobacillus, and Bifidobacterium have shown promising producing capacities. Current screening research works are generally based on direct analytical determination of production capacity (e.g., trial and error), representing an important bottleneck in these studies. This review aims to summarize the available information regarding identified genes and proteins involved in CLA/CLNA production by these groups of bacteria and, consequently, the possible enzymatic reactions behind such metabolic processes. Linoleate isomerase (LAI) was the first enzyme to be described to be involved in the microbiological transformation of linoleic acids (LAs) and linolenic acids (LNAs) into CFA isomers. Thus, the availability of lai gene sequences has allowed the development of genetic screening tools. Nevertheless, several studies have reported that LAIs have significant homology with myosin-cross-reactive antigen (MCRA) proteins, which are involved in the synthesis of hydroxy fatty acids, as shown by hydratase activity. Furthermore, it has been suggested that CLA and/or CLNA production results from a stress response performed by the activation of more than one gene in a multiple-step reaction. Studies on CFA biochemical pathways are essential to understand and characterize the metabolic mechanism behind this process, unraveling all the gene products that may be involved. As some of these bacteria have shown modulation of lipid metabolism in vivo, further research to be focused on this topic may help us to understand the role of the gut microbiota in human health.

Milk fat components with potential anticancer activity-a review.

During many years, the milk fat has been unfairly undervalued due to its association with higher levels of cardiovascular diseases, dyslipidaemia or obesity, among others. However, currently, this relationship is being re-evaluated because some of the dairy lipid components have been attributed potential health benefits. Due to this, and based on the increasing incidence of cancer in our society, this review work aims to discuss the state of the art concerning scientific evidence of milk lipid components and reported anticancer properties. Results from the in vitro and in vivo experiments suggest that specific fatty acids (FA) (as butyric acid and conjugated linoleic acid (CLA), among others), phospholipids and sphingolipids from milk globule membrane are potential anticarcinogenic agents. However, their mechanism of action remains still unclear due to limited and inconsistent findings in human studies.

Pedobacter lusitanus sp. nov., isolated from sludge of a deactivated uranium mine.

Strain NL19T is a Gram-stain-negative, aerobic bacterium that was isolated from sludge of a deactivated uranium mine in Portugal. 16S rRNA gene sequence analysis revealed that strain NL19T is a member of the genus Pedobacter and closely related to the strains Pedobacter himalayensis MTCC 6384T, Pedobacter cryoconitis DSM 14825T, Pedobacter westerhofensis DSM 19036T and Pedobacterhartonius DSM 19033T. It had a DNA G+C content of 40.8 mol%, which agreed with the genus description. The main fatty acids included C16 : 1ω7c, C14 : 1ω5c, C4 : 0, iso-C17 : 0, iso-C17 : 0 3-OH, C16 : 0, anteiso-C15 : 0 and iso-C15 : 0 3-OH. The main lipids present were phospholipids (60 %) and sphingolipids (35 %). The most abundant phospholipids included phosphatidylethanolamine, phosphatidylinositol and phosphatidylcholine. Menaquinone-7 (MK-7) was the only isoprenoid quinone detected. DNA-DNA hybridization similarities between strain NL19T and Pedobacter himalayensis MTCC 6384T, Pedobacter cryoconitis DSM 14825T, Pedobacter westerhofensis DSM 19036T and Pedobacter hartonius DSM 19033T were 15.3 , 16.2 , 11.5 and 16.0 %, respectively. Strain NL19T can also be distinguished from these four species based on gyrB and intergenic transcribed spacers (ITS) sequences and by some phenotypic traits such as NaCl tolerance, pH, growth temperature and carbon source utilization. Strain NL19Trepresents a novel species of the genus Pedobacter, for which the name Pedobacter lusitanus sp. nov. is proposed. The type strain is NL19T (=LMG 29220T=CECT 9028T). An amended description of Pedobacter himalayensis is also included.

Evidences and perspectives in the utilization of CLNA isomers as bioactive compounds in foods.

Conjugated alpha linolenic acid (CLNA) isomers are promising lipids owing to their similarities with conjugated linoleic acid (CLA) but exerting their bioactivity at lower doses; some isomers also belong to omega 3 family. This review aims to summarize the state of the art about the utilization of CLNA as a functional ingredient. Indeed, in vitro and in vivo studies reported that CLNA exerted anticancer, anti-inflammatory, anti-obese, and antioxidant activities. However, CLNA has not been tested in humans. These compounds are naturally present in meat and milk fat from ruminants but the highest concentrations are found in vegetable oils. Their incorporation in foodstuffs is one of the most effective strategies to elaborate CLNA-enriched products together with the microbiological production. Lactobacilli, propionibacteria, and bifidobacteria strains have been assayed to produce CLNA isomers but at the current moment there are not high CLNA concentration products elaborated using these strains. Furthermore, it is known that CLNA isomers are highly prone to oxidation when compared with linoleic acid and CLA, but the possible effects of elaboration and storage on high CLNA productsare unknown.The utilization of CLNA as a functional compound still remains a challenge and requires more research to address all of its technological and bioactivity aspects.

Endocrine Disruptor DDE Associated with a High-Fat Diet Enhances the Impairment of Liver Fatty Acid Composition in Rats.

The banned pesticide dichlorodiphenyltrichloroethane (DDT) and its main metabolite, p,p'-dichlorodiphenyldichloroethylene (DDE), are commonly found in the food chain and in all tissues of living organisms. DDE is associated with metabolic diseases acting as an endocrine disruptor and more recently with the obesity pandemic. This study focuses on using fatty acid analysis to relate DDE exposure and metabolic dysfunction: liver and adipose tissue (visceral and subcutaneous) composition from male Wistar rats fed a standard (STD) or high-fat (HF) diet versus the addition of DDE in water. DDE exposure increased liver levels of palmitic, stearic, oleic, trans fatty, and linoleic acids having altered the n6 and n3 pathways leading to high concentrations of arachidonic acid and DHA (C22:6 n3). The results of this study confirm the close relationship between this pesticide metabolite and hepatic lipid dysfunction, underscoring its role as an emerging target for the prevention and therapy of nonalcoholic fatty liver disease (NAFLD).

Considerations about the in situ derivatization and fractionation of EFA and NEFA in biological and food samples.

Despite their important role in tissues, fluids and foods, the analysis of non-esterified fatty acids (NEFA) as methyl esters (NEFAME) is performed using expensive, cumbersome and time-consuming procedures that needs of isolation, fractionation and derivatization steps. However, Yi et al. [1] proposed a promising in situ, single-step procedure to analyze esterified fatty acids (EFA) and NEFA from a same sample on the basis that acylglycerols and free fatty acids can be derivatized using specific reactions. However, according to the data presented in this research work, some modifications need to be performed to increase the reliability of the method:•Increment of the transesterification performance by adding hexane to the reaction mixture, decreasing the time for the derivatization of acylglycerols from 10 min to 3-4 min and stopping the reaction with sulfuric acid.•Avoid cross-contamination of the NEFAME extract by adding 500 µL of water after collection of EFA methyl esters (EFAME).•Samples are spiked with three internal standards: a triacylglycerol (to calculate the concentration of EFA), a free fatty acid (to calculate NEFA) and a FAME (to control isolation of FAME and cross-contamination).

Assessment of safety of enterococci isolated throughout traditional Terrincho cheesemaking: virulence factors and antibiotic susceptibility.

Enterococci account for an important fraction of the adventitious microflora of traditional cheeses manufactured in Mediterranean countries from small ruminants' raw milk and play an important role in the development of suitable organoleptic characteristics of the final product. It has been suggested that animals used for food or animals that supply edible products are a reservoir of antibiotic-resistant enterococci. The main purpose of this research effort was thus to identify, to the species level, a total of 73 enterococci with high tolerance to acidic pH and bile salts (as prevailing environmental conditions in the first portion of the gastrointestinal tract), which were previously isolated from the milk feedstock to the final product of Terrincho cheesemaking, and to determine their profiles of antibiotic susceptibility, coupled with the occurrence of specific virulence factors (especially in those that might eventually be claimed to exhibit suitable probiotic and technological performances). Isolates, identified by both API 20 STREP and PCR methods, were found to belong to the following Enterococcus species: E. casseliflavus, E. durans, E. faecalis, E. faecium, and E. gallinarum. Susceptibility of those isolates was observed to most antibiotics tested, whereas none harbored aminoglycoside resistance genes. PCR screenings for cytolysin genes (cylL(L), cylL(s), cylM, cylB, and cylA), surface adhesin genes (efaA(fs), efaA(fm), and esp), the aggregation protein gene (agg), and the extracellular metalloendopeptidase gene (gelE) were performed. All isolates proved negative for cylL(L), cylM, cylB, and agg genes. Both E. faecalis strains were positive for the cell wall-associated protein Esp and the cell wall adhesin efaA(fs), whereas the cell wall adhesin efaA(fm) was detected in 11 of the 12 E. faecium strains. Only one strain possessed the cylL(s) determinant, and another possessed the cylA gene. Incidence of virulence determinants was thus very low; hence, the enterococcal adventitious microflora tested is essentially safe.