Lipid Droplet Biogenesis.

Lipid droplets (LDs) are ubiquitous organelles that store neutral lipids for energy or membrane synthesis and act as hubs for metabolic processes. Cells generate LDs de novo, converting cells to emulsions with LDs constituting the dispersed oil phase in the aqueous cytoplasm. Here we review our current view of LD biogenesis. We present a model of LD formation from the ER in distinct steps and highlight the biology of proteins that govern this biophysical process. Areas of incomplete knowledge are identified, as are connections with physiology and diseases linked to alterations in LD biology.

Tailoring Highly Branched Poly(ß-amino ester)s for Efficient and Organ-Selective mRNA Delivery.

Development of mRNA therapeutics necessitates targeted delivery technology, while the clinically advanced lipid nanoparticles face difficulty for extrahepatic delivery. Herein, we design highly branched poly(ß-amino ester)s (HPAEs) for efficacious organ-selective mRNA delivery through tailoring their chemical compositions and topological structures. Using an "A2+B3+C2" Michael addition platform, a combinatorial library of 219 HPAEs with varied backbone structures, terminal groups, and branching degrees are synthesized. The branched topological structures of HPAEs provide enhanced serum resistance and significantly higher mRNA expression in vivo. The terminal amine structures of HPAEs determine the organ-selectivity of mRNA delivery following systemic administration: morpholine facilitates liver targeting, ethylenediamine favors spleen delivery, while methylpentane enables mRNA delivery to the liver, spleen, and lungs simultaneously. This study represents a comprehensive exploration of the structure-activity relationship governing both the efficiency and organ-selectivity of mRNA delivery by HPAEs, suggesting promising candidates for treating various organ-related diseases.

Temporal dissociation of COX-2-dependent arachidonic acid and 2-arachidonoylglycerol metabolism in RAW264.7 macrophages.

Cyclooxygenase-2 converts arachidonic acid to prostaglandins (PGs) and the endocannabinoid, 2-arachidonoylglycerol (2-AG), to PG glyceryl esters (PG-Gs). The physiological function of PG biosynthesis has been extensively studied, but the importance of the more recently discovered PG-G synthetic pathway remains incompletely defined. This disparity is due in part to a lack of knowledge of the physiological conditions under which PG-G biosynthesis occurs. We have discovered that RAW264.7 macrophages stimulated with Kdo2-lipid A (KLA) produce primarily PGs within the first 12 h followed by robust PG-G synthesis between 12 h and 24 h. We suggest that the amount of PG-Gs quantified is less than actually synthesized, because PG-Gs are subject to a significant level of hydrolysis during the time course of synthesis. Inhibition of cytosolic phospholipase A2 by giripladib does not accelerate PG-G synthesis, suggesting the differential time course of PG and PG-G synthesis is not due to the competition between arachidonic acid and 2-AG. The late-phase PG-G formation is accompanied by an increase in the level of 2-AG and a concomitant decrease in 18:0-20:4 diacylglycerol (DAG). Inhibition of DAG lipases by KT-172 decreases the levels of 2-AG and PG-Gs, indicating that the DAG-lipase pathway is involved in delayed 2-AG metabolism/PG-G synthesis. These results demonstrate that physiologically significant levels of PG-Gs are produced by activated RAW264.7 macrophages well after the production of PGs plateaus.

Sdr16c5 and Sdr16c6 control a dormant pathway at a bifurcation point between meibogenesis and sebogenesis.

Genes Sdr16c5 and Sdr16c6 encode proteins that belong to a superfamily of short-chain dehydrogenases/reductases (SDR16C5 and SDR16C6). Simultaneous inactivation of these genes in double-KO (DKO) mice was previously shown to result in a marked enlargement of the mouse Meibomian glands (MGs) and sebaceous glands, respectively. However, the exact roles of SDRs in physiology and biochemistry of MGs and sebaceous glands have not been established yet. Therefore, we characterized, for the first time, meibum and sebum of Sdr16c5/Sdr16c6-null (DKO) mice using high-resolution MS and LC. In this study, we demonstrated that the mutation upregulated the overall production of MG secretions (also known as meibogenesis) and noticeably altered their lipidomic profile, but had a more subtle effect on sebogenesis. The major changes in meibum of DKO mice included abnormal accumulation of shorter chain, sebaceous-type cholesteryl esters and wax esters (WEs), and a marked increase in the biosynthesis of monounsaturated and diunsaturated Meibomian-type WEs. Importantly, the MGs of DKO mice maintained their ability to produce typical extremely long chain Meibomian-type lipids at seemingly normal levels. These observations indicated preferential activation of a previously dormant biosynthetic pathway that produce shorter chain, and more unsaturated, sebaceous-type WEs in the MGs of DKO mice, without altering the elongation patterns of their extremely long chain Meibomian-type counterparts. We conclude that the Sdr16c5/Sdr16c6 pair may control a point of bifurcation in one of the meibogenesis subpathways at which biosynthesis of lipids can be redirected toward either abnormal sebaceous-type lipidome or normal Meibomian-type lipidome in WT mice.

The powerful potential of amino acid menthyl esters for anti-inflammatory and anti-obesity therapies.

Our newly developed menthyl esters of valine and isoleucine exhibit anti-inflammatory properties beyond those of the well-known menthol in macrophages stimulated by lipopolysaccharide (LPS) and in a mouse model of colitis induced by sodium dextran sulfate. Unlike menthol, which acts primarily through the cold-sensitive TRPM8 channel, these menthyl esters displayed unique mechanisms that operate independently of this receptor. They readily penetrated target cells and efficiently suppressed LPS-stimulated tumour necrosis factor-alpha (Tnf) expression mediated by liver X receptor (LXR), a key nuclear receptor that regulates intracellular cholesterol and lipid balance. The menthyl esters showed affinity for LXR and enhanced the transcriptional activity through their non-competitive and potentially synergistic agonistic effect. This effect can be attributed to the crucial involvement of SCD1, an enzyme regulated by LXR, which is central to lipid metabolism and plays a key role in the anti-inflammatory response. In addition, we discovered that the menthyl esters showed remarkable efficacy in suppressing adipogenesis in 3T3-L1 adipocytes at the mitotic clonal expansion stage in an LXR-independent manner as well as in mice subjected to diet-induced obesity. These multiple capabilities of our compounds establish them as formidable allies in the fight against inflammation and obesity, paving the way for a range of potential therapeutic applications.

Delta-6 desaturase FADS2 is a tumor-promoting factor in cholangiocarcinoma.

Cholangiocarcinoma is a fatal disease with limited therapeutic options. We screened genes required for cholangiocarcinoma tumorigenicity and identified FADS2, a delta-6 desaturase. FADS2 depletion reduced in vivo tumorigenicity and cell proliferation. In clinical samples, FADS2 was expressed in cancer cells but not in stromal cells. FADS2 inhibition also reduced the migration and sphere-forming ability of cells and increased apoptotic cell death and ferroptosis markers. Lipidome assay revealed that triglyceride and cholesterol ester levels were decreased in FADS2-knockdown cells. The oxygen consumption ratio was also decreased in FADS2-depleted cells. These data indicate that FADS2 depletion causes a reduction in lipid levels, resulting in decrease of energy production and attenuation of cancer cell malignancy.

The surface of lipid droplets constitutes a barrier for endoplasmic reticulum-resident integral membrane proteins.

Lipid droplets (LDs) are globular subcellular structures that store neutral lipids. LDs are closely associated with the endoplasmic reticulum (ER) and are limited by a phospholipid monolayer harboring a specific set of proteins. Most of these proteins associate with LDs through either an amphipathic helix or a membrane-embedded hairpin motif. Here, we address the question of whether integral membrane proteins can localize to the surface of LDs. To test this, we fused perilipin 3 (PLIN3), a mammalian LD-targeted protein, to ER-resident proteins. The resulting fusion proteins localized to the periphery of LDs in both yeast and mammalian cells. This peripheral LD localization of the fusion proteins, however, was due to a redistribution of the ER around LDs, as revealed by bimolecular fluorescence complementation between ER- and LD-localized partners. A LD-tethering function of PLIN3-containing membrane proteins was confirmed by fusing PLIN3 to the cytoplasmic domain of an outer mitochondrial membrane protein, OM14. Expression of OM14-PLIN3 induced a close apposition between LDs and mitochondria. These data indicate that the ER-LD junction constitutes a barrier for ER-resident integral membrane proteins.

Lipid droplets form a network interconnected by the endoplasmic reticulum through which their proteins equilibrate.

Lipid droplets (LDs) are globular intracellular structures dedicated to the storage of neutral lipids. They are closely associated with the endoplasmic reticulum (ER) and are delineated by a monolayer of phospholipids that is continuous with the cytoplasmic leaflet of the ER membrane. LDs contain a specific set of proteins, but how these proteins are targeted to the LD surface is not fully understood. Here, we devised a yeast mating-based microscopic readout to monitor the transfer of LD proteins upon zygote formation. The results of this analysis indicate that ER fusion between mating partners is required for transfer of LD proteins and that this transfer is continuous, bidirectional and affects most LDs simultaneously. These observations suggest that LDs do not fuse upon mating of yeast cells, but that they form a network that is interconnected through the ER membrane. Consistent with this, ER-localized LD proteins rapidly move onto LDs of a mating partner and this protein transfer is affected by seipin, a protein important for proper LD biogenesis and the functional connection of LDs with the ER membrane.

Solvent-free synthesis of hydrophobic and amphiphilic esters using a chemically modified lipase from Thermomyces lanuginosus: a comparative study with native and immobilized forms.

Using lipases to catalyze the synthesis of the most differentiated type of compounds remains one of the major challenges among scientists. Seeking more economic and advantageous catalysts is a current goal of green chemistry. In this work, we demonstrate the potential of a chemically modified form of lipase from Thermomyces lanuginosus (cmLTL) for the synthesis of both hydrophobic (heptyl heptanoate, heptyl octanoate, heptyl decanoate, decyl heptanoate, decyl octanoate and decyl decanoate) and amphiphilic (2-(2-ethoxyethoxy)ethyl oleate and 2-(2-ethoxyethoxy)ethyl linoleate) esters, in bulk. The results were compared with its native (LTL) and immobilized (imLTL) forms. The data revealed that LTL showed poor activity for all reactions performed with n-heptane (η<20 %). ImLTL was able to synthesize all hydrophobic esters (η>60 %), with exception of the short ester, heptyl heptanoate. cmLTL was the only form of LTL capable of producing hydrophobic and amphiphilic esters, without compromising the yield when the reactions were performed under solvent-free conditions (>50 %). Molecular modeling showed that the active pocket of cmLTL is able to deeply internalize transcutol, with stronger interactions, justifying the outstanding results obtained. Furthermore, owing to the possibility of cmLTL filtration, the reusability of the catalyst is ensured for at least 6 cycles, without compromising the reaction yields.

Biosensor-Guided Engineering of a Baeyer-Villiger Monooxygenase for Aliphatic Ester Production.

Esters are valuable aroma compounds and can be produced enzymatically by Baeyer-Villiger monooxygenases (BVMOs) from (aliphatic) ketone precursors. However, a genetically encoded biosensor system for the assessment of BVMO activity and the detection of reaction products is missing. In this work, we assembled a synthetic enzyme cascade - featuring an esterase, an alcohol dehydrogenase, and LuxAB - in the heterologous host Escherichia coli. Target esters are produced by a BVMO, subsequently cleaved, and the corresponding alcohol oxidized through the artificial pathway. Ultimately, aldehyde products are detected in vivo by LuxAB, a luciferase from Photorhabdus luminescens that emits bioluminescence upon the oxidation of aldehydes to the corresponding carboxylates. This biosensor system greatly accelerated the screening and selection of active BVMO variants from a focused library, omitting commonly used low-throughput chromatographic analysis. Engineered enzymes accepted linear aliphatic ketones such as 2-undecanone and 2-dodecanone and exhibited improved ester formation.

Unlocking lager's flavour palette by metabolic engineering of Saccharomyces pastorianus for enhanced ethyl ester production.

Despite being present in trace amounts, ethyl esters play a crucial role as flavour compounds in lager beer. In yeast, ethyl hexanoate, ethyl octanoate and ethyl decanoate, responsible for fruity and floral taste tones, are synthesized from the toxic medium chain acyl-CoA intermediates released by the fatty acid synthase complex during the fatty acid biosynthesis, as a protective mechanism. The aim of this study was to enhance the production of ethyl esters in the hybrid lager brewing yeast Saccharomyces pastorianus by improving the medium chain acyl-CoA precursor supply. Through CRISPR-Cas9-based genetic engineering, specific FAS1 and FAS2 genes harbouring mutations in domains of the fatty acid synthesis complex were overexpressed in a single and combinatorial approach. These mutations in the ScFAS genes led to specific overproduction of the respective ethyl esters: overexpression of ScFAS1I306A and ScFAS2G1250S significantly improved ethyl hexanoate production and ScFAS1R1834K boosted the ethyl octanoate production. Combinations of ScFAS1 mutant genes with ScFAS2G1250S greatly enhanced predictably the final ethyl ester concentrations in cultures grown on full malt wort, but also resulted in increased levels of free medium chain fatty acids causing alterations in flavour profiles. Finally, the elevated medium chain fatty acid pool was directed towards the ethyl esters by overexpressing the esterase ScEEB1. The genetically modified S. pastorianus strains were utilized in lager beer production, and the resulting beverage exhibited significantly altered flavour profiles, thereby greatly expanding the possibilities of the flavour palette of lager beers.

Short-term effects of gastric bypass versus sleeve gastrectomy on high LDL cholesterol: The BASALTO randomized clinical trial.

BACKGROUND: There has been a substantial increase in the use of laparoscopic sleeve gastrectomy (SG) to treat morbid obesity despite observational evidence demonstrating the superiority of Roux-en-Y gastric bypass (RYGB) for reducing low-density lipoprotein (LDL) cholesterol. The main aim was to ascertain whether high LDL cholesterol levels should be considered when selecting the most appropriate surgical procedure for each patient (RYGB or SG). METHODS: In this single-center, randomized clinical trial using intention-to-treat analysis, 38 patients with severe obesity and elevated levels of LDL cholesterol were randomly assigned to undergo RYGB or SG. The primary outcome was LDL cholesterol remission at 12 months, defined as LDL cholesterol < 3.36 nmol/l without lipid-lowering medications. Secondary outcomes included changes in weight, other comorbidities, qualitative lipoprotein traits, cholesterol esters, glycoproteins, cholesterol absorption and synthesis metabolites and complications. RESULTS: Intention-to-treat analysis revealed that LDL cholesterol remission occurred in 66.6% of RYGB patients compared to 27.8% of SG patients (p = 0.019). Among patients completing follow-up, RYGB demonstrated superior remission (80.0% vs. 29.4%, p = 0.005). Exclusive benefits of RYGB included a reduction in large, medium, and small LDL particles. Cholesterol absorption markers showed differential behavior after both techniques: campesterol (Δ -15.2 µg/mg, 95% CI -30.2 to -0.1) decreased after RYGB, and sitosterol (Δ 21.1 µg/mg, 95% CI 0.9 to 41.2), cholestanol (Δ 30.6 µg/mg, 95% CI 14.8 to 57.9) and campesterol (Δ 18.4 µg/mg, 95% CI 4.4 to 32.3) increased after SG. No differences in weight loss, cholesterol esters, glycoproteins, cholesterol synthesis metabolites or postoperative complications were observed between techniques. CONCLUSION: In conclusion, RYGB is superior to SG in terms of short-term of high LDL cholesterol remission. Furthermore, RYGB also led to a greater improvement in lipoprotein parameters that confer an atherogenic profile. Therefore, the presence of elevated levels of LDL cholesterol should be considered when determining the optimal bariatric surgery procedure for each patient. TRIAL REGISTRATION: Clinicaltrials.gov number, NCT03975478).

Generating high purity and yield of capsanthin and capsorubin carrot germplasm through synthetic metabolic engineering.

Capsanthin and capsorubin are red κ-xanthophylls exclusively found in a handful of other plant species. Currently, capsanthin and capsorubin are only extracted from red pepper. Here, high purity production of capsanthin and capsorubin has been achieved in carrot taproot by synthetic metabolic engineering strategy. Expression of a capsanthin-capsorubin synthase gene (CaCCS) from pepper resulted in dominant production of capsanthin whereas expression of a LiCCS gene from tiger lily resulted in production of both capsanthin and capsorubin in carrot taproot. The highest content of capsanthin and capsorubin was obtained in LiC-1 carrot taproot hosting the LiCCS gene, 150.09 µg/g DW (dry weight). Co-expression of DcBCH1 with CCS could improve the purity of capsanthin and capsorubin by eliminating the non-target carotenoids (eg. α-carotene and ß-carotene). The highest purity of capsanthin and capsorubin was obtained in BLiC-1 carrot taproot hosting DcBCH1+LiCCS genes, 91.10% of total carotenoids. The non-native pigments were esterified partially and stored in the globular chromoplast of carrot taproot. Our results demonstrated the possibility of employing carrot taproot as green factories for high purity production of capsanthin and capsorubin. The capsanthin/capsorubin carrot germplasms were also valuable materials for breeding colorful carrots cultivars.

Inhibitory effects of phthalate esters (PAEs) and phthalate monoesters towards human carboxylesterases (CESs).

Phthalate esters (PAEs), accompanied by phthalate monoesters as hydrolysis metabolites in humans, have been widely used as plasticizers and exhibited disruptive effects on the endocrine and metabolic systems. The present study aims to investigate the inhibition behavior of PAEs and phthalate monoesters on the activity of the important hydrolytic enzymes, carboxylesterases (CESs), to elucidate the toxicity mechanism from a new perspective. The results showed significant inhibition on CES1 and CES2 by most PAEs, but not by phthalate monoesters, above which the activity of CES1 was strongly inhibited by DCHP, DEHP, DiOP, DiPP, DNP, DPP and BBZP, with inhibition ratios exceeding 80%. Kinetic analyses and in vitro-in vivo extrapolation were conducted, revealing that PAEs have the potential to disrupt the metabolism of endogenous substances catalyzed by CES1 in vivo. Molecular docking results revealed that hydrogen bonds and hydrophobic contacts formed by ester bonds contributed to the interaction of PAEs towards CES1. These findings will be beneficial for understanding the adverse effect of PAEs and phthalate monoesters.

Metal-Free Decarboxylative Allylation of Oxime Esters under Light Irradiation.

Allylation reactions, often used as a key step for constructing complex molecules and drug candidates, typically rely on transition-metal (TM) catalysts. Even though TM-free radical allylations have been developed using allyl-stannanes, -sulfides, -silanes or -sulfones, much less procedures have been reported using simple and commercially available allyl halides, that are used for the preparation of the before-mentioned allyl derivatives. Here, we present a straightforward photocatalytic protocol for the decarboxylative allylation of oxime esters using allyl bromide derivatives under metal-free and mild conditions. This methodology yields a diverse variety of functionalized molecules including several pharmaceutically relevant molecules.

Direct Synthesis of Phosphoryltriacetates from White Phosphorus via Visible Light Catalysis.

Organophosphorus compounds (OPCs) are widely used in many fields. However, traditional synthetic routes in the industry usually involve multistep and hazardous procedures. Therefore, it's of great significance to construct such compounds in an environmentally-friendly and facile way. Herein, a photoredox catalytic method has been developed to construct novel phosphoryltriacetates. Using fac-Ir(ppy)3 (ppy=2-phenylpyridine) as the photocatalyst and blue LEDs (456 nm) as the light source, white phosphorus can react with α-bromo esters smoothly to generate phosphoryltriacetates in moderate to good yields. This one-step approach features mild reaction conditions and simple operational process without chlorination.

Photoinduced Decarboxylative Thioacylation of N-Hydroxyphthalimide Esters with Tetraalkylthiuram Disulfides.

Dithiocarbamate is a key structural sequence in pharmaceuticals and agrochemicals, and its synthesis is crucial in organic chemistry. Although significant progress has been made in related synthesis research, developing a practical and universal synthesis method remains fascinating. Herein, we report a new visible-light-induced decarboxylation coupling reaction between N-hydroxyphthalimide esters and tetraalkylthiuram disulfides, which uses Ir(ppy)3 as a photocatalyst to promote the generation of corresponding decarboxylation thioacylation product-dithiocarbamates in high yields. This redox-neutral protocol uses inexpensive and readily available starting material under mild reaction conditions, exhibiting broad substrate scope and wide functional group compatibility. This method can be further used for post modification of complex natural products and bioactive drugs.

Visible-Light-Induced Multi-Component Nitrooxylation Reactions of α-Diazoesters, Cyclic Ethers, and Tert-Butyl Nitrite Leading to Organic Nitrate Esters.

A simple and efficient strategy has been developed for the synthesis of organic nitrate esters via visible-light-induced multi-component nitrooxylation reactions of α-diazoesters, cyclic ethers, and tert-butyl nitrite under open air atmosphere. This transformation could be conducted under mild and metal-free conditions to provide a number of organic nitrate esters in moderate to good yields using air as the green oxidant.

Repeated High-Dose Vitamin A Supplements, Standard of Care for Treating Xerophthalmia, Leads to Hypervitaminosis A in Piglets.

BACKGROUND: Vitamin A (VA) deficiency and excess negatively affect development, growth, and bone health. The World Health Organization's standard of care for xerophthalmia due to VA deficiency, is 3 high-dose VA supplements of 50,000-200,000 IU, based on age, which may cause hypervitaminosis A in some individuals. OBJECTIVES: This study measured VA status following 3 VA doses in 2 piglet studies. METHODS: In Study 1, 5 groups of piglets (n = 10/group) were weaned 10 d postbirth to VA-free feed and orally administered 0; 25,000; 50,000; 100,000; or 200,000 IU VA ester on days 0, 1, and 7. On days 14 and 15, the piglets underwent the modified relative dose-response (MRDR) test for VA deficiency, and were killed. Tissues were collected for high-pressure liquid chromatography analysis. Study 2 used the same design in 3 groups (n = 13/group) weaned at 16 d and administered 0; 25,000; and 200,000 IU doses. RESULTS: In Study 1 (final weight: 3.6 ± 0.7 kg), liver VA concentration was hypervitaminotic in 40%, 90%, and 100% of 50,000; 100,000; and 200,000 IU groups, respectively. The 25,000 IU group was 100% adequate, and the placebo group was 40% deficient. In Study 2 (final weight: 8.7 ± 0.8 kg), where 200,000 IU could be prescribed to infants with a similar body weight, 31% of the piglets were hypervitaminotic, the 25,000 IU group was 100% VA adequate, and the placebo group was 100% deficient. The MRDR test measured deficiency in 50% and 70% of the placebo group in each study but had 3 false positives among hypervitaminotic piglets in Study 1. CONCLUSIONS: Repeated high-dose VA may cause hypervitaminosis, indicating dose sizes may need reduction. The MRDR resulted in false positives in a hypervitaminotic state during malnutrition and should be paired with serum retinyl ester evaluation to enhance VA status assessment in populations with overlapping interventions.

Aberrant lipid accumulation and retinal pigment epithelium dysfunction in PRCD-deficient mice.

Progressive Rod-Cone Degeneration (PRCD) is an integral membrane protein found in photoreceptor outer segment (OS) disc membranes and its function remains unknown. Mutations in Prcd are implicated in Retinitis pigmentosa (RP) in humans and multiple dog breeds. PRCD-deficient models exhibit decreased levels of cholesterol in the plasma. However, potential changes in the retinal cholesterol remain unexplored. In addition, impaired phagocytosis observed in these animal models points to potential deficits in the retinal pigment epithelium (RPE). Here, using a Prcd-/- murine model we investigated the alterations in the retinal cholesterol levels and impairments in the structural and functional integrity of the RPE. Lipidomic and immunohistochemical analyses show a 5-fold increase in the levels of cholesteryl esters (C.Es) and lipid deposits in the PRCD-deficient retina, respectively, indicating alterations in total retinal cholesterol. Furthermore, the RPE of Prcd-/- mice exhibit a 1.7-fold increase in the expression of lipid transporter gene ATP-binding cassette transporter A1 (Abca1). Longitudinal fundus and spectral domain optical coherence tomography (SD-OCT) examinations showed focal lesions and RPE hyperreflectivity. Strikingly, the RPE of Prcd-/- mice exhibited age-related pathological features such as lipofuscin accumulation, Bruch's membrane (BrM) deposits and drusenoid focal deposits, mirroring an Age-related Macular Degeneration (AMD)-like phenotype. We propose that the extensive lipofuscin accumulation likely impairs lysosomal function, leading to the defective phagocytosis observed in Prcd-/- mice. Our findings support the dysregulation of retinal cholesterol homeostasis in the absence of PRCD. Further, we demonstrate that progressive photoreceptor degeneration in Prcd-/- mice is accompanied by progressive structural and functional deficits in the RPE, which likely exacerbates vision loss over time.