Lipid Aggregates Research Articles

Druggability of lipid metabolism modulation against renal fibrosis.

Renal fibrosis contributes to progressive damage to renal structure and function. It is a common pathological process as chronic kidney disease develops into kidney failure, irrespective of diverse etiologies, and eventually leads to death. However, there are no effective drugs for renal fibrosis treatment at present. Lipid aggregation in the kidney and consequent lipotoxicity always accompany chronic kidney disease and fibrosis. Numerous studies have revealed that restoring the defective fatty acid oxidation in the kidney cells can mitigate renal fibrosis. Thus, it is an important strategy to reverse the dysfunctional lipid metabolism in the kidney, by targeting critical regulators of lipid metabolism. In this review, we highlight the potential "druggability" of lipid metabolism to ameliorate renal fibrosis and provide current pre-clinical evidence, exemplified by some representative druggable targets and several other metabolic regulators with anti-renal fibrosis roles. Then, we introduce the preliminary progress of noncoding RNAs as promising anti-renal fibrosis drug targets from the perspective of lipid metabolism. Finally, we discuss the prospects and deficiencies of drug targeting lipid reprogramming in the kidney.

Periodontal Pathogens Promote Foam Cell Formation by Blocking Lipid Efflux

Foam cells are one of the major cellular components of atherosclerotic plaques, within which the trace of periodontal pathogens has also been identified in recent studies. In line with these findings, the correlation between periodontitis and atherosclerotic cardiovascular incidences has been repetitively supported by evidence from a number of experimental studies. However, the direct role of periodontal pathogens in altered cellular signaling underlying such cardiovascular events has not been clearly defined. To determine the role of periodontal pathogens in the pathogenesis of atherosclerosis, especially in the evolution of macrophages into foam cells, we monitored the pattern of lipid accumulation within macrophages in the presence of periodontal pathogens, followed by characterization of these lipids and investigation of major molecules involved in lipid homeostasis. The cells were stained with the lipophilic fluorescent dye BODIPY 493/503 and Oil Red O to characterize the lipid profile. The amounts of Oil Red O–positive droplets, representing neutral lipids, as well as fluorescent lipid aggregates were prominently increased in periodontal pathogen–infected macrophages. Subsequent analysis allowed us to locate the accumulated lipids in the endoplasmic reticulum. In addition, the levels of cholesteryl ester in periodontal pathogen–infected macrophages were increased, implying disrupted lipid homeostasis. Further investigations to delineate the key messengers and regulatory factors involved in the altered lipid homeostasis have revealed alterations in cholesterol efflux–related enzymes, such as ABCG1 and CYP46A1, as contributors to foam cell formation, and increased Ca2+ signaling and reactive oxygen species (ROS) production as key events underlying disrupted lipid homeostasis. Consistently, a treatment of periodontal pathogen–infected macrophages with ROS inhibitors and nifedipine attenuated the accumulation of lipid droplets, further confirming periodontal pathogen–induced alterations in Ca2+ and ROS signaling and the subsequent dysregulation of lipid homeostasis as key regulatory events underlying the evolution of macrophages into foam cells.

Naphthalimide-Containing BP100 Leads to Higher Model Membranes Interactions and Antimicrobial Activity.

In a large variety of organisms, antimicrobial peptides (AMPs) are primary defenses against pathogens. BP100 (KKLFKKILKYL-NH2), a short, synthetic, cationic AMP, is active against bacteria and displays low toxicity towards eukaryotic cells. BP100 acquires a α-helical conformation upon interaction with membranes and increases membrane permeability. Despite the volume of information available, the action mechanism of BP100, the selectivity of its biological effects, and possible applications are far from consensual. Our group synthesized a fluorescent BP100 analogue containing naphthalimide linked to its N-terminal end, NAPHT-BP100 (Naphthalimide-AAKKLFKKILKYL-NH2). The fluorescence properties of naphthalimides, especially their spectral sensitivity to microenvironment changes, are well established, and their biological activities against transformed cells and bacteria are known. Naphthalimide derived compounds are known to interact with DNA disturbing related processes as replication and transcription, and used as anticancer agents due to this property. A wide variety of techniques were used to demonstrate that NAPHT-BP100 bound to and permeabilized zwitterionic POPC and negatively charged POPC:POPG liposomes and, upon interaction, acquired a α-helical structure. Membrane surface high peptide/lipid ratios triggered complete permeabilization of the liposomes in a detergent-like manner. Membrane disruption was driven by charge neutralization, lipid aggregation, and bilayer destabilization. NAPHT-BP100 also interacted with double-stranded DNA, indicating that this peptide could also affect other cellular processes besides causing membrane destabilization. NAPHT-BP100 showed increased antibacterial and hemolytic activities, compared to BP100, and may constitute an efficient antimicrobial agent for dermatological use. By conjugating BP100 and naphthalimide DNA binding properties, NAPHT-BP100 bound to a large extent to the bacterial membrane and could more efficiently destabilize it. We also speculate that peptide could enter the bacteria cell and interact with its DNA in the cytoplasm.

Lipid-Bilayer Assemblies on Polymer-Bearing Surfaces: The Nature of the Slip Plane in Asymmetric Boundary Lubrication.

Phospholipid–macromolecule complexes have been proposed to form highly efficient, lubricating boundary layers at artificial soft surfaces or at biological surfaces such as articular cartilage, where the friction reduction is attributed to the hydration lubrication mechanism acting at the exposed, hydrated head groups of the lipids. Here we measure, using a surface force balance, the normal and frictional interactions between model mica substrates across several different configurations of phosphatidylcholine (PC) lipid aggregates and adsorbed polymer (PEO) layers, to provide insight into the nature of such lubricating boundary layers in both symmetric and especially asymmetric configurations. Our results reveal that, irrespective of the configuration, the slip plane between the sliding surfaces reverts wherever possible to a bilayer–bilayer interface where hydration lubrication reduces the friction strongly. Where such an interface is not available, the sliding friction remains high. These findings may account for the low friction observed between both biological and synthetic hydrogel surfaces which may be asymmetrically coated with lipid-based boundary layers and fully support the hydration lubrication mechanism attributed to act at such boundary layers.

Benefits and Detriments of Gadolinium from Medical Advances to Health and Ecological Risks.

Gadolinium (Gd)-containing chelates have been established as diagnostics tools. However, extensive use in magnetic resonance imaging has led to increased Gd levels in industrialized parts of the world, adding to natural occurrence and causing environmental and health concerns. A vast amount of data shows that metal may accumulate in the human body and its deposition has been detected in organs such as brain and liver. Moreover, the disease nephrogenic systemic fibrosis has been linked to increased Gd3+ levels. Investigation of Gd3+ effects at the cellular and molecular levels mostly revolves around calcium-dependent proteins, since Gd3+ competes with calcium due to their similar size; other reports focus on interaction of Gd3+ with nucleic acids and carbohydrates. However, little is known about Gd3+ effects on membranes; yet some results suggest that Gd3+ interacts strongly with biologically-relevant lipids (e.g., brain membrane constituents) and causes serious structural changes including enhanced membrane rigidity and propensity for lipid fusion and aggregation at much lower concentrations than other ions, both toxic and essential. This review surveys the impact of the anthropogenic use of Gd emphasizing health risks and discussing debilitating effects of Gd3+ on cell membrane organization that may lead to deleterious health consequences.

Molecular mechanism of down-regulating adipogenic transcription factors in 3T3-L1 adipocyte cells by bioactive anti-adipogenic compounds.

Obesity is growing at an alarming rate, which is characterized by increased adipose tissue. It increases the probability of many health complications, such as diabetes, arthritis, cardiac disease, and cancer. In modern society, with a growing population of obese patients, several individuals have increased insulin resistance. Herbal medicines are known as the oldest method of health care treatment for obesity-related secondary health issues. Several traditional medicinal plants and their effective phytoconstituents have shown anti-diabetic and anti-adipogenic activity. Adipose tissue is a major site for lipid accumulation as well as the whole-body insulin sensitivity region. 3T3-L1 cell line model can achieve adipogenesis. Adipocyte characteristics features such as expression of adipocyte markers and aggregation of lipids are chemically induced in the 3T3-L1 fibroblast cell line. Differentiation of 3T3-L1 is an efficient and convenient way to obtain adipocyte like cells in experimental studies. Peroxisome proliferation activated receptor γ (PPARγ) and Cytosine-Cytosine-Adenosine-Adenosine-Thymidine/Enhancer-binding protein α(CCAAT/Enhancer-binding protein αor C/EBPα) are considered to be regulating adipogenesis at the early stage, while adiponectin and fatty acid synthase (FAS) is responsible for the mature adipocyte formation. Excess accumulation of these adipose tissues and lipids leads to obesity. Thus, investigating adipose tissue development and the underlying molecular mechanism is important in the therapeutical approach. This review describes the cellular mechanism of 3T3-L1 fibroblast cells on potential anti-adipogenic herbal bioactive compounds.

Prevalence of Dyslipidaemia among Type 2 Diabetes Mellitus Patients in the Western Cape, South Africa.

Dyslipidaemia, an irregular aggregate of lipids in the blood is common in diabetes and cardiovascular disease sufferers. A cross-sectional study on the prevalence of dyslipidaemia was performed among type 2 diabetes mellitus (T2DM) patients in the Western Cape, South Africa. Patients (n = 100) that participated in the study were within the age range of 19–68 years, of whom 89% were observed to have serum lipid abnormalities. Out of the 100 patients, 56%, 64%, 61%, and 65% were recorded to have high total cholesterol (TC), hypertriglycemia, increased low-density lipoproteins cholesterol (LDL-C), and reduced high-density lipoproteins cholesterol (HDL-C), respectively. In male diabetic patients, a marked prevalence of (94%) dyslipidemia was noted, of which 52% were affected by high TC (5.3–7.9 mmol/L), with 70% having a high level of triglyceride (TG) [1.72–7.34 mmol/L], while 60% had a high LDL-C (3.1–5.5 mmol/L), including 78% with low HDL-C (0.7–1.1 mmol/L). In comparison, 84% of diabetic females had dyslipidemia, with high TC (5.1–8.1 mmol/L), hypertriglycemia (1.73–8.63 mmol/L), high LDL-C (3.1–5.6 mmol/L), and low levels of HDL-C (0.8–1.1 mmol/L) affecting 60%, 58%, 62%, and 52% of the patients, respectively. This study showed the importance of screening and the regular surveillance of dyslipidaemia in T2DM patients as there is a paucity of data on it in Africa.

Exploring the Role of a Novel Peptide from Allomyrina dichotoma Larvae in Ameliorating Lipid Metabolism in Obesity.

The aim of this study was to identify an anti-obesity peptide from Allomyrina dichotoma and investigate the lipid metabolic mechanism. Enzymatically hydrolyzed A. dichotoma larvae were further separated using tangential flow filtration and consecutive chromatographic processes. Finally, an anti-obesity peptide that showed the highest inhibitory effect on lipid accumulation was obtained, and the sequence was Glu-Ile-Ala-Gln-Asp-Phe-Lys-Thr-Asp-Leu (EIA10). EIA10 decreased lipid aggregation in vitro and significantly reduced the accumulation of body weight gain, liver weight, and adipose tissue weight in high-fat-fed mice. Compared with the control group, the levels of total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL), insulin, and homeostasis model assessment of insulin resistance (HOMA-IR) in the high-fat diet (HFD) group increased significantly, and the content of high-density lipoprotein cholesterol (HDL) in the serum decreased significantly. On the contrary, the levels of TC, TG, and insulin in the EIA10 group decreased significantly, and the HDL content increased significantly compared with the HFD group. Additionally, EIA10 dramatically decreased mRNA and protein levels of transcription factors involved in lipid adipogenesis. Taken together, our results suggest that EIA10 could be a promising agent for the treatment and prevention of obesity.

Affinity of Lipophilic Drugs to Mixed Lipid Aggregates in Simulated Gastrointestinal Fluids

Mixed lipid aggregates, comprising of bile salts and phospholipids, present in the small intestine assist in drug solubilization and subsequent drug dissolution and absorption through the intestinal epithelium. The increased variability in their levels, observed physiologically, may create challenges not only for in vivo bioavailability and bioequivalence studies, but also for in vitro bio-predictive studies as correlations between in vitro and in vivo data are not always successful. The current study investigated the impact of biorelevant dissolution media, with physiologically relevant sodium taurocholate and lecithin levels, on the apparent solubility and affinity of lipophilic compounds with a wide range of physicochemical properties (drug ionization, drug lipophilicity, molecular weight) to mixed lipid aggregates. Apparent solubility data in biorelevant dissolution media for the studied neutral drugs, weak bases and weak acids were compared against a phosphate buffer pH 6.5 in the absence of these lipidic components. Presence of mixed lipid aggregates enhanced the apparent solubility of the majority of compounds and the use of multivariate data analysis identified the significant parameters affecting drug affinity to mixed lipid aggregates based on the chemical class of the drug. For neutral drugs, increasing bile salt concentrations and/or drug lipophilicity resulted in greater enhancement in apparent solubility at 24-hr. For weak bases and weak acids, the effect of increasing bile salt levels on apparent solubility depended mostly on an interplay between drug lipophilicity and drug ionization.

In situ monitoring of the formation of lipidic non-lamellar liquid crystalline depot formulations in synovial fluid

Administration of parenteral liquid crystalline phases, forming in-vivo with tunable nanostructural features and sustained release properties, offers an attractive approach for treatment of infections and local drug delivery. It has also a potential use for postoperative pain management after arthroscopic knee surgery. However, the optimal use of this drug delivery principle requires an improved understanding of the involved dynamic structural transitions after administration of low-viscous stimulus-responsive lipid precursors and their fate after direct contact with the biological environment. These precursors (preformulations) are typically based on a single biologically relevant lipid (or a lipid combination) with non-lamellar liquid crystalline phase forming propensity. In relation to liquid crystalline depot design for intra-articular drug delivery, it was our interest in the present study to shed light on such dynamic structural transitions by combining synchrotron SAXS with a remote controlled addition of synovial fluid (or buffer containing 2% (w/v) albumin). This combination allowed for monitoring in real-time the hydration-triggered dynamic structural events on exposure of the lipid precursor (organic stock solution consisting of the binary lipid mixture of monoolein and castor oil) to excess synovial fluid (or excess buffer). The synchrotron SAXS findings indicate a fast generation of inverse bicontinuous cubic phases within few seconds. The effects of (i) the organic solvent N-methyl-2-pyrolidone (NMP), (ii) the lipid composition, and (iii) the albumin content on modulating the structures of the self-assembled lipid aggregates and the implications of the experimental findings in the design of liquid crystalline depots for intra-articular drug delivery are discussed.

1-Deoxysphingolipids cause autophagosome and lysosome accumulation and trigger NLRP3 inflammasome activation

ABSTRACT 1-Deoxysphingolipids (deoxySLs) are atypical sphingolipids of clinical relevance as they are elevated in plasma of patients suffering from hereditary sensory and autonomic neuropathy (HSAN1) or type 2 diabetes. Their neurotoxicity is described best but they inflict damage to various cell types by an uncertain pathomechanism. Using mouse embryonic fibroblasts and an alkyne analog of 1-deoxysphinganine (doxSA), the metabolic precursor of all deoxySLs, we here study the impact of deoxySLs on macroautophagy/autophagy, the regulated degradation of dysfunctional or expendable cellular components. We find that deoxySLs induce autophagosome and lysosome accumulation indicative of an increase in autophagic flux. The autophagosomal machinery targets damaged mitochondria that have accumulated N-acylated doxSA metabolites, presumably deoxyceramide and deoxydihydroceramide, and show aberrant swelling and tubule formation. Autophagosomes and lysosomes also interact with cellular lipid aggregates and crystals that occur upon cellular uptake and N-acylation of monomeric doxSA. As crystals entering the lysophagosomal apparatus in phagocytes are known to trigger the NLRP3 inflammasome, we also treated macrophages with doxSA. We demonstrate the activation of the NLRP3 inflammasome by doxSLs, prompting the release of IL1B from primary macrophages. Taken together, our data establish an impact of doxSLs on autophagy and link doxSL pathophysiology to inflammation and the innate immune system. Abbreviations: alkyne-doxSA: (2S,3R)-2-aminooctadec-17yn-3-ol; alkyne-SA: (2S,3R)-2- aminooctadec-17yn-1,3-diol; aSA: alkyne-sphinganine; ASTM-BODIPY: azido-sulfo-tetramethyl-BODIPY; CerS: ceramide synthase; CMR: clonal macrophage reporter; deoxySLs: 1-deoxysphingolipids; dox(DH)Cer: 1-deoxydihydroceramide; doxCer: 1-deoxyceramide; doxSA: 1-deoxysphinganine; FB1: fumonisin B1; HSAN1: hereditary sensory and autonomic neuropathy type 1; LC3: MAP1LC3A and MAP1LC3B; LPS: lipopolysaccharide; MEF: mouse embryonal fibroblasts; MS: mass spectrometry; N3635P: azido-STAR635P; N3Cy3: azido-cyanine 3; N3picCy3: azido-picolylcyanine 3; NLRP3: NOD-like receptor pyrin domain containing protein 3; P4HB: prolyl 4-hydroxylase subunit beta; PINK1: PTEN induced putative kinase 1; PYCARD/ASC: PYD and CARD domain containing; SPTLC1: serine palmitoyltransferase long chain base subunit 1; SQSTM1: sequestosome 1; TLC: thin layer chromatography.

Viburnum tinus Fruits Use Lipids to Produce Metallic Blue Structural Color

Viburnum tinus is an evergreen shrub that is native to the Mediterranean region but cultivated widely in Europe and around the world. It produces ripe metallic blue fruits throughout winter [1]. Despite its limited fleshy pulp [2], its high lipid content [3] makes it a valuable resource to the small birds [4] that act as its seed-dispersers [5]. Here, we find that the metallic blue appearance of the fruits is produced by globular lipid inclusions arranged in a disordered multilayer structure. This structure is embedded in the cell walls of the epicarp and underlaid with a dark layer of anthocyanin pigments. The presence of such large, organized lipid aggregates in plant cell walls represents a new mechanism for structural coloration and may serve as an honest signal of nutritional content.

Gly14]-humanin restores cathepsin D function via FPRL1 and promotes autophagic degradation of Ox-LDL in HUVECs

Background and aimAbnormal aggregation of oxidized low-density lipoprotein (Ox-LDL) in vascular endothelial cells (VECs) is one of the major pathological changes in atherosclerotic lesions. Our research aimed to assess the mechanism of humanin (HN) in promoting autophagic degradation of Ox-LDL in HUVECs. Methods and resultsFlow cytometry and lipid quantitation results showed that Ox-LDL caused lipid and cholesterol accumulation in HUVECs. Western blot results showed that Ox-LDL increased the expression of autophagy-related proteins P62 and LC3-II in a concentration-dependent manner. The cathepsin D activity assay showed that Ox-LDL inhibited the function of cathepsin D. HNG pretreatment reduced lipid and cholesterol aggregation in HUVECs induced by Ox-LDL, increased LC3-II protein level, decreased P62 protein content, and reversed Ox-LDL-induced cathepsin D functional impairment. Inhibition of the FPRL1 pathway by FPRL1 siRNA or the FPRL1-specific inhibitor Boc-MLF blocked all HNG's protective effects. These results indicate that HNG could restore cathepsin D activity and protein level in HUVECs to repair lysosomal functional damage induced by Ox-LDL, further repairing Ox-LDL-induced autophagic damage in HUVECs. ConclusionHNG restores the activity of Ox-LDL-induced damaged lysosomal enzyme cathepsin D through its membrane protein receptor FPRL1 to promote autophagic degradation of Ox-LDL in HUVECs.

Ultrastructure of the Midgut Adult, Trachyderma philistina (Coleoptera: Tenebrionidae)

In the anterior part of the midgut adult Trachyderma philistina, the apical part showed microvilli in the form of membrane with folds and also microvilli appeared slender and uniform. Between these folds of microvilli appeared a lot of ribosomes and rough endoplasmic reticulum. Secretory vesicles are discharged from part of midgut. A well-developed oval nucleus appeared in the cytoplasm near the apical part of the cell. The nucleus is surrounded by a lot of free ribosomes and rough endoplasmic reticulum. Mitochondria appeared near the apical part of the cell. A well-developed thick basement membrane appeared in the basal part of the anterior midgut of adult beetle Trachyderma philistina. In the middle part of the midgut, a very thick well-developed brush border microvilli appeared. Mitochondria and numerous tracheae appeared through cytoplasm. Rough endoplasmic reticulum appeared throughout the cytoplasm. A lot of secretory vacuoles were observed directed apically. A well-developed oval and circular nucleus appeared near the basal part of the cell and surrounded by numerous rough endoplasmic reticulum A well-developed muscle fiber appeared apically and basally. A very thin basement membrane appeared in the basal part of the cell. The peritrophic membrane is absent in the midgut of adult stage Trachyderma philistine while regenerative cells appeared in the basal part of the middle midgut cell. In the posterior part of the midgut, microvilli appeared in the form of membranes due to the presence of numerous organelles that are discharging into the lumen. Lipid spheres and aggregations of glycogen are present throughout the cell. A thin basement membrane was observed.

Physical and rheological properties of mixed-component emulsion-based products: Influence of flaxseed gum concentration and pH on the aggregation of lipid droplets

The present study investigates the influence of different flaxseed gum (FG) concentrations on the physical and rheological properties of mixed-component emulsion-based products at specific pH values (3.0, 5.0, 7.0). The emulsion-based products included 2% (w/v) lipid droplets, 3% (w/v) modified cassava starch and 0−0.02% (w/v) FG. Either native or heated whey proteins were used as emulsifiers to stabilize lipid droplets. The formation of lipid aggregation was mainly due to electrostatic cross-linking between anionic FG and cationic whey protein. The results showed that the apparent viscosities and rheological moduli of emulsion mixtures obviously increased with increasing addition levels of FG (P < 0.05). However, higher concentration of FG (0.02 %, w/v) led to the formation of visibly large aggregates and coarse textures that negatively impacted the visual appearance. Moreover, at pH 5.0, extensive droplet aggregation of the emulsion mixture occurred near the isoelectric point (pH 4.6) of whey proteins, which resulted in a significant increase in the mixture’s particle size. Additionally, lipid droplets stabilized by heated whey proteins led to obviously higher apparent viscosities and rheological moduli of emulsion mixtures than their native protein form (P < 0.05). Our results potentially provide some useful information for the creation of mixed-component emulsion-based products with various desirable quality attributes.

From the Bench to the Bedside, the Determination of the Etiology of Dry Eye Using Spectroscopy

PurposeMeibum lipids are believed to be important to tear film stability, but tear film lipid compositional, structural and functional relationships are not well established.MethodsNMR, Fourier transform infrared and Bruster angle spectroscopies were used to quantify meibum lipid composition, structure, and film lipid rheology, respectively.ResultsThe molar ratio of Cholesterylesters/Wax ester increased with age, in cohorts aged 1 to 19 years old, respectively. The same molar ratio decreased for cohorts aged 20 to 88 years old with and without meibomian gland dysfunction (MGD) that leads to dry eye, respectively. The molar ratio of =C‐CH2 /ester increased with age for cohorts aged 1 to 19 years old, respectively. The same molar ratio decreased for cohorts aged 20 to 88 years old, with and without MGD, respectively. Compared with meibum from donors without dry eye, meibum from donors with MGD contained less straight‐chains, more iso‐branched chains, and the same amount of anteisobranched hydrocarbon chains. Human meibum order increases with age between 0 and 25 years old, meibomian gland dysfunction and with donors who have had hematopoietic stem cell transplantation. Interestingly, tear film stability also increases in the same order. When symptoms of dry eye were ameliorated with treatment, lipid order was restored. Principal component analysis scores correlated with age and dry eye by 93 %.ConclusionsThe changes in meibum composition with age and MGD contribute to the increase in lipid order of the same samples from donors without compared with those with MGD. Higher lipid order is associated with a higher reciprocal compressibility modulus that results in a tear film lipid layer that is not as compressible and not as viscoelastic. Strong lipid‐lipid interactions could contribute to the aggregation of tear film lipids and contribute to a decrease in tear film spreading and dry eye.Support or Funding InformationMajor support was obtained from the National Institute of Health [grant number EYO RO126180] and an unrestricted grant from Research to Prevent Blindness Inc. New York, NY, USA [grant number GN151619B].Lipid order or stiffness correlates with tear film stability with age and dry eye symptoms.Figure 1

Mixed polar-nonpolar lipid films as minimalistic models of Tear Film Lipid Layer: A Langmuir trough and fluorescence microscopy study

The Tear Film Lipid Layer (TFLL) covering the surface of the aqueous film at human cornea forms a first barrier between the eye and environment. Its alterations are related to dry eye disease. TFLL is formed by a complex mixture of lipids, with an excess of nonpolar components and a minor fraction of polar molecules. Its thickness is up to 160 nm, hence a multilayer-like structure of TFLL is assumed. However, details of TFLL organization are mostly unavailable in vivo due to the dynamic nature of the human tear film. To overcome this issue, we employ a minimalistic in vitro lipid model of TFLL. We study its biophysical characteristics by using a combination of the Langmuir trough with fluorescence microscopy. The model consists of two-component polar-nonpolar lipid films with a varying component ratio spread on the aqueous subphase at physiologically relevant temperature. We demonstrate that the model lipid mixture undergoes substantial structural reorganization as a function of lateral pressure and polar to nonpolar lipid ratio. In particular, the film is one-molecule-thick and homogenous under low lateral pressure. Upon compression, it transforms into a multilayer structure with inhomogeneities in the form of polar-nonpolar lipid assemblies. Based on this model, we hypothesize that TFLL in vivo has a duplex polar-nonpolar structure and it contains numerous mixed lipid aggregates formed because of film restructuring. These findings, despite the simplified character of the model, seem relevant for TFLL physiology as well as for understanding pathological conditions related to the lipids of the tear film.

Aloe-emodin exerts cholesterol-lowering effects by inhibiting proprotein convertase subtilisin/kexin type 9 in hyperlipidemic rats

Hyperlipidemia (HPL) characterized by metabolic disorder of lipids and cholesterol is one of the important risk factors for cardiovascular diseases. Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a potent circulating regulator of LDL through its ability to induce degradation of the low-density lipoprotein cholesterol receptor (LDLR) in the lysosome of hepatocytes. Aloe-emodin (AE) is one of potentially bioactive components of Chinese traditional medicine Daming capsule. In this study we evaluated the HPL-lowering efficacy of AE in both in vivo and in vitro HPL models. High-fat diet-induced rats were treated with AE (100 mg/kg per day, ig) for 6 weeks. We found that AE administration significantly decreased the levels of total cholesterol (TC) and LDL in the serum and liver tissues. Moreover, AE administration ameliorated HPL-induced hepatic lipid aggregation. But AE administration did not significantly inhibit HMG-CoA reductase activity in the liver of HPL rats. A cellular model of HPL was established in human hepatoma (HepG2) cells treated with cholesterol (20 μg/mL) and 25-hydroxycholesterol (2 μg/mL), which exhibited markedly elevated cholesterol levels. The increased cholesterol levels could be reversed by subsequent treatment with AE (30 μM). In both the in vivo and in vitro HPL models, we revealed that AE selectively suppressed the sterol-regulatory element-binding protein-2 (SREBP-2) and hepatocyte nuclear factor (HNF)1α-mediated PCSK9 signaling, which in turn upregulated LDL receptor (LDLR) and promoted LDL uptake. This study demonstrates that AE reduces cholesterol content in HPL rats by inhibiting the hepatic PCSK9/LDLR pathway.

Huntingtin Aggregation and Lipid Binding are Influenced by Physicochemical Properties of Membranes

Huntington's Disease is a fatal neurodegenerative disorder caused by an expanded glutamine repeat region (polyQ) within the huntingtin protein (htt), resulting in the misfolding and aggregation of htt into toxic aggregate species. The first 17 amino acids at the N-terminus of htt (Nt17) are intrinsically disordered in bulk solution, but adopt an amphipathic α-helical structure in the presence of binding partners, such as lipid membranes. In addition to comprising a lipid binding domain, this amphipathic α-helix has also been directly implicated in the initiation of htt aggregation via self-association with other Nt17 α-helices. Due to this, htt/lipid interaction likely has a large impact on the rate and extent of htt aggregate formation, with potential implications for HD pathogenesis. The interaction between Nt17 and lipid membranes is likely influenced by lipid properties, such as fluidity and lateral pressure. As a result, we undertook a series of experiments to determine the role that membrane physicochemical properties have on htt aggregation and lipid binding. Htt aggregation in the presence and absence of DMPC, POPC, and DOPC lipid vesicles was investigated using thioflavin T aggregation assays and atomic force microscopy (AFM). Huntingtin lipid binding was investigated using electrospray ionization mass spectrometry (ESI-MS), PDA lipid binding assays, and molecular dynamics simulations. Overall, our results suggest that membrane physicochemical properties associated with lipid tail groups alter htt lipid binding and aggregate formation.

Characterization of Small Objects in Homogenates of the Squid Optic Lobe

The squid (Loligo pealei) optic lobe is unique among sources of CNS synaptosomes via differential centrifugation, both in its ease and speed of dissection and preparation, and in the purity of the final result (Pekkurnaz et al. Biol. Bull. 2011). Despite the high purity of ∼1 µm-diameter synaptosomes in the synaptosome fraction, another class of 2-10 μm diameter objects were detected by one us (GP) in DIC microscopy of thin layers of this fraction. Recently, a publication assumed that these inclusions were large synaptosomes (Cefaliello et al. Molec. Neurobio. 2019). To test that assumption, and to further characterize these inclusions, we carried out a series of fluorescent labeling and dye exclusion experiments by direct imaging for size classification. Two major classes of objects were found, a larger class (L) 5-10 μm in diameter and a smaller class (S) that appeared more refractile, perfectly spherical, and averaged 2 μm in diameter. To identify some of the macromolecular content of these objects, we labeled them with fluorescent dyes for DNA, lipid, and protein aggregates. ‘L’ stained positive for the membrane dye BODIPY-sphingomyelin and the DNA marker Hoechst. The exclusion of rhodamine-dextran (10kDa) further corroborated the membrane-enclosed nature of the ‘L’ object. Electron microscopy showed a large ∼10 μm spheroid with a double membrane and dark material draping the internal membrane, suggesting that the ‘L’ inclusions were nuclei. The majority of the inclusions in the ‘S’ class did not exclude dyes, did not stain with the DNA dye Hoechst, and were positive for a rotamer fluorescent marker for intracellular aggregated misfolded proteins. Currently, efforts are underway to identify the protein composition of the ‘S’ droplets.