Cholesterol and Its Oxidation Derivatives Content in Market Dairy Products.

Cholesterol oxidation products (COPs) are contaminants of food of animal origin. Increased levels of these compounds in the human body are associated with an increased risk of many non-communicable diseases. Dairy products are mentioned among the main sources of these compounds in the diet. The objective of this study was to evaluate the contents of cholesterol and its oxidized derivatives in eleven groups of dairy products, willingly consumed in European countries. The levels of COPs were determined by applying the GC-TOF/MS method. In the tested products, cholesterol and its oxidation derivatives, such as 7-ketocholesterol, 7α-hydroxycholesterol, 7ß-hydroxycholesterol, 5,6ß-epoxycholesterol and 5,6α-epoxycholesterol, were determined. The studied dairy products differed in their contents and profiles of oxysterols. The highest contents of COPs were found in cheese with internal mold (13.8 ± 2.5 mg kg-1) and Cheddar (11.7 ± 3.5 mg kg-1), while the lowest levels were detected in yoghurt (0.94 ± 0.30 mg kg-1) and kefir (0.57 ± 0.11 mg kg-1). 7-ketocholesterol and 5,6ß-epoxycholesterol were the dominant oxysterols. The ratio of oxidized derivatives to total cholesterol was on average 1.7%. Our results confirmed that dairy products are an important dietary source of COPs. Their levels should be monitored in dairy products to provide the best health quality.

LC-MS/MS profiling of colon oxysterols and cholesterol precursors in mouse model of ulcerative colitis.

Oxysterols and cholesterol precursors are being increasingly investigated in humans and laboratory animals as markers for various diseases in addition to their important functions. However, the quantitative analysis of these bioactive molecules is obstructed by high structural similarity, poor ionization efficiency and low abundance. The current assay methods are still cumbersome to be of practical use, and their applicability in different bio-samples needs to be evaluated and optimized as necessary. In the present work, chromatographic separation conditions were carefully studied to achieve baseline separation of difficult-to-isolate compound pairs. On the other hand, an efficient sample purification method was established for colon tissue samples with good recoveries of sterols, demonstrating negligible autoxidation of cholesterol into oxysterols. The developed UPLC-APCI-MS/MS method was thoroughly validated and applied to measure oxysterols and cholesterol precursors in colon tissue of dextran sulfate sodium (DSS)-induced mouse colitis models, and it is expected to be successfully applied to the quantitative determination of such components in other tissue samples.

Squalene-epoxidase-catalyzed 24(S),25-epoxycholesterol synthesis promotes trained-immunity-mediated antitumor activity.

The importance of trained immunity in antitumor immunity has been increasingly recognized, but the underlying metabolic regulation mechanisms remain incompletely understood. In this study, we find that squalene epoxidase (SQLE), a key enzyme in cholesterol synthesis, is required for ß-glucan-induced trained immunity in macrophages and ensuing antitumor activity. Unexpectedly, the shunt pathway, but not the classical cholesterol synthesis pathway, catalyzed by SQLE, is required for trained immunity induction. Specifically, 24(S),25-epoxycholesterol (24(S),25-EC), the shunt pathway metabolite, activates liver X receptor and increases chromatin accessibility to evoke innate immune memory. Meanwhile, SQLE-induced reactive oxygen species accumulation stabilizes hypoxia-inducible factor 1α protein for metabolic switching into glycolysis. Hence, our findings identify 24(S),25-EC as a key metabolite for trained immunity and provide important insights into how SQLE regulates trained-immunity-mediated antitumor activity.

Phytosterols in human serum as measured using a liquid chromatography tandem mass spectrometry.

Phytosterols are lipophilic compounds found in plants with structural similarity to mammalian cholesterol. They cannot be endogenously produced by mammals and therefore always originate from diet. There has been increased interest in dietary phytosterols over the last few decades due to their association with a variety of beneficial health effects including low-density lipoprotein cholesterol lowering, anti-inflammatory and anti-cancerous effects. They are proposed as potential moderators for diseases associated with the central nervous system where cholesterol homeostasis is found to be imperative (multiple sclerosis, dementia, etc.) due to their ability to reach the brain. Here we utilised an enzyme-assisted derivatisation for sterol analysis (EADSA) in combination with a liquid chromatography tandem mass spectrometry (LC-MSn) to characterise phytosterol content in human serum. As little as 100 fg of plant sterol was injected on a reversed phase LC column. The method allows semi-quantitative measurements of phytosterols and their derivatives simultaneously with measurement of cholesterol metabolites. The identification of phytosterols in human serum was based on comparison of their LC retention times and MS2, MS3 spectra with a library of authentic standards. Free campesterol serum concentration was in the range from 0.30-4.10 µg/mL, ß-sitosterol 0.16-3.37 µg/mL and fucosterol was at lowest concentration range from 0.05-0.38 µg/mL in ten individuals. This analytical methodology could be applied to the analysis of other biological fluids and tissues.

Membrane lipid remodeling eradicates Helicobacter pylori by manipulating the cholesteryl 6'-acylglucoside biosynthesis.

BACKGROUND: Helicobacter pylori, the main cause of various gastric diseases, infects approximately half of the human population. This pathogen is auxotrophic for cholesterol which it converts to various cholesteryl α-glucoside derivatives, including cholesteryl 6'-acyl α-glucoside (CAG). Since the related biosynthetic enzymes can be translocated to the host cells, the acyl chain of CAG likely comes from its precursor phosphatidylethanolamine (PE) in the host membranes. This work aims at examining how the acyl chain of CAG and PE inhibits the membrane functions, especially bacterial adhesion. METHODS: Eleven CAGs that differ in acyl chains were used to study the membrane properties of human gastric adenocarcinoma cells (AGS cells), including lipid rafts clustering (monitored by immunofluorescence with confocal microscopy) and lateral membrane fluidity (by the fluorescence recovery after photobleaching). Cell-based and mouse models were employed to study the degree of bacterial adhesion, the analyses of which were conducted by using flow cytometry and immunofluorescence staining, respectively. The lipidomes of H. pylori, AGS cells and H. pylori-AGS co-cultures were analyzed by Ultraperformance Liquid Chromatography-Tandem Mass Spectroscopy (UPLC-MS/MS) to examine the effect of PE(10:0)2, PE(18:0)2, PE(18:3)2, or PE(22:6)2 treatments. RESULTS: CAG10:0, CAG18:3 and CAG22:6 were found to cause the most adverse effect on the bacterial adhesion. Further LC-MS analysis indicated that the treatment of PE(10:0)2 resulted in dual effects to inhibit the bacterial adhesion, including the generation of CAG10:0 and significant changes in the membrane compositions. The initial (1 h) lipidome changes involved in the incorporation of 10:0 acyl chains into dihydro- and phytosphingosine derivatives and ceramides. In contrast, after 16 h, glycerophospholipids displayed obvious increase in their very long chain fatty acids, monounsaturated and polyunsaturated fatty acids that are considered to enhance membrane fluidity. CONCLUSIONS: The PE(10:0)2 treatment significantly reduced bacterial adhesion in both AGS cells and mouse models. Our approach of membrane remodeling has thus shown great promise as a new anti-H. pylori therapy.

Modulating Lipid Nanoparticles with Histidinamide-Conjugated Cholesterol for Improved Intracellular Delivery of mRNA.

Recently, mRNA-based therapeutics, including vaccines, have gained significant attention in the field of gene therapy for treating various diseases. Among the various mRNA delivery vehicles, lipid nanoparticles (LNPs) have emerged as promising vehicles for packaging and delivering mRNA with low immunogenicity. However, while mRNA delivery has several advantages, the delivery efficiency and stability of LNPs remain challenging for mRNA therapy. In this study, an ionizable helper cholesterol analog, 3ß[L-histidinamide-carbamoyl] cholesterol (Hchol) lipid is developed and incorporated into LNPs instead of cholesterol to enhance the LNP potency. The pKa values of the Hchol-LNPs are ≈6.03 and 6.61 in MC3- and SM102-based lipid formulations. Notably, the Hchol-LNPs significantly improve the delivery efficiency by enhancing the endosomal escape of mRNA. Additionally, the Hchol-LNPs are more effective in a red blood cell hemolysis at pH 5.5, indicating a synergistic effect of the protonated imidazole groups of Hchol and cholesterol on endosomal membrane destabilization. Furthermore, mRNA delivery is substantially enhanced in mice treated with Hchol-LNPs. Importantly, LNP-encapsulated SARS-CoV-2 spike mRNA vaccinations induce potent antigen-specific antibodies against SARS-CoV-2. Overall, incorporating Hchol into LNP formulations enables efficient endosomal escape and stability, leading to an mRNA delivery vehicle with a higher delivery efficiency.

Combining Metabolic Engineering and Lipid Droplet Assembly to Achieve Campesterol Overproduction in Saccharomyces cerevisiae.

Campesterol is a kind of important functional food additive. Therefore, stable and efficient campesterol biosynthesis is significant. Herein, we first knocked out the sterol 22-desaturase gene in Saccharomyces cerevisiae and expressed sterol Δ7-reductase from Pangasianodon hypophthalmus, obtaining a strain that produced 6.6 mg/L campesterol. Then, the modular expression of campesterol synthesis enzymes was performed, and a campesterol titer of 88.3 mg/L was achieved. Because campesterol is a lipid-soluble macromolecule, we promoted lipid droplet formation by exploring regulatory factors, and campesterol production was improved to 169.20 mg/L. Next, triacylglycerol lipase was used to achieve compartment campesterol synthesis. After enhancing the expression of sterol Δ7-reductase and screening cations, the campesterol titer reached 438.28 mg/L in a shake flask and 1.44 g/L in a 5 L bioreactor, which represents the highest campesterol titer reported to date. Metabolic regulation combined with lipid droplet engineering may be useful for the synthesis of other steroids as well.

Plasma campesterol and ABCG5/ABCG8 gene loci on the risk of cholelithiasis and cholecystitis: evidence from Mendelian randomization and colocalization analyses.

The causal relationships between plasma metabolites and cholelithiasis/cholecystitis risks remain elusive. Using two-sample Mendelian randomization, we found that genetic proxied plasma campesterol level showed negative correlation with the risk of both cholelithiasis and cholecystitis. Furthermore, the increased risk of cholelithiasis is correlating with the increased level of plasma campesterol. Lastly, genetic colocalization study showed that the leading SNP, rs4299376, which residing at the ABCG5/ABCG8 gene loci, was shared by plasma campesterol level and cholelithiasis, indicating that the aberrant transportation of plant sterol/cholesterol from the blood stream to the bile duct/gut lumen might be the key in preventing cholesterol gallstone formation.

Network pharmacology-integrated molecular docking analysis of phytocompounds of Caesalpinia pulcherrima (peacock flower) as potential anti-metastatic agents.

Caesalpinia pulcherrima, or peacock flower, has been a subject of cancer therapeutics research, showing promising anti-cancer and anti-metastatic properties. The present research aims to investigate the anti-metastatic potential of the flower, through bioinformatics approaches. Metastasis targets numbering 471 were identified through overlap analysis following NCBI gene, Gene Card and OMIM query. Phytocompounds of the flower were retrieved from PubChem and their protein interactions predicted using Super-PRED and TargetNet. The 28 targets that overlapped with the predicted proteins were used to generate STRING >0.7. Enrichment analysis revealed that C. pulcherrima may inhibit metastasis through angiogenesis-related and leukocyte migration-related pathways. HSP90AA1, ESR1, PIK3CA, ERBB2, KDR and MMP9 were identified as potential core targets while and 6 compounds (3-[(4-Hydroxyphenyl)methylidene]-7,8-dimethoxychromen-4-one (163076213), clotrimazole (2812), Isovouacapenol A (636673), [(4aR,5R,6aS,7R,11aS,11bR)-4a-hydroxy-4,4,7,11b-tetramethyl-9-oxo-1,2,3,5,6,6a,7,11a-octahydronaphtho[2,1-f][1]benzofuran-5-yl] benzoate (163104827), Stigmast-5-en-3beta-ol (86821) and 4,2'-dihydroxy-4'-methoxychalcone (592216)) were identified as potential core compounds. Molecular docking analysis and molecular dynamics simulations investigations revealed that ERBB2, HSP90AA1 and KDR, along with the newly discovered 163076213 compound to be the most significant metastasis targets and bioactive compound, respectively. These three core targets demonstrated interactions consistent with angiogenesis and leukocyte migration pathways. Furthermore, potentially novel interactions, such as KDR-MMP9, KDR-PIK3CA, ERBB2-HSP90AA1, ERBB2-ESR1, ERBB2-PIK3CA and ERBB2-MMP9 interactions were identified and may play a role in crosslinking the aforementioned metastatic pathways. Therefore, the present study revealed the main mechanisms behind the anti-metastatic effects of C. pulcherrima, paving the path for further research on these compounds and proteins to accelerate the research of cancer therapeutics and application of C. pulcherrima.Communicated by Ramaswamy H. Sarma.

Cholesterol Hydroperoxide Co-trafficking in Testosterone-generating Leydig Cells: GPx4 Inhibition of Cytotoxic and Anti-steroidogenic Effects.

Trafficking of intracellular cholesterol (Ch) to and into mitochondria of steroidogenic cells is required for steroid hormone biosynthesis. This trafficking is typically mediated by one or more proteins of the steroidogenic acute regulatory (StAR) family. Our previous studies revealed that 7-OOH, a redox-active cholesterol hydroperoxide, could be co-trafficked with Ch to/into mitochondria of MA-10 Leydig cells, thereby inducing membrane lipid peroxidation (LPO) which impaired progesterone biosynthesis. These negative effects of 7-OOH were inhibited by endogenous selenoperoxidase GPx4, indicating that this enzyme could protect against 7-OOH-induced oxidative damage/dysfunction. In the present study, we advanced our Leydig focus to cultured murine TM3 cells and then to primary cells from rat testis, both of which produce testosterone. Using a fluorescent probe, we found that extensive free radical-mediated LPO occurred in mitochondria of stimulated primary Leydig cells during treatment with liposomal Ch+7-OOH, resulting in a significant decline in testosterone output relative to that with Ch alone. Strong enhancement of LPO and testosterone shortfall by RSL3 (a GPx4 inhibitor) and reversal thereof by Ebselen (a GPx4 mimetic), suggested that endogenous GPx4 was playing a key antioxidant role. 7-OOH in increasing doses was also cytotoxic to these cells, RSL3 exacerbating this in Ebselen-reversable fashion. Moreover, GPx4 knockdown increased cell sensitivity to LPO with reduced testosterone output. These findings, particularly with primary Leydigs (which best represent cells in intact testis) suggest that GPx4 plays a key protective role against peroxidative damage/dysfunction induced by 7-OOH co-trafficking with Ch.

Methionine sulfoxide reductases and cholesterol transporter STARD3 constitute an efficient system for detoxification of cholesterol hydroperoxides.

Methionine sulfoxide reductases (MSRs) are key enzymes in the cellular oxidative defense system. Reactive oxygen species oxidize methionine residues to methionine sulfoxide, and the methionine sulfoxide reductases catalyze their reduction back to methionine. We previously identified the cholesterol transport protein STARD3 as an in vivo binding partner of MSRA (methionine sulfoxide reductase A), an enzyme that reduces methionine-S-sulfoxide back to methionine. We hypothesized that STARD3 would also bind the cytotoxic cholesterol hydroperoxides and that its two methionine residues, Met307 and Met427, could be oxidized, thus detoxifying cholesterol hydroperoxide. We now show that in addition to binding MSRA, STARD3 binds all three MSRB (methionine sulfoxide reductase B), enzymes that reduce methionine-R-sulfoxide back to methionine. Using pure 5, 6, and 7 positional isomers of cholesterol hydroperoxide, we found that both Met307 and Met427 on STARD3 are oxidized by 6α-hydroperoxy-3ß-hydroxycholest-4-ene (cholesterol-6α-hydroperoxide) and 7α-hydroperoxy-3ß-hydroxycholest-5-ene (cholesterol-7α-hydroperoxide). MSRs reduce the methionine sulfoxide back to methionine, restoring the ability of STARD3 to bind cholesterol. Thus, the cyclic oxidation and reduction of methionine residues in STARD3 provides a catalytically efficient mechanism to detoxify cholesterol hydroperoxide during cholesterol transport, protecting membrane contact sites and the entire cell against the toxicity of cholesterol hydroperoxide.

Plasma Campesterol Is Positively Associated with Carotid Plaques in Asymptomatic Subjects.

BACKGROUND: Increased cholesterol absorption and reduced synthesis are processes that have been associated with cardiovascular disease risk in a controversial way. However, most of the studies involving markers of cholesterol synthesis and absorption include conditions, such as obesity, diabetes, dyslipidemia, which can be confounding factors. The present study aimed at investigating the relationships of plasma cholesterol synthesis and absorption markers with cardiovascular disease (CVD) risk factors, cIMT (carotid intima-media thickness), and the presence of carotid plaques in asymptomatic subjects. METHODS: A cross-sectional study was carried out in 270 asymptomatic individuals and anthropometrical parameters, fasting plasma lipids, glucometabolic profiles, high-sensitivity C-reactive protein (hs-CRP), markers of cholesterol synthesis (desmosterol and lathosterol), absorption (campesterol and sitosterol), cIMT, and the presence of atherosclerotic plaques were analyzed. RESULTS: Among the selected subjects aged between 19 and 75 years, 51% were females. Age, body mass index, systolic and diastolic blood pressure, total cholesterol, non-HDL-C, triglycerides, glucose, and lathosterol/sitosterol ratios correlated positively with cIMT (p ≤ 0.05). Atherosclerotic plaques were present in 19% of the subjects. A direct association of carotid plaques with campesterol, OR = 1.71 (95% CI = 1.04-2.82, p ≤ 0.05) and inverse associations with both ratios lathosterol/campesterol, OR = 0.29 (CI = 0.11-0.80, p ≤ 0.05) and lathosterol/sitosterol, OR = 0.45 (CI = 0.22-0.95, p ≤ 0.05) were observed in univariate logistic regression analysis. CONCLUSIONS: The findings suggested that campesterol may be associated with atherosclerotic plaques and the lathosterol/campesterol or sitosterol ratios suggested an inverse association. Furthermore, synthesis and absorption of cholesterol are inverse processes, and the absorption marker, campesterol, may reflect changes in body cholesterol homeostasis with atherogenic potential.

Anti-steroidogenic effects of cholesterol hydroperoxide trafficking in MA-10 Leydig cells: Role of mitochondrial lipid peroxidation and inhibition thereof by selenoperoxidase GPx4.

Steroid hormone synthesis in steroidogenic cells requires cholesterol (Ch) delivery to/into mitochondria via StAR family trafficking proteins. In previous work, we discovered that 7-OOH, an oxidative stress-induced cholesterol hydroperoxide, can be co-trafficked with Ch, thereby causing mitochondrial redox damage/dysfunction. We now report that exposing MA-10 Leydig cells to Ch/7-OOH-containing liposomes (SUVs) results in (i) a progressive increase in fluorescence probe-detected lipid peroxidation in mitochondrial membranes, (ii) a reciprocal decrease in immunoassay-detected progesterone generation, and ultimately (iii) loss of cell viability with increasing 7-OOH concentration. No significant effects were observed with a phospholipid hydroperoxide over the same concentration range. Glutathione peroxidase GPx4, which can catalyze lipid hydroperoxide detoxification, was detected in mitochondria of MA-10 cells. Mitochondrial lipid peroxidation and progesterone shortfall were exacerbated when MA-10 cells were treated with Ch/7-OOH in the presence of RSL3, a GPx4 inhibitor. However, Ebselen, a selenoperoxidase mimetic, substantially reduced RSL3's negative effects, thereby partially rescuing the cells from peroxidative damage. These findings demonstrate that co-trafficking of oxidative stress-induced 7-OOH can disable steroidogenesis, and that GPx4 can significantly protect against this.

The edible seaweed Laminaria japonica contains cholesterol analogues that inhibit lipid peroxidation and cyclooxygenase enzymes.

In this study, 5 sterols were isolated and purified from Laminaria japonica, commonly known as edible brown seaweed, and their structures were identified based on detailed chemical methods and spectroscopic analyses. Spectroscopic analyses characterized 5 sterols as 29-Hydroperoxy-stigmasta-5,24(28)-dien-3ß-ol, saringosterol (24-vinyl-cholest-5-ene-3ß,24-diol), 24-methylenecholesterol, fucosterol (stigmasta-5,24-diene-3ß-ol), and 24-Hydroperoxy-24-vinyl-cholesterol. The bioactivities of these sterols were tested using lipid peroxidation (LPO) and cyclooxygenase (COX-1 and -2) enzyme inhibitory assays. Fucosterol exhibited the highest COX-1 and -2 enzyme inhibitory activities at 59 and 47%, respectively. Saringosterol, 24-methylenecholesterol and fucosterol showed higher LPO inhibitory activity at >50% than the other compounds. In addition, the results of molecular docking revealed that the 5 sterols were located in different pocket of COX-1 and -2 and fucosterol with tetracyclic skeletons and olefin methine achieved the highest binding energy (-7.85 and -9.02 kcal/mol) through hydrophobic interactions and hydrogen bond. Our results confirm the presence of 5 sterols in L. japonica and its significant anti-inflammatory and antioxidant activity.

Plasma oxyphytosterols most likely originate from hepatic oxidation and subsequent spill-over in the circulation.

We evaluated oxyphytosterol (OPS) concentrations in plasma and various tissues of two genetically modified mouse models with either increased cholesterol (apoE KO mice) or increased cholesterol and plant sterol (PS) concentrations (apoExABCG8 dKO mice). Sixteen female apoE KO and 16 dKO mice followed the same standard, low OPS-chow diet. Animals were euthanized at 36 weeks to measure PS and OPS concentrations in plasma, brain, liver and aortic tissue. Cholesterol and oxysterol (OS) concentrations were analyzed as reference for sterol oxidation in general. Plasma campesterol (24.1 ± 4.3 vs. 11.8 ± 3.0 mg/dL) and sitosterol (67.4 ± 12.7 vs. 4.9 ± 1.1 mg/dL) concentrations were severely elevated in the dKO compared to the apoE KO mice (p < 0.001). Also, in aortic and brain tissue, PS levels were significantly elevated in dKO. However, plasma, aortic and brain OPS concentrations were comparable or even lower in the dKO mice. In contrast, in liver tissue, both PS and OPS concentrations were severely elevated in the dKO compared to apoE KO mice (sum OPS: 7.4 ± 1.6 vs. 4.1 ± 0.8 ng/mg, p < 0.001). OS concentrations followed cholesterol concentrations in plasma and all tissues suggesting ubiquitous oxidation. Despite severely elevated PS concentrations, OPS concentrations were only elevated in liver tissue, suggesting that OPS are primarily formed in the liver and plasma concentrations originate from hepatic spill-over into the circulation.

Plant Sterol-Poor Diet Is Associated with Pro-Inflammatory Lipid Mediators in the Murine Brain.

Plant sterols (PSs) cannot be synthesized in mammals and are exclusively diet-derived. PSs cross the blood-brain barrier and may have anti-neuroinflammatory effects. Obesity is linked to lower intestinal uptake and blood levels of PSs, but its effects in terms of neuroinflammation-if any-remain unknown. We investigated the effect of high-fat diet-induced obesity on PSs in the brain and the effects of the PSs campesterol and ß-sitosterol on in vitro microglia activation. Sterols (cholesterol, precursors, PSs) and polyunsaturated fatty acid-derived lipid mediators were measured in the food, blood, liver and brain of C57BL/6J mice. Under a PSs-poor high-fat diet, PSs levels decreased in the blood, liver and brain (>50%). This effect was reversible after 2 weeks upon changing back to a chow diet. Inflammatory thromboxane B2 and prostaglandin D2 were inversely correlated to campesterol and ß-sitosterol levels in all brain regions. PSs content was determined post mortem in human cortex samples as well. In vitro, PSs accumulate in lipid rafts isolated from SIM-A9 microglia cell membranes. In summary, PSs levels in the blood, liver and brain were associated directly with PSs food content and inversely with BMI. PSs dampen pro-inflammatory lipid mediators in the brain. The identification of PSs in the human cortex in comparable concentration ranges implies the relevance of our findings for humans.

Spectrally and Time-Resolved Fluorescence Imaging of 22-NBD-Cholesterol in Human Peripheral Blood Mononuclear Cells in Chronic Kidney Disease Patients.

The interaction of the fluorescent probe 22-NBD-cholesterol with membranes of human peripheral blood mononuclear cells (PBMC) was tested by time- and spectrally resolved fluorescence imaging to monitor the disturbance of lipid metabolism in chronic kidney disease (CKD) and its treatment with statins. Blood samples from healthy volunteers (HV) and CKD patients, either treated or untreated with statins, were compared. Spectral imaging was done using confocal microscopy at 16 spectral channels in response to 458 nm excitation. Time-resolved imaging was achieved by time-correlated single photon counting (TCSPC) following excitation at 475 nm. The fluorescence of 22-NBD-cholesterol was mostly integrated into plasmatic membrane and/or intracellular membrane but was missing from the nuclear region. The presence of two distinct spectral forms of 22-NBD-cholesterol was uncovered, with significant variations between studied groups. In addition, two fluorescence lifetime components were unmasked, changing in CKD patients treated with statins. The gathered results indicate that 22-NBD-cholesterol may serve as a tool to study changes in the lipid metabolism of patients with CKD to monitor the effect of statin treatment.

Increasing Transfection Efficiency of Lipoplexes by Modulating Complexation Solution for Transient Gene Expression.

Transient gene expression is a suitable tool for the production of biopharmaceutical candidates in the early stage of development and provides a simple and rapid alternative to the generation of stable cell line. In this study, an efficient transient gene expression methodology using DC-Chol/DOPE cationic liposomes and pDNA in Chinese hamster ovary suspension cells was established through screening of diverse lipoplex formation conditions. We modulated properties of both the liposome formation and pDNA solution, together called complexation solutions. Protein expression and cellular cytotoxicity were evaluated following transfection over the cell cultivation period to select the optimal complexation solution. Changes in hydrodynamic size, polydispersity index, and ζ potential of the liposomes and lipoplexes were analyzed depending on the various pH ranges of the complexation solutions using dynamic light scattering. The transfer of lipoplexes to the cytosol and their conformation were traced using fluorescence analysis until the early period of transfection. As a result, up to 1785 mg/L and 191 mg/L of human Fc protein and immunoglobulin G (bevacizumab), respectively, were successfully produced using acidic liposome formation and alkaline pDNA solutions. We expect that this lipoplex formation in acidic and alkaline complexation solutions could be an effective methodology for a promising gene delivery strategy.

Enhanced enzymatic production of cholesteryl 6'-acylglucoside impairs lysosomal degradation for the intracellular survival of Helicobacter pylori.

BACKGROUND: During autophagy defense against invading microbes, certain lipid types are indispensable for generating specialized membrane-bound organelles. The lipid composition of autophagosomes remains obscure, as does the issue of how specific lipids and lipid-associated enzymes participate in autophagosome formation and maturation. Helicobacter pylori is auxotrophic for cholesterol and converts cholesterol to cholesteryl glucoside derivatives, including cholesteryl 6'-O-acyl-α-D-glucoside (CAG). We investigated how CAG and its biosynthetic acyltransferase assist H. pylori to escape host-cell autophagy. METHODS: We applied a metabolite-tagging method to obtain fluorophore-containing cholesteryl glucosides that were utilized to understand their intracellular locations. H. pylori 26695 and a cholesteryl glucosyltransferase (CGT)-deletion mutant (ΔCGT) were used as the standard strain and the negative control that contains no cholesterol-derived metabolites, respectively. Bacterial internalization and several autophagy-related assays were conducted to unravel the possible mechanism that H. pylori develops to hijack the host-cell autophagy response. Subcellular fractions of H. pylori-infected AGS cells were obtained and measured for the acyltransferase activity. RESULTS: The imaging studies of fluorophore-labeled cholesteryl glucosides pinpointed their intracellular localization in AGS cells. The result indicated that CAG enhances the internalization of H. pylori in AGS cells. Particularly, CAG, instead of CG and CPG, is able to augment the autophagy response induced by H. pylori. How CAG participates in the autophagy process is multifaceted. CAG was found to intervene in the degradation of autophagosomes and reduce lysosomal biogenesis, supporting the idea that intracellular H. pylori is harbored by autophago-lysosomes in favor of the bacterial survival. Furthermore, we performed the enzyme activity assay of subcellular fractions of H. pylori-infected AGS cells. The analysis showed that the acyltransferase is mainly distributed in autophago-lysosomal compartments. CONCLUSIONS: Our results support the idea that the acyltransferase is mainly distributed in the subcellular compartment consisting of autophagosomes, late endosomes, and lysosomes, in which the acidic environment is beneficial for the maximal acyltransferase activity. The resulting elevated level of CAG can facilitate bacterial internalization, interfere with the autophagy flux, and causes reduced lysosomal biogenesis.

Synthesis and biological evaluation of cationic TopFluor cholesterol analogues.

Cholesterol is not only a major component of the cell membrane, but also plays an important role in a wide range of biological processes and pathologies. It is therefore crucial to develop appropriate tools for visualizing intracellular cholesterol transport. Here, we describe new cationic analogues of BODIPY-Cholesterol (TopFluor-Cholesterol, TF-Chol), which combine a positive charge on the sterol side chain and a BODIPY group connected via a C-4 linker. In contrast to TF-Chol, the new analogues TF-1 and TF-3 possessing acetyl groups on the A ring (C-3 position on steroid) internalized much faster and displayed slightly different levels of intracellular localization. Their applicability for cholesterol monitoring was indicated by the fact that they strongly label compartments with accumulated cholesterol in cells carrying a mutation of the Niemann-Pick disease-associated cholesterol transporter, NPC1.