Multi-Omics Reveals the Role of Arachidonic Acid Metabolism in the Gut-Follicle Axis for the Antral Follicular Development of Holstein Cows.

In vitro embryonic technology is crucial for improving farm animal reproduction but is hampered by the poor quality of oocytes and insufficient development potential. This study investigated the relationships among changes in the gut microbiota and metabolism, serum features, and the follicular fluid metabolome atlas. Correlation network maps were constructed to reveal how the metabolites affect follicular development by regulating gene expression in granulosa cells. The superovulation synchronization results showed that the number of follicle diameters from 4 to 8 mm, qualified oocyte number, cleavage, and blastocyst rates were improved in the dairy heifers (DH) compared with the non-lactating multiparous dairy cows (NDC) groups. The gut microbiota was decreased in Rikenellaceae_RC9_gut_group, Alistipes, and Bifidobacterium, but increased in Firmicutes, Cyanobacteria, Fibrobacterota, Desulfobacterota, and Verrucomicrobiota in the NDC group, which was highly associated with phospholipid-related metabolites of gut microbiota and serum. Metabolomic profiling of the gut microbiota, serum, and follicular fluid further demonstrated that the co-metabolites were phosphocholine and linoleic acid. Moreover, the expression of genes related to arachidonic acid metabolism in granulosa cells was significantly correlated with phosphocholine and linoleic acid. The results in granulosa cells showed that the levels of PLCB1 and COX2, participating in arachidonic acid metabolism, were increased in the DH group, which improved the concentrations of PGD2 and PGF2α in the follicular fluid. Finally, the expression levels of apoptosis-related proteins, cytokines, and steroidogenesis-related genes in granulosa cells and the concentrations of steroid hormones in follicular fluid were determinants of follicular development. According to our results, gut microbiota-related phosphocholine and linoleic acid participate in arachidonic acid metabolism in granulosa cells through the gut-follicle axis, which regulates follicular development. These findings hold promise for enhancing follicular development and optimizing oocyte quality in subfertile dairy cows.

Metabolic Perturbations associated with hIAPP-induced insulin resistance in Skeletal Muscles: Implications to the Development of Type 2 Diabetes.

The human islet amyloid polypeptide (hIAPP) tends to misfold and self-assemble to form amyloid fibrils, which has been associated with the loss of function and viability of pancreatic ß-cells in type 2 diabetes mellitus (T2DM). The role of hIAPP in the development of insulin resistance (a hallmark of T2DM) in skeletal muscles - the major sites for glucose utilization - needs further investigation. Even though, insulin-resistant conditions have been known to stimulate hIAPP aggregation, the events that lead to the development of insulin resistance due to hIAPP aggregation in skeletal muscles remain unidentified. Here, we have attempted to identify metabolic perturbations in L6 myotubes that were exposed to increasing concentrations of recombinant hIAPP for different time durations. It was observed that hIAPP exposure was associated with increased mitochondrial and cellular ROS levels, loss in mitochondrial membrane potential and viability of the myotubes. Metabolomic investigations of hIAPP-treated myotubes revealed significant perturbations in o-phosphocholine, sn-glycero-3-phosphocholine and dimethylamine levels (p < 0.05). Therefore, we anticipate that defects in glycerophospholipid metabolism and the associated oxidative stress and membrane damage may play key roles in the development of insulin resistance due to protein misfolding in skeletal muscles. In summary, the perturbed metabolites and their pathways have not only the potential to be used as early biomarkers to predict the onset of insulin resistance and T2DM but also as therapeutic targets for the effective management of the same.

Non-specific phospholipase C3 is involved in endoplasmic reticulum stress tolerance in Arabidopsis.

Non-specific phospholipase C (NPC) is an emerging family of lipolytic enzymes unique to plants and bacteria that play crucial roles in growth and stress responses. Among six copies of NPC isoforms found in Arabidopsis, the role of NPC3 remains elusive to date. Here, we show that NPC3 is a functional non-specific phospholipase C involved in tolerance to tunicamycin (TM)-induced endoplasmic reticulum (ER) stress through the synthesis of phosphocholine (PCho), a reaction product of NPC3. The npc3 mutant exhibited reduced sensitivity to TM treatment. Recombinant NPC3 possessed pronounced phospholipase C activity that hydrolyses phosphatidylcholine (PC). The hyposensitivity of npc3 to TM treatment was complemented by exogenous PCho, suggesting that NPC3-catalysed PCho production is involved in TM-induced ER stress tolerance. NPC3 was localized at the ER and was predominantly expressed in the roots, and it was further induced by TM-induced ER stress. Intriguingly, npc3 mutants showed a markedly reduced PCho content in shoots under ER stress. Our results indicate that ER stress induces NPC3 to produce PCho, which is involved in TM-induced ER stress tolerance.

Uncoated gold nanoparticles create fewer and less localized defects in model prokaryotic than in model eukaryotic lipid membranes.

The rise of the populations of antibiotic resistant bacteria represents an increasing threat to human health. In addition to the synthesis of new antibiotics, which is an extremely expensive and time-consuming process, one of the ways to combat bacterial infections is the use of gold nanoparticles (Au NPs) as the vehicles for targeted delivery of therapeutic drugs. Since such a strategy requires the investigation of the effect of Au NPs (with and without drugs) on both bacterial and human cells, we investigated how the presence of coating-free Au NPs affects the physicochemical properties of lipid membranes that model prokaryotic (PRO) and eukaryotic (EU) cells. PRO/EU systems prepared as multilamellar liposomes (MLVs) and hybrid structures (HSs) from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphatidylglycerol (DPPG)/1,2-dipalmitoyl-sn-glycero-3-phosphoserine (DPPS) in the absence (MLVs)/presence (HSs) of differently distributed Au NPs (sizes ∼20 nm) reported stabilization of the gel phase of PRO systems in comparison with EU one (DSC data of PRO/EU were Tm(MLVs) ≈ 41.8 °C/42.0 °C, Tm¯ (HSs) ≈ 43.1 °C/42.4 °C, whereas UV-Vis response Tm(MLVs) ≈ 41.5 °C/42.0 °C, Tm¯ (HSs) ≈ 42.9 °C/41.1 °C). Vibrational spectroscopic data unraveled a substantial impact of Au NPs on the non-polar part of lipid bilayers, emphasizing the increase of kink and gauche conformers of the hydrocarbon chain. By interpreting the latter as Au NPs-induced defects, which exert the greatest effect when Au NPs are found exclusively outside the lipid membrane, these findings suggested that Au NPs reduced the compactness of EU-based lipid bilayers much more than in analogous PRO systems. Since the uncoated Au NPs manifested adverse effects when applied as antimicrobials, the results obtained in this work contribute towards recognizing AuNP functionalization as a strategy in tuning and reversing this effect.

High-purity 1,2-dimyristoyl-sn-glycero-3-phosphocholine: synthesis and emulsifying performance evaluation.

Introduction: 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) is a promising emulsifier for bioactive delivery systems, but its industrial applications are limited by the lack of cost-effective and scalable synthetic routes. The purpose of this study was to economically produce high-purity DMPC by replacing commonly used column chromatography methods and to evaluate the emulsifying performance. Methods: DMPC was synthesized from sn-glycero-3-phosphocholine using Steglich esterification followed by sequential recrystallization from ethyl acetate and acetone. The structure of DMPC was identified and its purity was confirmed using various spectroscopy and chromatography techniques. The emulsifying performance was evaluated by examining the effects of storage on the properties of o/w emulsions prepared using soybean oil with (i) soy PC, (ii) soy PC + DMPC (1:1, w/w), and (iii) DMPC as emulsifiers. Results: The chemical impurities formed during the synthesis of DMPC was removed, and its final purity was 96%, and the melt transition temperature was 37.6°C. No visible difference between the three emulsions (soy PC, soy PC+DMPC, and DMPC) was observed during two-week storage, and the DMPC-based emulsion was more stable than soy PC emulsion, showing smaller particle size distribution during 6 months. Discussion: The highly pure DMPC was synthesized by an economical method, and DMPC-based emulsions demonstrated physicochemical stable, highlighting its potential for food and pharmaceutical industry-related applications. Our findings suggest that DMPC holds promise as an emulsifier with broad applications in the food industry.

Protonation of palmitic acid embedded in DPPC lipid bilayers obscures detection of ripple phase by FTIR spectroscopy.

The transformation of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) lipid bilayers from the gel (Lß') to the fluid (Lα) phase involves an intermediate ripple (Pß') phase forming a few degrees below the main transition temperature (Tm). While the exact cause of bilayer rippling is still debated, the presence of amphiphilic molecules, pH, and lipid bilayer architecture are all known to influence (pre)transition behavior. In particular, fatty acid chains interact with hydrophobic lipid tails, while the carboxylic groups simultaneously participate in proton transfer with interfacial water in the polar lipid region which is controlled by the pH of the surrounding aqueous medium. The molecular-level variations in the DPPC ripple phase in the presence of 2% palmitic acid (PA) were studied at pH levels 4.0, 7.3, and 9.1, where PA is fully protonated, partially protonated, or fully deprotonated. Bilayer thermotropic behavior was investigated by differential scanning calorimetry (DSC) and Fourier-transform infrared (FTIR) spectroscopy which agreed in their characterization of (pre)transition at pH of 9.1, but not at pH 4.0 and especially not at 7.3. Owing to the different insertion depths of protonated and deprotonated PA, along with the ability of protonated PA to undergo flip-flop in the bilayer, these two forms of PA show a different hydration pattern in the interfacial water layer. Finally, these results demonstrated the hitherto undiscovered potential of FTIR spectroscopy in the detection of the events occurring at the surface of lipid bilayers that obscure the low-cooperativity phase transition explored in this work.

Phosphatidylcholine head group chemistry alters the extrahepatic accumulation of lipid-conjugated siRNA.

Small interfering RNAs (siRNAs) are revolutionizing the treatment of liver-associated indications. Yet, robust delivery to extrahepatic tissues remains a challenge. Conjugating lipids (e.g., docosanoic acid [DCA]) to siRNA supports extrahepatic delivery, but tissue accumulation remains lower than that achieved in liver by approved siRNA therapeutics. Early evidence suggests that functionalizing DCA with a head group (e.g., phosphatidylcholine [PC]) may enhance delivery to certain tissues. Here, we report the first systematic evaluation of the effect of PC head group chemistry on the extrahepatic distribution of DCA-conjugated siRNAs. We show that functionalizing DCA with a PC head group enhances siRNA accumulation in heart, muscle, lung, pancreas, duodenum, urinary bladder, and fat. Varying the size of the linker between the phosphate and choline moiety of the PC head group altered the extrahepatic accumulation of siRNA, with the optimal linker length being different for different tissues. Increasing PC head group valency also improved extrahepatic accumulation in a tissue-specific manner. This study demonstrates the structural impact of the PC moiety on the biodistribution of lipid-conjugated siRNA and introduces multiple novel PC variants for the chemical optimization of DCA-conjugated siRNA. These chemical variants can be used in the context of other lipids to increase the repertoire of conjugates for the extrahepatic distribution of siRNAs.

Cross-species metabolomic profiling reveals phosphocholine-mediated liver protection from cold and ischemia/reperfusion.

Cold and ischemia/reperfusion (IR)-associated injuries are seemingly inevitable during liver transplantation and hepatectomy. Because Syrian hamsters demonstrate intrinsic tolerance to transplantation-like stimuli, cross-species comparative metabolomic analyses were conducted with hamster, rat, and donor liver samples to seek hepatic cold and IR-adaptive mechanisms. Lower hepatic phosphocholine contents were found in recipients with early graft-dysfunction and with virus-caused cirrhosis or high model for end-stage liver disease scores (≥30). Choline/phosphocholine deficiency in cultured human THLE-2 hepatocytes and animal models weakened hepatocellular cold tolerance and recovery of glutathione and ATP production, which was rescued by phosphocholine supplements. Among the biological processes impacted by choline/phosphocholine deficiency, 3 lipid-related metabolic processes were downregulated, whereas phosphocholine elevated the expression of genes in methylation processes. Consistently, in THLE-2, phosphocholine enhanced the overall RNA m6A methylation, among which the transcript stability of fatty acid desaturase 6 (FADS6) was improved. FADS6 functioned as a key phosphocholine effector in the production of polyunsaturated fatty acids, which may facilitate the hepatocellular recovery of energy and redox homeostasis. Thus, our study reveals the choline-phosphocholine metabolism and its downstream FADS6 functions in hepatic adaptation to cold and IR, which may inspire new strategies to monitor donor liver quality and improve recipient recovery from the liver transplantation process.

Identification and Quantification of a Pneumococcal Cell Wall Polysaccharide by Antibody-Enhanced Chromatography Assay.

Multivalent pneumococcal vaccines have been developed successfully to combat invasive pneumococcal diseases (IPD) and reduce the associated healthcare burden. These vaccines employ pneumococcal capsular polysaccharides (PnPs), either conjugated or unconjugated, as antigens to provide serotype-specific protection. Pneumococcal capsular polysaccharides used for vaccine often contain residual levels of cell wall polysaccharides (C-Ps), which can generate a non-serotype specific immune response and complicate the desired serotype-specific immunity. Therefore, the C-P level in a pneumococcal vaccine needs to be controlled in the vaccine process and the anti C-P responses need to be dialed out in clinical assays. Currently, two types of cell-wall polysaccharide structures have been identified: a mono-phosphocholine substituted cell-wall polysaccharide C-Ps1 and a di-phosphocholine substituted C-Ps2 structure. In our effort to develop a next-generation novel pneumococcal conjugate vaccine (PCV), we have generated a monoclonal antibody (mAb) specific to cell-wall polysaccharide C-Ps2 structure. An antibody-enhanced HPLC assay (AE-HPLC) has been established for serotype-specific quantification of pneumococcal polysaccharides in our lab. With the new anti C-Ps2 mAb, we herein extend the AE-HPLC assay to the quantification and identification of C-Ps2 species in pneumococcal polysaccharides used for vaccines.

Glutathione and a Pool of Metabolites Partly Related to Oxidative Stress Are Associated with Low and High Myopia in an Altered Bioenergetic Environment.

Oxidative stress forms part of the molecular basis contributing to the development and manifestation of myopia, a refractive error with associated pathology that is increasingly prevalent worldwide and that subsequently leads to an upsurge in degenerative visual impairment due to conditions that are especially associated with high myopia. The purpose of our study was to examine the interrelation of potential oxidative-stress-related metabolites found in the aqueous humor of high-myopic, low-myopic, and non-myopic patients within a clinical study. We conducted a cross-sectional study, selecting two sets of patients undergoing cataract surgery. The first set, which was used to analyze metabolites through an NMR assay, comprised 116 patients. A total of 59 metabolites were assigned and quantified. The PLS-DA score plot clearly showed a separation with minimal overlap between the HM and control samples. The PLS-DA model allowed us to determine 31 major metabolite differences in the aqueous humor of the study groups. Complementary statistical analysis of the data allowed us to determine six metabolites that presented significant differences among the experimental groups (p < 005). A significant number of these metabolites were discovered to have a direct or indirect connection to oxidative stress linked with conditions of myopic eyes. Notably, we identified metabolites associated with bioenergetic pathways and metabolites that have undergone methylation, along with choline and its derivatives. The second set consisted of 73 patients who underwent a glutathione assay. Here, we showed significant variations in both reduced and oxidized glutathione in aqueous humor among all patient groups (p < 0.01) for the first time. Axial length, refractive status, and complete ophthalmologic examination were also recorded, and interrelations among metabolic and clinical parameters were evaluated.

Phosphocholine-induced energy source shift alleviates mitochondrial dysfunction in lung cells caused by geospecific PM2.5 components.

Fine particulate matter (PM2.5) is globally recognized for its adverse implications on human health. Yet, remain limited the individual contribution of particular PM2.5 components to its toxicity, especially considering regional disparities. Moreover, prevention solutions for PM2.5-associated health effects are scarce. In the present study, we comprehensively characterized and compared the primary PM2.5 constituents and their altered metabolites from two locations: Taiyuan and Guangzhou. Analysis of year-long PM2.5 samples revealed 84 major components, encompassing organic carbon, elemental carbon, ions, metals, and organic chemicals. PM2.5 from Taiyuan exhibited higher contamination, associated health risks, dithiothreitol activity, and cytotoxicities than Guangzhou's counterpart. Applying metabolomics, BEAS-2B lung cells exposed to PM2.5 from both cities were screened for significant alterations. A correlation analysis revealed the metabolites altered by PM2.5 and the critical toxic PM2.5 components in both regions. Among the PM2.5-down-regulated metabolites, phosphocholine emerged as a promising intervention for PM2.5 cytotoxicities. Its supplementation effectively attenuated PM2.5-induced energy metabolism disorder and cell death via activating fatty acid oxidation and inhibiting Phospho1 expression. The highlighted toxic chemicals displayed combined toxicities, potentially counteracted by phosphocholine. Our study offered a promising functional metabolite to alleviate PM2.5-induced cellular disorder and provided insights into the geo-based variability in toxic PM2.5 components.

Identification of potential serum biomarkers associated with HbA1c levels in Indian type 2 diabetic subjects using NMR-based metabolomics.

BACKGROUND: The prevalence of type 2 diabetes mellitus (T2DM), a progressive metabolic disorder characterized by chronic hyperglycemia and the development of insulin resistance, has increased globally, with worrying statistics coming from children, adolescents, and young adults from developing countries like India. Here, we investigated unique circulating metabolic signatures associated with prediabetes and T2DM in an Indian cohort using NMR-based metabolomics. MATERIALS AND METHODS: The study subjects included healthy volunteers (N = 101), prediabetic subjects (N = 75), and T2DM patients (N = 108). Serum metabolic profiling was performed using 1H NMR spectroscopy and major perturbed metabolites were identified by multivariate analysis and receiver operating characteristic (ROC) modules. RESULTS: Of the 36 aqueous abundant metabolites, 24 showed a statistically significant difference between healthy volunteers, prediabetics, and established T2DM subjects. On performing multivariate ROC curve analysis with 5 commonly dysregulated metabolites (namely, glucose, pyroglutamate, o-phosphocholine, serine, and methionine) in prediabetes and T2DM, AUC values obtained were 0.96 (95 % confidence interval (CI) = 0.93, 0.98) for T2DM; and 0.88 (95 % CI = 0.81, 0.93) for prediabetic subjects, respectively. CONCLUSION: We propose that the identified metabolite panel can be used in the future as a biomarker for clinical diagnosis, patient surveillance, and for predicting individuals at risk for developing diabetes.

Phase-Dependent Adsorption of Myelin Basic Protein to Phosphatidylcholine Lipid Bilayers.

The dense packing of opposite cytoplasmic surfaces of the lipid-enriched myelin membrane, responsible for the proper saltatory conduction of nerve impulses through axons, is ensured by the adhesive properties of myelin basic protein (MBP). Although preferentially interacting with negatively charged phosphatidylserine (PS) lipids, as an intrinsically disordered protein, it can easily adapt its shape to its immediate environment and thus adsorb to domains made of zwitterionic phosphatidylcholine (PC) lipids. As the molecular-level interaction pattern between MBP and PC lipid membranes suffers from scarce characterization, an experimental and computational study of multilamellar liposomes (MLVs) composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in the presence of bovine MBP is presented here. Calorimetric and temperature-dependent UV-Vis measurements identified DPPC pretransition temperature (Tp) and calorimetric enthalpy (ΔHcal) as the physicochemical parameters most responsive to the presence of MBP. Besides suggesting an increase in ß-sheet fractions of structured MBP segments as DPPC lipids undergo from the gel (20 °C) to the fluid (50 °C) phase, FTIR spectra unraveled the significant contribution of lysine (Lys) residues in the adsorption pattern, especially when DPPC is in the fluid (50 °C) phase. In addition to highlighting the importance of Lys residues in the MBP adsorption on DPPC lipid bilayer, employing salt bridges (SBs) and hydrogen bonds (HBs), MD data suggest the crucial importance of the orientation of MBP with respect to the surface of the DPPC lipid bilayer.

The presence of uncoated gold nanoparticle aggregates may alter the phase of phosphatidylcholine lipid as evidenced by vibrational spectroscopies.

Spherical structures built from uni- and multilamellar lipid bilayers (LUV and MLV) are nowadays considered not just as nanocarriers of various kinds of therapeutics, but also as the vehicles that, when coupled with gold (Au) nanoparticles (NPs), can also serve as a tool for imaging and discriminating healthy and diseased tissues. Since the presence of Au NPs or their aggregates may affect the properties of the drug delivery vehicle, we investigated how the shape and position of Au NP aggregates adsorbed on the surface of MLV affect the arrangement and conformation of lipid molecules. By preparing MLVs constituted from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in the presence of uncoated Au NP aggregates found i) both within liposome core and on the surface of the outer lipid bilayer, or ii) adsorbed on the outer lipid bilayer surface only, we demonstrated the maintenance of lipid bilayer integrity by microscopic techniques (cryo-TEM, and AFM). The employment of SERS and FTIR-ATR techniques enabled us not only to elucidate the lipid interaction pattern and their orientation in regards to Au NP aggregates but also unequivocally confirmed the impact of Au NP aggregates on the persistence/breaking of van der Waals interactions between hydrocarbon chains of DPPC.

Coupling capillary electrophoresis with mass spectrometry for the analysis of oxidized phospholipids in human high-density lipoproteins.

The oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (ox-PAPC) products in human high-density lipoproteins (HDLs) were investigated by low-flow capillary electrophoresis-mass spectrometry (low-flow CE-MS). To accelerate the optimization, native PAPC (n-PAPC) standard was first analyzed by a commercial CE instrument with a photodiode array detector. The optimal separation buffer contained 60% (v/v) acetonitrile, 40% (v/v) methanol, 20 mM ammonium acetate, 0.5% (v/v) formic acid, and 0.1% (v/v) water. The selected separation voltage and capillary temperature were 20 kV and 23°C. The optimal CE separation buffer was then used for the low-flow CE-MS analysis. The selected MS conditions contained heated capillary temperature (250°C), capillary voltage (10 V), and injection time (1 s). No sheath gas was used for MS. The linear range for n-PAPC was 2.5-100.0 µg/mL. The coefficient of determination (R2 ) was 0.9918. The concentration limit of detection was 1.52 µg/mL, and the concentration limit of quantitation was 4.60 µg/mL. The optimal low-flow CE-MS method showed good repeatability and sensitivity. The ox-PAPC products in human HDLs were determined based on the in vitro ox-PAPC products of n-PAPC standard. Twenty-one ox-PAPC products have been analyzed in human HDLs. Uremic patients showed significantly higher levels of 15 ox-PAPC products than healthy subjects.

Biocompatibility and osteogenic potential of choline phosphate chitosan-coated biodegradable Zn1Mg.

Zinc alloys have demonstrated considerable potentials as implant materials for biodegradable vascular and orthopedic applications. However, the high initial release of Zn2+ can trigger intense immune responses that impede tissue healing. To address this challenge and enhance the osteogenic capacity of zinc alloys, the surface of Zn1Mg was subjected to CO2 plasma modification (Zn1Mg-PP) followed by grafting with choline phosphate chitosan (Zn1Mg-PP-PCCs). This study aims to investigate the in vitro and in vivo biocompatibility of the surface-modified Zn1Mg. The effect of the surface modification on the inflammatory response and osteogenic repair process was investigated. Compared with unmodified Zn1Mg, the degradation rate of Zn1Mg-PP-PCCs was significantly decreased, avoiding the cytotoxicity triggered by the release of large amounts of Zn2+. Moreover, PCCs significantly enhanced the cell-material adhesion, promoted the proliferation of osteoblasts (MC3T3-E1) and upregulated the expression of key osteogenic factors in vitro. Notably, the in vivo experiments revealed that the surface modification of Zn1Mg suppressed inhibited the expression of inflammatory cytokines, promoting the secretion of anti-inflammatory factors, thereby reducing inflammation and promoting bone tissue repair. Furthermore, histological analysis of tissue sections exhibited strong integration between the material and the bone, along with well-defined new bone formation and reduced osteoclast aggregation on the surface. This was attributed to the improved immune microenvironment by PCCs, which promoted osteogenic differentiation of osteoblasts. These findings highlight that the preparation of PCCs coatings on zinc alloy surfaces effectively inhibited ion release and modulated the immune environment to promote bone tissue repair. STATEMENT OF SIGNIFICANCE: Surface modification of biodegradable Zn alloys facilitates the suppression of intense immune responses caused by excessive ion release concentrations from implants. We modified the surface of Zn1Mg with choline phosphate chitosan (PCCs) and investigated the effects of surface modification on the inflammatory response and osteogenic repair process. In vitro results showed that the PCCs coating effectively reduced the degradation rate of Zn1Mg to avoid cytotoxicity caused by high Zn2+ concentration, favoring the proliferation of osteoblasts. In addition, in vivo results indicated that Zn1Mg-PP-PCCs attenuated inflammation to promote bone repair by modulating the release of inflammation-related factors. The surface-modified Zn1Mg implants demonstrated strong osseointegration, indicating that the PCCs coating effectively modulated the immune microenvironment and promoted bone healing.

Cholesterol-Enriched Hybrid Lipid Bilayer Formation on Inverse Phosphocholine Lipid-Functionalized Titanium Oxide Surfaces.

Hybrid lipid bilayers (HLBs) are rugged biomimetic cell membrane interfaces that can form on inorganic surfaces and be designed to contain biologically important components like cholesterol. In general, HLBs are formed by depositing phospholipids on top of a hydrophobic self-assembled monolayer (SAM) composed of one-tail amphiphiles, while recent findings have shown that two-tail amphiphiles such as inverse phosphocholine (CP) lipids can have advantageous properties to promote zwitterionic HLB formation. Herein, we explored the feasibility of fabricating cholesterol-enriched HLBs on CP SAM-functionalized TiO2 surfaces with the solvent exchange and vesicle fusion methods. All stages of the HLB fabrication process were tracked by quartz crystal microbalance-dissipation (QCM-D) measurements and revealed important differences in fabrication outcome depending on the chosen method. With the solvent exchange method, it was possible to fabricate HLBs with well-controlled cholesterol fractions up to ~65 mol% in the upper leaflet as confirmed by a methyl-ß-cyclodextrin (MßCD) extraction assay. In marked contrast, the vesicle fusion method was only effective at forming HLBs from precursor vesicles containing up to ~35 mol% cholesterol, but this performance was still superior to past results on hydrophilic SiO2. We discuss the contributing factors to the different efficiencies of the two methods as well as the general utility of two-tail CP SAMs as favorable interfaces to incorporate cholesterol into HLBs. Accordingly, our findings support that the solvent exchange method is a versatile tool to fabricate cholesterol-enriched HLBs on CP SAM-functionalized TiO2 surfaces.

Therapeutic role of 2-stearoxyphenethyl phosphocholine targeting microtubule dynamics and Wnt/ß-catenin/EMT signaling in human colorectal cancer cells.

The inhibition of cell death, perturbation of microtubule dynamics, and acceleration of Wnt/ß-catenin/epithelial-mesenchymal transition (EMT) signaling are fundamental processes in the progression and metastasis of colorectal cancer (CRC). To explore the role of 2-stearoxyphenethyl phosphocholine (stPEPC), an alkylphospholipid-based compound, in CRC, we conducted an MTT assay, cell cycle analysis, western blot analysis, immunoprecipitation, immunofluorescence staining, Annexin V/propidium iodide double staining, small interfering RNA gene silencing, a wound-healing assay, an invasion assay, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay in the human CRC cell lines HT29 and HCT116. stPEPC showed anti-proliferative properties and mitotic cell accumulation via upregulated phosphorylation of BUBR1 and an association between mitotic arrest deficiency 2 (MAD2) and cell division cycle protein 20 homolog (CDC20). These results suggest that activation of the mitotic checkpoint complex and tubulin polymerization occurred, resulting in mitotic catastrophe in HT29 and HCT116 cells. In addition, stPEPC attenuated cell migration and invasion by regulating proteins mediated by EMT, such as E-cadherin and occludin. stPEPC altered the protein expression of Wnt3a and phosphorylation of low-density lipoprotein receptor-related protein 6 (LRP6), glycogen synthase kinase 3ß (GSK3ß), and ß-catenin as well as their target genes, including cMyc and cyclin D1, in CRC cells. Thus, stPEPC may be useful for developing new drugs to treat human CRC.

Circulating choline and phosphocholine measurement by a hydrophilic interaction liquid chromatography-tandem mass spectrometry.

Background: Given the growing interest in studying the role of choline and phosphocholine in the development and progression of tumor pathology, in this study we describe the development and validation of a fast and robust method for the simultaneous analysis of choline and phosphocholine in human plasma. Methods: Choline and phosphocholine quantification in human plasma was obtained using a hydrophilic interaction liquid chromatography-tandem mass spectrometry technique. Assay performance parameters were evaluated using EMA guidelines. Results: Calibration curve ranged from 0.60 to 38.40 µmol/L (R2 = 0.999) and 0.08-5.43 µmol/L (R2 = 0.998) for choline and phosphocholine, respectively. The Limit Of Detection of the method was 0.06 µmol/L for choline and 0.04 µmol/L for phosphocholine. The coefficient of variation range for intra-assay precision is 2.2-4.1 % (choline) and 3.2-15 % (phosphocholine), and the inter-assay precision range is < 1-6.5 % (choline) and 6.2-20 % (phosphocholine). The accuracy of the method was below the ±20 % benchmarks at all the metabolites concentration levels. In-house plasma pool of apparently healthy adults was tested, and a mean concentration of 15.97 µmol/L for Choline and 0.34 µmol/L for Phosphocholine was quantified. Conclusions: The developed method shows good reliability in quantifying Choline and Phosphocholine in human plasma for clinical purposes.

The Equilibria of Triterpene Sapogenins-Phosphatidylcholine in Monolayers at the Air/Water Interface.

Sapogenins are the non-sugar parts of saponins (aglycones), high-molecular-weight glycosides linked to one or more sugar side chains. This group of compounds presents many properties, e.g., the potent properties of reducing surface tension and foaming properties, as evidenced by the amphipathic nature of these substances. They are used in the cosmetics industry, the washing and detergent industry, and the food industry. In addition, they have many healing properties. They lower blood cholesterol but are also used to synthesize steroid drugs or hormones. As reported in the literature, saponins also show antitumor activity, leading to cell cycle inhibition and apoptosis of various neoplastic cells. In this study, the influence of two sapogenins: asiatic acid (AA) and oleanolic acid (OA), on the properties of monolayers made of phosphatidylcholine (DPPC) was investigated. The method used in these studies was the Langmuir method with Brewster angle microscopy. The interactions between the tested compounds in mixed monolayers were described. Using mathematical equations, we established that oleanolic acid and asiatic acid formed complexes with DPPC at 1:1 ratios, characterized by high stability constants. We derived the parameters characterizing the formed complexes and described the phase transitions that occur during the formation of pure and mixed monolayers.