Phospholipids Phosphatidylcholine Research Articles

Formulation and validation on UV spectroscopy of Herbosomes loaded Mahonia aquifolium cream for psoriasis

Herbosome technology enhances the bioavailability of herbal excerpts. Herbosome act as the ground between the new delivery system and conventional delivery system. It's a complex fashion applied to phytopharmaceuticals for the improvement of bioavailability of herbal excerpt for medicinal operations. This composition overviews about herbosome technology, recent advance, their operation for colorful standardized herbal excerpts and aims to give complete of natural active constituents and phospholipid (phosphatidylcholine, phosphatidylserine etc.) which increases immersion of herbal excerpt. Herbosome is the new arising scientific information, characterization about herbosomes as a promising drug delivery system. Psoriasis is an inflammatory skin condition characterized by scaling with inflammation (pain, edema, warmth, and redness) that results in regions of thick, red skin covered in silvery scales. These spots can be itchy or painful. Systemic treatment, topical therapy, and phototherapy are all now used to treat psoriasis. These treatments have a variety of negative and perhaps fatal side effects. Patients with psoriasis are more likely to acquire other conditions such as psoriatic arthritis, anxiety and depression, cancer, metabolic syndrome, cardiovascular disease, and Crohn's disease. The majority of people use herbal medicine because it is readily available, inexpensive, and effective. Many plants have promising features, including significant results in the treatment of psoriasis. The present study plans to emphasize such plants, herbal formulations, and associated therapy, which could add value to the development of a better, safe, and efficacious formulation to treat psoriasis that may help new researchers in this field

Susceptibility of Legionella gormanii Membrane-Derived Phospholipids to the Peptide Action of Antimicrobial LL-37-Langmuir Monolayer Studies.

LL-37 is the only member of the cathelicidin-type host defense peptide family in humans. It exhibits broad-spectrum bactericidal activity, which represents a distinctive advantage for future therapeutic targets. The presence of choline in the growth medium for bacteria changes the composition and physicochemical properties of their membranes, which affects LL-37's activity as an antimicrobial agent. In this study, the effect of the LL-37 peptide on the phospholipid monolayers at the liquid-air interface imitating the membranes of Legionella gormanii bacteria was determined. The Langmuir monolayer technique was employed to prepare model membranes composed of individual classes of phospholipids-phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), cardiolipin (CL)-isolated from L. gormanii bacteria supplemented or non-supplemented with exogenous choline. Compression isotherms were obtained for the monolayers with or without the addition of the peptide to the subphase. Then, penetration tests were carried out for the phospholipid monolayers compressed to a surface pressure of 30 mN/m, followed by the insertion of the peptide into the subphase. Changes in the mean molecular area were observed over time. Our findings demonstrate the diversified effect of LL-37 on the phospholipid monolayers, depending on the bacteria growth conditions. The substantial changes in membrane properties due to its interactions with LL-37 enable us to propose a feasible mechanism of peptide action at a molecular level. This can be associated with the stable incorporation of the peptide inside the monolayer or with the disruption of the membrane leading to the removal (desorption) of molecules into the subphase. Understanding the role of antimicrobial peptides is crucial for the design and development of new strategies and routes for combating resistance to conventional antibiotics.

Comparative analysis of groundnut oil quality in the north-central zone of Nigeria: Determination and evaluation of heavy metals, fatty acids, Phospholipids, and iodine values in groundnut oil

The research presents a comparative analysis of the quality of locally produced groundnut oil (Arachis hypogaea) sold in the north-central zone of Nigeria markets (Benue, Nasarawa, Kogi, Kwara, Niger, Plateau States). The aim was to assess and compare the qualities of the oils and to know the safety of human consumption. The groundnut oil produced biodiesel, shampoo lubricants, and soap-making industries. The concentrations of the heavy metals were analyzed with atomic absorption spectrometry (AAS). It showed that the lead, zinc, and copper (Pb, Zn, Cu) were within the FAO/WHO recommended limit, while Cd (0.201-0.331 mg kg-1) was above the limit (0.07 mg kg-1). Also, the gas chromatography (GC-FID) results indicated that twelve fatty acids (linoleic > oleic > palmitic > stearic >lignoceric > arachidic acid > behenic > erucic> arachidonic > margaric > linolenic > palmitoleic) were obtained in the groundnut oils in all markets and fatty acids include caprylic acid, capric acid, lauric acid, and myristic acid were absent in oils. In addition, the magnitude of six Phospholipids (phosphatidylcholine > phosphatidylethanolamine> phosphatidylinositol > phosphatidylserine > phosphatidic acid > lysophosphatidylcholine) were also achieved, respectively. The iodine were below the FAO/WHO and the acid value was higher than the normal range.

Combination of quinoxaline with pentamethinium system: Mitochondrial staining and targeting

Pentamethinium indolium salts are promising fluorescence probes and anticancer agents with high mitochondrial selectivity. We synthesized two indolium pentamethinium salts: a cyclic form with quinoxaline directly incorporated in the pentamethinium chain (cPMS) and an open form with quinoxaline substitution in the γ-position (oPMS). To better understand their properties, we studied their interaction with mitochondrial phospholipids (cardiolipin and phosphatidylcholine) by spectroscopic methods (UV–Vis, fluorescence, and NMR spectroscopy). Both compounds displayed significant affinity for cardiolipin and phosphatidylcholine, which was associated with a strong change in their UV–Vis spectra. Nevertheless, we surprisingly observed that fluorescence properties of cPMS changed in complex with both cardiolipin and phosphatidylcholine, whereas those of oPMS only changed in complex with cardiolipin. Both salts, especially cPMS, display high usability in mitochondrial imaging and are cytotoxic for cancer cells. The above clearly indicates that conjugates of pentamethinium and quinoxaline group, especially cPMS, represent promising structural motifs for designing mitochondrial-specific agents.

Tumor Lipid Signatures Are Descriptive of Acquisition of Therapy Resistance in an Endocrine-Related Breast Cancer Mouse Model.

The lipid metabolism adaptations of estrogen and progesterone receptor-positive breast cancer tumors from a mouse syngeneic model are investigated in relation to differences across the transition from hormone-dependent (HD) to hormone-independent (HI) tumor growth and the acquisition of endocrine therapy (ET) resistance (HIR tumors). Results are articulated with reported polar metabolome results to complete a metabolic picture of the above transitions and suggest markers of tumor progression and aggressiveness. Untargeted nuclear magnetic resonance metabolomics was used to analyze tumor and mammary tissue lipid extracts. Tumor progression (HD-HI-HIR) was accompanied by increased nonesterified cholesterol forms and phospholipids (phosphatidylcholine, phosphatidylethanolamine, sphingomyelins, and plasmalogens) and decreased relative contents of triglycerides and fatty acids. Predominating fatty acids became shorter and more saturated on average. These results were consistent with gradually more activated cholesterol synthesis, β-oxidation, and phospholipid biosynthesis to sustain tumor growth, as well as an increase in cholesterol (possibly oxysterol) forms. Particular compound levels and ratios were identified as potential endocrine tumor HD-HI-HIR progression markers, supporting new hypotheses to explain acquired ET resistance.

Sublimation application of 5-chloro-2-mercaptobenzothiazole matrix for matrix-assisted laser desorption/ionization mass spectrometry imaging of mouse kidney.

Sublimation is a solvent-free technique used to apply a uniform matrix coating over a large sample plate, improving the matrix's purity and enhancing the analyte signal. Although the 5-chloro-2-mercaptobenzothiazole (CMBT) matrix was introduced years ago, there are no reports of its application via sublimation. We investigated the experimental parameters that are optimal for CMBT matrix sublimation on mouse kidney samples. We also evaluated the stability of the sublimed CMBT matrix under a vacuum environment. Using kidney samples prepared with a sublimated CMBT matrix, we conducted matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) analysis of specific phospholipids (phosphatidylcholine and phosphatidylglycerol in the positive ion mode and phosphatidylinositol in the negative ion mode). We also explored various spatial resolutions (50, 20, and 10μm) and performed sequential MALDI-hematoxylin and eosin (H&E) staining. The CMBT matrix was applied to kidney samples using a sublimation apparatus connected to a vacuum pump to achieve a pressure of 0.05 Torr. The matrix was then subjected to different temperatures and sublimation times to determine the optimal conditions for matrix application. Subsequently, a Q-Exactive mass spectrometer equipped with a Spectroglyph MALDI ion source was employed for MALDI-MSI experiments. Standard protocols were followed for H&E staining after MALDI analysis. A matrix thickness of 0.15 mg/cm2 yielded high-quality images. The sublimated matrix exhibited minimal loss after approximately 20 h of exposure to a vacuum of 7Torr, indicating its stability under these conditions. Ion images were successfully obtained at spatial resolutions of 50, 20, and 10μm. Furthermore, orthogonal histological information was obtained through sequential MALDI-H&E staining. We demonstrate that samples prepared for MALDI-MSI using sublimation to apply the CMBT matrix yield high-quality mass spectrometric images of mouse kidney sections. We also provide data for the impact of various experimental parameters on image quality (e.g., temperature, time, matrix thickness, and spatial resolution).

Lipid composition of blood plasma and epithelium of the jejunal mucosa in calves with dyspepsia and its correction

Enteropathology in newborn ruminants significantly affects the functional formation of the digestive tract, which induces complications in other organs and systems, reducing the resistance and productivity in recovered animals. It is also characterized by metabolic disorders, particularly lipids, which adversely affects the regenerative processes at the cellular level. The material for the study was 2-day-old calves, from which 3 groups of 6 animals each were formed. 1st group – control was made up of clinically healthy animals, 2nd group – calves with toxic dyspepsia which received traditional treatment and 3rd group – calves which were additionally orally administered a liposomal form of a 1% solution of BAD "FLP-MD". The lipid composition of blood plasma and the epithelium of the mucous membrane of the jejunum of calves was studied by the method of thin-layer chromatography on standard Silufol plates. The lipidogram of blood plasma in 30-day-old calves recovered from dyspepsia under traditional treatment regimen was characterized by dyslipidemia, which was manifested by an increase in total lipid content by 10.0% amid hypercholesterolemia and hypertriacylglycerolemia and a decrease in free fatty acids by 24.6% and phospholipids by 11.8%. Also, phosphatidylcholine and sphingomyelin content decreasesd in the blood plasma. In epithelium of the jejunal mucosa, total lipid content decreased due to total cholesterol, triacylglycerols, and phospholipids. The restoration of total lipid content and individual fractions was found in blood plasma of calves that received additionally phospholipid-containing bio-additive "FLP-MD". Among the individual phospholipids phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine prevailed. In these calves, most of the lipid components in the epithelium of the jejunal mucosa were stabilized. In the future, we plan to investigate the content of total lipids in the blood and mucous membrane of the small intestine of calves.

Amphipathic Liponecrosis Impairs Bacterial Clearance and Causes Infection During Sterile Inflammation

Although transient bacteremia is common during dental and endoscopic procedures, infections developing during sterile diseases like acute pancreatitis (AP) can have grave consequences. We examined how impaired bacterial clearance may cause this transition. Blood samples from patients with AP, normal controls, and rodents with pancreatitis or those administered different nonesterified fatty acids (NEFAs) were analyzed for albumin-unbound NEFAs, microbiome, and inflammatory cell injury. Macrophage uptake of unbound NEFAs using a novel coumarin tracer were done and the downstream effects-NEFA-membrane phospholipid (phosphatidylcholine) interactions-were studied on isothermal titration calorimetry. Patients with infected AP had higher circulating unsaturated NEFAs; unbound NEFAs, including linoleic acid (LA) and oleic acid (OA); higher bacterial 16S DNA; mitochondrial DNA; altered β-diversity; enrichment in Pseudomonadales; and increased annexin V-positive myeloid (CD14) and CD3-positive T cells on admission. These, and increased circulating dead inflammatory cells, were also noted in rodents with unbound, unsaturated NEFAs. Isothermal titration calorimetry showed progressively stronger unbound LA interactions with aqueous media, phosphatidylcholine, cardiolipin, and albumin. Unbound NEFAs were taken into protein-free membranes, cells, and mitochondria, inducing voltage-dependent anion channel oligomerization, reducing ATP, and impairing phagocytosis. These were reversed by albumin. Invivo, unbound LA and OA increased bacterial loads and impaired phagocytosis, causing infection. LA and OA were more potent for these amphipathic interactions than the hydrophobic palmitic acid. Release of stored LA and OA can increase their circulating unbound levels and cause amphipathic liponecrosis of immune cells via uptake by membrane phospholipids. This impairs bacterial clearance and causes infection during sterile inflammation.

Lipid Deposition Profiles Influence Foreign Body Responses.

Fibrosis remains a significant cause of failure in implanted biomedical devices and early absorption of proteins on implant surfaces has been shown to be a key instigating factor. However, lipids can also regulate immune activity and their presence may also contribute to biomaterial-induced foreign body responses (FBR) and fibrosis. Here it is demonstrated that the surface presentation of lipids on implant affects FBR by influencing reactions of immune cells to materials as well as their resultant inflammatory/suppressive polarization. Time-of-flight secondary ion mass spectroscopy (ToF-SIMS) is employed to characterize lipid deposition on implants that are surface-modified chemically with immunomodulatory small molecules. Multiple immunosuppressive phospholipids (phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine, and sphingomyelin) are all found to deposit preferentially on implants with anti-FBR surface modifications in mice. Significantly, a set of 11 fatty acids is enriched on unmodified implanted devices that failed in both mice and humans, highlighting relevance across species. Phospholipid deposition is also found to upregulate the transcription of anti-inflammatory genes in murine macrophages, while fatty acid deposition stimulated the expression of pro-inflammatory genes. These results provide further insights into how to improve the design of biomaterials and medical devices to mitigate biomaterial material-induced FBR and fibrosis.

Membranes and Transport Mechanisms

Cellular life depends on the movement of membrane ions. For cells to continue functioning as they go through their life cycle, there must be a tremendous amount of exchange. Cell membranes are active structures rather than merely passive barriers separating cells from their surroundings. Cell membranes are known to be selectively permeable, allowing some chemicals to pass through while preventing others from doing so. Therefore, all cellular exchange activities can have active participation from cell membranes. Through the membrane interface, elements needed for cellular existence, communication among cells, and interaction between cells and their environment are taken in. Additionally, cells have the ability to direct and influence input and export processes, as well as moderate the subsequent signal response. Effective drug transportation across cellular membranes is essential for all treatment procedures. The majority of medications used in cancer therapy require carrier proteins for their transmembrane transport, even though hydrophobic small molecules can enter the cell membrane through straightforward diffusion. As a result, many scientists concentrate on creating novel strategies to improve drug delivery and uptake into desired cells. A live cell's plasma membrane serves as its outer wall. Although it acts as a barrier, it also permits transport between the cell and its surroundings.
 While the hydrophilic head groups are in contact with the surrounding water, the bilayer's hydrophobic interior, which is made up of fatty acid chains, renders the membrane impermeable to water-soluble compounds. To reduce the accessibility of hydrophobic fatty acid chains, phospholipids can spontaneously form stable bilayers in an aqueous environment due to their amphipathic nature. As lipids and proteins positioned in the plasma membrane can rotate or diffuse laterally and are distributed asymmetrically in the two leaflets, membranes are extremely dynamic structures. In addition to glycolipids, the outer leaflet of the plasma membrane mostly consists of the phospholipids phosphatidylcholine and sphingomyelin. Phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol are the three main substances that make up the inner leaflet.
 Additionally, both leaflets contain cholesterol, which affects the fluidity of the membrane. Lipid rafts are particular domains abundant in sphingolipids and cholesterol. The plasma membrane's basic role as a selectively permeable wall dividing the inside from the outside of the cell is determined by the structure of the phospholipid bilayer. Membrane proteins are essential for guaranteeing the selective translocation of molecules across the membrane as well as the regulation of cellular connections. They can function as energy converters, ion channels, pumps, receptors, or enzymes. The maintenance of the osmotic pressure and cellular pH, as well as the regulation of drug uptake mechanisms, account for the regulation of fundamental cellular activities.

O074 Metabolic phenotyping in diabetic foot ulceration

Abstract Introduction Diabetic foot ulceration is a devasting complication of diabetes contributing to significant morbidity. Delayed wound healing leads to limb loss and associated stress, anxiety and depression. Apart from systemic inflammatory markers and qualitative assessment of the ulcers, there is no molecular biomarker that can help prognosticate the fate of a diabetic foot ulcer. Metabonomics is a powerful toolset employed to describe metabolic signatures of diabetes in various biological systems. The aim of this systematic review is to summarise the existing evidence on the subject. Methods A systematic review was performed following a predefined protocol. Searches of Embase and MEDLINE by two independent authors, retrieved 292 articles, of which two were included in this study. A qualitative synthesis was performed due to heterogeneity between the studies. Result Two articles pertaining to metabonomics in diabetic foot ulcers were included. One study found that phospholipids (phosphatidylcholine, phosphatidylethanolamine and lysophosphatidylethanolamine) were associated with the development of diabetic foot ulcers. In the second study, amino acids such as arginine, isoleucine, leucine and threonine were found to be in greater concentration in the healed ulcer group as compared to the non-healed ulcer group. Conclusion The studies have highlighted disruption in the phospholipid pathway. They also suggest that specific amino acids are indeed deficient in non-healing ulcers. There is limited published literature on the application of metabolic phenotyping platforms in DFU. The methodology is heterogeneous and hence not directly comparable. Further studies are needed that utilize a biological approach, standardized design and additionally have translational application. Take-home message Diabetic foot ulcer healing is not fully understood. More study designs using metabolic phenotyping should be developed as they provide the most patient specific phenotype.

Unusual Lipid Components of Legionella gormanii Membranes.

Legionella spp. cause Legionnaires’ disease with pneumonia as the predominant clinical symptom. L. gormanii is the second most prevalent causative agent of community-acquired pneumonia after L. pneumophila. The study aimed to characterize the lipidome of L. gormanii membranes and the importance of these analyses in bacterial chemotaxonomy. Lipidomic analyses based on ultra-high performance liquid chromatography-mass spectrometry allowed the detection of individual molecular species of a wide range of L. gormanii membrane lipids contained in the outer (OM) and inner membranes (IM). The lipid profile comprised glycerolipids (triglycerides, diglycerides), phospholipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, cardiolipin), and sphingolipids (ceramides, hexosylceramides). The most abundant lipid fraction in the IM and OM were phospholipids. The lipidomic analysis showed that two independent phosphatidylcholine (PC) synthesis pathways operating in L. gormanii: the PE-methylation (PmtA) pathway and the PC synthase (Pcs) pathway. Comparison of the molecular profile of PC species contained in the lipids of L. gormanii membranes cultured on the medium, with and without exogenous choline, showed quantitative differences in the PC pool. An unusual feature of the L. gormanii lipids was the presence of ceramides and hexosylceramides, which are typical components of eukaryotic cells and a very small group of bacteria. To the best of our knowledge, this is the first report of the occurrence of ceramides in Legionella bacteria.

Yeast Pah1 PA phosphatase contains a novel domain within its N‐terminal intrinsically disordered region

Pah1 PA phosphatase catalyzes the dephosphorylation of PA to produce diacylglycerol. In yeast and higher eukaryotes, the diacylglycerol produced in the reaction is used for the synthesis of triacylglycerol or the membrane phospholipids phosphatidylcholine and phosphatidylethanolamine. Pah1 is inactive as a phosphorylated enzyme in the cytosol and becomes active after its recruitment and dephosphorylation by the ER‐associated Nem1‐Spo7 protein phosphatase complex. Conserved N‐LIP and HAD‐like domains are required for PA phosphatase activity and a conserved tryptophan within the WRDPLVDID domain is required for its in vivo function in lipid metabolism. Intrinsically disordered regions, which are located between the conserved catalytic domains and at the C terminus, contain multiple sites for phosphorylation and regulation of Pah1 location and function. Prediction of Pah1 structure by AlphaFold identifies a novel domain contained within the N‐terminal intrinsically disordered region for which its function is unknown. A truncation mutant that lacks amino acid residues 186‐266 have been constructed and expressed in a pah1D and pah1D nem1D mutants to assess the function of the novel domain for regulating Pah1 phosphorylation, recruitment and dephosphorylation by the Nem1‐Spo7 protein phosphatase complex, and function in lipid metabolism.

Choline kinase inhibition promotes ER-phagy

The major membrane phospholipid phosphatidylcholine is predominantly synthesized through the CDP-choline (Kennedy) pathway at the endoplasmic reticulum (ER). The first step in this pathway is the phosphorylation of choline by choline kinase. Humans contain two choline kinase isoforms, CHKA and CHKB. Variants in the CHKA gene that decrease enzymatic activity have been shown to cause an inherited neurodevelopmental disorder with epilepsy, while recessive inheritance of nonfunctional CHKB alleles causes an inherited rostrocaudal muscular dystrophy (1, 2).

Single Molecule-Level Detection via Liposome-Based Signal Amplification Mass Spectrometry Counting Assay.

Because of the low atomization and/or ionization efficiencies of many biological macromolecules, the application of mass spectrometry to the direct quantitative detection of low-abundance proteins and nucleic acids remains a significant challenge. Herein, we report mass spectrum tags (MS-tags) based upon gold nanoparticle (AuNP)-templated phosphatidylcholine phospholipid (DSPC) liposomes, which exhibit high and reliable signals via electrospray ionization (ESI). Using these MS-tags, we constructed a liposome signal amplification-based mass spectrometric (LSAMS) "digital" counting assay to enable ultrasensitive detection of target nucleic acids. The LSAMS system consists of liposomes modified with a gold nanoparticle core and surface-anchored photocleavable DNA. In the presence of target nucleic acids, the modified liposome and a magnetic bead simultaneously hybridize with the target nucleic acid. After magnetic separation and photolysis, the MS-tag is released and can be analyzed by ESI-MS. At very low target concentrations, one liposome particle corresponds to one target molecule; thus, the concentration of the target can be estimated by counting the number of liposomes. With this assay, hepatitis C (HCV) virus RNA was successfully analyzed in clinical samples.

Endo- and Exometabolome Crosstalk in Mesenchymal Stem Cells Undergoing Osteogenic Differentiation.

This paper describes, for the first time to our knowledge, a lipidome and exometabolome characterization of osteogenic differentiation for human adipose tissue stem cells (hAMSCs) using nuclear magnetic resonance (NMR) spectroscopy. The holistic nature of NMR enabled the time-course evolution of cholesterol, mono- and polyunsaturated fatty acids (including ω-6 and ω-3 fatty acids), several phospholipids (phosphatidylcholine, phosphatidylethanolamine, sphingomyelins, and plasmalogens), and mono- and triglycerides to be followed. Lipid changes occurred almost exclusively between days 1 and 7, followed by a tendency for lipidome stabilization after day 7. On average, phospholipids and longer and more unsaturated fatty acids increased up to day 7, probably related to plasma membrane fluidity. Articulation of lipidome changes with previously reported polar endometabolome profiling and with exometabolome changes reported here in the same cells, enabled important correlations to be established during hAMSC osteogenic differentiation. Our results supported hypotheses related to the dynamics of membrane remodelling, anti-oxidative mechanisms, protein synthesis, and energy metabolism. Importantly, the observation of specific up-taken or excreted metabolites paves the way for the identification of potential osteoinductive metabolites useful for optimized osteogenic protocols.

Mechanism of action and therapeutic route for a muscular dystrophy caused by a genetic defect in lipid metabolism

CHKB encodes one of two mammalian choline kinase enzymes that catalyze the first step in the synthesis of the membrane phospholipid phosphatidylcholine. In humans and mice, inactivation of the CHKB gene (Chkb in mice) causes a recessive rostral-to-caudal muscular dystrophy. Using Chkb knockout mice, we reveal that at no stage of the disease is phosphatidylcholine level significantly altered. We observe that in affected muscle a temporal change in lipid metabolism occurs with an initial inability to utilize fatty acids for energy via mitochondrial β-oxidation resulting in shunting of fatty acids into triacyglycerol as the disease progresses. There is a decrease in peroxisome proliferator-activated receptors and target gene expression specific to Chkb−/− affected muscle. Treatment of Chkb−/− myocytes with peroxisome proliferator-activated receptor agonists enables fatty acids to be used for β-oxidation and prevents triacyglyerol accumulation, while simultaneously increasing expression of the compensatory choline kinase alpha (Chka) isoform, preventing muscle cell injury.

Stage related metabolic profile of the synovial fluid in patients with acute flares of knee osteoarthritis.

Osteoarthritis (OA) is the most common joint condition and the leading cause of pain and disability in elderly patients. Currently, there is no biomarker available for the early diagnosis of OA, and limited data is available regarding the molecular basis of progression for OA. For this reason, this study aimed to identify the metabolomic profile of early and late OA using high-performance liquid chromatography coupled with untargeted mass spectrometry (LC-MS). 31 patients with knee OA and joint effusion were enrolled. Based on Kellgren/Laurence scale, 12 patients were classified as early OA (eOA) and 19 as late OA (lOA). The synovial fluid (SF) was collected and characterized by untargeted LC-MS. Only the metabolites identified in more than 25% of each group were kept for further analysis. Principal component analysis (PCA) enabled the unsupervised clustering of the eOA and lOA groups. Further, for classification, the best three principal components (PCs) were used as input for two machine learning algorithms (random forest and naïve Bayes), which were trained to discriminate between the eOA and lOA groups. 43 metabolites were identified in both eOA and lOA, but after selecting the metabolites present in at least 25% of the patients in each group, the metabolomics analysis yielded a panel of only nine metabolites: four metabolites related to phospholipids (phosphatidylcholine 20:0/18:2 and 18:0/20:2, sphingomyelin, and ceramide), three metabolites belonging to purine metabolites (inosine 5'-phosphate, adenosine thiamine diphosphate, and diadenosine 5',5'-diphosphate), one metabolite was a gonadal steroid hormone (estrone 3-sulfate), and one metabolite represented by heme, with all but ceramide (d18:1/20:0) being enriched in the lOA group. By using as features the best three PCs (PC2, PC8 and PC9), random forest and naïve Bayes machine learning algorithms yielded a classification accuracy of 0.81 and 0.78, respectively. Our LC-MS analysis of SF from patients with eOA and lOA indicates stage-dependent differences, lOA being associated with a perturbed metabolome of phospholipids, purine metabolites, gonadal steroid hormones (estrone 3-sulfate) and a heme molecule. Specific questions need to be answered regarding the biosynthesis and function of these metabolites in osteoarthritic joints, with the aim of developing new relevant biomarkers and therapeutic strategies.

A mouse model of inherited choline kinase β-deficiency presents with specific cardiac abnormalities and a predisposition to arrhythmia

The CHKB gene encodes choline kinase β, which catalyzes the first step in the biosynthetic pathway for the major phospholipid phosphatidylcholine. Homozygous loss-of-function variants in human CHKB are associated with a congenital muscular dystrophy. Dilated cardiomyopathy is present in some CHKB patients and can cause heart failure and death. Mechanisms underlying a cardiac phenotype due to decreased CHKB levels are not well characterized. We determined that there is cardiac hypertrophy in Chkb−/− mice along with a decrease in left ventricle size, internal diameter, and stroke volume compared with wildtype and Chkb+/− mice. Unlike wildtype mice, 60% of the Chkb+/− and all Chkb−/− mice tested displayed arrhythmic events when challenged with isoproterenol. Lipidomic analysis revealed that the major change in lipid level in Chkb+/− and Chkb−/− hearts was an increase in the arrhythmogenic lipid acylcarnitine. An increase in acylcarnitine level is also associated with a defect in the ability of mitochondria to use fatty acids for energy and we observed that mitochondria from Chkb−/− hearts had abnormal cristae and inefficient electron transport chain activity. Atrial natriuretic peptide (ANP) is a hormone produced by the heart that protects against the development of heart failure including ventricular conduction defects. We determined that there was a decrease in expression of ANP, its receptor NPRA, as well as ventricular conduction system markers in Chkb+/− and Chkb−/− mice.

ANALYSIS OF SPERMATOZOA QUALITY USING PERCOLL DENSITY GRADIENT CENTRIFUGATION THROUGH THE ADMINISTRATION OF PHOSPHOLIPID + EGTA

The Percoll density gradient centrifugation (PDGC) method is frequently used in the sexing of spermatozoa. However, this method causes damage to the spermatozoa membranes, resulting in a decreased quality of spermatozoa. We analysed the impacts of phospholipid PC (phosphatidylcholine) and EGTA (ethylene glycol bis (β-aminoethyl ether) N,N,N',N'-tetraacetic acid) Ca2+ free buffer on the quality of bovine spermatozoa after the PDGC process, using semen from Friesian Holstein (FH) bulls aged 5 - 8 years. The following variables were observed: spermatozoa motility, spermatozoa viability, spermatozoa membrane integrity, spermatozoa that have not experienced capacitation, spermatozoa that have experienced capacitation and spermatozoa that have undergone acrosomal reaction. The results showed that the administration of phospholipid PC + EGTA Ca2+ free buffer to spermatozoa, followed by the PDGC process, could maintain or further improve the values of all variables. In the PDGC process, phospholipid PC 10% + EGTA Ca2+ free buffer at 1 mM was most suitable.