Lactational Changes of Phospholipids Content and Composition in Chinese Breast Milk.

Phospholipids are pivotal polar lipids in human milk and essential for infants' growth and development, especially in the brain and cognitive development. Its content and composition are affected by multiple factors and there exist discrepancies in different studies. In this study, we determined five major phospholipids classes (phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylcholine, and sphingomyelin) in 2270 human milk samples collected from 0 to 400 days postpartum in six regions of China. The high-performance liquid chromatography coupled with an evaporative light scattering detector (HPLC-ELSD) was performed to quantify the phospholipids. Total phospholipid median (IQR) content was in a range between 170.38 ± 96.52 mg/L to 195.69 ± 81.80 mg/L during lactation and was higher concentrated in colostrum milk and later stage of lactation (after 200 days postpartum) compared with that in the samples collected between 10 to 45 days postpartum. Variations in five major sub-class phospholipids content were also observed across lactation stages (phosphatidylethanolamine: 52.61 ± 29.05 to 59.95 ± 41.74 mg/L; phosphatidylinositol: 17.65 ± 10.68 to 20.38 ± 8.55 mg/L; phosphatidylserine: 15.98 ± 9.02 to 22.77 ± 11.17 mg/L; phosphatidylcholine: 34.13 ± 25.33 to 48.64 ± 19.73 mg/L; sphingomyelin: 41.35 ± 20.31 to 54.79 ± 35.26 mg/L). Phosphatidylethanolamine (29.18-32.52%), phosphatidylcholine (19.90-25.04%) and sphingomyelin (22.39-29.17%) were the dominant sub-class phospholipids in Chinese breast milk during the whole lactation period. These results updated phospholipids data in Chinese human milk and could provide evidence for better development of secure and effective human milk surrogates for infants without access to breast milk.

Application of hybrid surface technology for improving sensitivity and peak shape of phosphorylated lipids such as phosphatidic acid and phosphatidylserine.

The use of hybrid surface technology (HST), applied to the metal surfaces of an ACQUITY™ UPLC™ system and column, designed to mitigate the chelation, poor peak shape and analyte loss seen with acidic phospholipids was investigated. Compared to a conventional system significant improvements in both sensitivity, recovery and peak shape were obtained following UPLC on a CSH C18 column when the HST was used for the analysis of lysophosphatidic acid (LPA), phosphatidic acid (PA), lysophosphatidylserine (LPS), phosphatidylserine (PS), phosphatidylinositol-monophosphates (PIP), ceramide phosphate (CerP) and sphingoid base phosphate (SPBP). The benefits in chromatographic performance provided by the HST were seen particularly at low concentrations of these analytes. The HST system and column reduced peak tailing by 65-80% and peak width by 70-86% for LPA and PA. Moreover, increased signal intensities of up to 12.7 times were observed for LPA with the HST approach compared to the equivalent untreated LC system and column. The application of this methodology to the analysis of chicken egg PA and brain porcine PS extracts were accompanied by similar improvements in data quality.

Lipid Dynamics due to Muscle Atrophy Induced by Immobilization.

Muscle atrophy refers to skeletal muscle loss and dysfunction that affects glucose and lipid metabolism. Moreover, muscle atrophy is manifested in cancer, diabetes, and obesity. In this study, we focused on lipid metabolism during muscle atrophy. We observed that the gastrocnemius muscle was associated with significant atrophy with 8 days of immobilization of hind limb joints and that muscle atrophy occurred regardless of the muscle fiber type. Further, we performed lipid analyses using thin layer chromatography, liquid chromatography-mass spectrometry, and mass spectrometry imaging. Total amounts of triacylglycerol, phosphatidylserine, and sphingomyelin were found to be increased in the immobilized muscle. Additionally, we found that specific molecular species of phosphatidylserine, phosphatidylcholine, and sphingomyelin were increased by immobilization. Furthermore, the expression of adipose triglyceride lipase and the activity of cyclooxygenase-2 were significantly reduced by atrophy. From these results, it was revealed that lipid accumulation and metabolic changes in specific fatty acids occur during disuse muscle atrophy. The present study holds implications in validating preventive treatment strategies for muscle atrophy.

Apoptosis in platelets from adult patients with chronic idiopathic thrombocytopenic purpura.

Adult chronic idiopathic thrombocytopenic purpura (cITP) is a chronic and usually life-long haemorrhagic disorder in which enhanced platelet destruction and weakened platelet production lead to thrombocytopenia. Platelets were isolated from blood samples collected from 40 adult patients with cITP and 40 healthy volunteers. Mitochondrial membrane potential (ΔΨm) and plasma membrane phosphatidylserine externalization were determined by flow cytometry, and activation of caspase-3 and expressions of Bax, Bak and Bcl-xL were analysed by western blotting. Flow cytometry showed increased mitochondrial depolarization and lower ΔΨm in platelets from adult patients with cITP. In addition, plasma membrane phosphatidylserine externalization was observed on platelets from adult patients with cITP, but rarely from healthy volunteers. Western blot analysis of platelet proteins revealed that, in adult cITP patients, caspase-3 was activated, which cleaved gelsolin and to release a 47-kDa fragment. Moreover, the expressions of Bax and Bak were elevated, and Bcl-xL was decreased markedly in platelets from adult patients with cITP. Our findings reveal, based on loss of mitochondrial membrane potential (Δψm), phosphatidylserine exposure, caspase-3 activation, enhanced expression of Bax and Bak, and attenuated expression of Bcl-xL, that platelet death in the pathogenesis of thrombocytopenia in chronic ITP in adults is apoptotic.

Phosphatidylserine-exposing tumor-derived microparticles exacerbate coagulation and cancer cell transendothelial migration in triple-negative breast cancer.

Background: Neoadjuvant chemotherapy is relevant to the formation of thromboembolism and secondary neoplasms in triple-negative breast cancer (TNBC). Chemotherapy-induced breast cancer cell-derived microparticles (BCMPs) may have important thrombogenic and pro-metastatic effects on platelets and endothelium, which may be related to the expression and distribution of phosphatidylserine (PS). However, investigating these interactions is challenging due to technical limitations. Methods: A study was conducted in 20 healthy individuals and 18 patients who had been recently diagnosed with TNBC and were undergoing neoadjuvant chemotherapy with doxorubicin and cyclophosphamide. BCMPs were isolated from patient blood samples and doxorubicin-treated breast cancer cell lines. Their structure and morphology were studied by electron microscopy and antigen levels were measured by fluorescence-activated cell sorting. In an inhibition assay, isolated BCMPs were pretreated with lactadherin or tissue factor antibodies. Platelets isolated from healthy subjects were treated with BCMPs and coagulation time, fibrin formation, and expression of intrinsic/extrinsic factor Xase (FXa) and thrombin were evaluated. The effects of BCMPs on endothelial thrombogenicity and integrity were assessed by confocal microscopy, electron microscopy, measurement of intrinsic/extrinsic FXa, prothrombinase assay, and transwell permeability assay. Results: Neoadjuvant chemotherapy significantly increased the expression of PS+ BCMPs in patient plasma. Its expression was associated with a rapid increase in procoagulant activity. Treatment with lactadherin, a PS-binding scavenging molecule, markedly reduced the adhesion of BCMPs and abolished their procoagulant activity, but this was not observed with tissue factor antibody treatment. Intravenous injection of BCMPs in mice induced a significant hypercoagulable state, reducing the extent of plasma fibrinogen and promoting the appearance of new thrombus. Cancer cells incubated with doxorubicin released large numbers of PS+ BCMPs, which stimulated and transformed endothelial cells into a procoagulant phenotype and increased the aggregation and activation of platelets. Moreover, cancer cells exploited this BCMP-induced endothelial leakiness and showed promoted metastasis. Pretreatment with lactadherin increased uptake of both PS+ BCMPs and cancer cells by endothelial cells and limited the transendothelial migration of cancer cells. Conclusion: Lactadherin, a biosensor that we developed, was used to study the extracellular vesicle distribution of PS, which revealed a novel PS+ BCMPs administrative axis that initiated a local coagulation cascade and facilitated metastatic colonization of circulating cancer cells.

Quantitative and comparative study of plasmalogen molecular species in six edible shellfishes by hydrophilic interaction chromatography mass spectrometry.

Shellfishes contain plasmalogens correlating to the functions of brain, heart, etc. Herein, a mild acid hydrolysis and hydrophilic interaction chromatography (HILIC) tandem mass spectrometry method was developed for analyzing plasmalogens in six shellfish species. A total of 19 plasmalogen molecular species were successfully identified, including nine phosphatidylcholine plasmalogen (plasPC), seven phosphatidylethanolamine plasmalogen (plasPE), and three phosphatidylserine plasmalogen (plasPS). The quantitative results indicated that mussel (32 µg·mg-1) possessed the highest content of plasmalogens, followed by oyster (21 µg·mg-1) and razor clam (15 µg·mg-1). The statistic models showed that the plasPE P-18:0/20:5 (m/z 748), plasPE P-16:0/22:2 & P-18:0/20:2 (m/z 754) and plasPS were the most contributing difference between shellfishes. The results indicated that this method was sensitive and precise to determine plasmalogens in shellfish, and mussel was demonstrated to be a good choice for the large-scale preparation of plasmalogens.

Ketogenic Diet: Impact on Cellular Lipids in Hippocampal Murine Neurons.

BACKGROUND: The mechanism of action of the ketogenic diet (KD), an effective treatment for pharmacotherapy refractory epilepsy, is not fully elucidated. The present study examined the effects of two metabolites accumulating under KD-beta-hydroxybutyrate (ßHB) and decanoic acid (C10) in hippocampal murine (HT22) neurons. METHODS: A mouse HT22 hippocampal neuronal cell line was used in the present study. Cellular lipids were analyzed in cell cultures incubated with high (standard) versus low glucose supplemented with ßHB or C10. Cellular cholesterol was analyzed using HPLC, while phospholipids and sphingomyelin (SM) were analyzed using HPTLC. RESULTS: HT22 cells showed higher cholesterol, but lower SM levels in the low glucose group without supplements as compared to the high glucose groups. While cellular cholesterol was reduced in both ßHB- and C10-incubated cells, phospholipids were significantly higher in C10-incubated neurons. Ratios of individual phospholipids to cholesterol were significantly higher in ßHB- and C10-incubated neurons as compared to controls. CONCLUSION: Changes in the ratios of individual phospholipids to cholesterol in HT22 neurons suggest a possible alteration in the composition of the plasma membrane and organelle membranes, which may provide insight into the working mechanism of KD metabolites ßHB and C10.

A near-infrared light-controlled, ultrasensitive one-step photoelectrochemical detection of dual cell apoptosis indicators in living cancer cells.

Here, a near-infrared (NIR) light-controlled, ultrasensitive one-step photoelectrochemical (PEC) strategy was constructed to simultaneously detect cell apoptosis indicators, phosphatidylserine (Pho) and sodium-potassium adenosine triphosphatase (Sat), on living cancer cells. Using NIR light as excitation, the signal probe methylene blue (Tagkinetic) could be released, leading to a gradually decreased photocurrent signal Ikinetic; meanwhile, the photocurrent Istable of the signal probe carbon quantum dots (Tagstable) remained stable. The simultaneous detection of Pho and Sat could be achieved based on rapid one-step PEC detection under single NIR light with the assistance of a smart signal decryption strategy with Ikinetic and Istable. Importantly, this proposal provides more effective drug candidates with milder pharmaceutical effect but improved safety.

Recovery of brain DHA-containing phosphatidylserine and ethanolamine plasmalogen after dietary DHA-enriched phosphatidylcholine and phosphatidylserine in SAMP8 mice fed with high-fat diet.

BACKGROUND: Glycerophospholipids were the main components of cerebral cortex lipids, and there was a close association between lipid homeostasis and human health. It has been reported that dietary DHA-enriched phosphatidylcholine (DHA-PC) and phosphatidylserine (DHA-PS) could improve brain function. However, it was unclear that whether supplementation of DHA-PC and DHA-PS could change lipid profiles in the brain of dementia animals. METHODS: SAMP8 mice was fed with different diet patterns for 2 months, including high-fat diet and low-fat diet. After intervention with DHA-PC and DHA-PS for another 2 months, the lipid profile in cerebral cortex was determined by lipidomics in dementia mice. RESULTS: High-fat diet could significantly decrease the levels of DHA-containing PS/pPE, DPA-containing PS, and AA-containing PE, which might exhibit the potential of lipid biomarkers for the prevention and diagnosis of AD. Notably, DHA-PC and DHA-PS remarkably recovered the lipid homeostasis in dementia mice. These might provide a potential novel therapy strategy and direction of dietary intervention for patients with cognitive decline. CONCLUSIONS: DHA-PC and DHA-PS could recover the content of brain DHA-containing PS and pPE in SAMP8 mice fed with high-fat diet.

Affordable, Reliable Dual-Platform Approach to Quantitating Phosphatidylserine-Exposing Platelets in Platelet Components.

OBJECTIVE: To compare the number of phosphatidylserine (PS)-exposing platelets obtained using the dual-platform approach and bead-based flow cytometry. METHODS: Platelets were enumerated using the ADVIA 2010i instrument (Siemens AG). The numbers and percentages of PS-exposing platelets in 175 platelet products were determined using a FACSCalibur flow cytometer (Becton, Dickinson and Company) and counting beads. RESULTS: Our results showed good correlation (r2 = 0.96; P <.001) between the PS-exposing platelets obtained using counting beads and the dual-platform approach. The results of Bland-Altman analysis showed a bias of +46,449 cells per µL and a limit of agreement (LOA) from -197,863 to 290,762 cells per µL. Also, 8 measurements (5.0%) revealed a number of PS-exposing platelets outside the LOA ranges. Further, 21 measurements (12.0%) revealed greater than 2-fold changes in the number of PS-exposing platelets. CONCLUSIONS: The results suggest that the dual-platform approach is affordable and reliable for quantitating PS-exposing platelets as part of monitoring the quality of platelet products.

Selected CD molecules and the phagocytosis of microvesicles released from erythrocytes ex vivo.

BACKGROUND AND OBJECTIVES: The accumulation of microvesicles in erythrocyte concentrates during storage or irradiation may be responsible for clinical symptoms such as inflammation, coagulation and immunization. Our aim was to determine whether any of the cluster of differentiation (CD) molecules responsible for important functions are present on microvesicles, and if their expression level is dependent on the storage period of erythrocyte concentrates. MATERIAL AND METHODS: Erythrocyte microvesicles were isolated from 'fresh' (2nd day) and 'old' (42nd day) stored erythrocyte concentrates. Qualitative cytometric analysis of 0·5 µm, erythrocyte-derived, PS-exposing vesicles was performed using the annexin V-FITC, anti-CD235a-PE antibody and calibrated beads. The microvesicles were also visualized under a confocal microscope. The expression of the molecules CD235a, CD44, CD47, CD55, CD59 and of phosphatidylserine (PS) was compared using flow cytometry. Measurements of microvesicle phagocytosis by human monocytes were carried out using a flow cytometer and a confocal microscope. RESULTS: The analysis of the microvesicles with calibration beads allowed us to identify these structures with a diameter of about 0·5 µm in the 'fresh' and 'old' samples. At day 2, the microvesicles had elevated expression levels of CD47, reduced expression levels of PS, CD55 and CD59. The phagocytosis index was higher for the microvesicles isolated from the 42-day-old erythrocyte concentrates. CONCLUSION: This research may bring us closer to understanding the factors responsible for erythrocyte ageing and to evaluate the quality of stored red blood concentrates intended for transfusion.

Analysis of oxidised and glycated aminophospholipids: Complete structural characterisation by C30 liquid chromatography-high resolution tandem mass spectrometry.

The aminophospholipids (APL), phosphatidylethanolamine (PE) and phosphatidylserine (PS) are widely present in cell membranes and lipoproteins. Glucose and reactive oxygen species (ROS), such as the hydroxyl radical (•OH), can react with APL leading to an array of oxidised, glycated and glycoxidised derivatives. Modified APL have been implicated in inflammatory diseases and diabetes, and were identified as signalling molecules regulating cell death. However, the biological relevance of these molecules has not been completely established, since they are present in very low amounts, and new sensitive methodologies are needed to detect them in biological systems. Few studies have focused on the characterisation of APL modifications using liquid chromatography-tandem mass spectrometry (LC-MS/MS), mainly using C5 or C18 reversed phase (RP) columns. In the present study, we propose a new analytical approach for the characterisation of complex mixtures of oxidised, glycated and glycoxidised PE and PS. This LC approach was based on a reversed-phase C30 column combined with high-resolution MS, and higher energy C-trap dissociation (HCD) MS/MS. C30 RP-LC separated short and long fatty acyl oxidation products, along with glycoxidised APL bearing oxidative modifications on the glucose moiety and the fatty acyl chains. Functional isomers (e.g. hydroxy-hydroperoxy-APL and tri-hydroxy-APL) and positional isomers (e.g. 9-hydroxy-APL and 13-hydroxy-APL) were also discriminated by the method. HCD fragmentation patterns allowed unequivocal structural characterisation of the modified APL, and are translatable into targeted MS/MS fingerprinting of the modified derivatives in biological samples.

Increased Phosphatidylserine on Blood Cells in Oral Squamous Cell Carcinoma.

The specific function of phosphatidylserine (PS) in the context of the development of a hypercoagulable state among individuals with oral squamous cell carcinoma (OSCC) is uncertain. The goal of this study was therefore to assess the exposure of PS on microparticles (MPs) as well as on endothelial and blood cells and to assess procoagulant activity (PCA) as a function of the stage of OSCC progression. We recruited patients with OSCC (n = 63) as well as healthy controls (n = 26) to participate in this study. PS exposure was then assessed via confocal microscopy and flow cytometry, revealing that patients with stage III/IV OSCC exhibited higher frequencies of PS-exposing blood cells, MPs, and serum-cultured endothelial cells (ECs) than did patients with stage I/II OSCC or healthy controls. When we conducted functional coagulation assays, we discovered that PS+ blood cells, MPs, and serum-cultured ECs from patients with stage III/IV OSCC mediated more rapid coagulation and more substantial production of FXa, thrombin, and fibrin as compared with controls. When samples were treated with the PS antagonist lactadherin, this resulted in an 80% disruption of PCA. Strikingly, when pre- and postoperative samples were compared from patients with stage III/IV OSCC undergoing resective surgery, PCA was significantly reduced in the postoperative samples. After stimulating ECs with inflammatory cytokines, we found by confocal microscopy that they expose PS on their cell membranes, thus generating FVa and FXa binding sites and mediating the formation of fibrin. Together our findings provide evidence that PS+ blood cells and MPs are important mediators of the development of a hypercoagulable and prothrombotic state among individuals afflicted by advanced-stage OSCC. As such, a PS blockade may be a viable therapeutic strategy for treating such patients.

Calcium electroporation and electrochemotherapy for cancer treatment: Importance of cell membrane composition investigated by lipidomics, calorimetry and in vitro efficacy.

Calcium electroporation is a novel anti-cancer treatment investigated in clinical trials. We explored cell sensitivity to calcium electroporation and electroporation with bleomycin, using viability assays at different time and temperature points, as well as heat calorimetry, lipidomics, and flow cytometry. Three cell lines: HT29 (colon cancer), MDA-MB231 (breast cancer), and HDF-n (normal fibroblasts) were investigated for; (a) cell survival dependent on time of addition of drug relative to electroporation (1.2 kV/cm, 8 pulses, 99 µs, 1 Hz), at different temperatures (37 °C, 27 °C, 17 °C); (b) heat capacity profiles obtained by differential scanning calorimetry without added calcium; (c) lipid composition by mass spectrometry; (d) phosphatidylserine in the plasma membrane outer leaflet using flow cytometry. Temperature as well as time of drug administration affected treatment efficacy in HT29 and HDF-n cells, but not MDA-MB231 cells. Interestingly the HT29 cell line displayed a higher phase transition temperature (approximately 20 °C) versus 14 °C (HDF-n) and 15 °C (MDA-MB231). Furthermore the HT29 cell membranes had a higher ratio of ethers to esters, and a higher expression of phosphatidylserine in the outer leaflet. In conclusion, lipid composition and heat capacity of the membrane might influence permeabilisation of cells and thereby the effect of calcium electroporation and electrochemotherapy.

Single cell analysis of aged RBCs: quantitative analysis of the aged cells and byproducts.

This study initially focused on characterizing the aging process of red blood cells by correlating the loss of hemoglobin and the translocation of phosphatidylserine (PS) in expired human red blood cells, hRBCs. Five pre-storage, leukoreduced hRBC units in AS-5 solution were stored between 1 and 6 °C for 42 days. Aliquots from each of these units were stained with Annexin-V FLUOS, which binds to externalized PS, and the hemoglobin within the cells was placed in a methemoglobin state with sodium nitrite, metHb. These aliquots were subsequently sorted into four sub-populations, ranging from no PS expression to high PS expression using a BD FACS ARIAIII. Each of these sub-fractions were introduced into the cell tracking velocimetry apparatus which measured both the magnetically-induced and the gravity-induced velocity. Subsequently, the samples were removed from the cell tracking velocimetry instrument and characterized using the Multisizer 4e Coulter Counter. From the magnetically-induced velocity, the amount of hemoglobin, in pg Hb per cell can be determined, and using an average value of the density of RBCs, the size can be determined. For the PS negative sub-fraction of RBCs, the size of the RBC was as expected but the average hemoglobin, Hb, content was below the threshold which defines anemia. In contrast, unexpected results were observed with the various levels of expression of PS. First, virtually all of the PS expressing cells were significantly smaller, on the order of 1 micron, than a normal RBC after 42 days of storage; yet the density of these small cells/microvesicles was such that they had settling velocities similar to normal-sized RBCs. Further, while the total amount of Hb per small cell/microvesicle was only approximately 25% of the full-sized RBCs, the volume of these small cells/microvesicles is only 1/200 of the PS negative RBCs. This suggests that these PS expressing cells are shrunken RBCs, or shrunken microvesicles from RBCs that concentrated the Hb internally. These results suggest not only a relationship between the loss of hemoglobin and the amount of PS exposed on the cellular outer wall, but also a mechanism by which these aged RBCs break down. It is not known at this time whether this is an artifact of storage or similar mechanisms occur in circulation within the human body.

Evaluation of circulating microparticles in healthy medical workers occupationally exposed to ionizing radiation: A preliminary study.

OBJECTIVES: Ionizing radiation was known to cause disruption of cytoskeleton. However, the disorganization of the cytoskeleton leads to form microparticles (MP) that carry membrane and cytoplasmic constituents from their parent cells they are released from. Therefore, authors investigated the effect of the occupational exposure to low doses of ionizing radiation on MP levels. MATERIAL AND METHODS: The current study was conducted on 38 healthy medical workers occupationally exposed to low doses of ionizing radiation and 29 controls matched by gender, age, and smoking habits. The MP levels measured by flow cytometry were classified as positive or negative phosphatidylserine (PS+ or PS-), and phenotyped according to their cellular origin. RESULTS: Total MP (PS-/PS+) levels, regardless of phenotype, were significantly higher in workers occupationally exposed to ionizing radiation than in healthy individuals (p = 0.0004). Negative phosphatidylserine microparticles were predominant in medical exposed workers and, to a lesser extent, in controls (68% and 57%, respectively). With regard to phenotypic characterization of cellular origin, MP derived from platelets (CD41a+), endothelial (CD146+), leucocytes (CD45+) and erythrocytes (CD235a+) MP were significantly enhanced in exposed workers compared with controls (p < 0.0001). However, no significant difference was found in the proportion of the other blood elements in the peripheral circulation between the 2 groups. Serum levels of C-reactive protein were normal for all individuals. In addition, no association was observed between MP levels and the studied confounding factors. CONCLUSIONS: The results suggest that elevated circulating MP levels represent an indicator of cellular damage caused by medical exposure to low doses of ionizing radiation. By consequence, the quantification of MP seems to be a useful biomarker for assessing the negative effects of occupational exposure to ionizing radiation. Int J Occup Med Environ Health 2018;31(6):783-793.

Inhibition of Erythrocyte Cell Membrane Scrambling Following Energy Depletion and Hyperosmotic Shock by Alectinib.

BACKGROUND/AIMS: The anaplastic lymphoma kinase (ALK) inhibitor alectinib is clinically used for the treatment of ALK positive non-small-cell lung cancer. At least in part the substance is effective by triggering suicidal death or apoptosis of tumor cells. Erythrocytes are lacking mitochondria and nuclei, key organelles of apoptosis but are, similar to apoptosis of nucleated cells, able to enter suicidal erythrocyte death or eryptosis. Stimulators of eryptosis include energy depletion, hyperosmotic shock, oxidative stress, and increase of cytosolic Ca2+ activity ([Ca2+]i). The present study explored, whether alectinib influences eryptosis. METHODS: Flow cytometry was employed to quantify phosphatidylserine exposure at the cell surface from annexin-V-binding and cell volume from forward scatter. Measurements were made without or with energy depletion (glucose deprivation for 48 hours), hyperosmotic shock (+550mM sucrose for 6 hours), oxidative stress (50 min exposure to 0.3 mM tert-butylhydroperoxide), and Ca2+ loading (60 minutes treatment with 1 µM Ca2+ ionophore ionomycin). RESULTS: A 48 hours exposure of human erythrocytes to alectinib (150-600 ng/ml) did not significantly modify the percentage of annexin-V-binding cells and forward scatter. Energy depletion, hyperosmotic shock, oxidative stress and Ca2+ loading were each followed by profound and significant increase of the percentage annexin-V-binding erythrocytes and a significant decrease of forward scatter. The effects of energy depletion and hyperosmotic shock, but not of oxidative stress or Ca2+ loading on annexin-V-binding were significantly blunted in the presence of alectinib (150-600 ng/ml). In none of the conditions was forward scatter significantly modified by alectinib. CONCLUSION: Alectinib inhibits cell membrane scrambling following energy depletion and hyperosmotic shock.

Phosphatidylserine expressing microvesicles in relation to microvascular complications in type 1 diabetes.

INTRODUCTION: Type 1 diabetes is a prothrombotic state strongly linked to vascular complications. The role of microvesicles (MVs) as mediators and potential biomarkers in microangiopathy in type 1 diabetes remains unclear. MATERIALS AND METHODS: MV levels in plasma samples from 106 patients with type 1 diabetes with microangiopathy, 130 patients without microangiopathy and 100 healthy controls were analysed using flow cytometry. Phosphatidylserine (PS) expression in MVs was assessed by lactadherin, and the ability of MVs to induce thrombin generation was investigated in vitro. Endogenous plasma lactadherin levels were measured using ELISA. RESULTS: Patients with type 1 diabetes had higher MV levels compared to healthy controls, with no significant differences between patients with and without microangiopathy. MV-induced thrombin generation in normal-pooled plasma was blocked by addition of lactadherin. Endogenous lactadherin levels were higher in patients compared to controls, and the highest levels were found in patients with microangiopathy. Plasma lactadherin levels did not correlate with levels of PS positive/negative MVs. CONCLUSION: Patients with type 1 diabetes with and without microangiopathy have higher levels of circulating MVs than healthy controls, probably reflecting higher cellular activation and turnover. However, we found no associations between clinical microangiopathy and levels of MVs in total or PS-expressing MVs. Plasma levels of lactadherin, which is a glycoprotein important in the clearance of cells and MVs, are increased in patients with type 1 diabetes and correlate with microangiopathy.

Polyhexamethylene biguanide and chloroquine induce programmed cell death in Acanthamoeba castellanii.

Several chemotherapeutic drugs have been described as amoebicidal agents acting against Acanthamoeba trophozoites and cysts. However, the underlying mechanism of action is poorly characterized. Here, we describe programmed cell death (PCD) in A. castellanii induced by polyhexamethylene biguanide (PHMB) and chloroquine. We used four types of amoebicidal agents including 0.02% PHMB, 0.02% chlorhexidine digluconate, 100 µM chloroquine, and 100 µM 2,6-dichlorobenzonitrile to kill Acanthamoeba trophozoites and cysts. Exposure to PHMB and chloroquine induced cell shrinkage and membrane blebbing in Acanthamoeba, observed microscopically. Externalization of phosphatidyl serine on the membranes of Acanthamoeba was detected by annexin V staining. Apoptotic cell death of Acanthamoeba by PHMB and chloroquine was confirmed by FACS analysis. Nuclear fragmentation of Acanthamoeba was demonstrated by DAPI staining. PHMB induced PCD in trophozoites and cysts, and chloroquine induced PCD in cysts. These findings are discussed to establish the most effective treatment for Acanthamoeba-induced keratitis.

Lipidomics reveals skin surface lipid abnormity in acne in young men.

BACKGROUND: Acne is a complex and multifactorial skin disorder. Alterations in skin surface lipid (SSL) are believed to be an important factor in the pathogenesis of acne and SSL plays a key role in the initiation of acne lesions. OBJECTIVES: To analyse the lipidome profiles of SSL in patients with acne and in healthy controls in order to understand SSL abnormity in acne in young men. METHODS: Ultraperformance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) and multivariate data analysis were used to investigate the SSL variations in main lipid classes, subclasses and individual species. RESULTS: Results showed that there were significant differences in the lipidome between the two groups. Significantly increased levels of three main classes of glycerophospholipids, fatty acyls and sterol lipids and significantly decreased levels of two main classes of prenol lipids and saccharolipids were observed in patients with acne. Subsequent analysis showed that there were 18 subclasses, which varied significantly and shared the same changing trends of the main classes to which they belonged. Multivariate data analysis indicated that 36 individual species were mostly responsible for this discrimination and the majority of differentiating lipid species were phosphatidylserines. Furthermore, it was observed that the chain length of ceramides were reduced and unsaturated free fatty acids were increased in patients with acne. CONCLUSIONS: SSL sampled from young male patients with acne had significantly higher levels of phosphatidylserines. Additionally, the reduction in the chain length of ceramides and the increase in unsaturated free fatty acids contributed to an altered lipid organization and decreased skin barrier function in acne.