Assessment of plasma amino acids, purines, tricarboxylic acid cycle metabolites, and lipids levels in NSCLC patients based on LC-MS/MS quantification.

Non-small cell lung cancer (NSCLC) is the most common type of malignant tumor of the lung with poor prognosis. Currently, there is still no effective strategy for diagnosing lung cancer from the perspective of multiple biomarkers containing both polar and nonpolar molecules. In order to explore the pathological changes of NSCLC at the endogenous molecule levels, and further establish the strategy for identifying and monitoring drug efficacy of NSCLC, targeted metabolomics and lipidomics studies were established with NSCLC patients. Polar metabolites including 21 amino acids, 7 purines, 6 tricarboxylic acid (TCA) cycle metabolites, and nonpolar lipids like phosphatidylcholine (PC), phosphatidylethanolamine (PE), lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE), sphingomyelin (SM), and ceramide (Cer), diacylglycerol (DG), triacylglycerol (TG), were quantitatively determined based on LC-MS/MS, taking into account their metabolism were significantly concerned with the occurrence of lung cancer in previous study. As a result, 14 polar metabolites and 16 lipids were prominently altered in the plasma of NSCLC patients, among which, after multivariate statistical analysis, LPC 18:0 (sn-2), L-Phenylalanine (Phe), oxaloacetic acid (OAA) and xanthine (XA) were screened out as potential small molecules and lipid biomarkers for NSCLC. Furthermore, a new strategy for formulating equation of NSCLC identification was proposed and clinical utility was successfully evaluated through Kangai injection treatment to NSCLC patients. Taking together, this study investigated the pathological changes of NSCLC from the perspective of endogenous polar and nonpolar molecules, and shed a light on identification of NSCLC.

Assessment of a combination of plasma anti-histone autoantibodies and PLA2/PE ratio as potential biomarkers to clinically predict autism spectrum disorders.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by deficiencies in social interaction and repetitive behaviors. Multiple studies have reported abnormal cell membrane composition and autoimmunity as known mechanisms associated with the etiopathogenesis of ASD. In this study, multiple regression and combined receiver operating characteristic (ROC) curve as statistic tools were done to clarify the relationship between phospholipase A2 and phosphatidylethanolamine (PE) ratio (PLA2/PE) as marker of lipid metabolism and membrane fluidity, and antihistone-autoantibodies as marker of autoimmunity in the etiopathology of ASD. Furthermore, the study intended to define the linear combination that maximizes the partial area under an ROC curve for a panel of markers. Forty five children with ASD and forty age- and sex-matched controls were enrolled in the study. Using ELISA, the levels of antihistone-autoantibodies, and PLA2 were measured in the plasma of both groups. PE was measured using HPLC. Statistical analyses using ROC curves and multiple and logistic regression models were performed. A notable rise in the area under the curve was detected using combined ROC curve models. Additionally, higher specificity and sensitivity of the combined markers were documented. The present study indicates that the measurement of the predictive value of selected biomarkers related to autoimmunity and lipid metabolism in children with ASD using a ROC curve analysis should lead to a better understanding of the pathophysiological mechanism of ASD and its link with metabolism. This information may enable the early diagnosis and intervention.

Neurotoxicity assessment of QoI strobilurin fungicides azoxystrobin and trifloxystrobin in human SH-SY5Y neuroblastoma cells: Insights from lipidomics and mitochondrial bioenergetics.

Strobilurin fungicides are quinone outside inhibitors (QoI) used to treat fungal pathogens for agricultural and residential use. Here, we compared the potential for neurotoxicity of the widely used strobilurins, azoxystrobin (AZS) and trifloxystrobin (TFS), in differentiated human SH-SY5Y cells. Fungicides did not include cytotoxicity up to 200 µM but both induced loss of cell viability at 48 h, with TFS showing slightly higher toxicity that AZS. Caspase 3/7 activity was induced in SH-SY5Y cells by both fungicides at 48 h (50 µM for AZS and 25 µM for TFS). ATP levels were reduced following a 24-hour exposure to > 25 µM AZS and > 6.25 µM TFS and both fungicides rapidly impaired oxidative respiration (~12.5 µM for AZS and ~3.125 µM TFS) and decreased oligomycin-induced ATP production, maximal respiration, and mitochondrial spare capacity. AZS at 100 µM showed a continual impairment of mitochondrial membrane potential (MMP) between 4 and 48 h while TFS at > 50 µM decreased MMP at 24 h. Taken together, TFS exerted higher mitochondrial toxicity at lower concentrations compared to AZS in SH-SY5Y cells. To discern toxicity mechanisms of strobilurin fungicides, lipidomics was conducted in SH-SY5Y cells following exposure to 6.25 µM and 25 µM AZS, and a total of 1595 lipids were detected, representing 49 different lipid classes. Lipid classes with the largest proportion of lipids detected in SH-SY5Y cells included triglycerides (17%), phosphatidylethanolamines (8%), ether-linked triglycerides (8%), phosphatidylcholines (7%), ether-linked phosphatidylethanolamines (6%), and diacylglycerols (5%). Together, these 5 lipid classes accounted for over 50% of the total lipids measured in SH-SY5Y cells. Lipids that were increased by AZS included acyl carnitine, which plays a role in long chain fatty acid utilization for mitochondrial ß-oxidation, as well as non-modified, ether linked, and oxidized triacylglycerols, suggesting compensatory upregulation of triglyceride biosynthesis. The ceramide HexCer-NS, linked to neurodegenerative diseases, was decreased in abundance following AZS exposure. In summary, strobilurin fungicides rapidly inhibit mitochondrial oxidative respiration and alter the abundance of several lipids in neuronal cells, relevant for understanding environmental exposure risks related to their neurotoxicity.

A novel experimental workflow to determine the impact of storage parameters on the mass spectrometric profiling and assessment of representative phosphatidylethanolamine lipids in mouse tissues.

Evaluation of signaling lipids is essential for measuring biological processes. There is a lack of experimental data regarding the proper storage of extracts for signaling lipid analysis, potentially impacting the procedures that can lead to accurate and reproducible evaluation. In this study, the importance of pre-analytical conditions for analyzing ion transitions for phosphatidylethanolamines (PEs), an abundant signaling phospholipid, was systematically assessed. A novel workflow was utilized involving an MRM-based experimental approach followed by statistical analysis. Specifically, lipids were extracted from the brain, heart, lungs, and serum of C57BL/6 mice. Extract subsets were resuspended in organic solvents prior to storage in various temperature conditions. Mass spectrometry analysis by multiple reaction monitoring (MRM) profiling was performed at four time points (1 day, 2 weeks, 2 months, or 6 months) to measure relative amounts of PEs in distinct lipid extract aliquots. We introduce an innovative statistical workflow to measure the changes in relative amounts of PEs in the profiles over time to determine lipid extract storage conditions in which fewer profile changes occur. Results demonstrated that time is the most significant factor affecting the changes in lipid samples, with temperature and solvent having comparatively minor effects. We conclude that for lipid extracts obtained by Bligh & Dyer extraction, storage at - 80.0 °C without solvent for less than 2 weeks before analysis is ideal. By considering the data generated by this study, lipid extract storage practices may be optimized and standardized, enhancing the validity and reproducibility of lipid assessments.

Differences in oxidative susceptibilities between glycerophosphocholine and glycerophosphoethanolamine in dried scallop (Argopecten irradians) adductor muscle during storage: an oxidation kinetic assessment.

BACKGROUND: Phospholipids, the main lipid component in marine shellfish, mainly comprise glycerophosphocholine (GPC) and glycerophosphoethanolamine (GPE). GPC and GPE in marine shellfish, especially scallop, carry n-3 long-chain polyunsaturated fatty acids, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), although different types of glycerophospholipids (GP) have different health benefits on human health. Moreover, different GP subclasses such as GPC and GPE have different oxidative susceptibilities in complex food systems. The present study compared the oxidative susceptibilities of GPC and GPE in dried scallop during storage by high-performance liquid chromatography-tandem mass spectrometry and kinetic models, and also investigated the effects of natural phenolic antioxidant on their susceptibilities. RESULTS: The results showed that GPC and GPE molecular species (carrying EPA or DHA) contents in samples continuously reduced during storage at two different temperatures. The first-order kinetic model better reflected the changes of GPC and GPE molecular species (carrying EPA or DHA) in samples than the zero-order kinetic model during storage. According to the oxidation rate (k) obtained from first-order kinetic models, GPE possessed a greater oxidation rate than GPC during storage. Moreover, the results showed that antioxidants of bamboo leaves (AOB, polar polyphenolic antioxidants) significantly decreased the oxidation rates of GPC and GPE molecular species (carrying EPA or DHA) in samples during storage, and GPC could be more effectively protected by AOB compared to GPE. CONCLUSION: The present study provides a practical method for accurately evaluating the oxidative susceptibility of different phospholipid classes in complex food systems. © 2020 Society of Chemical Industry.

Regiochemical Assignment of N-Acylphosphatidylethanolamines (NAPE) by Liquid Chromatography/Electrospray Ionization with Multistage Mass Spectrometry and Its Application to Extracts of Lupin Seeds.

1,2-Diacyl-sn-glycero-3-phospho-N-acyl-ethanolamines (NAPE) are low abundance phospholipids but important constituents of intracellular membranes of plant tissues, responsible for generating bioactive N-acylethanolamine (NAE), which participates in several physiological processes such as regulation of seed germination and protection against pathogenic attacks. From an analytical point of view, the critical aspect of these bioactive lipids lies in the determination of fatty acyl chains located in sn-1/sn-2 position on the glycerol backbone (O-linked), along with the amide-bound (N-linked) fatty acyl chain. Here, the identity and occurrence of NAPE in lipid extracts of lupin seeds (Lupinus luteus L.) was assessed by electrospray ionization in negative ion mode upon reversed-phase liquid chromatography (RPLC-ESI) coupled to mass spectrometry (MS) either at high- (i.e., Orbitrap FTMS) or low- (linear ion trap, LIT) resolution/accuracy. Collisional induced dissociation (CID)-tandem MS and MS3 acquisitions of chemically prepared NAPE allowed to unequivocally recognize the N-linked fatty acyl chain and to establish the diagnostic product ions that were successfully applied to identify NAPE in lipid extracts of yellow lupin seeds. The most abundant NAPE species were those containing N-acyl groups C18:1, C18:2; a minor prevalence was found for C16:0, C18:0, and C18:3, and almost the same acyl chains O-linked on the glycerol backbone in several sn-1/sn-2 combinations were observed. The positional isomers of NAPE species were identified as deprotonated molecules ([M-H]-) at m/z 978.7541 (three isomers 52:3), m/z 980.7694 (two isomers 52:2), m/z 1002.7535 (four isomers 54:5), m/z 1004.7686 (two isomers 54:4), m/z 1006.7837 (two isomers 54:3), and m/z 1008.8026 (single isomer 54:2). The total amount of NAPE in lupin seeds ranged in the interval of 2.00 ± 0.13 mg/g dw, in agreement with other edible legumes. We anticipate our approach to be a robust assessment method potentially applicable to biological extracts containing NAPE species and can provide comprehensive profiles and contents.

High-Speed Microscopy of Diffusion in Pore-Spanning Lipid Membranes.

Pore-spanning membranes (PSMs) provide a highly attractive model system for investigating fundamental processes in lipid bilayers. We measure and compare lipid diffusion in the supported and suspended regions of PSMs prepared on a microfabricated porous substrate. Although some properties of the suspended regions in PSMs have been characterized using fluorescence studies, it has not been possible to examine the mobility of membrane components on the supported membrane parts. Here, we resolve this issue by employing interferometric scattering microscopy (iSCAT). We study the location-dependent diffusion of DOPE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) lipids (DOPE) labeled with gold nanoparticles in (1,2-dioleoyl-sn-glycero-3-phosphocholine) (DOPC) bilayers prepared on holey silicon nitride substrates that were either (i) oxygen-plasma-treated or (ii) functionalized with gold and 6-mercapto-1-hexanol. For both substrate treatments, diffusion in regions suspended on pores with diameters of 5 µm is found to be free. In the case of functionalization with gold and 6-mercapto-1-hexanol, similar diffusion coefficients are obtained for both the suspended and the supported regions, whereas for oxygen-plasma-treated surfaces, diffusion is almost 4 times slower in the supported parts of the membranes. We attribute this reduced diffusion on the supported parts in the case of oxygen-plasma-treated surfaces to larger membrane-substrate interactions, which lead to a higher membrane tension in the freestanding membrane parts. Furthermore, we find clear indications for a decrease of the diffusion constant in the freestanding regions away from the pore center. We provide a detailed characterization of the diffusion behavior in these membrane systems and discuss future directions.

Assessment of dually labelled PEGylated liposomes transplacental passage and placental penetration using a combination of two ex-vivo human models: the dually perfused placenta and the suspended villous explants.

Uptake and passage of nanocarriers through the placenta are critical information to develop new therapeutic approaches during pregnancy. In order to assess nanocarriers transplacental passage and penetration into the placenta, we studied and optimized two ex-vivo human models: the dually perfused placenta and the placenta explants. Doubly labelled PEGylated liposomes were used as models to provide data on the penetration and transplacental passage of drugs and liposomes. A HPLC method was set-up to quantify both carboxyfluorescein and lipid-rhodamine. Transplacental passage was then quantified using HPLC and placental penetration was assessed using spinning disk microscopy. We found a similar transplacental passage rate for both free and encapsulated carboxyfluorescein as well as a homogeneous fluorescence intensity in the outer cell layer of the placental villous, the syncytiotrophoblast, and the mesenchyma. Besides, liposome-rhodamine was not detected in the fetal circulation. The absence of transplacental passage of PEGylated liposomes is also supported by their detection in the sole syncytiotrophoblast. The combination of two ex-vivo models and the monitoring of both the drug and the carrier provided consistent and complementary information. Overall, we suggest combining the perfused human placenta and the human explants villous models to evaluate nanocarriers designed for treatments during pregnancy.

MALDI-MS Patterning of Caspase Activities and Its Application in the Assessment of Drug Resistance.

Mass spectrometry (MS) has been widely used for enzyme activity assays. Herein, we propose a MALDI-MS patterning strategy for the convenient visual presentation of multiple enzyme activities with an easy-to-prepare chip. The array-based caspase-activity patterned chip (Casp-PC) is fabricated by hydrophobically assembling different phospholipid-tagged peptide substrates on a modified ITO slide. The advantages of amphipathic phospholipids lead to high-quality mass spectra for imaging analysis. Upon the respective cleavage of these substrates by different caspases, such as caspase-1, -2, -3, and -8, to produce a mass shift, the enzyme activities can be directly evaluated by MALDI-MS patterning by m/z-dependent imaging of the cleavage products. The ability to identify drug-sensitive/resistant cancer cells and assess the curative effects of anticancer drugs is demonstrated, indicating the applicability of the method and the designed chip.

Hepatotoxicity assessment of Mn-doped ZnS quantum dots after repeated administration in mice.

Doped ZnS quantum dots (QDs) have a longer dopant emission lifetime and potentially lower cytotoxicity compared to other doped QDs. The liver is the key organ for clearance and detoxification of xenobiotics by phagocytosis and metabolism. The present study was designed to synthesize and evaluate the hepatotoxicity of Mn-doped ZnS QDs and their polyethylene glycol-coated counterparts (1 mg/kg and 5 mg/kg) in mice. The results demonstrated that daily injection of Mn-doped ZnS QDs and polyethylene glycol-coated QDs via tail vein for 7 days did not influence body weight, relative liver weight, serum aminotransferases (alanine aminotransferase and aspartate aminotransferase), the levels of antioxidant enzymes (catalase, glutathione peroxidase, and superoxide dismutase), or malondialdehyde in the liver. Analysis of hepatocyte ultrastructure showed that Mn-doped ZnS QDs and polyethylene glycol-coated QDs mainly accumulated in mitochondria at 24 hours after repeated intravenous injection. No damage to cell nuclei or mitochondria was observed with either of the QDs. Our results indicate that Mn-doped ZnS QDs did not cause obvious damage to the liver. This study will assist in the development of Mn-doped ZnS QDs-based bioimaging and biomedical applications in the future.

Lifetime effectiveness of mifamurtide addition to chemotherapy in nonmetastatic and metastatic osteosarcoma: a Markov process model analysis.

The mortality and progression rates in osteosarcoma differ depending on the presence of metastasis. A decision model would be useful for estimating long-term effectiveness of treatment with limited clinical trial data. The aim of this study was to explore the lifetime effectiveness of the addition of mifamurtide to chemotherapy for patients with metastatic and nonmetastatic osteosarcoma. The target population was osteosarcoma patients with or without metastasis. A Markov process model was used, whose time horizon was lifetime with a starting age of 13 years. There were five health states: disease-free (DF), recurrence, post-recurrence disease-free, post-recurrence disease-progression, and death. Transition probabilities of the starting state, DF, were calculated from the INT-0133 clinical trials for chemotherapy with and without mifamurtide. Quality-adjusted life-years (QALY) increased upon addition of mifamurtide to chemotherapy by 10.5 % (10.13 and 9.17 QALY with and without mifamurtide, respectively) and 45.2 % (7.23 and 4.98 QALY with and without mifamurtide, respectively) relative to the lifetime effectiveness of chemotherapy in nonmetastatic and metastatic osteosarcoma, respectively. Life-years gained (LYG) increased by 10.1 % (13.10 LYG with mifamurtide and 11.90 LYG without mifamurtide) in nonmetastatic patients and 42.2 % (9.43 LYG with mifamurtide and 6.63 LYG without mifamurtide) in metastatic osteosarcoma patients. The Markov model analysis showed that chemotherapy with mifamurtide improved the lifetime effectiveness compared to chemotherapy alone in both nonmetastatic and metastatic osteosarcoma. Relative effectiveness of the therapy was higher in metastatic than nonmetastatic osteosarcoma over lifetime. However, absolute lifetime effectiveness was higher in nonmetastatic than metastatic osteosarcoma.

Orphan drugs revisited: cost-effectiveness analysis of the addition of mifamurtide to the conventional treatment of osteosarcoma.

INTRODUCTION: Mepact(®) (mifamurtide) is the first drug approved for the treatment of high-grade resectable non-metastatic osteosarcoma in patients aged 2-30 in the last 20 years. It follows a randomized clinical trial showing a statistically-significant and clinically-relevant decrease in the risk of death without compromising safety. AIM: This study assessed the cost-effectiveness and budget impact of mifamurtide. METHODS: The economic evaluation was done on a hypothetical cohort of young patients under the age of 30 with high-grade, non-metastatic, resectable osteosarcoma. Standard chemotherapy without mifamurtide was used as comparator. A Markov model was adapted using Spanish costs and the results of the INT-0133 Phase III study. The analysis has been carried out from the perspective of the Spanish National Health Service, with a time horizon of up to 60 years in the base analysis. RESULTS: The observed greater efficacy of mifamurtide in the trial translates into a gain of 3.03 (undiscounted) and 1.33 (discounted) quality-adjusted life years at an additional cost of €102,000. The estimated budgetary impact of using mifamurtide in 10-100% of the potential population would cost €671,000 and €6.7 million respectively. CONCLUSION: The cost per quality-adjusted life years gained with mifamurtide in Spain is in the low band (<€100,000) of the iCERs obtained by other orphan drugs and would have a limited and affordable cost in Spain.

High resolution in vivo 31P-MRS of the liver: potential advantages in the assessment of non-alcoholic fatty liver disease.

BACKGROUND: Biopsy remains the current gold-standard for assessing non-alcoholic fatty liver disease (NAFLD). To develop a non-invasive means of assessing the disease, 31P magnetic resonance spectroscopy (31P-MRS) has been explored, but the severe spectral overlaps and low signal-to-noise-ratio in 31P-MRS spectra at clinical field strength are clearly limiting factors. PURPOSE: To investigate potential advantages of high resolution in vivo 31P-MRS in assessing NAFLD. MATERIAL AND METHODS: The study was conducted at 9.4T in control and carbon tetrachloride (CCl4)-treated rats. Rats were divided according to histopathologic findings into a control group (n = 15), a non-alcoholic steatohepatitis group (n = 17), and a cirrhosis group (n = 12). Data were presented with different reference peaks that are commonly used for peak normalization such as total phosphorous signal, phosphomonoester + phosphodiester (PME + PDE), and nucleotide triphosphate (NTP). Then, multivariate analyses were performed. RESULTS: In all spectra PME and PDE were well resolved into phosphoethanolamine (PE) and phosphocholine (PC), and into glycerophosphorylethanolamine (GPE) and glycerophosphorylcholine (GPC), respectively. Those MRS measures quantifiable only in highly resolved spectra had higher correlations with histology than those conventional MRS measures such as PME, PDE, and NTP. The optimized partial least-squares discriminant analysis (PLS-DA) model correctly classified 79% (22/28) of the rats in the training set and correctly predicted 69% (11/16) of the rats in the test set. CONCLUSION: PE, PC, GPE, GPC, and nicotinamide adenine dinucleotide phosphate (NADP) that can be separately quantifiable in highly resolved spectra may further improve the potential efficacy of 31P-MRS in the diagnosis of NAFLD.

The addition of mifamurtide to chemotherapy improves lifetime effectiveness in children with osteosarcoma: a Markov model analysis.

In the absence of long-term clinical trials that compare mifamurtide plus chemotherapy versus chemotherapy only for treatment of osteosarcoma, decision analysis is a useful tool that helps to determine the optimal treatment strategy. We analyzed the differences between mifamurtide plus chemotherapy versus chemotherapy only by using modeling to determine the treatment approach that results in longer life expectancy among children with osteosarcoma. We used the Markov model to compare the expected lifetime quality-adjusted life years (QALYs) between mifamurtide plus chemotherapy versus chemotherapy only. Our target cohort consisted of children with osteosarcoma. The starting age of the cohort was 12 years and cycle length was 3 months. The transition probabilities for each disease state and death were calculated using overall survival or progression free survival data from randomized controlled trials. Utility weights from scenario-based survey for 303 Korean general populations were applied to the model. Based on the base case analysis, the incremental benefit analysis indicated that mifamurtide plus chemotherapy resulted in an incremental QALY increase of 1.57 (a relative increase of 16.3 % in QALY expectancy) compared to chemotherapy only. Also, the incremental life years gained (LYG) from mifamurtide plus chemotherapy was 1.96 on comparison with chemotherapy only; this is a relative increase of 15.7 % in LYG expectancy. The decision analysis model indicated that mifamurtide plus chemotherapy was associated with a substantially longer survival than chemotherapy only among children with osteosarcoma during their lifetime.

Mifamurtide for high-grade, resectable, nonmetastatic osteosarcoma following surgical resection: a cost-effectiveness analysis.

OBJECTIVES: Mifamurtide is an immune macrophage stimulant that when added to standard chemotherapy has demonstrated survival benefit for newly diagnosed osteosarcoma. The objectives of this study were to investigate the cost-effectiveness of adding mifamurtide to standard three- or four-agent chemotherapy for high-grade, resectable, nonmetastatic osteosarcoma following surgical resection and the issues of obtaining robust cost-effectiveness estimates for ultra-orphan drugs, given the shortage of data. METHODS: An economic evaluation was conducted from the perspective of the UK's National Health Service as part of the manufacturer's submission to the National Institute for Health and Care Excellence. The disease process was simplified to a transition through a series of health states, modeled by using a Markov approach. Data to inform the model were derived from patient-level data of Study INT-0133, published literature, and expert opinion. The final efficacy measure was life-years gained (LYG), and utilities were used to obtain quality-adjusted life-years (QALYs). RESULTS: For a 60-year time frame and a discount rate of 3.5% for outcomes, patients receiving mifamurtide benefited from an average additional 1.57 years of life and 1.34 QALYs, compared with patients receiving chemotherapy alone, giving an incremental cost-effectiveness ratio (ICER) of £58,737 per LYG and £68,734 per QALY. Because treatment effects were both substantial in restoring health and sustained over a very long period, the National Institute for Health and Care Excellence changed its guidance to allow a discount of 1.5% for outcomes to be applied in these special circumstances. By using this discount factor, it was found that patients receiving mifamurtide had an average additional 2.58 years of life and 2.20 QALYs compared with patients receiving chemotherapy alone, resulting in an ICER of £35,765 per LYG and £41,933 per QALY. CONCLUSION: Mifamurtide's ICER is cost-effective compared with that of other orphan and ultra-orphan drugs, for which prices and corresponding cost-effectiveness estimates are high.

External surface area determination of lipid vesicles using trinitrobenzene sulfonate and ultraviolet/visible spectrophotometry.

The lamellarity of liposomes is an important parameter to be controlled in liposomal delivery-release applications. A practical estimate of the degree of liposome lamellarity can be obtained by measuring the relative external surface area of the liposomes using a chemical assay. All such assays are based on a signal change caused by exposed marker lipids on reaction with a specific externally added reagent. However, a quantitative determination is often distorted by background reactions and contributions of internal lipid labeling. In the so-called TNBS assay, the marker lipid is phosphatidylethanolamine (PE) and the externally added reagent is TNBS (2,4,6-trinotrobenzene sulfonate). Mechanistic aspects of the TNBS assay were considered for improving the assay. Internal lipid labeling via PE flip-flop and/or TNBS permeation was minimal not only in cholesterol-containing liposomes but also in cholesterol-free liposomes if in the latter case membrane fluidity was decreased by slightly increasing the PE content. Compared with earlier versions of the TNBS assay, the amount of marker lipid and the time for analysis could be reduced considerably. The elaborated protocol was also applied to liposomes prepared from lipidic egg yolk isolates, offering a simple and inexpensive method for the development and in-process control of new liposome formation technologies.

The cost of living in the membrane: a case study of hydrophobic mismatch for the multi-segment protein LeuT.

Many observations of the role of the membrane in the function and organization of transmembrane (TM) proteins have been explained in terms of hydrophobic mismatch between the membrane and the inserted protein. For a quantitative investigation of this mechanism in the lipid-protein interactions of functionally relevant conformations adopted by a multi-TM segment protein, the bacterial leucine transporter (LeuT), we employed a novel method, Continuum-Molecular Dynamics (CTMD), that quantifies the energetics of hydrophobic mismatch by combining the elastic continuum theory of membrane deformations with an atomistic level description of the radially asymmetric membrane-protein interface from MD simulations. LeuT has been serving as a model for structure-function studies of the mammalian neurotransmitter:sodium symporters (NSSs), such as the dopamine and serotonin transporters, which are the subject of intense research in the field of neurotransmission. The membrane models in which LeuT was embedded for these studies were composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid, or 3:1 mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) lipids. The results show that deformation of the host membrane alone is not sufficient to alleviate the hydrophobic mismatch at specific residues of LeuT. The calculations reveal significant membrane thinning and water penetration due to the specific local polar environment produced by the charged K288 of TM7 in LeuT, that is membrane-facing deep inside the hydrophobic milieu of the membrane. This significant perturbation is shown to result in unfavorable polar-hydrophobic interactions at neighboring hydrophobic residues in TM1a and TM7. We show that all the effects attributed to the K288 residue (membrane thinning, water penetration, and the unfavorable polar-hydrophobic interactions at TM1a and TM7), are abolished in calculations with the K288A mutant. The involvement of hydrophobic mismatch is somewhat different in the functionally distinct conformations (outward-open, occluded, inward-open) of LeuT, and the differences are shown to connect to structural elements (e.g., TM1a) known to play key roles in transport. This finding suggests a mechanistic hypothesis for the enhanced transport activity observed for the K288A mutant, suggesting that the unfavorable hydrophobic-hydrophilic interactions hinder the motion of TM1a in the functionally relevant conformational transition to the inward-open state. Various extents of such unfavorable interactions, involving exposure to the lipid environment of adjacent hydrophobic and polar residues, are common in multi-segment transmembrane proteins, and must be considered to affect functionally relevant conformational transitions.

Growth and spectral assessment of Yb(3+)-doped KBaGd(MoO4)3 crystal: a candidate for ultrashort pulse and tunable lasers.

In order to explore new more powerful ultrashort pulse laser and tunable laser for diode-pumping, this paper reports the growth and spectral assessment of Yb(3+)-doped KBaGd(MoO(4))(3) crystal. An Yb(3+):KBaGd(MoO(4))(3) crystal with dimensions of 50×40×9 mm(3) was grown by the TSSG method from the K(2)Mo(2)O(7) flux. The investigated spectral properties indicated that Yb(3+):KBaGd(MoO(4))(3) crystal exhibits broad absorption and emission bands, except the large emission and gain cross-sections. This feature of the broad absorption and emission bands is not only suitable for the diode pumping, but also for the production of ultrashort pulses and tunability. Therefore, Yb(3+):KBaGd(MoO(4))(3) crystal can be regarded as a candidate for the ultrashort pulse and tunable lasers.

Structural elucidation of molecular species of pacific oyster ether amino phospholipids by normal-phase liquid chromatography/negative-ion electrospray ionization and quadrupole/multiple-stage linear ion-trap mass spectrometry.

Although marine oysters contain abundant amounts of ether-linked aminophospholipids, the structural identification of the various molecular species has not been reported. We developed a normal-phase silica liquid chromatography/negative-ion electrospray ionization/quadrupole multiple-stage linear ion-trap mass spectrometric (NPLC-NI-ESI/Q-TRAP-MS(3)) method for the structural elucidation of ether molecular species of serine and ethanolamine phospholipids from marine oysters. The major advantages of the approach are (i) to avoid incorrect selection of isobaric precursor ions derived from different phospholipid classes in a lipid mixture, and to generate informative and clear MS(n) product ion mass spectra of the species for the identification of the sn-1 plasmanyl or plasmenyl linkages, and (ii) to increase precursor ion intensities by "concentrating" lipid molecules of each phospholipid class for further structural determination of minor molecular species. Employing a combination of NPLC-NI-ESI/MS(3) and NPLC-NI-ESI/MS(2), we elucidated, for the first time, the chemical structures of docosahexaenoyl and eicosapentaenoyl plasmenyl phosphatidylserine (PS) species and differentiated up to six isobaric species of diacyl/alkylacyl/alkenylacyl phosphatidylethanolamine (PE) in the US pacific oysters. The presence of a high content of both omega-3 plasmenyl PS/plasmenyl PE species and multiple isobaric molecular species isomers is the noteworthy characteristic of the marine oyster. The simple and robust NPLC-NI-ESI/MS(n)-based methodology should be particularly valuable in the detailed characterization of marine lipid dietary supplements with respect to omega-3 aminophospholipids.

Assessment of the roles of ordered lipid microdomains in post-endocytic trafficking of glycosyl-phosphatidylinositol-anchored proteins in mammalian fibroblasts.

We have used artificial phosphatidylethanolamine-polyethylene glycol (PE-PEG)-anchored proteins, incorporated into living mammalian cells, to evaluate previously proposed roles for ordered lipid 'raft' domains in the post-endocytic trafficking of glycosylphosphatidylinositol (GPI)-anchored proteins in CHO and BHK cells. In CHO cells, endocytosed PE-PEG protein conjugates colocalized strongly with the internalized GPI-anchored folate receptor, concentrating in the endosomal recycling compartment, regardless of the structure of the hydrocarbon chains of the PE-PEG 'anchor'. However, internalized PE-PEG protein conjugates with long-chain saturated anchors recycled to the plasma membrane at a slow rate comparable to that measured for the GPI-anchored folate receptor, whereas conjugates with short-chain or unsaturated anchors recycled at a faster rate similar to that observed for the transferrin receptor. These findings support the proposal (Mayor et al. Cholesterol-dependent retention of GPI-anchored proteins in endosomes. EMBO J 1998;17:4628-4638) that the slow recycling of GPI proteins in CHO cells rests on their affinity for ordered lipid domains. In BHK cells, internalized PE-PEG protein conjugates with either saturated or unsaturated 'anchors' colocalized strongly with simultaneously endocytosed folate receptor and, like the folate receptor, gradually accumulated in late endosomes/lysosomes. These latter findings do not support previous suggestions that the sorting of GPI proteins to late endosomes in BHK cells depends on their association with lipid rafts.