ABSTRACT
INTRODUCTION: Onychomycosis is a fungal infection of the nails that can be challenging to treat. Here, matrix-assisted laser desorption ionization-Fourier transform ion cyclotron resonance (MALDI-FTICR) imaging was applied to the quantitative analysis of the penetration profile of the antifungal compound, amorolfine, in human mycotic toenails. The amorolfine profile was compared with those of three other antifungals, ciclopirox, naftifine, and tioconazole. METHODS: Antifungal compounds (amorolfine 5% lacquer, ciclopirox 8% lacquer, naftifine 1% solution, and tioconazole 28% solution) were applied to mycotic nails (n = 42). Nail sections were prepared, and MALDI-FTICR analysis was performed on the sections at a spatial resolution of 70 µm to compare the distribution profiles. Based on the minimum inhibitory concentrations of the four test compounds needed to kill 90% (MIC90) of the fungal organism, Trichophyton rubrum, the fold differences between the MIC90 and the antifungal concentrations in the nails (termed the multiplicity of the MIC90) were calculated for each. RESULTS: The penetration profiles indicated higher concentrations of amorolfine and ciclopirox in the deeper layers of the nails 3 h after treatment, compared with naftifine and tioconazole. The mean concentrations across the entire nail sections at 3 h were significantly different among the four antifungals: amorolfine, 2.46 mM; ciclopirox, 0.95 mM; naftifine, 0.63 mM; and tioconazole, 1.36 mM (p = 0.016; n = 8 per compound). The median multiplicity of the MIC90 at 3 h was 191-fold for amorolfine, tenfold for ciclopirox, 52-fold for naftifine, and 208-fold for tioconazole. CONCLUSION: In this study, MALDI-FTICR was successfully applied to the quantitative analysis of antifungal distribution in human mycotic nails. The findings suggest that amorolfine penetrates deeper layers of the nail and accumulates at concentrations far exceeding the MIC needed to exert antimycotic activity.
ABSTRACT
INTRODUCTION: Amorolfine 5% lacquer is an established topical treatment for fungal infection of the nails. The success of topical therapy for onychomycosis depends on whether the permeated drug concentration in the deep nail bed is retained above the effective antifungal minimum inhibitory concentration (MIC). We compared the penetration profile of amorolfine and a new topical formula of terbinafine in human mycotic toenails using matrix-assisted laser desorption ionization mass spectrometry imaging-Fourier transform ion cyclotron resonance (MALDI-FTICR) imaging. METHODS: Amorolfine 5% lacquer and terbinafine 7.8% lacquer were applied to mycotic nails (n = 17); nail sections were prepared, and MALDI-FTICR analysis was performed. Based on the MICs of amorolfine and terbinafine needed to kill 90% (MIC90) of Trichophyton rubrum, the fold differences between the MIC90 and the antifungal concentrations in the nails (the multiplicity of the MIC90) were calculated overall and for the keratin-unbound fractions. RESULTS: Both amorolfine and terbinafine penetrated the entire thickness of the nail. The mean concentration across the entire nail section 3 h following terbinafine treatment was 1414 µg/g of tissue (equivalent to 4.9 mM) compared with 780 µg/g (2.5 mM) following amorolfine treatment (not significantly different; p = 0.878). The median multiplicity of the MIC90 was significantly higher in amorolfine- than terbinafine-treated nails overall (191 vs. 48; p = 0.010) and for the keratin-unbound fractions only (7.4 vs. 0.8; p = 0.002). CONCLUSION: In this ex vivo study, MALDI-FTICR demonstrated that, although amorolfine 5% and terbinafine 7.8% had similar distribution profiles, both penetrating from the surface to the nail bed, the concentration of amorolfine in the nail was significantly higher than that of terbinafine relative to their respective MIC90 values. Clinical studies are required to determine whether these effects translate to a clinical difference in treatment success.
ABSTRACT
BACKGROUND: Lactic acid is a common active ingredient in many topical skincare products; however, measuring its delivery into the skin is challenging due to the presence of a large level of endogenous lactic acid. In this study, matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) was used to quantitatively and qualitatively measure the delivery of lactic acid into the skin from a range of topical skincare products. MATERIALS AND METHODS: Porcine skin samples were treated with various skincare products containing lactic acid. After 24 h, skin samples were sectioned and treated via H&E staining or prepared for MALDI-MSI using chemical derivatization. Samples were then analyzed by MALDI-MSI imaging to obtain lactic acid distribution in the entire skin section. RESULTS: Due to the high level of endogenous lactic acid in the skin, a "triple isotope" of lactic acid (L-Lactic acid-13 C3 ), was needed to provide full resolution from the skin's background signal with MALDI-MSI. With this approach, the topically delivered lactic acid could be quantitatively and qualitatively analyzed from a variety of skincare products. CONCLUSIONS: The combination of L-Lactic acid-13 C3 and MALDI-MSI was successfully used to quantitatively and qualitatively measure the topical delivery of lactic acid from a variety of skincare products. This approach could be used in future work to better understand the mode of action of lactic acid as an active ingredient in skincare products.
Subject(s)
Lactic Acid , Skin , Animals , Swine , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Skin/diagnostic imaging , Diagnostic ImagingABSTRACT
The lipid composition of the brain is well regulated during development, and the specific temporospatial distribution of various lipid species is essential for the development of optimal neural functions. Dietary lipids are the main source of brain lipids and thus contribute to the brain lipidome. Human milk is the only source of a dietary lipids for exclusively breastfed infant. Notably, it contains milk fat globule membrane (MFGM) enriched in polar lipids (PL). While early life is a key for early brain development, the interplay between dietary intake of polar lipids and spatial dynamics of lipid distribution during brain development is poorly understood. Here, we carried out an exploratory study to assess the early postnatal temporal profiling of brain lipidome between postnatal day (PND) 7 and PND 50 using matrix-assisted laser desorption ionization as a mass spectrometry imaging (MALDI-MSI) in an in vivo preclinical model. We also assessed the effect of chronic supplementation with PL extracted from alpha-lactalbumin-enriched whey protein concentrate (WPC) containing 10% lipids, including major lipid classes found in the brain (37% phospholipids and 15% sphingomyelin). MALDI-MSI of the spatial and temporal accretion of lipid species during brain development showed that the brain lipidome is changing heterogeneously along time during brain development. In addition, increases in 400+ PL supplement-dependent lipids were observed. PL supplementation had significant spatial and temporal effect on specific fatty esters, glycerophosphocholines, glycerophosphoethanolamines, and phosphosphingolipids. Interestingly, the average levels of these lipids per brain area tended to be constant in various brain structures across the age groups, paralleling the general brain growth. In contrast, other lipids, such as cytidine diphosphate diacylglycerol, diacylglycerophosphates, phosphocholines, specific ether-phosphoethanolamines, phosphosphingolipids, glycerophosphoinositols, and glycerophosphoserines showed clear age-dependent changes uncoupled from the general brain growth. These results suggest that the dietary PL supplementation may preferentially provide the building blocks for the general brain growth during development. Our findings add to the understanding of brain-nutrient relations, their temporospatial dynamics, and potential impact on neurodevelopment.
ABSTRACT
Hyaluronic acid (HA) is a major component of the skin, contributing to tissue hydration and biomechanical properties. As HA content in the skin decreases with age, formulas containing HA are widely used in cosmetics and HA injections in aesthetic procedures to reduce the signs of aging. To prove the beneficial effects of these treatments, efficient quantification of HA levels in the skin is necessary, but remains difficult. A new analytical method has been developed based on matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to quantify HA content in cross sections of human skin explants. A standardized and reproducible chemical entity (3 dimeric motifs or 6-mer) quantifiable by MALDI-MSI was produced by enzymatic hydrolysis using a specific hyaluronidase (H1136) in HA solution. This enzymatic digestion was carried out on skin sections before laser desorption, enabling the detection of HA. Histological coloration allowed us to localize the epidermis and the dermis on skin sections and, by comparison with the MALDI molecular image, to calculate the relative HA concentrations in these tissue areas. Skin explants were treated topically using a formula containing HA or its placebo, and the HA distribution profiles were compared with those obtained from untreated explants. A significant increase in HA was shown in each skin layer following topical application of the formula containing HA versus placebo and untreated samples (average of 126±40% and 92±40%, respectively). The MALDI-MSI technique enabled the quantification and localization of all HA macromolecules (endogenous and exogenous) on skin sections and could be useful for determining the efficacy of new cosmetic products designed to fight the signs of aging.
Subject(s)
Hyaluronic Acid , Skin , Epidermis , Humans , Hyaluronoglucosaminidase , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methodsABSTRACT
Impairment in cholesterol metabolism is associated with many neurodegenerative disorders including Alzheimer's disease (AD). However, the lipid alterations underlying neurodegeneration and the connection between altered cholesterol levels and AD remains not fully understood. We recently showed that cholesterol accumulation in hippocampal neurons, induced by silencing Cyp46a1 gene expression, leads to neurodegeneration with a progressive neuronal loss associated with AD-like phenotype in wild-type mice. We used a targeted and non-targeted lipidomics approach by liquid chromatography coupled to high-resolution mass spectrometry to further characterize lipid modifications associated to neurodegeneration and cholesterol accumulation induced by CYP46A1 inhibition. Hippocampus lipidome of normal mice was profiled 4 weeks after cholesterol accumulation due to Cyp46a1 gene expression down-regulation at the onset of neurodegeneration. We showed that major membrane lipids, sphingolipids and specific enzymes involved in phosphatidylcholine and sphingolipid metabolism, were rapidly increased in the hippocampus of AAV-shCYP46A1 injected mice. This lipid accumulation was associated with alterations in the lysosomal cargoe, accumulation of phagolysosomes and impairment of endosome-lysosome trafficking. Altogether, we demonstrated that inhibition of cholesterol 24-hydroxylase, key enzyme of cholesterol metabolism leads to a complex dysregulation of lipid homeostasis. Our results contribute to dissect the potential role of lipids in severe neurodegenerative diseases like AD.
ABSTRACT
BACKGROUND: Oxylipins are potent lipid mediators demonstrated to initiate and regulate inflammation yet little is known regarding their involvement in the response to surgical trauma. As key modulators of the inflammatory response, oxylipins have the potential to provide novel insights into the physiological response to surgery and the pathophysiology of post-operative complications. We aimed to investigate the effects of major surgery on longitudinal oxylipin profile. METHODS: Adults patients undergoing elective laparoscopic or open colorectal resections were included. Primary outcomes were serum oxylipin profile quantified by ultra high-performance liquid chromatography-mass spectrometry, serum white cell count and C-reactive protein concentration. Serum samples were taken at three time-points: pre-operative (day zero), early post-operative (day one) and late post-operative (day four/five). RESULTS: Some 55 patients were included, of which 33 (60%) underwent surgery that was completed laparoscopically. Pre-operative oxylipin profiles were characterised by marked heterogeneity but surgery induced a common shift resulting in more homogeneity at the early post-operative time-point. By the late post-operative phase, oxylipin profiles were again highly variable. This evolution was driven by time-dependent changes in specific oxylipins. Notably, the levels of several oxylipins with anti-inflammatory properties (15-HETE and four regioisomers of DHET) were reduced at the early post-operative point before returning to baseline by the late post-operative period. In addition, levels of the pro-inflammatory 11-HETE rose in the early post-operative phase while levels of anti-thrombotic mediators (9-HODE and 13-HODE) fell; concentrations of all three oxylipins then remained fairly static from early to late post-operative phases. Compared to those undergoing laparoscopic surgery, patients undergoing open surgery had lower levels of some anti-inflammatory oxylipins (8,9-DHET and 17-HDoHE) in addition to reduced concentrations of anti-thrombotic mediators (9-HODE and 13-HODE) with increased concentration of their pro-thrombotic counterpart (TxB2). CONCLUSIONS: Serum oxylipin profile is modified by surgical intervention and may even be sensitive to the degree of surgical trauma and therefore represents a novel descriptor of the surgical systemic inflammatory response.
Subject(s)
Inflammation/blood , Laparoscopy , Oxylipins/blood , Aged , C-Reactive Protein/metabolism , Demography , Fatty Acids, Unsaturated/blood , Female , Humans , Leukocyte Count , Longitudinal Studies , Male , Middle Aged , Principal Component Analysis , Time FactorsABSTRACT
Lipid mediators, highly bioactive compounds synthesized from polyunsaturated fatty acids (PUFAs), have a fundamental role in the initiation and signaling of the inflammatory response. Although extensively studied in isolation, only a limited number of analytical methods offer a comprehensive coverage of the oxylipin synthetic cascade applicable to a wide range of human biofluids. We report the development of an ultrahigh-performance liquid chromatography-electrospray ionization triple quadrupole mass spectrometry (UHPLC-MS) assay to quantify oxylipins and their PUFA precursors in 100 µL of human serum, plasma, urine, and cell culture supernatant. A single 15 min UHPLC run enables the quantification of 43 oxylipins and 5 PUFAs, covering pro and anti-inflammatory lipid mediators synthesized across the cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 (CYP450) pathways. The method was validated in multiple biofluid matrixes (serum, plasma, urine, and cell supernatant) and suppliers, ensuring its suitability for large scale metabonomic studies. The approach is accurate, precise, and reproducible (RSD < 15%) over multiple days and concentrations. Very high sensitivity is achieved with limits of quantification inferior to picograms for the majority of analytes (0.05-125 pg) and linear range spanning up to 5 orders of magnitude. This enabled the quantification of the great majority of these analytes at their low endogenous level in human biofluids. We successfully applied the procedure to individuals undergoing a fasting intervention; oxylipin profiles highlighted significantly altered PUFA and inflammatory profiles in accordance with previously published studies as well as offered new insight on the modulation of the biosynthetic cascade responsible for the regulation of oxylipins.
Subject(s)
Fatty Acids, Unsaturated/analysis , High-Throughput Screening Assays , Oxylipins/analysis , Cells, Cultured , Chromatography, High Pressure Liquid , Humans , Mass SpectrometryABSTRACT
The development of 'omics' sciences offers new opportunities for the study of neurodegenerative diseases but increases at the same time the sample demand on brain banks that collect and store valuable human post-mortem tissue. Our study aims to evaluate in lipidomics the potential of formalin-fixed tissue compared with the cryopreservation method, considered as the gold standard for biochemical research. Two complementary liquid chromatography/mass spectrometry analytical platforms were used on the basis of hybrid quadrupole time-of-flight and triple quadrupole mass spectrometers. Untargeted fingerprinting, semitargeted profiling of specific lipid classes and targeted monitoring of lipid species were performed in formalin-fixed and cryopreserved samples to provide detailed information at the molecular level on the formalin-induced alterations of the brain tissue. In vitro incubations of lipid standards were also performed to further describe the degradation processes induced by formaldehyde. Phospholipid compounds were found to be extensively hydrolysed, whilst the sphingolipid ones were preserved. N-methylation and N-formylation of amine-containing phospholipids have also been evidenced. These findings show that the potential detrimental effect of formalin on the analytes of interest must be taken into account when analysing formalin-fixed samples.
Subject(s)
Brain Chemistry/drug effects , Chromatography, High Pressure Liquid/methods , Formaldehyde/chemistry , Lipids/analysis , Mass Spectrometry/methods , Cryopreservation , Humans , Principal Component Analysis , Tissue FixationABSTRACT
Exploratory or untargeted ultra performance liquid chromatography-mass spectrometry (UPLC-MS) profiling offers an overview of the complex lipid species diversity present in blood plasma. Here, we evaluate and compare eight sample preparation protocols for optimized blood plasma lipid extraction and measurement by UPLC-MS lipid profiling, including four protein precipitation methods (i.e., methanol, acetonitrile, isopropanol, and isopropanol-acetonitrile) and four liquid-liquid extractions (i.e., methanol combined with chloroform, dichloromethane, and methyl-tert butyl ether and isopropanol with hexane). The eight methods were then benchmarked using a set of qualitative and quantitative criteria selected to warrant compliance with high-throughput analytical workflows: protein removal efficiency, selectivity, repeatability, and recovery efficiency of the sample preparation. We found that protein removal was more efficient by precipitation (99%) than extraction (95%). Additionally, isopropanol appeared to be the most straightforward and robust solvent (61.1% of features with coefficient of variation (CV) < 20%) while enabling a broad coverage and recovery of plasma lipid species. These results demonstrate that isopropanol precipitation is an excellent sample preparation procedure for high-throughput untargeted lipid profiling using UPLC-MS. Isopropanol precipitation is not limited to untargeted profiling and could also be of interest for targeted UPLC-MS/MS lipid analysis. Collectively, these data show that lipid profiling greatly benefits from an isopropanol precipitation in terms of simplicity, protein removal efficiency, repeatability, lipid recovery, and coverage.
Subject(s)
High-Throughput Screening Assays , Lipids/blood , Humans , PlasmaABSTRACT
Alzheimer's disease (AD) is a progressive brain disease that leads to an irreversible loss of neurons and cognition. It is the most common cause of dementia and can be considered as a major public health problem. At the histological level, AD is characterized by senile plaques and neurofibrillary tangles. Numerous studies involving genomic, transcriptomic and proteomic approaches have been published in order to understand the molecular mechanisms involved in AD, and to find new biomarkers. Metabolomics, and in particular lipidomics, have recently offered new possibilities due to the development of robust and sensitive analytical methods, such as LC-MS. This review aims to illustrate how lipidomics can help understand the biological mechanisms inherent to AD and how lipids can be considered as relevant biomarkers of AD at early stages.
Subject(s)
Alzheimer Disease/metabolism , Lipids/analysis , Alzheimer Disease/pathology , Animals , Biomarkers/analysis , Biomarkers/metabolism , Chromatography, Gas , Chromatography, High Pressure Liquid , Chromatography, Thin Layer , Glycolipids/analysis , Glycolipids/metabolism , Humans , Lipids/chemistry , Mass Spectrometry , Sphingolipids/analysis , Sphingolipids/metabolism , Sterols/analysis , Sterols/metabolismABSTRACT
The senile plaque is a hallmark lesion of Alzheimer disease (AD). We compared, without a priori, the lipidome of the senile plaques and of the adjacent plaque-free neuropil. The analysis by liquid chromatography coupled with electrospray ionization mass spectrometry revealed that laser microdissected senile plaques were enriched in saturated ceramides Cer(d18:1/18:0) and Cer(d18:1/20:0) by 33 and 78% respectively with respect to the surrounding neuropil. This accumulation of ceramides was not explained by their affinity for Aß deposits: no interaction between ceramide-liposomes and Aß fibrils was observed in vitro by surface plasmon resonance and fluorescent ceramide-liposomes showed no affinity for the senile plaques in AD brain tissue. Accumulation of ceramides could be, at least partially, the result of a local production by acid and neutral sphingomyelinases that we found to be present in the corona of the senile plaques.
Subject(s)
Alzheimer Disease/pathology , Ceramides/metabolism , Plaque, Amyloid/metabolism , Sphingomyelin Phosphodiesterase/metabolism , Aged , Aged, 80 and over , Alzheimer Disease/complications , Alzheimer Disease/genetics , Amyloid beta-Peptides/metabolism , Apolipoproteins E/genetics , Chromatography, Liquid , Female , Humans , Male , Mass Spectrometry , Microdissection , Middle Aged , Plaque, Amyloid/etiology , Surface Plasmon ResonanceABSTRACT
Cholesterol and oxysterols are involved as key compounds in the development of severe neurodegenerative diseases and in neuroinflammation processes. Monitoring their concentration changes under pathological conditions is of interest to get insights into the role of lipids in diseases. For numerous years, liquid chromatography coupled to mass spectrometry has been the method of choice for metabolites identification and quantification in biological samples. However, sterols and oxysterols are relatively apolar molecules poorly adapted to electrospray ionization (ESI). To circumvent this drawback, we developed a novel and robust analytical method involving derivatization of these analytes in cholesteryl N-4-(N,N-dimethylamino)phenyl carbamates under alkaline conditions followed by ultra-performance liquid chromatography-high resolution mass spectrometry analysis (UPLC-HRMS). Optimized UPLC conditions led to the separation of a mixture of 11 derivatized sterols and oxysterols especially side chain substituted derivatives after 6 min of chromatographic run. High sensitivity time-of-flight mass analysis allowed analytes to be detected in the nanomolar range. The method was validated for the analysis of oxysterols and sterols in mice brain in respect of linearity, limits of quantification, accuracy, precision, analyte stability, and recovery according to the Food and Drug Administration (FDA) guidelines. The developed method was successfully applied to investigate the impact of lipopolysaccharide (LPS) treatment on the rat cerebral steroidome.
Subject(s)
Brain Chemistry , Spectrometry, Mass, Electrospray Ionization/methods , Sterols/analysis , Animals , Brain/immunology , Carbamates/chemistry , Chromatography, High Pressure Liquid/methods , Inflammation/immunology , Lipopolysaccharides/immunology , Rats , Rats, Wistar , Sensitivity and Specificity , Sterols/immunologyABSTRACT
BACKGROUND: Lipidomic studies related to Alzheimer's disease have been reported on either biological fluids or large human brain samples. For a better understanding of the role of lipids, especially during the amyloid-ß peptide aggregation, it is crucial to determine the composition of the senile plaque versus the surrounding tissue, that is, the neuropil. RESULTS: A laser microdissection step was added to the analysis by UPLC-MS/MS. Despite the very low amount of sample, two phosphatidylcholines that were significantly depleted in the senile plaque were identified. CONCLUSION: Changes in the phospholipid content have been shown between senile plaque versus neuropil. Nano HPLC, allowing a complete lipidomic profile, should further improve the results.
Subject(s)
Alzheimer Disease/metabolism , Brain/metabolism , Choline/metabolism , Neuropil/metabolism , Phospholipids/metabolism , Plaque, Amyloid/metabolism , Aged , Aged, 80 and over , Alzheimer Disease/pathology , Brain/pathology , Brain Chemistry , Choline/analysis , Chromatography, High Pressure Liquid , Female , Humans , Immunohistochemistry , Male , Microdissection , Phospholipids/analysis , Plaque, Amyloid/chemistry , Tandem Mass SpectrometryABSTRACT
A comparison of electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI) for the analysis of a wide range of lipids has been performed on standard mixtures and extracts of Leishmania donovani promastigotes resistant to Amphotericin B (AmB). Calibration model, precision, limits of detection and quantification (LOD and LOQ) were assessed for each source. APPI provided the highest signal, signal-to-noise (S/N), and sensitivity for non-polar and low-polarity lipids, while ESI and APCI gave better results for the most polar ones. The linear model was valid for all lipids, except for one class with APPI, six classes with ESI, and eleven classes with APCI. LODs ranged from 0.2 to 20 µg mL(-1) for ESI, from 0.1 to 10 µg mL(-1) for APCI, and from 0.02 to 9.5 µg mL(-1) for APPI. LOQs ranged from 0.2 to 61 µg mL(-1) for ESI, from 0.4 to 31 µg mL(-1) for APCI, and from 0.1 to 29 µg mL(-1) for APPI. Each source provided similar lipid composition and variations in a comparison of three different L. donovani samples: miltefosine-treated, miltefosine-resistant and treated miltefosine-resistant parasites. A treated miltefosine-resistant sample was finally analyzed with each ion source in order to verify that the same lipid molecular species are detected.
Subject(s)
Leishmania donovani/chemistry , Lipids/analysis , Photochemistry/methods , Spectrometry, Mass, Electrospray Ionization/methods , Atmospheric Pressure , Drug Resistance , Leishmania donovani/drug effects , Leishmania donovani/metabolism , Limit of Detection , Lipids/chemistry , Metabolome/drug effects , Phosphorylcholine/analogs & derivatives , Phosphorylcholine/pharmacology , Reproducibility of Results , Signal-To-Noise RatioABSTRACT
In brome mosaic virus, both the replication of the genomic (+)-RNA strands and the transcription of the subgenomic RNA are carried out by the viral replicase. The production of (-)-RNA strands is dependent on the formation of an AUA triloop in the stem-loop C (SLC) hairpin in the 3'-untranslated region of the (+)-RNA strands. Two alternate hypotheses have been put forward for the mechanism of subgenomic RNA transcription. One posits that transcription commences by recognition of at least four key nucleotides in the subgenomic promoter by the replicase. The other posits that subgenomic transcription starts by binding of the replicase to a hairpin formed by the subgenomic promoter that resembles the minus strand promoter hairpin SLC. In this study, we have determined the three-dimensional structure of the subgenomic promoter hairpin using NMR spectroscopy. The data show that the hairpin is stable at 30°C and that it forms a pseudo-triloop structure with a transloop base pair and a nucleotide completely excluded from the helix. The transloop base pair is capped by an AUA triloop that possesses an extremely well packed structure very similar to that of the AUA triloop of SLC, including the formation of a so-called clamped-adenine motif. The similarities of the NMR structures of the hairpins required for genomic RNA and subgenomic RNA synthesis show that the replicase recognizes structure rather than sequence-specific motifs in both promoters.
Subject(s)
Bromovirus/genetics , Genome, Viral , Inverted Repeat Sequences , Promoter Regions, Genetic , RNA, Viral/chemistry , Base Pairing , Base Sequence , Models, Molecular , Mutation , Nuclear Magnetic Resonance, Biomolecular , Nucleic Acid Conformation , ThermodynamicsABSTRACT
Lipidomic studies often use liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) for separation, identification, and quantification. However, due to the wide structural diversity of lipids, the most apolar part of the lipidome is often detected with low sensitivity in ESI. Atmospheric pressure (APPI) can be an alternative ionization source since normal-phase solvents are known to enhance photoionization of these classes. In this paper, we intend to show the efficiency of APPI to identify different lipid classes, with a special interest on sphingolipids. In-source APPI fragmentation appears to be an added value for the structural analysis of lipids. It provides a detailed characterization of both the polar head and the non polar moiety of most lipid classes, and it makes possible the detection of all lipids in both polarities, which is not always possible with ESI.