LIPID MAPS: update to databases and tools for the lipidomics community.

LIPID MAPS (LIPID Metabolites and Pathways Strategy), www.lipidmaps.org, provides a systematic and standardized approach to organizing lipid structural and biochemical data. Founded 20 years ago, the LIPID MAPS nomenclature and classification has become the accepted community standard. LIPID MAPS provides databases for cataloging and identifying lipids at varying levels of characterization in addition to numerous software tools and educational resources, and became an ELIXIR-UK data resource in 2020. This paper describes the expansion of existing databases in LIPID MAPS, including richer metadata with literature provenance, taxonomic data and improved interoperability to facilitate FAIR compliance. A joint project funded by ELIXIR-UK, in collaboration with WikiPathways, curates and hosts pathway data, and annotates lipids in the context of their biochemical pathways. Updated features of the search infrastructure are described along with implementation of programmatic access via API and SPARQL. New lipid-specific databases have been developed and provision of lipidomics tools to the community has been updated. Training and engagement have been expanded with webinars, podcasts and an online training school.

Methods for the relative quantitation of human plasma lipidome using liquid chromatography coupled with mass spectrometry using minimal sample manipulation.

Extraction protocols and liquid chromatography coupled with mass spectrometry (LC-MS) and tandem mass spectrometry (LC-MS/MS) methods for the measurement of four lipid categories, namely glycerophospholipids, glycerolipids, sphingolipids and sterol lipids in human plasma, are described here. Step-by-step instructions are provided for the liquid-liquid extraction methods, including solution preparation and the non-endogenous lipid internal standards used. All instrumental conditions, chromatography columns and solutions required for the LC-MS and LC-MS/MS methods are also provided in detail.

Comprehensive lipidome of human plasma using minimal sample manipulation by liquid chromatography coupled with mass spectrometry.

RATIONALE: The present work shows comprehensive chromatographic methods and MS conditions that have been developed based on the chemical properties of each lipid subclass to detect low-abundance molecular species. This study shows that the developed methods can detect low- and/or very-low-abundant lipids like phosphatidic acid (PA) in the glycerophospholipid (GP) method; dihydroceramide (dhCer) and dihydrosphingosine/sphinganine (dhSPB) in the sphingolipid (SP) method; and lysophosphatidic acid (LPA), LPI, LPG and sphingosine-1-phosphate (SPBP) in the lysolipid method. METHODS: An optimised method for the extraction of lysolipids in plasma is used in addition to Folch extraction. Then, four chromatographic methods coupled with mass spectrometry using targeted and untargeted approaches are described here. Three of the methods use a tertiary pumping system to enable the inclusion of a gradient for analyte separation (pumps A and B) and an isocratic wash (pump C). This wash solution elutes interfering compounds that could cause background signal in the subsequent injections, reducing column lifetime. RESULTS: Semi-quantitative values for 37 lipid subclasses are reported for a plasma sample (NIST SRM 1950). Furthermore, the methods presented here enabled the identification of 338 different lipid molecular species for GPs (mono- and diacyl-phospholipds), SPs, sterols and glycerolipids. The methods have been validated, and the reproducibility is presented here. CONCLUSIONS: The comprehensive analysis of the lipidome addressed here of glycerolipids, GPs, sterols and SPs is in good agreement with previously reported results, in the NIST SRM 1950 sample, by other laboratories. Ten lipid subclasses LPS, LPI, alkyl-lysophosphatidic acid/alkenyl-lysophosphatidic acid, alkyl-lysophosphatidylethanolamine/alkenyl-lysophosphatidylethanolamine, dhCer (d18:0), SPB (d18:1), dhSPB (d18:0) and SPBP (d18:2) have been detected using this comprehensive method and are uniquely reported here.

Guiding the choice of informatics software and tools for lipidomics research applications.

Progress in mass spectrometry lipidomics has led to a rapid proliferation of studies across biology and biomedicine. These generate extremely large raw datasets requiring sophisticated solutions to support automated data processing. To address this, numerous software tools have been developed and tailored for specific tasks. However, for researchers, deciding which approach best suits their application relies on ad hoc testing, which is inefficient and time consuming. Here we first review the data processing pipeline, summarizing the scope of available tools. Next, to support researchers, LIPID MAPS provides an interactive online portal listing open-access tools with a graphical user interface. This guides users towards appropriate solutions within major areas in data processing, including (1) lipid-oriented databases, (2) mass spectrometry data repositories, (3) analysis of targeted lipidomics datasets, (4) lipid identification and (5) quantification from untargeted lipidomics datasets, (6) statistical analysis and visualization, and (7) data integration solutions. Detailed descriptions of functions and requirements are provided to guide customized data analysis workflows.

The Effect of a Pex3 Mutation on Hearing and Lipid Content of the Inner Ear.

Peroxisome biogenesis disorders (due to PEX gene mutations) are associated with symptoms that range in severity and can lead to early childhood death, but a common feature is hearing impairment. In this study, mice carrying Pex3 mutations were found to show normal auditory development followed by an early-onset progressive increase in auditory response thresholds. The only structural defect detected in the cochlea at four weeks old was the disruption of synapses below inner hair cells. A conditional approach was used to establish that Pex3 expression is required locally within the cochlea for normal hearing, rather than hearing loss being due to systemic effects. A lipidomics analysis of the inner ear revealed a local reduction in plasmalogens in the Pex3 mouse mutants, comparable to the systemic plasmalogen reduction reported in human peroxisome biogenesis disorders. Thus, mice with Pex3 mutations may be a useful tool to understand the physiological basis of peroxisome biogenesis disorders.

Caracterización de la apnea del sueño en pacientes cardiópatas e internación por enfermedad grave por COVID-19 / Characterization of sleep apnea and hospitalization for severe illness due to COVID-19

La COVID-19 ha generado mundialmente una morbi-mortalidad considerable, particularmente entre aquellos con comorbilidades crónicas: hipertensión, diabetes y enfermedad cardiovascular. Investigaciones han demostrado que la apnea del sueño puede agravar el pronóstico vital, al causar o agudizar la disfunción endotelial, inflamación, estrés oxidativo, microaspiración y lesiones pulmonares, lo que amplifica el riesgo de hospitalización e incluso de sufrir de insuficiencia respiratoria. Se realizó un estudio descriptivo retrospectivo y transversal, se evaluaron 187 pacientes cardiópatas con clínica sugestiva a apnea obstructiva del sueño con resultaron positivo para SARS-CoV-2, entre 2020-2021.Se aplicaron la metodología de Castro et al., 2021 y cuestionario de STOP-BANG para valorar la disnea y caracterizar la AOS respectivamente. Se corroboró la fuerte asociación entre la enfermedad cardíaca y la presencia de AOS; más de la mitad de los pacientes estudiados presentaron, enfermedad grave por COVID-19, con disnea moderada-grave, que amerito hospitalización con cuidados intensivos, observándose mayor frecuencia en el sexo masculino, con más de 50 años y con IMC >30. Sin embargo, las féminas presentaron valores significativos de STOP-BANG, lo que sugiere a la HTA y obesidad como factores de riesgo para AOS, independiente al sexo. Se recomienda realizar el descarte de AOS como rutina médica, que permita establecer la epidemiología y estrategias para abordaje adecuado de estos pacientes(AU)

COVID-19 has generated considerable morbidity and mortality worldwide, particularly among those with chronic comorbidities: hypertension, diabetes, and cardiovascular disease. Research has shown that sleep apnea can worsen the vital prognosis, by causing or exacerbating endothelial dysfunction, inflammation, oxidative stress, microaspiration and lung damage, which amplifies the risk of hospitalization and even respiratory failure. A retrospective and cross-sectional descriptive study was carried out, 187 heart patients with clinical signs suggestive of obstructive sleep apnea were evaluated and were positive for SARS-CoV-2, between 2020-2021. The methodology of Castro et al., 2021 and questionnaire were applied. STOP-BANG to assess dyspnea and characterize OSA, respectively. The strong association between heart disease and the presence of OSA was confirmed; More than half of the patients studied presented severe disease due to COVID-19, with moderate-severe dyspnea, which required hospitalization with intensive care, with a higher frequency observed in males, over 50 years of age and with BMI >30. However, females presented significant STOP-BANG values, which suggests hypertension and obesity as risk factors for OSA, regardless of gender. It is recommended to rule out OSA as a medical routine, which allows establishing the epidemiology and strategies for an adequate approach to these patients(AU)

Neuronal HSF-1 coordinates the propagation of fat desaturation across tissues to enable adaptation to high temperatures in C. elegans.

To survive elevated temperatures, ectotherms adjust the fluidity of membranes by fine-tuning lipid desaturation levels in a process previously described to be cell autonomous. We have discovered that, in Caenorhabditis elegans, neuronal heat shock factor 1 (HSF-1), the conserved master regulator of the heat shock response (HSR), causes extensive fat remodeling in peripheral tissues. These changes include a decrease in fat desaturase and acid lipase expression in the intestine and a global shift in the saturation levels of plasma membrane's phospholipids. The observed remodeling of plasma membrane is in line with ectothermic adaptive responses and gives worms a cumulative advantage to warm temperatures. We have determined that at least 6 TAX-2/TAX-4 cyclic guanosine monophosphate (cGMP) gated channel expressing sensory neurons, and transforming growth factor ß (TGF-ß)/bone morphogenetic protein (BMP) are required for signaling across tissues to modulate fat desaturation. We also find neuronal hsf-1 is not only sufficient but also partially necessary to control the fat remodeling response and for survival at warm temperatures. This is the first study to show that a thermostat-based mechanism can cell nonautonomously coordinate membrane saturation and composition across tissues in a multicellular animal.

Local Fractal Connections to Characterize the Spatial Processes of Deforestation in the Ecuadorian Amazon.

Deforestation by human activities is a common issue in Amazonian countries. This occurs at different spatial and temporal scales causing primary forest loss and land fragmentation issues. During the deforestation process as the forest loses connectivity, the deforested patches create new intricate connections, which in turn create complex networks. In this study, we analyzed the local connected fractal dimension (LCFD) of the deforestation process in the Sumaco Biosphere Reserve (SBR) with two segmentation methods, -CA-wavelet and K-means-to categorize the complexity of deforested patches' connections and then relate these with the spatial processes. The results showed an agreement with both methods, in which LCFD values below 1 corresponded to isolated patches with simple shapes and those above 1 signified more complex and connected patches. From CA-wavelet a threshold of 1.57 was detected allowing us to identify and discern low and high land transformation, while the threshold for K-means was 1.61. Both values represent the region from which deforestation performs local aggressive expansion networks. The thresholds were used to map the LCFD in which all spatial processes were visually detected. However, the threshold of 1.6 ± 0.03 was more effective in discerning high land transformation. such as shrinkage and attrition, in the deforestation process in the SBR.

Integrated lipidomics and proteomics reveal cardiolipin alterations, upregulation of HADHA and long chain fatty acids in pancreatic cancer stem cells.

Pancreatic cancer stem cells (PCSCs) play a key role in the aggressiveness of pancreatic ductal adenocarcinomas (PDAC); however, little is known about their signaling and metabolic pathways. Here we show that PCSCs have specific and common proteome and lipidome modulations. PCSCs displayed downregulation of lactate dehydrogenase A chain, and upregulation of trifunctional enzyme subunit alpha. The upregulated proteins of PCSCs are mainly involved in fatty acid (FA) elongation and biosynthesis of unsaturated FAs. Accordingly, lipidomics reveals an increase in long and very long-chain unsaturated FAs, which are products of fatty acid elongase-5 predicted as a key gene. Moreover, lipidomics showed the induction in PCSCs of molecular species of cardiolipin with mixed incorporation of 16:0, 18:1, and 18:2 acyl chains. Our data indicate a crucial role of FA elongation and alteration in cardiolipin acyl chain composition in PCSCs, representing attractive therapeutic targets in PDAC.

BioPAN: a web-based tool to explore mammalian lipidome metabolic pathways on LIPID MAPS.

Lipidomics increasingly describes the quantification using mass spectrometry of all lipids present in a biological sample.  As the power of lipidomics protocols increase, thousands of lipid molecular species from multiple categories can now be profiled in a single experiment.  Observed changes due to biological differences often encompass large numbers of structurally-related lipids, with these being regulated by enzymes from well-known metabolic pathways.  As lipidomics datasets increase in complexity, the interpretation of their results becomes more challenging.  BioPAN addresses this by enabling the researcher to visualise quantitative lipidomics data in the context of known biosynthetic pathways.  BioPAN provides a list of genes, which could be involved in the activation or suppression of enzymes catalysing lipid metabolism in mammalian tissues.

Non-canonical autophagy drives alternative ATG8 conjugation to phosphatidylserine.

Autophagy is a fundamental catabolic process that uses a unique post-translational modification, the conjugation of ATG8 protein to phosphatidylethanolamine (PE). ATG8 lipidation also occurs during non-canonical autophagy, a parallel pathway involving conjugation of ATG8 to single membranes (CASM) at endolysosomal compartments, with key functions in immunity, vision, and neurobiology. It is widely assumed that CASM involves the same conjugation of ATG8 to PE, but this has not been formally tested. Here, we discover that all ATG8s can also undergo alternative lipidation to phosphatidylserine (PS) during CASM, induced pharmacologically, by LC3-associated phagocytosis or influenza A virus infection, in mammalian cells. Importantly, ATG8-PS and ATG8-PE adducts are differentially delipidated by the ATG4 family and bear different cellular dynamics, indicating significant molecular distinctions. These results provide important insights into autophagy signaling, revealing an alternative form of the hallmark ATG8 lipidation event. Furthermore, ATG8-PS provides a specific "molecular signature" for the non-canonical autophagy pathway.

Espirometría forzada en estudiantes de 18 a 25 años de la Universidad de Guayaquil: Valores referenciales a considerar en la post pandemia COVID 19 / Forced spirometry in students aged 18 to 25 at the University of Guayaquil: Reference values to consider in the post-COVID 19 pandemic

Espirometría Forzada (EF), técnica focalizada en explorar la función ventilatoria pulmonar medida a partir de la cantidad de aire que pueden retener durante el simulacro de espiración obligada. Objetivo: determinar los valores de Volumen Espiratorio Forzado en el primer segundo (FEV1) y Capacidad Vital Forzada (FVC) normales/estándar en estudiantes de la Universidad de Guayaquil, entre 18 a 25 años de edad, identificando los principales factores de riesgo causantes de la disminución de la función pulmonar, comparables en la postpandemia COVID 19. Estudio epidemiológico de corte trasversal desarrollado durante Octubre 2018 - Marzo 2019. De un universo de 63.000 estudiantes, la muestra seleccionada al azar según los criterios de inclusión de Mart Elkin fueron 715 estudiantes de Administración, Ciencias Médicas y Jurisprudencia. La técnica de recolección de datos fue la guía de observación, siendo tabulados en Microsoft Excel2010, aplicando estadística descriptiva con medidas de tendencia central. Resultados reflejaron que 83,644% oscila entre 18 y 23 años, un 50,77% fueron del sexo femenino siendo este grupo etario el de menor estatura de los grupos estudiados. El 32,87% tiene sobrepeso u obesidad. Aunque el 75% no tiene el hábito de fumar, Ciencias Médicas mostró 0,44% de los fumadores. La escala de severidad FVC es normal ubicándose >80 con coeficiente FVC1/ FVC para ambos sexos. Los valores espirométricos permiten decidir a partir de criterios clínicos cuál, cuándo y por qué aplicar una terapia respiratoria lo que constituye un recurso valioso en tiempos de postpandemia Covid 19.

Forced Spirometry (PE), a technique focused on exploring pulmonary ventilation function measured from the amount of air that can be retained during forced expiration simulation. Objective: to determine the normal / standard Forced Expiratory Volume in the first second (FEV1) and Forced Vital Capacity (FVC) values in students of the University of Guayaquil, between 18 and 25 years of age, identifying the main risk factors causing the decrease in lung function, comparable in the post-pandemic COVID 19. Epidemiological cross-sectional study developed during October 2018 - March 2019. From a universe of 63,000 students, the randomly selected sample according to the inclusion criteria of Mart Elkin was 715 students of Administration, Medical Sciences and Jurisprudence. The data collection technique was the observation guide, being tabulated in Microsoft Excel2010, applying descriptive statistics with measures of central tendency. Results reflected in that 83.644% ranged between 18 and 23 years, 50.77% were female, this age group being the smallest of the groups studied. 32.87% are overweight or obese. Although 75% do not have the habit of smoking, Medical Sciences showed 0.44% of smokers. The FVC severity scale is normal, being> 80 with a FVC1 / FVC coefficient for both sexes. Spirometric values make it possible to decide based on clinical criteria which, when and why to apply respiratory therapy, which constitutes a valuable resource in times of post-Covid 19 pandemic.

Intubación rápida en pacientes críticos COVID-19 en Guayaquil Ecuador, 2020 / Rapid intubation in critical COVID-19 patients in Guayaquil Ecuador, 2020

La neumonía COVID-19, puede condicir a insuficiencia respiratoria aguda, en pacientes críticos se requiera de la intubación orotraqueal con la finalidad de aislar la vía aérea y permitir la protección y el con¬trol o asistencia de la ventilación, siendo la secuencia rápida una alternativa para garantizar el éxito terapéutico y seguridad del personal. Bajo estudio prospectivo de cohorte, desde junio a diciembre de 2020, en pacientes con diagnóstico confirmado de SARS-CoV2, ingresados en la Unidad de Cuidados Intensivos, se incluyeron 72, 45 fueron masculino (62,50%) y 27 femeninos (37,50%), con un rango de edad entre 27 a 64 años, siendo el grupo mas vulnerable de 55 a 64 años con un 59,72% (43/72). La maniobra fue excelente en 47 ocasiones (65,28+12,28%), buena en 25 (31,94+6,04%) y en dos oportuniddes imposible, la secuencia de Fentanilo-propofol succinilcolina para la inducción rápida se ejecuto en el 50,00%, con efectividad de 100,00%; sin embargo, no hubo diferencia con las otras secuencias. Antes de proceder a la inducción farmacológica de la inconsciencia y del bloqueo neuromuscular, es necesario evaluar minuciosamente ciertos aspectos de la historia clínica y del examen físico del paciente(AU)

COVID-19 pneumonia can lead to acute respiratory failure, in critical patients orotracheal intubation is required in order to isolate the airway and allow protection and control or assistance of ventilation, the rapid sequence being an alternative to guarantee therapeutic success and staff safety. Under a prospective cohort study, from June to December 2020, in patients with a confirmed diagnosis of SARS-CoV2, admitted to the Intensive Care Unit, 72 were included, 45 were male (62.50%) and 27 were female (37, 50%), with an age range between 27 to 64 years, the most vulnerable group being 55 to 64 years with 59.72% (43/72). The maneuver was excellent on 47 occasions (65.28 + 12.28%), good on 25 (31.94 + 6.04%) and on two occasions impossible, the Fentanyl-propofol succinylcholine sequence for rapid induction was performed in 50.00%, with effectiveness of 100.00%; however, there was no difference with the other sequences. Before proceeding with the pharmacological induction of unconsciousness and neuromuscular blockade, it is necessary to carefully evaluate certain aspects of the patient's medical history and physical examination(AU)

THP-1 macrophage cholesterol efflux is impaired by palmitoleate through Akt activation.

Lipoprotein lipase (LPL) is upregulated in atherosclerotic lesions and it may promote the progression of atherosclerosis, but the mechanisms behind this process are not completely understood. We previously showed that the phosphorylation of Akt within THP-1 macrophages is increased in response to the lipid hydrolysis products generated by LPL from total lipoproteins. Notably, the free fatty acid (FFA) component was responsible for this effect. In the present study, we aimed to reveal more detail as to how the FFA component may affect Akt signalling. We show that the phosphorylation of Akt within THP-1 macrophages increases with total FFA concentration and that phosphorylation is elevated up to 18 hours. We further show that specifically the palmitoleate component of the total FFA affects Akt phosphorylation. This is tied with changes to the levels of select molecular species of phosphoinositides. We further show that the total FFA component, and specifically palmitoleate, reduces apolipoprotein A-I-mediated cholesterol efflux, and that the reduction can be reversed in the presence of the Akt inhibitor MK-2206. Overall, our data support a negative role for the FFA component of lipoprotein hydrolysis products generated by LPL, by impairing macrophage cholesterol efflux via Akt activation.

Structural Analysis of 14-3-3-ζ-Derived Phosphopeptides Using Electron Capture Dissociation Mass Spectrometry, Traveling Wave Ion Mobility Spectrometry, and Molecular Modeling.

Previously, we have demonstrated the effect of salt bridges on the electron capture dissociation mass spectrometry behavior of synthetic model phosphopeptides and applied an ion mobility spectrometry/molecular modeling approach to rationalize the findings in terms of peptide ion structure. Here, we develop and apply the approach to a biologically derived phosphopeptide. Specifically, we have investigated variants of a 15-mer phosphopeptide VVGARRSsWRVVSSI (s denotes phosphorylated Ser) derived from Akt1 substrate 14-3-3-ζ, which contains the phosphorylation motif RRSsWR. Variants were generated by successive arginine-to-leucine substitutions within the phosphorylation motif. ECD fragmentation patterns for the eight phosphopeptide variants show greater sequence coverage with successive R → L substitutions. Peptides with two or more basic residues had regions with no sequence coverage, while full sequence coverage was observed for peptides with one or no basic residues. For three of the peptide variants, low-abundance fragments were observed between the phosphoserine and a basic residue, possibly due to the presence of multiple conformers with and without noncovalent interactions between these residues. For the five variants whose dissociation behavior suggested the presence of intramolecular noncovalent interactions, we employed ion mobility spectrometry and molecular modeling to probe the nature of these interactions. Our workflow allowed us to propose candidate structures whose noncovalent interactions were consistent with the ECD data for all of the peptides modeled. Additionally, the AMBER parameter sets created for and validated by this work are presented and made available online ( http://www.biosciences-labs.bham.ac.uk/cooper/datasets.php ).

The circadian clock components BMAL1 and REV-ERBα regulate flavivirus replication.

The circadian clock regulates immune responses to microbes and affects pathogen replication, but the underlying molecular mechanisms are not well understood. Here we demonstrate that the circadian components BMAL1 and REV-ERBα influence several steps in the hepatitis C virus (HCV) life cycle, including particle entry into hepatocytes and RNA genome replication. Genetic knock out of Bmal1 and over-expression or activation of REV-ERB with synthetic agonists inhibits the replication of HCV and the related flaviruses dengue and Zika via perturbation of lipid signaling pathways. This study highlights a role for the circadian clock component REV-ERBα in regulating flavivirus replication.

Structural analysis of peptides modified with organo-iridium complexes, opportunities from multi-mode fragmentation.

The most widely used anticancer drugs are platinum complexes, but complexes of other transition metals also show promise and may widen the spectrum of activity, reduce side-effects, and overcome resistance. The latter include organo-iridium(iii) 'piano-stool' complexes. To understand their mechanism of action, it is important to discover how they bind to biomolecules and how binding is affected by functionalisation of the ligands bound to iridium. We have characterised, by MS and MS/MS techniques, unusual adducts from reactions between 3 novel iridium(iii) anti-cancer complexes each possessing reactive sites both at the metal (coordination by substitution of a labile chlorido ligand) and on the ligand (covalent bond formation involving imine formation by one or two aldehyde functions). Peptide modification by the metal complex had a drastic effect on both Collisonally Activated Dissociation (CAD) and Electron Capture Dissociation (ECD) MS/MS behaviour, tuning requirements, and fragmentation channels. CAD MS/MS was effective only when studying the covalent condensation products. ECD MS/MS, although hindered by electron-quenching at the Iridium complex site, was suitable for studying many of the species observed, locating the modification sites, and often identifying them to within a single amino acid residue.

Glucosepane is associated with changes to structural and physical properties of collagen fibrils.

Collagen glycation, and in particular the formation of advanced glycation end-product (AGE) crosslinks, plays a central role in the ageing process and in many of the long-term complications of diabetes. Glucosepane, the most abundant and relevant AGE crosslink, has been suggested to increase the stiffness of tissue and reduce its solubility, although no evidence is available concerning the mechanisms. We have used a combination of computational and experimental techniques to study a collagen-rich tissue with a relatively simple organisation to further our understanding of the impact of glucosepane on the structural and physical properties of collagen fibrils. Our work shows that glucosepane levels increase dramatically in aged tendon tissue and are associated with the reduced density of collagen packing and increased porosity to water molecules. Our studies provide the basis to understand many of the tissue dysfunctions associated with ageing and diabetes across a range of different tissues types.

CD151 regulates expression of FGFR2 in breast cancer cells via PKC-dependent pathways.

Expression of the tetraspanin CD151 is frequently upregulated in epithelial malignancies and correlates with poor prognosis. Here, we report that CD151 is involved in regulation of the expression of fibroblast growth factor receptor 2 (FGFR2). Depletion of CD151 in breast cancer cells resulted in an increased level of FGFR2. Accordingly, an inverse correlation between CD151 and FGFR2 was observed in breast cancer tissues. CD151-dependent regulation of the FGFR2 expression relies on post-transcriptional mechanisms involving HuR (also known as ELAVL1), a multifunctional RNA-binding protein, and the assembly of processing bodies (P-bodies). Depletion of CD151 correlated with inhibition of PKC, a well-established downstream target of CD151. Accordingly, the levels of dialcylglycerol species were decreased in CD151-negative cells, and inhibition of PKC resulted in the increased expression of FGFR2. Whereas expression of FGFR2 itself did not correlate with any of the clinicopathological data, we found that FGFR2-/CD151+ patients were more likely to have developed lymph node metastasis. Conversely, FGFR2-/CD151- patients demonstrated better overall survival. These results illustrate functional interdependency between CD151 complexes and FGFR2, and suggest a previously unsuspected role of CD151 in breast tumorigenesis.

Liquid Extraction Surface Analysis (LESA) Electron-Induced Dissociation and Collision-Induced Dissociation Mass Spectrometry of Small Molecule Drug Compounds.

Here, we present liquid extraction surface analysis (LESA) coupled with electron-induced dissociation (EID) mass spectrometry in a Fourier-transform ion cyclotron resonance mass spectrometer for the analysis of small organic pharmaceutical compounds directly from dosed tissue. First, the direct infusion electrospray ionisation EID and collision-induced dissociation (CID) behaviour of erlotinib, moxifloxacin, clozapine and olanzapine standards were compared. EID mass spectra were also compared with experimental or reference electron impact ionisation mass spectra. The results show that (with the exception of erlotinib) EID and CID result in complementary fragment ions. Subsequently, we performed LESA EID MS/MS and LESA CID MS/MS on singly charged ions of moxifloxacin and erlotinib extracted from a thin tissue section of rat kidney from a cassette-dosed animal. Both techniques provided structural information, with the majority of peaks observed for the drug standards also observed for the tissue-extracted species. Overall, these results demonstrate the feasibility of LESA EID MS/MS of drug compounds from dosed tissue and extend the number of molecular structures for which EID behaviour has been determined. Graphical Abstract ᅟ.