Directly Infused Research Articles

The Therapeutic Potential of Oxyhydrogen Gas in Oncology: A Study on Epstein–Barr Virus-Immortalised B-Lymphoblastoid (TK6) Cells

Cancer is a leading cause of mortality worldwide. B-cells are a keystone of the adaptive immune response and are essential for the presentation of tumor-associated antigens to various types of T-cells. Approximately 1.5% of global cancer cases, including breast and gastric carcinomas and both Hodgkin’s and non-Hodgkin’s lymphomas, are linked with prior Epstein–Barr Virus (EBV) infection. Such properties make EBV-infected lymphocytes ideal models for understanding the effect of oxyhydrogen gas on dysfunctional cell cycling. The aim of this study is to assess the effects of the direct infusion of oxyhydrogen gas on the replicative capacity of EBV-immortalised B-lymphocytes. Oxyhydrogen gas was directly infused into cell culture media. Cells were incubated in 95% air and 5% CO2 for up to 72 h. Cell enumeration was assessed with and without the addition of mitogenic growth stimuli, and subsequent cell-cycle analysis was performed. Cell enumeration: An initial trend of replicative inhibition of TK6 cells is noted with a single oxyhydrogen treatment at the 24 and 48 h time points. The daily addition of oxyhydrogen-infused media showed statistically relevant data at 24 and 48 h but not at 72 h. In mitogen-stimulated cells, a non-statistical trend of inhibition was observed at 24, 48 and 72 h. Analysis details a significant increase in DNA in the Sub G1 phase, indicating increased apoptosis.

A comparative study of a rabbit spinal tuberculosis model constructed by local direct infection via the posterior lateral approach

The present study aims to establish a method of constructing a New Zealand rabbit spinal tuberculosis model by direct local infusion of M. tuberculosis H37Rv strain into the intervertebral disc space through the posterior lateral approach. Sixty-six New Zealand rabbits were pretreated with complete Freund's adjuvant and randomly divided into 4 group: the posterolateral approach model group (Group A, 25), ventral transverse process approach model group (Group B, 25), control group (Group C, 10), and blank group (Group D, 6). In Groups A and B, the bone holes were filled with gelatin sponge after drilling, and the local area was directly infused with 0.1 ml of M. tuberculosis H37Rv strain suspension. In Group C, the gelatin sponge was filled through the posterolateral approach and the local area was infused with 0.1 ml of normal saline suspension. In Group D, No specific treatment was performed. The general conditions of the experimental rabbits in each group were compared to those of a control group; the degree of vertebral body exposure, incision length, and complications of the two methods were compared; and the tuberculosis models were evaluated by imaging, histopathology, and bacterial culture. In Group A, the lateral side of the vertebral body was well exposed, the damage was mild, and no peritoneal rupture or gastrointestinal complications were observed. In Group B, the ventral side of the vertebral body and the intervertebral disc were exposed, and abdominal complications were more likely to occur. The survival rates of the experimental rabbits at 8 weeks after surgery were 92.0% in Group A, 88.00% in Group B, 90.0% in Group C, and 100% in Group D. MRI examinations showed that in Group A, the positive rate of radiographic bone findings was 86.9% at 4 weeks after surgery and 100% at 8 weeks after surgery; in Group B, the positive rate of radiographic bone findings was 78.2% at 4 weeks after surgery and 95.4% at 8 weeks after surgery. There was no significant difference between Groups A and B in the radiographic bone findings rate detected by the same imaging method at the same time point (P > 0.05). Eight weeks after surgery, bone destruction, paravertebral abscess, and caseous necrosis occurred in the vertebral bodies of surviving rabbits in Groups A and B. The BacT/ALERT 3D rapid culture system was used to culture the pus in the lesion, and the results showed that the positive rate of tuberculosis was 52.17% in Group A and 54.54% in Group B, and the difference was not statistically significant (P > 0.05). After pretreatment with complete Freund's adjuvant, direct infusion of the H37Rv strain of M. tuberculosis into the intervertebral disc space of New Zealand rabbits via the posterolateral approach and the ventral transverse process approach can successfully establish rabbit spinal tuberculosis models.

Abstract CT216: Phase I study of intraventricular infusions of autologous ex vivo expanded NK cells in children with recurrent/refractory malignant posterior fossa tumors of the central nervous system

Abstract Scientific background/rationale: The prognosis of recurrent/refractory medulloblastoma (MB), ependymoma (EP) and atypical teratoid/rhabdoid neoplasms (AT/RT) which constitutes the majority of malignant posterior fossa tumors (MPFT) in children, remains dismal with no defined effective therapy. Current treatment strategies have significant toxicities, thus profiling newer trials with clinical efficacy and minimal side-effects are imperative. Advantages of direct infusion of Natural Killer (NK) cells into the ventricles could enhance therapeutic efficacy by maximally concentrating them in the brain in close proximity to the tumor area, bypassing the blood brain barrier and alleviating toxicity often seen with systemic chemotherapy. We have shown efficacy of activated and propagated NK cells to lyse and kill the MPFT in vitro and tumor explants in mice models. We demonstrated in a pilot study for the first time in humans, the feasibility of infusions of biologic agents into the fourth ventricle. Infusing biologic agents into the lateral ventricles are well established in neurooncology. The current ongoing study is the first-in-human, loco-regional infusion of NK cells directly into the brain evaluating safety and efficacy in the context of a phase I trial with an intent to avoid the significant side-effects of systemic therapy. This novel trial, will hopefully be able to provide therapeutic efficacy and a meaningful increase in the survival of these children. Correlative studies: Cerebrospinal fluid (CSF) withdrawn during infusions of NK cells will be evaluated for the immunophenotype, function, persistence of NK cells and influx or presence of any other immune cells. Methods: NK cells are directly infused into the lateral or fourth ventricles, after placement of a catheter at that location. Recurrent or refractory MB, AT/RT and EP involving the brain and/or spine at original diagnosis or relapse with histological verification are eligible. Three to six evaluable patients, are entered at a dose level in this study for determination of maximum tolerated dose (MTD). A minimum of 9 patients and a maximum of 24 patients are planned to be enrolled. After autologous expanded NK cell product have been manufactured as per good manufacturing guidelines (GMP) and released, patients receive 3 cycles of NK cell infusions over 12 weeks. Each cycle consists of 1 infusion per week for 3 weeks followed by a rest week (week 4). Dosing is based on recipient body surface area (BSA) and 3 dose levels will be evaluated. Currently the trial is at the final cohort (third dose level), with no dose limiting toxicity (DLT) noted with the previous dose levels. 80% of the initial minimal accrual have been met. Also as a secondary aim we will evaluate the anti-tumor activity of the NK cell infusions within the confines of a phase I study. Trial (NCT02271711-recruiting) Citation Format: Soumen Khatua, Dean Lee, Laurence Cooper, Judy Moyes, David Sandberg, Zsila Sadighi, Heather Meador, Leena Ketonen, Michael E. Rytting, Jason M. Johnson, Dristhi Raagonanan, Diane D. Liu, Vidya Gopalakrishnan, Wafik Zaky. Phase I study of intraventricular infusions of autologous ex vivo expanded NK cells in children with recurrent/refractory malignant posterior fossa tumors of the central nervous system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr CT216.

Direct target and non-target analysis of urban aerosol sample extracts using atmospheric pressure photoionisation high-resolution mass spectrometry

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous atmospheric pollutants of high concern for public health. In the atmosphere they undergo oxidation, mainly through reactions with ·OH and NOx to produce nitro- and oxygenated (oxy-) derivatives. In this study, we developed a new method for the detection of particle-bound PAHs, nitro-PAHs and oxy-PAHs using direct infusion into an atmospheric pressure photoionisation high-resolution mass spectrometer (APPI-HRMS). Method optimisation was done by testing different source temperatures, gas flow rates, mobile phases and dopants. Samples were extracted with methanol, concentrated by evaporation and directly infused in the APPI source after adding toluene as dopant. Acquisition was performed in both polarity modes. The method was applied to target analysis of seasonal PM2.5 samples from an urban background site in Padua (Italy), in the Po Valley, in which a series of PAHs, nitro- and oxy-PAHs were detected. APPI-HRMS was then used for non-target analysis of seasonal PM2.5 samples and results compared with nano-electrospray ionisation (nanoESI) HRMS. The results showed that, when samples were characterised by highly oxidised organic compounds, including S-containing compounds, like in summer samples, APPI did not bring any additional information with respect to nanoESI in negative polarity (nanoESI(-)). Conversely, for winter samples, APPI(-) could detect a series of aromatic and poly-aromatic compounds, mainly oxidised and nitrogenated aromatics, that were not otherwise detected with nanoESI.

Intravenously-injected gold nanoparticles (AuNPs) access intracerebral F98 rat gliomas better than AuNPs infused directly into the tumor site by convection enhanced delivery.

BackgroundIntravenously (IV)-injected gold nanoparticles (AuNPs) powerfully enhance the efficacy of X-ray therapy of tumors including advanced gliomas. However, pharmacokinetic issues, such as slow tissue clearance and skin discoloration, may impede clinical translation. The direct infusion of AuNPs into the tumor might be an alternative mode of delivery.Materials and methodsUsing the advanced, invasive, and difficult-to-treat F98 rat glioma model, we have studied the biodistribution of the AuNPs in the tumor and surrounding brain after either IV injection or direct intratumoral infusion by convection-enhanced delivery using light microscopy immunofluorescence and direct gold visualization.ResultsIV-injected AuNPs localize more specifically to intracerebral tumor cells, both in the main tumor mass and in the migrated tumor cells as well as the tumor edema, than do the directly infused AuNPs. Although some of the directly infused AuNPs do access the main tumor region, such access is largely restricted.ConclusionThese data suggest that IV-injected AuNPs are likely to have a greater therapeutic benefit when combined with radiation therapy than after the direct infusion of AuNPs.

Discovery and Confirmation of Diagnostic Serum Lipid Biomarkers for Alzheimer's Disease Using Direct Infusion Mass Spectrometry.

Alzheimer's disease (AD) is a neurodegenerative disorder lacking early biochemical diagnosis and treatment. Lipids have been implicated in neurodegenerative disorders including AD. A shotgun lipidomic approach was undertaken to determine if lipid biomarkers exist that can discriminate AD cases from controls. The discovery study involved sera from 29different stage AD cases and 32 controls. Lipid extraction was performed using organic solvent and the samples were directly infused into a time-of-flight mass spectrometer. Differences between AD cases and controls were detected with 87 statistically significant lipid candidate markers found. These potential lipid markers were reevaluated in a second confirmatory study involving 27 cases and 30 controls. Of the 87 candidates from the first study, 35 continued to be statistically significant in the second confirmatory set. Tandem MS studies were performed and almost all confirmed markers were characterized and classified. Using a Bayesian lasso probit regression model on the confirmed markers, a multi-marker set with AUC = 0.886 was developed comparing all stages of AD with controls. Additionally, using confirmed biomarkers, multi-marker sets with AUCs >0.90 were developed for each specific AD Clinical Dementia Rating versus controls, including the earliest stage of AD. More conservative and likely more realistic statistical analyses still found multi-marker sets that appeared useful in diagnosing AD. Finally, using ordinal modeling a set of markers was developed that staged AD accurately 70% of the time, p = 0.0079. These results suggest that these serum lipidomic biomarkers may help diagnose and perhaps even stage AD.

Direct brain infusion can be enhanced with focused ultrasound and microbubbles

The delivery of most therapeutic agents is rendered ineffective for the treatment of brain diseases due to the presence of the blood–brain barrier (BBB). The goal of this study was to investigate the effect of pre-infusion focused ultrasound (FUS) and microbubbles on the distribution of direct brain infusion in vivo. A single-element FUS transducer was used in all sonications, which were carried out immediately prior to direct infusion procedures. Mice received direct infusion of either Gadolinium-labeled albumin (Gd-albumin, 74 kDa) or adeno-associated virus (AAV, ∼4 MDa). The volumes of Gd-albumin at 30 min were deemed comparable (P = 0.334) between the direct infusion (DI)-only group and the FUS + DI group. At 120 min, the FUS + DI group showed significantly higher contrast-enhanced volume (9.76 ± 0.74 mm3) than the DI-only group (7.14 ± 0.34 mm3). For mice infused with AAV, the total volume of transduction was estimated as GFP-positive regions and FUS + DI group demonstrated significantly higher (P = 0.017) transduction efficiency in vivo. In conclusion, enhanced bio-distribution of directly infused agents was observed when the targeted region was pre-conditioned with FUS and microbubbles. Focused ultrasound has the potential, as an adjuvant technique, to significantly enhance direct brain infusion and achieve the desired therapeutic outcomes.

Classification of Tunisian extra virgin olive oils according to their genetic variety and maturity index using fatty acid profiles established by direct infusion mass spectrometry

The usefulness of fatty acid profiles established by direct infusion mass spectrometry (DIMS) as a tool to discriminate between seven genetic varieties of Tunisian extra virgin olive oils (EVOOs) was evaluated in this work. Moreover, the discrimination of EVOOs from the genetic varieties Chemchali, Fouji, and Zarrazi, characterized with different maturity indices, was also studied. For DIMS, EVOO samples were diluted with an 85:15 propanol/methanol (v/v) mixture containing 40 mM ammonia and directly infused into the mass spectrometer using a syringe pump. The establishment of ratios of the peak abundances of the free fatty acids followed by linear discriminant analysis was employed to predict both genetic variety and maturity index. In all cases, an excellent resolution between all category pairs was achieved, which demonstrated that fatty acid profiles are a good marker of both genetic variety and maturity index of EVOOs.Practical applications: As EVOO quality can be affected by different parameters, such as cultivar and maturity index, among others, it is necessary to develop analytical methods able to verify EVOO quality, and also to control its origin as olive oil producers have to include in their manufactured products the cultivar (monovarietal oils) from which the oil was obtained. Currently, almost all the research related to Tunisian EVOOs has been mainly focused on the improvement and characterization of the two main cultivars (Chétoui and Chemlali). Thus, to diversify Tunisian olive oil resources and improve the quality of olive oil produced in Tunisia, research on additional cultivars needs to be conducted. Thus, the present method could be used as an authentication tool for olive oil industry to asses both cultivar and maturity index of Tunisian EVOO, which could be extended to EVOO from all around the world.DIMS data of Tunisian EVOOs were used to predict EVOO genetic variety and maturity index using LDA.

Statistical classification of pumpkin seed oils by direct infusion mass spectrometry: Correlation with GC‐FID profiles

A method to classify pumpkin seed oils according to their species (Cucurbita maxima, Cucurbita pepo, and Cucurbita moschata), and C. moschata oils according to their genetic variety (RG, Inivit C‐88 and Inivit C‐2000) using fatty acid profiles obtained by direct infusion mass spectrometry (DIMS) has been developed. For DIMS, pumpkin seed oils were 1:4 (v/v) diluted with an 85:15 propanol/methanol (v/v) mixture containing 40 mM ammonia and directly infused into the mass spectrometer using a syringe pump. The establishment of ratios of the peak abundances of the free fatty acids followed by linear discriminant analysis was employed to predict both pumpkin seed oil species and C. moschata genetic variety. Moreover, the profiles obtained with this methodology were compared with the profiles obtained by the traditional GC‐FID. Since a good correlation between both methodologies was obtained, the proposed DIMS method is a promising alternative for fatty acid fingerprinting of pumpkin oils, with the advantage that prior chromatographic separation is not required.Practical applications: Pumpkin seed oils from several species and variety, differing in their organoleptic, antioxidant, and pharmaceutical properties, are found at high prices within the organic farming and gastronomical commercial circuits. Therefore, authentication methods capable of establishing the origin and other quality parameters of pumpkin seed oils are required. In this work, a simple and quick DIMS method, capable of predicting the species and genetic variety of pumpkin seed oils has been developed. The MS intensities of the free fatty acids can be measured after a simple dilution of the oils; in this way, the conventional GC‐FID separation is avoided, being only necessary to rinse the spectrometer for 5 min between successive infusions to achieve low backgrounds and reproducible oil profiles. Thus, the proposed method is a promising alternative to the traditional GC‐FID method for fatty acid fingerprinting, with the additional advantage that previous chromatographic separation is not required.Fatty acid profiles obtained by direct infusion MS were used to predict the species and genetic variety of pumpkin seed oils.

The efficacy of superselective intra-arterial chemo-radiotherapy for cervical neck lymph node metastases

We report herein on 32 cases of head and neck carcinoma with cervical lymph node metastases treated by radiotherapy and concomitant intraarterial cisplatin (RADPLAT) from April 2009 to May 2013. N3 cases revealed residual disease of the cervical lymph nodes in 7/9 cases. Among the 22 patients excluding N1 and N3 cases, the pathological CR rate was 63.6%. Among the 13 patients in whom the anticancer drug was directly infused into the cervical lymph nodes, the pathological CR rate was 76.9%, whereas in the 9 patients without direct infusion of the cervical lymph nodes, the pathological CR rate was 44.4%. Therefore, we recommend the direct infusion into cervical lymph node metastases for not only N3 cases but also N2 cases if a feeding artery is identified easily. When clinical examination after RADPLAT leads to suspected residual disease, neck dissection should be adapted. If the clinical examination leads to a diagnosis of CR, we recommend a biopsy of the original cervical lymph nodes because the cases which we diagnosed as CR revealed residual disease of the cervical lymph nodes in 4/16.

Distinct and opposing roles for the phosphatidylinositol 3-OH kinase catalytic subunits p110α and p110β in the regulation of insulin secretion from rodent and human beta cells

Phosphatidylinositol 3-OH kinases (PI3Ks) regulate beta cell mass, gene transcription, and function, although the contribution of the specific isoforms is unknown. As reduced type 1A PI3K signalling is thought to contribute to impaired insulin secretion, we investigated the role of the type 1A PI3K catalytic subunits α and β (p110α and -β) in insulin granule recruitment and exocytosis in rodent and human islets. The p110α and p110β subunits were inhibited pharmacologically or by small hairpin (sh)RNA-mediated knockdown, and were directly infused or overexpressed in mouse and human islets, beta cells and INS-1 832/13 cells. Glucose-stimulated insulin secretion (GSIS), single-cell exocytosis, Ca(2+) signalling, plasma membrane granule localisation, and actin density were monitored. Inhibition or knockdown of p110α increased GSIS. This was not due to altered Ca(2+) responses, depolymerisation of cortical actin or increased cortical granule density, but to enhanced Ca(2+)-dependent exocytosis. Intracellular infusion of recombinant PI3Kα (p110α/p85β) blocked exocytosis. Conversely, knockdown (but not pharmacological inhibition) of p110β blunted GSIS, reduced cortical granule density and impaired exocytosis. Exocytosis was rescued by direct intracellular infusion of recombinant PI3Kβ (p110β/p85β) even when p110β catalytic activity was inhibited. Conversely, both the wild-type p110β and a catalytically inactive mutant directly facilitated exocytosis. Type 1A PI3K isoforms have distinct and opposing roles in the acute regulation of insulin secretion. While p110α acts as a negative regulator of beta cell exocytosis and insulin secretion, p110β is a positive regulator of insulin secretion through a mechanism separate from its catalytic activity.

Generation of dyspeptic symptoms by direct acid and water infusion into the stomachs of functional dyspepsia patients and healthy subjects

The mechanisms of the development of symptoms in functional dyspepsia (FD) patients have not been fully elucidated. We previously reported that acid directly infused into the stomach causes dyspeptic symptoms in asymptomatic healthy controls (HCs); however, the response to acid infusion of FD patients was not determined. To investigate the severity of dyspeptic symptoms induced by direct acid infusion in FD subjects and HCs. This was a multi-centre, cross-over, randomised, double-blind study in 23 FD subjects and 32 HCs. FD was defined using the Rome III criteria. All subjects were Helicobacter pylori negative. Each subject received two tests; 0.1 mol/L hydrochloric acid and water infused into the stomach. The presence and severity of 12 dyspeptic symptoms were assessed using a visual analogue scale. The proportion of subjects developing symptoms by acid or water infusion was significantly greater in FD subjects than HCs. All of the FD subjects experienced at least one symptom by water or acid infusion. In the FD subjects, the severity of symptoms was significantly greater with acid infusion than water infusion. The severity of symptoms in total and the scores for eight of the 12 symptoms induced by acid infusion was significantly greater in FD subjects than in HCs. The severity of dyspeptic symptom generation induced by direct acid infusion into the stomach was significantly greater in functional dyspepsia subjects than in healthy controls, suggesting that hypersensitivity to acid is one of the important mechanisms of the development of symptoms in functional dyspepsia patients.

A Membrane Cell for On-line Hydrogen/Deuterium Exchange to Study Protein Folding and Protein-Protein Interactions by Mass Spectrometry

A membrane cell for hydrogen and deuterium exchange on-line with mass spectrometry has been developed to monitor protein-protein interactions and protein conformations. It consists of two channels separated by a semipermeable membrane, where one channel carries the protein sample and the other deuterium oxide. The membrane allows transfer of deuterium oxide into the sample flow. The labeling time is controlled via the flow rate in the sample channel. This cell was validated against three models commonly used in hydrogen-deuterium exchange mass spectrometry: monitoring of folded and unfolded states in a protein, mapping the protein secondary structure at the peptide level, and detection of protein and antibody interactions. The system avoids the conventionally used sample dilution and handling, allowing for potential automation.

N-Glycosylation at the SynCAM (Synaptic Cell Adhesion Molecule) Immunoglobulin Interface Modulates Synaptic Adhesion

Select adhesion molecules connect pre- and postsynaptic membranes and organize developing synapses. The regulation of these trans-synaptic interactions is an important neurobiological question. We have previously shown that the synaptic cell adhesion molecules (SynCAMs) 1 and 2 engage in homo- and heterophilic interactions and bridge the synaptic cleft to induce presynaptic terminals. Here, we demonstrate that site-specific N-glycosylation impacts the structure and function of adhesive SynCAM interactions. Through crystallographic analysis of SynCAM 2, we identified within the adhesive interface of its Ig1 domain an N-glycan on residue Asn(60). Structural modeling of the corresponding SynCAM 1 Ig1 domain indicates that its glycosylation sites Asn(70)/Asn(104) flank the binding interface of this domain. Mass spectrometric and mutational studies confirm and characterize the modification of these three sites. These site-specific N-glycans affect SynCAM adhesion yet act in a differential manner. Although glycosylation of SynCAM 2 at Asn(60) reduces adhesion, N-glycans at Asn(70)/Asn(104) of SynCAM 1 increase its interactions. The modification of SynCAM 1 with sialic acids contributes to the glycan-dependent strengthening of its binding. Functionally, N-glycosylation promotes the trans-synaptic interactions of SynCAM 1 and is required for synapse induction. These results demonstrate that N-glycosylation of SynCAM proteins differentially affects their binding interface and implicate post-translational modification as a mechanism to regulate trans-synaptic adhesion.

The N-Glycome of Human Plasma

N-linked glycans isolated from human plasma proteins have been profiled and sequenced by mass spectrometry using an ion trap instrument (ITMSn). The released glycans were prepared as reduced, methylated analogues and directly infused into a chip-based nanoelectrospray ionization system and analyzed by ITMSn. The resulting mass profiles (MS1) of IgG-depleted and nondepleted plasma samples were contrasted and these results were again compared with recent literature reports. Before depletion, approximately 50 independent glycan ions were detected; this more than doubled to 106 after depletion. The mass range profiled was 1-5 kDa which included many doubly and triply charged ions that were resolved by higher MS resolution. Selected ions in the depleted sample were disassembled to define their detailed structure providing a high-performance sequencing result. The simplicity of this nonchromatographic, direct infusion and gas-phase structural characterization compares most favorably with the latest reports using alternative instrumentation and adjunct techniques.

Activation of N-Methyl-d-aspartate (NMDA) Receptors in the Dorsal Vagal Complex Lowers Glucose Production

Diabetes is characterized by hyperglycemia due partly to increased hepatic glucose production. The hypothalamus regulates hepatic glucose production in rodents. However, it is currently unknown whether other regions of the brain are sufficient in glucose production regulation. The N-methyl-D-aspartate (NMDA) receptor is composed of NR1 and NR2 subunits, which are activated by co-agonist glycine and glutamate or aspartate, respectively. Here we report that direct administration of either co-agonist glycine or NMDA into the dorsal vagal complex (DVC), targeting the nucleus of the solitary tract, lowered glucose production in vivo. Direct infusion of the NMDA receptor blocker MK-801 into the DVC negated the metabolic effect of glycine. To evaluate whether NR1 subunit of the NMDA receptor mediates the effect of glycine, NR1 in the DVC was inhibited by DVC NR1 antagonist 7-chlorokynurenic acid or DVC shRNA-NR1. Pharmacological and molecular inhibition of DVC NR1 negated the metabolic effect of glycine. To evaluate whether the NMDA receptors mediate the effects of NR2 agonist NMDA, DVC NMDA receptors were inhibited by antagonist D-2-amino-5-phosphonovaleric acid (D-APV). DVC D-APV fully negated the ability of DVC NMDA to lower glucose production. Finally, hepatic vagotomy negated the DVC glycine ability to lower glucose production. These findings demonstrate that activation of NR1 and NR2 subunits of the NMDA receptors in the DVC is sufficient to trigger a brain-liver axis to lower glucose production, and suggest that DVC NMDA receptors serve as a therapeutic target for diabetes and obesity.

Evaluation of the oxidative status of virgin olive oils with different phenolic content by direct infusion atmospheric pressure chemical ionization mass spectrometry

Atmospheric pressure chemical ionization mass spectrometry was used to predict the oxidative status of virgin olive oils (VOO) during their storage. VOO samples, with and without phenolic compounds, were stored in the dark at 60 degrees C up to 7 weeks. The VOO samples were diluted in an alkaline propanol/methanol mixture and directly infused into an ion-trap mass spectrometer. The abundances of the [M-H](-) peaks of free fatty acids, oxidized fatty acids, tocopherols and phenolic compounds, jointly with their oxidized forms, were measured and used as predictors. Two linear discriminant analysis (LDA) models were constructed in order to classify samples according to their oxidative levels. The first model was constructed using both VOO samples (with and without phenols), considering as predictors only fatty acids and their oxidized products. The second LDA model was constructed with the VOO sample with phenolic compounds considering as predictors all the peaks measured. In both models, the samples divided in the eight different storage times were correctly classified (100%) by leave-one-out cross-validation with an excellent resolution among all the category pairs (for the first model Wilks' lambda, lambda(w) = 0.229 and for the second lambda(w) = 0.928). This method is a very fast tool for on-line monitoring of VOO oxidation status.

Identification of phosphatidylserylglutamate: a novel minor lipid in Escherichia coli

Advances in mass spectrometry have facilitated the identification of novel lipid structures. In this work, we fractionated the lipids of Escherichia coli B and analyzed the fractions using negative-ion electrospray ionization mass spectrometry to reveal unknown lipid structures. Analysis of a fraction eluting with high salt from DEAE cellulose revealed a series of ions not corresponding to any of the known lipids of E. coli. The ions, with m/z 861.5, 875.5, 887.5, 889.5, and 915.5, were analyzed using collision-induced dissociation mass spectrometry (MS/MS) and yielded related fragmentation patterns consistent with a novel diacylated glycerophospholipid. Product ions arising by neutral loss of 216 mass units were observed with all of the unknowns. A corresponding negative product ion was also observed at m/z 215.0. Additional ions at m/z 197.0, 171.0, 146.0, and 128.0 were used to propose the novel structure phosphatidylserylglutamate (PSE). The hypothesized structure was confirmed by comparison with the MS/MS spectrum of a synthetic standard. Normal phase liquid chromatography-mass spectrometry analysis further showed that the endogenous PSE and synthetic PSE eluted with the same retention times. PSE was also observed in the equivalent anion exchange fractions of total lipids extracted from the wild-type E. coli K-12 strain MG1655.

Evaluation of the quality of olive oil using fatty acid profiles by direct infusion electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry is used to predict the olive oil quality according to European Union marketing standards. Samples were 1:50 diluted in an alkaline 85:15 (v/v) propanol/methanol mixture and directly infused into the electrospray ionization source of an ion trap mass spectrometer. The establishment of ratios of the peak abundances of the free fatty acids followed by linear discriminant analysis was employed to predict the olive oil quality grade. In addition, using multiple linear regression and partial least-squares regression, the percentages of extra virgin and virgin olive oils in binary mixtures were predicted with 5–11% average prediction errors.

Lipids of human meibum: mass-spectrometric analysis and structural elucidation

The purpose of this study was to structurally characterize the major lipid species present in human meibomian gland secretions (MGS) of individual subjects by means of ion trap atmospheric pressure ionization mass spectrometry analysis (API MS(n)). The samples of MGS and authentic lipid standards were analyzed in direct infusion and high-pressure liquid chromatography (HPLC) experiments with API MS(n) detection of the analytes (HPLC API MS(n)). The major precursor ions were isolated and subjected to further sequential fragmentation in MS(n) experiments, and their fragmentation patterns were compared with those of authentic lipid standards. Multiple precursor ions were observed in the positive-ion mode. Among those, previously identified cholesterol (Chl; m/z 369; [M - H(2)O + H](+)) and oleic acid (OA; m/z 283; [M + H](+)) were found. The other major compounds of the general molecular formula C(n)H(2n-2)O(2) were consistent with wax esters (WEs), with OA as fatty acyl component. Accompanying them were two homologous series of compounds that fit the molecular formulas C(n)H(2n-4)O(2) and C(n)H(2n)O(2). Subset 2 was found to be a homolog series of linoleic acid-based WEs, whereas subset 3 was, apparently, a mixture of stearic acid-based WEs. HPLC API MS(n) analysis revealed the presence of large quantities of cholesteryl esters (Chl-Es) in all of the tested samples. Less than 0.1% (w/w) of oleamide was detected in human MGS. In the negative-ion mode, three major compounds with m/z values of 729, 757, and 785 that were apparently related to anionogenic lipids of the diacylglyceryl family were found in all of the samples. Common phospholipids and ceramides (Cers) were not present among the major MGS lipids. Phosphocholine-based lipids were found in MGS in quantities less than 0.01% (w/w), if at all. This ratio is two orders of magnitude lower than reported previously. These observations suggest that MGS are a major source of nonpolar lipids of the WE and Chl-E families for the tear film lipid layer, but not of its previously reported (phospho)lipid, Cer, and fatty acid amide components.