Trapped ion mobility spectrometry and PASEF enable in-depth lipidomics from minimal sample amounts

A comprehensive characterization of the lipidome from limited starting material remains very challenging. Here we report a high-sensitivity lipidomics workflow based on nanoflow liquid chromatography and trapped ion mobility spectrometry (TIMS). Taking advantage of parallel accumulation-serial fragmentation (PASEF), we fragment on average 15 precursors in each of 100 ms TIMS scans, while maintaining the full mobility resolution of co-eluting isomers. The acquisition speed of over 100 Hz allows us to obtain MS/MS spectra of the vast majority of isotope patterns. Analyzing 1 mu L of human plasma, PASEF increases the number of identified lipids more than three times over standard TIMS-MS/MS, achieving attomole sensitivity. Building on high intra- and inter-laboratory precision and accuracy of TIMS collisional cross sections (CCS), we compile 1856 lipid CCS values from plasma, liver and cancer cells. Our study establishes PASEF in lipid analysis and paves the way for sensitive, ion mobility-enhanced lipidomics in four dimensions

Role of Epigenetic Modification and Immunomodulation in a Murine Prostate Cancer Model

INTRODUCTION. Decreased expression of highly immunogenic cancer-testis antigens (CTA) might help tumor to achieve low immunogenicity, escape immune surveillance and grow unimpeded. Our aim was to evaluate CTA expression in tumor and normal tissues and to investigate possible means of improving the immune response in a murine prostate cancer (CaP) model by using the combination of epigenetic modifier 5-azacitidine (5-AzaC) and immunomodulator lenalidomide. No study to date has examined the effect of this combination on the prostate cancer or its impact on antigen-presenting cells (APC). MATERIALS AND METHODS. Gene microarrays were performed to compare expression of several CTA in murine prostate cancer (RM-1 cells) and normal prostate. RM-1 cells were treated with 5-AzaC and real-time PCR was performed to investigate the expression of several CTA. Western blotting was used to determine whether expression of CTA-specific mRNA induced by 5-AzaC resulted in increase in the corresponding protein. Effect of the epigenetic agents and immunomodulators was assessed on dendritic cells (DC) using flow cytometry, ELISA and T-cell proliferation assay. RESULTS. Gene arrays demonstrated decreased expression of 35 CTA in CaP tissue compared to normal prostate. 5-AzaC treatment of RM-1 prostate cancer cells upregulated the expression of all 13 CTA tested in a dose-dependent fashion. DC were treated with 5-AzaC and lenalidomide and the expression of surface markers MHC Class I, MHC Class II, CD80, CD86, CD 205, and CD40 was increased. Combination of 5-AzaC and lenalidomide enhances the ability of DC to stimulate T-cell proliferation in mixed leukocyte reaction. Secretion of IL-12 and IL-15 by DC increased significantly with addition of 5-AzaC or 5-AzaC and lenalidomide. CONCLUSIONS. Decreased expression of CTA by prostate cancer may be a means of escaping immune monitoring. Combination of epigenetic modifications and immunomodulation by 5-AzaC and lenalidomide increased tumor immunogenicity and enhanced DC function and may be used in the treatment of advanced prostate cancer

Peptide functionalized superparamagnetic iron oxide nanoparticles as MRI contrast agents

Cataloged from PDF version of article.Magnetic resonance imaging (MRI) attracts great attention in cellular and molecular imaging due to its non-invasive and multidimensional tomographic capabilities. Development of new contrast agents is necessary to enhance the MRI signal in tissues of interest. Superparamagnetic iron oxide nanoparticles (SPIONs) are used as contrast agents for signal enhancement as they have revealed extraordinary magnetic properties at the nanometre size and their toxicity level is very low compared to other commercial contrast agents. In this study, we developed a new method to functionalize the surface of SPIONs. Peptide amphiphile molecules are used to coat SPIONs non-covalently to provide water solubility and to enhance biocompatibility. Superparamagnetic properties of the peptide-SPION complexes and their ability as contrast agents are demonstrated. In vitro cell culture experiments reveal that the peptide-SPION complexes are biocompatible and are localized around the cells due to their peptide coating

Direct-hydrogen-fueled proton-exchange-membrane fuel cell system for transportation applications: Conceptual vehicle design report pure fuel cell powertrain vehicle

In partial fulfillment of the Department of Energy (DOE) Contract No. DE-AC02-94CE50389, {open_quotes}Direct-Hydrogen-Fueled Proton-Exchange-Membrane (PEM) Fuel Cell for Transportation Applications{close_quotes}, this preliminary report addresses the conceptual design and packaging of a fuel cell-only powered vehicle. Three classes of vehicles are considered in this design and packaging exercise, the Aspire representing the small vehicle class, the Taurus or Aluminum Intensive Vehicle (AIV) Sable representing the mid-size vehicle and the E-150 Econoline representing the van-size class. A fuel cell system spreadsheet model and Ford`s Corporate Vehicle Simulation Program (CVSP) were utilized to determine the size and the weight of the fuel cell required to power a particular size vehicle. The fuel cell power system must meet the required performance criteria for each vehicle. In this vehicle design and packaging exercise, the following assumptions were made: fuel cell power system density of 0.33 kW/kg and 0.33 kg/liter, platinum catalyst loading less than or equal to 0.25 mg/cm{sup 2} total and hydrogen tanks containing gaseous hydrogen under 340 atm (5000 psia) pressure. The fuel cell power system includes gas conditioning, thermal management, humidity control, and blowers or compressors, where appropriate. This conceptual design of a fuel cell-only powered vehicle will help in the determination of the propulsion system requirements for a vehicle powered by a PEMFC engine in lieu of the internal combustion (IC) engine. Only basic performance level requirements are considered for the three classes of vehicles in this report. Each vehicle will contain one or more hydrogen storage tanks and hydrogen fuel for 560 km (350 mi) driving range. Under these circumstances, the packaging of a fuel cell-only powered vehicle is increasingly difficult as the vehicle size diminishes

The LHC Continuous Cryostat Interconnections: The Organization of a Logistically Complex Worksite Requiring Strict Quality Standards and High Output

The interconnections of the Large Hadron Collider (LHC) continuous cryostat have been completed in fall 2007: 1695 interconnections magnet to magnet and 224 interconnections between the continuous cryostat and the cryogenic distribution line have been executed along the 27 km of the LHC. The very tight schedule, the complexity of the interconnection sequence, the strict quality standards applied have required the creation of an ad hoc organization in order to steer and coordinate the activities on the worksite dispersed along the whole accelerator ring. The concatenation of construction and test phases carried out by CERN staff, CERN collaborating institutes and contractors have led to the necessity of a common approach and of a very effective information flow. In this paper, after having recalled the main technical challenges, we review the organizational choices that have been taken and we briefly analyze the development of the worksite in term of allocated resources and production

The Quality Control of the LHC Continuous Cryostat Interconnections

The interconnections between the Large Hadron Collider (LHC) magnets have required some 40 000 TIG welded joints and 65 000 electrical splices. At the level of single joints and splices, non-destructive techniques find limited application: quality control is based on the qualification of the process and of operators, on the recording of production parameters and on production samples. Visual inspection and process audits were the main techniques used. At the level of an extended chain of joints and splices - from a 53.5 m half-cell to a complete 2.7 km arc sector - quality control is based on vacuum leak tests, electrical tests and RF microwave reflectometry that progressively validated the work performed. Subsequent pressure tests, cryogenic circuits flushing with high pressure helium and cool-downs revealed a few unseen or new defects. This paper presents an overview of the quality control techniques used, seeking lessons applicable to similar large, complex projects

Original Article

Objective: Glucagon is well known to regulate blood glucose but may be equally important for amino acid metabolism. Plasma levels of amino acids are regulated by glucagon-dependent mechanism(s), while amino acids stimulate glucagon secretion from alpha cells, completing the recently described liver-alpha cell axis. The mechanisms underlying the cycle and the possible impact of hepatic steatosis are unclear. Methods: We assessed amino acid clearance in vivo in mice treated with a glucagon receptor antagonist (GRA), transgenic mice with 95% reduction in alpha cells, and mice with hepatic steatosis. In addition, we evaluated urea formation in primary hepatocytes from ob/ob mice and humans, and we studied acute metabolic effects of glucagon in perfused rat livers. We also performed RNA sequencing on livers from glucagon receptor knock-out mice and mice with hepatic steatosis. Finally, we measured individual plasma amino acids and glucagon in healthy controls and in two independent cohorts of patients with biopsy-verified non-alcoholic fatty liver disease (NAFLD). Results: Amino acid clearance was reduced in mice treated with GRA and mice lacking endogenous glucagon (loss of alpha cells) concomitantly with reduced production of urea. Glucagon administration markedly changed the secretion of rat liver metabolites and within minutes increased urea formation in mice, in perfused rat liver, and in primary human hepatocytes. Transcriptomic analyses revealed that three genes responsible for amino acid catabolism (Cps1, Slc7a2, and Slc38a2) were downregulated both in mice with hepatic steatosis and in mice with deletion of the glucagon receptor. Cultured ob/ob hepatocytes produced less urea upon stimulation with mixed amino acids, and amino acid clearance was lower in mice with hepatic steatosis. Glucagon-induced ureagenesis was impaired in perfused rat livers with hepatic steatosis. Patients with NAFLD had hyperglucagonemia and increased levels of glucagonotropic amino acids, including alanine in particular. Both glucagon and alanine levels were reduced after diet-induced reduction in Homeostatic Model Assessment for Insulin Resistance (HOMA-IR, a marker of hepatic steatosis). Conclusions: Glucagon regulates amino acid metabolism both non-transcriptionally and transcriptionally. Hepatic steatosis may impair glucagon-dependent enhancement of amino acid catabolism. (C) 2020 The Author(s). Published by Elsevier GmbH