Norharmane Matrix Enhances Detection of Endotoxin by MALDI-MS for Simultaneous Profiling of Pathogen, Host, and Vector Systems.

The discovery of novel pathogenic mechanisms engaged during bacterial infections requires the evolution of advanced techniques. Here, we evaluate the dual polarity matrix norharmane (NRM) to improve detection of bacterial lipid A (endotoxin), from host and vector tissues infected with Francisella novicida (Fn). We evaluated NRM for improved detection and characterization of a wide range of lipids in both positive and negative polarities, including lipid A and phospholipids across a range of matrix assisted laser desorption-ionization (MALDI)-coupled applications. NRM matrix improved the limit of detection (LOD) for monophosphoryl lipid A (MPLA) down to picogram-level representing a ten-fold improvement of LOD versus 2,5-dihydroxybenzoic acid (DHB) and 100-fold improvement of LOD versus 9-aminoacridine (9-AA). Improved LOD for lipid A subsequently facilitated detection of the Fn lipid A major ion (m/z 1665) from extracts of infected mouse spleen and the temperature-modified Fn lipid A at m/z 1637 from infected D. variabilis ticks. Finally, we simultaneously mapped bacterial phospholipid signatures within an Fn infected spleen along with exclusively host-derived inositol-based phospholipid (m/z 933) demonstrating co-profiling for the host-pathogen interaction. Expanded use of NRM matrix in other infection models and endotoxin-targeting imaging experiments will improve our understanding of the lipid interactions at the host-pathogen interface.

More from less: high-throughput dual polarity lipid imaging of biological tissues.

The high ion signals produced by many lipids in mass spectrometry imaging (MSI) make them an ideal molecular class to study compositional changes throughout tissue sections and their relationship with disease. However, the large extent of structural diversity observed in the lipidome means optimal ion signal for different lipid classes is often obtained in opposite polarities. In this work we demonstrate how new high speed MALDI-MSI technologies combined with precise laser position control enables the acquisition of positive and negative ion mode lipid data from the same tissue section much faster than is possible with other MSI instruments. Critically, using this approach we explicitly demonstrate how such dual polarity acquisitions provide more information regarding molecular composition and spatial distributions throughout biological tissues. For example, in applying this approach to the zebra finch songbird brain we reveal the high abundance of DHA containing phospholipids (PC in positive mode and PE, PS in negative ion mode) in the nuclei that control song learning behaviour. To make the most of dual polarity data from single tissues we have also developed a pLSA-based multivariate analysis technique that includes both positive and negative ion data in the classification approach. In doing so the correlation amongst different lipid classes that ionise best in opposite polarities and contribute to certain spatial patterns within the tissue can be directly revealed. To demonstrate we apply this approach to studying the lipidomic changes throughout the tumor microenvironment within xenografts from a lung cancer model.

Neuronal expression of GalNAc transferase is sufficient to prevent the age-related neurodegenerative phenotype of complex ganglioside-deficient mice.

Gangliosides are widely expressed sialylated glycosphingolipids with multifunctional properties in different cell types and organs. In the nervous system, they are highly enriched in both glial and neuronal membranes. Mice lacking complex gangliosides attributable to targeted ablation of the B4galnt1 gene that encodes ß-1,4-N-acetylegalactosaminyltransferase 1 (GalNAc-transferase; GalNAcT(-/-)) develop normally before exhibiting an age-dependent neurodegenerative phenotype characterized by marked behavioral abnormalities, central and peripheral axonal degeneration, reduced myelin volume, and loss of axo-glial junction integrity. The cell biological substrates underlying this neurodegeneration and the relative contribution of either glial or neuronal gangliosides to the process are unknown. To address this, we generated neuron-specific and glial-specific GalNAcT rescue mice crossed on the global GalNAcT(-/-) background [GalNAcT(-/-)-Tg(neuronal) and GalNAcT(-/-)-Tg(glial)] and analyzed their behavioral, morphological, and electrophysiological phenotype. Complex gangliosides, as assessed by thin-layer chromatography, mass spectrometry, GalNAcT enzyme activity, and anti-ganglioside antibody (AgAb) immunohistology, were restored in both neuronal and glial GalNAcT rescue mice. Behaviorally, GalNAcT(-/-)-Tg(neuronal) retained a normal "wild-type" (WT) phenotype throughout life, whereas GalNAcT(-/-)-Tg(glial) resembled GalNAcT(-/-) mice, exhibiting progressive tremor, weakness, and ataxia with aging. Quantitative electron microscopy demonstrated that GalNAcT(-/-) and GalNAcT(-/-)-Tg(glial) nerves had significantly increased rates of axon degeneration and reduced myelin volume, whereas GalNAcT(-/-)-Tg(neuronal) and WT appeared normal. The increased invasion of the paranode with juxtaparanodal Kv1.1, characteristically seen in GalNAcT(-/-) and attributed to a breakdown of the axo-glial junction, was normalized in GalNAcT(-/-)-Tg(neuronal) but remained present in GalNAcT(-/-)-Tg(glial) mice. These results indicate that neuronal rather than glial gangliosides are critical to the age-related maintenance of nervous system integrity.

Anti-GM2 ganglioside antibodies are a biomarker for acute canine polyradiculoneuritis.

Acute canine polyradiculoneuritis (ACP) is considered to be the canine equivalent of the human peripheral nerve disorder Guillain-Barré syndrome (GBS); an aetiological relationship, however, remains to be demonstrated. In GBS, anti-glycolipid antibodies (Abs) are considered as important disease mediators. To address the possibility of common Ab biomarkers, the sera of 25 ACP dogs, 19 non-neurological, and 15 epileptic control dogs were screened for IgG Abs to 10 glycolipids and their 1 : 1 heteromeric complexes using combinatorial glycoarrays. Anti-GM2 ganglioside Abs were detected in 14/25 ACP dogs, and anti-GA1 Abs in one further dog. All controls except for one were negative for anti-glycolipid Abs. In this cohort of cases and controls, the glycoarray screen reached a diagnostic sensitivity of 60% and a specificity of 97%; a lower sensitivity (32%) was reported using a conventional glycolipid ELISA. To address the possible pathogenic role for anti-GM2 Abs in ACP, we identified GM2 in canine sciatic nerve by both mass spectrometry and thin layer chromatography overlay. In immunohistological studies, GM2 was localized predominantly to the abaxonal Schwann cell membrane. The presence of anti-GM2 Abs in ACP suggests that it may share a similar pathophysiology with GBS, for which it could thus be considered a naturally occurring animal model.

Conference report: Seventh Exploratory Measurement Science Group Symposium.

The impressive 18th Century Ardgour House again played host to the seventh annual Exploratory Measurement Science Group Symposium. The Symposium was organised as a study retreat for young and established scientists who share a common interest in the development and application of advanced analytical instrumentation. Speakers from a wide range of backgrounds in academia, industry and government were invited to present and discuss their research interests surrounded by the stunning Highland scenery of Fort William.