Stephen Fai

Performing vaginal lavage, crystal violet staining, and vaginal cytological evaluation for mouse estrous cycle staging identification.

A rapid means of assessing reproductive status in rodents is useful not only in the study of reproductive dysfunction but is also required for the production of new mouse models of disease and investigations into the hormonal regulation of tissue degeneration (or regeneration) following pathological challenge. The murine reproductive (or estrous) cycle is divided into 4 stages: proestrus, estrus, metestrus, and diestrus. Defined fluctuations in circulating levels of the ovarian steroids 17-β-estradiol and progesterone, the gonadotropins luteinizing and follicle stimulating hormones, and the luteotropic hormone prolactin signal transition through these reproductive stages. Changes in cell typology within the murine vaginal canal reflect these underlying endocrine events. Daily assessment of the relative ratio of nucleated epithelial cells, cornified squamous epithelial cells, and leukocytes present in vaginal smears can be used to identify murine estrous stages. The degree of invasiveness, however, employed in collecting these samples can alter reproductive status and elicit an inflammatory response that can confound cytological assessment of smears. Here, we describe a simple, non-invasive protocol that can be used to determine the stage of the estrous cycle of a female mouse without altering her reproductive cycle. We detail how to differentiate between the four stages of the estrous cycle by collection and analysis of predominant cell typology in vaginal smears and we show how these changes can be interpreted with respect to endocrine status.

Platelet activating factors in depression and coronary artery disease: A potential biomarker related to inflammatory mechanisms and neurodegeneration

The persistence of a depressive episode in coronary artery disease (CAD) patients not only heightens the risk of acute ischemic events, but it is also associated with accelerated cognitive decline. Antidepressant interventions for depression in CAD have only modest effects and novel approaches are limited by a poor understanding of etiological mechanisms. This review proposes that the platelet activating factor (PAF) family of lipids might be associated with the persistence of a depressive episode and related neurodegenerative pathology in CAD due to their association with leading etiological mechanisms for depression in CAD such as inflammation, oxidative and nitrosative stress, vascular endothelial dysfunction, and platelet reactivity. The evidence implicating PAFs in CAD, vascular pathology, and neurodegenerative processes is also presented. We also propose future directions for the investigation of PAFs as mediators of persistent depression. In summary, PAFs are implicated in leading mechanisms associated with depression in CAD. PAFs may therefore be associated with the persistence of depression in CAD and related to neurodegenerative and cognitive sequelae.

Using neurolipidomics to identify phospholipid mediators of synaptic (dys)function in Alzheimer's Disease.

Not all of the mysteries of life lie in our genetic code. Some can be found buried in our membranes. These shells of fat, sculpted in the central nervous system into the cellular (and subcellular) boundaries of neurons and glia, are themselves complex systems of information. The diversity of neural phospholipids, coupled with their chameleon-like capacity to transmute into bioactive molecules, provides a vast repertoire of immediate response second messengers. The effects of compositional changes on synaptic function have only begun to be appreciated. Here, we mined 29 neurolipidomic datasets for changes in neuronal membrane phospholipid metabolism in Alzheimers Disease (AD). Three overarching metabolic disturbances were detected. We found that an increase in the hydrolysis of platelet activating factor precursors and ethanolamine-containing plasmalogens, coupled with a failure to regenerate relatively rare alkyl-acyl and alkenyl-acyl structural phospholipids, correlated with disease severity. Accumulation of specific bioactive metabolites [i.e., PC(O-16:0/2:0) and PE(P-16:0/0:0)] was associated with aggravating tau pathology, enhancing vesicular release, and signaling neuronal loss. Finally, depletion of PI(16:0/20:4), PI(16:0/22:6), and PI(18:0/22:6) was implicated in accelerating Aβ42 biogenesis. Our analysis further suggested that converging disruptions in platelet activating factor, plasmalogen, phosphoinositol, phosphoethanolamine (PE), and docosahexaenoic acid metabolism may contribute mechanistically to catastrophic vesicular depletion, impaired receptor trafficking, and morphological dendritic deformation. Together, this analysis supports an emerging hypothesis that aberrant phospholipid metabolism may be one of multiple critical determinants required for Alzheimer disease conversion.

Targeted lipidomics – advances in profiling lysophosphocholine and platelet‐activating factor second messengers

Glycerophosphocholines are the major building blocks of biological membranes. They are also precursors of low‐molecular‐weight second messengers with mass to charge ratios of 450–600. These messengers include lysophosphatidylcholines (LPCs) and lyso‐platelet activating factors (PAFs) that may be further processed into PAFs. Often considered as a single species, LPCs, PAFs and lyso‐PAFs are, in fact, families of glycerophosphocholine‐derived lipids distinguished by the linkage of their sn‐1 carbon chains to the phosphoglyceride backbone (ester or ether), their sn‐1 carbon chain length and degree of unsaturation, and the identity of their sn‐2 constituents (a hydroxyl or acetyl group). Each LPC and PAF species exhibits a different affinity for its cognate G‐protein‐coupled receptors, and each species elicits receptor‐independent actions that play critical signalling roles. Targeted mass spectrometry‐based lipidomic approaches are enabling the molecular identification and quantification of these low‐abundance second messengers. Variations between datasets map the temporal landscape of second messengers available for signalling, and provide snapshots of the state of structural membrane compositional remodelling at the time of extraction. Here, we review a number of advances in lipidomic methodologies used to identify LPCs, lyso‐PAFs and PAFs, and highlight how these targeted approaches are providing valuable insight into the roles played by the cellular lipidome in cell function and disease susceptibility.

Visualization and Phospholipid Identification (VaLID)

Motivation: Establishing phospholipid identities in large lipidomic datasets is a labour-intensive process. Where genomics and proteomics capitalize on sequence-based signatures, glycerophospholipids lack easily definable molecular fingerprints. Carbon chain length, degree of unsaturation, linkage, and polar head group identity must be calculated from mass to charge (m/z) ratios under defined mass spectrometry (MS) conditions. Given increasing MS sensitivity, many m/z values are not represented in existing prediction engines. To address this need, Visualization and Phospholipid Identification is a web-based application that returns all theoretically possible phospholipids for any m/z value and MS condition. Visualization algorithms produce multiple chemical structure files for each species. Curated lipids detected by the Canadian Institutes of Health Research Training Program in Neurodegenerative Lipidomics are provided as high-resolution structures. Availability: VaLID is available through the Canadian Institutes of Health Research Training Program in Neurodegenerative Lipidomics resources web site at https://www.med.uottawa.ca/lipidomics/resources.html. Contacts: lipawrd@uottawa.ca Supplementary Information: Supplementary data are available at Bioinformatics online.

Predicting Glycerophosphoinositol Identities in Lipidomic Datasets Using VaLID (Visualization and Phospholipid Identification)—An Online Bioinformatic Search Engine

The capacity to predict and visualize all theoretically possible glycerophospholipid molecular identities present in lipidomic datasets is currently limited. To address this issue, we expanded the search-engine and compositional databases of the online Visualization and Phospholipid Identification (VaLID) bioinformatic tool to include the glycerophosphoinositol superfamily. VaLID v1.0.0 originally allowed exact and average mass libraries of 736,584 individual species from eight phospholipid classes: glycerophosphates, glyceropyrophosphates, glycerophosphocholines, glycerophosphoethanolamines, glycerophosphoglycerols, glycerophosphoglycerophosphates, glycerophosphoserines, and cytidine 5′-diphosphate 1,2-diacyl-sn-glycerols to be searched for any mass to charge value (with adjustable tolerance levels) under a variety of mass spectrometry conditions. Here, we describe an update that now includes all possible glycerophosphoinositols, glycerophosphoinositol monophosphates, glycerophosphoinositol bisphosphates, and glycerophosphoinositol trisphosphates. This update expands the total number of lipid species represented in the VaLID v2.0.0 database to 1,473,168 phospholipids. Each phospholipid can be generated in skeletal representation. A subset of species curated by the Canadian Institutes of Health Research Training Program in Neurodegenerative Lipidomics (CTPNL) team is provided as an array of high-resolution structures. VaLID is freely available and responds to all users through the CTPNL resources web site.

Localizing Protein in 3D Neural Stem Cell Culture: a Hybrid Visualization Methodology

The importance of 3-dimensional (3D) topography in influencing neural stem and progenitor cell (NPC) phenotype is widely acknowledged yet challenging to study. When dissociated from embryonic or post-natal brain, single NPCs will proliferate in suspension to form neurospheres. Daughter cells within these cultures spontaneously adopt distinct developmental lineages (neurons, oligodendrocytes, and astrocytes) over the course of expansion despite being exposed to the same extracellular milieu. This progression recapitulates many of the stages observed over the course of neurogenesis and gliogenesis in post-natal brain and is often used to study basic NPC biology within a controlled environment. Assessing the full impact of 3D topography and cellular positioning within these cultures on NPC fate is, however, difficult. To localize target proteins and identify NPC lineages by immunocytochemistry, free-floating neurospheres must be plated on a substrate or serially sectioned. This processing is required to ensure equivalent cell permeabilization and antibody access throughout the sphere. As a result, 2D epifluorescent images of cryosections or confocal reconstructions of 3D Z-stacks can only provide spatial information about cell position within discrete physical or digital 3D slices and do not visualize cellular position in the intact sphere. Here, to reiterate the topography of the neurosphere culture and permit spatial analysis of protein expression throughout the entire culture, we present a protocol for isolation, expansion, and serial sectioning of post-natal hippocampal neurospheres suitable for epifluorescent or confocal immunodetection of target proteins. Connexin29 (Cx29) is analyzed as an example. Next, using a hybrid of graphic editing and 3D modelling softwares rigorously applied to maintain biological detail, we describe how to re-assemble the 3D structural positioning of these images and digitally map labelled cells within the complete neurosphere. This methodology enables visualization and analysis of the cellular position of target proteins and cells throughout the entire 3D culture topography and will facilitate a more detailed analysis of the spatial relationships between cells over the course of neurogenesis and gliogenesis in vitro. Both Imbeault and Valenzuela contributed equally and should be considered joint first authors.

The VR Kiosk

Since 2013, the Carleton Immersive Media Studio (CIMS) has partnered with the Canadian government to document the West Block, East Block, and Centre Block of the Parliamentary Precinct. Using laser scanning, photogrammetry, and photography, an accurate recording of the current condition is created and translated into a building information model (BIM). This data will aid in the planning and management of a multi-year rehabilitation project that will close the Centre Block building. CIMS saw a need to take the existing content generated from the documentation work and BIM and find a way to disseminate it to the general public through digitally assisted storytelling, thus creating the project: The VR Kiosk. Beginning in May 2017, an installation containing five Virtual Reality (VR) stations was located in front of the Capital Information Kiosk, also known as the visitor’s centre, where tourists go before touring the Parliament buildings. Five short experiences were available that show, through 360-degree videos, aspects of the Centre Block building and rehabilitation project that are not accessible during the physical tour. Careful selection of the equipment, interface, and style of experience was made to ensure a smooth and quick event for the thousands of expected tourists who ranged in age, mobility, and technological knowledge. The content of The VR Kiosk leveraged existing gathered data from the documentation and BIM conducted by CIMS, through this new application, revisions to existing protocols improved the transition from data acquisition, building information model and digitally assisted storytelling through virtual reality.