Sphingosine-1-Phosphate Lyase Deficient Cells as a Tool to Study Protein Lipid Interactions

Cell membranes contain hundreds to thousands of individual lipid species that are of structural importance but also specifically interact with proteins. Due to their highly controlled synthesis and role in signaling events sphingolipids are an intensely studied class of lipids. In order to investigate their metabolism and to study proteins interacting with sphingolipids, metabolic labeling based on photoactivatable sphingoid bases is the most straightforward approach. In order to monitor protein-lipid-crosslink products, sphingosine derivatives containing a reporter moiety, such as a radiolabel or a clickable group, are used. In normal cells, degradation of sphingoid bases via action of the checkpoint enzyme sphingosine-1-phosphate lyase occurs at position C2-C3 of the sphingoid base and channels the resulting hexadecenal into the glycerolipid biosynthesis pathway. In case the functionalized sphingosine looses the reporter moiety during its degradation, specificity towards sphingolipid labeling is maintained. In case degradation of a sphingosine derivative does not remove either the photoactivatable or reporter group from the resulting hexadecenal, specificity towards sphingolipid labeling can be achieved by blocking sphingosine-1-phosphate lyase activity and thus preventing sphingosine derivatives to be channeled into the sphingolipid-to-glycerolipid metabolic pathway. Here we report an approach using clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated nuclease Cas9 to create a sphingosine-1-phosphate lyase (SGPL1) HeLa knockout cell line to disrupt the sphingolipid-to-glycerolipid metabolic pathway. We found that the lipid and protein compositions as well as sphingolipid metabolism of SGPL1 knock-out HeLa cells only show little adaptations, which validates these cells as model systems to study transient protein-sphingolipid interactions.     

The Positive and Negative Syndrome Scale (PANSS): A Three-Factor Model of Psychopathology in Marginally Housed Persons with Substance Dependence and Psychiatric Illness

Rates of psychopathology are elevated in marginalized and unstably housed persons, underscoring the need for applicable clinical measures for these populations. The Positive and Negative Syndrome Scale (PANSS) is a clinical instrument principally developed for use in schizophrenia to identify the presence and severity of psychopathology symptoms. The current study investigates whether a reliable and valid PANSS factor structure emerges in a marginally housed, heterogeneous sample recruited from the Downtown Eastside of Vancouver where substance use disorders and psychiatric illness are pervasive. Participants (n = 270) underwent structured clinical assessments including the PANSS and then were randomly assigned to either exploratory (EFA) or confirmatory factor analytic (CFA) subsamples. EFA pointed to a novel three factor PANSS. This solution was supported by CFA. All retained items (28 out of 30) load significantly upon hypothesized factors and model goodness of fit analyses are in the acceptable to good range. Each of the three first-order factor constructs, labeled Psychosis/Disorganized, Negative Symptoms/Hostility, and Insight/Awareness, contributed significantly to measurement of a higher-order psychopathology construct. Further, the latent structure of this 3-factor solution appears temporally consistent over one-year. This PANSS factor structure appears valid and reliable for use in persons with multimorbidity, including substance use disorders. The structure is somewhat distinct from existing solutions likely due to the unique characteristics of this marginally housed sample.     

Microparticle-Induced Coagulation Relates to Coronary Artery Atherosclerosis in Severe Aortic Valve Stenosis

Background

Circulating microparticles (MPs) derived from endothelial cells and blood cells bear procoagulant activity and promote thrombin generation. Thrombin exerts proinflammatory effects mediating the progression of atherosclerosis. Aortic valve stenosis may represent an atherosclerosis-like process involving both the aortic valve and the vascular system. The aim of this study was to investigate whether MP-induced thrombin generation is related to coronary atherosclerosis and aortic valve calcification.

Methods

In a cross-sectional study of 55 patients with severe aortic valve stenosis, we assessed the coronary calcification score (CAC) as indicator of total coronary atherosclerosis burden, and aortic valve calcification (AVC) by computed tomography. Thrombin-antithrombin complex (TATc) levels were measured as a marker for thrombin formation. Circulating MPs were characterized by flow cytometry according to the expression of established surface antigens and by measuring MP-induced thrombin generation.

Results

Patients with CAC score below the median were classified as patients with low CAC, patients with CAC Score above the median as high CAC. In patients with high CAC compared to patients with low CAC we detected higher levels of TATc, platelet-derived MPs (PMPs), endothelial-derived MPs (EMPs) and MP-induced thrombin generation. Increased level of PMPs and MP-induced thrombin generation were independent predictors for the severity of CAC. In contrast, AVC Score did not differ between patients with high and low CAC and did neither correlate with MPs levels nor with MP-induced thrombin generation.

Conclusion

In patients with severe aortic valve stenosis MP-induced thrombin generation was independently associated with the severity of CAC but not AVC indicating different pathomechanisms involved in coronary artery and aortic valve calcification.     

Literature-Informed Analysis of a Genome-Wide Association Study of Gestational Age in Norwegian Women and Children Suggests Involvement of Inflammatory Pathways

BackgroundFive-to-eighteen percent of pregnancies worldwide end in preterm birth, which is the major cause of neonatal death and morbidity. Approximately 30% of the variation in gestational age at birth can be attributed to genetic factors. Genome-wide association studies (GWAS) have not shown robust evidence of association with genomic loci yet.MethodsWe separately investigated 1921 Norwegian mothers and 1199 children from pregnancies with spontaneous onset of delivery. Individuals were further divided based on the onset of delivery: initiated by labor or prelabor rupture of membranes. Genetic association with ultrasound-dated gestational age was evaluated using three genetic models and adaptive permutations. The top-ranked loci were tested for enrichment in 12 candidate gene-sets generated by text-mining PubMed abstracts containing pregnancy-related keywords.ResultsThe six GWAS did not reveal significant associations, with the most extreme empirical p = 5.1 × 10⁻⁷. The top loci from maternal GWAS with deliveries initiated by labor showed significant enrichment in 10 PubMed gene-sets, e.g., p = 0.001 and 0.005 for keywords "uterus" and "preterm" respectively. Enrichment signals were mainly caused by infection/inflammation-related genes TLR4, NFKB1, ABCA1, MMP9. Literature-informed analysis of top loci revealed further immunity genes: IL1A, IL1B, CAMP, TREM1, TFRC, NFKBIA, MEFV, IRF8, WNT5A.ConclusionOur analyses support the role of inflammatory pathways in determining pregnancy duration and provide a list of 32 candidate genes for a follow-up work. We observed that the top regions from GWAS in mothers with labor-initiated deliveries significantly more often overlap with pregnancy-related genes than would be expected by chance, suggesting that increased sample size would benefit similar studies.     

Localization of the human neonatal Fc receptor (FcRn) in human nasal epithelium

The airway epithelium is a central player in the defense against pathogens including efficient mucociliary clearance and secretion of immunoglobulins, mainly polymeric IgA, but also IgG. Pulmonary administration of therapeutic antibodies on one hand, and intranasal immunization on the other, are powerful tools to treat airway infections. In either case, the airway epithelium is the primary site of antibody transfer. In various epithelia, bi-polar transcytosis of IgG and IgG immune complexes is mediated by the human neonatal Fc receptor, FcRn, but FcRn expression in the nasal epithelium had not been demonstrated, so far. We prepared affinity-purified antibodies against FcRn α-chain and confirmed their specificity by Western blotting and immunofluorescence microscopy. These antibodies were used to study the localization of FcRn α-chain in fixed nasal tissue. We here demonstrate for the first time that ciliated epithelial cells, basal cells, gland cells, and endothelial cells in the underlying connective tissue express the receptor. A predominant basolateral steady state distribution of the receptor was observed in ciliated epithelial as well as in gland cells. Co-localization of FcRn α-chain with IgG or with early sorting endosomes (EEA1-positive) but not with late endosomes/lysosomes (LAMP-2-positive) in ciliated cells was observed. This is indicative for the presence of the receptor in the recycling/transcytotic pathway but not in compartments involved in lysosomal degradation supporting the role of FcRn in IgG transcytosis in the nasal epithelium.     

Metformin inhibits proliferation and migration of glioblastoma cells independently of TGF-β2

To this day, glioblastoma (GBM) remains an incurable brain tumor. Previous research has shown that metformin, an oral anti-diabetic drug, may decrease GBM cell proliferation and migration especially in brain tumor initiating cells (BTICs). As transforming growth factor β 2 (TGF-β₂) has been reported to promote high-grade glioma and is inhibited by metformin in other tumors, we explored whether metformin directly interferes with TGF-β₂-signaling. Functional investigation of proliferation and migration of primary BTICs after treatment with metformin+/?TGF-β₂ revealed that metformin doses as low as 0.01 mM metformin thrice a day were able to inhibit proliferation of susceptible cell lines, whereas migration was impacted only at higher doses. Known cellular mechanisms of metformin, such as increased lactate secretion, reduced oxygen consumption and activated AMPK-signaling, could be confirmed. However, TGF-β₂ and metformin did not act as functional antagonists, but both rather inhibited proliferation and/or migration, if significant effects were present. We did not observe a relevant influence of metformin on TGF-β₂ mRNA expression (qRT-PCR), TGF-β₂ protein expression (ELISA) or SMAD-signaling (Western blot). Therefore, it seems that metformin does not exert its inhibitory effects on GBM BTIC proliferation and migration by altering TGF-β₂-signaling. Nonetheless, as low doses of metformin are able to reduce proliferation of certain GBM cells, further exploration of predictors of BTICs' susceptibility to metformin appears justified.     

The anterior visceral endoderm of the mouse embryo is established from both preimplantation precursor cells and by de novo gene expression after implantation

Initiation of the development of the anterior-posterior axis in the mouse embryo has been thought to take place only when the anterior visceral endoderm (AVE) emerges and starts its asymmetric migration. However, expression of Lefty1, a marker of the AVE, was recently found to initiate before embryo implantation. This finding has raised two important questions: are the cells that show such early, preimplantation expression of this AVE marker the real precursors of the AVE and, if so, how does this contribute to the establishment of the AVE? Here, we address both of these questions. First, we show that the expression of another AVE marker, Cer1, also commences before implantation and its expression becomes consolidated in the subset of ICM cells that comprise the primitive endoderm. Second, to determine whether the cells showing this early Cer1 expression are true precursors of the AVE, we set up conditions to trace these cells in time-lapse studies from early periimplantation stages until the AVE emerges and becomes asymmetrically displaced. We found that Cer1-expressing cells are asymmetrically located after implantation and, as the embryo grows, they become dispersed into two or three clusters. The expression of Cer1 in the proximal domain is progressively diminished, whilst it is reinforced in the distal-lateral domain. Our time-lapse studies demonstrate that this distal-lateral domain is incorporated into the AVE together with cells in which Cer1 expression begins only after implantation. Thus, the AVE is formed from both part of an ancestral population of Cerl-expressing cells and cells that acquire Cer1 expression later. Finally, we demonstrate that when the AVE shifts asymmetrically to establish the anterior pole, this occurs towards the region where the earlier postimplantation expression of Cer1 was strongest. Together, these results suggest that the orientation of the anterior-posterior axis is already anticipated before AVE migration.