Autophagy and its role in synapse function

The regulated turnover of synaptic vesicle (SV) proteins is thought to involve the ubiquitin- dependent tagging and degradation of SV proteins through endo-lysosomal and autophagy pathways. Yet, it remains unclear which of these pathways are used, when they become activated and whether SVs are cleared en-mass together with SV proteins or whether both are degraded selectively. Equally puzzling is how quickly these systems can be activated and whether they function in real-time to support synaptic health. To address these questions, I have developed an imaging based system that simultaneously labels presynaptic proteins while monitoring the appearance of autophagosomes and/or late endosomes within the presynaptic bouton. Moreover, by tagging three synaptic proteins (Synaptophysin, Synaptotagmin, Synapsin) with a light-activated reactive oxygen species (ROS) generator, Supernova, it was possible to temporally control the damage of specific SV proteins and assess its consequence to autophagy mediated clearance mechanisms and synaptic function. My results show that, in mouse hippocampal neurons, presynaptic autophagy can be induced in as little as 5-10 minutes (after Synaptophysin-Supernova bleaching) and eliminates primarily the damaged protein rather than the SV en-mass. Additionally, I could show that induction of ROS-activated presynaptic autophagy requires the close association of Supernova with SVs. Importantly, I also found that autophagy is essential for synaptic function, as ROS damage to e.g. Synaptophysin only compromises synaptic function when autophagy is simulaneously blocked, as measured in FM dye uptake and electrophysiology experiments. These data support the concept that presynaptic boutons have a robust, highly regulated clearance system to react in real-time to maintain not only synapse integrity, but also synapse function. Additionally, I was able to further characterize neurons from Bassoon KO mice, which depict higher autophagy levels at boutons and along axons compared to neurons from WT mice. Therefore, they may be a useful tool to study presynaptic autophagy. In my study, imaging based experiments confirmed that the autophagy-related phenotype in Bassoon KO neurons does not show a neurodegenerative progression and that the increase in autophagy is dependent on poly- ubiquitination as well as the autophagy-related protein Atg5. To summarize, I was able to provide new insights into the timing, the cargo and the function of presynaptic autophagy. Furthermore, my findings support the concept that functional autophagy is important for the clearance of damaged proteins, whereas excessively induced autophagy can be detrimental for synapse health. Thus, synaptic autophagy needs to be in balance

Single-Mindedness and Self-Reflectiveness: Laboratory Studies

Rechtschaffen (1978) has suggested that dreams are categorically single-minded and isolated. The phenomenon of lucid dreaming, however, suggests that his conclusion is overstated. Furthermore, the empirical status of Rechtschaffen’s claim is uncertain. The data on which his claim is based are personal and impressionistic. We view single-mindedness and lucidity as related along a continuum of self-reflectiveness, as suggested by Rossi (1972) and as operationalized in a scale of self-reflectiveness we derived from his work. In order to examine his assertion we conducted two laboratory experimental studies to examine the distribution of self-reflectiveness and singlemindedness in the dream reports of high and low frequency dream recallers awakened from Stages REM, 2 and 4. Self-reflectiveness of dream reports was quantified using the nine-step scale presented below

Single-Mindedness and Self-Reflectiveness: Laboratory Studies

Rechtschaffen (1978) has suggested that dreams are categorically single-minded and isolated. The phenomenon of lucid dreaming, however, suggests that his conclusion is overstated. Furthermore, the empirical status of Rechtschaffen’s claim is uncertain. The data on which his claim is based are personal and impressionistic. We view single-mindedness and lucidity as related along a continuum of self-reflectiveness, as suggested by Rossi (1972) and as operationalized in a scale of self-reflectiveness we derived from his work. In order to examine his assertion we conducted two laboratory experimental studies to examine the distribution of self-reflectiveness and single-mindedness in the dream reports of high and low frequency dream recallers awakened from stages REM, 2 and 4 Self-reflectiveness of dream reports was quantified using the 9-step scale presented below

Skin and Systemic Inflammation in Schnitzler's Syndrome Are Associated With Neutrophil Extracellular Trap Formation

Schnitzler's syndrome is a rare autoinflammatory disorder characterized by interleukin-1ß-mediated and neutrophil-dominated inflammation. Neutrophil extracellular traps (NETs) are web-like structures of decondensed chromatin, histones, and antimicrobial peptides released by neutrophils. NETs were initially described in the context of pathogen defense but are also involved in autoimmune-mediated skin diseases. Here, we assessed the role of neutrophil extracellular trap formation (NETosis) in Schnitzler's syndrome. Immunofluorescence co-staining of myeloperoxidase and subnucleosomal complex was performed on lesional skin samples from patients with Schnitzler's syndrome, other neutrophilic dermatoses (cryopyrin-associated periodic syndrome, Sweet syndrome, and pyoderma gangrenosum), urticarial vasculitis and chronic spontaneous urticaria as well as healthy control skin. Blood neutrophils from patients with Schnitzler's syndrome and controls were isolated, and NETosis was induced by phorbol 12-myristate 13-acetate (PMA). Also, NETosis of control neutrophils induced by symptomatic Schnitzler's syndrome sera, cytokines and sub-threshold PMA doses was studied. Immunofluorescence co-staining revealed widespread and substantial NET formation in lesional skin of Schnitzler's syndrome patients but absence of NETs in chronic spontaneous urticaria and control skin. Neutrophils undergoing NETosis were observed in the skin of other neutrophilic diseases too. Correspondingly, blood neutrophils from Schnitzler's syndrome patients showed significantly elevated NETosis rates compared to control neutrophils following stimulation with PMA. Increased NETosis correlated well with high levels of C-reactive protein (CRP). SchS patients with the lowest NETosis rates had persistent joint and bone pain despite IL-1 blockade. Stimulation of control neutrophils and sub-threshold PMA with sera of symptomatic Schnitzler's syndrome patients disclosed enhanced NETosis as compared to control sera. Our results suggest that the induction of NET formation by neutrophils contributes to skin and systemic inflammation and may support the resolution of local inflammation in Schnitzler's syndrome.</p

Post-ingestive effects of flavonoids in the midgut epithelium of Anticarsia gemmatalis (Hübner 1818) (Lepidoptera: Erebidae) larvae

An alternative control to soybean caterpillar has been the use of insect resistant plants that present phenolic flavonoids. The midgut is the main access pathway of food and chemical substances ingested. The present study examined morphological alterations in the midgut epithelium of the soybean caterpillar, after the ingestion of soybean genotypes containing the flavonoids rutin and genistin. The caterpillars and genotypes (BRS 257 - control, BR 16, Dowling, PI 229358, IAC 100 and PI 227687) were obtained from the insect rearing facility at the Embrapa Soja. The midguts were collected, fixed in Karnovsky, processed and analyzed under a light microscope. All treatments caused alterations in the midgut epithelial cells. These alterations in the columnar cells were more assiduous than the on other cell types, showing an increase of cytoplasmic protrusions and vacuoles. The goblet cells showed few changes for all genotypes tested, while the regenerative cells presented alterations mainly for the Dowling and PI 227687 treatments. The peritrophic membrane was absent for genotypes IAC100 and PI 227687. We conclude that the Dowling and PI 227687 genotypes were effective and promoted great morphological alterations in the midgut of the soybean caterpillars, being able to be very effective for the control of this plague

Studies of jet quenching within a partonic transport model

Background: Finite mixture models posit the existence of a latent categorical variable and can be used for probabilistic classification. The authors illustrate the use of mixture models for dietary pattern analysis. An advantage of this approach is taking classification uncertainty into account. Methods: Participants were a random sample of women from the European Prospective Investigation into Cancer. Food consumption was measured using dietary questionnaires. Mixture models identified latent classes in food consumption data, which were interpreted as dietary patterns. Results: Among various assumptions examined, models allowing the variance of foods to vary within and between classes fit better than alternatives assuming constant variance (the K-means method of cluster analysis also makes the latter assumption). An eight-class model was best fitting and five patterns validated well in a second random sample. Patterns with lower classification uncertainty tended to be better validated. One pattern showed low consumption of foods despite being associated with moderate body mass index. Conclusion: Mixture modelling for dietary pattern analysis has advantages over both factor and cluster analysis. In contrast to these other methods, it is easy to estimate pattern prevalence, to describe patterns and to use patterns to predict disease taking classification uncertainty into account. Owing to substantial error in food consumptions, any analysis will usually find some patterns that cannot be well validated. While knowledge of classification uncertainty may aid pattern evaluation, any method will better identify patterns from food consumptions measured with less error. Mixture models may be useful to identify individuals who under-report food consumption

Skin and Systemic Inflammation in Schnitzler's Syndrome Are Associated With Neutrophil Extracellular Trap Formation

Schnitzler's syndrome is a rare autoinflammatory disorder characterized by interleukin-1ß-mediated and neutrophil-dominated inflammation. Neutrophil extracellular traps (NETs) are web-like structures of decondensed chromatin, histones, and antimicrobial peptides released by neutrophils. NETs were initially described in the context of pathogen defense but are also involved in autoimmune-mediated skin diseases. Here, we assessed the role of neutrophil extracellular trap formation (NETosis) in Schnitzler's syndrome. Immunofluorescence co-staining of myeloperoxidase and subnucleosomal complex was performed on lesional skin samples from patients with Schnitzler's syndrome, other neutrophilic dermatoses (cryopyrin-associated periodic syndrome, Sweet syndrome, and pyoderma gangrenosum), urticarial vasculitis and chronic spontaneous urticaria as well as healthy control skin. Blood neutrophils from patients with Schnitzler's syndrome and controls were isolated, and NETosis was induced by phorbol 12-myristate 13-acetate (PMA). Also, NETosis of control neutrophils induced by symptomatic Schnitzler's syndrome sera, cytokines and sub-threshold PMA doses was studied. Immunofluorescence co-staining revealed widespread and substantial NET formation in lesional skin of Schnitzler's syndrome patients but absence of NETs in chronic spontaneous urticaria and control skin. Neutrophils undergoing NETosis were observed in the skin of other neutrophilic diseases too. Correspondingly, blood neutrophils from Schnitzler's syndrome patients showed significantly elevated NETosis rates compared to control neutrophils following stimulation with PMA. Increased NETosis correlated well with high levels of C-reactive protein (CRP). SchS patients with the lowest NETosis rates had persistent joint and bone pain despite IL-1 blockade. Stimulation of control neutrophils and sub-threshold PMA with sera of symptomatic Schnitzler's syndrome patients disclosed enhanced NETosis as compared to control sera. Our results suggest that the induction of NET formation by neutrophils contributes to skin and systemic inflammation and may support the resolution of local inflammation in Schnitzler's syndrome

A Systematic Review of Mosquito Coils and Passive Emanators: Defining Recommendations for Spatial Repellency Testing Methodologies.

Mosquito coils, vaporizer mats and emanators confer protection against mosquito bites through the spatial action of emanated vapor or airborne pyrethroid particles. These products dominate the pest control market; therefore, it is vital to characterize mosquito responses elicited by the chemical actives and their potential for disease prevention. The aim of this review was to determine effects of mosquito coils and emanators on mosquito responses that reduce human-vector contact and to propose scientific consensus on terminologies and methodologies used for evaluation of product formats that could contain spatial chemical actives, including indoor residual spraying (IRS), long lasting insecticide treated nets (LLINs) and insecticide treated materials (ITMs). PubMed, (National Centre for Biotechnology Information (NCBI), U.S. National Library of Medicine, NIH), MEDLINE, LILAC, Cochrane library, IBECS and Armed Forces Pest Management Board Literature Retrieval System search engines were used to identify studies of pyrethroid based coils and emanators with key-words "Mosquito coils" "Mosquito emanators" and "Spatial repellents". It was concluded that there is need to improve statistical reporting of studies, and reach consensus in the methodologies and terminologies used through standardized testing guidelines. Despite differing evaluation methodologies, data showed that coils and emanators induce mortality, deterrence, repellency as well as reduce the ability of mosquitoes to feed on humans. Available data on efficacy outdoors, dose-response relationships and effective distance of coils and emanators is inadequate for developing a target product profile (TPP), which will be required for such chemicals before optimized implementation can occur for maximum benefits in disease control

No evidence for cardiac dysfunction in Kif6 mutant mice.

A KIF6 variant in man has been reported to be associated with adverse cardiovascular outcomes after myocardial infarction. No clear biological or physiological data exist for Kif6. We sought to investigate the impact of a deleterious KIF6 mutation on cardiac function in mice. Kif6 mutant mice were generated and verified. Cardiac function was assessed by serial echocardiography at baseline, after ageing and after exercise. Lipid levels were also measured. No discernable adverse lipid or cardiac phenotype was detected in Kif6 mutant mice. These data suggest that dysfunction of Kif6 is linked to other more complex biological/biochemical parameters or is unlikely to be of material consequence in cardiac function

Genetic association study of QT interval highlights role for calcium signaling pathways in myocardial repolarization.

The QT interval, an electrocardiographic measure reflecting myocardial repolarization, is a heritable trait. QT prolongation is a risk factor for ventricular arrhythmias and sudden cardiac death (SCD) and could indicate the presence of the potentially lethal mendelian long-QT syndrome (LQTS). Using a genome-wide association and replication study in up to 100,000 individuals, we identified 35 common variant loci associated with QT interval that collectively explain ∼8-10% of QT-interval variation and highlight the importance of calcium regulation in myocardial repolarization. Rare variant analysis of 6 new QT interval-associated loci in 298 unrelated probands with LQTS identified coding variants not found in controls but of uncertain causality and therefore requiring validation. Several newly identified loci encode proteins that physically interact with other recognized repolarization proteins. Our integration of common variant association, expression and orthogonal protein-protein interaction screens provides new insights into cardiac electrophysiology and identifies new candidate genes for ventricular arrhythmias, LQTS and SCD