Borna Disease Virus Blocks Potentiation of Presynaptic Activity through Inhibition of Protein Kinase C Signaling

Infection by Borna disease virus (BDV) enables the study of the molecular mechanisms whereby a virus can persist in the central nervous system and lead to altered brain function in the absence of overt cytolysis and inflammation. This neurotropic virus infects a wide variety of vertebrates and causes behavioral diseases. The basis of BDV-induced behavioral impairment remains largely unknown. Here, we investigated whether BDV infection of neurons affected synaptic activity, by studying the rate of synaptic vesicle (SV) recycling, a good indicator of synaptic activity. Vesicular cycling was visualized in cultured hippocampal neurons synapses, using an assay based on the uptake of an antibody directed against the luminal domain of synaptotagmin I. BDV infection did not affect elementary presynaptic functioning, such as spontaneous or depolarization-induced vesicular cycling. In contrast, infection of neurons with BDV specifically blocked the enhancement of SV recycling that is observed in response to stimuli-induced synaptic potentiation, suggesting defects in long-term potentiation. Studies of signaling pathways involved in synaptic potentiation revealed that this blockade was due to a reduction of the phosphorylation by protein kinase C (PKC) of proteins that regulate SV recycling, such as myristoylated alanine-rich C kinase substrate (MARCKS) and Munc18–1/nSec1. Moreover, BDV interference with PKC-dependent phosphorylation was identified downstream of PKC activation. We also provide evidence suggesting that the BDV phosphoprotein interferes with PKC-dependent phosphorylation. Altogether, our results reveal a new mechanism by which a virus can cause synaptic dysfunction and contribute to neurobehavioral disorders

Identification of bitter pit markers in Malus domestica

Bitter pit is a physiological disorder occurring in apple, pear, and quince and has been associated with calcium uptake or lack thereof. Studied for over a century, little is known regarding the development of bitter pit, what triggers its occurrence, and why there are no completely effective preventative treatments to reduce fruit loss. In the present study, Malus domestica proteins were extracted, injected, and analyzed on a LC Q Exactive mass spectrometer. More than 1500 proteins were identified between bitter pit and healthy samples. To establish quality markers of bitter pit, proteins were filtered using p<0.05 (95 % significance). A total of 170 proteins were identified as quality markers for bitter pit (Whether significantly greater or lower). This study is first to report the bitter pit protein profile in Malus domestica and identify significantly different markers between bitter pit and healthy samples that indicate the occurrence of bitter pit.Peer Reviewe

Neurons are MHC Class I-Dependent Targets for CD8 T Cells upon Neurotropic Viral Infection

Following infection of the central nervous system (CNS), the immune system is faced with the challenge of eliminating the pathogen without causing significant damage to neurons, which have limited capacities of renewal. In particular, it was thought that neurons were protected from direct attack by cytotoxic T lymphocytes (CTL) because they do not express major histocompatibility class I (MHC I) molecules, at least at steady state. To date, most of our current knowledge on the specifics of neuron-CTL interaction is based on studies artificially inducing MHC I expression on neurons, loading them with exogenous peptide and applying CTL clones or lines often differentiated in culture. Thus, much remains to be uncovered regarding the modalities of the interaction between infected neurons and antiviral CD8 T cells in the course of a natural disease. Here, we used the model of neuroinflammation caused by neurotropic Borna disease virus (BDV), in which virus-specific CTL have been demonstrated as the main immune effectors triggering disease. We tested the pathogenic properties of brain-isolated CD8 T cells against pure neuronal cultures infected with BDV. We observed that BDV infection of cortical neurons triggered a significant up regulation of MHC I molecules, rendering them susceptible to recognition by antiviral CTL, freshly isolated from the brains of acutely infected rats. Using real-time imaging, we analyzed the spatio-temporal relationships between neurons and CTL. Brain-isolated CTL exhibited a reduced mobility and established stable contacts with BDV-infected neurons, in an antigen- and MHC-dependent manner. This interaction induced rapid morphological changes of the neurons, without immediate killing or impairment of electrical activity. Early signs of neuronal apoptosis were detected only hours after this initial contact. Thus, our results show that infected neurons can be recognized efficiently by brain-isolated antiviral CD8 T cells and uncover the unusual modalities of CTL-induced neuronal damage

Extracorporeal Membrane Oxygenation for Severe Acute Respiratory Distress Syndrome associated with COVID-19: An Emulated Target Trial Analysis.

RATIONALE: Whether COVID patients may benefit from extracorporeal membrane oxygenation (ECMO) compared with conventional invasive mechanical ventilation (IMV) remains unknown. OBJECTIVES: To estimate the effect of ECMO on 90-Day mortality vs IMV only Methods: Among 4,244 critically ill adult patients with COVID-19 included in a multicenter cohort study, we emulated a target trial comparing the treatment strategies of initiating ECMO vs. no ECMO within 7 days of IMV in patients with severe acute respiratory distress syndrome (PaO2/FiO2 <80 or PaCO2 ≥60 mmHg). We controlled for confounding using a multivariable Cox model based on predefined variables. MAIN RESULTS: 1,235 patients met the full eligibility criteria for the emulated trial, among whom 164 patients initiated ECMO. The ECMO strategy had a higher survival probability at Day-7 from the onset of eligibility criteria (87% vs 83%, risk difference: 4%, 95% CI 0;9%) which decreased during follow-up (survival at Day-90: 63% vs 65%, risk difference: -2%, 95% CI -10;5%). However, ECMO was associated with higher survival when performed in high-volume ECMO centers or in regions where a specific ECMO network organization was set up to handle high demand, and when initiated within the first 4 days of MV and in profoundly hypoxemic patients. CONCLUSIONS: In an emulated trial based on a nationwide COVID-19 cohort, we found differential survival over time of an ECMO compared with a no-ECMO strategy. However, ECMO was consistently associated with better outcomes when performed in high-volume centers and in regions with ECMO capacities specifically organized to handle high demand. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Identification and quantification of proteins in Malus domestica affected by bitter pit

Bitter pit is a physiological disorder that occurs in apple, pear, and quince and has been associated with calcium uptake or lack thereof. Although bitter pit has been studied for over a century, there is still not enough knowledge about bitter pit and why there are no completely effective preventive treatments to reduce fruit loss. In a previous group publication (Val et al. 2006), it was conjectured after SDS-PAGE that an unknown 18 kDa protein might contribute to bitter pito The objective of the present study was to identify this 18 kDa protein and get more extensive data on the proteomic changes associated to bitter pit using the latest mass spectrometry-based proteomics. Healthy and bitter pit fruit samples (Malus domesica 'Reinette gris du Canada' and Malus domestica 'Golden Smoothee') were collected near Zaragoza (Aragón, Spain). Following phenol extraction, ten µg protein were allowed to run on SDS-PAGE, trypsin digested, and digestion products were analyzed on a Q-Exactive mass spectrometer (Thermo Scientific). Proteins were identified with X!Tandem pipeline software (http://pappso.inra.fr/) and relative quantification was performed by spectral counting. More than two hundred proteins were identified in the range of 12-24 kDa. We focused on 35 proteins that varied significantly between the two conditions (bitter pit vs. healthy) being over/under expressed. There were 26 and 22 bitter pit proteins ('Reinette gris du Canada' and 'Smoothee Golden Delicious,' respectively) detected with at least 50% greater abundance when compared to their respective healthy counterparts. Among these proteins, 14 found in both cultivars, were identified as Pathogenesis-related protein Bet v 1, a major allergen found in trees within the order Fagales. In both apple cultivars, 2 proteins were identified as thaumatin-like protein (TLP), a group of proteins responsible for several fruit allergies. Glutathione S-transferase, linked to protein binding and heat shock transcriptional factors (Hsfs) in Malus, was abundantly detected in bitter pit samples for both cultivars. Several proteins identified near or at 18-kDa, are related to Malus domestica' response to stress, desiccation, and increased protein binding. Considerable differences were found in allergen concentrations between bitter pit and healthy samples, suggesting an increased allergen risk for consumers who ingest bitter pit affected fruit.Peer Reviewe

PMA, but Not Forskolin-Induced Potentiation of Vesicular Recycling Is Impaired in BDV-Infected Neurons

<p>Cumulative probability distributions of fluorescence ratios (F1/F2) for individual synapses for the analysis of SV recycling in spontaneous conditions and after (A) PMA treatment or (B) forskolin treatment. The overview of the experimental stimulation protocol is depicted above each graph. The level of SV recycling is significantly lower in BDV-infected neurons compared to NI neurons after stimulation with PMA (<i>p</i> < 0.001 using KS test). Pairwise comparisons of mean presynaptic activities in independent experiments involved at least 180 synapses (small symbols). Bigger squares correspond to mean values of all independent experiments. Similar results were found in four independent experiments. Also shown are results for NI neurons treated with the PKC inhibitor Bis or the PKA inhibitor H89. Hash mark (#) indicates <i>p</i> < 0.05. n.s., not significant, by paired <i>t</i> test.</p

Analysis of PKC Activation

<div><p>(A) Cytosol-to-membrane recruitment of PKC. Subcellular distribution of PKC isoforms in NI and BDV-infected neurons after treatment with PMA. The detergent-soluble and particulate fractions, corresponding to cytosolic and membrane fractions, were subjected to Western blots for the detection of PKCα, PKCγ, PKCɛ, and PKCδ. Note the shift in the subcellular distribution of PKC isoforms after treatment with PMA. Results are representative of three independent experiments.</p><p>(B) In vitro measurements of basal and cofactor-stimulated PKC activities. The cofactor-stimulated specific activity represents kinase activity in the presence of Ca²⁺ and lipid activators. Results are expressed as percentage of increase over the mean of unstimulated controls.</p></div

Analysis of PKC Signaling in NI and BDV-Infected Neurons

<p>Western blot analyses of (A) MARCKS and (B) Munc18 phosphorylation by PKC. β-Tubulin III and total Munc18 were used to normalize expression. Results are representative of three to five independent experiments. Double asterisks (**) indicate <i>p</i> < 0.01. n.s., not significant, using unpaired <i>t</i> test.</p

PKC Signaling Is Impaired in BDV-Infected Neurons

<p>Western blots of neuronal extracts from NI and BDV-infected cultures stimulated with glycine using antibodies specific for: (A) Diphosphorylated ERK 1/2 (pERK) and total ERK 1/2; (B) phospho-synapsin I (pSynapsin I; Site 3, a site specific for CaMK II), and total synapsin I; (C) phospho-synapsin I (Site 1, a site specific for PKA and CamK I) and total synapsin I; (D) phospho-MARCKS (pMARCKS; PKC site at Ser152/156) and β-tubulin III. Results are representative of four independent experiments. Single asterisk (*) indicates <i>p</i> < 0.05; double asterisks (**) indicate <i>p</i> < 0.01 by unpaired <i>t</i> test.</p