Correlative super-resolution and electron microscopy to resolve protein localization in zebrafish retina

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and scanning electron microscopy. The sub-diffraction limit resolution capabilities of super-resolution light microscopes allow improving the accuracy of the correlated data. Briefly, 110 nanometer thick cryo-sections are transferred to a silicon wafer and, after immunofluorescence staining, are imaged by super-resolution light microscopy. Subsequently, the sections are preserved in methylcellulose and platinum shadowed prior to imaging in a scanning electron microscope (SEM). The images from these two microscopy modalities are easily merged using tissue landmarks with open source software. Here we describe the adapted method for the larval zebrafish retina. However, this method is also applicable to other types of tissues and organisms. We demonstrate that the complementary information obtained by this correlation is able to resolve the expression of mitochondrial proteins in relation with the membranes and cristae of mitochondria as well as to other compartments of the cell

Topographic contrast of ultrathin cryo-sections for correlative super-resolution light and electron microscopy

Fluorescence microscopy reveals molecular expression at nanometer resolution but lacks ultrastructural context information. This deficit often hinders a clear interpretation of results. Electron microscopy provides this contextual subcellular detail, but protein identification can often be problematic. Correlative light and electron microscopy produces complimentary information that expands our knowledge of protein expression in cells and tissue. Inherent methodological difficulties are however encountered when combining these two very different microscopy technologies. We present a quick, simple and reproducible method for protein localization by conventional and super-resolution light microscopy combined with platinum shadowing and scanning electron microscopy to obtain topographic contrast from the surface of ultrathin cryo-sections. We demonstrate protein distribution at nuclear pores and at mitochondrial and plasma membranes in the extended topographical landscape of tissue

Nuclear envelope impairment is facilitated by the herpes simplex virus 1 Us3 kinase

Background: Capsids of herpes simplex virus 1 (HSV-1) are assembled in the nucleus, translocated either to the perinuclear space by budding at the inner nuclear membrane acquiring tegument and envelope, or released to the cytosol in a “naked” state via impaired nuclear pores that finally results in impairment of the nuclear envelope. The Us3 gene encodes a protein acting as a kinase, which is responsible for phosphorylation of numerous viral and cellular substrates. The Us3 kinase plays a crucial role in nucleus to cytoplasm capsid translocation. We thus investigate the nuclear surface in order to evaluate the significance of Us3 in maintenance of the nuclear envelope during HSV-1 infection. Methods: To address alterations of the nuclear envelope and capsid nucleus to cytoplasm translocation related to the function of the Us3 kinase we investigated cells infected with wild type HSV-1 or the Us3 deletion mutant R7041(∆Us3) by transmission electron microscopy, focused ion-beam electron scanning microscopy, cryo-field emission scanning electron microscopy, confocal super resolution light microscopy, and polyacrylamide gel electrophoresis. Results: Confocal super resolution microscopy and cryo-field emission scanning electron microscopy revealed decrement in pore numbers in infected cells. Number and degree of pore impairment was significantly reduced after infection with R7041(∆Us3) compared to infection with wild type HSV-1. The nuclear surface was significantly enlarged in cells infected with any of the viruses. Morphometric analysis revealed that additional nuclear membranes were produced forming multiple folds and caveolae, in which virions accumulated as documented by three-dimensional reconstruction after ion-beam scanning electron microscopy. Finally, significantly more R7041(∆Us3) capsids were retained in the nucleus than wild-type capsids whereas the number of R7041(∆Us3) capsids in the cytosol was significantly lower. Conclusions: The data indicate that Us3 kinase is involved in facilitation of nuclear pore impairment and, concomitantly, in capsid release through impaired nuclear envelope

GABA neurons of the VTA drive conditioned place aversion

Salient but aversive stimuli inhibit the majority of dopamine (DA) neurons in the ventral tegmental area (VTA) and cause conditioned place aversion (CPA). The cellular mechanism underlying DA neuron inhibition has not been investigated and the causal link to behavior remains elusive. Here, we show that GABA neurons of the VTA inhibit DA neurons through neurotransmission at GABA(A) receptors. We also observe that GABA neurons increase their firing in response to a footshock and provide evidence that driving GABA neurons with optogenetic effectors is sufficient to affect behavior. Taken together, our data demonstrate that synaptic inhibition of DA neurons drives place aversion

Mid-regional pro-atrial natriuretic peptide as a prognostic marker for all-cause mortality in patients with symptomatic coronary artery disease

In the present study, we investigated the prognostic value of MR-proANP (mid-regional pro-atrial natriuretic peptide). We consecutively evaluated a catheterization laboratory cohort of 2700 patients with symptomatic CAD (coronary artery disease) [74.1% male; ACS (acute coronary syndrome), n=1316; SAP (stable angina pectoris), n=1384] presenting to the Cardiology Department of a large primary care hospital, all of whom underwent coronary angiography. Serum MR-proANP and other laboratory markers were sampled at the time of presentation or in the catheterization laboratory. Clinical outcome was assessed by hospital chart analysis and telephone interviews. The primary end point was all-cause death at 3 months after enrolment. Follow-up data were complete in 2621 patients (97.1%). Using ROC (receiver operating characteristic) curves, the AUC (area under the curve) of 0.73 [95% CI (confidence interval), 0.67–0.79] for MR-proANP was significantly higher compared with 0.58 (95% CI, 0.55–0.62) for Tn-I (troponin-I; DeLong test, P=0.0024). According to ROC analysis, the optimal cut-off value of MR-proANP was at 236 pmol/l for all-cause death, which helped to find a significantly increased rate of all-cause death (n=76) at 3 months in patients with elevated baseline concentrations (≥236 pmol/l) compared with patients with a lower concentration level in Kaplan–Meier survival analysis (log rank, P&amp;lt;0.001). The predictive performance of MR-proANP was independent of other clinical variables or cardiovascular risk factors, and superior to that of Tn-I or other cardiac biomarkers (all: P&amp;lt;0.0001). MR-proANP may help in the prediction of all-cause death in patients with symptomatic CAD. Further studies should verify its prognostic value and confirm the appropriate cut-off value.</jats:p

Copeptin as a prognostic factor for major adverse cardiovascular events in patients with coronary artery disease

Background C-terminal portion of provasopressin (copeptin) has recently been discussed as a novel biomarker for the early rule-out of acute myocardial infarction (AMI). The aim is to investigate the prognostic value of copeptin with regard to mortality and morbidity in patients with symptomatic coronary artery disease (CAD). Methods We consecutively recruited a cath lab cohort of 2,700 patients (74.1% male; AMI, n = 1316; stable angina pectoris, n = 1384) presenting to the emergency department of a large primary care hospital. All patients received coronary angiography. Copeptin and other laboratory markers were sampled at the time of presentation or in the cath lab. Clinical outcomes were assessed by hospital chart analysis and telephone interviews. 2621 patients (97.1%) have been successfully followed-up at three months. The primary endpoint was a combined endpoint of rehospitalization for cardiovascular events, stroke, and all-cause death. Results Using receiver operating characteristic curves, we calculated areas under the curve of 0.703 (95%confidence interval(CI):0.681–0.725) for the composite endpoint after three months (myocardial reinfarction, stroke, all-cause death;n = 183), and 0.770 (95%CI:0.736–0.803) for all-cause death (n = 76) for copeptin. A cutoff value of 21.6 pmol/L for the composite endpoint yielded a sensitivity of 56.3% and a specificity of 78.6%. The predictive performance of copeptin was independent of other clinical variables or cardiovascular risk factors, and superior to that of troponin I or other cardiac biomarkers (all:P < 0.0001). Conclusions Copeptin may help in the prediction of major adverse cardiovascular events in patients with symptomatic CAD. Further studies should substantiate the findings and support the suggested cutoff value of the present study

Systemic immune challenges trigger and drive Alzheimer-like neuropathology in mice

BACKGROUND: Alzheimer's disease (AD) is the most prevalent form of age-related dementia, and its effect on society increases exponentially as the population ages. Accumulating evidence suggests that neuroinflammation, mediated by the brain's innate immune system, contributes to AD neuropathology and exacerbates the course of the disease. However, there is no experimental evidence for a causal link between systemic inflammation or neuroinflammation and the onset of the disease. METHODS: The viral mimic, polyriboinosinic-polyribocytidilic acid (PolyI:C) was used to stimulate the immune system of experimental animals. Wild-type (WT) and transgenic mice were exposed to this cytokine inducer prenatally (gestation day (GD)17) and/or in adulthood. Behavioral, immunological, immunohistochemical, and biochemical analyses of AD-associated neuropathologic changes were performed during aging. RESULTS: We found that a systemic immune challenge during late gestation predisposes WT mice to develop AD-like neuropathology during the course of aging. They display chronic elevation of inflammatory cytokines, an increase in the levels of hippocampal amyloid precursor protein (APP) and its proteolytic fragments, altered Tau phosphorylation, and mis-sorting to somatodendritic compartments, and significant impairments in working memory in old age. If this prenatal infection is followed by a second immune challenge in adulthood, the phenotype is strongly exacerbated, and mimics AD-like neuropathologic changes. These include deposition of APP and its proteolytic fragments, along with Tau aggregation, microglia activation and reactive gliosis. Whereas Aβ peptides were not significantly enriched in extracellular deposits of double immune-challenged WT mice at 15 months, they dramatically increased in age-matched immune-challenged transgenic AD mice, precisely around the inflammation-induced accumulations of APP and its proteolytic fragments, in striking similarity to the post-mortem findings in human patients with AD. CONCLUSION: Chronic inflammatory conditions induce age-associated development of an AD-like phenotype in WT mice, including the induction of APP accumulations, which represent a seed for deposition of aggregation-prone peptides. The PolyI:C mouse model therefore provides a unique tool to investigate the molecular mechanisms underlying the earliest pathophysiological changes preceding fibrillary Aβ plaque deposition and neurofibrillary tangle formations in a physiological context of aging. Based on the similarity between the changes in immune-challenged mice and the development of AD in humans, we suggest that systemic infections represent a major risk factor for the development of AD