Long-term potentiation through calcium-mediated N-Cadherin interaction is tightly controlled by the three-dimensional architecture of the synapse

Poster presentation: Twenty Second Annual Computational Neuroscience Meeting: CNS*2013. Paris, France. 13-18 July 2013. The synaptic cleft is an extracellular domain that is capable of relaying a presynaptically received electrical signal by diffusive neurotransmitters to the postsynaptic membrane. The cleft is trans-synaptically bridged by ring-like shaped clusters of pre- and postsynaptically localized calcium-dependent adhesion proteins of the N-Cadherin type and is possibly the smallest intercircuit in nervous systems [1]. The strength of association between the pre- and postsynaptic membranes can account for synaptic plasticity such as long-term potentiation [2]. Through neuronal activity the intra- and extracellular calcium levels are modulated through calcium exchangers embedded in the pre- and postsynaptic membrane. Variations of the concentration of cleft calcium induces changes in the N-Cadherin-zipper, that in synaptic resting states is rigid and tightly connects the pre- and postsynaptic domain. During synaptic activity calcium concentrations are hypothesized to drop below critical thresholds which leads to loosening of the N-Cadherin connections and subsequently "unzips" the Cadherin-mediated connection. These processes may result in changes in synaptic strength [2]. In order to investigate the calcium-mediated N-Cadherin dynamics at the synaptic cleft, we developed a three-dimensional model including the cleft morphology and all prominent calcium exchangers and corresponding density distributions [3-6]. The necessity for a fully three-dimensional model becomes apparent, when investigating the effects of the spatial architecture of the synapse [7], [8]. Our data show, that the localization of calcium channels with respect to the N-Cadherin ring has substantial effects on the time-scales on which the Cadherin-zipper switches between states, ranging from seconds to minutes. This will have significant effects on synaptic signaling. Furthermore we see, that high-frequency action potential firing can only be relayed to the Calcium/N-Cadherin-system at a synapse under precise spatial synaptic reorganization

Correlative Light- and Electron Microscopy with chemical tags

AbstractCorrelative microscopy incorporates the specificity of fluorescent protein labeling into high-resolution electron micrographs. Several approaches exist for correlative microscopy, most of which have used the green fluorescent protein (GFP) as the label for light microscopy. Here we use chemical tagging and synthetic fluorophores instead, in order to achieve protein-specific labeling, and to perform multicolor imaging. We show that synthetic fluorophores preserve their post-embedding fluorescence in the presence of uranyl acetate. Post-embedding fluorescence is of such quality that the specimen can be prepared with identical protocols for scanning electron microscopy (SEM) and transmission electron microscopy (TEM); this is particularly valuable when singular or otherwise difficult samples are examined. We show that synthetic fluorophores give bright, well-resolved signals in super-resolution light microscopy, enabling us to superimpose light microscopic images with a precision of up to 25nm in the x–y plane on electron micrographs. To exemplify the preservation quality of our new method we visualize the molecular arrangement of cadherins in adherens junctions of mouse epithelial cells

A Deep-Learning Algorithm to Predict Short-Term Progression to Geographic Atrophy on Spectral-Domain Optical Coherence Tomography

IMPORTANCE: The identification of patients at risk of progressing from intermediate age-related macular degeneration (iAMD) to geographic atrophy (GA) is essential for clinical trials aimed at preventing disease progression. DeepGAze is a fully automated and accurate convolutional neural network-based deep learning algorithm for predicting progression from iAMD to GA within 1 year from spectral-domain optical coherence tomography (SD-OCT) scans. OBJECTIVE: To develop a deep-learning algorithm based on volumetric SD-OCT scans to predict the progression from iAMD to GA during the year following the scan. DESIGN, SETTING, AND PARTICIPANTS: This retrospective cohort study included participants with iAMD at baseline and who either progressed or did not progress to GA within the subsequent 13 months. Participants were included from centers in 4 US states. Data set 1 included patients from the Age-Related Eye Disease Study 2 AREDS2 (Ancillary Spectral-Domain Optical Coherence Tomography) A2A study (July 2008 to August 2015). Data sets 2 and 3 included patients with imaging taken in routine clinical care at a tertiary referral center and associated satellites between January 2013 and January 2023. The stored imaging data were retrieved for the purpose of this study from July 1, 2022, to February 1, 2023. Data were analyzed from May 2021 to July 2023. EXPOSURE: A position-aware convolutional neural network with proactive pseudointervention was trained and cross-validated on Bioptigen SD-OCT volumes (data set 1) and validated on 2 external data sets comprising Heidelberg Spectralis SD-OCT scans (data sets 2 and 3). MAIN OUTCOMES AND MEASURES: Prediction of progression to GA within 13 months was evaluated with area under the receiver-operator characteristic curves (AUROC) as well as area under the precision-recall curve (AUPRC), sensitivity, specificity, positive predictive value, negative predictive value, and accuracy. RESULTS: The study included a total of 417 patients: 316 in data set 1 (mean [SD] age, 74 [8]; 185 [59%] female), 53 in data set 2, (mean [SD] age, 83 [8]; 32 [60%] female), and 48 in data set 3 (mean [SD] age, 81 [8]; 32 [67%] female). The AUROC for prediction of progression from iAMD to GA within 1 year was 0.94 (95% CI, 0.92-0.95; AUPRC, 0.90 [95% CI, 0.85-0.95]; sensitivity, 0.88 [95% CI, 0.84-0.92]; specificity, 0.90 [95% CI, 0.87-0.92]) for data set 1. The addition of expert-annotated SD-OCT features to the model resulted in no improvement compared to the fully autonomous model (AUROC, 0.95; 95% CI, 0.92-0.95; P = .19). On an independent validation data set (data set 2), the model predicted progression to GA with an AUROC of 0.94 (95% CI, 0.91-0.96; AUPRC, 0.92 [0.89-0.94]; sensitivity, 0.91 [95% CI, 0.74-0.98]; specificity, 0.80 [95% CI, 0.63-0.91]). At a high-specificity operating point, simulated clinical trial recruitment was enriched for patients progressing to GA within 1 year by 8.3- to 20.7-fold (data sets 2 and 3). CONCLUSIONS AND RELEVANCE: The fully automated, position-aware deep-learning algorithm assessed in this study successfully predicted progression from iAMD to GA over a clinically meaningful time frame. The ability to predict imminent GA progression could facilitate clinical trials aimed at preventing the condition and could guide clinical decision-making regarding screening frequency or treatment initiation

Temas y problemas en Antropología Social

El presente texto tiene que ver con el programa de la materia Antropología Cultural y Social dictada en la Facultad de Psicología de nuestra Universidad de La Plata. Sus diversos capítulos cubren varios temas del curso y fueron antecedidos por otros textos temáticos menos formalizados, editados anteriormente por la Cátedra. Nos ha parecido siempre importante adaptar los conocimientos de la Antropología en el marco de las Ciencias Sociales y de la Antropología Social en particular -que constituyen el eje de la materia- con la intención de conformar un eje didáctico de materiales que sean de fácil comprensión y permitan una lectura ulterior de mayor profundidad y continuidad, según el avance en la construcción de los conocimientos por parte de alumnos. Esto es importante por cuanto la disciplina constituye, de acuerdo al nuevo perfil del Plan de Estudios, uno de los cuatro pilares de conocimiento básico de la Psicología. En este sentido, se la considera un ámbito disciplinar académico destacado que aporta a los estudiantes herramientas conceptual-metodológicas básicas para la lectura y comprensión crítica del contexto sociohistórico, cultural y político en el que desarrollan sus prácticas actuales y su futura práctica profesional. Con una perspectiva más amplia se ha pensado, al redactar los capítulos, en el posible interés que puedan tener su lectura en el ámbito general de la Universidad.Facultad de Psicologí

Direct visualization of newly synthesized target proteins in situ

Protein synthesis is a dynamic process that tunes the cellular proteome in response to internal and external demands. Metabolic labeling approaches identify the general proteomic response but cannot visualize specific newly synthesized proteins within cells. Here we describe a technique that couples noncanonical amino acid tagging or puromycylation with the proximity ligation assay to visualize specific newly synthesized proteins and monitor their origin, redistribution and turnover in situ

EuReCa ONE—27 Nations, ONE Europe, ONE Registry A prospective one month analysis of out-of-hospital cardiac arrest outcomes in 27 countries in Europe

AbstractIntroductionThe aim of the EuReCa ONE study was to determine the incidence, process, and outcome for out of hospital cardiac arrest (OHCA) throughout Europe.MethodsThis was an international, prospective, multi-centre one-month study. Patients who suffered an OHCA during October 2014 who were attended and/or treated by an Emergency Medical Service (EMS) were eligible for inclusion in the study. Data were extracted from national, regional or local registries.ResultsData on 10,682 confirmed OHCAs from 248 regions in 27 countries, covering an estimated population of 174 million. In 7146 (66%) cases, CPR was started by a bystander or by the EMS. The incidence of CPR attempts ranged from 19.0 to 104.0 per 100,000 population per year. 1735 had ROSC on arrival at hospital (25.2%), Overall, 662/6414 (10.3%) in all cases with CPR attempted survived for at least 30 days or to hospital discharge.ConclusionThe results of EuReCa ONE highlight that OHCA is still a major public health problem accounting for a substantial number of deaths in Europe.EuReCa ONE very clearly demonstrates marked differences in the processes for data collection and reported outcomes following OHCA all over Europe. Using these data and analyses, different countries, regions, systems, and concepts can benchmark themselves and may learn from each other to further improve survival following one of our major health care events

Influence of Ca²⁺-chelation on the individual binding interfaces of N-cadherin.

<p>(<b>A</b>) Disruption of the <i>trans</i> interaction of N-cadherin by BAPTA. Upon disruption of this interaction the fluorophores move further apart, reducing FRET. Examples for ratiometric FRET measurements of the WT (<b>B</b>), W2A (<b>C</b>), R14E (<b>D</b>) and V81D/V174D (<b>E</b>) before and after Ca²⁺-chelation are shown. After baseline recording, the Ca²⁺-chelator BAPTA (20 mM final concentration) was added. The fluorescence of the FRET acceptor Venus (yellow) and the donor Cerulean (cyan) as well as the ratio of the two (blue) are shown. (<b>F</b>) The quantification of the effect of BAPTA on all mutants is shown. While the mutants W2A and V81D/V174D showed a significantly higher decrease of the FRET signal due to Ca²⁺-chelation than the WT, no Ca²⁺-sensitivity was observed for the X-dimer mutant R14E. This confirms the hypothesis that the X-dimer is a Ca²⁺-dependent intermediate step in the formation of the strand-swapped dimer. n(WT) = 8, n(W2A) = 8, n(R14E) = 9, n(V81D/V174D) = 7 junctions. Statistics were conducted using either paired or unpaired t-test.</p

Analysis of the <i>trans</i> interaction of N-cadherin in living cells by acceptor bleach FRET experiments.

<p>(<b>A</b>) Scheme depicting the insertion of Venus (FRET acceptor) and Cerulean (FRET donor) and the interactions of cadherin molecules across a cellular junction. If the two opposing molecules interact with each other in <i>trans</i>, the donor and acceptor are within the FRET distance, resulting in an increased acceptor emission and a donor quenching (upper panel). Upon bleaching of the FRET acceptor, the FRET donor is dequenched, leading to an increase in its fluorescence (lower panel). (<b>B</b>) Example for an acceptor bleach experiment with COS7-cells expressing either N-cadherin-Venus or –Cerulean. The upper two images show the Venus-channel before and after bleaching of the Venus signal in the junction (boxed region). The Cerulean channel is depicted in the lower two images. Bleaching of the Venus fluorescence leads to a dequenching of the FRET donor Cerulean, which can be observed in the lower two images. An enlargement of the junction (boxed region) is shown next to the images. (<b>C</b>) Quantitative comparison of the acceptor bleach experiments for N-cadherin-WT and its mutants. The bars represent the mean ± SEM. The mean of the W2A (n = 32) is significantly increased compared to the WT (n = 35, p < 0.0001, Mann Whitney, ***), while the mean of the mutant R14E was significantly lower (n = 29, p=0.0001, two-tailed unpaired t-test , ***). The <i>cis</i> mutant V81D/V174D (n = 33) does not show a significant difference (p=0.1158, two-tailed unpaired t-test). Scale bar = 20 µm.</p

All mutants exhibit sensitivity to prolonged Ca²⁺-chelation.

<p>(<b>A</b>) Example images for WT and all mutants before and 1 h after BAPTA addition. Scale bar = 10 µm. (<b>B</b>) Quantification of junction disassembly. Mean fluorescence intensity of multiple junctions from the different genotypes was measured over time. The drop in mean fluorescence intensity is specific to the addition of BAPTA (blue), indicated by the arrow, as application of a vehicle (green) did not lead to a decrease in junction intensity. (<b>C</b>) Comparison of junction disassembly between different mutants and WT. Although the time course and degree of disassembly differed for the various mutants, with W2A junctions displaying the highest degree of disassembly; on a longer timescale, none of them were completely Ca²⁺-insensitive. N-cadherin-R14E, which appeared Ca²⁺-insensitive on a short timescale (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081517#pone-0081517-g003" target="_blank">Fig. 3 D</a>) also showed a drop in junction intensity 10 min after BAPTA application. Error bars indicate SEM. n(WT) = 84, n(W2A) = 56, n(R14E) = 84, n(V81D/V174D) = 52 junctions. </p