Protein nanobarcodes enable single-step multiplexed fluorescence imaging

Multiplexed cellular imaging typically relies on the sequential application of detection probes, as antibodies or DNA barcodes, which is complex and time-consuming. To address this, we developed here protein nanobarcodes, composed of combinations of epitopes recognized by specific sets of nanobodies. The nanobarcodes are read in a single imaging step, relying on nanobodies conjugated to distinct fluorophores, which enables a precise analysis of large numbers of protein combinations. Fluorescence images from nanobarcodes were used as input images for a deep neural network, which was able to identify proteins with high precision. We thus present an efficient and straightforward protein identification method, which is applicable to relatively complex biological assays. We demonstrate this by a multicell competition assay, in which we successfully used our nanobarcoded proteins together with neurexin and neuroligin isoforms, thereby testing the preferred binding combinations of multiple isoforms, in parallel

Detektion und funktionelle Analyse von Ca2+-Mikrodomänen und BK-Kanälen in olfaktorischen Rezeptorzellen der Xenopus laevis Larve

Olfaktorische Rezeptorneurone (ORNs) nehmen Gerüche durch die Interaktion von Geruchsstoffen mit ihren Rezeptoren wahr und führen über die Generierung von Aktionspotentialen (APs) den ersten Schritt der olfaktorischen Signaltransduktion aus. Es ist bekannt, dass BK-Kanäle und spannungsabhängige Ca2+-Kanäle (VGCCs) an der Feinabstimmung von APs in vielen Neuronen beteiligt sind. Aufgrund der geringen Expression der beiden Kanäle in ORNs ist es jedoch nicht bekannt, ob sie in diesen Zellen von funktioneller Bedeutung sind. In der aktuellen Studie wird eine Kolokalisierung von BK-Kanälen und spannungsabhängigen Ca2+-Mikrodomänen auf der Oberfläche der ORNs von Xenopus laevis beobachtet. Die funktionellen Ca2+-Mikrodomänen können vor allem Cluster von VGCC statt einzelner Ca2+-Kanäle umfassen. Darüber hinaus wird der Abstand zwischen den BK-Kanälen und den VGCCs durch den Einsatz verschiedener Ca2+-Puffer auf ungefähr 50 bis 200 nm, jedoch nicht geringer als 30 nm geschätzt. Außerdem führt das Blockieren von BK-Kanälen mit Iberiotoxin (IbTx) oder das Chelatisieren von Ca2+-Ionen durch BAPTA zu einer signifikanten Zunahme der Abklingzeit für die Repolarisation von APs. Die Feuerrate von ORNs als Antwort auf eine Stimulation durch Geruchsstoffe wird auch durch IbTx gesenkt. Zusammengenommen zeigen die hier präsentierten Ergebnisse, dass trotz des geringen Expressionslevels BK-Kanäle und VGCCs durch die Bildung von funktionalen Ca2+-Mikrodomänen eine Schlüsselfunktion in der olfaktorischen Signalweiterleitung einnehmen, indem sie die Form von APs definieren und hohe Feuerraten ermöglichen

The Effects of Sodium Ions, Phosphorus, and Silicon on the Eco-Friendly Process of Vanadium Precipitation by Hydrothermal Hydrogen Reduction

The effects of sodium ions, phosphorus, and silicon on the eco-friendly process of vanadium precipitation by hydrothermal hydrogen reduction were investigated to establish the suitable concentrated solution system for this eco-friendly process. The results showed that sodium ions had no negative effects on the vanadium precipitation process. Phosphorus can reduce vanadate ion activity, and results in the decrease of vanadium precipitation percentage from 99.5% to 61.3%, as the phosphorus concentration in the feed solution increased from 0.05 g/L to 3 g/L. As a result, the aimed products of V2O3 were hard to be obtained, and the purity of the precipitates was lowered. Silicon can absorb in the form of H3Si3O7 on the surface of the precipitates, thus it was difficult for H (activity hydrogen atom) to react with the intermediate vanadium-bearing precipitates. As a result, the vanadium precipitation percentage decreased from 99.5% to 86.2% as the silicon concentration in the feed solution increased from 0.1 g/L to 3 g/L. The aimed products of V2O3 were not easy to be obtained, and only the intermediate vanadium-bearing precipitates containing sodium ions were obtained. The upper limits of the concentrations of phosphorus and silicon in the feed V (V) solution were ascertained as 0.5 g/L and 0.1 g/L, respectively. As the concentrations of phosphorus and silicon in the purified alkaline-concentrated V (V) solution extracted from vanadium-bearing shale are usually below the upper limits of the concentrations, the eco-friendly process of vanadium precipitation by hydrothermal hydrogen reduction has a great application prospect in the field of vanadium extraction from vanadium-bearing shale

Double exponential decay analysis in time- and Legendre-domain.

<p>(<b>a,b</b>) The interaction of Ru(II) complexes with DNA (same as in in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090500#pone-0090500-g001" target="_blank">Fig. 1</a>) shows double exponential decay(<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090500#pone.0090500-Bazzicalupi1" target="_blank">[12]</a>, data courtesy of F. Secco, Univ Pisa,I). Same data fitted in t- (<b>a</b>) and L-domain (<b>b</b>). (<b>c</b>) Probability for the fitting error in the L-domain, , to be smaller than that in the t-domain, , represented as a function of and . Color code of the probability is shown beneath the plot. For each pair () 1000 trials were computed. (<b>d</b>) Relative difference of fitting errors, , as a function of and . Color code beneath the plot. 1000 trials per pixel. Left of the solid and dashed white lines in <b>a</b> and <b>b</b>, the success rate of the fit in the L-domain is larger that and , respectively. Left of the solid and dashed black lines in <b>c</b> and <b>d</b>, the success rate of the fit in the t-domain is larger that 50% and 95%, respectively. Relative error differences were calculated only for successful trials. (<b>e</b>) Success rate of fitting in the L-domain (solid) and t-domain (dashed) along the vertical line in <b>c</b>, i.e., as a function of with kept at .</p

Comparison of fitting in L-domain and t-domain of simulated noisy exponentials defined by parameters and , superimposed Poisson noise and stationary gaussian noise with different standard deviations .

<p> is the probability that the fitting parameters obtained in the L-domain approximate the true values better than those obtained in the t-domain (i.e., ) under the indicated noise condition. With optimal importance-weighting for Poisson noise (), the comparison is carried out for four different levels of gaussian noise, given as a ().</p

- dependence of the required number of Legendre components.

<p>(<b>a</b>) Legendre spectra of noisy exponentials with (gray) and (red). Shown are the average Legendre amplitudes obtained from trials. Error bars, standard deviation of the respective component. (<b>b</b>) Coefficient of variation of the components for the two spectra shown in <b>a</b>.</p

Legendre lowpass-filtered EPSP.

<p>(<b>a</b>) Noisy EPSP and its mean simulated as , with , and . (<b>b, d</b>) Legendre spectra of the noisy EPSP (<b>b</b>), and its mean (<b>d</b>). (<b>c</b>) Inverse transform of through of <b>b</b> approximating the EPSP's mean (dashed, red). Gray curve in <b>c</b>, Fourier lowpass () of the noisy EPSP.</p

Filtering exponentials and Legendre lowpass.

<p>(<b>a</b>) Double exponential decay during a stopped-flow recording. The reaction monitored is the interaction of ruthenium complexes with DNA, scaled to the interval [−1, 1]. For the experimental details of the system, see [12]. (<b>b</b>) The first 17 components of the Legendre spectrum of . The inverse fLT of the components through of the spectrum (<b>b</b>) gives the red curve in (<b>a</b>). Note that the sharp peak in the noisy trace is virtually not reflected in the filtered curve. (<b>c</b>) Autocorrelation curve (gray) resulting from an experiment where the diffusion constant of tetramethylrhodamine was measured (own data). In this example, fLT and ifLT are performed for non-equidistant samples, and we re-scaled the x-axis to correlation delays. (<b>d</b>) Legendre spectrum of the ACF shown in (<b>c</b>). The red curve in (<b>c</b>) is the inverse fLT of the components through of the Legendre spectrum.</p

Filtering exponentials convolved with a system response function.

<p>(<b>a</b>) Exponential on the interval [−1, 1] with Poisson noise added. Amplitude, , time constant . (<b>b</b>) Legendre spectrum of x as resulting from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090500#pone.0090500.e283" target="_blank">eq. 5</a>. (<b>c</b>) Mean of (continuous) and inverse fLT (dashed, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090500#pone.0090500.e306" target="_blank">eq. 6</a>) of through of the spectrum shown in <b>b</b>. (<b>d</b>) Legendre spectrum of the mean , largely lacking higher noise components. (<b>e</b>) Noisy curve is the convolution of with and . was chosen such that the curve overlaps with for large . (<b>f</b>) Legendre spectrum (gray bars) of convoluted noisy exponential shown in <b>e</b> (continuous curve). The lowpass-filtered inverse transform is shown in <b>e</b> (continuous curve) and approximates the convoluted noisy exponential. In addition, <b>f</b> shows the Legendre spectrum of , obtained through <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090500#pone.0090500.e321" target="_blank">eq. 9</a>. The lowpass-filtered inverse transform of this spectrum is shown as the red dashed curve in <b>e</b> and approximates the original non-convoluted exponential, from which the noisy convoluted curve was generated.</p