120 research outputs found

    Multiplex identification of proteins using a neural network–based spectral analysis.

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    (A) Experimental design of a co-seeding assay including 11 different cell types, labeled with specific nanobarcodes (see Methods section for details). (B) Example of an Nrxn-2ß (SS#4(+))/Nlgn-1 (SS#AB) and an Nrxn-2ß (SS#4(−))/Nlgn-2 (−) pair (red boxes depict typical cell contacts). (C) Overlay of cells containing nanobarcode proteins and Nrxn- or Nlgn-positive cells. Nanobarcode proteins are shown in green (anti-ALFA-Atto488). Nrxn or Nlgn isoforms are shown in magenta (anti-HA and anti-goat-Cy3). See S21 Fig for example images of all proteins. Scale bars: 20 μm. (D) Interaction preferences of Nrxn/Nlgn isoforms. A total of 4,569 cell contacts, 147 images, 4 independent co-seeding experiments. The Nrxn/Nlgn codes, such as SS#4(+) refer to the respective splicing sites of the proteins, according to the literature (e.g., [24]). The data underlying this Figure can be found in the S1 Data file, Sheet “Fig 4D”, available from http://dx.doi.org/10.17169/refubium-40101.</p

    Design and topology of protein constructs.

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    (A-C) Legends for expected topology (A), protein length (B), and construct epitopes (C). (D) Protein topology schemes for the 15 constructs used. Below is a list with detailed information about the respective topology scheme of each construct depicted in (B). Uniprot accession numbers (acc.nr.) are available under https://www.uniprot.org/uniprot/. Sequences of all constructs are listed in “plasmid_sequence_information.xlsx” stored in “Plasmid_design.zip” available from http://dx.doi.org/10.17169/refubium-40101. No protein, used for background signals. (TIF)</p

    Table_1_Tracking cell turnover in human brain using 15N-thymidine imaging mass spectrometry.docx

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    Microcephaly is often caused by an impairment of the generation of neurons in the brain, a process referred to as neurogenesis. While most neurogenesis in mammals occurs during brain development, it thought to continue to take place through adulthood in selected regions of the mammalian brain, notably the hippocampus. However, the generality of neurogenesis in the adult brain has been controversial. While studies in mice and rats have provided compelling evidence for neurogenesis occurring in the adult rodent hippocampus, the lack of applicability in humans of key methods to demonstrate neurogenesis has led to an intense debate about the existence and, in particular, the magnitude of neurogenesis in the adult human brain. Here, we demonstrate the applicability of a powerful method to address this debate, that is, the in vivo labeling of adult human patients with 15N-thymidine, a non-hazardous form of thymidine, an approach without any clinical harm or ethical concerns. 15N-thymidine incorporation into newly synthesized DNA of specific cells was quantified at the single-cell level with subcellular resolution by Multiple-isotype imaging mass spectrometry (MIMS) of brain tissue resected for medical reasons. Two adult human patients, a glioblastoma patient and a patient with drug-refractory right temporal lobe epilepsy, were infused for 24 h with 15N-thymidine. Detection of 15N-positive leukocyte nuclei in blood samples from these patients confirmed previous findings by others and demonstrated the appropriateness of this approach to search for the generation of new cells in the adult human brain. 15N-positive neural cells were easily identified in the glioblastoma tissue sample, and the range of the 15N signal suggested that cells that underwent S-phase fully or partially during the 24 h in vivo labeling period, as well as cells generated therefrom, were detected. In contrast, within the hippocampus tissue resected from the epilepsy patient, none of the 2,000 dentate gyrus neurons analyzed was positive for 15N-thymidine uptake, consistent with the notion that the rate of neurogenesis in the adult human hippocampus is rather low. Of note, the likelihood of detecting neurogenesis was reduced because of (i) the low number of cells analyzed, (ii) the fact that hippocampal tissue was explored that may have had reduced neurogenesis due to epilepsy, and (iii) the labeling period of 24 h which may have been too short to capture quiescent neural stem cells. Yet, overall, our approach to enrich NeuN-labeled neuronal nuclei by FACS prior to MIMS analysis provides a promising strategy to quantify even low rates of neurogenesis in the adult human hippocampus after in vivo15N-thymidine infusion. From a general point of view and regarding future perspectives, the in vivo labeling of humans with 15N-thymidine followed by MIMS analysis of brain tissue constitutes a novel approach to study mitotically active cells and their progeny in the brain, and thus allows a broad spectrum of studies of brain physiology and pathology, including microcephaly.</p

    Transferrin and EGF imaging assays, tested for nanobarcoded syntaxin 13.

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    (A) Visualization of transferrin-Alexa488 (green) and EGF-Alexa647 (magenta), as well as the transfected protein, visualized with the ALFA nanobody (NbALFA) conjugated to AZdye568 (white). The 3 rows show the 10-minute pulse with the ligands (endocytosis), followed by the 10- and 20-minute chase (wash-off). To enable optimal visualization, the images are scaled differently, with the image scaling indicated in all panels. Scale bar: 20 μm. (B) The nanobarcoding scheme and the expected localization of the protein. (C) The NbALFA fluorescence intensity is plotted against the transferrin (green) and EGF (magenta) intensity, for all signals measured in 2 independent experiments, for all conditions. All intensities were normalized to the medians of the distributions and were then grouped in 20 bins of ALFA intensity, each containing similar numbers of values. The mean and SEM of each bin in the respective channels are plotted. The data underlying this Figure can be found in the S1 Data file, Sheet “SFig 14C_STX13,” available from http://dx.doi.org/10.17169/refubium-40101. (D) The Pearson’s correlation coefficients for the distributions from panel C are shown, with the p-values corrected for multiple testing using a Bonferroni correction. (TIF)</p

    Design of protein constructs with nanobarcodes using 4 nanobody epitopes.

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    (A, B) Scheme of the 4 nanobarcode epitopes (A) and the fluorescent nanobodies used for recognizing them (B). (B) NbRFP-Atto565 in red, NbEGFP-Atto488 in green, NbSyn87-Dylight405 in cyan, NbSyn2-Star635P in magenta. (C) Design of the protein construct VAMP4(1111). Each protein construct contains a target protein (the protein to identify) and a barcode. In this example, the target protein is VAMP4, and its barcode contains the following nonfluorescent epitopes: mCherry (Y71L), GFP (Y66L), syn87, and syn2. The ALFA-tag [10] is present for testing purposes. See S1 Fig for further sequence information. Barcode epitopes recognized by fluorescent nanobodies shown as “ones” in pseudocolors that correspond to the fluorophores used. (D) Nanobarcodes, 15 in total, resulting from a binary combination of 4 nanobarcode-epitopes. Epitopes from left to right: mCherry(Y71L), GFP(Y66L), syn87 and syn2. The nanobody scheme is the same as in (B). (E) The expected cellular protein distribution for the proteins used, according to the literature. (F) Nanobarcode-based identification of the proteins STX6(0011), GFP(0100), and SNAP25(1100). The pseudocolors for merged images correspond to the fluorescence channels of the nanobodies: NbRFP-Atto565 in red, NbGFP-Atto488 in green, NbSyn87-Dylight405 in cyan, and NbSyn2-Star635P in magenta. Scale bar: 20 μm.</p

    Deep neural network for nanobarcode identification.

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    (A) Schematic representation of experimental protocol for obtaining multichannel images of HEK293 cells transfected with a single protein construct. HEK293 cells are seeded (1) and transfected with the necessary DNA plasmids. After an incubation of at least 14 hours, the HEK293 cells, now expressing the protein constructs, are fixed and stained with nanobodies (2). Multichannel images from the respective cells (3) are used for the training of a neuronal network. Wavelengths of excitation lasers used: λ = 405 nm, λ = 488 nm, λ = 561 nm, and λ = 633 nm. Emission channels used: 417–485 nm (CH1), 495–553 nm (CH2), 573–631 nm (CH3), and 641–729 nm (CH4). (B) Architecture of the deep network used for protein identification from channel intensity values pertaining to each pixel. For the dense layers, given numbers indicate input and output dimensions. The network contains 4 parallel branches in the middle (2 are shown), the outputs of which are summed and processed by the final layers. The branches are composed of sequential residual blocks with skip connections bypassing triplets of layers, as shown in the expansion panel to the left (further details in Methods section “Deep neural network-based protein identification”). (C) The output probability distributions of the network are used to render false color images that contain information on the identified proteins in each pixel. Scale bars: 50 μm. (TIF)</p

    Information about antibodies used for target protein validation purposes.

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    The first 19 antibodies listed are primary antibodies. Antibodies 20–23 are secondary antibodies. See Methods section for further information about the staining procedures. (DOCX)</p

    Visualization of 15 nanobarcode epitopes using 4 spectrally distinct nanobodies.

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    (A-D) Nanobody-based identification of the 4 genetically encoded nanobarcode epitopes mCherry(Y71L), GFP(Y66L), syn87, and syn2 and the ALFA-tag epitope by their corresponding nanobodies NbRFP, NbEGFP, NbSyn87, NbSyn2, and NbALFA. Scaling was optimized for each protein. (D) VAMP4(1111) example (all epitopes present) and negative control condition: mock transfection (no DNA, no epitopes present) using same intensity scale. (E) As in (D), now with upscale intensities. Scale bar: 50 μm. (TIFF)</p

    Transferrin and EGF imaging assays, tested for nanobarcoded endobrevin.

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    (A) Visualization of transferrin-Alexa488 (green) and EGF-Alexa647 (magenta), as well as the transfected protein, visualized with the ALFA nanobody (NbALFA) conjugated to AZdye568 (white). The 3 rows show the 10-minute pulse with the ligands (endocytosis), followed by the 10- and 20-minute chase (wash-off). To enable optimal visualization, the images are scaled differently, with the image scaling indicated in all panels. Scale bar: 20 μm. (B) The nanobarcoding scheme and the expected localization of the protein. (C) The NbALFA fluorescence intensity is plotted against the transferrin (green) and EGF (magenta) intensity, for all signals measured in 2 independent experiments, for all conditions. All intensities were normalized to the medians of the distributions and were then grouped in 20 bins of ALFA intensity, each containing similar numbers of values. The mean and SEM of each bin in the respective channels are plotted. The data underlying this Figure can be found in the S1 Data file, Sheet “SFig 11C_Endo,” available from http://dx.doi.org/10.17169/refubium-40101. (D) The Pearson’s correlation coefficients for the distributions from panel C are shown, with the p-values corrected for multiple testing using a Bonferroni correction. (PNG)</p
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