Journal: bioRxiv

Article Title: Humanized antibody potently neutralizes all SARS-CoV-2 variants by a novel mechanism

doi: 10.1101/2022.06.26.497634

Figure Lengend Snippet: ( A ) Immunization scheme. Prime plus boost immunizations were performed at an interval of four weeks. Table shows the V H 1-2/Vκ1-33 antibodies isolated from SARS-CoV-2 spike-specific or RBD-specific IgG + B cells. The antibody sequences and sequence features were shown in . ( C ) Table shows the neutralization activities of three mAbs against VOCs and VOIs in pseudovirus neutralization assays. Experiments were done in 293T/ACE2 cells. The data are also shown in and . Data are representative of 2 biologically independent experiments for most VOCs and VOIs, but one experiment for BA.2 and BA.3. Each independent experiment contains 2 technical replicates. IC 50 values were color-coded based on the key shown at the right. ( D ) Table shows the neutralization activities of three mAbs against VOCs in PRNT live virus neutralization assays. Data are representative of one independent experiment with 2 technical replicates. IC 50 and IC 90 values were color-coded based on the key shown at the right.

Article Snippet: Next, 1:1000 diluted AP-conjugated anti-mouse IgG antibody (Southern Biotech, 1030-04) (Pooled sera: reacts with the heavy chains of mouse IgG1, IgG2a, IgG2b, IgG2c, and IgG3) was added and plates were incubated for 1-hour at room temperature.

Techniques: Isolation, Sequencing, Neutralization, Virus

Journal: bioRxiv

Article Title: Humanized antibody potently neutralizes all SARS-CoV-2 variants by a novel mechanism

doi: 10.1101/2022.06.26.497634

Figure Lengend Snippet: ( A ) Binding curves showing reactivity of sera to SARS-CoV-2 related antigens. The upper panel shows the sera from the SARS-CoV-2 Spike immunized mice at week 0, 2 and 6. The bottom panel shows the sera from VHH7-RBD immunized mice. Data were mean ± SD of three mice. ( B ) Gating strategy for single cell sorting of SARS-CoV2 spike-specific or RBD-specific IgG + B cells after immunization. The upper panel showed the mice immunized with SARS-CoV2 spike plus poly I:C adjuvant. The bottom panels showed the mice immunized with poly I:C (control) or VHH7-RBD plus poly I:C adjuvant. ( C ) Table shows the binding affinities of nine mAbs to the SARS-CoV-2 spike protein, RBD, NTD. Data are representative of two independent experiments. EC 50 values were color-coded based on the key shown at the right.

Article Snippet: Next, 1:1000 diluted AP-conjugated anti-mouse IgG antibody (Southern Biotech, 1030-04) (Pooled sera: reacts with the heavy chains of mouse IgG1, IgG2a, IgG2b, IgG2c, and IgG3) was added and plates were incubated for 1-hour at room temperature.

Techniques: Binding Assay, FACS, Adjuvant, Control

Journal: bioRxiv

Article Title: Humanized antibody potently neutralizes all SARS-CoV-2 variants by a novel mechanism

doi: 10.1101/2022.06.26.497634

Figure Lengend Snippet: ( A ) Example image of Atto 565 fluorescence of virus adsorbed for 10 minutes on a glass coverslip coated with poly-D-lysine coated slides and imaged and imaged with spinning disc confocal microscopy. Photobleaching (B), used to determine the intensity of single Atto 565 dye intensities allowed to establish that ~20-40 dyes were attached to each virus. ( C ) Histogram and ( D ) data summary of the number of Atto 565 molecules on VSV-SARS-CoV-2-Atto 565 determined by single molecule counting. Treatments prior to adsorption included none (control), incubation with 1 µg/mL trypsin for 30 min at 37°C, treatment without or with trypsin followed by an incubation with 0.5 µM of recombinant ACE2 for 10 min at 37°C, or treatment with trypsin then incubated with 100 ng/mL SP1-77 IgG or SP1-77 Fab for 1 hour at 37°C followed by incubation with ACE2. ( D ) Statistics are averages of peak distribution determined by a Gaussian fit of 3 independent experiments. ( E ) Infection assays showing neutralization of VSV-SARS-CoV-2-Atto 565 infection with SP1-77 IgG or SP1-77 Fab. A soluble eGFP reporter genetically encoded into this VSV chimera allowed for infection to be determined by fluorescence imaging on a spinning disc confocal microscope as shown in the representative examples obtained 7 hr post infection. Cell outlines were obtained with a WGA-Alexa 647 membrane stain applied to cells immediately prior to fixation. Each condition was measured with 3 independent experiments.

Article Snippet: Next, 1:1000 diluted AP-conjugated anti-mouse IgG antibody (Southern Biotech, 1030-04) (Pooled sera: reacts with the heavy chains of mouse IgG1, IgG2a, IgG2b, IgG2c, and IgG3) was added and plates were incubated for 1-hour at room temperature.

Techniques: Fluorescence, Virus, Confocal Microscopy, Single Molecule Counting, Adsorption, Control, Incubation, Recombinant, Infection, Neutralization, Imaging, Microscopy, Membrane, Staining

Journal: Communications Biology

Article Title: Polarized ATP synthase in synaptic mitochondria induced by learning and plasticity signals

doi: 10.1038/s42003-025-08963-3

Figure Lengend Snippet: a – c Labeling of memory engram and non-engram cells in the hippocampal DG region by AAV viral vectors. a Expression of EGFP protein driven by the CaMKII promoter for labeling excitatory neurons in the hippocampal DG region. mCherry protein expression driven by the Syn promoter, with loxP sequences, labels activity-dependent memory engram cells. b Overview of the TRAP technique. By combining TRAP with tamoxifen (TM) injection, active neurons within a specific time window can be fluorescently labeled. c Schematic representation of the behavioral protocol for labeling in cFos-CreER mice. d Representative confocal images showing memory engram and non-engram cells. Blue: DAPI, Red: mCherry, Green: EGFP. Scale bar: 200 μm. e , f Observation and statistical analysis of dendritic spine density in memory engram and non-engram cells. e Cells with co-localized red and green signals, along with dendrites, are classified as memory engram cells, while cells with only green fluorescence are categorized as non-engram cells. Scale bar (cell images):10 μm, Scale bar (dendrite images): 2 μm. f Quantification of spine density and spine width (n-engram cells: 21 dendritic segments, n-non-engram cells: 36 dendritic segments, from three mice). Significance: ** P < 0.01, *** P < 0.001 (spine density: Unpaired t test, spine width: Mann–Whitney U test), P = 0.007 for spine density quantification and P = 0.0026 for spine width quantification. g 3D volume view of dendrites and mitochondria in each group. Mitochondria labeled with anti-TOMM20-AF647 (validated in Supplementary Fig. ). h Quantification of mitochondrial localization within dendritic spines of memory engram and non-engram cells (n-engram: 27 dendritic segments; n-non-engram: 35 dendritic segments; from 11 brain sections/4 mice). Significance: ** P < 0.01 (Mann–Whitney U test), P = 0.0015. i Representative 3D volume renderings of dendritic spines (with/without mitochondria) in engram cells. j 3D MINFLUX imaging of ATP5a protein in mitochondria located in mCherry-positive dendrite (left) and EGFP-positive dendrite (right). The z-axis is color-coded. ATP5a was first labeled using a primary antibody, followed by secondary antibody conjugation with AF647. Scale bar (confocal images): 2 μm, Scale bar (3D MINFLUX images): 500 nm. In the box-and-whisker plot, the box represents the interquartile range (IQR, 25th–75th percentiles), divided by the median. The whiskers span the full data range, terminating at the minimum and maximum observed values, individual data points are displayed in dot plots while violin plots depict probability density distributions.

Article Snippet: Recent advancements in single-molecule localization microscopy techniques, such as MINFLUX, offer high-resolution insights into cellular structures and molecular dynamics , .

Techniques: Labeling, Expressing, Activity Assay, Injection, Fluorescence, MANN-WHITNEY, Imaging, Conjugation Assay, Whisker Assay

Journal: Communications Biology

Article Title: Polarized ATP synthase in synaptic mitochondria induced by learning and plasticity signals

doi: 10.1038/s42003-025-08963-3

Figure Lengend Snippet: a , b Representative MINFLUX imaging of ATP5a in dendritic spines of memory engram cells. a Left: mCherry-labeled dendritic spines (Gray). Middle: ATP5a (red) and synaptophysin (green). Right: line intensity profile. b Z-projection of 3D MINFLUX data for mitochondrial ATP5a in the region indicated, green arrows highlight regions of high syn intensity. Confocal scale bar, 500 nm, MINFLUX scale bar, 200 nm. c , d Representative MINFLUX imaging of ATP5a in dendritic spines of non-engram cells. c Left: EGFP-labeled dendritic spines (Gray). Middle: ATP5a (red) and synaptophysin (green). Right: line intensity profile. d Z-projection of 3D MINFLUX data for mitochondrial ATP5a in the region indicated, green arrows highlight regions of high syn intensity. Confocal scale bar, 500 nm, MINFLUX scale bar, 200 nm. e , f Analysis pipeline for correlating ATP5a MINFLUX local density with syn intensity in dendritic spines. e Regions of interest (ROIs) containing mitochondria within dendritic spines of an engram cell were selected for MINFLUX imaging. Clusters of ATP5a molecules were identified using DBSCAN-based clustering, and local densities were calculated to assess ATP5a distribution. Scale bar, 200 nm. f ATP5a local density and syn intensity data were measured using an 8-segment division centered on the mitochondrial staining centroid. Regions with the highest synaptic intensity (syn+) and the lowest synaptic intensity (syn−) were compared to analyze differences in ATP5a molecular density. g Spatial redistribution of mitochondrial ATP5a relative to synaptic contact regions in dendritic spines of memory engram cells versus non-engram cells. Statistical analysis revealed significant differences in the spatial distribution of ATP5a between these two groups. (n-memory engram = 26 spine mitochondria from 12 brain sections from nine mice; n-non-engram = 13 spine mitochondria from 6 brain sections from 4 mice). Significance: ** P , ## P < 0.01,*** P < 0.001 (Aligned Rank Transformation ANOVA, Group factor: F(1, 77) = 11.3, P = 0.0012; Region factor: F(1, 77) = 6.96, P = 0.0102; Interaction, F(1, 77) = 9.53, P = 0.0028; Bonferroni post-hoc test, P non-engram_syn– vs. non-engram_syn+ > 0.9999; P engram_syn– vs. engram_syn+ < 0.001; P engram_syn+ vs. non-engram_syn+ = 0.0034). h Proposed molecular model of ATP5a reorganization in dendritic spines of memory engram cells during learning-related processes.

Article Snippet: Recent advancements in single-molecule localization microscopy techniques, such as MINFLUX, offer high-resolution insights into cellular structures and molecular dynamics , .

Techniques: Imaging, Labeling, Staining, Transformation Assay

Journal: Communications Biology

Article Title: Polarized ATP synthase in synaptic mitochondria induced by learning and plasticity signals

doi: 10.1038/s42003-025-08963-3

Figure Lengend Snippet: a Representative images of DIV21 cultured primary cortical neurons expressing mGFP. Scale bar: 50 μm. b Mitochondria were labeled with anti-ATP5a and AF647-conjugated secondary antibodies. Presynaptic sites were labeled with anti-synaptophysin and AF555-conjugated secondary antibodies. Red: ATP5a, Green: Syn, Gray: mGFP. c Representative images showing postsynaptic mitochondria in spine synapses and shaft synapses. Line profiles show ATP5a and Syn confocal signals across the postsynaptic sites in spine and shaft synapses. Scale bar: 1 μm. d – f Reorganization of mitochondrial ATP5a in dendritic spines under Control ( d ) and cLTP ( e ) conditions. Confocal images show mitochondrial ATP5a (red) and Syn (green) in dendritic spines of cultured neurons. The white dashed line indicates the mitochondria area. The bottom panel shows z-projection of MINFLUX data for ATP5a localization. Confocal scale bar: 400 nm; MINFLUX scale bar: 200 nm. f Quantification of ATP5a local density enrichment near synaptic contact regions in Control and cLTP conditions. (n-cLTP = 51 spine mitochondria from 34 neurons/4 cultures, n-Control = 47 spine mitochondria from 32 neurons/ 5 cultures). Significance: # P < 0.05,** P < 0.01. (ART ANOVA, Group factor: F(1, 195) = 1.14, P = 0.2873; Region factor: F(1, 195) = 0.55, P = 0.4573; Interaction, F(1, 195) = 6.60, P = 0.0110; Bonferroni post hoc test, P Control_syn– vs. Control_syn+ = 0.5784; P cLTP_syn– vs. cLTP_syn+ = 0.0047; P cLTP_syn+ vs. Control_syn+ = 0.0159). g , h Distribution of mitochondrial ATP5a protein in dendritic spines at different time points after cLTP induction. g Top: confocal images showing mitochondrial ATP5a (red) and adjacent Syn (green) in dendritic spines. Bottom: z projection of MINFLUX data showing ATP5a localization in the same dendritic spines. Confocal scale bar: 400 nm; MINFLUX scale bar: 200 nm. h Quantification of local density enrichment of ATP5a near synaptic contact regions. (n-Control = 40 spine mitochondria from 27 neurons/3 cultures, n-cLTP = 22 spine mitochondria from 15 neurons/3 cultures, n-30 min = 40 spine mitochondria from 20 neurons/4 cultures, n-12 h = 28 spine mitochondria from 19 neurons/3 cultures and n-3days = 38 spine mitochondria from 25 neurons/3 cultures). Significance: * P , # P < 0.05, ** P , ## P < 0.01. (ART ANOVA, Group factor: F(4, 339) = 4.08, P = 0.0031; Region factor: F(1, 339) = 0.24, P = 0.6227; interaction, F(4, 339) = 4.57, P = 0.0013; Bonferroni post hoc test: P Ctrl_syn–vs. Ctrl_syn+ > 0.9999; P cLTP_syn–vs. cLTP_syn+ = 0.0084; P 30min_syn–vs. 30 min_syn+ = 0.0212; P 12hrs_syn–vs. 12 h_syn+ = 0.0025; P 3days_syn–vs. 3days syn+ > 0.9999; P Ctrl_syn+ vs. cLTP_syn+ = 0.0032; P Ctrl_syn+ vs. 30 min_syn+ = 0.0426; P Ctrl_syn+ vs. 12 h_syn+ = 0.0056; P Ctrl_syn+ vs. 3days_syn+ > 0.9999;).

Article Snippet: Recent advancements in single-molecule localization microscopy techniques, such as MINFLUX, offer high-resolution insights into cellular structures and molecular dynamics , .

Techniques: Cell Culture, Expressing, Labeling, Control

Journal: Communications Biology

Article Title: Polarized ATP synthase in synaptic mitochondria induced by learning and plasticity signals

doi: 10.1038/s42003-025-08963-3

Figure Lengend Snippet: a Representative 3D dual-color MINFLUX imaging of ATP5a and TOMM20 in dendritic spines under Control and cLTP conditions. Confocal images show the inner mitochondrial membrane protein ATP5a (red), outer membrane protein TOMM20 (green), and Syn (blue). MINFLUX imaging reveals the distribution of TOMM20 and ATP5a. Maximum z-projection (z-proj.) and 3D reconstructions were generated using ImageJ. Pseudo-coloring is consistent across all modalities. b , c Quantification of local density enrichment of TOMM20 ( b ) and ATP5a ( c ) near synaptic contact regions in the Control and cLTP groups. Local density calculations for dual-color imaging are provided in the Supplementary Fig. . (n-cLTP: 20 spine mitochondria from 14 neurons/3 cultures, n-Control: n = 16 spine mitochondria from 11 neurons/3 cultures) Significance: ** P , ## P < 0.01. (TOMM20:ART ANOVA, Group factor: F(1, 71) = 0.7498, P = 0.3896; Region factor: F(1, 71) = 0.9172, P = 0.3416; Interaction (Group×Region): F(1, 71) = 1.3784, P = 0.2445. Bonferroni post hoc test: P Control_syn–vs. Control_syn+ <0.9999; P cLTP_syn– vs. cLTP_syn+ = 0.5494; P cLTP_syn+ vs. Control_syn+ = 0.3472; ATP5a:ART ANOVA, Group factor: F(1, 71) = 10.0817, P = 0.0023; Region factor: F(1, 71) = 1.9934, P = 0.1625; Interaction F(1, 71) = 5.9228, P = 0.0176. Bonferroni post hoc test: P Control_syn-vs. Control_syn+ > 0.9999; P cLTP_syn–vs. cLTP_syn+ = 0.0098; P cLTP_syn+ vs. Control_syn+ = 0.0066). d Proposed model showing that mitochondrial inner membrane protein ATP5a, but not outer membrane protein TOMM20, relocates within synapses in response to cLTP induction.

Article Snippet: Recent advancements in single-molecule localization microscopy techniques, such as MINFLUX, offer high-resolution insights into cellular structures and molecular dynamics , .

Techniques: Imaging, Control, Membrane, Generated

Journal: Cell Genomics

Article Title: A gene with a thousand alleles: The hyper-variable effectors of plant-parasitic nematodes

doi: 10.1016/j.xgen.2024.100580

Figure Lengend Snippet: Figure of hypotheses The primary observations that (1) when sequencing a pool of individuals from a population, we observe many unique alleles (with a long tail of rare alleles) but (2) when we sequence individuals from that same population and pool their dominant allele per individual, we get very few unique alleles (with no long tail of rare alleles). Four potential hypotheses are discussed (preferential mating, preferential zygote abortion, gene conversion, and somatic hypermutation). Hypothesis 4, somatic hypermutation, is the most parsimonious explanation, as it alone explains all phenomena observed.

Article Snippet: Due to the microscopic size of each animal, we employed single-molecule read sequencing (Oxford Nanopore and PacBio) of a population.

Techniques: Sequencing

Journal: Cell Genomics

Article Title: A gene with a thousand alleles: The hyper-variable effectors of plant-parasitic nematodes

doi: 10.1016/j.xgen.2024.100580

Figure Lengend Snippet:

Article Snippet: Due to the microscopic size of each animal, we employed single-molecule read sequencing (Oxford Nanopore and PacBio) of a population.

Techniques: Recombinant, CRISPR, Ligation, Sequencing, Nanopore Sequencing, Amplification, In Silico, Software