Journal: Npj Biosensing

Article Title: Microfluidic nanomagnetically isolated neuron- and astrocyte-derived extracellular vesicles to differentiate Lewy body and Alzheimer’s disease

doi: 10.1038/s44328-026-00086-x

Figure Lengend Snippet: A Outline of study time-course and sample processing for EV isolation. Antemortem plasma samples with postmortem pathological confirmation of neurological diagnoses were processed to isolate cell-specific EVs using our mTENPO microfluidic platform, alongside plasma protein biomarkers using commercial digital ELISA, for patients with LBD ( n = 30), AD ( n = 31), AD/LBD ( n = 30), AD/ALB ( n = 19), and controls ( n = 27). B The mTENPO platform, illustrating the external magnet, inlet reservoir, outlet ports, and tubing connections to syringe pumps. Syringes are connected to the waste outlet for blocking, washing, and sample addition steps, and then replaced and switched to the lysate outlet before captured EVs are lysed on-chip. The inset shows a photo of the mTENPO chip with a quarter for scale. C Schematic of operation of the mTENPO platform for cell-specific EV isolation using antibody-labeled magnetic nanoparticles (MNPs) for GluR2+ (top) and GLAST+ (bottom) EV pulldowns. D Scanning electron microscopy (SEM) images of GluR2+ EVs immobilized on the edges of pores of the mTENPO device’s surface. E SEM images of GLAST+ EVs immobilized on the edges of pores of the mTENPO device’s surface. F Representative cropped western blot images showing protein expression of GluR2, GLAST, and EV-associated marker TSG101 using mTENPO-isolated GluR2+ or GLAST + EV lysates from n = 2 human plasma samples. Full-length western blot images are shown in Supplementary Fig. .

Article Snippet: Briefly, 500 μL of patient plasma was incubated for 20 min at a concentration of 1 μg/mL with either biotinylated GluR2 capture antibody (GluR1 + GluR2 polyclonal antibody, Bioss bs-10042R-Biotin) for neuron-derived EVs per our previous work or biotinylated GLAST capture antibody [GLAST (ACSA-1) antibody, anti-human/mouse/rat Biotin, Miltenyi Biotec, 130-118-984] for astrocyte-derived EVs, where the use of GLAST as a protein target for astrocyte EV isolation has been previously reported , – , .

Techniques: Isolation, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Blocking Assay, Labeling, Electron Microscopy, Western Blot, Expressing, Marker

Journal: Npj Biosensing

Article Title: Microfluidic nanomagnetically isolated neuron- and astrocyte-derived extracellular vesicles to differentiate Lewy body and Alzheimer’s disease

doi: 10.1038/s44328-026-00086-x

Figure Lengend Snippet: A Heatmap of z-score of log 2 (expression) for biomarkers with Benjamini-Hochberg FDR-corrected P value < 0.1. Subjects (columns) are hierarchically clustered within cohort and biomarkers within each compartment (rows) are sorted by descending fold-change. B Volcano plot demonstrating differential expression of GluR2+ EV miRNAs, GLAST + EV miRNAs, and plasma proteins. C Venn diagram showing overlap in FDR P value significant miRNAs ( P value < 0.1) between GluR2+ EVs and GLAST+ EVs. D Top 30 biomarkers in all compartments ranked by descending area under the curve (AUC). Error bars represent standard error from bootstrapping 10x.

Article Snippet: Briefly, 500 μL of patient plasma was incubated for 20 min at a concentration of 1 μg/mL with either biotinylated GluR2 capture antibody (GluR1 + GluR2 polyclonal antibody, Bioss bs-10042R-Biotin) for neuron-derived EVs per our previous work or biotinylated GLAST capture antibody [GLAST (ACSA-1) antibody, anti-human/mouse/rat Biotin, Miltenyi Biotec, 130-118-984] for astrocyte-derived EVs, where the use of GLAST as a protein target for astrocyte EV isolation has been previously reported , – , .

Techniques: Expressing, Quantitative Proteomics, Clinical Proteomics

Journal: Npj Biosensing

Article Title: Microfluidic nanomagnetically isolated neuron- and astrocyte-derived extracellular vesicles to differentiate Lewy body and Alzheimer’s disease

doi: 10.1038/s44328-026-00086-x

Figure Lengend Snippet: GO and KEGG pathway analyses were performed on differentially expressed miRNAs using DIANA miRPath v4.0 using the TarBase v8.0 database. FDR P values for identified GO terms and KEGG pathways were calculated using a one-sided Fisher’s exact test and considered significant at P value < 0.05. The top 10 (ranked by number of target genes) terms within each of the three GO categories (BP, CC, MF) and top 10 (ranked by number of target genes) KEGG pathways were identified for each pulldown. A Top 10 terms within each GO category for GluR2+ EV miRNAs. B Top 10 KEGG pathways for GluR2+ EV miRNAs. C Top 10 terms within each GO category for GLAST + EV miRNAs. D Top 10 KEGG pathways for GLAST + EV miRNAs. In all panels, each bar is labeled to the right with the number of differentially expressed miRNAs associated with the given GO term or KEGG pathway.

Article Snippet: Briefly, 500 μL of patient plasma was incubated for 20 min at a concentration of 1 μg/mL with either biotinylated GluR2 capture antibody (GluR1 + GluR2 polyclonal antibody, Bioss bs-10042R-Biotin) for neuron-derived EVs per our previous work or biotinylated GLAST capture antibody [GLAST (ACSA-1) antibody, anti-human/mouse/rat Biotin, Miltenyi Biotec, 130-118-984] for astrocyte-derived EVs, where the use of GLAST as a protein target for astrocyte EV isolation has been previously reported , – , .

Techniques: Labeling

Journal: Npj Biosensing

Article Title: Microfluidic nanomagnetically isolated neuron- and astrocyte-derived extracellular vesicles to differentiate Lewy body and Alzheimer’s disease

doi: 10.1038/s44328-026-00086-x

Figure Lengend Snippet: A Heatmap of z-score of log 2 (expression) for LASSO-selected biomarkers. Subjects (columns) are hierarchically clustered within cohort and biomarkers within each compartment (rows) are sorted by descending AUC. B Kendall correlation staircase plots identifying the extent to which biomarker information was correlated between the LASSO-selected GluR2+ EV, GLAST + EV, and protein biomarkers. Biomarkers are sorted within compartments by AUC. The inset shows the correlation distribution of Kendall’s τ, where the dotted line represents the median count. C LASSO panel accuracy versus panel size for classifying LBD versus AD, shown in blue; accuracy is assessed through tenfold cross-validation, with error bars representing standard error from 5 repeats of panel training on the LBD vs AD patient groups. Average accuracy and standard error for control experiments performed by scrambling patient labels 10x are shown in orange. D LASSO panel AUC versus panel size for classifying LBD versus AD, shown in blue with error bars as described in ( C ). Average AUC and standard error for the same control experiments described in ( C ) are shown in orange. E AUCs for the 15-marker LASSO panel and individual LASSO biomarkers, sorted by descending AUC. Error bars represent 95% confidence intervals, calculated from 5x repeats of tenfold cross-validation for the 15-marker panel or from bootstrapping 10x for individual markers.

Article Snippet: Briefly, 500 μL of patient plasma was incubated for 20 min at a concentration of 1 μg/mL with either biotinylated GluR2 capture antibody (GluR1 + GluR2 polyclonal antibody, Bioss bs-10042R-Biotin) for neuron-derived EVs per our previous work or biotinylated GLAST capture antibody [GLAST (ACSA-1) antibody, anti-human/mouse/rat Biotin, Miltenyi Biotec, 130-118-984] for astrocyte-derived EVs, where the use of GLAST as a protein target for astrocyte EV isolation has been previously reported , – , .

Techniques: Expressing, Biomarker Discovery, Control, Marker

Journal: bioRxiv

Article Title: Expansion microscopy at one nanometer resolution

doi: 10.1101/2022.08.03.502284

Figure Lengend Snippet: a-c , Synaptic vesicles were labeled live using an antibody against a luminal epitope of synaptotagmin 1 (Syt1, magenta). The vesicular glutamate transporter (vGluT1, blue) and PSD95 (gray) were immunostained using an antibody and a nanobody, respectively. a , Recently endocytosed vesicle exhibiting circular morphology. b , Readily retrievable pool molecules form patches containing Syt1/vGluT1 (top), which are dispersed by cholesterol extraction using MβCD (bottom). c , MβCD causes molecules to spread across larger areas (left: N = 22-19, 2 independent experiments, p < 0.0044, Mann-Whitney test; right: N = 22-22, 2 independent experiments, p = 0.8937), although the signal per vesicle (the Syt1 copy number) remains unchanged. d , A visualization of PSDs (top and side views), after immunostaining PSD95 with the same nanobody used in a-c, and Shank2 and Homer1 with specific antibodies. The graph indicates the axial positioning, which agrees well with the literature . N = 11 measurements for each protein, 2 independent experiments; symbols show the medians, SEM and SD. e , Side view of a postsynapse displaying PSD95, MAP2 and two glutamate receptors (GluR2, AMPA type, and GluN2b, NMDA type). f , ONE images of PSD95 (top views), before or after the addition of 10% 1,6-hexanediol (Hex). g , Line scans through the PSD95 stainings shown in panel f. h , An analysis of PSD95 spot profiles; N = 10-7 synapses, Friedman test followed by Dunn-Sidak testing, p = 0.0027; the error bars show the SEM. For details on the analysis, see .

Article Snippet: The primary antibodies used were anti synaptotagmin1 (SYT1, #105011 Synaptic Systems), anti Homer1 (#160 003, Synpatic Systems), anti Shank2 (#162204 Synaptic Systems), anti GluR2 (Alomone Labs, #AGC-005, Jerusalem, Israel), anti GluN2b (Neuromab 75-101, California, USA), anti MAP2 (Novus Biologicals #NB300-213), anti vGluT1 (#135304, Synaptic Systems), anti Bassoon (#ADI-VAM-PS003-F, Enzo, New York, USA).

Techniques: Labeling, Extraction, MANN-WHITNEY, Immunostaining

Journal: Neuron

Article Title: Regulation of Thalamic and Cortical Network Synchrony by Scn8a

doi: 10.1016/j.neuron.2017.01.031

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Monoclonal mouse anti-GluR2 , Neuromab , Cat# 75-002; RRID: AB_2232661.

Techniques: Plasmid Preparation, Virus, Recombinant, Avidin-Biotin Assay, Software, Imaging

Journal: Molecular Metabolism

Article Title: Prolyl carboxypeptidase in Agouti-related Peptide neurons modulates food intake and body weight

doi: 10.1016/j.molmet.2018.02.003

Figure Lengend Snippet: PRCP expression in NPY/AgRP neurons and its secretion. ( A – C ) Representative photographs of the hypothalamic arcuate nucleus showing GFP (NPY-GFP mouse; A ) and PRCP immunolabeling ( B ). Analysis of double labeled cells ( C ) showed that 77.8 ± 2.39% of NPY neurons were positive for PRCP (arrowheads). ( D ) Graph showing the result of the PRCP measurements in hypothalamic explants of mice containing the PVN and the anterior arcuate nucleus. Ghrelin addition (100 nM) to the basal (8 mM glucose) aCSF induced a significant increase of PRCP in the medium compared to either basal aCSF or 15 mM glucose-containing aCSF (n = 7 per treatment). ( E ) Graph showing the result of the PRCP measurements in hypothalamic explants of Prcp AgRPKO and control mice (n = 5 per group) containing the PVN and the anterior arcuate nucleus. Compared to controls, PRCP levels in the medium after ghrelin incubation were significantly lower in Prcp AgRPKO mice. ( F and G ) Western blot images ( F ) and quantification of the density ( G ) of PRCP in cytoplasmic extracts from hypothalamic explants (n = 4 per group) incubated with either 8 mM Glucose, 15 mM Glucose or 8 mM Glucose plus 100 nM Ghrelin for PRCP, β-actin and GluR2. PRCP density was normalized to β-actin density. ( H and I ) Western blot images ( H ) and quantification of the density ( I ) of PRCP in synaptic extracts from hypothalamic explants (n = 4 per group) incubated with either 8 mM Glucose, 15 mM Glucose or 8 mM Glucose plus 100 nM Ghrelin for PRCP, β-actin and GluR2. PRCP density was normalized to β-actin density. 3v = third ventricle; ARC = arcuate nucleus; ME = median eminence. Bar scale in A (for all panels) represents 50 μm. Data represent the mean ± SEM.* = P < 0.05; ** = P < 0.01; *** = P < 0.001.

Article Snippet: After 3 times washing with TBST (TBS including 0.05% Tween 20), anti-goat IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Cat# sc-2768) for anti-PRCP antibody or anti-mouse IgG-HRP (Santa Cruz Biotechnology, Cat# sc-2005) for anti-GluR2 antibody was incubated for 1 h and developed by ECL kit (Thermo scientific, Cat# 32016).

Techniques: Expressing, Immunolabeling, Labeling, Control, Incubation, Western Blot

Journal: Annals of Clinical and Translational Neurology

Article Title: Blocking GluR2–GAPDH ameliorates experimental autoimmune encephalomyelitis

doi: 10.1002/acn3.182

Figure Lengend Snippet: GluR2–GAPDH complex in multiple sclerosis (MS). (A, B) GluR2–GAPDH complex formation is significantly increased in the plaque of multiple sclerosis (MS). Postmortem samples from control, MS plaque (plaque+) area, and MS nonplaque (plaque−) area were incubated with GluR2 antibody and the precipitated proteins were immunoblotted with GAPDH antibody or GluR2 antibody. The intensity of each protein band for GAPDH (A), GluR2 (B) from all three groups was quantified by densitometry (AIS software, Imaging Research Inc.). Results for each sample are presented as the percentage of the mean of the control samples on the same blot. (* P < 0.05, n = 8, one-way ANOVA). (C–E) GluR2–GAPDH complex formation is significantly increased in experimental autoimmune encephalitis (EAE) mice compared to sham mice. (A) Representative image of western blot analysis of GAPDH (top) and GluR2 (bottom) levels precipitated by GluR2 antibody in extract prepared from mouse spinal cord tissue were incubated with GluR2 antibody. Precipitated proteins were subject to SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and immunoblotted with GAPDH or GluR2 antibody. (D, E) Densitometric analysis of the level of GAPDH and GluR2. The intensity of GAPDH and GluR2 was quantified by densitometry (software: Image J, NIH). Data were analyzed by t -test. (** P < 0.01, n = 3). (F) Clinical EAE scores (mean ± SEM) over time of four groups vaccinated with MOG 35-55 on day 0 and treated intraperitoneally daily with TAT-G-Gpep and TAT-G-Gpep-Sc from day 10 (arrow). Starting from day 12, P < 0.05, data were analyzed by Mann–Whitney U test.

Article Snippet: For co-immunoprecipitation experiments, solubilized spinal cord protein (500–700 μ g) was incubated in the presence of primary antibodies anti-GluR2 (Novus Biologicals, Oakville, ON, Canada), or rabbit IgG (2–4 μ g) for 3 h at 4°C, followed by the addition of 25 μ L of protein A/G plus agarose (Santa Cruz Biotechnology, Santa Cruz, CA) for 12 h before subjecting to SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) for western blot analysis.

Techniques: Control, Incubation, Software, Imaging, Western Blot, SDS Page, Polyacrylamide Gel Electrophoresis, MANN-WHITNEY