Journal: Molecular Therapy. Nucleic Acids

Article Title: Identification and characterization of a MAPT-targeting locked nucleic acid antisense oligonucleotide therapeutic for tauopathies

doi: 10.1016/j.omtn.2022.07.027

Figure Lengend Snippet: ASO-001933 is a highly potent and selective ASO targeting MAPT on human neurons (A) Schematic diagram of ASO treatment schedule in hESCs for (B)–(F). (B) Dose-dependent reduction of MAPT expression at RNA and protein level upon ASO-001933 or BIIB080 treatment. hESC-derived neurons were treated with ASO at indicated concentrations. RNA and protein levels were assessed by qRT-PCR and AlphaLISA respectively. Absolute IC 50 values are reported in the figure (n = 3 /treatment, mean +/- SEM). (C) Quantification of 3R and 4R Tau mRNA by qPCR after ASO treatment at 1 μM (n = 3 /treatment, mean +/- SEM). (D and E) Representative images of RNAscope ISH and ICC using probes against MAPT CDS, MAPT 3′ UTR (D) and antibody against Tau (Tau HT7) (E). (F) Volcano plot illustrating differentially regulated proteins from proteomics analysis between ASO-001933-treated cells versus NT ASO-treated neurons (n = 3). MAPT is the only significantly regulated protein (Log2 FC = −2.94, adjusted Log10 p value = −4.54). (G) Evaluation of off-target effects of ASO-001933 in hiPSC-derived neurons. Volcano plot showing differentially expressed genes (Log2FC > 1 or Log2FC< −1 and −Log10 adjusted p >20) from RNA-seq analysis after 72 h of treatment (n = 3).

Article Snippet: Cell extracts were diluted 20-fold into AlphaLISA HiBlock buffer (PerkinElmer) for assay.

Techniques: Expressing, Derivative Assay, Quantitative RT-PCR, RNAscope, RNA Sequencing

Journal: Frontiers in Cardiovascular Medicine

Article Title: Utility of atherosclerosis-associated serum antibodies against colony-stimulating factor 2 in predicting the onset of acute ischemic stroke and prognosis of colorectal cancer

doi: 10.3389/fcvm.2023.1042272

Figure Lengend Snippet: Comparison of the s-CSF2-Ab and s-CSF2pep-Ab levels in patients with AIS and TIA and in HDs. (A–B) Levels of serum antibodies against (A) s-CSF2-Ab and (B) s-CSF2pep-Ab determined using amplified luminescence proximity homogeneous assay-linked immunosorbent assay (AlphaLISA), calculated by subtracting the levels of antibodies against the control GST. Scatter dot plots of the s-CSF2-Ab and s-CSF2pep-Ab levels are also presented. Bars represent averages ± standard deviation (SD). ** P < 0.05; * P < 0.01; ns, not significant, Kruskal–Wallis test. (C–E) A receiver operating characteristic (ROC) curve analysis was conducted to assess the ability of s-CSF2-Ab to detect AIS (C) and that of s-CSF2pep-Ab to detect AIS (D) and TIA (E) . The numbers indicate the cutoff values for the indicated markers, and the numbers in parentheses indicate sensitivity (left) and specificity (right). The areas under the ROC curve (AUC) and 95% confidence intervals (CIs) are also shown. AIS, acute ischemic stroke; HD, healthy donor; CSF2-Ab, CSF2 antibody; CSF2pep-Ab, CSF2 peptide antibody; TIA, transient ischemic attack.

Article Snippet: The reaction mixture containing 2.5 μL of serum samples diluted at 1:100 in AlphaLISA buffer (25 mM HEPES pH 7.4, 0.1% casein, 0.5% Triton X-100, 1 mg/mL of dextran-500, and 0.05% Proclin-300) and 2.5 μL of GST or GST-CSF2 proteins (10 μg/ml) or 400 ng/mL of bCSF2-77 was incubated in 384-well white opaque OptiPlate microtiter plates (PerkinElmer, Beaconsfield, United Kingdom) at room temperature for 6–8 h. Next, 2.5 μL of anti-human immunoglobulin G (IgG)-conjugated acceptor beads (40 μg/ml) and 2.5-μL glutathione-conjugated donor beads (40 μg/ml) or 2.5 μL of streptavidin-conjugated donor beads (40 μg/ml) were added.

Techniques: Comparison, Amplification, Control, Standard Deviation

Journal: Frontiers in Cardiovascular Medicine

Article Title: Utility of atherosclerosis-associated serum antibodies against colony-stimulating factor 2 in predicting the onset of acute ischemic stroke and prognosis of colorectal cancer

doi: 10.3389/fcvm.2023.1042272

Figure Lengend Snippet: Comparison of the s-CSF2-Ab and s-CSF2pep-Ab levels between HDs and patients with AMI and DM. ( A,B ) The levels of s-CSF2-Ab and s-CSF2pep-Ab in HDs and patients with acute myocardial infarction (AMI) ( A ) and diabetes mellitus (DM) ( B ) were determined using AlphaLISA. The bars represent averages ± SD. *** P < 0.001, Mann–Whitney U test. ( D,E ) The ROC curves to assess the ability of s-CSF2-Ab to predict AMI and DM are presented in ( D ) and ( E ), respectively. ( F,G ) The ability of s-CSF2pep-Ab to predict AMI ( E ) and DM ( F ) was also evaluated via ROC analysis. Comparison of the overall survival in patients with DM between the s-CSF2-Ab positive (s-CSF2-Ab + ) and s-CSF2-Ab negative (s-CSF2-Ab − ) groups ( P = 0.019; C ) and between the s-CSF2pep-Ab positive (s-CSF2-Ab + ) and s-CSF2pep-Ab negative (s-CSF2-Ab − ) groups ( P = 0.034; C ). Statistical analyzes were performed by the Log-Rank test between two groups.

Article Snippet: The reaction mixture containing 2.5 μL of serum samples diluted at 1:100 in AlphaLISA buffer (25 mM HEPES pH 7.4, 0.1% casein, 0.5% Triton X-100, 1 mg/mL of dextran-500, and 0.05% Proclin-300) and 2.5 μL of GST or GST-CSF2 proteins (10 μg/ml) or 400 ng/mL of bCSF2-77 was incubated in 384-well white opaque OptiPlate microtiter plates (PerkinElmer, Beaconsfield, United Kingdom) at room temperature for 6–8 h. Next, 2.5 μL of anti-human immunoglobulin G (IgG)-conjugated acceptor beads (40 μg/ml) and 2.5-μL glutathione-conjugated donor beads (40 μg/ml) or 2.5 μL of streptavidin-conjugated donor beads (40 μg/ml) were added.

Techniques: Comparison, MANN-WHITNEY

Journal: Frontiers in Cardiovascular Medicine

Article Title: Utility of atherosclerosis-associated serum antibodies against colony-stimulating factor 2 in predicting the onset of acute ischemic stroke and prognosis of colorectal cancer

doi: 10.3389/fcvm.2023.1042272

Figure Lengend Snippet: Association of the s-CSF2-Ab and s-CSF2pep-Ab levels with CKD. The levels of s-CSF-Ab ( A ) and s-CSF2pep-Ab ( B ) in HDs and in patients with diabetic CKD (CKD type 1), nephrosclerosis (CKD type 2), and glomerulonephritis (CKD type 3) were examined using AlphaLISA. The bars represent averages ± SD. * P < 0.05; ** P < 0.01; *** P < 0.001, Kruskal–Wallis test. The data are summarized in . ( C–E ) ROC analysis to determine the ability of s-CSF2-Ab to predict CKD type 1 ( C ), type 2 ( D ), and type 3 ( E ). ( F–H ) ROC analysis to determine the ability of s-CSF2pep-Ab to predict CKD type 1 ( F ), type 2 ( G ), and type 3 ( H ). The numbers in the graphs are the same as those presented . CKD, chronic kidney disease.

Article Snippet: The reaction mixture containing 2.5 μL of serum samples diluted at 1:100 in AlphaLISA buffer (25 mM HEPES pH 7.4, 0.1% casein, 0.5% Triton X-100, 1 mg/mL of dextran-500, and 0.05% Proclin-300) and 2.5 μL of GST or GST-CSF2 proteins (10 μg/ml) or 400 ng/mL of bCSF2-77 was incubated in 384-well white opaque OptiPlate microtiter plates (PerkinElmer, Beaconsfield, United Kingdom) at room temperature for 6–8 h. Next, 2.5 μL of anti-human immunoglobulin G (IgG)-conjugated acceptor beads (40 μg/ml) and 2.5-μL glutathione-conjugated donor beads (40 μg/ml) or 2.5 μL of streptavidin-conjugated donor beads (40 μg/ml) were added.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: The carboxyl-terminal TSP1-homology domain is the biologically active effector peptide of matricellular protein CCN5 that counteracts profibrotic CCN2

doi: 10.1016/j.jbc.2022.102803

Figure Lengend Snippet: The TSP1 domain of CCN5 is sufficient to inhibit phosphorylation of AKT and CCN2-induced cell migration. Panel ( A ) demonstrates concentration-effect curves of HHS-CCN5(III)- and HHS-CCN5(FL)-mediated inhibition of phospho-AKT (Ser473) levels in A549 human adenocarcinoma epithelial cells following 60 min incubation with the indicated proteins. The His-Halo-Sumo (HHS) fusion partner alone did not display any efficacy. Panel ( B ) demonstrates concentration-effect curves of Alb-CCN5(III)- and Alb-CCN3(III)-stimulated inhibition of phospho-AKT (Ser473) levels in A549 cells following 60 min incubation with the indicated proteins. The data in panels ( A and B ) represent the mean ± SEM (n = 3 independent experiments assayed in three replicates for each data point) using a TR-FRET–based or AlphaLISA-based immunoassay of cellular phospho-AKT (Ser473) contents. The data were subjected to curve fitting by four-variable nonlinear regression in GraphPad Prism. Panels ( C ) and ( D ) demonstrate concentration-effect curves of HHS-CCN5(III)- and Alb-CCN3(III)-mediated inhibition of CCN2 (5 μg/ml)-stimulated migration of Rat2 fibroblasts following 20 h incubation. The HHS fusion partner alone did not show any efficacy in the migration of Rat2 fibroblasts. Cell migration was determined using multiscreen migration assay (Boyden chamber principle). The data represent the mean ± SEM (n = 3 independent experiments assayed in four replicates for each data point). The data were subjected to curve fitting by four-variable nonlinear regression in GraphPad Prism. Alb, albumin; CCN, Cellular Communication Network; HHS, His-Halo-Sumo; TSP1, thrombospondin type 1 repeat.

Article Snippet: The cells were subsequently lysed in lysis buffer provided by the AlphaLISA SureFire Ultra phospho-AKT (Ser473) and phospho-ERK1/2 (Thr202/Tyr204 and Thr185/Tyr187) Assay Kits (PerkinElmer, Cat. No ALSU-PAKT-B-HV 100 test and ALSU-PERK-A-HV 100 test), transferred to a 96-well AlphaPlate (PerkinElmer), and analyzed with the sandwich bead–based AlphaLISA immunoassay (AlphaLISA Surefire Ultra immunoassay, PerkinElmer), according to the manufacturer’s instructions.

Techniques: Migration, Concentration Assay, Inhibition, Incubation

Journal: Toxins

Article Title: Detection of Shiga Toxin 2 Produced by Escherichia coli in Foods Using a Novel AlphaLISA

doi: 10.3390/toxins10110422

Figure Lengend Snippet: Schematic representation of the sandwich amplified luminescent proximity homogenous assay-linked immunosorbent assay (AlphaLISA) for Shiga toxin detection. Two sets of monoclonal antibodies were employed in this assay (orange and gray Y’s): one specific for the ( A ) subunit (M-1005) and other for the ( B ) subunit (M-1003) of Stx2. One set of antibodies was biotinylated (green hemisphere) to allow association with streptavidin-coated donor beads (blue sphere), while the other antibody set was directly conjugated to the acceptor beads (yellow sphere). When Stx2 is present, the donor and acceptor beads are colocalized through binding of the antibodies to the different subunits of the common antigen. Upon excitation by an Alpha laser at 680 nm (lightning bolt), the donor beads emit singlet oxygen molecules that react with the acceptor beads. Ultimately, energy transfer results in the emission of light by the acceptor beads, thus creating a detectable fluorescence signal at 615 nm. The following two formats were analyzed during assay development: ( A ) Antibody M-1003 was biotinylated and thus associated with the donor beads while M-1005 was conjugated to the acceptor beads and ( B ) antibody M-1005 was biotinylated and thus associated with the donor beads while M-1003 was conjugated to the acceptor beads.

Article Snippet: STX-2), at 0.5 mg/mL, and diluted to 3, 30, and 300 ng/mL in AlphaLISA buffer (25 mM HEPES pH 7.4 (VWR International, Radnor, PA, USA; prod.

Techniques: Amplification, Binding Assay, Fluorescence

Journal: Toxins

Article Title: Detection of Shiga Toxin 2 Produced by Escherichia coli in Foods Using a Novel AlphaLISA

doi: 10.3390/toxins10110422

Figure Lengend Snippet: Optimization of donor/acceptor bead pairing and antibody concentration. A multifactorial test involving the donor/acceptor bead pairing, donor antibody concentration, and Stx2 concentration was performed to identify the optimal assay parameters for the AlphaLISA. AlphaLISA signals were recorded for the following factors: (a) bead pairings consisting of donor M-1003 with 25 µg acceptor M-1005 (left panel) versus donor M-1005 with 25 µg acceptor M-1003 (right panel); (b) donor antibody concentrations of 0 (black bars), 0.3 (dark gray bars), 1.0 (medium gray bars), and 3.0 nM (light gray bars); and (c) Stx2 concentrations of 0, 3 ng/mL, 30 ng/mL, and 300 ng/mL. Emitted light was measured on a BioTek Cytation 5 in Alpha mode at a gain setting of 100. Bars represent the mean values ( n = 4) with error bars denoting the standard deviation.

Article Snippet: STX-2), at 0.5 mg/mL, and diluted to 3, 30, and 300 ng/mL in AlphaLISA buffer (25 mM HEPES pH 7.4 (VWR International, Radnor, PA, USA; prod.

Techniques: Concentration Assay, Standard Deviation

Journal: Toxins

Article Title: Detection of Shiga Toxin 2 Produced by Escherichia coli in Foods Using a Novel AlphaLISA

doi: 10.3390/toxins10110422

Figure Lengend Snippet: Detection of purified Stx2 using the AlphaLISA and the enzyme-linked immunosorbent assay (ELISA). Varying concentrations of purified Stx2 ranging from 0–10 ng/mL were analyzed using both the AlphaLISA (black line) and the ELISA (gray line). The response from both assays were quantified on a BioTek Cytation 5 with the emitted light from the AlphaLISA measured via the Alpha mode at a gain setting of 100 (left y -axis) and the absorbance values of the ELISA measured at 450 nm (right y -axis). Mean values from 6 trials were plotted with error bars denoting the standard deviation.

Article Snippet: STX-2), at 0.5 mg/mL, and diluted to 3, 30, and 300 ng/mL in AlphaLISA buffer (25 mM HEPES pH 7.4 (VWR International, Radnor, PA, USA; prod.

Techniques: Purification, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Toxins

Article Title: Detection of Shiga Toxin 2 Produced by Escherichia coli in Foods Using a Novel AlphaLISA

doi: 10.3390/toxins10110422

Figure Lengend Snippet: Detection of Stx2 in Shiga toxin-producing Escherichia coli (STEC)-inoculated foods using the AlphaLISA and ELISA. Stx2 production was induced using mitomycin C in STEC-inoculated ( A ) Romaine lettuce and ( B ) ground beef. Undiluted (1×) or diluted (10× and 100×) samples were subsequently assayed for the presence of Stx2 using both the AlphaLISA (dark gray bars) and the ELISA (light gray bars). Control samples contained uninoculated lettuce and ground beef samples, respectively. Responses were quantified on a BioTek Cytation 5 with the emitted light from the AlphaLISA measured via Alpha mode at a gain setting of 100 (left y -axis) and the absorbance values of the ELISA measured at 450 nm (right y -axis). Mean values from 3 independent trials containing 2 replicates were plotted with error bars denoting the standard deviation. Significance between AlphaLISA signal or absorbance values are denoted by dissimilar letters as determined by a Student’s t -test at a 95% confidence level.

Article Snippet: STX-2), at 0.5 mg/mL, and diluted to 3, 30, and 300 ng/mL in AlphaLISA buffer (25 mM HEPES pH 7.4 (VWR International, Radnor, PA, USA; prod.

Techniques: Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Toxins

Article Title: Detection of Shiga Toxin 2 Produced by Escherichia coli in Foods Using a Novel AlphaLISA

doi: 10.3390/toxins10110422

Figure Lengend Snippet: The effect of increasing reaction components on the sensitivity of the AlphaLISA for Stx2 in food matrices. Purified Stx2 (0, 0.5, 1.0, 3.0 or 5.0 ng/mL) was added to uninoculated lettuce ( left ) panel or ground beef ( right ) panel and the effect of increasing all of the reaction components (1×-blue line, 2×-red line, or 3×-green line) in the AlphaLISA signal was determined. Responses were quantified on a BioTek Cytation 5 with the emitted light from the AlphaLISA measured via Alpha mode at a gain setting of 100. Mean values from 2 independent trials, each containing 2 replicates are plotted with error bars denoting the standard deviation.

Article Snippet: STX-2), at 0.5 mg/mL, and diluted to 3, 30, and 300 ng/mL in AlphaLISA buffer (25 mM HEPES pH 7.4 (VWR International, Radnor, PA, USA; prod.

Techniques: Purification, Standard Deviation

Journal: Toxins

Article Title: Detection of Shiga Toxin 2 Produced by Escherichia coli in Foods Using a Novel AlphaLISA

doi: 10.3390/toxins10110422

Figure Lengend Snippet: Comparisons of the limit of detection and signal-to-noise ratios of the AlphaLISA versus the ELISA.

Article Snippet: STX-2), at 0.5 mg/mL, and diluted to 3, 30, and 300 ng/mL in AlphaLISA buffer (25 mM HEPES pH 7.4 (VWR International, Radnor, PA, USA; prod.

Techniques: Enzyme-linked Immunosorbent Assay