Journal: International Journal of Molecular Sciences

Article Title: Neuroprotective Action of Coumarin Derivatives through Activation of TRKB-CREB-BDNF Pathway and Reduction of Caspase Activity in Neuronal Cells Expressing Pro-Aggregated Tau Protein

doi: 10.3390/ijms232112734

Figure Lengend Snippet: Cellular ΔK280 tau RD aggregation and oxidative stress inhibitory effects of coumarin compounds in ΔK280 tau RD -DsRed SH-SY5Y cells. ( A ) Experimental flow chart. On day 1, cells were plated with retinoic acid (RA, 10 µM) added to the culture medium. On day 2, Congo red, LM-031 or LMDS-1 to -4 (2.5–10 µM) was added to the cells for 8 h, followed by inducing ΔK280 tau RD -DsRed expression with doxycycline (Dox, 2 µg/mL) for 6 days. On day 8, ΔK280 tau RD -DsRed fluorescence, tau RD -DsRed RNA and ROS (DCF stain) were measured. ( B ) Assessment of DsRed fluorescence with Congo red, LM-031 or LMDS-1 to -4 (2.5–10 µM) treatment ( n = 3). Shown below are cell number analyzed in each treatment. The relative DsRed fluorescence/cell number of untreated cells (Untr) was normalized as 100% (two-tailed Student’s t -test; *: p < 0.05). ( C ) tau RD -DsRed RNA of ΔK280 tau RD -DsRed cells untreated or treated with Congo red, LM-031 or LMDS-1 to -4 at 10 µM ( n = 3). HPRT1 was used for normalization. ( D ) Images of DCF stain (green) and ROS assay of ΔK280 tau RD -DsRed cells uninduced, untreated or treated with Congo red, LM-031 or LMDS-1 to -4 at 10 µM ( n = 3). The relative ROS of uninduced cells was normalized (100%). ( C , D ) p values: comparisons between induced (Dox+) vs. uninduced (Dox−) cells ( # : p < 0.05, ### : p < 0.001), or compound-treated vs. untreated (induced) (Dox+) cells (*: p < 0.05, **: p < 0.01, ***: p < 0.001) (one-way ANOVA with post hoc Tukey test).

Article Snippet: Quantitative PCR was performed with 50 ng cDNA and customized Assays-by-Design probe for DsRed [ ] and HPRT1 (4326321E, endogenous control) in a 96-well real-time PCR instrument (StepOnePlus TM Real-time PCR system; Applied Biosystems, Foster City, CA, USA).

Techniques: Expressing, Fluorescence, Staining, Two Tailed Test, ROS Assay

Journal: medRxiv

Article Title: Immune responses in COVID-19 respiratory tract and blood reveal mechanisms of disease severity

doi: 10.1101/2021.09.01.21262715

Figure Lengend Snippet: a Number of patients recruited in the DRASTIC cohort and samples collected (top panel) and days post disease onset of COVID-19 blood samples (bottom panel). b Collection timepoints of respiratory samples (endotracheal tube aspirate (ETA), sputum, and pleural fluid) and paired blood samples from the same patients (left panel). Comparison of days post disease onset between blood and respiratory samples for COVID-19 and non-COVID-19 patients (right panel) using Mann-Whitney test. c Maximum likelihood phylogenetic tree of SARS-CoV-2 sequences from Victoria from 28 January 2020 to 28 October 2020 (including context sequences from rest of Australia and New Zealand). Phylogenetic tree includes randomly subsampled sequences from transmission networks (TN) A and TN B in Victoria, with a total number of 10941 and 145 cases respectively. The outermost tip of each radial line represents a single sequence; the sum of each radial line between two tips represents the genetic distance between two sequences. Each radial stepwise progression represents approximately one single nucleotide polymorphism (SNP). Sequences from study patients are shown as open circles (patient with blood samples only) or solid-coloured circles (patients with blood and respiratory samples, same colours as in section b). Half-filled circles are used when samples are located close to each other. d Distribution of clinical data in ward and intensive care unit (ICU) COVID-19 patients. Box and bars indicate first and third quartiles, and range respectively. Statistical significance was determined with the Fisher’s exact test for the National Institutes of Health (NIH) score, and Mann-Whitney test for age, weight, height, and body weight index (BMI). Correlation was determined with Spearman’s correlation.

Article Snippet: As previously described , a custom multiplex bead array was designed and coupled with SARS-CoV-2 spike 1 (Sino Biological), spike 2 (ACRO Biosystems), RBD (BEI Resources) and nucleoprotein (ACRO Biosystems), as well as SARS and hCoV (229E, NL63, HKU1, OC43) spikes and nucleoproteins (Sino Biological) (Supplementary Fig. 5).

Techniques: MANN-WHITNEY, Transmission Assay, Sequencing

Journal: medRxiv

Article Title: Immune responses in COVID-19 respiratory tract and blood reveal mechanisms of disease severity

doi: 10.1101/2021.09.01.21262715

Figure Lengend Snippet: a ELISA titration curves against the SARS-CoV-2 receptor-binding domain (RBD) for IgM, IgG, and IgA in COVID-19 respiratory and paired blood samples and non-COVID-19 respiratory samples. Dotted lines within each graph indicates the cut-off used to determine end-point titres. b End-point titres of SARS-CoV-2 RBD antibodies between (i) respiratory samples of COVID-19 and non-COVID-19 patients, and (ii) plasma and respiratory samples of COVID-19 patients. (i) Bars indicate median with interquartile range. Dotted line indicates the detection level. (ii) Dotted lines connect the most closely matched plasma and respiratory samples from each patient. Statistical significance was determined with Mann-Whitney test. c ELISA titration curves against the SARS-CoV-2 RBD for 3 COVID-19 patients with serial respiratory samples. d Heatmap of percentage (%) inhibition tested by surrogate virus neutralization test (sVNT) and anti-RBD ELISA titres. e Correlation between anti-RBD antibody titres and (%) sVNT inhibition. Correlation was determined with Spearman’s correlation. f Number of (i) samples and (ii) patients with seroconverted anti-RBD IgM, IgG, IgA and positive % sVNT inhibition. Pink curved lines surrounding the donut graphs indicate the samples/patients with seroconverted IgM. Earliest samples were used for each patient when determining seroconversion which was defined as average titre +2×SD of non-COVID-19 samples. Positive % sVNT inhibition was defined as % sVNT inhibition ≥ 20%.

Article Snippet: As previously described , a custom multiplex bead array was designed and coupled with SARS-CoV-2 spike 1 (Sino Biological), spike 2 (ACRO Biosystems), RBD (BEI Resources) and nucleoprotein (ACRO Biosystems), as well as SARS and hCoV (229E, NL63, HKU1, OC43) spikes and nucleoproteins (Sino Biological) (Supplementary Fig. 5).

Techniques: Enzyme-linked Immunosorbent Assay, Titration, Binding Assay, MANN-WHITNEY, Inhibition, Neutralization

Journal: medRxiv

Article Title: Immune responses in COVID-19 respiratory tract and blood reveal mechanisms of disease severity

doi: 10.1101/2021.09.01.21262715

Figure Lengend Snippet: a Heatmaps with unsupervised clustering of SARS-CoV-2-specific antibodies in COVID-19 respiratory and plasma samples. b median fluorescence intensity of IgM, IgG, IgA1, and IgA2 antibodies between COVID-19 and non-COVID-19 respiratory samples. Statistical significance was determined with Mann-Whitney test. c Partial Least-Squares Discriminant Analysis (PLSDA) scores and loading plots of ETA and plasma from five COVID-19 and five non-COVID-19 patients with the smallest difference in days post disease onset between ETA and plasma samples.

Article Snippet: As previously described , a custom multiplex bead array was designed and coupled with SARS-CoV-2 spike 1 (Sino Biological), spike 2 (ACRO Biosystems), RBD (BEI Resources) and nucleoprotein (ACRO Biosystems), as well as SARS and hCoV (229E, NL63, HKU1, OC43) spikes and nucleoproteins (Sino Biological) (Supplementary Fig. 5).

Techniques: Fluorescence, MANN-WHITNEY

Journal: Scientific Reports

Article Title: N-cadherin is Key to Expression of the Nucleus Pulposus Cell Phenotype under Selective Substrate Culture Conditions

doi: 10.1038/srep28038

Figure Lengend Snippet: ( A ) Representative immunostaining of phalloidin and cadherins for porcine NP cells on soft and stiff PEG-LM (green = protein, red = cell nuclei, scale bar = 50 μm). ( B ) Changes in sGAG production for porcine NP cells on soft and stiff PEG-LM. ( C ) Quantification of gene expression for juvenile NP cell phenotype markers in porcine NP cells on soft, relative to NP cells on stiff (CDH2 = N-cadherin, T = brachyury, LM1 = Laminin1, AGC = aggrecan, COL2 = type II collagen). ( D ) Representative immunostaining of phalloidin and cadherins for juvenile (juv) and degenerate (deg) human NP cells on soft PEG-LM (green = protein, red = cell nuclei, scale bar = 50 μm, CDH2 + = CDH2 positive, CDH2− = CDH2 negative). ( E ) Same as B but with human NP cells on PEG-LM. ( F ) Same as C but with human NP cells on PEG-LM (additional marker CDH1 was quantified in human). ( G ) Representative immunostaining for β-catenin (green) in NP cells on soft PEG-LM with associated changes in phosphorylated β-catenin, fold-change for β-catenin in the cytoplasm to nucleus, with corresponding western blot images for β-catenin (immunostaining images scale bar = 50 μm, with higher magnification inset scale bar = 20 μm). ( H ) Western blot images for LMNA expression on soft and stiff PEG-LM (All western blot images were cropped to display protein expression concisely; see for full western blots) (For all studies: 2-way ANOVA with Tukey’s post-hoc analysis, *p < 0.05).

Article Snippet: The genes analyzed via qRT-PCR were: CDH2 (porcine: custom-designed by Life Technologies, human: Hs00983056_m1), CDH1 (human only: Hs01023894_m1), T-brachyury (porcine: Ss03374654_g1, human: Hs00610080_m1), type II collagen (porcine: Ss03373344_g1, human: Hs00156568_m1), laminin β1 (porcine: Ss03375563_u1), laminin a1 (human: Hs00300550_m1), and aggrecan (porcine: Ss03374823_m1, human: Hs00153936_m1).

Techniques: Immunostaining, Expressing, Marker, Western Blot

Journal: Scientific Reports

Article Title: N-cadherin is Key to Expression of the Nucleus Pulposus Cell Phenotype under Selective Substrate Culture Conditions

doi: 10.1038/srep28038

Figure Lengend Snippet: ( A ) CRISPRi construct sequence. ( B ) Representative images of successful CDH2 knockdown analyzed by fluorescence microscopy. ( C ) Validation of successful CDH2 knockdown via flow cytometry. ( D ) Quantification of CDH2 knockdown via qRT-PCR; arrows indicate successful CDH2 knockdown using species-specific CRISPRs (porcine seq = porcine sequence, human seq = human sequence). ( E ) Representative immunostaining for phalloidin, CDH2 and β-catenin in porcine NP cells after CDH2 knockdown, compared to scramble control and no treatment (blue = protein, pink/red = cell nuclei, scale bar = 50 μm). ( F ) Quantification of total phosphorylated β-catenin levels in porcine NP cells (1 = CDH2 (−), 2 = scramble, 3 = no treatment). ( G ) Changes in sGAG in CDH2 knockdown porcine NP cells on soft. ( H ) Changes in gene expression for CDH2 knockdown porcine NP cells on soft, compared to stiff (CDH2 = N-cadherin, T = brachyury, LM1 = laminin, COL2 = type II collagen, AGC = aggrecan; * denotes that all genes are significantly different from scramble and no treatment controls) ( I–L ) same as ( E – H ) respectively, but with CDH2 knockdon in juvenile human NP cells (For all studies: 2-way ANOVA with Tukey’s post-hoc analysis, *p < 0.05).

Article Snippet: The genes analyzed via qRT-PCR were: CDH2 (porcine: custom-designed by Life Technologies, human: Hs00983056_m1), CDH1 (human only: Hs01023894_m1), T-brachyury (porcine: Ss03374654_g1, human: Hs00610080_m1), type II collagen (porcine: Ss03373344_g1, human: Hs00156568_m1), laminin β1 (porcine: Ss03375563_u1), laminin a1 (human: Hs00300550_m1), and aggrecan (porcine: Ss03374823_m1, human: Hs00153936_m1).

Techniques: Construct, Sequencing, Fluorescence, Microscopy, Flow Cytometry, Quantitative RT-PCR, Immunostaining, Expressing

Journal: Scientific Reports

Article Title: N-cadherin is Key to Expression of the Nucleus Pulposus Cell Phenotype under Selective Substrate Culture Conditions

doi: 10.1038/srep28038

Figure Lengend Snippet: ( A,B ) Representative immunostaining of phalloidin, CDH2, and CDH1 for NP and AF cells, respectively (green = protein, red = cell nuclei, scale bar = 50 μm). ( C ) Changes in sGAG production for porcine NP cells on soft and stiff PEG-LM after blocking antibody treatment. ( D ) Quantification of gene expression for juvenile NP cell phenotype markers in porcine NP cells on soft, relative to NP cells on stiff after blocking antibody treatment (2-way ANOVA with Tukey’s post-hoc analysis, *p < 0.05) (Key: 1 = No treatment condition, 2 = CDH2 blocking antibody treatment, 3 = CDH1 blocking antibody treatment; CDH2 = N-cadherin, T = brachyury, LM1 = Laminin1, AGC = aggrecan, COL2 = type II collagen).

Article Snippet: The genes analyzed via qRT-PCR were: CDH2 (porcine: custom-designed by Life Technologies, human: Hs00983056_m1), CDH1 (human only: Hs01023894_m1), T-brachyury (porcine: Ss03374654_g1, human: Hs00610080_m1), type II collagen (porcine: Ss03373344_g1, human: Hs00156568_m1), laminin β1 (porcine: Ss03375563_u1), laminin a1 (human: Hs00300550_m1), and aggrecan (porcine: Ss03374823_m1, human: Hs00153936_m1).

Techniques: Immunostaining, Blocking Assay, Expressing

Journal: Scientific Reports

Article Title: N-cadherin is Key to Expression of the Nucleus Pulposus Cell Phenotype under Selective Substrate Culture Conditions

doi: 10.1038/srep28038

Figure Lengend Snippet: (A) Representative immunostaining of CDH2 (a,b), type I collagen (Col I) (c,d), type II collagen (Col II) (e,f), and aggrecan (agg) (g,h) in rat tail discs 2 weeks after intradiscal injection of CDH2 blocking antibody (Cdh2 Ab) or control immunoglobulin (IgG) (red = protein, blue = cell nuclei, scale bar = 100 μm, NP = nucleus pulposus; AF = anulus fibrosus). ( B) Representative immunostaining of non-phosphorylated β-catenin (a,b), brachyury (c,d), and E-cadherin (e,f) and representative histological assessment of H&E staining (g,h) 2 weeks after intradiscal injection of Cdh2 Ab or IgG (red = protein, blue = cell nuclei, scale bar = 50 μm, NP tissue only). (C,D) Similar to panel A and B, but assessed 8 weeks after blocking antibody delivery.

Article Snippet: The genes analyzed via qRT-PCR were: CDH2 (porcine: custom-designed by Life Technologies, human: Hs00983056_m1), CDH1 (human only: Hs01023894_m1), T-brachyury (porcine: Ss03374654_g1, human: Hs00610080_m1), type II collagen (porcine: Ss03373344_g1, human: Hs00156568_m1), laminin β1 (porcine: Ss03375563_u1), laminin a1 (human: Hs00300550_m1), and aggrecan (porcine: Ss03374823_m1, human: Hs00153936_m1).

Techniques: Immunostaining, Injection, Blocking Assay, Staining