Journal: iScience

Article Title: CD28 signaling domain boosts persistence and in vivo anti-tumor activity of stem cell-derived CD19-CAR-NK cells

doi: 10.1016/j.isci.2025.112548

Figure Lengend Snippet: The GS lentiviral plasmid supports sustained transgene expression during CD34 + HSC differentiation to NK cells under multiple promoters (A) Linear map of the GS lentiviral plasmid backbone. Five different promoters (CMV, MNDU3, PGK, EF1a, and SV40) driving gene expression were compared. Ori: origin of replication; CMV: cytomegalovirus; LTR: long terminal repeat; HIV-1: human immunodeficiency virus type-1; Psi: viral packaging signal sequence; RRE: Rev response element; GOI: gene of interest; SV40: simian vacuolating virus 40; poly(A): polyadenylation signal; KanR: kanamycin resistance. (B) Workflow of the generation of genetically modified (GM) NK cells from CD34 + HSCs. (C and D) eGFP expression, driven by one of the five promoters, during weeks 1–5 of NK cell differentiation after HSC transduction at MOI20, expressed as (C) percentage of eGFP expressing CD45 + cells and as (D) intensity of eGFP signal (ΔMFI) ( n = 3 donors). (E) NK differentiation measured as CD56 expression % of transduced cells, between week 3–5 ( n = 3 donors). (F and G) The tables represent p values of the statistically significant differences between week 2 and every other week (1–5) post-transduction for (F) percentage of eGFP and (G) intensity of eGFP signal (ΔMFI) calculated using Two-way ANOVA with Dunnett’s multiple comparisons test. (H) Representative flow cytometry dot plots of eGFP expression (FITC channel) under different promoters at week 3 post-transduction of CD34 + cells. Mock cells went through the same transduction process (minus lentiviral supernatant) and culture scheme as transduced cells and served as a negative control. Data are shown as mean ± SD.

Article Snippet: Human monoclonal IgG1 isotype control conjugated to FITC (clone REA293) , Miltenyi Biotec B.V. , Cat#130-113-437; RRID: AB_2733689.

Techniques: Plasmid Preparation, Expressing, Gene Expression, Virus, Sequencing, Genetically Modified, Cell Differentiation, Transduction, Flow Cytometry, Negative Control

Journal: Journal of medical microbiology

Article Title: Comprehensive analysis of human coronavirus antibody responses in ICU and non-ICU COVID-19 patients reveals IgG3 against SARS-CoV-2 spike protein as a key biomarker of disease severity.

doi: 10.1099/jmm.0.002012

Figure Lengend Snippet: Fig. 2. Total IgG, IgM and IgA antibody responses against SARS-CoV-2 antigens in ICU and non-ICU patients. (a) Frequency of positive Ig response in ICU and non-ICU patients. IgM, IgG and IgA RIs to SARS-CoV-2 antigens for (b) Envelope, (c) Nucleo, (d) Spike protein trimer and its fragments, (e) S1 and (f) RBD. A horizontal straight line represents the means, and the bars represent the sem. Unpaired two-tailed t-tests corrected for multiple comparisons by the Holm–Šidák correction were used to compare the ICU and non-ICU patients. (g) Heatmap analysis of the average of total IgG, IgM and IgA antibody responses against HCoVs S1 subunits. Yellow, green and blue indicate high, medium and low positivity index average values according to the scale of log2 of RIs. The significance symbols are plotted on the highest RI for each reactivity. Significance levels *: 0.01≤P≤ 0.05, **: 0.001≤P<0.01 and ***: P<0.001.

Article Snippet: After three vacuum washes with assay buffer using a PALL vacuum manifold (PALL), the microspheres in one plate were incubated with 50 μl of assay buffer containing 1 μg ml−1 of AlexaFluor 488- labelled mouse monoclonal antibody 4E3 anti- IgG1 hinge (SouthernBiotech, catalogue # 9052–30), 1 μg ml−1 of PE- labelled mouse monoclonal antibody HP605 anti- IgG3 hinge (SouthernBiotech, catalogue # 9210–09) and 1 μg ml−1 mouse monoclonal antibody HP602 anti- IgG2 Fc (SouthernBiotech, catalogue # 9710–31) for 20 min at room temperature under agitation at 800 r.p.m.

Techniques: Two Tailed Test

Journal: Journal of medical microbiology

Article Title: Comprehensive analysis of human coronavirus antibody responses in ICU and non-ICU COVID-19 patients reveals IgG3 against SARS-CoV-2 spike protein as a key biomarker of disease severity.

doi: 10.1099/jmm.0.002012

Figure Lengend Snippet: Fig. 3. Subclass of IgG and IgA antibody responses against SARS-CoV-2 antigens in ICU and non-ICU patients. (a) Frequency of positive Ig response to SARS-CoV-2 antigens. RIs to SARS-CoV-2 antigens for (b) Envelope, (c) Nucleo, (d) Spike protein trimer and its fragments, (e) S1 and (f) RBD. A horizontal straight line represents means, and the bars represent the sem. Unpaired two-tailed t-tests corrected for multiple comparisons by the Holm–Šidák correction were used to compare the ICU and non-ICU patients. (g) Heatmap analysis of the average of total IgG, IgM and IgA antibody responses against HCoVs S1 subunits. Yellow, green and blue indicate high, medium and low positivity index average values according to the scale of log2 of RIs. Significance levels *: 0.01≤P≤ 0.05, **: 0.001≤P<0.01 and ***: P<0.001.

Article Snippet: After three vacuum washes with assay buffer using a PALL vacuum manifold (PALL), the microspheres in one plate were incubated with 50 μl of assay buffer containing 1 μg ml−1 of AlexaFluor 488- labelled mouse monoclonal antibody 4E3 anti- IgG1 hinge (SouthernBiotech, catalogue # 9052–30), 1 μg ml−1 of PE- labelled mouse monoclonal antibody HP605 anti- IgG3 hinge (SouthernBiotech, catalogue # 9210–09) and 1 μg ml−1 mouse monoclonal antibody HP602 anti- IgG2 Fc (SouthernBiotech, catalogue # 9710–31) for 20 min at room temperature under agitation at 800 r.p.m.

Techniques: Two Tailed Test

Journal: Journal of medical microbiology

Article Title: Comprehensive analysis of human coronavirus antibody responses in ICU and non-ICU COVID-19 patients reveals IgG3 against SARS-CoV-2 spike protein as a key biomarker of disease severity.

doi: 10.1099/jmm.0.002012

Figure Lengend Snippet: Fig. 4. Total IgG, IgM and IgA antibody responses against HCoVs S1 proteins in ICU and non-ICU patients. (a) Frequency of positive Ig response. IgM, IgG and IgA RIs to HCoVs S1 proteins for (b) SARS-CoV-2, (c) SARS, (d) MERS, (e) 229E, (f) NL63, (g) HUK1 and (h) OC43. A horizontal straight line represents the mean, and the bars represent the sem. Unpaired two-tailed t-tests corrected for multiple comparisons by the Holm–Šidák correction were used to compare the ICU and non-ICU patients. (i) Heatmap analysis of the average of total IgG, IgM and IgA antibody responses against HCoVs S1 subunits. Yellow, green and blue indicate high, medium and low positivity index average values according to the scale of log2 of RIs. Significance levels *: 0.01≤P≤0.05, **: 0.001≤P<0.01 and ***: P<0.000.1.

Article Snippet: After three vacuum washes with assay buffer using a PALL vacuum manifold (PALL), the microspheres in one plate were incubated with 50 μl of assay buffer containing 1 μg ml−1 of AlexaFluor 488- labelled mouse monoclonal antibody 4E3 anti- IgG1 hinge (SouthernBiotech, catalogue # 9052–30), 1 μg ml−1 of PE- labelled mouse monoclonal antibody HP605 anti- IgG3 hinge (SouthernBiotech, catalogue # 9210–09) and 1 μg ml−1 mouse monoclonal antibody HP602 anti- IgG2 Fc (SouthernBiotech, catalogue # 9710–31) for 20 min at room temperature under agitation at 800 r.p.m.

Techniques: Two Tailed Test

Journal: Journal of medical microbiology

Article Title: Comprehensive analysis of human coronavirus antibody responses in ICU and non-ICU COVID-19 patients reveals IgG3 against SARS-CoV-2 spike protein as a key biomarker of disease severity.

doi: 10.1099/jmm.0.002012

Figure Lengend Snippet: Fig. 5. Subclass of IgG and IgA antibody responses against HCoVs S1 proteins in ICU and non-ICU patients. (a) Frequency of positive Ig subclass response to S1 proteins. RIs to HCoVs S1 proteins for (b) SARS-CoV-2, (c) SARS, (d) MERS, (e) 229E, (f) NL63, (g) HUK1 and (h) OC43. A horizontal straight line represents the mean, and the bars represent the sem. Unpaired two-tailed t-tests corrected for multiple comparisons by the Holm–Šidák correction were used to compare the ICU and non-ICU patients. (i) Heatmap analysis of the average of total IgG, IgM and IgA antibody responses against HCoVs S1 subunits. Yellow, green and blue indicate high, medium and low positivity index average values according to the scale of log2 of RIs. Significance levels *: 0.01≤P≤ 0.05, **: 0.001≤P<0.01 and ***: P<0.001.

Article Snippet: After three vacuum washes with assay buffer using a PALL vacuum manifold (PALL), the microspheres in one plate were incubated with 50 μl of assay buffer containing 1 μg ml−1 of AlexaFluor 488- labelled mouse monoclonal antibody 4E3 anti- IgG1 hinge (SouthernBiotech, catalogue # 9052–30), 1 μg ml−1 of PE- labelled mouse monoclonal antibody HP605 anti- IgG3 hinge (SouthernBiotech, catalogue # 9210–09) and 1 μg ml−1 mouse monoclonal antibody HP602 anti- IgG2 Fc (SouthernBiotech, catalogue # 9710–31) for 20 min at room temperature under agitation at 800 r.p.m.

Techniques: Two Tailed Test

Journal: bioRxiv

Article Title: Inhibiting SOX18 with propranolol restores vascular integrity in NR2F2-driven malformations

doi: 10.1101/2025.04.25.650344

Figure Lengend Snippet: A Immunostaining of sagittal sections from a CS17 human embryo. Co-expression of NR2F2 (green) and SOX18 (blue) in the gut. scale bars 50 μm. B-B ”,and C-C ” Sections of proband liver (B) and duodenum (C) stained for CD31 (cyan), SOX18 (red arrows), NR2F2 (green arrows), and DAPI (purple). Orange and gold boxes denote zoomed in regions of individual vessels. Orange box represents vessel that shows cells co-expressing both NR2F2 and SOX18 in the same nucleus, whereas the gold box is a vessel with cells either expressing NR2F2 or SOX18. Scale bar = 50μm (B-C) and 10 μm (B’”-C””).

Article Snippet: Blocked for 30 min was performed in 10% donkey serum followed by incubation overnight at 4C with monoclonal IgG1 mouse anti-human SOX18 (D-8) (1:50, Santa Cruz Biotechnology, sc-166025), monoclonal IgG 2A mouse anti-human COUP-TF II (1:100, R&D, PP-H7147-00), and UEA1 fluorescently labeled with Alexa Fluor 649 (1:50, Vector Laboratories, DL1068).

Techniques: Immunostaining, Expressing, Staining

Journal: bioRxiv

Article Title: Inhibiting SOX18 with propranolol restores vascular integrity in NR2F2-driven malformations

doi: 10.1101/2025.04.25.650344

Figure Lengend Snippet: A Hetero-dimer formation measured by N&B. B Hetero-dimer co-diffusion measured by cRICS. C Diffusion mobility graph from SMT comparing NR2F2 WT (purple) and NR2F2 WT + SOX18 (orange). Pie charts represent the proportion of the population that falls into either confined mobility or non-confined mobility based on diffusion coefficient. SMT performed on NR2F2. D Ratio of non-confined to confined molecules per cell from C. E Diffusion mobility graph from SMT comparing SOX18 (orange), SOX18 + NR2F2 WT (purple), and SOX18 + NR2F2 c994del (green). Pie charts represent the proportion of the population that falls into either confined mobility or non-confined mobility based on diffusion coefficient. SMT performed on SOX18. F Ratio of non-confined to confined molecules per cell from E. G Layering of SOX18, NR2F2 ChIP-seq datasets from Sissaoui et al. (NR2F2) and Overman et al. (SOX18) (black bars), with ATAC-seq (purple), histone marks (light blue), and ENCODE cis regulatory elements map (red/orange/yellow) upstream of the DCBLD2 gene locus in HUVECs. Green boxes denote co-binding locations at distal enhancer regions by both NR2F2 and SOX18. H Proportion and expression of known arterio-venous identity markers. Yellow box denotes a subset of genes that are regulated by both NR2F2 and SOX18. I) Relative gene expression of treated HUVECS measured by qPCR. For panel D, n > 20 cells, statistical significance was determined by Welch’s T-test, * p < 0.05. For panels F (n > 9 cells) and I (n = 3 repeats), statistical significance was determined by ANOVA, * p < 0.05, ** p < 0.005, *** p < 000.5, **** p < 0.0001.

Article Snippet: Blocked for 30 min was performed in 10% donkey serum followed by incubation overnight at 4C with monoclonal IgG1 mouse anti-human SOX18 (D-8) (1:50, Santa Cruz Biotechnology, sc-166025), monoclonal IgG 2A mouse anti-human COUP-TF II (1:100, R&D, PP-H7147-00), and UEA1 fluorescently labeled with Alexa Fluor 649 (1:50, Vector Laboratories, DL1068).

Techniques: Diffusion-based Assay, ChIP-sequencing, Binding Assay, Expressing, Gene Expression

Journal: bioRxiv

Article Title: Inhibiting SOX18 with propranolol restores vascular integrity in NR2F2-driven malformations

doi: 10.1101/2025.04.25.650344

Figure Lengend Snippet:

Article Snippet: Blocked for 30 min was performed in 10% donkey serum followed by incubation overnight at 4C with monoclonal IgG1 mouse anti-human SOX18 (D-8) (1:50, Santa Cruz Biotechnology, sc-166025), monoclonal IgG 2A mouse anti-human COUP-TF II (1:100, R&D, PP-H7147-00), and UEA1 fluorescently labeled with Alexa Fluor 649 (1:50, Vector Laboratories, DL1068).

Techniques:

Journal: bioRxiv

Article Title: Inhibiting SOX18 with propranolol restores vascular integrity in NR2F2-driven malformations

doi: 10.1101/2025.04.25.650344

Figure Lengend Snippet: A SMT performed on NR2F2. Diffusion mobility graph from SMT comparing NR2F2 WT + PBS (purple), NR2F2 WT + SOX18 + PBS (orange), and NR2F2 WT + SOX18 + R(+)prop (red). Pie charts represent the proportion of the population that falls into either confined mobility or non-confined mobility based on diffusion coefficient. B Ratio of non-confined to confined molecules per cell from A. C Schematic of hESC to EC differentiation timeline with drug treatment. D Histogram plots of venous derived populations, WT c994del and corresponding quantification of MFI. An increase of the CD73 expression occurs in the presence of SOX18 blockers despite the NR2F2 mutation. E Relative gene expression measured by qPCR from day 4 venous cells. F Model of the proposed molecular mechanism underlying the NR2F2 c994del rescue. Under physiological conditions in venous cells, NR2F2 and SOX18 are co-expressed at a specific ratio. This ratio allows for an antagonistic co-regulation of arteriovenous markers in vein ECs that is essential to maintain venous identity. In the probands’s scenario NR2F2 heterozygous mutation alters the proper ratio between NR2F2 WT and SOX18 that then becomes unbalanced in favor of SOX18 activity, leading to an upregulation of arterial markers and a perturbation of venous identity. Treatment with a SOX18 inhibitor enables the restoration of balance between SOX18 and NR2F2 activity, allowing a smaller proportion of both TF populations to function properly at physiological levels. Panels B (n > 16 cells) and E (n = 3 repeats), statistical significance was determined by ANOVA, * p < 0.05, ** p < 0.005, *** p < 000.5.

Article Snippet: Blocked for 30 min was performed in 10% donkey serum followed by incubation overnight at 4C with monoclonal IgG1 mouse anti-human SOX18 (D-8) (1:50, Santa Cruz Biotechnology, sc-166025), monoclonal IgG 2A mouse anti-human COUP-TF II (1:100, R&D, PP-H7147-00), and UEA1 fluorescently labeled with Alexa Fluor 649 (1:50, Vector Laboratories, DL1068).

Techniques: Diffusion-based Assay, Derivative Assay, Expressing, Mutagenesis, Gene Expression, Activity Assay