Journal: Journal of Cancer

Article Title: Serum Adiponectin Level May be an Independent Predictor of Clear Cell Renal Cell Carcinoma

doi: 10.7150/jca.14716

Figure Lengend Snippet: Serum adiponectin level, leptin level and VFA in different subtypes of renal cell carcinoma. The mean and standard deviation data were plotted.

Article Snippet: The detection of plasma leptin concentration was the same as that described above, using a human-specific leptin ELISA kit (EK1972, Multisciences, Shanghai, China).

Techniques: Standard Deviation

Journal: Journal of Cancer

Article Title: Serum Adiponectin Level May be an Independent Predictor of Clear Cell Renal Cell Carcinoma

doi: 10.7150/jca.14716

Figure Lengend Snippet: Differences of serum adiponectin, leptin level and VFA between ccRCC patients and non-ccRCC patients.

Article Snippet: The detection of plasma leptin concentration was the same as that described above, using a human-specific leptin ELISA kit (EK1972, Multisciences, Shanghai, China).

Techniques:

Journal: Journal of Cancer

Article Title: Serum Adiponectin Level May be an Independent Predictor of Clear Cell Renal Cell Carcinoma

doi: 10.7150/jca.14716

Figure Lengend Snippet: The association of VFA with serum adiponectin and leptin level. Pearson's tests show that the correlation between VFA and serum adiponectin level (p < 0.001) and the link between VFA and serum leptin concentration (p < 0.001) are statistically significant.

Article Snippet: The detection of plasma leptin concentration was the same as that described above, using a human-specific leptin ELISA kit (EK1972, Multisciences, Shanghai, China).

Techniques: Concentration Assay

Journal: Journal of Cancer

Article Title: Serum Adiponectin Level May be an Independent Predictor of Clear Cell Renal Cell Carcinoma

doi: 10.7150/jca.14716

Figure Lengend Snippet: Univariate analysis and multivariate analysis of predictors for different subtypes of RCC.

Article Snippet: The detection of plasma leptin concentration was the same as that described above, using a human-specific leptin ELISA kit (EK1972, Multisciences, Shanghai, China).

Techniques:

Journal: Journal of Cancer

Article Title: Serum Adiponectin Level May be an Independent Predictor of Clear Cell Renal Cell Carcinoma

doi: 10.7150/jca.14716

Figure Lengend Snippet: Association between clinical parameters and plasma adiponectin/leptin levels in the ccRCC subgroup.

Article Snippet: The detection of plasma leptin concentration was the same as that described above, using a human-specific leptin ELISA kit (EK1972, Multisciences, Shanghai, China).

Techniques: Clinical Proteomics

Journal: Journal of Cancer

Article Title: Serum Adiponectin Level May be an Independent Predictor of Clear Cell Renal Cell Carcinoma

doi: 10.7150/jca.14716

Figure Lengend Snippet: Mann-Whitney U test of serum adiponectin level, Student's t test of VFA and serum leptin level in patients with ccRCC and non-ccRCC. Patients with ccRCC have a significantly lower plasma adiponectin level (p = 0.004) and higher VFA (p = 0.044) than non-ccRCC patients, while no differences are seen in mean plasma leptin level (p = 0.940).

Article Snippet: The detection of plasma leptin concentration was the same as that described above, using a human-specific leptin ELISA kit (EK1972, Multisciences, Shanghai, China).

Techniques: MANN-WHITNEY, Clinical Proteomics

Journal: Journal of Cancer

Article Title: Serum Adiponectin Level May be an Independent Predictor of Clear Cell Renal Cell Carcinoma

doi: 10.7150/jca.14716

Figure Lengend Snippet: Patient demographic and clinical characteristics.

Article Snippet: The detection of plasma leptin concentration was the same as that described above, using a human-specific leptin ELISA kit (EK1972, Multisciences, Shanghai, China).

Techniques:

Journal: eBioMedicine

Article Title: Exercise-derived peptide protects against pathological cardiac remodeling

doi: 10.1016/j.ebiom.2022.104164

Figure Lengend Snippet: The primers used in this study.

Article Snippet: The concentrations of CCDC80tide in EVs were measured by ELISA (Human CCDC80 ELISA Kit, EK1962, Boster Bio, USA) combined with immunoblotting assay.

Techniques:

Journal: eBioMedicine

Article Title: Exercise-derived peptide protects against pathological cardiac remodeling

doi: 10.1016/j.ebiom.2022.104164

Figure Lengend Snippet: CCDC80 is a potential exerkine involved in cardiovascular pathophysiology . a. Circos plot visualizes the overlap between upregulated gene lists from three databases, and the blue curves link genes that belong to the same enriched GO term. b. Pie charts represent the network of enriched terms in upregulated genes. Each circle node represents an enriched term and is clustered by its identity, where the size of a slice represents the percentage of genes under the term that originated from the corresponding database. c. Heatmap of enriched terms across the three upregulated gene lists, colored by p -values. d. Heatmap of enrichment analysis in DisGeNET, colored by p-values. e. Circos plot displays the overlap between upregulated gene lists, and the purple curves link identical genes. The five genes within the black box represent the genes with significant upregulation throughout the three databases. f. RT-PCR analysis of the expression levels of CCDC80 in various tissues of mice. g, h. After overexpression of PGC-1α in C2C12 cells, the expressions of Pgc-1α (g) and Ccdc80 (h) was determined by qPCR. i, j and k. Eight-week-old male mice were forced to swim 5 days per week for 3 months. Control groups mice were raised normally without swimming training. After complete swimming training, the skeletal muscles of mice were collected for western blot analysis of CCDC80 and PGC-1α ( n= 7). Data are presented as the average of four biological replicates (n = 4) ± SD. Statistical significance was determined using Student's t-test, *** p < 0.001.

Article Snippet: The concentrations of CCDC80tide in EVs were measured by ELISA (Human CCDC80 ELISA Kit, EK1962, Boster Bio, USA) combined with immunoblotting assay.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Over Expression, Control, Muscles, Western Blot

Journal: eBioMedicine

Article Title: Exercise-derived peptide protects against pathological cardiac remodeling

doi: 10.1016/j.ebiom.2022.104164

Figure Lengend Snippet: CCDC80 generates a peptide encapsulated in EVs in response to PGC-1α overexpression . a. The cell lysates and culture supernatants collected from vehicle- or Flag-PGC-1α-overexpressing C2C12 cells (treated or not with BFA, 1 μg/mL, 16 h) were prepared and analyzed for CCDC80 protein levels by WB. N= 3 b. The supernatants of CCDC80-EGFP-overexpressing C2C12 cells were harvested and filtered through a sterile 0.22-μm filter and then added to H9c2 cells. After 12-h incubation, the cell fluorescence was detected. The fluorescent spots indicated by white arrows were supposedly EVs. N= 3 c, d, e. The supernatants of CCDC80-overexpressing C2C12 cells were collected, and EVs were extracted for TEM (c), NTA (d), and EVs markers (e) determinations. N= 3. WCL: whole cell lysates. f. The cell lysates and EVs isolated from the supernatants of vehicle- or CCDC80-HA-overexpressing C2C12 cells were prepared and analyzed for CCDC80 protein levels by WB. g. The schematic diagram of CCDC80tide in CCDC80.

Article Snippet: The concentrations of CCDC80tide in EVs were measured by ELISA (Human CCDC80 ELISA Kit, EK1962, Boster Bio, USA) combined with immunoblotting assay.

Techniques: Over Expression, Sterility, Incubation, Fluorescence, Isolation

Journal: medRxiv

Article Title: RIPK1 Biallelic Activating Variants lead to Autoinflammatory Disease Driven by T cell Death

doi: 10.1101/2024.03.28.24304774

Figure Lengend Snippet: Cytokine array analysis of patient’s serum. Images of representative cytokines in group 1 ( a ), group 2 ( c ) and group 3 ( e-f ) clustered from cytokine array data according to were presented. Typical cytokines were re-validated with ELISA ( b, d ) and integrated relative MFI plots of group 3 cytokines were shown.

Article Snippet: Human or mouse serum cytokines were measured by enzyme-linked immunosorbent assay (ELISA) (Multi Sciences: 70-EK1280, 70-EK194, 70-EK182HS, 70-EK106HS, 70-EK191, 70-EK180HS, 70-EK1F01, 70-EK1F02, EK206HS, EK282HS/2) according to the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: medRxiv

Article Title: RIPK1 Biallelic Activating Variants lead to Autoinflammatory Disease Driven by T cell Death

doi: 10.1101/2024.03.28.24304774

Figure Lengend Snippet: Increased cell death and CD4/CD8 ratio in T cells caused by RIPK1 activation. a , Average expression values (arcsinh transformed) of cl. Casp-3 across identified peripheral immune cell lineages in PBMCs freshly isolated from four unaffected controls (C) and two patients (P, 1 sample from P1 and 2 samples from P2) by mass cytometry by time-of-flight (CyTOF) analysis. b-e, Representative contour plots of cl. Casp-3 ( b-c ) and histograms of total RIPK1, pRIPK1(S166) ( d-e ) levels in CD4 + T and CD8 + T cells in freshly isolated PBMCs from unaffected controls (n=10-12) and patients (n=4 for P1 and P2) (left panel). Summary histograms of cl. Casp-3 and pRIPK1(S166) (right panel) were shown. f, Immunoblotting analysis of primary CD3 + T cells isolated from two unaffected controls (C1 and C2) and P1 treated with T and S with or without Z, N for 6 hours. Antibodies as indicated were immunoblotted to show the activation of apoptosis or necroptosis. g, CellTiter-Glo luminescent cell viability detection of primary CD3 + T cells isolated from three unaffected controls (C1-C3), two patients (P1 and P2) and their parents treated with TS or TSZ for indicated times, n=4. h, Representative histogram and statistical analysis of surface CD69 expression on CD3 + T cells from unaffected controls (n=7) and two patients (n=3 for P1 and P2). i, Serum IFNγ, sCD25, sCD95 and CD95L of unaffected controls (C, n=10), two patients (P1, n=5-7; P2, n=4-8) and their parents (n=6-7) as well as two CRIA syndrome patients (n=6) determined by ELISA. j, CD4/CD8 ratio in T cells, percentages and absolute counts of CD3 + T, CD4 + T, CD8 + T cells in whole blood sample of P1 (red dots) and P2 (blue dots) measured using immunophenotyping. k, In vitro expansion of primary T cells isolated from unaffected controls (n=7) and patients (n=3 for P1 and P2) as indicated. Cell numbers were counted every two days post anti-CD3/CD28 dynabeads stimulation. l, Proliferation of CD4 + T and CD8 + T cells isolated from three unaffected controls (C1-C3) and P1 were analyzed using CFSE staining upon stimulation with anti-CD3/CD28 dynabeads for 96 hours. m, Representative histograms of surface CD95 expression on CD3 + T cells analyzed with flow cytometry after culturing in vitro for 24 hours. n, Representative contour plots depicting the percentages of CD4 + T, CD8 + T, double negative T (DNT) and double positive T (DPT) in CD3 + T cells gated from PBMCs of P2 culturing in vitro for 24 hours. Histograms displaying cl. Casp-3 and RIPK1 expression levels in CD8 hi T and CD8 lo T cells. o, Representative contour plots and summary histograms showing percentages of CD4 + T, CD8 + T and CD8 hi T cells and CD4/CD8 ratio in CD3 + T cells isolated from P2 cultured for indicated times. p-q, Representative contour plots showing percentages of CD4 + T, CD8 + T in CD3 + T cells of P2 with (TurboGFP + ) or without (TurboGFP - ) restoration using CRISPR/Cas9-mediated in situ knock-in of WT RIPK1-P2A-TurboGFP under stimulation of T with or without S, Z for 6 hours ( p ). CD4/CD8 ratio in CD3 + T cells of unaffected controls (n=7) and two patients (n=3 for P1 and P2) were quantified ( q ). r-s, Representative contour plots ( r ) and statistical analysis ( s ) showing percentages of CD4 + T, CD8 + T, DNT and DPT cell in CD3 + T cells isolated from unaffected control (n=7) and two patients (n=9 for P1 and P2) under the stimulation of anti-CD3 antibody (5 μg ml - ) for 72 hours. t, Statistical analysis of fold change of CD4 /CD8 ratio in CD3 + T cells isolated from two patients (n=4 for P1 and P2) under stimulation of anti-CD3 antibody (5 μg ml - ) with or without 250 nM Ponatinib or 10 μM Nec-1s for 24 hours. For representative contour plots, see Extended Data Fig. 6h. T denotes 10 ng ml - TNF; S denotes 250 nM SM164; Z denotes 25 μM z-VAD-fmk; N denotes 10 μM Nec-1s. All histogram graphs show mean±SD. Statistical significance was determined by one-way ANOVA ( g) or two-tailed unpaired t-test in the rest, * P <0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Human or mouse serum cytokines were measured by enzyme-linked immunosorbent assay (ELISA) (Multi Sciences: 70-EK1280, 70-EK194, 70-EK182HS, 70-EK106HS, 70-EK191, 70-EK180HS, 70-EK1F01, 70-EK1F02, EK206HS, EK282HS/2) according to the manufacturer’s instructions.

Techniques: Activation Assay, Expressing, Transformation Assay, Isolation, Mass Cytometry, Western Blot, Enzyme-linked Immunosorbent Assay, In Vitro, Staining, Flow Cytometry, Cell Culture, CRISPR, In Situ, Knock-In, Control, Two Tailed Test

Journal: medRxiv

Article Title: RIPK1 Biallelic Activating Variants lead to Autoinflammatory Disease Driven by T cell Death

doi: 10.1101/2024.03.28.24304774

Figure Lengend Snippet: RIPK1 activation causes inflammation through T-Mono axis. a , Schematic diagram of T cell-MDM co-culture system. b-c, qRT-PCR analysis of TNF, IL1B and IL6 mRNA levels in GM-CSF-activated monocytes derived macrophages (MDMs) co-cultured with or without T cells for 6 hours. GM-CSF-activated MDMs generated from unaffected control 1 (C1) (left panel) or unaffected control 3 (C3) (right panel) ( b ) or P1 ( c ) were co-cultured with donor-matched T cells as well as T cells from other unaffected controls (C2 and C4) or patients as indicated. d, Dot plot of IFNG, TNF in cell subtypes found in scRNA-seq of PBMCs from three unaffected controls and two patients. The size of the circle indicates the percentage of cells positive for gene expression and the color represents the average expression level. e, Representative histograms and statistical analysis of TNF and IFNγ expression in CD8 + T cells in PBMCs isolated from unaffected controls (n=6) and patients (n=3 for P1 and P2) treated with 10 ng ml - TNF (T) with or without 250 nM SM-164 (S), 25 μM z-VAD-fmk (Z) for 24 hours. f, T cells isolated from P2 were co-cultured with MDMs generated from unaffected control in the presence of 10 μg ml - adalimumab, 10 μg ml - emapalumab alone or in combination for 6 hours. TNF, IL1B and IL6 mRNA levels of MDMs were analyzed by qRT-PCR. g, Experimental timeline for generation of HSCs-transplanted mice with conditional overexpression of human WT or mutated RIPK1 K377E, R390G or D324V in CD8α + T cell- or myeloid cell-specific lineage. h-i, ELISA measurement of serum Tnf and Il6 levels in mice specifically overexpressing WT or mutated hRIPK1 in CD8α + T cells ( h ) or myeloid cells ( i ). j-m, Pedigrees of two families with CRIA patients (CRIA-1 and CRIA-2) ( j ). Patients were represented with filled symbols. Summary histograms of CD4/CD8 ratio in CD3 + T cells of unaffected controls (n=12), CRIA-1 (n=7) and CRIA-2 (n=5) were shown ( k). Representative contour plots of cl. Casp-3 ( l ) and histograms of pRIPK1(S166) ( m ) protein levels in CD8 + T cells in freshly isolated PBMCs from CRIA patients. n-o, Whole blood cell counts (WBC), cell counts and proportions of neutrophil (NE#-NE%) and lymphocyte (LYMPH# -LYMPH%) ( n) , Serum C-reactive protein (CRP), TNF and IL-6 ( o ) of P1 (red dots) and P2 (blue dots) measured before and after tocilizumab or adalimumab treatments. The grey area represents the range of unaffected controls. Dots represent each sample and graphs show mean±SD. Statistical significance was determined by two-tailed unpaired t-test, * P <0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Human or mouse serum cytokines were measured by enzyme-linked immunosorbent assay (ELISA) (Multi Sciences: 70-EK1280, 70-EK194, 70-EK182HS, 70-EK106HS, 70-EK191, 70-EK180HS, 70-EK1F01, 70-EK1F02, EK206HS, EK282HS/2) according to the manufacturer’s instructions.

Techniques: Activation Assay, Co-Culture Assay, Quantitative RT-PCR, Derivative Assay, Cell Culture, Generated, Control, Gene Expression, Expressing, Isolation, Over Expression, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: medRxiv

Article Title: RIPK1 Biallelic Activating Variants lead to Autoinflammatory Disease Driven by T cell Death

doi: 10.1101/2024.03.28.24304774

Figure Lengend Snippet: Efficiency of conditionally induced overexpression of RIPK1 in mice. a-c, loxp-GFP-stop-loxp-TagBFP2-P2A-WT/mutated human RIPK1 were lentivirally transduced into HSCs isolated from Lyz2 -CreERT2 ( a ) or Cd8a -CreERT2 donor mice ( c ). Histogram graphs showing expression levels of GFP in HSCs. Whole blood cells isolated from recipient mice with Lyz2 -CreERT2 ( b ) and Cd8a -CreERT2 ( d ) HSCs transplantation before and after tamoxifen induction. Histogram graphs showing expression levels of TagBFP2 in myeloid cells ( b ) and CD8α + T cells ( d ). e-f, ELISA measurement of serum Tnf and Il-6 levels in mice specifically overexpressing WT or mutated hRIPK1 in myeloid cells ( e ) or CD8α + T cells ( f ) with 0.5 mg/kg LPS challenge for 2 hours. Statistical significance was determined by two-tailed unpaired t-test.

Article Snippet: Human or mouse serum cytokines were measured by enzyme-linked immunosorbent assay (ELISA) (Multi Sciences: 70-EK1280, 70-EK194, 70-EK182HS, 70-EK106HS, 70-EK191, 70-EK180HS, 70-EK1F01, 70-EK1F02, EK206HS, EK282HS/2) according to the manufacturer’s instructions.

Techniques: Over Expression, Isolation, Expressing, Transplantation Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal:

Article Title: NAD-Dependent DNA-Binding Activity of the Bifunctional NadR Regulator of Salmonella typhimurium

doi:

Figure Lengend Snippet: Strains used

Article Snippet: EK193 , EK192(DE3) λ lysogen; lacUV5 controlled expression of T7 RNA polymerase , Novagen.

Techniques: Expressing, Transformation Assay

Journal: Nature Communications

Article Title: Single-cell transcriptomics analysis of bullous pemphigoid unveils immune-stromal crosstalk in type 2 inflammatory disease

doi: 10.1038/s41467-024-50283-3

Figure Lengend Snippet: a Volcano plot of gene features of the CCL19 + FB cluster in BP patients compared to HC. b Volcano plot of gene features of the APCDD1 + FB cluster in BP patients compared to HC. P -values in a and b were obtained using the two-sided Likelihood-ratio test and Bonferroni corrected (Seurat 4). c The feature plot and the bar chart showing PLA2G2A mRNA expression within total skin cells between BP ( n = 5) and HC ( n = 8). d Protein level of PLA2G2A in serum from BP ( n = 73) and HC ( n = 31) examined by ELISA. e Transwell assays were used to measure cell migration of THP1 ( n = 9) and Jurkat ( n = 9) cells treated by PLA2G2A recombinant protein. P -values in c – e were calculated using two-sided Mann–Whitney U-test. * P < 0.05, ** P < 0.01, **** P < 0.0001. In the box plot c – e : Minima: Lower limit of the whisker. Maxima: Upper limit of the whisker. Center: Median line inside the box. The upper and lower box bounds represent the 25% and 75% percentile of data. f Representative images of a BP patient and HC stained by multicolored IHC; green represents PLA2G2A + fibroblasts, red represents macrophages, and yellow represents CD3 T cells. scale bar = 50 μm. Data are from three independent experiments. g Circle plots of the inferred CXCL12 - CXCR4 pathway among major cell types in the BP and HC groups. h Hierarchical plot showing inferred intercellular communication network of CXCL12 - CXCR4 signaling in BP skin. i The PLA2G2A / CXCL12 gene pair co-expression (upper panel), and the gene expression correlation analysis in expressing both genes (lower panel) in fibroblasts. The correlation was measured using the Pearson correlation coefficient. P -values were calculated using two-sided Pearson correlation test. j Flow plots of CD3 + T cells from PBMCs showing the expression of CXCR4 treated by PLA2G2A recombinant protein (upper panel), and the frequency of CXCR4 (lower panel) in BP ( n = 21) and HC ( n = 13) groups. P -values were calculated using paired two-sided Student’s t -test. ** P < 0.01. Each sample is represented as one dot.

Article Snippet: The serum levels of IL-13 (EK0424), IL-4 (EK0404), IL-5 (EK0407), PLA2G2A (EK1944), and CCL17 (EK0684) in both patients and controls were quantified using ELISA Development Kits from BosterBio, USA, following the manufacturer’s instructions.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Migration, Recombinant, MANN-WHITNEY, Whisker Assay, Staining, Gene Expression

Journal: Nature Communications

Article Title: Single-cell transcriptomics analysis of bullous pemphigoid unveils immune-stromal crosstalk in type 2 inflammatory disease

doi: 10.1038/s41467-024-50283-3

Figure Lengend Snippet: a Hierarchical plot showing inferred intercellular communication network of CCL17 - CCR4 signaling. b The feature plot and the bar chart showing CCL17 mRNA expression within total skin cells between BP ( n = 5) and HC ( n = 8). c Protein level of CCL17 in serum from BP ( n = 73) and HC ( n = 32) by ELISA. d The feature plot and the bar chart showing CCR4 mRNA expression within total skin cells between BP ( n = 5) and HC ( n = 8). In the box plot b – d : Minima: Lower limit of the whisker. Maxima: Upper limit of the whisker. Center: Median line inside the box. The upper and lower box bounds represent the 25% and 75% percentile of data. e Flow plots of CD3 + T cells from PBMCs showing the expression of CCR4 treated by CCL17 recombinant protein (left panel), and the frequency of CCR4 (right panel) in BP ( n = 30) and HC ( n = 16) groups. f The positive correlation between the level of PLA2G2A and CCL17 in serum from in BP patients ( n = 73). P -value was calculated using two-sided Pearson correlation test. r -value was Pearson correlation coefficient. g The level of CCL17 in PBMC from BP patients ( n = 26) and HC ( n = 7) treated with PLA2G2A recombinant protein. h Effect of CCL17 treatment on the IL-13 secretion from BP patients ( n = 26) and HC ( n = 13). i Effect of PLA2G2A treatment on the IL-13 secretion from BP patients ( n = 20) and HC ( n = 13). j , k ELISA analysis of anti-BP180-NC16A antibody titers in supernatants of CCL17 ( j ) or PLA2G2A ( k ) stimulated PBMCs from BP patients ( n = 26) and HC ( n = 22). P -values in b–d were calculated using two-sided Mann–Whitney U-test. P -values in e and g – k were calculated using paired two-sided Student’s t -test. * P < 0.05, ** P < 0.01, **** P < 0.0001, only P -values < 0.05 are shown. Each sample is represented as one dot.

Article Snippet: The serum levels of IL-13 (EK0424), IL-4 (EK0404), IL-5 (EK0407), PLA2G2A (EK1944), and CCL17 (EK0684) in both patients and controls were quantified using ELISA Development Kits from BosterBio, USA, following the manufacturer’s instructions.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Whisker Assay, Recombinant, MANN-WHITNEY

Journal: Nature Communications

Article Title: Single-cell transcriptomics analysis of bullous pemphigoid unveils immune-stromal crosstalk in type 2 inflammatory disease

doi: 10.1038/s41467-024-50283-3

Figure Lengend Snippet: It illustrates a positive feedback loop where fibroblasts respond to Th2 cells through the IL13RA1 - IL13 pair, leading to increased secretion of PLA2G2A and CXCL12. This, in turn, amplifies the Th2-mediated response. (i) The lesional fibroblasts respond to IL-13 and induce the overexpression of PLA2G2A, which promotes the expression of CXCR4 on the surface of immune cells to recruit the immune cells from peripheral blood into skin lesions. (ii) Fibroblasts-derived PLA2G2A and myeloid cells-derived CCL17 elevate the secretion of IL-13, and fibroblast and myeloid cells further respond to IL-13, forming a positive feedback loop between immune cells and fibroblasts. (iii) IL-13 activates B cells to secrete autoantibodies. (iv) In blister, the secretion of autoantibodies recruits T cells, myeloid cells, mast cells, neutrophils and eosinophils. T cell-derived IL-13 activates eosinophils to secrete various cytokines (including IL-13), which further promotes Th2 polarization and mediates the crosstalk between immune cells.

Article Snippet: The serum levels of IL-13 (EK0424), IL-4 (EK0404), IL-5 (EK0407), PLA2G2A (EK1944), and CCL17 (EK0684) in both patients and controls were quantified using ELISA Development Kits from BosterBio, USA, following the manufacturer’s instructions.

Techniques: Over Expression, Expressing, Derivative Assay

Journal: medRxiv

Article Title: RIPK1 Biallelic Activating Variants lead to Autoinflammatory Disease Driven by T cell Death

doi: 10.1101/2024.03.28.24304774

Figure Lengend Snippet: Cytokine array analysis of patient’s serum. Images of representative cytokines in group 1 ( a ), group 2 ( c ) and group 3 ( e-f ) clustered from cytokine array data according to were presented. Typical cytokines were re-validated with ELISA ( b, d ) and integrated relative MFI plots of group 3 cytokines were shown.

Article Snippet: Human or mouse serum cytokines were measured by enzyme-linked immunosorbent assay (ELISA) (Multi Sciences: 70-EK1280, 70-EK194, 70-EK182HS, 70-EK106HS, 70-EK191, 70-EK180HS, 70-EK1F01, 70-EK1F02, EK206HS, EK282HS/2) according to the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: medRxiv

Article Title: RIPK1 Biallelic Activating Variants lead to Autoinflammatory Disease Driven by T cell Death

doi: 10.1101/2024.03.28.24304774

Figure Lengend Snippet: Increased cell death and CD4/CD8 ratio in T cells caused by RIPK1 activation. a , Average expression values (arcsinh transformed) of cl. Casp-3 across identified peripheral immune cell lineages in PBMCs freshly isolated from four unaffected controls (C) and two patients (P, 1 sample from P1 and 2 samples from P2) by mass cytometry by time-of-flight (CyTOF) analysis. b-e, Representative contour plots of cl. Casp-3 ( b-c ) and histograms of total RIPK1, pRIPK1(S166) ( d-e ) levels in CD4 + T and CD8 + T cells in freshly isolated PBMCs from unaffected controls (n=10-12) and patients (n=4 for P1 and P2) (left panel). Summary histograms of cl. Casp-3 and pRIPK1(S166) (right panel) were shown. f, Immunoblotting analysis of primary CD3 + T cells isolated from two unaffected controls (C1 and C2) and P1 treated with T and S with or without Z, N for 6 hours. Antibodies as indicated were immunoblotted to show the activation of apoptosis or necroptosis. g, CellTiter-Glo luminescent cell viability detection of primary CD3 + T cells isolated from three unaffected controls (C1-C3), two patients (P1 and P2) and their parents treated with TS or TSZ for indicated times, n=4. h, Representative histogram and statistical analysis of surface CD69 expression on CD3 + T cells from unaffected controls (n=7) and two patients (n=3 for P1 and P2). i, Serum IFNγ, sCD25, sCD95 and CD95L of unaffected controls (C, n=10), two patients (P1, n=5-7; P2, n=4-8) and their parents (n=6-7) as well as two CRIA syndrome patients (n=6) determined by ELISA. j, CD4/CD8 ratio in T cells, percentages and absolute counts of CD3 + T, CD4 + T, CD8 + T cells in whole blood sample of P1 (red dots) and P2 (blue dots) measured using immunophenotyping. k, In vitro expansion of primary T cells isolated from unaffected controls (n=7) and patients (n=3 for P1 and P2) as indicated. Cell numbers were counted every two days post anti-CD3/CD28 dynabeads stimulation. l, Proliferation of CD4 + T and CD8 + T cells isolated from three unaffected controls (C1-C3) and P1 were analyzed using CFSE staining upon stimulation with anti-CD3/CD28 dynabeads for 96 hours. m, Representative histograms of surface CD95 expression on CD3 + T cells analyzed with flow cytometry after culturing in vitro for 24 hours. n, Representative contour plots depicting the percentages of CD4 + T, CD8 + T, double negative T (DNT) and double positive T (DPT) in CD3 + T cells gated from PBMCs of P2 culturing in vitro for 24 hours. Histograms displaying cl. Casp-3 and RIPK1 expression levels in CD8 hi T and CD8 lo T cells. o, Representative contour plots and summary histograms showing percentages of CD4 + T, CD8 + T and CD8 hi T cells and CD4/CD8 ratio in CD3 + T cells isolated from P2 cultured for indicated times. p-q, Representative contour plots showing percentages of CD4 + T, CD8 + T in CD3 + T cells of P2 with (TurboGFP + ) or without (TurboGFP - ) restoration using CRISPR/Cas9-mediated in situ knock-in of WT RIPK1-P2A-TurboGFP under stimulation of T with or without S, Z for 6 hours ( p ). CD4/CD8 ratio in CD3 + T cells of unaffected controls (n=7) and two patients (n=3 for P1 and P2) were quantified ( q ). r-s, Representative contour plots ( r ) and statistical analysis ( s ) showing percentages of CD4 + T, CD8 + T, DNT and DPT cell in CD3 + T cells isolated from unaffected control (n=7) and two patients (n=9 for P1 and P2) under the stimulation of anti-CD3 antibody (5 μg ml - ) for 72 hours. t, Statistical analysis of fold change of CD4 /CD8 ratio in CD3 + T cells isolated from two patients (n=4 for P1 and P2) under stimulation of anti-CD3 antibody (5 μg ml - ) with or without 250 nM Ponatinib or 10 μM Nec-1s for 24 hours. For representative contour plots, see Extended Data Fig. 6h. T denotes 10 ng ml - TNF; S denotes 250 nM SM164; Z denotes 25 μM z-VAD-fmk; N denotes 10 μM Nec-1s. All histogram graphs show mean±SD. Statistical significance was determined by one-way ANOVA ( g) or two-tailed unpaired t-test in the rest, * P <0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Human or mouse serum cytokines were measured by enzyme-linked immunosorbent assay (ELISA) (Multi Sciences: 70-EK1280, 70-EK194, 70-EK182HS, 70-EK106HS, 70-EK191, 70-EK180HS, 70-EK1F01, 70-EK1F02, EK206HS, EK282HS/2) according to the manufacturer’s instructions.

Techniques: Activation Assay, Expressing, Transformation Assay, Isolation, Mass Cytometry, Western Blot, Enzyme-linked Immunosorbent Assay, In Vitro, Staining, Flow Cytometry, Cell Culture, CRISPR, In Situ, Knock-In, Control, Two Tailed Test

Journal: medRxiv

Article Title: RIPK1 Biallelic Activating Variants lead to Autoinflammatory Disease Driven by T cell Death

doi: 10.1101/2024.03.28.24304774

Figure Lengend Snippet: RIPK1 activation causes inflammation through T-Mono axis. a , Schematic diagram of T cell-MDM co-culture system. b-c, qRT-PCR analysis of TNF, IL1B and IL6 mRNA levels in GM-CSF-activated monocytes derived macrophages (MDMs) co-cultured with or without T cells for 6 hours. GM-CSF-activated MDMs generated from unaffected control 1 (C1) (left panel) or unaffected control 3 (C3) (right panel) ( b ) or P1 ( c ) were co-cultured with donor-matched T cells as well as T cells from other unaffected controls (C2 and C4) or patients as indicated. d, Dot plot of IFNG, TNF in cell subtypes found in scRNA-seq of PBMCs from three unaffected controls and two patients. The size of the circle indicates the percentage of cells positive for gene expression and the color represents the average expression level. e, Representative histograms and statistical analysis of TNF and IFNγ expression in CD8 + T cells in PBMCs isolated from unaffected controls (n=6) and patients (n=3 for P1 and P2) treated with 10 ng ml - TNF (T) with or without 250 nM SM-164 (S), 25 μM z-VAD-fmk (Z) for 24 hours. f, T cells isolated from P2 were co-cultured with MDMs generated from unaffected control in the presence of 10 μg ml - adalimumab, 10 μg ml - emapalumab alone or in combination for 6 hours. TNF, IL1B and IL6 mRNA levels of MDMs were analyzed by qRT-PCR. g, Experimental timeline for generation of HSCs-transplanted mice with conditional overexpression of human WT or mutated RIPK1 K377E, R390G or D324V in CD8α + T cell- or myeloid cell-specific lineage. h-i, ELISA measurement of serum Tnf and Il6 levels in mice specifically overexpressing WT or mutated hRIPK1 in CD8α + T cells ( h ) or myeloid cells ( i ). j-m, Pedigrees of two families with CRIA patients (CRIA-1 and CRIA-2) ( j ). Patients were represented with filled symbols. Summary histograms of CD4/CD8 ratio in CD3 + T cells of unaffected controls (n=12), CRIA-1 (n=7) and CRIA-2 (n=5) were shown ( k). Representative contour plots of cl. Casp-3 ( l ) and histograms of pRIPK1(S166) ( m ) protein levels in CD8 + T cells in freshly isolated PBMCs from CRIA patients. n-o, Whole blood cell counts (WBC), cell counts and proportions of neutrophil (NE#-NE%) and lymphocyte (LYMPH# -LYMPH%) ( n) , Serum C-reactive protein (CRP), TNF and IL-6 ( o ) of P1 (red dots) and P2 (blue dots) measured before and after tocilizumab or adalimumab treatments. The grey area represents the range of unaffected controls. Dots represent each sample and graphs show mean±SD. Statistical significance was determined by two-tailed unpaired t-test, * P <0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Human or mouse serum cytokines were measured by enzyme-linked immunosorbent assay (ELISA) (Multi Sciences: 70-EK1280, 70-EK194, 70-EK182HS, 70-EK106HS, 70-EK191, 70-EK180HS, 70-EK1F01, 70-EK1F02, EK206HS, EK282HS/2) according to the manufacturer’s instructions.

Techniques: Activation Assay, Co-Culture Assay, Quantitative RT-PCR, Derivative Assay, Cell Culture, Generated, Control, Expressing, Isolation, Over Expression, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: medRxiv

Article Title: RIPK1 Biallelic Activating Variants lead to Autoinflammatory Disease Driven by T cell Death

doi: 10.1101/2024.03.28.24304774

Figure Lengend Snippet: Efficiency of conditionally induced overexpression of RIPK1 in mice. a-c, loxp-GFP-stop-loxp-TagBFP2-P2A-WT/mutated human RIPK1 were lentivirally transduced into HSCs isolated from Lyz2 -CreERT2 ( a ) or Cd8a -CreERT2 donor mice ( c ). Histogram graphs showing expression levels of GFP in HSCs. Whole blood cells isolated from recipient mice with Lyz2 -CreERT2 ( b ) and Cd8a -CreERT2 ( d ) HSCs transplantation before and after tamoxifen induction. Histogram graphs showing expression levels of TagBFP2 in myeloid cells ( b ) and CD8α + T cells ( d ). e-f, ELISA measurement of serum Tnf and Il-6 levels in mice specifically overexpressing WT or mutated hRIPK1 in myeloid cells ( e ) or CD8α + T cells ( f ) with 0.5 mg/kg LPS challenge for 2 hours. Statistical significance was determined by two-tailed unpaired t-test.

Article Snippet: Human or mouse serum cytokines were measured by enzyme-linked immunosorbent assay (ELISA) (Multi Sciences: 70-EK1280, 70-EK194, 70-EK182HS, 70-EK106HS, 70-EK191, 70-EK180HS, 70-EK1F01, 70-EK1F02, EK206HS, EK282HS/2) according to the manufacturer’s instructions.

Techniques: Over Expression, Isolation, Expressing, Transplantation Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal:

Article Title: NAD-Dependent DNA-Binding Activity of the Bifunctional NadR Regulator of Salmonella typhimurium

doi:

Figure Lengend Snippet: Strains used

Article Snippet: EK194 , EK193/pLysS; T7 lysozyme under the control of the φ T7 3.8 promoter , Novagen.

Techniques: Expressing, Transformation Assay