Journal: International Journal of Molecular Sciences

Article Title: Circulatory Neutrophils Exhibit Enhanced Neutrophil Extracellular Trap Formation in Early Puerperium: NETs at the Nexus of Thrombosis and Immunity

doi: 10.3390/ijms222413646

Figure Lengend Snippet: Markers of NETosis and neutrophil degranulation are substantially increased 48 h post-delivery ( a ) Cell-free DNA levels in serum from donors during pregnancy ( n = 12), 48 h postpartum ( n = 9) and healthy matched blood donors ( n = 11), determined by GAPDH real-time PCR (left panel) and dsDNA-binding fluorescent dye quantification (right panel). ( b ) Cell-free nucleosome levels in serum from healthy donor controls, donors during gestation and postpartum, determined by ELISA and NET-associated MPO/DNA complexes quantified utilizing a modified capture ELISA. ( c ) Neutrophil and peripheral blood mononuclear differential cell counts in healthy non-pregnant blood donors, donors during pregnancy and 48 h postpartum. P: pregnancy; PD: post-delivery. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The concentrations of thrombin-antithrombin (TAT) complexes and D-dimers were measured by sandwich ELISA, utilizing the human TAT Complexes ELISA Kit (Assaypro, St. Charles, MO, USA) and the Imuclone D-Dimer ELISA Kit (American Diagnostica, Pfungstadt, Germany) respectively.

Techniques: Real-time Polymerase Chain Reaction, Binding Assay, Enzyme-linked Immunosorbent Assay, Modification

Journal: International Journal of Molecular Sciences

Article Title: Circulatory Neutrophils Exhibit Enhanced Neutrophil Extracellular Trap Formation in Early Puerperium: NETs at the Nexus of Thrombosis and Immunity

doi: 10.3390/ijms222413646

Figure Lengend Snippet: Excessive NET formation by postpartum circulating neutrophils is complemented by activation of the coagulation cascade and alterations in the platelet activity status. ( a ) Immunohistochemical staining for TF (green), and DNA counterstain with DAPI (blue) after a 3 h in vitro culture. Magnification: 20×; Scale bars: 50 μm. ( b ) Platelet differential counts in healthy non-pregnant blood donors, donors during pregnancy and 48 h postpartum. ( c ) Comparative analysis between healthy donor controls, donors during gestation and postpartum, concerning the platelet morphology and activation indices mean platelet volume (MPV) and platelet distribution width (PDW). ( d ) TAT complexes concentration in citrate plasma from healthy donor controls, donors during pregnancy and postpartum, determined by ELISA. ( e ) Spearman correlation between TAT complexes concentration and NET-associated MPO/DNA complexes. ( f ) D-dimer levels in citrate plasma from healthy donor controls, donors during gestation and postpartum, determined by ELISA. P: pregnancy; PD: post-delivery. Data are presented as mean ± SEM. * p < 0.05, ** p < 0.01 (one way ANOVA followed by Bonferroni’s multiple comparison post-test). All experiments were performed at least 6 times with consistent results.

Article Snippet: The concentrations of thrombin-antithrombin (TAT) complexes and D-dimers were measured by sandwich ELISA, utilizing the human TAT Complexes ELISA Kit (Assaypro, St. Charles, MO, USA) and the Imuclone D-Dimer ELISA Kit (American Diagnostica, Pfungstadt, Germany) respectively.

Techniques: Activation Assay, Coagulation, Activity Assay, Immunohistochemical staining, Staining, In Vitro, Concentration Assay, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Neural regeneration research

Article Title: Thrombin increases the expression of cholesterol 25-hydroxylase in rat astrocytes after spinal cord injury.

doi: 10.4103/1673-5374.357905

Figure Lengend Snippet: Figure 1 ｜Quantitative changes in thrombin production and cholesterol hydroxylase expression at the lesion site following spinal cord injury (SCI) in a rat model. (A) Experimental design. Experiment I was designed to investigate the effects of the protease-activated receptor 1 (PAR1) inhibitor SCH79797 on thrombin- induced cholesterol-25-hydroxylase (CH25H) expression by astrocytes. For quantitative polymerase chain reaction, enzyme-linked immunosorbent assay, tissue immunofluorescence, and hematoxylin-eosin staining, 1 cm spinal cord segments were harvested from rats at each time point. Experiment II was designed to investigate the effects of rat motor function after SCH79797 treatment. (B) Enzyme-linked immunosorbent assay of thrombin production at 0, 1, 4, and 7 days following SCI. Day 0 was used as the control. (C–E) RNA levels of ch25h, cyp46a1, and cyp27a1 at the lesion site were determined by quantitative polymerase chain reaction at different time points with or without injection of 4.5 μL of 5 mM SCH79797. Quantities were normalized to gapdh levels on day 0. Data are expressed as mean ± SEM (n = 3). *P < 0.05 (independent sample t-test). DMSO: Dimethyl sulfoxide; gapdh: glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: After centrifuging at 2750 × g for 5–10 minutes at 2–8°C, the supernatant was subjected to thrombin enzyme-linked immunosorbent assay (ELISA) using a kit (Rat TAT ELISA Kit, Elabscience) according to the manufacturer’s directions.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Control, Injection

Journal: Cell Death & Disease

Article Title: YOD1 protects against MRSA sepsis-induced DIC through Lys33-linked deubiquitination of NLRP3

doi: 10.1038/s41419-024-06731-5

Figure Lengend Snippet: A , B Immunoblot analysis of supernatants (SN) or cell lysates (CL) of BMDMs from WT and Yod1 −/− mice, then treated with PBS or MRSA for 4 h. NLRP3, p20 and p17 expression levels were quantitated by measuring band intensities using “ImageJ” software. The values were normalized to β-actin. C , D Representative images of ASC specks in MRSA-infected WT and Yod1 −/− BMDMs. ASC, green; nuclei, blue. Scale bar, 50 μm. The percentage of cells containing an ASC speck was quantified. E – G ELISA of IL-1β ( E ), IL-6 ( F ), and TNF-α ( G ) concentration in supernatants of BMDMs from WT and Yod1 −/− mice after MRSA infection. H , I Immunoblot analysis of liver tissue from WT and Yod1 −/− mice. NLRP3 expression levels were quantitated by measuring band intensities using “ImageJ” software. The values were normalized to β-actin. J ELISA analysis of plasma levels of IL-1β from WT and Yod1 −/− mice after MRSA injection. Data are presented as mean ± SD. ** p < 0.01, *** p < 0.001, **** p < 0.0001, NS means no significance.

Article Snippet: Mouse D-Dimer ELISA Kit (E-EL-M0400c), Mouse thrombin-antithrombin (TAT) ELISA Kit (E-EL-M1138c), Mouse interleukin 1 Beta (IL-1β) ELISA Kit (E-EL-M0037c), Mouse plasminogen activator inhibitor 1 (PAI-1) ELISA Kit (E-EL-M3041) were purchased from Elabscience (Wuhan, China).

Techniques: Western Blot, Expressing, Software, Infection, Enzyme-linked Immunosorbent Assay, Concentration Assay, Clinical Proteomics, Injection

Journal: Small (Weinheim an Der Bergstrasse, Germany)

Article Title: Multifunctional Co‐Delivery Systems with Downregulation of the Novel Target PIM1 in Macrophages to Ameliorate TF‐Mediated Coagulopathy in Sepsis

doi: 10.1002/smll.202412688

Figure Lengend Snippet: PIM1 inhibitor SMI‐4a protects septic mice against coagulation activation and sepsis‐induced acute lung injury. A) Schematic representation of animal experimental procedures. B) Representative western blot membranes and corresponding densitometric analyses of (C) PIM1 in lung tissue (n = 6/group). D) mRNA levels of PIM1 in murine lung tissues (n = 5/group). E) Platelet count in mice (n = 4/group). F–H) Plasma levels of coagulation‐related factors in mice plasma by ELISA, including (F) TAT, (G) D‐dimer, and (H) fibrinogen (n = 4/group). I) The lung sections were subjected to hematoxylin and eosin (HE) staining, F4/80, fibrin, and PIM1 immunohistochemical analysis (n = 4/group; scale bar:50 µm). J) Representative western blot membranes and corresponding densitometric analyses of (K) TF, (L) PAI‐1, (M) Thrombin in lung tissue (n = 6/group). N) mRNA levels of TF in murine lung tissues (n = 5/group). O–Q) mRNA levels of (O) IL‐1β, (P) IL‐6, and (Q) TNF‐ɑ in murine lung tissues (n = 5/group). R) Survival curves of mice in all groups (n = 10/group). Each bar represents the mean ± SD. Statistical analysis for three or more groups was carried out using one‐way ANOVA (C‐H and K‐Q). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: The concentrations of PIM1 (CSB‐ E11825 h, Cusabio, China) in human plasma and TAT (CSB‐ E08433 m, Cusabio, China), Fbg (CSB‐ E08202 m, Cusabio, China), and D2D (CSB‐ E13584 m, Cusabio, China) in mouse plasma were assessed using ELISA kits according to the guidelines outlined in the respective ELISA kits.

Techniques: Coagulation, Activation Assay, Western Blot, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Staining, Immunohistochemical staining