Journal: Indian Journal of Clinical Biochemistry

Article Title: Evaluation of C4b as an adjunct marker in symptomatic RT-PCR negative Covid-19 cases

doi: 10.1007/s12291-022-01033-z

Figure Lengend Snippet: Evaluation of plasma levels of (A) C5b-9 and (B) C1-INH. (A) Mean comparison of plasma C5b-9 levels (ng/ml) between Covid19 cases (1) and controls (0): Mean C5b-9 levels with 95% CI in cases and controls were 257.90 (223.47–292.32) and 235.10 (205.23–264.95) respectively, and were not significantly different (t-stat = 1.01; p -value = 0.3144). (B) Mean comparison of plasma C1-INH levels (ng/ml) between Covid19 cases (1) and controls (0): Mean C1-INH levels with 95% CI in cases and controls were 510.09 (383.94–636.24) and 1124.20 (959.10 − 1289.30) respectively, and were significantly lower in cases (t-stat=-5.97; p - value = 0.0001)

Article Snippet: Levels of C4b, C5b-9 and C1-INH were evaluated by ELISA kits (Bioassay Technology, Shanghai Korain Biotech Co) according to manufacturer’s guidelines with some minor modifications.

Techniques: Comparison

Journal: Journal of Neuroinflammation

Article Title: β-arrestin1 regulates astrocytic reactivity via Drp1-dependent mitochondrial fission: implications in postoperative delirium

doi: 10.1186/s12974-023-02794-x

Figure Lengend Snippet: Orthopedic surgery induces glial activation and pro-inflammatory phenotypes in the hippocampus. A Mice were established orthopedic surgery-induced POD model. Levels of pro-inflammatory cytokines in the hippocampus were analyzed by ELISA. B Levels of anti-inflammatory and neurotrophic cytokines in the hippocampus were analyzed by ELISA. C mRNA levels of pro-inflammatory and anti-inflammatory genes as well as neurotrophic genes in the hippocampus were analyzed by RT-PCR. D Immunohistochemical staining of Iba-1 in the hippocampus of CON and POD mice. E Immunohistochemical staining of GFAP in the hippocampus of CON and POD mice. F Analysis of Iba-1-positive area and cell numbers in the hippocampus. G Analysis of GFAP-positive area and cell numbers in the hippocampus. H Expression of GFAP and Iba-1 in the hippocampus of CON and POD mice. I Densitometric analysis of GFAP and Iba-1. J Heatmap of neurotoxic astrocytes transcripts in the hippocampus of CON and POD mice. K Heatmap of neuroprotective astrocytes transcripts in the hippocampus of CON and POD mice. L Expression of C3, Serping1 and Psmb8 in the hippocampus of CON and POD mice. M Densitometric analysis of C3, Serping1 and Psmb8. N Double immunofluorescent staining of astrocytic pan-active marker GFAP and neurotoxic astrocytic marker C3 in hippocampus of CON and POD mice. O Relative co-localized signals of the GFAP-positive and C3-positive immunofluorescent particles between CON and POD group. Data are analyzed by unpaired Student’s t-test. * P < 0.05, ** P < 0.01 and *** P < 0.001 vs . the CON group. Values are presented as means ± SEM from at least three independent experiments

Article Snippet: After blocking with 10% nonfat dry milk in Tris-buffered saline (20 mM Tris–HCl, 500 mM NaCl, pH 7.4) with Tween 20 (Aladdin, #T104863), the membranes were then probed with the following primary antibodies overnight at 4 °C: mouse anti-GFAP antibody (1:1000, Cell Signaling Technology, #3670), rabbit anti-Iba-1 antibody (1:1000, Wako, #019-19741), rabbit anti-β-arrestin1 (1:1000, Cell Signaling Technologies, #12697), rabbit anti-β-arrestin2 (1:200, Cell Signaling Technologies, #3857), rabbit anti-C3 antibody (1:1000, abcam, #ab11887), rabbit anti-Serping1 antibody (1:1000, proteintech, #12259-1-AP), rabbit anti-Psmb8 antibody (1:1000, proteintech, #14859-1-AP), mouse anti-Fis1 antibody (1:1000, Santa Cruz, #sc-376447), mouse anti-Drp1 antibody (1:1000, Santa Cruz, #sc-101270), rabbit anti-Drp1 antibody (1:1000, proteintech, #12957-1-AP), rabbit anti-COX IV antibody (1:1000, Cell Signaling Technology, #4850), mouse anti-β-actin antibody (1:3000, sigma, #a1978).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction, Immunohistochemical staining, Staining, Expressing, Marker

Journal: Journal of Neuroinflammation

Article Title: β-arrestin1 regulates astrocytic reactivity via Drp1-dependent mitochondrial fission: implications in postoperative delirium

doi: 10.1186/s12974-023-02794-x

Figure Lengend Snippet: β-arrestin1 in astrocytes modulates cognitive impairments and astrocytic reactivity in the mouse model for POD. A β-arrestin1 protein levels in brain lysate of WT and β-arrestin1 −/− mice. B Experimental protocol and timeline of the POD mouse model. C Representative moving track plots (red curve) of mice in the second trial of Y-maze test. Blue box represents the novel arm. D Time (%) spent in the novel arm in the Y-maze test. E Bouts of novel arm entry in the Y-maze test. F Representative moving track plots (red curve) of mice in the probe trial of Morris water maze test. Black circle represents the invisible platform. G Latency (s) to reach the hidden platform in the probe test of Morris water maze test. H Crossing times in target quadrant in the probe test of Morris water maze test. I Representative immunofluorescent staining of GFAP in the hippocampus. J Analysis of GFAP-positive cell body area in the hippocampus. K Analysis of GFAP-positive cell numbers in the hippocampus. L Heatmap of the expression level of the neurotoxic astrocytes-specific transcripts in the hippocampus. M Expression of C3, Serping1 and Psmb8 in the hippocampus. N Densitometric analysis of C3, Serping1 and Psmb8. O Schematic diagram of the mice model with micro-injection of AAV-siβ-arrestin1 into the hippocampus. P Immunofluorescent co-localization of GFP and GFAP (red) after the AAV micro-injection. Q Representative immunofluorescent staining of GFAP (red) in the hippocampus. R Analysis of astrocytic reactivity by GFAP-positive cell body area and GFAP-positive cell numbers in the hippocampus. S Bouts of novel arm entry in the Y-maze test. T Time (%) spent in the novel arm in the Y-maze test. U Latency (s) to reach the hidden platform in the probe test of Morris water maze test. V Crossing times in target quadrant in the probe test of Morris water maze test. For C-M except for R, data were analyzed by two-way ANOVA followed by Tukey's multiple comparisons test. * P < 0.05, ** P < 0.01 and *** P < 0.001 vs. the WT-CON group or NC AAV CON group. # P < 0.05, ## P < 0.01 and ### P < 0.001 vs. the WT-POD mice or NC AAV POD. For R, data are analyzed by unpaired Student’s t-test. * P < 0.05 and *** P < 0.001 vs. the NC AAV POD group. n = 6 mice per group for immunofluorescent staining. n = 3 for western blotting. n = 7–10 mice for behavioral tests. Values are presented as means ± SEM

Article Snippet: After blocking with 10% nonfat dry milk in Tris-buffered saline (20 mM Tris–HCl, 500 mM NaCl, pH 7.4) with Tween 20 (Aladdin, #T104863), the membranes were then probed with the following primary antibodies overnight at 4 °C: mouse anti-GFAP antibody (1:1000, Cell Signaling Technology, #3670), rabbit anti-Iba-1 antibody (1:1000, Wako, #019-19741), rabbit anti-β-arrestin1 (1:1000, Cell Signaling Technologies, #12697), rabbit anti-β-arrestin2 (1:200, Cell Signaling Technologies, #3857), rabbit anti-C3 antibody (1:1000, abcam, #ab11887), rabbit anti-Serping1 antibody (1:1000, proteintech, #12259-1-AP), rabbit anti-Psmb8 antibody (1:1000, proteintech, #14859-1-AP), mouse anti-Fis1 antibody (1:1000, Santa Cruz, #sc-376447), mouse anti-Drp1 antibody (1:1000, Santa Cruz, #sc-101270), rabbit anti-Drp1 antibody (1:1000, proteintech, #12957-1-AP), rabbit anti-COX IV antibody (1:1000, Cell Signaling Technology, #4850), mouse anti-β-actin antibody (1:3000, sigma, #a1978).

Techniques: Staining, Expressing, Microinjection, Western Blot

Journal: Journal of Neuroinflammation

Article Title: β-arrestin1 regulates astrocytic reactivity via Drp1-dependent mitochondrial fission: implications in postoperative delirium

doi: 10.1186/s12974-023-02794-x

Figure Lengend Snippet: β-arrestin1 deletion aggravates the neurotoxic reactivity of primary astrocytes. A Schematic of the experimental design. B Expression of C3, Serping1 and Psmb8 in the primary astrocytes. C Densitometric analysis of C3, Serping1 and Psmb8. D Heat map of A1 astrocytic genes in primary cell cultures. E Immunofluorescent staining of C3 (green) and GFAP (red) in primary astrocytes. F Immunofluorescent staining of Serping1 (red) and GFAP (green) in primary astrocytes. G Relative co-localized signals of the GFAP-positive and C3-positive immunofluorescent particles between groups. H Relative co-localized signals of the GFAP-positive and Serping1-positive immunofluorescent particles between groups. I Astrocytes were stained with MitoSOX and analyzed by flow cytometry. J JC-1 staining in astrocytes were analyzed by flow cytometry. K Quantification of the mitochondrial ROS in MitoSOX staining. L Quantification of the loss of mitochondrial membrane potential in JC-1 staining measured by flow cytometry. M Oxygen consumption rates were evaluated by Seahorse. N Quantification of oxygen consumption for ATP production, basal respiration and proton leak. O ATP levels in astrocytes. Data were analyzed by two-way ANOVA followed by Tukey's multiple comparisons test. * P < 0.05, ** P < 0.01 and *** P < 0.001 vs. the WT CON-MCM group. # P < 0.05, ## P < 0.01 and ### P < 0.001 vs. the WT LPS-MCM group. Values are presented as means ± SEM from at least three independent experiments

Article Snippet: After blocking with 10% nonfat dry milk in Tris-buffered saline (20 mM Tris–HCl, 500 mM NaCl, pH 7.4) with Tween 20 (Aladdin, #T104863), the membranes were then probed with the following primary antibodies overnight at 4 °C: mouse anti-GFAP antibody (1:1000, Cell Signaling Technology, #3670), rabbit anti-Iba-1 antibody (1:1000, Wako, #019-19741), rabbit anti-β-arrestin1 (1:1000, Cell Signaling Technologies, #12697), rabbit anti-β-arrestin2 (1:200, Cell Signaling Technologies, #3857), rabbit anti-C3 antibody (1:1000, abcam, #ab11887), rabbit anti-Serping1 antibody (1:1000, proteintech, #12259-1-AP), rabbit anti-Psmb8 antibody (1:1000, proteintech, #14859-1-AP), mouse anti-Fis1 antibody (1:1000, Santa Cruz, #sc-376447), mouse anti-Drp1 antibody (1:1000, Santa Cruz, #sc-101270), rabbit anti-Drp1 antibody (1:1000, proteintech, #12957-1-AP), rabbit anti-COX IV antibody (1:1000, Cell Signaling Technology, #4850), mouse anti-β-actin antibody (1:3000, sigma, #a1978).

Techniques: Expressing, Staining, Flow Cytometry, Membrane

Journal: Journal of Neuroinflammation

Article Title: β-arrestin1 regulates astrocytic reactivity via Drp1-dependent mitochondrial fission: implications in postoperative delirium

doi: 10.1186/s12974-023-02794-x

Figure Lengend Snippet: β-arrestin1-biased ligand Carvedilol recovers the neurotoxic astrocytes reactivity. A Heat map of the expression levels of the signature genes for neurotoxic astrocytes in primary cell cultures. B Expression of C3, Serping1 and Psmb8 in primary cell cultures. C Densitometric analysis of C3, Serping1 and Psmb8. D Immunofluorescent staining of GFAP (red) and C3 (green) in primary astrocytes. E Immunofluorescent staining of GFAP (green) and Serping1 (red) in primary astrocytes. F Relative co-localized signals of the GFAP-positive and C3-positive immunofluorescent particles between groups. G Relative co-localized signals of the GFAP-positive and Serping1-positive immunofluorescent particles between groups. H Astrocytes were stained with MitoSOX and analyzed by flow cytometry. I JC-1 staining in astrocytes was analyzed by flow cytometry. J Quantification of the mitochondrial ROS in MitoSOX staining. K Quantification of the loss of mitochondrial membrane potential in JC-1 staining measured by flow cytometry. L Oxygen consumption rates were evaluated by Seahorse. M Quantification of oxygen consumption for ATP production, basal respiration and proton leak. N ATP levels in astrocytes. Data were analyzed by one-way ANOVA followed by Dunnet’s post-hoc test. * P < 0.05, ** P < 0.01 and *** P < 0.001 vs. the CON group. # P < 0.05, ## P < 0.01 and ### P < 0.01 vs. the LPS-MCM group. Values are presented as means ± SEM from at least three independent experiments

Article Snippet: After blocking with 10% nonfat dry milk in Tris-buffered saline (20 mM Tris–HCl, 500 mM NaCl, pH 7.4) with Tween 20 (Aladdin, #T104863), the membranes were then probed with the following primary antibodies overnight at 4 °C: mouse anti-GFAP antibody (1:1000, Cell Signaling Technology, #3670), rabbit anti-Iba-1 antibody (1:1000, Wako, #019-19741), rabbit anti-β-arrestin1 (1:1000, Cell Signaling Technologies, #12697), rabbit anti-β-arrestin2 (1:200, Cell Signaling Technologies, #3857), rabbit anti-C3 antibody (1:1000, abcam, #ab11887), rabbit anti-Serping1 antibody (1:1000, proteintech, #12259-1-AP), rabbit anti-Psmb8 antibody (1:1000, proteintech, #14859-1-AP), mouse anti-Fis1 antibody (1:1000, Santa Cruz, #sc-376447), mouse anti-Drp1 antibody (1:1000, Santa Cruz, #sc-101270), rabbit anti-Drp1 antibody (1:1000, proteintech, #12957-1-AP), rabbit anti-COX IV antibody (1:1000, Cell Signaling Technology, #4850), mouse anti-β-actin antibody (1:3000, sigma, #a1978).

Techniques: Expressing, Staining, Flow Cytometry, Membrane

Journal: Journal of Neuroinflammation

Article Title: β-arrestin1 regulates astrocytic reactivity via Drp1-dependent mitochondrial fission: implications in postoperative delirium

doi: 10.1186/s12974-023-02794-x

Figure Lengend Snippet: β-arrestin1-biased ligand Carvedilol protects POD mice from pro-inflammatory phenotypes. A Experimental protocol and timeline of the mouse model. B Representative immunofluorescent staining of GFAP in the hippocampus. C Relative GFAP-positive cell body area in the hippocampus. D Relative GFAP-positive cell numbers in the hippocampus. E Heatmap of the expression level of the A1-specific transcripts in hippocampal samples. F Expression of C3, Serping1 and Psmb8 in the hippocampus. G Densitometric analysis of C3, Serping1 and Psmb8. H Representative moving track plots (red curve) of mice in the second trial of Y-maze test. Blue box represents the novel arm. I Time (%) spent in the novel arm in the Y-maze test. J Bouts of novel arm entry in the Y-maze test. K Representative moving track plots (red curve) of mice in the probe trial of Morris water maze test. Black circle represents the invisible platform. L Latency (s) to reach the hidden platform in the probe test of Morris water maze test. M Crossing times in target quadrant in the probe test of Morris water maze test. Data were analyzed by one-way ANOVA followed by Dunnet’s post-hoc test. * P < 0.05, ** P < 0.01 and *** P < 0.001 vs. the CON group. # P < 0.05, ## P < 0.01 and ### P < 0.01 vs. the POD group. n = 6 mice per group for immunofluorescent staining. n = 3 for western blotting. n = 10 mice for behavioral tests. Values are presented as means ± SEM

Article Snippet: After blocking with 10% nonfat dry milk in Tris-buffered saline (20 mM Tris–HCl, 500 mM NaCl, pH 7.4) with Tween 20 (Aladdin, #T104863), the membranes were then probed with the following primary antibodies overnight at 4 °C: mouse anti-GFAP antibody (1:1000, Cell Signaling Technology, #3670), rabbit anti-Iba-1 antibody (1:1000, Wako, #019-19741), rabbit anti-β-arrestin1 (1:1000, Cell Signaling Technologies, #12697), rabbit anti-β-arrestin2 (1:200, Cell Signaling Technologies, #3857), rabbit anti-C3 antibody (1:1000, abcam, #ab11887), rabbit anti-Serping1 antibody (1:1000, proteintech, #12259-1-AP), rabbit anti-Psmb8 antibody (1:1000, proteintech, #14859-1-AP), mouse anti-Fis1 antibody (1:1000, Santa Cruz, #sc-376447), mouse anti-Drp1 antibody (1:1000, Santa Cruz, #sc-101270), rabbit anti-Drp1 antibody (1:1000, proteintech, #12957-1-AP), rabbit anti-COX IV antibody (1:1000, Cell Signaling Technology, #4850), mouse anti-β-actin antibody (1:3000, sigma, #a1978).

Techniques: Staining, Expressing, Western Blot

Journal: Journal of Neuroinflammation

Article Title: β-arrestin1 regulates astrocytic reactivity via Drp1-dependent mitochondrial fission: implications in postoperative delirium

doi: 10.1186/s12974-023-02794-x

Figure Lengend Snippet:

Article Snippet: After blocking with 10% nonfat dry milk in Tris-buffered saline (20 mM Tris–HCl, 500 mM NaCl, pH 7.4) with Tween 20 (Aladdin, #T104863), the membranes were then probed with the following primary antibodies overnight at 4 °C: mouse anti-GFAP antibody (1:1000, Cell Signaling Technology, #3670), rabbit anti-Iba-1 antibody (1:1000, Wako, #019-19741), rabbit anti-β-arrestin1 (1:1000, Cell Signaling Technologies, #12697), rabbit anti-β-arrestin2 (1:200, Cell Signaling Technologies, #3857), rabbit anti-C3 antibody (1:1000, abcam, #ab11887), rabbit anti-Serping1 antibody (1:1000, proteintech, #12259-1-AP), rabbit anti-Psmb8 antibody (1:1000, proteintech, #14859-1-AP), mouse anti-Fis1 antibody (1:1000, Santa Cruz, #sc-376447), mouse anti-Drp1 antibody (1:1000, Santa Cruz, #sc-101270), rabbit anti-Drp1 antibody (1:1000, proteintech, #12957-1-AP), rabbit anti-COX IV antibody (1:1000, Cell Signaling Technology, #4850), mouse anti-β-actin antibody (1:3000, sigma, #a1978).

Techniques:

Journal: F1000Research

Article Title: Recent advances in (therapeutic protein) drug development

doi: 10.12688/f1000research.9970.1

Figure Lengend Snippet: Protein-engineering platform technologies.

Article Snippet: Production of proteins in transgenic animals , C1 esterase inhibitor (Ruconest) produced in transgenic rabbit milk .

Techniques: Drug discovery, Transgenic Assay, Produced, Recombinant

Journal: F1000Research

Article Title: Recent advances in (therapeutic protein) drug development

doi: 10.12688/f1000research.9970.1

Figure Lengend Snippet: U.S. Food and Drug Administration-approved protein therapeutics (2011–2016).

Article Snippet: Production of proteins in transgenic animals , C1 esterase inhibitor (Ruconest) produced in transgenic rabbit milk .

Techniques: Injection, Coagulation, Recombinant, Clinical Proteomics

Journal: F1000Research

Article Title: Recent advances in (therapeutic protein) drug development

doi: 10.12688/f1000research.9970.1

Figure Lengend Snippet: Therapeutic proteins granted orphan designation upon original submission (2011–2016).

Article Snippet: Production of proteins in transgenic animals , C1 esterase inhibitor (Ruconest) produced in transgenic rabbit milk .

Techniques: Coagulation, Recombinant, Control, Clinical Proteomics