Journal: Vaccines

Article Title: SARS-CoV-2: Immunity, Challenges with Current Vaccines, and a Novel Perspective on Mucosal Vaccines

doi: 10.3390/vaccines11040849

Figure Lengend Snippet: Mucosal SARS-CoV-2 Vaccines in Clinical trials.

Article Snippet: , Human adenovirus type 5 vector , AdCOVID , Altimmune , Receptor-binding domain (RBD) , IN , NCT04679909 (phase 1) (Discontinued).

Techniques: Plasmid Preparation, Modification, Recombinant

Journal: Frontiers in Cell and Developmental Biology

Article Title: Upregulation of Parkin Accelerates Osteoblastic Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells and Bone Regeneration by Enhancing Autophagy and β-Catenin Signaling

doi: 10.3389/fcell.2020.576104

Figure Lengend Snippet: Overexpression of Parkin enhances osteoblastic differentiation of bone marrow mesenchymal stem cells (BMSCs). (A,B) Protein expression levels of Parkin were determined at 0, 6, 12, 24, 48, 72, and 96 h after induction of osteoblastic differentiation. (C) mRNA expression of Parkin was analyzed by quantitative real-time polymerase chain reaction (RT-qPCR) at days 1 and 6 after infection. (D,E) Protein expression of Parkin was analyzed by western blot (WB) analysis at days 1 and 6 after infection. (F) Protein expression of Parkin was analyzed by immunofluorescence staining at day 6 after infection. (G) Proliferation was detected by Cell Counting Kit-8 in the Parkin-overexpressed and control group at days 1, 2, 3, 4, and 5. (H) Osteo-specific genes Alp, Runx2, and Col1 were analyzed by RT-qPCR at day 1 after osteogenic induction. (I) Osteo-specific genes Alp, Runx2, and Col1 were analyzed by RT-qPCR at day 3 after osteogenic induction. (J,K) Mineral deposits were determined by Alizarin Red staining at day 12 after osteogenic induction. (L) Alkaline phosphatase activity at day 3 of osteogenic differentiation of BMSCs. (M,N) Osteo-specific proteins Alp, Runx2, and Col1 were analyzed by WB at day 3 after osteogenic induction. Data are expressed as the mean ± standard deviation (SD) ( n = 3). NC, BMSCs transfected with negative control adenovirus vectors; OE, BMSCs transfected with Parkin-overexpressed adenovirus vectors; * P < 0.05 vs. BMSCs in the NC group.

Article Snippet: Parkin overexpression adenovirus vectors (OE; AV-CMV > Mouse Parkin/T2A/PolyA) and negative control adenovirus vectors (NC; AV-CMV > PolyA) were purchased from Cyagen Biosciences Inc., and the viral titers were l × 10 10 PFU/mL. mRNA and protein levels of Parkin were analyzed by quantitative real-time polymerase chain reaction (RT-qPCR) and western blot (WB) analysis, respectively.

Techniques: Over Expression, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Infection, Western Blot, Immunofluorescence, Staining, Cell Counting, Control, Activity Assay, Standard Deviation, Transfection, Negative Control

Journal: Journal of the American Society of Nephrology : JASN

Article Title: SIRT2 Regulates LPS-Induced Renal Tubular CXCL2 and CCL2 Expression

doi: 10.1681/ASN.2014030226

Figure Lengend Snippet: Sirt2 and LPS-induced CXCL2/CCL2 are expressed in the proximal tubular epithelial cells. (A) Immunofluorescence staining of SIRT2 and Lectin in the kidney of Sirt2+/+ and Sirt2−/− mice. Kidney sections from Sirt2+/+ and Sirt2−/− mice were stained with SIRT2 antibody and Lectin. Lectin was used as a marker of proximal tubular epithelial cell. (B and C) Immunofluorescence findings of CXCL2 (B), CCL2 (C), and Lectin in the kidneys. After stimulation with LPS or control buffer (CB), kidney sections of mice were stained with a CXCL2 or CCL2 antibody. Scale bar=100 µm. G, glomerulus. SIRT2-, CXCL2- and CCL2-postive cells were also stained with Lectin.

Article Snippet: Replication-deficient recombinant adenovirus vectors containing the SIRT2 gene were purchased from ViraQuest, Inc. (North Liberty, IA).

Techniques: Immunofluorescence, Staining, Marker, Control

Journal: Journal of the American Society of Nephrology : JASN

Article Title: SIRT2 Regulates LPS-Induced Renal Tubular CXCL2 and CCL2 Expression

doi: 10.1681/ASN.2014030226

Figure Lengend Snippet: Knockout of SIRT2 decreases LPS-induced CXCL2 and CCL2 expression in the kidney. (A and B) Immunohistochemical findings of CXCL2 and CCL2. Kidneys were harvested at 3 hours after intraperitoneal LPS injection. Tissues were fixed in 4% formaldehyde solution and kidney sections were then stained with CXCL2 (A) or CCL2 (B) antibody. Scale bar=100 µm. (C and D) Quantitative score of CXCL2 (C) or CCL2-positive (D) area in the kidney. (E and F) Tissue levels of CXCL2 (E) and CCL2 (F) in the kidney. Cytokines were quantified by ELISA. n=5 per group. (G and H) CXCL2 (G) and CCL2 (H) mRNA expression analysis by qRT-PCR in the kidneys of Sirt2+/+ and Sirt2−/− mice at 1 and 3 hours after LPS or control buffer (CB) treatment. Fold changes of CXCL2 and CCL2 expression are relative to CB-treated kidneys. Bars represent the mean±SD from six mice per group. The relative ratio measured in the CB-treated kidney of Sirt2+/+ mice is arbitrarily presented as 1. Data are expressed as mean±SD. *P<0.05 versus CB-treated Sirt2+/+ mice; **P<0.01 versus CB-treated Sirt2+/+ mice; ***P<0.001 versus CB-treated Sirt2+/+ mice; ‡P<0.01 versus LPS-treated Sirt2+/+ mice. Closed arrows in A and open arrows in B indicate CXCL2 and CCL2 expression in tubular epithelial cells, respectively.

Article Snippet: Replication-deficient recombinant adenovirus vectors containing the SIRT2 gene were purchased from ViraQuest, Inc. (North Liberty, IA).

Techniques: Knock-Out, Expressing, Immunohistochemical staining, Injection, Staining, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Control

Journal: Journal of the American Society of Nephrology : JASN

Article Title: SIRT2 Regulates LPS-Induced Renal Tubular CXCL2 and CCL2 Expression

doi: 10.1681/ASN.2014030226

Figure Lengend Snippet: Sirt2 deficiency decreases neutrophil and macrophage infiltration in the kidney and increases renal function after LPS administration. (A and B) Immunofluorescence staining of Gr-1 (A) and ER-HR3 (B) in the kidney of Sirt2+/+ and Sirt2−/− mice. Kidneys were harvested at 3 and 72 hours after LPS administration, and kidney sections were stained with Gr-1 or ER-HR3 antibody. Gr-1 was used as a marker of neutrophils and ER-HR3 was used as a marker of macrophages. Scale bar=100 µm. (C and D) Quantification of Gr-1–positive neutrophils (C) and ER-HR3–positive macrophages (D) in the kidneys of Sirt2+/+ and Sirt2−/− mice. Value is shown as number per high power field. n=6 per group. (E) MPO activity in the kidney measured at 3 hours after LPS treatment in Sirt2−/− and Sirt2+/+ mice. n=6 per group. (F) Urinary NGAL levels in Sirt2−/− and Sirt2+/+ mice. Urinary NGAL were measured at 3 hours after LPS treatment. n=6 per group. (G) Effect of SIRT2 deficiency on inulin clearance. Inulin clearance was measured with Sirt2+/+ and Sirt2−/− mice 18 hours after LPS treatment (10 mg/kg intraperitoneally). Inulin clearance was significantly higher in Sirt2−/− mice than in Sirt2+/+ mice after LPS administration. n=3 per group. Data are expressed as mean±SD. **P<0.01 versus control buffer (CB)–treated Sirt2+/+ mice; †P<0.01 versus LPS-treated Sirt2+/+ mice; ‡P<0.01 versus LPS-treated Sirt2+/+ mice. Opened arrows in A indicate Gr-1–positive neutrophils and closed arrows in B indicate ER-HR3–positive macrophages in kidney tissue.

Article Snippet: Replication-deficient recombinant adenovirus vectors containing the SIRT2 gene were purchased from ViraQuest, Inc. (North Liberty, IA).

Techniques: Immunofluorescence, Staining, Marker, Activity Assay, Control

Journal: Journal of the American Society of Nephrology : JASN

Article Title: SIRT2 Regulates LPS-Induced Renal Tubular CXCL2 and CCL2 Expression

doi: 10.1681/ASN.2014030226

Figure Lengend Snippet: Sirt2 deletion suppresses pro-inflammatory cytokine production triggered by LPS. (A–C) Tissue levels of TNF-α (A), IL-1β (B), and IL-6 (C) in the kidney of Sirt2+/+ and Sirt2−/− mice. Kidney sections from Sirt2+/+ and Sirt2−/− mice were harvested at 3 and 6 hours after LPS or control buffer (CB) injection. Cytokines were quantified by ELISA. n=6 for each experimental group. (D–G) Serum levels of TNF-α (D), IL-1β (E), and IL-12 (F) and RANTES (G) from Sirt2+/+ and Sirt2−/− mice. Blood samples were harvested at 3 and 6 hours after LPS or CB injection. Cytokines were quantified by ELISA. n=6 per group. Data are expressed as mean±SD. *P<0.05 versus CB-treated Sirt2+/+ mice; **P<0.01 versus CB-treated Sirt2+/+ mice; ***P<0.001 versus CB-treated Sirt2+/+ mice; †P<0.01 versus LPS-treated Sirt2+/+ mice; ‡P<0.01 versus LPS-treated Sirt2+/+ mice.

Article Snippet: Replication-deficient recombinant adenovirus vectors containing the SIRT2 gene were purchased from ViraQuest, Inc. (North Liberty, IA).

Techniques: Control, Injection, Enzyme-linked Immunosorbent Assay

Journal: Journal of the American Society of Nephrology : JASN

Article Title: SIRT2 Regulates LPS-Induced Renal Tubular CXCL2 and CCL2 Expression

doi: 10.1681/ASN.2014030226

Figure Lengend Snippet: SIRT2 regulates the acetylation level of MKP-1 after LPS stimulation. (A) MPT cells were transfected with Ad-SIRT2 or Ad-control and treated with or without LPS (10 μg/ml). Cell lysates were immunoprecipitated with an MKP-1 antibody followed by SDS/PAGE and immunoblotted with an SIRT2 antibody. Representative immunoblots of total SIRT2 and actin are shown in the lower two panels. Endogenous actin was used as a loading control before immunoprecipitation. Band densities of SIRT2 are expressed as a percentage of MKP-1 in control buffer (CB) plus Ad-control transfected cells. (B and C) Western blot analysis of MKP-1 acetylation in SIRT2-siRNA or Cont-siRNA at 1 hour after LPS treatment (B). Western blot analysis of MKP-1 acetylation in Ad-SIRT2– or Ad-control–transfected MPT cells at 1 hour after LPS treatment (C). Transfected MPT cell lysates were immunoprecipitated against MKP-1 antibody, and immunoprecipitates were immunoblotted with an acetyl-lysine antibody. *P<0.05 versus MPT cells treated with CB plus Ad-control; **P<0.01 versus MPT cells treated with CB plus control-siRNA or Ad-control; †P<0.05 versus MPT cells treated with LPS plus control-siRNA or Ad-control; ‡P<0.01 versus LPS-treated MPT cells with Cont-siRNA or Ad-control.

Article Snippet: Replication-deficient recombinant adenovirus vectors containing the SIRT2 gene were purchased from ViraQuest, Inc. (North Liberty, IA).

Techniques: Transfection, Control, Immunoprecipitation, SDS Page, Western Blot

Journal: Journal of the American Society of Nephrology : JASN

Article Title: SIRT2 Regulates LPS-Induced Renal Tubular CXCL2 and CCL2 Expression

doi: 10.1681/ASN.2014030226

Figure Lengend Snippet: SIRT2 regulates the phosphorylation level of JNK and p38 after LPS treatment. (A and B) Western blot analyses of JNK phosphorylation in SIRT2-siRNA– or Ad-SIRT2–transfected MPT cells. siRNA- or adenovirus-transfected MPT cells were treated with LPS for 15 minutes. Densitometric analyses are presented as the relative ratio of phospho-JNK to JNK. Bars represent the mean±SD from four independent experiments. (C and D) Western blot analyses of p38 phosphorylation in SIRT2-siRNA– (C) or Ad-SIRT2–transfected (D) MPT cells. siRNA- or adenovirus-transfected MPT cells were treated with LPS for 15 minutes. Densitometry analyses are presented as the relative ratio of phospho-p38 to p38. Bars represent the mean±SD from four independent experiments. (E and F) CXCL2 (E) and CCL2 (F) mRNA expression analysis by qRT-PCR in MPT cells after treatment with a p38 inhibitor, SB203580 (SB), or JNK inhibitor SP600125 (SP) at 1 hour after LPS treatment. Fold changes of CXCL2 and CCL2 expression are relative to control buffer (CB)–treated MPT cells. Bars represent the mean±SD from six independent experiments. The relative ratio measured in the CB-treated control-siRNA– or Ad-control–transfected MPT cells is arbitrarily presented as 1. **P<0.01 versus MPT cells treated with CB plus control-siRNA, Ad-control, or CB; †P<0.05 versus LPS-treated MPT cells with Cont-siRNA; ‡P<0.01 versus LPS-treated MPT cells with Cont-siRNA, Ad-control, or CB.

Article Snippet: Replication-deficient recombinant adenovirus vectors containing the SIRT2 gene were purchased from ViraQuest, Inc. (North Liberty, IA).

Techniques: Phospho-proteomics, Western Blot, Transfection, Expressing, Quantitative RT-PCR, Control

Journal: Journal of the American Society of Nephrology : JASN

Article Title: SIRT2 Regulates LPS-Induced Renal Tubular CXCL2 and CCL2 Expression

doi: 10.1681/ASN.2014030226

Figure Lengend Snippet: Binding of p65 at the CXCL2 and CCL2 gene promoter is regulated by SIRT2. SIRT2-siRNA or Cont-siRNA transfected MPT cells (A and C) were treated with LPS for 1 hour. Ad-SIRT2– or Ad-control–transfected MPT cells (B and D) were incubated with LPS for 1 hour. The ChIP assay was performed with an antibody against p65. qRT-PCR analysis was performed using primers to the mouse CXCL2 and CCL2 initiation site. The PCR amplification of input chromatin (INPUT 2%) before immunoprecipitation was used as a positive control. All results were normalized to input levels. Results are the mean enrichment levels relative to MPT cells treated with Cont-siRNA or Ad-control. Bars represent the means±SD from three independent experiments. **P<0.01 versus MPT treated with CB plus cont-siRNA or CB plus Ad-control; †P<0.05 versus MPT cells treated with LPS plus cont-siRNA or LPS plus Ad-control; ‡P<0.01 versus MPT cells treated with LPS plus cont-siRNA or LPS plus Ad-control. (E) Schematic diagram of signal pathway of SIRT2 on CXCL2 and CCL2 expression.

Article Snippet: Replication-deficient recombinant adenovirus vectors containing the SIRT2 gene were purchased from ViraQuest, Inc. (North Liberty, IA).

Techniques: Binding Assay, Transfection, Control, Incubation, Quantitative RT-PCR, Amplification, Immunoprecipitation, Positive Control, Expressing