Journal: PLoS Pathogens

Article Title: Induction of influenza-specific local CD8 T-cells in the respiratory tract after aerosol delivery of vaccine antigen or virus in the Babraham inbred pig

doi: 10.1371/journal.ppat.1007017

Figure Lengend Snippet: Babraham pig 625 (left panel of 21 plots) and 650 (right panel of 21 plots) received H5N1 S-FLU intranasally and inactivated H1N1 virus [A/Swine/Spain/SF11131/2007] with montanide adjuvant intramuscularly, followed by a boost at day 25 using the same preparation. Pigs were culled at day 38 (day 13 post boost) and blood, bronchoalveolar lavage (BAL) and tracheobronchial lymph nodes (TBLNs) harvested and frozen as single cell suspensions. Tetramer staining was performed on thawed cells from the blood, BAL and TBLN using a no tetramer control, and staining with Irrelevant and nucleoprotein peptide tetramers. The sequences for the nucleoprotein peptides are shown. Irrelevant tetramers: SLA-1*14:02-AFAAAAAAL, SLA-2*11:04-AGAAAAAAI (pig 625) and SLA-2*11:04-GAGGGGGGI (pig 650). Gating strategy: lymphocytes, single cells, viability (Vivid neg ) CD3 + CD14 neg then CD8β + CD4 + and displayed as CD8β versus tetramer .

Article Snippet: The Biorad TUK4 anti-human CD14 clone cross-reacts with pig CD14.

Techniques: Staining

Journal: PLoS Pathogens

Article Title: Induction of influenza-specific local CD8 T-cells in the respiratory tract after aerosol delivery of vaccine antigen or virus in the Babraham inbred pig

doi: 10.1371/journal.ppat.1007017

Figure Lengend Snippet: Babraham pigs were either left unvaccinated (1 and 2) or received H1N1 S-FLU via aerosol administration (6, 7, 8). H1N1 S-FLU vaccinated animals received a boost at day 28 with the same vaccine. Animals were culled and bronchoalveolar lavage harvested at day 57. Nucleoprotein and irrelevant peptide SLA-I tetramer staining was performed on thawed bronchoalveolar lavage samples and the percentage of tetramer + cells of CD8β + cells displayed in red. The sequences of the nucleoprotein peptides are shown. Irrelevant tetramers: SLA-1*14:02-AFAAAAAAL and SLA-2*11:04-GAGGGGGGI. Gating strategy: lymphocytes, single cells, viability (Vivid neg ) CD3 + CD14 neg then CD8β + CD4 + and displayed as CD8β versus tetramer .

Article Snippet: The Biorad TUK4 anti-human CD14 clone cross-reacts with pig CD14.

Techniques: Staining

Journal: PLoS Pathogens

Article Title: Induction of influenza-specific local CD8 T-cells in the respiratory tract after aerosol delivery of vaccine antigen or virus in the Babraham inbred pig

doi: 10.1371/journal.ppat.1007017

Figure Lengend Snippet: Babraham pigs were either left uninfected (741) or infected intranasally with pandemic H1N1 [A/sw/Eng/1353/09] (742, 744, 745). The pigs were culled on day 0 (741), 5 (744) or 14 (742 and 745) post infection. ( A ) 200,000 bronchoalveolar lavage cells from pig 745 (infected, day 14 cull) were incubated alone, with 10 -5 M peptide, or virus for 16–18 h. A Babraham kidney cell line was included in each well (15,000 per well) to act as antigen presenting cells. All conditions were performed in duplicate and spot forming cells (SFCs) detected by IFNγ ELISPOT and displayed as mean +SEM and scaled (X5) to 10 6 BAL cells. ( B ) Irrelevant and nucleoprotein peptide-SLA-1*14:02 and SLA-2*11:04 tetramer staining was performed on thawed bronchoalveolar lavage samples and the percentage of tetramer + cells of CD8β cells displayed in red. Nucleoprotein peptide sequences are shown. Irrelevant tetramers were SLA-1*14:02-AFAAAAAAL and SLA-2*11:04-AGAAAAAAI. Gating strategy: lymphocytes, single cells, viability (Vivid neg ) CD3 + CD14 neg then CD8β + CD4 + and displayed as CD8β versus tetramer .

Article Snippet: The Biorad TUK4 anti-human CD14 clone cross-reacts with pig CD14.

Techniques: Infection, Incubation, Enzyme-linked Immunospot, Staining

Journal: Antioxidants

Article Title: Functional and Structural Insights into the Human PPARα/δ/γ Targeting Preferences of Anti-NASH Investigational Drugs, Lanifibranor, Seladelpar, and Elafibranor

doi: 10.3390/antiox12081523

Figure Lengend Snippet: TR-FRET-based LBD-mediated human PPARα/δ/γ coactivator recruitment assay. The LBD-mediated PPARα/δ/γ recruitment of coactivator peptides, PGC1α and SRC1, was induced by PPARα-selective GW7647, PPARδ-selective GW501516, and PPARγ-selective GW1929 in a concentration-dependent manner, and their maximal responses (all at 1 µM) were considered as the 100% responses (dashed lines). The PGC1α ( A , C , E , G , I ) or the SRC1 ( B , D , F , H , J ) recruitment activities by lanifibranor ( A , B ), seladelpar ( C , D ), elafibranor ( E , F ), saroglitazar ( G , H ), and pioglitazone ( I , J ) were investigated. Data are presented as the mean ± SE of three or four independent experiments with duplicate samples, and the calculated EC 50 values are shown.

Article Snippet: The activation status of each PPARα/δ/γ subtype was also determined by a time-resolved fluorescence resonance energy transfer (TR-FRET) assay that detects physical interactions between His-tagged hPPARα/δ/γ-LBD proteins and a biotin-labeled PPARγ coactivator 1α (PGC1α) coactivator peptide (biotin-EAEEPSLLKKLLLAPANTQ (amino acids 137–155) synthesized by GenScript) or a steroid receptor coactivator 1 (SRC1) peptide (biotin-CPSSHSSLTERHKILHRLLQEGSPS (amino acids 676–700) from GenScript) using the LANCE Ultra TR-FRET assay (PerkinElmer, Waltham, MA, USA) [ , , ].

Techniques: Concentration Assay

Journal: Antioxidants

Article Title: Functional and Structural Insights into the Human PPARα/δ/γ Targeting Preferences of Anti-NASH Investigational Drugs, Lanifibranor, Seladelpar, and Elafibranor

doi: 10.3390/antiox12081523

Figure Lengend Snippet: PPARα/δ/γ-LBD–lanifibranor cocrystal structures. Cocrystals of lanifibranor and PPARα-LBD ( A – C ), PPARδ-LBD ( D – F ), or PPARγ-LBD ( G – I ) were analyzed using X-ray diffraction. ( A , D , G ) Overall structures of the complexes deposited in PDB with IDs: 8HUK, 8HUL, and 8HUM, respectively. The SRC1 peptide (α-helix in magenta) and the AF-2 helix 12 (α-helix in red) are indicated by arrows (only in ( G )) and arrowheads, respectively. The highest resolutions are labeled. ( B , E , H ) Magnified views of lanifibranor located in the “Center” region of PPARα/δ/γ-LBD. The electron density is shown in the mesh via F o – F c omit maps contoured at +3.0 σ. A water molecule is presented as a cyan sphere in ( H ). ( C , F , I ) Hydrogen bonds and electrostatic interactions between lanifibranor and the four consensus amino acid residues (that recognize the carboxyl moiety of lanifibranor) are indicated by red and blue dotted lines, respectively, along with their distances (in Å). ( J ) Superposed view of lanifibranor in PPARα (magenta)/PPARδ (green)/PPARγ (cyan)-LBD cocrystal structures. ( K ) Superposed view of our F o – F c omit maps of PPARγ-LBD–lanifibranor (the same with ( H )) and lanifibranor in a previous PDB submission (ID: 6ENQ).

Article Snippet: The activation status of each PPARα/δ/γ subtype was also determined by a time-resolved fluorescence resonance energy transfer (TR-FRET) assay that detects physical interactions between His-tagged hPPARα/δ/γ-LBD proteins and a biotin-labeled PPARγ coactivator 1α (PGC1α) coactivator peptide (biotin-EAEEPSLLKKLLLAPANTQ (amino acids 137–155) synthesized by GenScript) or a steroid receptor coactivator 1 (SRC1) peptide (biotin-CPSSHSSLTERHKILHRLLQEGSPS (amino acids 676–700) from GenScript) using the LANCE Ultra TR-FRET assay (PerkinElmer, Waltham, MA, USA) [ , , ].

Techniques: Labeling

Journal: Antioxidants

Article Title: Functional and Structural Insights into the Human PPARα/δ/γ Targeting Preferences of Anti-NASH Investigational Drugs, Lanifibranor, Seladelpar, and Elafibranor

doi: 10.3390/antiox12081523

Figure Lengend Snippet: PPARα/δ/γ-LBD–seladelpar cocrystal structures. Cocrystals of seladelpar and PPARα-LBD ( A – C ), PPARδ-LBD ( D – F ), or PPARγ-LBD ( G – I ) were analyzed using X-ray diffraction. ( A , D , G ) Overall structures of the complexes deposited in PDB with IDs 8HUN, 8HUO, and 8HUP, respectively. The SRC1 peptide (α-helix in magenta) and the AF-2 helix 12 (α-helix in red) are indicated by arrows (only in ( G )) and arrowheads, respectively. The highest resolutions are labeled. ( B , E , H ) Magnified views of seladelpar located in the “Center” and the “Arm II” regions of PPARα/δ/γ-LBD. The electron density is shown in the mesh via F o – F c omit maps contoured at +3.0 σ. Water molecules are presented as cyan spheres in ( B ). ( C , F , I ) Hydrogen bonds and electrostatic interactions between seladelpar and the four consensus amino acid residues (that recognize the carboxyl moiety of seladelpar) are indicated by red and blue dotted lines, respectively, along with their distances (in Å). ( J ) Superposed view of seladelpar in PPARα (magenta)/PPARδ (green)/PPARγ (cyan)-LBD cocrystal structures.

Article Snippet: The activation status of each PPARα/δ/γ subtype was also determined by a time-resolved fluorescence resonance energy transfer (TR-FRET) assay that detects physical interactions between His-tagged hPPARα/δ/γ-LBD proteins and a biotin-labeled PPARγ coactivator 1α (PGC1α) coactivator peptide (biotin-EAEEPSLLKKLLLAPANTQ (amino acids 137–155) synthesized by GenScript) or a steroid receptor coactivator 1 (SRC1) peptide (biotin-CPSSHSSLTERHKILHRLLQEGSPS (amino acids 676–700) from GenScript) using the LANCE Ultra TR-FRET assay (PerkinElmer, Waltham, MA, USA) [ , , ].

Techniques: Labeling

Journal: Antioxidants

Article Title: Functional and Structural Insights into the Human PPARα/δ/γ Targeting Preferences of Anti-NASH Investigational Drugs, Lanifibranor, Seladelpar, and Elafibranor

doi: 10.3390/antiox12081523

Figure Lengend Snippet: PPARα-LBD–elafibranor cocrystal structures. A cocrystal of elafibranor and PPARα-LBD ( A – C ) was analyzed using X-ray diffraction. ( A ) The 2.36 Å resolution overall structure of the complex deposited in PDB ID: 8HUQ. The SRC1 peptide (α-helix in magenta) and the AF-2 helix 12 (α-helix in red) are indicated by an arrow and an arrowhead, respectively. ( B ) Magnified view of elafibranor located in the “Center” and the “Arm II” regions of PPARα-LBD. The electron density is shown in the mesh via F o - F c omit maps contoured at +3.0 σ. Water molecules are presented as cyan spheres. ( C ) Hydrogen bonds and electrostatic interactions between elafibranor and the four consensus amino acid residues (that recognize the carboxyl moiety of elafibranor) are indicated by red and blue dotted lines, respectively, along with their distances (in Å).

Article Snippet: The activation status of each PPARα/δ/γ subtype was also determined by a time-resolved fluorescence resonance energy transfer (TR-FRET) assay that detects physical interactions between His-tagged hPPARα/δ/γ-LBD proteins and a biotin-labeled PPARγ coactivator 1α (PGC1α) coactivator peptide (biotin-EAEEPSLLKKLLLAPANTQ (amino acids 137–155) synthesized by GenScript) or a steroid receptor coactivator 1 (SRC1) peptide (biotin-CPSSHSSLTERHKILHRLLQEGSPS (amino acids 676–700) from GenScript) using the LANCE Ultra TR-FRET assay (PerkinElmer, Waltham, MA, USA) [ , , ].

Techniques:

Journal: Acta Histochemica et Cytochemica

Article Title: Involvement of Leptin in the Progression of Experimentally Induced Peritoneal Fibrosis in Mice

doi: 10.1267/ahc.13005

Figure Lengend Snippet: The list of primary antibodies for immunohistochemistry

Article Snippet: F4/80 (macrophages) , rat , 1:100 , MCA497; Serotec, Oxford, UK.

Techniques:

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Krüppel‐Like Factor 15/Interleukin 11 Axis‐Mediated Adventitial Remodeling Depends on Extracellular Signal‐Regulated Kinases 1 and 2 Activation in Angiotensin II–Induced Hypertension

doi: 10.1161/JAHA.120.020554

Figure Lengend Snippet: A , Representative hematoxylin and eosin (H&E) and picrosirius red staining of the thoracic aorta tissues in wild type (WT) and IL‐11‐/‐ mice after Ang II infusion for 14 days. Bar=50 µm. B through D , Quantitative analysis of wall thickness ( B ), wall area ( C ), and collagen area ( D ). E , Systolic blood pressure of the 4 groups. F , Representative immunofluorescence staining and quantitative analysis of ER‐TR7 (red), α‐smooth muscle actin (ACTA2) (green), CD68 (green), and Krüppel‐like factor 15 (KLF15) (red) in thoracic aorta. The 4′,6‐diamidino‐2‐phenylindole was used to stain the nucleus in blue. Bar=50 µm. G , The mRNA levels of KLF15 (1.00‐, 0.27‐, 1.07‐, and 0.22‐fold), collagen, type I, α 1 (COL1a1) (1.00‐, 14.52‐, 0.43‐, and 1.74‐fold), collagen, type III, α 1 (COL3a1) (1.00‐, 1.69‐, 0.86‐, and 1.17‐fold), interleukin 6 (IL‐6) (1.00‐, 1.93‐, 0.33‐, and 0.43‐fold) and chemokine (C‐C motif) ligand 2 (CCL2) (1.00‐, 2.11‐, 0.42‐, and 0.53‐fold) were measured by quantitative reverse transcription–polymerase chain reaction. A indicates adventitia. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Immunofluorescence staining was performed with primary antibodies against ER‐TR7 (Abcam; ab51824), KLF15 (Millipore Sigma; ABC471), ACTA2 (Millipore Sigma; A2547), phosphorylated ERK1/2 (Cell Signaling Technology, Danvers, MA; 4695), and CD68 (Bio‐Rad, Hercules, CA; MCA341GA) at a dilution of 1:100.

Techniques: Staining, Immunofluorescence, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Krüppel‐Like Factor 15/Interleukin 11 Axis‐Mediated Adventitial Remodeling Depends on Extracellular Signal‐Regulated Kinases 1 and 2 Activation in Angiotensin II–Induced Hypertension

doi: 10.1161/JAHA.120.020554

Figure Lengend Snippet: Wild‐type mice were randomly divided into 4 groups treated with rmIL‐11 or IL‐11 neutralizing antibody with or without Ang II infusion. A , Representative hematoxylin and eosin (H&E) and picrosirius red staining of the thoracic aorta tissues. Bar=50 µm. B through D , Quantitative analysis of wall thickness ( B ), wall area ( C ), and collagen area ( D ). E , Systolic blood pressure of the 4 groups. F , Representative immunofluorescence staining and quantitative analysis of ER‐TR7 (red), α‐smooth muscle actin (ACTA2) (green), CD68 (green), and Krüppel‐like factor 15 (KLF15) (red) in thoracic aorta. The 4′,6‐diamidino‐2‐phenylindole was used to stain the nucleus in blue. Bar=50 µm. G , The mRNA levels of KLF15 (1.00‐, 0.47‐, 0.39‐, and 0.40‐fold), collagen, type I, α 1 (COL1a1) (1.00‐, 2.85‐, 0.81‐, and 3.70‐fold), collagen, type III, α 1 (COL3a1) (1.00‐, 2.69‐, 0.85‐, and 6.67‐fold), interleukin‐6 (IL‐6) (1.00‐, 1.75‐, 0.23‐, and 1.95‐fold), and chemokine (C‐C motif) ligand 2 (CCL2) (1.00‐, 1.83‐, 0.39‐, and 2.76‐fold) were measured by quantitative reverse transcription–polymerase chain reaction. A indicates adventitia. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Immunofluorescence staining was performed with primary antibodies against ER‐TR7 (Abcam; ab51824), KLF15 (Millipore Sigma; ABC471), ACTA2 (Millipore Sigma; A2547), phosphorylated ERK1/2 (Cell Signaling Technology, Danvers, MA; 4695), and CD68 (Bio‐Rad, Hercules, CA; MCA341GA) at a dilution of 1:100.

Techniques: Staining, Immunofluorescence, Reverse Transcription Polymerase Chain Reaction