Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Small molecule inhibition of gut microbial choline trimethylamine lyase activity alters host cholesterol and bile acid metabolism

doi: 10.1152/ajpheart.00584.2019

Figure Lengend Snippet: Primers used for real-time qPCR and antibodies used for Western blot analysis

Article Snippet: Information of antibodies is available in . table ft1 table-wrap mode="anchored" t5 Table 2. caption a7 No. Antibody Vendor Catalog No. Primary antibody information 1 ABCA1 Novus-Biological NB400-105 2 NPC1L1 Gift from Dr. Joyce Repa (Univ. of Texas–Southwestern Medical Center Not applicable 3 HMGCOA-reductase Gift from Dr. Russell DeBose-Boyd (Univ. of Texas–Southwestern Medical Center) Not applicable 4 CYP7A1 Abcam Ab65596 5 Calnexin Cell Signaling Technologies (C5C9)#2679 6 GAPDH Cell Signaling Technologies (D16H11)#8884 Secondary antibody information 1 Anti-rabbit HRP-conjugated Cell Signaling Technologies 7074 2 Anti-mouse HRP-conjugated Cell Signaling Technologies 7076 Open in a separate window ABCA1, ATP-binding cassette transporter; CYP7A1, bile acid modifying cytochrome-P450 family member; NPC1L1, Niemann-pick C1-like 1.

Techniques: Western Blot

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Small molecule inhibition of gut microbial choline trimethylamine lyase activity alters host cholesterol and bile acid metabolism

doi: 10.1152/ajpheart.00584.2019

Figure Lengend Snippet: Primary and secondary antibodies

Article Snippet: Information of antibodies is available in . table ft1 table-wrap mode="anchored" t5 Table 2. caption a7 No. Antibody Vendor Catalog No. Primary antibody information 1 ABCA1 Novus-Biological NB400-105 2 NPC1L1 Gift from Dr. Joyce Repa (Univ. of Texas–Southwestern Medical Center Not applicable 3 HMGCOA-reductase Gift from Dr. Russell DeBose-Boyd (Univ. of Texas–Southwestern Medical Center) Not applicable 4 CYP7A1 Abcam Ab65596 5 Calnexin Cell Signaling Technologies (C5C9)#2679 6 GAPDH Cell Signaling Technologies (D16H11)#8884 Secondary antibody information 1 Anti-rabbit HRP-conjugated Cell Signaling Technologies 7074 2 Anti-mouse HRP-conjugated Cell Signaling Technologies 7076 Open in a separate window ABCA1, ATP-binding cassette transporter; CYP7A1, bile acid modifying cytochrome-P450 family member; NPC1L1, Niemann-pick C1-like 1.

Techniques:

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Small molecule inhibition of gut microbial choline trimethylamine lyase activity alters host cholesterol and bile acid metabolism

doi: 10.1152/ajpheart.00584.2019

Figure Lengend Snippet: Trimethylamine (TMA) lyase inhibition alters the hepatic expression of key genes involved in sterol and bile acid metabolism. At 8 wk of age, wild-type C57BL/6J male mice were switched from standard rodent chow to 1 of 4 experimental synthetic diets containing low (0.02%, wt/wt) or high (0.2%, wt/wt) levels of dietary cholesterol with or without the microbe-targeted TMA lyase inhibitor iodomethylcholine (IMC; 0.06%, wt/wt). After 4 wk on these diets, liver RNA was extracted and gene expression was measured using quantitative real-time PCR. A: relative mRNA expression of sterol regulatory-binding protein 2 (Srebp2), HMG-CoA reductase (HMG CoA-red), HMG-CoA synthase (HMG CoA-syn), squalene synthase (Squalene-syn), ATP-binding cassette transporters (Abca1, Abcg5, and Abcg8), bile acid modifying cytochrome-P450 family members (Cyp7a1, Cyp8b1, and Cyp27a1), bile salt export pump (Bsep), and small heterodimeric partner (Shp) were quantified using the ΔΔCT method. B and C: Western blot analysis HMG-CoA reductase and Cyp7a1 with densitometric quantification. Data shown in A represent the means ± SE for n = 5 mice per group, whereas data in B and C represent the means ± SE for n = 3 mice per group; *significantly different than the nondrug-treated mice within each diet group. AU, arbitrary units.

Article Snippet: Information of antibodies is available in . table ft1 table-wrap mode="anchored" t5 Table 2. caption a7 No. Antibody Vendor Catalog No. Primary antibody information 1 ABCA1 Novus-Biological NB400-105 2 NPC1L1 Gift from Dr. Joyce Repa (Univ. of Texas–Southwestern Medical Center Not applicable 3 HMGCOA-reductase Gift from Dr. Russell DeBose-Boyd (Univ. of Texas–Southwestern Medical Center) Not applicable 4 CYP7A1 Abcam Ab65596 5 Calnexin Cell Signaling Technologies (C5C9)#2679 6 GAPDH Cell Signaling Technologies (D16H11)#8884 Secondary antibody information 1 Anti-rabbit HRP-conjugated Cell Signaling Technologies 7074 2 Anti-mouse HRP-conjugated Cell Signaling Technologies 7076 Open in a separate window ABCA1, ATP-binding cassette transporter; CYP7A1, bile acid modifying cytochrome-P450 family member; NPC1L1, Niemann-pick C1-like 1.

Techniques: Inhibition, Expressing, Real-time Polymerase Chain Reaction, Binding Assay, Western Blot

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Small molecule inhibition of gut microbial choline trimethylamine lyase activity alters host cholesterol and bile acid metabolism

doi: 10.1152/ajpheart.00584.2019

Figure Lengend Snippet: Trimethylamine (TMA) lyase inhibition alters the host intestinal expression of key genes involved in sterol and bile acid transport and metabolism. At 8 wk of age, wild-type C57BL/6J male mice were switched from standard rodent chow to 1 of 4 experimental synthetic diets containing low (0.02%, wt/wt) or high (0.2%, wt/wt) levels of dietary cholesterol with or without the microbe-targeted TMA lyase inhibitor iodomethylcholine (IMC; 0.06%, wt/wt). After 4 wk on these diets, RNA was extracted from either the jejunum (A) or ileum (B), and gene expression was measured using quantitative real-time PCR. The mRNA abundance of HMG-CoA reductase (HMG Reduc.), HMG-CoA synthase (HMG Synth.), ATP-binding cassette transporters (Abca1, Abcg5, and Abcg8), Niemann-pick C1-like 1 (Npc1l1), farnesoid X receptor (Fxr), apical sodium bile acid transporter (Asbt), organic solute transporter α and β (Ostα and Ostβ), fibroblast growth factor 15 (Fgf15), and ileal bile acid-binding protein (I-babp) were quantified using the ΔΔCT method. C: Western blot analysis of Abca1, Npc1l1, and Gapdh with densitometric quantification. Data shown in A represent the means ± SE for n = 5 mice per group, whereas data in B and C represent the means ± SE for n = 3 mice per group; *significantly different than the nondrug-treated mice within each diet group.

Article Snippet: Information of antibodies is available in . table ft1 table-wrap mode="anchored" t5 Table 2. caption a7 No. Antibody Vendor Catalog No. Primary antibody information 1 ABCA1 Novus-Biological NB400-105 2 NPC1L1 Gift from Dr. Joyce Repa (Univ. of Texas–Southwestern Medical Center Not applicable 3 HMGCOA-reductase Gift from Dr. Russell DeBose-Boyd (Univ. of Texas–Southwestern Medical Center) Not applicable 4 CYP7A1 Abcam Ab65596 5 Calnexin Cell Signaling Technologies (C5C9)#2679 6 GAPDH Cell Signaling Technologies (D16H11)#8884 Secondary antibody information 1 Anti-rabbit HRP-conjugated Cell Signaling Technologies 7074 2 Anti-mouse HRP-conjugated Cell Signaling Technologies 7076 Open in a separate window ABCA1, ATP-binding cassette transporter; CYP7A1, bile acid modifying cytochrome-P450 family member; NPC1L1, Niemann-pick C1-like 1.

Techniques: Inhibition, Expressing, Real-time Polymerase Chain Reaction, Binding Assay, Western Blot

Journal: PloS one

Article Title: A spinal cord window chamber model for in vivo longitudinal multimodal optical and acoustic imaging in a murine model.

doi: 10.1371/journal.pone.0058081

Figure Lengend Snippet: Figure 2. Visual and histological confirmation that SCWC implantation does not damage the spinal cord structure or cause significant inflammation or infection. (A) White light images following SCWC implantation at 0, 1, 3 and 7 days showed no signs of local infection, excessive bleeding around the installation site, or device rejection. SCWC remained optically clear for 29 days, permitting long-term high-resolution imaging of cord and vascular structures. Yellow arrows indicate the location of the spinal cord. (B–E) Histological analysis and quantification of spinal cord tissue cross- sections cut directly below the caudal edge of the implanted SCWC. (B) H&E staining confirmed tissue morphology was intact after WC implantation. (C) Representative Iba-1 immunohistochemistry images from spinal cords 24, 48 and 72 hours following SCWC implantation. Sham and spinal cord injury (SCI) (Iba-1 positive control) animals are also shown for comparison. SCI positive control animals showed a significant increase in Iba-1 expression, * p,0.001. No notable changes in Iba-1 expression in ex vivo spinal cord were observed between the SCWC implanted groups). (D) Western blot for Iba-1 prior to SCWC implant (sham), and at 24, 48, and 72 hours post-implantation

Article Snippet: Mouse Spinal Cord Window Chamber Installation Female athymic nude mice (NCRNU-F, Taconic, Hudson, NY, USA) or C57BL6 (Jackson Laboratories, Bar Harbor, Maine, USA) at 15–20 weeks, were anesthetized using a mixture of ketamine (80 mg/kg) and xylazine (5 mg/kg) prior to surgical installation of the SCWCs.

Techniques: Infection, Imaging, Staining, Immunohistochemistry, Positive Control, Comparison, Expressing, Ex Vivo, Western Blot

Journal: PloS one

Article Title: A spinal cord window chamber model for in vivo longitudinal multimodal optical and acoustic imaging in a murine model.

doi: 10.1371/journal.pone.0058081

Figure Lengend Snippet: Figure 7. Intravital multispectral fluorescence microscopic imaging of medulloblastoma tumor metastasis to the spinal cord. (A) In vivo bioluminescence images of mice 7 days following intracranial tumor implantation of human WW426 medulloblastoma tumor cells, demonstrating local tumor growth. (B) SCWC was implanted 27 days after tumor implantation, when metastatic GFP+ tumor cells to the spinal cord could be seen using both BLI and intravital two-photon images (color bar indicates bioluminescence signal intensity; BLI units are photons/s/cm2/Sr). The head of the mouse was covered in ‘‘B’’ to reduce the bioluminescence signal from the brain in order to detect lower bioluminescence from the tumor micrometastases. (C) Wide-field fluorescence imaging and (D) confocal fluorescence microscopy of the SCWC-bearing mouse 28 days after initial tumor implantation (1 day post-SCWC installation). The outline of the spinal cord is highlighted with the orange dotted line in ‘‘C’’. TRITC- dextran shows the posterior spinal cord vein. The arrows in ‘‘C’’ and ‘‘D’’ indicate the location of multiple tumor micrometastases in close proximity to the spinal cord vasculature. Scale bars = 1 mm. SCWC = spinal cord window chamber. BLI = bioluminescence imaging. doi:10.1371/journal.pone.0058081.g007

Article Snippet: Mouse Spinal Cord Window Chamber Installation Female athymic nude mice (NCRNU-F, Taconic, Hudson, NY, USA) or C57BL6 (Jackson Laboratories, Bar Harbor, Maine, USA) at 15–20 weeks, were anesthetized using a mixture of ketamine (80 mg/kg) and xylazine (5 mg/kg) prior to surgical installation of the SCWCs.

Techniques: Fluorescence, Imaging, In Vivo, Intracranial Tumor Implantation, Tumor Implantation, Microscopy