Journal: eGastroenterology

Article Title: Gut cannabinoid receptor 1 regulates alcohol binge-induced intestinal permeability

doi: 10.1136/egastro-2024-100173

Figure Lengend Snippet: Figure 1 Measurement of gut endocannabinoids and generation of intestinal-specific CB1R KO mice. Different parts of the intestine were collected in C57BL/6 J mice (n=4). Endocannabinoids were extracted and quantified by LC-MS/MS (created with Biorender.com) (A). Analysis of endocannabinoids in different segments of the intestine (B). Schematic of the crossing strategy between VillinCre and CB1f/f mice to obtain intestinal-specific CB1R KO mice (CB1IEC−/−) (C). Duodenal gene expression of CB1R in CB1f/f and CB1IEC−/− mice (D). Saturation binding of the tritiated antagonist [3H]-CP55,940 to wild-type CB1f/f and CB1IEC−/− mice brain membrane homogenate. Error bars represent SEM for three separate experiments, each performed in duplicate. The fitted Kd and Bmax values are in the table below (E). CB1R binding affinity of the cannabinoid peripheral antagonist (S)-MRI-1891 in [3H]-CP55.940 competition binding assays to wild-type CB1f/f and CB1IEC−/− mice brain membrane homogenate. Figures represent the specific binding of the radioligand in percentage in the presence of increasing concentrations (10−12–10−6 M) of the indicated ligand. Each experiment was performed in duplicate. Data are expressed as percentage of mean specific binding±SEM (n=3) (F). [35S]-GTPγS signal properties: inhibition of CB1R agonist (CP55,940)-induced [35S]-GTPγS binding by CB1R antagonist (S)-MRI-1891 using wild-type CB1f/f and CB1IEC−/− mice brain membrane homogenate. Values represent mean±SEM from three independent experiments. Below, binding affinity (Ki) and [35S]-GTPγS signal property IC50 of cannabinoid receptor antagonist (S)-MRI-1891 mice brain homogenate (G). Values represent mean±SEM; *p<0.05, **p<0.01, ***p<0.001. AEA, endocannabinoid anandamide; 2-AG, 2-arachidonoylglycerol; Bmax, maximal number of binding sites; CB1f/f, CB1 floxed/floxed; CB1IEC−/−, intestinal epithelial-specific CB1R; CB1R, cannabinoid receptor 1; IC50, inhibitory concentration; Kd, dissociation constant; KO, knockout; LC-MS/MS, liquid chromatography-tandem mass spectrometry; mRNA, messenger RNA.

Article Snippet: Importantly, (S)MRI- 1891 did not induce anxiogenic behaviour at 3 mg/kg or even at a high dose of 30 mg/kg during chronic treatment, in contrast to the brain- penetrant CB1R antagonist rimonabant, which induced significant anxiety at a dose of 3 mg/kg.21 The mitogen activated protein kinase kinase 1/2 (MEK1/2) inhibitor U0126 (20 mg/kg; MedChemExpress) was delivered intraperitoneally 1.5 hours before the alcohol binge.

Techniques: Liquid Chromatography with Mass Spectroscopy, Gene Expression, Binding Assay, Membrane, Inhibition, Concentration Assay, Knock-Out, Liquid Chromatography, Mass Spectrometry

Journal: eGastroenterology

Article Title: Gut cannabinoid receptor 1 regulates alcohol binge-induced intestinal permeability

doi: 10.1136/egastro-2024-100173

Figure Lengend Snippet: Figure 2 Intestinal CB1R and intestinal permeability. Schematic of the experimental protocol used to evaluate intestinal permeability in vivo. Mice were gavaged with the fluorescent probe FITC-dextran 4 kDa and binged with alcohol 5 g/kg 1 hour later. Control groups were treated with an isocaloric maltose solution of 9 g/kg (not shown in this representative schematic). Three hours later, blood was collected and centrifuged and fluorescence was measured in the plasma (created with Biorender. com) (A). Measurement of alcohol-induced intestinal permeability in male and female CB1f/f and CB1IEC−/− mice (n=3/group maltose, n=7–9/group ethanol in male, n=9–10/group female mice) (B). CB1f/f and CB1IEC−/− mice were randomly subjected to ND (n=4–8) and 60% HFD (n=7–10/group) for 2 weeks. After fasting overnight, mice were gavaged with FITC-dextran 4 kDa and intestinal permeability was measured (C). Different parts of the intestine were collected in C57BL/6 J mice (n=4/group) binged with alcohol versus control group. Endocannabinoids were extracted and quantified by LC-MS/MS (created with Biorender.com) (D). Analysis of endocannabinoids levels in alcohol-treated mice versus controls in the proximal small intestine (E). Values represent mean±SEM; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. AEA, endocannabinoid anandamide; 2-AG, 2-arachidonoylglycerol; CB1f/f, CB1 floxed/floxed; CB1IEC−/−, intestinal epithelial-specific CB1R; CB1R, cannabinoid receptor 1; EtOH, ethanol; FITC, fluorescein isothicoyanate; HFD, high-fat diet; LC-MS/MS, liquid chromatography-tandem mass spectrometry; ND, normal chow diet.

Article Snippet: Importantly, (S)MRI- 1891 did not induce anxiogenic behaviour at 3 mg/kg or even at a high dose of 30 mg/kg during chronic treatment, in contrast to the brain- penetrant CB1R antagonist rimonabant, which induced significant anxiety at a dose of 3 mg/kg.21 The mitogen activated protein kinase kinase 1/2 (MEK1/2) inhibitor U0126 (20 mg/kg; MedChemExpress) was delivered intraperitoneally 1.5 hours before the alcohol binge.

Techniques: Permeability, In Vivo, Control, Fluorescence, Clinical Proteomics, Liquid Chromatography with Mass Spectroscopy, Liquid Chromatography, Mass Spectrometry

Journal: eGastroenterology

Article Title: Gut cannabinoid receptor 1 regulates alcohol binge-induced intestinal permeability

doi: 10.1136/egastro-2024-100173

Figure Lengend Snippet: Figure 3 Differentiation, tight junctions and CB1R in alcohol-induced intestinal permeability. Hematoxylin & Eosin staining in CB1f/f and CB1IEC−/− mice treated with maltose or with ethanol (A). Morphometric analysis of villi length in the four groups of mice (n=3 to 5/group) (B). Serum iFABP, a marker of intestinal epithelial damage, in CB1f/f and CB1IEC−/− mice treated with maltose or with ethanol (n=3–7/group) (C). Immunohistochemistry staining of p-ERK1/2 in ethanol-treated CB1f/f and CB1IEC−/− mice (D). Duodenal gene expression of tight junctions ocln and cldn15 (n=3–4/group) (E). Mice were treated with vehicle or with the MEK1/2 inhibitor U0126 (n=5–7/group). Intestinal permeability was measured in vivo with the FITC-dextran 4 kDa method (figure created with Biorender.com). Duodenum was collected and stained with p-ERK1/2 antibody (F). Values represent mean±SEM; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. CB1f/f, CB1 floxed/floxed; CB1IEC−/−, intestinal epithelial-specific CB1R; CB1R, cannabinoid receptor 1; cldn15, claudin 15; EtOH, ethanol; FITC, fluorescein isothicoyanate; iFABP, intestinal fatty acid binding protein; MEK, mitogen activated protein kinase kinase; mRNA, messenger RNA; ocln, occludin; pERK1/2, phosphorlated mitogen-activated protein kinase 1/2.

Article Snippet: Importantly, (S)MRI- 1891 did not induce anxiogenic behaviour at 3 mg/kg or even at a high dose of 30 mg/kg during chronic treatment, in contrast to the brain- penetrant CB1R antagonist rimonabant, which induced significant anxiety at a dose of 3 mg/kg.21 The mitogen activated protein kinase kinase 1/2 (MEK1/2) inhibitor U0126 (20 mg/kg; MedChemExpress) was delivered intraperitoneally 1.5 hours before the alcohol binge.

Techniques: Permeability, Staining, Marker, Immunohistochemistry, Gene Expression, In Vivo, Binding Assay

Journal: eGastroenterology

Article Title: Gut cannabinoid receptor 1 regulates alcohol binge-induced intestinal permeability

doi: 10.1136/egastro-2024-100173

Figure Lengend Snippet: Figure 4 Pharmacological in vivo proof of the role of CB1R in alcohol-induced intestinal permeability. Mice (n=6–8/group) were treated orally by gavage with the peripheral-restricted CB1R selective antagonist (S)-MRI-1891 at the effective dose of 3 mg/ kg. Intestinal permeability was assessed in vivo in ethanol-treated mice with the FITC-dextran 4 kDa method (figure created with Biorender.com) (A). Measurement of intestinal permeability in vehicle (Veh) and (S)-MRI-1891-treated CB1f/f and CB1IEC−/− mice (B). Duodenum tissues were collected, RNA extracted and gene expression of tight junctions-related genes ocln, cldn2, cldn3, cldn7 and cldn15 evaluated by RT-qPCR in CB1f/f (C) and CB1IEC−/− mice (D). Duodenal expression of p-ERK1/2 in vehicle and (S)-MRI-1891-treated CB1f/f and CB1IEC−/− mice (E). Values represent mean±SEM; *p<0.05, **p<0.01. CB1f/f, CB1 floxed/floxed; CB1IEC−/−, intestinal epithelial-specific CB1R; CB1R, cannabinoid receptor 1; cldn, claudin; FITC, fluorescein isothiocyanate; mRNA, messenger RNA; ocln, occludin; pERK1/2, phosphorlated mitogen-activated protein kinase 1/2; RT-qPCR, quantitative reverse transcription PCR.

Article Snippet: Importantly, (S)MRI- 1891 did not induce anxiogenic behaviour at 3 mg/kg or even at a high dose of 30 mg/kg during chronic treatment, in contrast to the brain- penetrant CB1R antagonist rimonabant, which induced significant anxiety at a dose of 3 mg/kg.21 The mitogen activated protein kinase kinase 1/2 (MEK1/2) inhibitor U0126 (20 mg/kg; MedChemExpress) was delivered intraperitoneally 1.5 hours before the alcohol binge.

Techniques: In Vivo, Permeability, Gene Expression, Quantitative RT-PCR, Expressing, Reverse Transcription

Journal: eGastroenterology

Article Title: Gut cannabinoid receptor 1 regulates alcohol binge-induced intestinal permeability

doi: 10.1136/egastro-2024-100173

Figure Lengend Snippet: Figure 5 Intestinal CB1R in ALD and MASLD. CB1f/f and CB1IEC−/− mice (n=8/group) were subjected to the chronic-plus-binge (10d+1B) model. Serum ALT was measured as a marker of liver damage (A). CB1f/f and CB1IEC−/− mice (n=4–6/group) aged 6–8 weeks old were randomly fed with a ND or HFD for 14 weeks. Liver weight and ALT (B) as well as longitudinal body weight (BW) and ipGTT, OGTT and ITT (C) were measured. Representative staining of IBA1+ (macrophages) and S100A9+ (neutrophils) cells in livers from CB1f/f and CB1IEC−/− mice subjected to the 10d+1B model (D, E). Values represent mean±SEM; ****p<0.0001. ALD, alcohol-associated liver disease; ALT, alanine aminotransferase; CB1f/f, CB1 floxed/floxed; CB1IEC−/−, intestinal epithelial- specific CB1R; CB1R, cannabinoid receptor 1; EtOH, ethanol; HFD, high-fat diet; ipGTT, intraperitoneal glucose tolerance test; ITT, insulin tolerance test; MASLD, metabolic dysfunction-associated steatotic liver disease; ND, normal chow diet; OGTT, oral glucose tolerance test.

Article Snippet: Importantly, (S)MRI- 1891 did not induce anxiogenic behaviour at 3 mg/kg or even at a high dose of 30 mg/kg during chronic treatment, in contrast to the brain- penetrant CB1R antagonist rimonabant, which induced significant anxiety at a dose of 3 mg/kg.21 The mitogen activated protein kinase kinase 1/2 (MEK1/2) inhibitor U0126 (20 mg/kg; MedChemExpress) was delivered intraperitoneally 1.5 hours before the alcohol binge.

Techniques: Marker, Staining

Journal: eGastroenterology

Article Title: Gut cannabinoid receptor 1 regulates alcohol binge-induced intestinal permeability

doi: 10.1136/egastro-2024-100173

Figure Lengend Snippet: Figure 6 A schematic of the role of intestinal CB1R in alcohol-induced leaky gut. Alcohol binge increases intestinal permeability by activating intestinal epithelial CB1R-ERK1/2 signalling with subsequently reduced differentiation and downregulation of tight junctions (left). Genetic or pharmacological inhibition of intestinal CB1R-ERK1/2 signalling restored normal intestinal permeability and epithelial differentiation (right). Figure created with Biorender.com. CB1R, cannabinoid receptor 1; ERK1, mitogen-activated protein kinase 1/2.

Article Snippet: Importantly, (S)MRI- 1891 did not induce anxiogenic behaviour at 3 mg/kg or even at a high dose of 30 mg/kg during chronic treatment, in contrast to the brain- penetrant CB1R antagonist rimonabant, which induced significant anxiety at a dose of 3 mg/kg.21 The mitogen activated protein kinase kinase 1/2 (MEK1/2) inhibitor U0126 (20 mg/kg; MedChemExpress) was delivered intraperitoneally 1.5 hours before the alcohol binge.

Techniques: Permeability, Inhibition

Journal: Pediatric research

Article Title: Concentrations of remdesivir and GS-441524 in human milk from lactating individuals diagnosed with COVID-19.

doi: 10.1038/s41390-024-03053-2

Figure Lengend Snippet: Fig. 1 Remdesivir and GS-441524 concentrations determined in human human milk as measured by isotope dilution LC-MS/MS. The lower limit of quantification (LLOQ) of the assay for both parent and metabolite was set at 100 ng/mL in human milk. Orange and blue bars represent concentrations less than the LLOQ for each analyte. The total number of specimens tested is indicated on the x- axis below each individual analyte, while the number of specimens that were measured to be

Article Snippet: In adults, 48.6% of the remdesivir dose is recovered in urine as GS-441524.

Techniques: Isotope Dilution, Liquid Chromatography with Mass Spectroscopy

Journal: Molecules

Article Title: Methods of Increasing the Performance of Radionuclide Generators Used in Nuclear Medicine: Daughter Nuclide Build-Up Optimisation, Elution-Purification-Concentration Integration, and Effective Control of Radionuclidic Purity

doi: 10.3390/molecules19067714

Figure Lengend Snippet: Detection limits of photo peaks, L D (not corrected with detector counting efficiency and gamma ray yield) evaluated from a background spectrum of blank sample measured for 36,000 s on HP Ge detector coupled ORTEC gamma-ray spectrometer.

Article Snippet: Capintec dose calibrator and Ortec gamma-ray spectrometer coupled with HP Ge detector were used for radioactivity measurement.

Techniques: