Journal: Biomolecules

Article Title: Epileptic Encephalopathy GABRB Structural Variants Share Common Gating and Trafficking Defects.

doi: 10.3390/biom13121790

Figure Lengend Snippet: Figure 3. GABRB3 mutations altered the stability of GABAA receptors. (A) Cryo-EM structure of the pentameric α1β3γ2 GABAA receptor (6HUP) viewed from the side, with the β subunits in red, the α subunits in blue, and the γ subunit in gray. GABRB3 mutations are mapped onto the structure and represented as green sticks and numbered according to the panels in (B). Network of interatomic interactions predicted by wild-type (wt, left panels) and β3 mutant (mut, right panels) 6HUP receptors. The panels are numbered according to the labels in panel (A), where the mutations are mapped. wt and mut amino acids are colored in light green, and the neighborhood residues are in CPK representation. All residues are represented as sticks. In brackets, the residues are numbered based on the 6HUP structure. For correspondence with the GABRB3 protein sequence deposited in UniProt-P28472, 25 must be added to the number indicated on the panel. Structural domains involved in the network of interactions are labeled, and residues are equally colored. ∆∆G values are shown on the corresponding panels.

Article Snippet: The primary antibodies used to detect GABAA receptors were as follows: mouse anti-α1 subunit antibody (1:500; NeuroMab, 75-136, Davis, CA, USA), rabbit anti-β3 subunit antibody (1:500; Novus, NB300199, St. Louis, MO, USA), rabbit anti-β2 subunit antibody (1:1000; Millipore, AB5561, Burlington, MA, USA), and rabbit anti-γ2 subunit antibody (1:1000; Millipore, AB5559).

Techniques: Cryo-EM Sample Prep, Mutagenesis, Sequencing, Labeling

Journal: Biomolecules

Article Title: Epileptic Encephalopathy GABRB Structural Variants Share Common Gating and Trafficking Defects.

doi: 10.3390/biom13121790

Figure Lengend Snippet: Figure 5. GABRB3 mutations altered the flexibility and function of GABAA receptors. (A) Structural representation of the GABAA receptor flexible conformation based on the vibrational entropy energy change (∆∆SVib ENCoM) predicted between the wild-type and the mutant β3 subunit in the 6HUP structure. Mutant β3 subunits are colored according to the vibrational entropy perturbation upon mutation. Shades of blue represent a loss of flexibility (rigidification of the structure), and shades of red mean an increase in structural flexibility. ∆∆SVib ENCoM values are shown at the bottom of the corresponding mutation. In brackets, the variants are numbered based on the 6HUP structure. (B) In the upper panel, concentration–response curves of wild-type and mutant α1β3γ2 receptors are shown. GABA-evoked currents were normalized to the maximal response to 1 mM GABA. Lower panel, Pearson correlation coefficients obtained from the ∆∆G of the mutant β3 structures were plotted against surface (left graph) and total (right graph) expression levels of α1, β3, and γ2 subunits according to the data displayed in panels (C,D), respectively. Lines represent linear regression fits. (C,D) Surface and total expression levels of wild-type and mutant β3 subunits coexpressed with α1 and γ2 subunits, respectively.

Article Snippet: The primary antibodies used to detect GABAA receptors were as follows: mouse anti-α1 subunit antibody (1:500; NeuroMab, 75-136, Davis, CA, USA), rabbit anti-β3 subunit antibody (1:500; Novus, NB300199, St. Louis, MO, USA), rabbit anti-β2 subunit antibody (1:1000; Millipore, AB5561, Burlington, MA, USA), and rabbit anti-γ2 subunit antibody (1:1000; Millipore, AB5559).

Techniques: Mutagenesis, Concentration Assay, Expressing

Journal: Acta Physiologica (Oxford, England)

Article Title: Reversal of Diabesity Through Modulating Sympathetic Inputs to Adipose Tissue Following Carotid Body Resection

doi: 10.1111/apha.70074

Figure Lengend Snippet: Ablation of carotid body (CB) activity through carotid sinus nerve (CSN) resection heightens visceral white adipose tissue (WAT) metabolism in obese dysmetabolic rodents. (A) Panel shows a schematic illustration of the protocol used to evaluate mice WAT oxygen consumption rate (OCR). From (B) to (H) is described the effect of high fat (HF) diet and of CSN resection on the: (B) OCR per minute, reflecting adipose tissue metabolism, before and after stimulation with norepinephrine [15 μM] (left panel) or dopamine [100 nM] (right panel) in mice (3 pieces of tissue from 4–6 animals); (C) average basal OCR in mice (15–27 pieces of tissue from 4–6 animals) before the stimulation with norepinephrine and dopamine; (D) average OCR after stimulation with norepinephrine [15 μM] or dopamine [100 nM] (3 pieces of tissue from 4–6 animals); (E) Illustration of the molecular markers involved in brown adipocytes differentiation as well as the stimuli involved in the beiging of WAT; (F) average expression of PGC1α (92 kDa) ( n = 3–5); (G) average expression of PPARγ (53‐57 kDa) on visceral WAT of rats and mice ( n = 4–5)—representative western blots are shown on the top of the graphs; and (H) percentage of UCP1 protein labeled cells and percentage of mitotrackerTM Red CMXRos labeled cells (top panels) in the perienteric depot ( n = 4–5) in rats and mice; right panels show representative images of UCP1 and MitotrackerTM Red CMXRos labeled cells, Green—UCP1 labeled adipocytes; Red—MitotrackerTM Red CMXRos labeled adipocytes; Blue—DAPI labeled nuclei of the adipocytes; Yellow—Merge of UCP1 and MitotrackerTM Red CMXRos labeled cells. Gray and blue colors represent, respectively, normal chow (NC) and HF rats. Gray and red colors show NC and HF mice, respectively. Den—means animals submitted to CSN denervation/resection. Bars represent mean values ± SEM. Two‐Way ANOVA with Bonferroni multicomparison test. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 comparing NC vs. HF groups, # p < 0.05, ## p < 0.01, ### p < 0.001 and ### p < 0.0001 comparing values with and without CSN resection.

Article Snippet: After 1 h of blocking in milk, the membranes were incubated overnight at 4°C with the primary antibodies against β2 receptors (1:200; 47 kDa; Alomone, Jerusalem, Israel), β3 receptors (1:200; 45KDa; Alomone, Jerusalem, Israel), D1R (1:200; 48 kDa; Abcam, Cambridge, UK), D2R (1:200; 49 kDa, Sigma‐Aldrich, Madrid, Spain), Dopamine β hydroxylase (DβH) (1:1000; Merck, Darmstadt, Germany), HSL (1:1000; 83 KDa; Cell Signaling Technology, Massachusetts, EUA), pAMPK (phospho Thr172) (1:1000; 60 kDa; Cell Signaling Technology, Massachusetts, EUA), pATGL (phospho S406) (1:1000; 55 kDa; Abcam, Cambridge, UK), PGC‐1α (1:1000; 92 kDa; Santa Cruz Biotechnology INC, Texas, EUA), PPARγ (1:1000; 53–57 kDa, Cell Signaling Technology, Massachusetts, EUA), TH (1:1000; 60 kDa; Abcam, Cambridge, UK).

Techniques: Activity Assay, Expressing, Western Blot, Labeling

Journal: Acta Physiologica (Oxford, England)

Article Title: Reversal of Diabesity Through Modulating Sympathetic Inputs to Adipose Tissue Following Carotid Body Resection

doi: 10.1111/apha.70074

Figure Lengend Snippet: Carotid body (CB) modulates lipid fluxes in white adipose tissue (WAT) in rodents. (A) Illustration of the molecular markers involved in adipose tissue energy expenditure. From (B) to (F) is described the effect of high fat (HF) diet and of CSN resection on: (B) Rg′ values, reflecting glucose uptake on WAT depots in rats ( n = 4–7); (C) WAT average expression of HSL (83 kDa) on WAT of rats ( n = 5–8); (D) WAT average expression of phosphorylated ATGL (pATGL) (55 kDa) in rats ( n = 5–7)—representative western blots are shown on the top of the graphs; (E) glycerol levels, as an index of lipolysis ( n = 6–8) in WAT in rats; (F) WAT average expression of phosphorylated AMPK (pAMPK) (60 kDa) in rats and mice ( n = 3–6)—representative western blots are shown on the top of the graphs. Gray and blue colors represent, respectively, normal chow (NC) and HF diet rats. Gray and red colors show NC and HF mice, respectively. Den—means animals submitted to CSN denervation/resection. Bars represent mean values ± SEM. Two‐Way ANOVA with Bonferroni multicomparison test. ** p < 0.01 comparing NC vs. HF groups; # p < 0.05, ## p < 0.01 and ### p < 0.001 comparing values with and without CSN resection.

Article Snippet: After 1 h of blocking in milk, the membranes were incubated overnight at 4°C with the primary antibodies against β2 receptors (1:200; 47 kDa; Alomone, Jerusalem, Israel), β3 receptors (1:200; 45KDa; Alomone, Jerusalem, Israel), D1R (1:200; 48 kDa; Abcam, Cambridge, UK), D2R (1:200; 49 kDa, Sigma‐Aldrich, Madrid, Spain), Dopamine β hydroxylase (DβH) (1:1000; Merck, Darmstadt, Germany), HSL (1:1000; 83 KDa; Cell Signaling Technology, Massachusetts, EUA), pAMPK (phospho Thr172) (1:1000; 60 kDa; Cell Signaling Technology, Massachusetts, EUA), pATGL (phospho S406) (1:1000; 55 kDa; Abcam, Cambridge, UK), PGC‐1α (1:1000; 92 kDa; Santa Cruz Biotechnology INC, Texas, EUA), PPARγ (1:1000; 53–57 kDa, Cell Signaling Technology, Massachusetts, EUA), TH (1:1000; 60 kDa; Abcam, Cambridge, UK).

Techniques: Expressing, Western Blot

Journal: Acta Physiologica (Oxford, England)

Article Title: Reversal of Diabesity Through Modulating Sympathetic Inputs to Adipose Tissue Following Carotid Body Resection

doi: 10.1111/apha.70074

Figure Lengend Snippet: Carotid sinus nerve (CSN) resection improves brown adipose tissue (BAT) metabolism in rodents. Effect of high fat (HF) diet and of CSN resection on: (A) Curves of oxygen consumption rate (OCR) per minute, reflecting adipose tissue metabolism, before and after stimulation with norepinephrine [15 μM] (left panel) or dopamine [100 nM] (right panel) in the BAT of mice ( n = 18–27 pieces of tissue from 6–8 animals). (B) Average basal OCR in the BAT in mice ( n = 18–27 pieces of tissue from 6–8 animals) before the stimulation with norepinephrine and dopamine; (C) Average OCR after stimulation with norepinephrine [15 μM] or dopamine [100 nM] ( n = 5–8 animals); (D) percentage of UCP1 protein labeled cells and percentage of mitotrackerTM Red CMXRos labeled cells (top panels) in BAT ( n = 4–5) in rats and mice; bottom panels show representative images of UCP1 and MitotrackerTM Red CMXRos labeled cells, Green—UCP1 labeled adipocytes; Red—MitotrackerTM Red CMXRos labeled adipocytes; Blue—DAPI labeled nuclei of the adipocytes; Yellow—Merge of UCP1 and MitotrackerTM Red CMXRos labeled cells; (E) Rg′ values, reflecting glucose uptake on BAT depots in rats ( n = 5–7); (F) BAT average expression of pATGL (55 kDa) in rats ( n = 4–5); (G) average expression of pAMPK (60 kDa) on BAT of rats and mice ( n = 3–8)—representative western blots are shown on the top of the graphs. Gray and blue colors represent, respectively, normal chow (NC) and HF diet rats. Gray and red colors show NC and HF mice, respectively. Den—means animals submitted to CSN denervation/resection. Bars represent mean values ± SEM. Two‐Way ANOVA with Bonferroni multicomparison test. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 comparing NC vs. HF groups; # p < 0.05, ## p < 0.01, ### p < 0.001 and #### p < 0.0001 comparing values with and without CSN resection.

Article Snippet: After 1 h of blocking in milk, the membranes were incubated overnight at 4°C with the primary antibodies against β2 receptors (1:200; 47 kDa; Alomone, Jerusalem, Israel), β3 receptors (1:200; 45KDa; Alomone, Jerusalem, Israel), D1R (1:200; 48 kDa; Abcam, Cambridge, UK), D2R (1:200; 49 kDa, Sigma‐Aldrich, Madrid, Spain), Dopamine β hydroxylase (DβH) (1:1000; Merck, Darmstadt, Germany), HSL (1:1000; 83 KDa; Cell Signaling Technology, Massachusetts, EUA), pAMPK (phospho Thr172) (1:1000; 60 kDa; Cell Signaling Technology, Massachusetts, EUA), pATGL (phospho S406) (1:1000; 55 kDa; Abcam, Cambridge, UK), PGC‐1α (1:1000; 92 kDa; Santa Cruz Biotechnology INC, Texas, EUA), PPARγ (1:1000; 53–57 kDa, Cell Signaling Technology, Massachusetts, EUA), TH (1:1000; 60 kDa; Abcam, Cambridge, UK).

Techniques: Labeling, Expressing, Western Blot

Journal: Acta Physiologica (Oxford, England)

Article Title: Reversal of Diabesity Through Modulating Sympathetic Inputs to Adipose Tissue Following Carotid Body Resection

doi: 10.1111/apha.70074

Figure Lengend Snippet: Ablation of carotid body (CB) activity through carotid sinus nerve (CSN) resection restores sympathetic inputs in visceral white adipose tissue (WAT). (A) schematic representation of the catecholamine biosynthesis pathways; (B) effect of HF diet and CSN resection on the levels of dopamine + DOPAC, norepinephrine and epinephrine in the WAT. From (C) to (E)—Sympathetic innervation in the WAT presented by: (C) WAT average tyrosine hydroxylase (TH) levels (60 kDa) in rats and mice ( n = 5–7); (D) WAT average dopamine β hydroxylase (DβH) levels (75 kDa) in rats; (E) from the left to the right: Intensity and fibers volume of TH immunolabeling ( n = 4–5) in WAT of rats—representative images are shown at the right panel, for animated gif of the images consult the videos White adipose tissue (WAT ); (F) average β2 receptors (β2R, 47 kDa) in rats (left panel) and β3 receptors (β3R, 45 kDa) in rats and mice (right panel) ( n = 3–7); and (G) average dopamine type 1 receptors (D1R, 48 kDa) in rats (left panel) and dopamine type 2 receptors (D2R, 49 kDa) in rats and mice (left panel) ( n = 4–6). Representative western blots are shown on the top of the graphs. Gray and blue colors represent, respectively, normal chow (NC) and HF diet rats. Gray and red colors show NC and HF mice, respectively. Bars represent mean values ± SEM. Two‐Way ANOVA with Bonferroni multicomparison test. * p < 0.05, ** p < 0.01 and **** p < 0.0001 comparing NC vs. HF groups; # p < 0.05; ## p < 0.01; ### p < 0.001 and #### p < 0.0001 comparing values with and without CSN resection.

Article Snippet: After 1 h of blocking in milk, the membranes were incubated overnight at 4°C with the primary antibodies against β2 receptors (1:200; 47 kDa; Alomone, Jerusalem, Israel), β3 receptors (1:200; 45KDa; Alomone, Jerusalem, Israel), D1R (1:200; 48 kDa; Abcam, Cambridge, UK), D2R (1:200; 49 kDa, Sigma‐Aldrich, Madrid, Spain), Dopamine β hydroxylase (DβH) (1:1000; Merck, Darmstadt, Germany), HSL (1:1000; 83 KDa; Cell Signaling Technology, Massachusetts, EUA), pAMPK (phospho Thr172) (1:1000; 60 kDa; Cell Signaling Technology, Massachusetts, EUA), pATGL (phospho S406) (1:1000; 55 kDa; Abcam, Cambridge, UK), PGC‐1α (1:1000; 92 kDa; Santa Cruz Biotechnology INC, Texas, EUA), PPARγ (1:1000; 53–57 kDa, Cell Signaling Technology, Massachusetts, EUA), TH (1:1000; 60 kDa; Abcam, Cambridge, UK).

Techniques: Activity Assay, Immunolabeling, Western Blot

Journal: Acta Physiologica (Oxford, England)

Article Title: Reversal of Diabesity Through Modulating Sympathetic Inputs to Adipose Tissue Following Carotid Body Resection

doi: 10.1111/apha.70074

Figure Lengend Snippet: Carotid body (CB) regulation of norepinephrine (NE) action on β3 adrenergic receptor is key to brown adipose tissue (BAT) metabolic function. (A) effect of high fat (HF) diet and carotid sinus nerve (CSN) resection on the levels of dopamine + DOPAC, NE and epinephrine in the BAT. From (B) to (D): Sympathetic innervation to BAT assessed as: (B) BAT average TH expression (60 kDa) in rats and mice ( n = 4–6); (C) BAT average D𝛃H levels (60 kDa) in rats and mice ( n = 4–6); (D) BAT average 𝛃2 receptors (𝛃2R, 47 kDa) in rats (left panel) and 𝛃3 receptors (𝛃3R, 45 kDa) in rats and mice (right panel) ( n = 3–7); Representative western blots are shown on the top of the graphs. Gray and blue colors represent, respectively, normal chow (NC) and HF diet rats. Gray and red colors show NC and HF mice, respectively. Den—means animals submitted to CSN denervation/resection. Bars represent mean values ± SEM. Two‐Way ANOVA with Bonferroni multicomparison test. * p < 0.05; ** p < 0.01 and *** p < 0.001 comparing NC vs. HF groups; # p < 0.05 and ## p < 0.01 comparing values with and without CSN resection.

Article Snippet: After 1 h of blocking in milk, the membranes were incubated overnight at 4°C with the primary antibodies against β2 receptors (1:200; 47 kDa; Alomone, Jerusalem, Israel), β3 receptors (1:200; 45KDa; Alomone, Jerusalem, Israel), D1R (1:200; 48 kDa; Abcam, Cambridge, UK), D2R (1:200; 49 kDa, Sigma‐Aldrich, Madrid, Spain), Dopamine β hydroxylase (DβH) (1:1000; Merck, Darmstadt, Germany), HSL (1:1000; 83 KDa; Cell Signaling Technology, Massachusetts, EUA), pAMPK (phospho Thr172) (1:1000; 60 kDa; Cell Signaling Technology, Massachusetts, EUA), pATGL (phospho S406) (1:1000; 55 kDa; Abcam, Cambridge, UK), PGC‐1α (1:1000; 92 kDa; Santa Cruz Biotechnology INC, Texas, EUA), PPARγ (1:1000; 53–57 kDa, Cell Signaling Technology, Massachusetts, EUA), TH (1:1000; 60 kDa; Abcam, Cambridge, UK).

Techniques: Expressing, Western Blot