Journal: Nature chemical biology

Article Title: The morphogen Sonic hedgehog inhibits its receptor Patched by a pincer grasp mechanism

doi: 10.1038/s41589-019-0370-y

Figure Lengend Snippet: a, Schematic of the various SHH proteins used in signalling assays. b, Two different amounts of each SHH variant from (h) were analyzed by immunoblotting. Full gels can be found on Supplementary Figure 7. c and d, Fold-increase in Gli1 mRNA (relative to the no SHH added condition) was used as a metric for signalling strength after treatment of NIH/3T3 cells with various concentrations of the indicated ligands. e, HH signalling strength at increasing concentrations of the N-terminal palmitoylated peptide (Palm-ShhN15) in the absence or presence (100 μM) of a cholesteroylated C-terminal peptide (ShhN7-chol). In c-e, the mean is depicted and error bars reflect standard deviation (n=4). Each experiment in c-e was repeated at least 3 times.

Article Snippet: ShhN7-chol (VAAKSGG-cholesterol), a cholesteroylated peptide containing the C-terminal 7 aars of human SHH, was kindly provided by Professor Herbert Waldmann.

Techniques: Variant Assay, Western Blot, Standard Deviation

Journal: Scientific Reports

Article Title: Global loss of acetylcholinesterase activity with mitochondrial complexes inhibition and inflammation in brain of hypercholesterolemic mice

doi: 10.1038/s41598-017-17911-z

Figure Lengend Snippet: High-cholesterol diet causes hypercholesterolemia, increases cholesterol level in brain and disrupts blood-brain barrier (BBB). Control (CON) mice received normal diet and the HCD group received high-cholesterol diet (at 5% w/w mixed with normal diet) for 12 weeks and sacrificed on the last day. ( A ) Cholesterol level in blood serum (n = 8). ( B ) Cholesterol level in brain. Brain cholesterol level was assayed histologically in paraformaldehyde fixed tissue sections following Schultz’s method. Photographs are representative sections showing the cortex (CX), striatum (NCP), hippocampus (HP) and substantia nigra (SN) from control and HCD mice. The blue or greenish-blue colour denotes cholesterol. The dark circles denote air bubbles formed as a result of acid (sulphuric acid: acetic acid) reaction. Photographs were taken at 4× magnification. ( C ) Estimation of brain cholesterol level. Cholesterol level was estimated from tissue homogenates of CX, NCP, HP and SN regions of brain by using kit (n = 6). ( D ) BBB disruption. Evans Blue dye (EBD) extraversion in brain tissues were analysed for possible disruption of BBB using a Spectrophotometric method (n = 4). The results are mean ± S.E.M. * P ≤ 0.05 or ** P ≤ 0.01 as compared to control. Data of cholesterol level in serum ( A ) and brain EBD were analyzed using an unpaired Student’s t-test ( D ), and brain cholesterol levels were analyzed using a two-way repeated measure ANOVA ( C ).

Article Snippet: Serum cholesterol estimation kit (CHOL, Autopak) was obtained from Siemens Ltd., India.

Techniques: Control, Disruption

Journal: Scientific Reports

Article Title: Global loss of acetylcholinesterase activity with mitochondrial complexes inhibition and inflammation in brain of hypercholesterolemic mice

doi: 10.1038/s41598-017-17911-z

Figure Lengend Snippet: Hypercholesterolemia reduces acetylcholinesterase (AChE) activity in brain. Animals were sacrificed and perfused with 10% glycerol on the last day of 12 weeks on normal diet (control; CON/CS) or high-cholesterol diet (HCD). 20 µm coronal serial sections of different brain regions were processed for AChE histoenzymology. Photographs are representative sections of AChE staining showing the cortex (CX; A1 and A2), striatum (NCP; B1 and B2), hippocampus (HP; C1 and C2) and substantia nigra (SN; D1 and D2) of CON and HCD mice. Photographs were taken at 4× magnification. Activity of AChE in CX (A3), NCP (B3), HP (C3) and SN (D3) were quantified using Ellman’s method. Intended brain regions were dissected out and processed for the estimation of AChE activity by taking absorbency at 412 nm (n = 6). AChE activity decreased significantly in all the regions. Data represent AChE activity in mu/mg protein; and mean ± SEM. * P ≤ 0.05 and ** P ≤ 0.01 as compared to CON of the respective regions of brain. P-values were calculated using a two-way repeated measure ANOVA.

Article Snippet: Serum cholesterol estimation kit (CHOL, Autopak) was obtained from Siemens Ltd., India.

Techniques: Activity Assay, Control, Staining

Journal: Scientific Reports

Article Title: Global loss of acetylcholinesterase activity with mitochondrial complexes inhibition and inflammation in brain of hypercholesterolemic mice

doi: 10.1038/s41598-017-17911-z

Figure Lengend Snippet: Hypercholesterolemia ( A ) diminishes mitochondrial complex-I activity and ( B ) elevates hydroxyl radical (•OH) level in brain. Mice were subjected to standard diet (control; CON) or high cholesterol diet (HCD) daily for 12 weeks. Animals were sacrificed by decapitation on the last day of treatment, different regions of brain, such as cortex (CX), striatum (NCP), hippocampus (HP) and substantia nigra (SN) were quickly dissected out and processed for specific activity assay of mitochondrial complex-I using NADH as a substrate and represented as nmol of NADH oxidized/min/mg protein (n = 6). For estimating the levels of •OH, animals were sacrificed 2 h post administration of salicylic acid (100 mg/kg; i.p.) on the last day of treatment. 2,3- and 2,5-dihydroxy benzoic acid (DHBA; •OH adducts of salicylate) formed were measured from tissue homogenates of intended brain regions using a sensitive HPLC-ECD method to measure the level of total DHBA. Data are expressed as pmol of DHBA per mg tissue. Data represented mean ± S.E.M. *P ≤ 0.05 as compared to CON (n = 6). A two-way repeated measures ANOVA was used to calculate P-values.

Article Snippet: Serum cholesterol estimation kit (CHOL, Autopak) was obtained from Siemens Ltd., India.

Techniques: Activity Assay, Control

Journal: Scientific Reports

Article Title: Global loss of acetylcholinesterase activity with mitochondrial complexes inhibition and inflammation in brain of hypercholesterolemic mice

doi: 10.1038/s41598-017-17911-z

Figure Lengend Snippet: Hypercholesterolemia diminishes mitochondrial complex-II activity in brain. Representative photographs of histoenzymological staining of mitochondrial complex-II activity in cortex (CX; A1 and A2), striatum (NCP; B1and B2), hippocampus (HP; C1 and C2) and substantia nigra (SN; D1 and D2) regions of brain of control (CON/CS) and high-cholesterol diet (HCD) groups of mice. Photographs were taken at 4× magnification. Densitometric analysis of the photographs of complex-II staining (A3,B3,C3,D3). Photographs of four serial sections of CX (A3), NCP (B3), HP (C3) and SN (D3) of each group (n = 4) were analyzed using ImageJ software for determination of optical density. Data represented mean ± S.E.M. *P ≤ 0.05 as compared to CON. P-values were calculated using an unpaired Student’s t-test (two-tailed).

Article Snippet: Serum cholesterol estimation kit (CHOL, Autopak) was obtained from Siemens Ltd., India.

Techniques: Activity Assay, Staining, Control, Software, Two Tailed Test

Journal: Scientific Reports

Article Title: Global loss of acetylcholinesterase activity with mitochondrial complexes inhibition and inflammation in brain of hypercholesterolemic mice

doi: 10.1038/s41598-017-17911-z

Figure Lengend Snippet: Hypercholesterolemia diminishes mitochondrial complex-III activity in brain. Representative photographs of histoenzymological staining of complex-III activity in cortex (CX; A 1 and A 2), striatum (NCP; B1 and B2), hippocampus (HP; C 1 and C 2) and substantia nigra (SN; D 1 and D 2) of control (CON/CS) and high-cholesterol diet (HCD) groups of mice. Photographs were taken at 4× magnification. Densitometric analysis of the photographs of complex-III staining (A3,B3,C3,D3). Optical density was analyzed from four serial sections of CX (A3), NCP (B3), HP (C3) and SN (D3) regions of brain of each group (n = 4). Data represented mean ± S.E.M. *P ≤ 0.05 as compared to CON. An unpaired Student’s t-test (two-tailed) was used to calculate P-values.

Article Snippet: Serum cholesterol estimation kit (CHOL, Autopak) was obtained from Siemens Ltd., India.

Techniques: Activity Assay, Staining, Control, Two Tailed Test

Journal: Scientific Reports

Article Title: Global loss of acetylcholinesterase activity with mitochondrial complexes inhibition and inflammation in brain of hypercholesterolemic mice

doi: 10.1038/s41598-017-17911-z

Figure Lengend Snippet: Hypercholesterolemia causes inflammation in glia. Animals were sacrificed and perfused with 4% PFA following 12 weeks (84 th day) on normal diet (control; CON) or high-cholesterol diet (HCD). The coronal sections passing through CX ( A , B ), NCP ( C , D ), HP CA1 (E1,F1), HP CA3 (E2,F2), HP DG (E3,F3) and SN ( G–H ) were processed for Glial fibrillary acidic protein (GFAP)-immunoreactivity. GFAP-reactivity was increased in all the brain regions of hypercholesterolemic mice ( B , B 1, D , D 1, F , F 1– F 3, H , H 1) compared to the corresponding brain regions of control mice ( A , A 1, C , C 1, E , E 1– E 3, G,G1 ), which signifies astrocytosis due to inflammation. Photographs were taken at 4× and 20× magnification. CX = cortex; NCP = striatum; HP = hippocampus; SN = substantia nigra; CA = Cornus ammonis; DG = Dentate Gyrus.

Article Snippet: Serum cholesterol estimation kit (CHOL, Autopak) was obtained from Siemens Ltd., India.

Techniques: Control