Journal: European Thyroid Journal

Article Title: Histological, functional and transcriptomic alterations in the juvenile hippocampus in a mouse model of thyroid hormone resistance

doi: 10.1530/ETJ-21-0097

Figure Lengend Snippet: Spatial and temporal expression of Thra1 mRNA in mouse hippocampus by RNAscope. (A) RNAscope hybridization demonstrates the dynamic change of Thra1 mRNAs in mouse hippocampus during the embryonic period. Formalin-perfused, fixed mouse embryo (E10.5 and E14.5) and brain (E16.5, E18.5, P3, P7, P14, and P56) were dehydrated in gradient ethanol, transparent in xylene, embedded in paraffin, and cut at a setting of 5 µm in the sagittal plane. The cytoarchitecture of the hippocampus in b, d, f (100×) is corresponding to the red squares in the sagittal plane of brain (a, c, e, 10×), respectively. The cytoarchitecture of the hippocampus in h and i (100×) is corresponding to the red squares in the sagittal plane of brain (g, 10×), with h denoting CA1 and i denoting DG area. The red arrow shows the Thra1 mRNA hybridized to the probes. Scale bar: 200 μm for a, 1 mm for c, 100 μm for e, g, and 10 μm for b, d, f, h, i. (B) RNAscope hybridization demonstrates the dynamic change of Thra1 mRNAs in mouse hippocampus during the postnatal period. A1, B1, C1, and D1 show the overall structure of the hippocampus at P3, P7, P14, and P56. A2-A8, B2-B8, C2-C8, and D2-D8 show structure of the sublayers in the hippocampus at P3, P7, P14, and P56, respectively. Taking the hippocampal structure of P56 as an example, the names and boundaries of the layers of hippocampal CA and DG are marked in D1. The red arrow shows the Thra1 mRNA hybridized to the probes. E, embryonic day; P, postnatal day; CA1, cornu ammonis area 1; DG, dentate gyrus; so, stratum oriens; sp, stratum pyramidale; sr, stratum radiatum; slm, stratum lacunosum moleculare; mo, moleculare layer; sg, stratum granulosum; po, polymorph layer.

Article Snippet: The probes for Thra1 (nine synthetic oligonucleotides complementary to the nucleotide sequence 1836–2336 of Thra1 ) were provided by Advanced Cell Diagnostics (#531731, ACD, San Francisco, CA, USA), which only detect Thra1 and do not cross detect Thra2.

Techniques: Expressing, RNAscope, Hybridization

Journal: European Thyroid Journal

Article Title: Histological, functional and transcriptomic alterations in the juvenile hippocampus in a mouse model of thyroid hormone resistance

doi: 10.1530/ETJ-21-0097

Figure Lengend Snippet: Representative photomicrographs of Nissl-stained coronal sections of the hippocampus. (A) The cytoarchitecture of the hippocampus (A1-I1, 4×; A2-I2, 10×) at postnatal day (P) 0 (A–C), P7 (D–F), and P21 (G–I). A3-I3 and A4-I4 showing the cytoarchitecture of the DG and CA1 area of hippocampus, respectively, which is corresponding to the yellow rectangle area in A1-I1. The yellow arrow denotes subventricular zone. Scale bar: 200 μm for A1-I1; 100 μm for A2-I2; and 20 μm for A3-I3 and A4-I4. All the brain tissues were cut at a setting of 5 µm in the coronal plane, and n = 3 for each genotype male mice at P0, P7, and P21, respectively. (B) Quantitation of the hippocampal sublayer thickness and the cell numbers in the pyramidal cell layer of the CA1 and granular cell layer in the DG at postnatal day 21. The maximal coronal section at the hippocampal level (corresponding to −2.92 mm from Bregma in adult mice (Paxinos brain atlas)) was used to analyze the morphology of hippocampus. The measurement of the layer thickness and the cell number were performed in the region where the highest point of the outer arm of the DG area, and the corresponding CA1 area. Two slides of each mouse were selected for the measurement and cell count of each sublayer in the CA1 or DG region of the hippocampus, average as the final result of each mouse. Three male mice were used for this experiment for each genotype. The number of cells in CA1 pyramidal cell layer and DG granule cell layer were corrected for the area of the counting region: cell counts/mm 2 at 40× objective. Data were presented as the mean ± s.e.m . Statistical analysis was calculated by one-way ANOVA. *Compared with Thra +/+ , P < 0.05; #Compared with Thra E403X/+ , P < 0.05.

Article Snippet: The probes for Thra1 (nine synthetic oligonucleotides complementary to the nucleotide sequence 1836–2336 of Thra1 ) were provided by Advanced Cell Diagnostics (#531731, ACD, San Francisco, CA, USA), which only detect Thra1 and do not cross detect Thra2.

Techniques: Staining, Quantitation Assay, Cell Counting

Journal: European Thyroid Journal

Article Title: Histological, functional and transcriptomic alterations in the juvenile hippocampus in a mouse model of thyroid hormone resistance

doi: 10.1530/ETJ-21-0097

Figure Lengend Snippet: Impaired long-term potentiation. Long-term potentiation (LTP) was performed in the male Thra E403X/E403X mice ( n = 7) and male WT mice ( n = 5) at postnatal day 21. LTP was induced by HFS, measured as an increase in f-EPSP slope and amplitude, expressed as a percentage of the baseline of the f-EPSP slope and amplitude after HFS in mice. f-EPSP slope and amplitude were reduced in the Thra E403X/E403X group. Data are expressed as the mean ± s.e.m. *Compared with the Thra +/+ , P < 0.05. Statistical analysis was calculated by the Student’s t test. f-EPSP, feld excitatory postsynaptic potential; HFS, high-frequency stimulation; TET, tetanic high-frequency stimulation.

Article Snippet: The probes for Thra1 (nine synthetic oligonucleotides complementary to the nucleotide sequence 1836–2336 of Thra1 ) were provided by Advanced Cell Diagnostics (#531731, ACD, San Francisco, CA, USA), which only detect Thra1 and do not cross detect Thra2.

Techniques:

Journal: European Thyroid Journal

Article Title: Histological, functional and transcriptomic alterations in the juvenile hippocampus in a mouse model of thyroid hormone resistance

doi: 10.1530/ETJ-21-0097

Figure Lengend Snippet: Behavioral abnormalities of hippocampal origin. (A) Learning and memory performance in Morris Water Maze test. Morris water maze (MWM) test was performed in the Thra E403X/+ mice and Thra +/+ mice at 16 weeks. n = 11 for male Thra E403X/+ mice and male Thra +/+ mice, respectively. (A) (a) Time of escape latency. (b) Times of crossing of target quadrant and platform area in the spatial probe trial. Data are presented as mean ± s.e.m. .Statistical analysis was calculated by the Student’s t test. Compared with the Thra +/+ mice: *P < 0.05; ** P < 0.01; *** P < 0.001. (B) Anxiety-like behavior in Elevated zero maze test. Elevated Zero Maze Test was performed in the Thra E403X/+ mice and Thra +/+ mice at 16 weeks. n =11 for male Thra E403X/+ mice and male Thra +/+ mice, respectively. (B) (a) Total distance of traveling. (b) Latency to visit the open arms. (c) Percentage of freezing time. (d) Number of entries into the open arms. (e) Percentage of time spent in the open arms. (f) Number of rearing and head dipping events. Data are expressed as the mean ± s.e.m. Statistical analysis was calculated by the Student’s t test. Compared with the Thra +/+ mice: * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The probes for Thra1 (nine synthetic oligonucleotides complementary to the nucleotide sequence 1836–2336 of Thra1 ) were provided by Advanced Cell Diagnostics (#531731, ACD, San Francisco, CA, USA), which only detect Thra1 and do not cross detect Thra2.

Techniques:

Journal: European Thyroid Journal

Article Title: Histological, functional and transcriptomic alterations in the juvenile hippocampus in a mouse model of thyroid hormone resistance

doi: 10.1530/ETJ-21-0097

Figure Lengend Snippet: Transcriptomic changes induced by Thra E403X . (A) Volcano plot for differentially expressed gene mRNAs. (B) Hierarchical clustering analysis for differentially expressed gene mRNAs. Pheatmap is the abbreviation of pretty heatmap (a function of R). (C) Gene ontology analysis for differentially expressed gene mRNAs. (D) KEGG pathway analysis for differentially expressed gene mRNAs. The lower q-value indicates the more significant enrichment. Point size indicates DEG number (The bigger the ball, the more numbers the detected genes). Rich Factor refers to the value of enrichment factor, which is the quotient of foreground value (the number of DEGs) and background value (total gene amount). The larger the value, the more significant enrichment. Eight samples were codified as follows: HOM_3W, hippocampus sample from male Thra E403X/E403X mice at 3-week-old ( n = 4); WT_3 W, hippocampus sample from male Thra +/+ mice at 3-week-old ( n = 4).

Article Snippet: The probes for Thra1 (nine synthetic oligonucleotides complementary to the nucleotide sequence 1836–2336 of Thra1 ) were provided by Advanced Cell Diagnostics (#531731, ACD, San Francisco, CA, USA), which only detect Thra1 and do not cross detect Thra2.

Techniques:

Journal: European Thyroid Journal

Article Title: Histological, functional and transcriptomic alterations in the juvenile hippocampus in a mouse model of thyroid hormone resistance

doi: 10.1530/ETJ-21-0097

Figure Lengend Snippet: Validation by qPCR analyses. Relative mRNA levels of 25 DEGs were evaluated in Thra +/+ ( n = 4) and Thra E403X/ E403X mice ( n = 4). The results were calculated using the comparative Ct method formula 2−ΔΔCt method and normalized against the housekeeping gene Gapdh. Data were presented as the mean ± s.e.m. Thra +/+ control was set to 1. Statistical analysis was calculated by the Student’s t test. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The probes for Thra1 (nine synthetic oligonucleotides complementary to the nucleotide sequence 1836–2336 of Thra1 ) were provided by Advanced Cell Diagnostics (#531731, ACD, San Francisco, CA, USA), which only detect Thra1 and do not cross detect Thra2.

Techniques: Biomarker Discovery, Control

Journal: Nature structural & molecular biology

Article Title: Regulation of miRNA-mediated gene silencing by miRNA precursors

doi: 10.1038/nsmb.2862

Figure Lengend Snippet: Mmu-miR-151-5p cleaves E2f6 in the absence of a seed match. (a) Genomic locus of mmu-miR-151 encoded by a LINE2 repeat element. (b) Schematic of the binding site of mmu-miR-151-5p to E2f6 3′UTR. (c) Dual-luciferase reporter assay for the wildtype E2f6 3′UTR (wt) or other mutants (per 5p and mut 5p) in presence of miR-151-5p overexpression (sh-151-5p). (d) Western blot for E2f6in presence of sh-151-5p or a scrambled control (sh-scr). Actin serves as a loading control. Uncropped blot in . (e) E2f6 qPCR on miR-151-5p overexpression. Error bars, s.e.m. (n = 3 replicates). (f) Dual-luciferase reporter assay for E2f6 3′UTR or control ( Sox4 3′UTR) in presence of an increasing dosage of miR-151-5p inhibitor. (g) Dual-luciferase reporter assay in Ago2 −/− cells for E2f6 3′UTR in presence of sh-151-5p and a functional copy of Ago2 or cleavage deficient Ago2 (D597A) or Ago1. (h) 5′-RACE of E2f6 . Arrowhead in the E2f6 3′UTR sequence (schematic) indicates the 5′ end of majority of E2f6 cleavage products in mouse lung tissue. Agarose gel showing E2f6 cleaved products (shown by an asterisk) is shown in the top gel (uncropped gel in ). The bottom gel serves as a RACE reaction control to detect the presence of E2f6 and ARHGDIA cDNAs. (c,f, g) For reporter assays, normalization was done with respect to sh-scr. Error bars, s.e.m. (n = 2 biological replicates, each with 4 technical replicates), ns denotes not significant,*** P = 0.001 by two-tailed Student′s t test.

Article Snippet: Two micrograms of total RNA were reverse-transcribed using superscript II RT kit (Life technologies) and subjected to gene expression analyses using gene specific Taqman probes (Mm01270320_m1 for E2f6 and Mm03306373_pri for pri-miR-151).

Techniques: Binding Assay, Luciferase, Reporter Assay, Over Expression, Western Blot, Control, Functional Assay, Sequencing, Agarose Gel Electrophoresis, Two Tailed Test

Journal: Nature structural & molecular biology

Article Title: Regulation of miRNA-mediated gene silencing by miRNA precursors

doi: 10.1038/nsmb.2862

Figure Lengend Snippet: miR-151-3p suppresses E2f6 expression by binding to E2f6 3′UTR adjacent to where miR-151-5p binds. (a) Schematic of a putative binding site of the miR-151-3p to the E2f6 3′UTR region adjacent to where 5p strand binds, in both mice and humans. The seed regions (nucleotides 2-8) are indicated for both the 5p and 3p arms. (b) Dual-luciferase reporter assay for the wildtype E2f6 3′UTR (wt) or other mutants (mut 3p and seed 3p as shown in the schematic) in presence of a miR-151-3p overexpression (sh-151-3p) or a scrambled control(sh-scr). A reporter construct with deletion of the entire miR-151-3p binding site in E2f6 3′UTR was also included. (c) Dual-luciferase reporter assay in wildtype MEF and Ago2 −/− cells for E2f6 3′UTR in presence of sh-151-3p. For comparision, dual-luciferase reporter assay for E2f6 3′UTR in presence of sh-151-5p is also shown. (b,c) For reporter assays, normalization was done with respect to sh-scr. Error bars, s.e.m. (n = 2 biological replicates, each with 4 technical replicates).

Article Snippet: Two micrograms of total RNA were reverse-transcribed using superscript II RT kit (Life technologies) and subjected to gene expression analyses using gene specific Taqman probes (Mm01270320_m1 for E2f6 and Mm03306373_pri for pri-miR-151).

Techniques: Expressing, Binding Assay, Luciferase, Reporter Assay, Over Expression, Control, Construct

Journal: Nature structural & molecular biology

Article Title: Regulation of miRNA-mediated gene silencing by miRNA precursors

doi: 10.1038/nsmb.2862

Figure Lengend Snippet: Precursor miR-151 competes with the mature miR-151-5p for binding to E2f6 3′UTR. (a) Thermodynamics of pre-miR-151 binding to E2f6 . (b) Schematic of the stem-loop structure of the pre-miR-151 with the 5p arm (blue), 3p arm (purple) and two adenosines (green) substituted to guanosines (orange). (c) Northern analysis of miR-151 processing from pre-miR-151 overexpression plasmid (pEZX-151) or the double mutant form of pre-miR-151 (pEZX-DM). Let-7a serves as a loading control. (d) Dual-luciferase reporter assay for E2f6 3′UTR in presence of only the mature miR-151-5p (sh-miR-151-5p), or both the pre-miR-151 and mature miR-151-5p (pEZX-151 and pEZX-DM). (e) Schematic of the binding site of pre-miR-151 in E2f6 3′UTR and its modifications ( E2f6 3p del and E2f6 3p-5p swap). (f) In-vitro gel shift assay with radiolabed (denoted by an asterisk) synthetic pre-miR-151(I) or a control pre-miR-122 and increasing molar concentrations of wildtype E2f6 3′UTR (1, 10 and 100 nM) or its modified forms. (g) Dual-luciferase analysis for E2f6 3′UTR (wt), 3p del or 3p-5p swap reporters with pEZX-151 or pEZX-DM. (h) In-vitro gel shift assay of E2f6 3′UTR bound to miR-151-5p with increasing molar concentrations of a synthetic pre-miR-151 or a control pre-miR-122. Bands below the blue star and orange star represent radiolabeled pre-miR-151 and pre-miR-122 respectively. “*” denotes radiolabeled oligos. (d, g) For reporter assays, normalization was done with respect to a scrambled control (sh-scr). Error bars, s.e.m. (n = 3 biological replicates, each with 3 technical replicates). * P = 0.05, ** P = 0.01 by two-tailed Student′s t test.

Article Snippet: Two micrograms of total RNA were reverse-transcribed using superscript II RT kit (Life technologies) and subjected to gene expression analyses using gene specific Taqman probes (Mm01270320_m1 for E2f6 and Mm03306373_pri for pri-miR-151).

Techniques: Binding Assay, Northern Blot, Over Expression, Plasmid Preparation, Mutagenesis, Control, Luciferase, Reporter Assay, In Vitro, Gel Shift, Modification, Two Tailed Test

Journal: Nature structural & molecular biology

Article Title: Regulation of miRNA-mediated gene silencing by miRNA precursors

doi: 10.1038/nsmb.2862

Figure Lengend Snippet: Pre-miR-151 binds to E2f6 in vivo and may protect the E2f6 transcript in quiescent tissues. (a) Schematic of the ChIRP method used to pull-down E2f6 mRNA from mouse brain. Quantitative PCR of (b) E2f6 mRNA and a control Ctdnep1 mRNA, (c) pre-miR-151 and a control pre-miR-124, (d) mature miR-151-5p and a control miR-124, pulled down by the biotinylated tiling oligonucleotides against the E2f6 3′UTR or a control lacZ mRNA. Quantitative PCR of (e) E2f6 mRNA, (f) pri-miR-151, (g) mature miR-151-5p in quiescent and non-quiescent tissues. In each case ( e — g ), the data are presented as fold induction after normalization to the liver sample (value = 1). (h) Northern analysis of miR-151 processing in various tissues. The blot on the left was probed with a LNA probe against mature miR-151-5p as shown by the schematic above the blot. The primary or intermediate product in the miR-151 biogenesis pathway is indicated by an arrowhead (→) and the mature miR-151-5p is indicated by a circle (○). U6 serves as a loading control. The blot on the right was probed (sequence is provided in ) for a region just outside the annotated stem loop structure of mmu-miR-151 (as shown by the schematic above the blot). (i) Quantitative PCR analyses of E2f6 , pri-miR-151 and miR-151-5pduring differentiation of muscle cells (C2C12) ( b — g, i ) For qPCR data, error bars, s.e.m. (n = 2 biological replicates, each with 3 technical replicates).

Article Snippet: Two micrograms of total RNA were reverse-transcribed using superscript II RT kit (Life technologies) and subjected to gene expression analyses using gene specific Taqman probes (Mm01270320_m1 for E2f6 and Mm03306373_pri for pri-miR-151).

Techniques: In Vivo, Real-time Polymerase Chain Reaction, Control, Northern Blot, Sequencing

Journal: Arquivos Brasileiros de Cardiologia

Article Title: Congenital Heart Disease as a Warning Sign for the Diagnosis of the 22q11.2 Deletion

doi: 10.5935/abc.20140145

Figure Lengend Snippet: Demonstration of the deletion in region 22q11.2. A. FISH technique. B. MLPA technique.

Article Snippet: Commercial probes of unique sequences were used for the specific region in 22q11.2 (probes DGS/VCFS, TUPLE1 and N25 D22S75, Cytocell, Cambridge, UK) , and/or MLPA using various kits (P036-E1, P070-B2, P064-B3, MRC-Holland, Amsterdam, Netherlands - www.mlpa.com ).

Techniques:

Journal: Arquivos Brasileiros de Cardiologia

Article Title: Congenital Heart Disease as a Warning Sign for the Diagnosis of the 22q11.2 Deletion

doi: 10.5935/abc.20140145

Figure Lengend Snippet: Congenital heart diseases in 47 patients with the 22q11.2 deletion syndrome and the surgical corrections performed

Article Snippet: Commercial probes of unique sequences were used for the specific region in 22q11.2 (probes DGS/VCFS, TUPLE1 and N25 D22S75, Cytocell, Cambridge, UK) , and/or MLPA using various kits (P036-E1, P070-B2, P064-B3, MRC-Holland, Amsterdam, Netherlands - www.mlpa.com ).

Techniques:

Journal: Arquivos Brasileiros de Cardiologia

Article Title: Congenital Heart Disease as a Warning Sign for the Diagnosis of the 22q11.2 Deletion

doi: 10.5935/abc.20140145

Figure Lengend Snippet: Phenotypic characteristics of 60 patients with the 22q11.2 deletion syndrome

Article Snippet: Commercial probes of unique sequences were used for the specific region in 22q11.2 (probes DGS/VCFS, TUPLE1 and N25 D22S75, Cytocell, Cambridge, UK) , and/or MLPA using various kits (P036-E1, P070-B2, P064-B3, MRC-Holland, Amsterdam, Netherlands - www.mlpa.com ).

Techniques:

Journal: Arquivos Brasileiros de Cardiologia

Article Title: Congenital Heart Disease as a Warning Sign for the Diagnosis of the 22q11.2 Deletion

doi: 10.5935/abc.20140145

Figure Lengend Snippet: Main phenotypic characteristics of patients with the 22q11.2 deletion syndrome. A) Narrow palpebral fissure. B) Elongated face and/or nose. C) Thin lips.

Article Snippet: Commercial probes of unique sequences were used for the specific region in 22q11.2 (probes DGS/VCFS, TUPLE1 and N25 D22S75, Cytocell, Cambridge, UK) , and/or MLPA using various kits (P036-E1, P070-B2, P064-B3, MRC-Holland, Amsterdam, Netherlands - www.mlpa.com ).

Techniques:

Journal: Arquivos Brasileiros de Cardiologia

Article Title: Congenital Heart Disease as a Warning Sign for the Diagnosis of the 22q11.2 Deletion

doi: 10.5935/abc.20140145

Figure Lengend Snippet: Pictures showing evolving features of patients with the 22q11.2 deletion syndrome at different ages. A) Newborn with thin lips and dysplasic ears. These phenotypic features become more characteristic at school age. B) Newborn with facial dysmorphism (elongated face and nose, narrow palpebral fissure, thin lips). C) Infant with elongated face and nose more evident during development

Article Snippet: Commercial probes of unique sequences were used for the specific region in 22q11.2 (probes DGS/VCFS, TUPLE1 and N25 D22S75, Cytocell, Cambridge, UK) , and/or MLPA using various kits (P036-E1, P070-B2, P064-B3, MRC-Holland, Amsterdam, Netherlands - www.mlpa.com ).

Techniques:

Journal: Arquivos Brasileiros de Cardiologia

Article Title: Congenital Heart Disease as a Warning Sign for the Diagnosis of the 22q11.2 Deletion

doi: 10.5935/abc.20140145

Figure Lengend Snippet: Distribution of values of total lymphocytes, CD4 + , CD8 + and CD19 + in patients with the 22q11.2 deletion syndrome. A) Number of total lymphocytes. B) CD4 + count. C) CD8 + count. D) CD19 + count. Each dot ( • ) corresponds to an individual patient. Max: Maximum; Min: Mimum

Article Snippet: Commercial probes of unique sequences were used for the specific region in 22q11.2 (probes DGS/VCFS, TUPLE1 and N25 D22S75, Cytocell, Cambridge, UK) , and/or MLPA using various kits (P036-E1, P070-B2, P064-B3, MRC-Holland, Amsterdam, Netherlands - www.mlpa.com ).

Techniques: