Journal: Cell Reports

Article Title: Microglia contribute to the postnatal development of cortical somatostatin-positive inhibitory cells and to whisker-evoked cortical activity

doi: 10.1016/j.celrep.2022.111209

Figure Lengend Snippet: CX3CL1 and CXCL12 are key molecular players of microglia-dependent development of SST + cells (A) Schematics of the in silico screening. (B) Lists of putative interactors. Line thickness is proportional to STRING confidence score (a score of 1,000 indicates the highest confidence of interaction). (C) Cx3cl1 and Cxcl12 expression levels (RNA sequencing data from Favuzzi et al., 2019 ). (D) Schematics of the subpial electroporation protocol and timeline. Below are the maps of the plasmids. (E) Examples of parts of traced axons (scale bar: 12.5 μm). Puncta density is calculated as total number of counted puncta divided by total length of traced axons per cell (each dot is a cell; CTRL n = 8, Cx3cl1 cKO n = 11, Cxcl12 cKO n = 12; t test: CTRL versus Cx3cl1 cKO p = 0.55, CTRL versus Cxcl12 cKO p = 0.29; one-way ANOVA test p = 0.53). (F) Examples of traced cells (axons in red, dendrites in blue). Below is the total length of traced axons (each dot is a cell; CTRL n = 11, Cx3cl1 cKO n = 9, t test p = 0.019) and Sholl analysis (t test p values are plotted below; two-way ANOVA RM test from 8 to 200 μm p = 0.018). (G) Examples of traced cells. Below is the total length of traced axons (CTRL n = 11, Cxcl12 cKO n = 12, t test p = 0.08) and Sholl analysis (t test p-values are plotted below; two-way ANOVA RM test from 90 to 250 μm p = 0.05). (H) Examples of traced cells. Below is the total length of traced axons (CTRL + PLX n = 12, Cx3cl1 cKO + PLX n = 9, Cxcl12 cKO + PLX n = 12; t test: CTRL + PLX versus Cx3cl1 cKO + PLX p = 0.43, CTRL + PLX versus Cxcl12 cKO + PLX p = 0.37; one-way ANOVA test p = 0.27) and Sholl analysis (t test p values are plotted below; two-way ANOVA RM test from 8 to 300 μm p = 0.3). (I) Schematics of the subpial electroporation protocol. Below are the maps of the plasmids. (J) An example of an electroporated cell at P15 (scale bar: 50 μm; pia surface is indicated by the dotted line). (K) Schematics of the optogenetics experiment. (L) Average traces of maximum amplitude of evoked IPSCs. (M) Peak amplitude quantification (each dot is a cell; CTRL n = 6 cells from 4 mice, Cx3cl1 cKO n = 11 cells from 6 mice, Cxcl12 cKO n = 7 cells from 4 mice; t test: CTRL versus Cx3cl1 cKO p = 0.79, CTRL versus Cxcl12 cKO p = 0.29; one-way ANOVA test p = 0.7). (N) Quantification of the number of pyramidal neurons responding to a single activated SST + cell (each dot is the percentage of connected pyramidal neurons per single stimulated SST + cell; CTRL n = 9, Cx3cl1 cKO n = 8, Cxcl12 cKO n = 7; Wilcoxon test: CTRL versus Cx3cl1 cKO p = 0.004, CTRL versus Cxcl12 cKO p = 0.26; Kruskal-Wallis test p = 0.032). All data are represented as mean ± SEM, and the exact values are reported in Table S1 . See also Figure S2 .

Article Snippet: Cx3cl1 target DNA amplification RV: 5′-GACAGGAAACGCTGGACTTC-3′ , Microsynth AG , N/A.

Techniques: In Silico, Expressing, RNA Sequencing Assay, Electroporation, Optogenetics

Journal: Cell Reports

Article Title: Microglia contribute to the postnatal development of cortical somatostatin-positive inhibitory cells and to whisker-evoked cortical activity

doi: 10.1016/j.celrep.2022.111209

Figure Lengend Snippet:

Article Snippet: Cx3cl1 target DNA amplification RV: 5′-GACAGGAAACGCTGGACTTC-3′ , Microsynth AG , N/A.

Techniques: Recombinant, Blocking Assay, Amplification, DNA Sequencing, Software

Journal: Nucleic Acids Research

Article Title: Structural insights into the DNA recognition mechanism by the bacterial transcription factor PdxR

doi: 10.1093/nar/gkad552

Figure Lengend Snippet: Cryo-EM structures of the PLP-bound PdxR in complex with its target DNA. ( A ) Representative cryo-EM micrograph for the holo-PdxR–DNA complex. ( B ) Representative 2D class averages of the open, the half-closed, and the closed conformer of the complex as calculated with Relion . ( C ) Side and top views of the cryo-EM 3D reconstructions of the holo-PdxR–DNA complex.

Article Snippet: Various DNA fragments encompassing the wild type and modified PdxR target sequences were designed and purchased from Eurofins Genomics (Ebersberg, Germany) or Metabion International AG (Steinkirchen, Germany) as lyophilized forward and reverse single-stranded oligonucleotides (sequences are listed and schematically displayed in ).

Techniques: Cryo-EM Sample Prep

Journal: Nucleic Acids Research

Article Title: Structural insights into the DNA recognition mechanism by the bacterial transcription factor PdxR

doi: 10.1093/nar/gkad552

Figure Lengend Snippet: Sequence-specific and non-specific interactions guide the holo-PdxR–DNA recognition. ( A ) The wHTH domain forms specific interaction with the target DNA. Representative ribbon depiction of the wHTH domain of the holo-PdxR–DNA in the closed conformation (light pink). Residues interacting with the DNA are represented as sticks, labelled, and differently colored according to the secondary structure element they belong to (α(H)2, green; α(H)3, orange; w = wing, red). DNA is shown as gray ribbon. ( B ) Schematic summary of the holo-PdxR–DNA main contacts formed through the wHTH domain. The different distribution of the identified contacts on motifs 1 and 3 is likely due to coordinate uncertainties associated with the local resolution of the EM maps. ( C ) Non-specific contacts formed with the AAT-like domain. Basic residues of the PdxR AAT-like domain interacting with the DNA backbone are highlighted and represented in dark and light pink sticks, depending on the PdxR monomer. In all panels motifs 1, 2, and 3 are colored in dark green, blue and light green respectively.

Article Snippet: Various DNA fragments encompassing the wild type and modified PdxR target sequences were designed and purchased from Eurofins Genomics (Ebersberg, Germany) or Metabion International AG (Steinkirchen, Germany) as lyophilized forward and reverse single-stranded oligonucleotides (sequences are listed and schematically displayed in ).

Techniques: Sequencing

Journal: Nucleic Acids Research

Article Title: Structural insights into the DNA recognition mechanism by the bacterial transcription factor PdxR

doi: 10.1093/nar/gkad552

Figure Lengend Snippet: DNA structural dynamics upon holo-PdxR binding. ( A ) The predicted curvature of the 48-bp DNA containing PdxR target sequences generated by (C. Gohlke, www.lfd.uci.edu/∼gohlke/dnacurve/ ) is shown. ( B ) Side and top views of the holo-PdxR–DNA complex in the open (top), half-closed (middle), and closed (bottom) conformers. In both panels the DNA molecule is depicted in gray ribbon representation. PdxR cognate binding sites are colored in dark green (motif 1) and light green (motif 3); motif 2 is colored in blue. The protein is represented as white transparent ribbons.

Article Snippet: Various DNA fragments encompassing the wild type and modified PdxR target sequences were designed and purchased from Eurofins Genomics (Ebersberg, Germany) or Metabion International AG (Steinkirchen, Germany) as lyophilized forward and reverse single-stranded oligonucleotides (sequences are listed and schematically displayed in ).

Techniques: Binding Assay, Generated

Journal: Nucleic Acids Research

Article Title: Structural insights into the DNA recognition mechanism by the bacterial transcription factor PdxR

doi: 10.1093/nar/gkad552

Figure Lengend Snippet: DNA binding experiments and mutational analysis. The K Dapp values are the mean ± SEM of at least three independent fluorescence binding assays. For those fragments whose binding could not be detected, K D was not determined (nd). The experiments were performed using the holo form of PdxR (PdxR) variants

Article Snippet: Various DNA fragments encompassing the wild type and modified PdxR target sequences were designed and purchased from Eurofins Genomics (Ebersberg, Germany) or Metabion International AG (Steinkirchen, Germany) as lyophilized forward and reverse single-stranded oligonucleotides (sequences are listed and schematically displayed in ).

Techniques: Binding Assay, Fluorescence

Journal: Nucleic Acids Research

Article Title: Structural insights into the DNA recognition mechanism by the bacterial transcription factor PdxR

doi: 10.1093/nar/gkad552

Figure Lengend Snippet: The multi-step mechanism of binding of holo-PdxR to DNA. Scheme of the binding mechanism of holo-PdxR–DNA. The process involves conformational changes of both binding partners, which are allosterically driven by electrostatic interactions. The two subunits of the PdxR dimer are shown in dark and light gray, the linker is represented as a black line. PdxR binding sites are colored in dark green (motif 1) and light green (motif 3); motif 2 is colored in blue.

Article Snippet: Various DNA fragments encompassing the wild type and modified PdxR target sequences were designed and purchased from Eurofins Genomics (Ebersberg, Germany) or Metabion International AG (Steinkirchen, Germany) as lyophilized forward and reverse single-stranded oligonucleotides (sequences are listed and schematically displayed in ).

Techniques: Binding Assay

Journal: Frontiers in Pharmacology

Article Title: An Improved Genetically Encoded Fluorescent cAMP Indicator for Sensitive cAMP Imaging and Fast Drug Screening

doi: 10.3389/fphar.2022.902290

Figure Lengend Snippet: cAMP diffusion from cytosol to mitochondrial matrix in HeLa cells. (A) Schematic of mitochondrial matrix-localized G-Flamp2 (Mito-G-Flamp2) biosensor. The mitochondrial-targeting sequence from subunit VIII of the human cytochrome oxidase (COX) was fused to the N-terminus of G-Flamp2 with a flexible (G4S) 10 linker. (B) Epifluorescence images of Mito-G-Flamp2 (Green) and Mito-Tracker (Red) in HeLa cells. (Scale bars, 5 μm). (C) Epifluorescence images of G-Flamp2 (GF2), Mito-G-Flamp2 (Mito-GF2) and G-Flamp2 NC (GF2 NC) to 60 µM Fsk stimulation in HeLa cells. Response of GF2, Mito-GF2 and GF2 NC to 60 µM Fsk stimulation. Quantification of peak ∆F/F 0 are on the right. n = 11, 12 and 11 cells for GF2, Mito-GF2 and GF2 NC from 3 cultures for each sensor. (Scale bars, 10 μm). (D) Epifluorescence images of GF2, Mito-GF2 and GF2 NC to cAMP that is generated by bPACm in HeLa cells. The absorption peak of purified bPAC with and without light is at 453 and 441 nm, respectively, which is coincidentally consistent with the spectral-dependent G-Flamp sensors that the excitation peak at ∼ 450 nm. Response of GF2, Mito-GF2 and GF2 NC to cAMP that is generated by bPAC mutation. Quantification of peak ∆F/F 0 are on the right. n = 12, 14 and 12 cells for GF2, Mito-GF2 and GF2 NC from 3 cultures for each sensor. (Scale bars, 10 μm). Data are presented as the mean ± standard deviation. *Significantly different from GF2 NC, *** p < 0.001, **** p < 0.0001 as measured by one-way analysis of variance (ANOVA) with post hoc Tukey’s test. See .

Article Snippet: DNA sequence for Green cGull and mitochondrial targeting peptide was synthesized by Genscript.

Techniques: Diffusion-based Assay, Sequencing, Generated, Purification, Mutagenesis, Standard Deviation

Journal: Molecular Genetics & Genomic Medicine

Article Title: Adult‐onset Niemann–Pick disease type C masquerading as spinocerebellar ataxia

doi: 10.1002/mgg3.1906

Figure Lengend Snippet: Adult sibling pair affected with cerebellar ataxia. (a) Pedigree (proband indicated with arrow). (b) MRI of one sibling (AR18054a2) showing cerebellar degeneration (white arrow). (c) Cartoon of NPC1 protein with identity and location of the pathogenic alleles. d. Patient fibroblasts showing lipid accumulation within cells (Filipin staining). (e) Defective cholesteryl esterification in affected sibs. n = 3 biological replicates. CTD, C‐terminal domain; MLD, middle luminal domain; NTD, N‐terminal domain; TM, transmembrane domain; WT, wildtype control. NPC1 structure taken from Li et al.

Article Snippet: Variants were phased through targeted DNA sequencing at GeneDx of NPC1 performed on a buccal sample from the proband's son.

Techniques: Staining, Control