Journal: Molecular therapy. Nucleic acids

Article Title: Regulating PCCA gene expression by modulation of pseudoexon splicing patterns to rescue enzyme activity in propionic acidemia.

doi: 10.1016/j.omtn.2023.102101

Figure Lengend Snippet: Figure 4. Investigating splice-switching antisense oligonucleotide-mediated modulation of PCCA pseudoexon splicing patterns in a cellular model (A) Schematic representation of the HepG2 PCCA cell lines generated with CRISPR gene editing. (B) Endogenous PCCA pseudoexon splicing patterns from untransfected (UT), control- and SSO-treated (ctrl SSO and SSO 2) HepG2 PCCA WT (WT), HepG2 PCCA MUT (MUT), and HepG2 PCCA Del7 (Del7) cell lines. Error bars indicate SEMs, n = 6 culture wells from 3 individual experiments. (C) Representative western blots from investigation of PCCA and PCCB protein levels between untransfected, control-treated, and SSO-treated HepG2 PCCA cell lines, and protein quantification by densitometry normalized to the immunodetection of GAPDH used as loading control, indicated as the change in protein levels relative to untransfected HepG2 PCCA WT. Comparisons between control- and SSO-treated samples by 2-sample t test in R. *p < 0.05; **p < 0.005; ***p < 0.0005. Error bars indicate SEMs, n = 3 from individual experiments. (D) PCC enzyme activity between untransfected, control-treated, and SSO-treated HepG2 PCCA cell lines, indicated as the change in PCC activity relative to untransfected HepG2 PCCA WT. Comparisons between control- and SSO-treated samples by 2-sample t test in R. *p < 0.05; **p < 0.005; ***p < 0.0005. Error bars indicate SEMs, n = 3 from individual experiments.

Article Snippet: Immunodetection was carried out using commercially available primary antibodies against PCCA (1:500; sc-374341; Santa Cruz Biotechnology, Santa Cruz, CA) or PCCB (1:500; sc-393929; Santa Cruz Biotechnology), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:5,000; ab8245; Abcam; Cambridge, UK) as loading control, with a secondary antibody against mouse immunoglobulin G (IgG) (1:2,000; no. 7076; Cell Signaling Technology, Danvers, MA).

Techniques: Generated, CRISPR, Control, Western Blot, Immunodetection, Activity Assay

Journal: Molecular therapy. Nucleic acids

Article Title: Regulating PCCA gene expression by modulation of pseudoexon splicing patterns to rescue enzyme activity in propionic acidemia.

doi: 10.1016/j.omtn.2023.102101

Figure Lengend Snippet: Figure 5. Investigating transcriptomic and metabolomic effects of the pathogenic PCCA pseudoexon activation event and splice-switching antisense oligonucleotide-mediated modulation of pseudoexon splicing patterns (A) Principal-component analysis (PCA) plot of the variation between control- and SSO-treated (ctrl SSO and SSO 2) HepG2 PCCA WT (WT), HepG2 PCCA MUT (MUT), and HepG2 PCCA Del7 (Del7) cell lines in RNA sequencing data (n = 3 from individual experiments). (B) Relative expression of PCCA and PCCB between control- and SSO- treated HepG2 PCCA cell lines indicated as log-transformed normalized read counts from RNA sequencing data. Adjusted p values (p.adj.) from Wald test with post-hoc Benjamini-Hochberg test for differential gene expression between treatment conditions: p.adj. = 1.742 107 (PCCA) and p.adj. = 0.690 (PCCB). (C) Venn diagrams with overlap of significant differentially expressed genes (p.adj. < 0.05) and scatterplots comparing the log2 fold changes (log2FC) of the overlapping genes between genotypes; control-treated HepG2 PCCA MUT/Del7 versus WT, and between treatment conditions; SSO- versus control-treated HepG2 PCCA MUT/Del7. Pearson’s correlation, r, is indicated for the overlapping genes in both scatterplots. (D) Scatterplot of 2,578 identified features in metabolomics data (n = 3 from individual experiments), indicating features with a significant difference in abundance (orange) between control- and SSO-treated HepG2 PCCA cell lines from 1-way ANOVA with post-hoc Fisher’s least- significant difference test; p.adj. < 0.05. Significant features: neostigmine bromide (p.adj. = 3.195 105), isovalerylcarnitine (p.adj. = 1.609 103), pyrene-4,5-dione (p.adj. = 6.627 103), 2-methylcitrate (p.adj. = 0.016), L-propionylcarnitine (p.adj. = 0.045), and N-3-hydroxyoctanoyl-L-homoserine lactone (p.adj. = 0.045). PCA plot of the variation between control- and SSO-treated HepG2 PCCA cell lines in Figure S14. (E) Relative difference in abundance of 2-methylcitrate between control- and SSO-treated HepG2 PCCA cell lines indicated as log-transformed signals from metabolomics data. (F) Relative difference in abundance of L-propionylcarnitine.

Article Snippet: Immunodetection was carried out using commercially available primary antibodies against PCCA (1:500; sc-374341; Santa Cruz Biotechnology, Santa Cruz, CA) or PCCB (1:500; sc-393929; Santa Cruz Biotechnology), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:5,000; ab8245; Abcam; Cambridge, UK) as loading control, with a secondary antibody against mouse immunoglobulin G (IgG) (1:2,000; no. 7076; Cell Signaling Technology, Danvers, MA).

Techniques: Activation Assay, Control, RNA Sequencing, Expressing, Transformation Assay, Gene Expression

Journal: Molecular therapy. Nucleic acids

Article Title: Regulating PCCA gene expression by modulation of pseudoexon splicing patterns to rescue enzyme activity in propionic acidemia.

doi: 10.1016/j.omtn.2023.102101

Figure Lengend Snippet: Figure 6. Investigating the therapeutic potential of splice-switching antisense oligonucleotide-mediated modulation of PCCA pseudoexon splicing patterns (A) Endogenous PCCA pseudoexon splicing patterns from control- and SSO-treated (ctrl SSO and SSO 2) control fibroblasts and patient fibroblasts homozygous for PCCA c.1285-1416A>G. Error bars indicate range, n = 2 culture wells. (B) Representative western blots from investigation of PCCA protein levels between control- and SSO-treated control fibroblasts and patient fibroblasts homozygous for PCCA c.1285-1416A>G (PCCA-def.). (C) PCC enzyme activity between control- and SSO-treated control fi- broblasts, PCCA-deficient patient fibroblasts homozygous for PCCA c.1285-1416A>G, homozygous for PCCA c.412G>A (p.A138T), and compound heterozygous for PCCA c.229C>T (p.R77W)/c.1846-2_1852del9, and PCCB-deficient patient fibroblasts homozygous for PCCB c.1228C>T (p.R410W), indicated as the change in PCC activity relative to control-treated samples. Comparisons by 2-sample t test in R. *p < 0.05; **p < 0.005; ***p < 0.0005. Error bars indicate SEMs, n = 15, 4, 6, 5, and 4, respectively. (D) Representative western blots from investigation of PCCB protein levels between control- and SSO-treated control fibroblasts and patient fibroblasts ho- mozygous for PCCB c.1228C>T (PCCB-def.).

Article Snippet: Immunodetection was carried out using commercially available primary antibodies against PCCA (1:500; sc-374341; Santa Cruz Biotechnology, Santa Cruz, CA) or PCCB (1:500; sc-393929; Santa Cruz Biotechnology), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:5,000; ab8245; Abcam; Cambridge, UK) as loading control, with a secondary antibody against mouse immunoglobulin G (IgG) (1:2,000; no. 7076; Cell Signaling Technology, Danvers, MA).

Techniques: Control, Western Blot, Activity Assay

Journal:

Article Title: The Gas Vesicle Gene Cluster from Microcystis aeruginosa and DNA Rearrangements That Lead to Loss of Cell Buoyancy †

doi: 10.1128/JB.186.8.2355-2365.2004

Figure Lengend Snippet: Electron micrographs of the wild-type strains (A and E) and the GV-deficient mutant strains (B, C, D, and F) of M. aeruginosa. (A) PCC 7806 wild type; (B) PCC 7806 M1; (C) PCC 7806 M2; (D) PCC 7806 M3; (E) PCC 9354 wild type; (F) PCC 9354 M4. gv-l, GV in longitudinal section; gv-c, GV in cross-section. Bars, 500 nm.

Article Snippet: This database sequence is the preliminary result of shotgun sequencing of 700-bp fragments of M. aeruginosa PCC 7806 genomic DNA cloned in the pcDNA-2.1 vector (Invitrogen Life Technologies, Rockville, Md.) and forms part of a current sequencing genome project at the Génopole-Ile de France, Institut Pasteur, Paris, France.

Techniques: Mutagenesis

Journal:

Article Title: The Gas Vesicle Gene Cluster from Microcystis aeruginosa and DNA Rearrangements That Lead to Loss of Cell Buoyancy †

doi: 10.1128/JB.186.8.2355-2365.2004

Figure Lengend Snippet: The gvp gene cluster region in M. aeruginosa and IS found in the GV-deficient mutants. (A) Strain PCC 7806; (B) strain PCC 9354. From top to bottom, names and sizes of the IS, localizations of the insertions, names of the putative genes, maps of the ORFs and their orientations, and intergenic regions (ig) in base pairs (bp) are shown. Arrows, ORFs; black arrows and lines, sequenced regions; grey-shaded arrows and lines, nonsequenced regions. Precise locations (with respect to the sequence submitted to DDBJ/EMBL/GenBank under no AJ577136) of the insertion elements are as follows: between nucleotides 115 and 2206 for ISMae1, between nucleotides 4033 and 4034 for ISMae2, between nucleotides 7633 and 7634 for ISMae3, and between nucleotides 8301 and 8302 for ISMae4.

Article Snippet: This database sequence is the preliminary result of shotgun sequencing of 700-bp fragments of M. aeruginosa PCC 7806 genomic DNA cloned in the pcDNA-2.1 vector (Invitrogen Life Technologies, Rockville, Md.) and forms part of a current sequencing genome project at the Génopole-Ile de France, Institut Pasteur, Paris, France.

Techniques: Sequencing

Journal:

Article Title: The Gas Vesicle Gene Cluster from Microcystis aeruginosa and DNA Rearrangements That Lead to Loss of Cell Buoyancy †

doi: 10.1128/JB.186.8.2355-2365.2004

Figure Lengend Snippet: Transcription analysis of the gvp cluster. (A) RNA blot analysis of the gvpA, gvpC, and rnpB transcripts from M. aeruginosa PCC 7806 grown under a day-night cycle (16 h-8 h). The cells were harvested for RNA extraction after 1 h of light when the culture reached OD750 = 0.4. The same blot (15 μg of total RNA per lane) was successively hybridized with different probes: a 180-bp gvpA fragment (obtained by PCR amplification with primers 635 and 636), a 400-bp gvpC fragment (amplified with primers 870 and 871), and (as a control for RNA loading and transfer) a 0.6-kb rnpB fragment (obtained by PCR with the primers mentioned in Materials and Methods). The sizes of the different transcripts are indicated in kilobases (kb). (B) RT-PCR analysis showing cotranscription between the genes of the gvp cluster. The gvpC-N fragment was amplified with primers 893 and 973, the gvpN-J fragment was amplified with primers 975 and 1108, the gvpJ-X fragment was amplified with primers 974 and 1110, the gvpX-K fragment was amplified with primers 978 and 1109, the gvpK-F fragment was amplified with primers 1111 and 1113, the gvpF-G fragment was amplified with primers 965 and 1018, and the gvpK-G fragment was amplified with primers 965 and 1111. s, analyzed sample (with cDNA as a template); −, negative control (with RNA as a template); +, positive control (with genomic DNA as a template). A DNA ladder (100 bp [Amersham Biosciences] or 1 kb [Invitrogen Life Technologies]) was used as a size marker (lanes M).

Article Snippet: This database sequence is the preliminary result of shotgun sequencing of 700-bp fragments of M. aeruginosa PCC 7806 genomic DNA cloned in the pcDNA-2.1 vector (Invitrogen Life Technologies, Rockville, Md.) and forms part of a current sequencing genome project at the Génopole-Ile de France, Institut Pasteur, Paris, France.

Techniques: Northern blot, RNA Extraction, Amplification, Reverse Transcription Polymerase Chain Reaction, Negative Control, Positive Control, Marker

Journal:

Article Title: The Gas Vesicle Gene Cluster from Microcystis aeruginosa and DNA Rearrangements That Lead to Loss of Cell Buoyancy †

doi: 10.1128/JB.186.8.2355-2365.2004

Figure Lengend Snippet: Transcription analysis of gvp genes in the wild-type and the GV-deficient mutant strains of M. aeruginosa. (A) RT-PCR with the gvpA-specific primers 994 and 995 (see Table ​Table1).1). The results for PCC 7806 wild-type (WT) and mutant (M2 and M3) strains and PCC 9354 wild-type and mutant (M4) strains are shown. (B) RT-PCR results for the three genes carrying an IS (gvpN, gvpV, and gvpW) and for gvpJ and gvpX. The results for PCC 7806 wild-type (WT) and mutant (M3 and M2) strains and PCC 9354 wild-type and mutant (M4) strains are shown. The gvpV gene was amplified with primers 1081 and 1082, gvpN was amplified with primers 949 and 972, gvpJ was amplified with primers 974 and 975, gvpX was amplified with primers 1109 and 1110, and gvpW was amplified with primers 983 and 1077. A DNA ladder (100 bp [Amersham Biosciences] or 1 kb [Invitrogen Life Technologies]) was used as a size marker (lanes M).

Article Snippet: This database sequence is the preliminary result of shotgun sequencing of 700-bp fragments of M. aeruginosa PCC 7806 genomic DNA cloned in the pcDNA-2.1 vector (Invitrogen Life Technologies, Rockville, Md.) and forms part of a current sequencing genome project at the Génopole-Ile de France, Institut Pasteur, Paris, France.

Techniques: Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Amplification, Marker

Journal:

Article Title: The Gas Vesicle Gene Cluster from Microcystis aeruginosa and DNA Rearrangements That Lead to Loss of Cell Buoyancy †

doi: 10.1128/JB.186.8.2355-2365.2004

Figure Lengend Snippet: Immunodetection of GvpA on isolated GV and cell extracts from the wild-type and the GV-deficient mutant strains of M. aeruginosa. GV, enriched GV fraction (20 μg) from PCC 7806 wild-type strain. Other lanes show the results for crude extracts (200 μg) from PCC 7806 wild-type (WT) and mutant (M1, M2, and M3) strains and from PCC 9354 wild-type and mutant (M4) strains.

Article Snippet: This database sequence is the preliminary result of shotgun sequencing of 700-bp fragments of M. aeruginosa PCC 7806 genomic DNA cloned in the pcDNA-2.1 vector (Invitrogen Life Technologies, Rockville, Md.) and forms part of a current sequencing genome project at the Génopole-Ile de France, Institut Pasteur, Paris, France.

Techniques: Immunodetection, Isolation, Mutagenesis

Journal: Bioinformatics and Biology Insights

Article Title: Effect of Diet Supplementation on the Expression of Bovine Genes Associated with Fatty Acid Synthesis and Metabolism

doi:

Figure Lengend Snippet: Enzymes involved in the conversion of substrates to citric acid cycle intermediates (gluconeogenesis) that were differentially expressed among the four dietary treatments in microarray analysis and their P -value. C

Article Snippet: Potential genes involved in this process like propionyl—CoA carboxylase alpha (PCCA), methylmalonyl—CoA mutase (MUT), methylmalonyl—CoA epimerase (MCE), succinate dehydrogenase complex (SDH), phosphoenolpyruvate carboxylase 1 (PCK1), phosphoenolpyruvate carboxylase 2 (PCK2) and fructose—1,6-bisphosphate (FBP2) were found to be differentially expressed among the various treatment comparisons.

Techniques: Microarray