Journal: International Journal of Molecular Sciences

Article Title: Bioprospecting Antimicrobials from Lactiplantibacillus plantarum : Key Factors Underlying Its Probiotic Action

doi: 10.3390/ijms222112076

Figure Lengend Snippet: Probiotic L. plantarum strains with documented in vitro antibacterial activity.

Article Snippet: Faeces of healthy infants , Lp 34-5 , CFS (pH acid) , n.i , S. flexneri ATCC 12022, ETEC H10407 enteropathogenic bacteria , Pazhoohan M, 2020.

Techniques: In Vitro, Activity Assay, Membrane, Lysis, Sequencing, Expressing, Inhibition, Purification, Bacteria, Disruption, Binding Assay, Over Expression, Permeability, Modification, Concentration Assay

Journal: Nature

Article Title: PIK3CA and CCM mutations fuel cavernomas through a cancer-like mechanism

doi: 10.1038/s41586-021-03562-8

Figure Lengend Snippet: a, Schematic of neonatal Krit1BECKO animals with a susceptible microbiome administered a single dose treatment with Rapamycin or vehicle on P2. b, Representative visual and microCT images of the hindbrains of littermates treated with either vehicle or Rapamycin. Scale bars, 1mm. c, MicroCT quantitation of lesion volumes normalized to total brain volume following treatment with vehicle or Rapamycin. (Vehicle, n=13; Rapamycin, n=16). p=0.0009. d, Experimental design for Rapamycin or vehicle treatment of adult animals with combined CCM LOF and PIK3CA GOF (Krit1fl/fl;iPik3caH1047R) using cranial window surgery and AAV injection is shown. e, Representative visual and microCT images of brains harvested 21 days after injection of AAV-Cre into littermate animals. Dotted circles indicate the site of cranial window and AAV-Cre injection. Scale bars, 1mm. f, MicroCT quantitation of lesion volumes 21 days after creation of the cranial window and injection of AAV-Cre is shown. (Vehicle, n=6; Rapamycin, n=7). p=0.0358. g, Schematic of neonatal KLF4iBECGOF animals administered a single dose treatment with Rapamycin or vehicle followed by induction of KLF4 expression on P10. h, Representative visual and microCT images of the hindbrains of littermates treated with either vehicle or Rapamycin. Scale bars, 1mm. i, MicroCT quantitation of lesion volumes normalized to total brain volume following treatment of the indicated mice with vehicle or Rapamycin. (Vehicle, n=20; Rapamycin, n=22). p=4e−5. Data are mean ± s.e.m. Unpaired, two-tailed Welch’s t-test. *indicates p<0.05; ***indicates p<0.001; ****indicates p<0.0001.

Article Snippet: These steps were repeated for three assays testing the presence of the three most common PIK3CA mutations: E542K, E545K, and H1047 (ThermoFisher assay IDs: Hs000000085_rm, Hs000000086_rm, Hs000000088_rm, respectively).

Techniques: Quantitation Assay, Injection, Expressing, Two Tailed Test

Journal: Nature

Article Title: PIK3CA and CCM mutations fuel cavernomas through a cancer-like mechanism

doi: 10.1038/s41586-021-03562-8

Figure Lengend Snippet: a, Schematic of neonatal endothelial induction of Krit1 deletion and/or PIK3CAH1047R expression. b & c, Hematoxylin-Eosin (H-E) staining of saggital hindbrain sections from P6 control, Pik3caiBECGOF, Krit1iBECKO;Pik3caiBECGOF (b and b’) and Krit1iBECKO (c and c’) animals with a resistant (Res) or susceptible (Susc) microbiome. b’and c’ samples were obtained from animals distinct from those in b and c. Note that lesions form in the white matter with both CCM loss of function and PIK3CA gain of function. Boxes in upper images denote area of magnified image immediately below. Dotted lines outline the white matter of the cerebellum. Arrows indicate lesions in white matter veins and venules. H-E images representative of 6 tissue sections from n=4 animals/genotype. wm, white matter. Scale bars, 0.1mm.

Article Snippet: These steps were repeated for three assays testing the presence of the three most common PIK3CA mutations: E542K, E545K, and H1047 (ThermoFisher assay IDs: Hs000000085_rm, Hs000000086_rm, Hs000000088_rm, respectively).

Techniques: Expressing, Staining, Control

Journal: Nature

Article Title: PIK3CA and CCM mutations fuel cavernomas through a cancer-like mechanism

doi: 10.1038/s41586-021-03562-8

Figure Lengend Snippet: a, A schematic summary of the somatic PIK3CA mutations, the germline mutations in KRIT1, CCM2 and PDCD10, and the somatic mutations in KRIT1, CCM2 and PDCD10 as identified in 79 human CCMs via bulk sequencing of frozen resected tissue is shown. Color denotes the affected CCM gene. Samples listed as neither familial nor sporadic are deidentified banked CCMs lacking either clinical information or genetic evidence supporting either classification. ‡ indicates familial CCMs with an activating mutation in PIK3CA and both germline and somatic mutations in a CCM gene. indicates known or presumed sporadic CCMs with an activating mutation in PIK3CA and two somatic mutations in a CCM gene. b, Percentage of lesions with an activating mutation in PIK3CA present in all sequenced CCMs vs. control brain AVMs, all three forms of familial CCMs and sporadic CCMs. The value inside the bar shows the number of samples in the corresponding group. c, The distributions of the 16 most common somatic PIK3CA mutations identified in human CCMs (top) and cancer (bottom) as reported in the COSMIC database are shown. Colored boxes represent domains in PIK3CA in order from left to right: Adaptor BD, RAS BD, C2, Kinase. d, Schematic of workflow for processing frozen surgically-resected human CCM lesions for single-nucleus DNA sequencing. e, Representative data for sporadic and familial CCMs detailing the number of nuclei with each combination of PIK3CA and CCM mutations. p was determined by two-tailed chi-squared test between the observed and expected triple mutant nuclei predicted by a Poisson distribution (see Methods). ns indicates p not significant, p>0.05; ***indicates p<1e−16.

Article Snippet: These steps were repeated for three assays testing the presence of the three most common PIK3CA mutations: E542K, E545K, and H1047 (ThermoFisher assay IDs: Hs000000085_rm, Hs000000086_rm, Hs000000088_rm, respectively).

Techniques: Sequencing, Mutagenesis, Control, DNA Sequencing, Two Tailed Test

Journal: Nature

Article Title: PIK3CA and CCM mutations fuel cavernomas through a cancer-like mechanism

doi: 10.1038/s41586-021-03562-8

Figure Lengend Snippet: a, The relationship between somatic PIK3CA and CCM mutations detected in bulk sequencing is graphed. Points indicate individual mutations in either a CCM gene or PIK3CA. Lines connect the CCM gene and PIK3CA gene mutations present in a single sample. Box plots show the aggregate frequencies of PIK3CA and CCM mutations. Center lines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range from the 25th to 75th percentiles, outliers are represented by dots. n = 21 sample points for both plots. b, Representative FANS plots of unstained (top row) and DAPI stained (bottom row) CCM homogenate. Doublet discrimination by forward scatter profile for DAPI stained sample. Plots consist of 100,000 events. The unstained sample contains 1 event (0%) in the DAPI+ singlet gate. The DAPI stained samples contains 2,414 events (2.4%) in the DAPI+ singlet gate. c, Total reads and average coverage per nucleus from snDNAseq for each mutation detected by bulk sequencing. Dotted line shows 20x coverage, the minimum cutoff used for establishing genotype. d, Pseudobulk allele frequency from snDNA-seq for each mutation detected by bulk sequencing. Dotted line shows 1% allele frequency. Note the data point with the arrow in c-d shows a mutation in sample 5079 detected in bulk sequencing which, due to poor amplification during snDNA-seq, received insufficient coverage per nucleus (4.5x) to establish nuclear genotypes however is clearly present in pseudobulk reads (1849/9814). e, Comparison of mutation allele frequency as detected by bulk and snDNA-seq. As nuclei are diploid for the relevant autosomes, the x-axis is equal to the fraction of mutant nuclei divided by two. Dotted line shows perfect correlation at x=y. R and p were calculated by Pearson’s correlation coefficient. f, A summary of snDNA-seq results for 3 sporadic and 2 familial CCMs analyzed is shown. The number of nuclei with each possible genotype are listed. + indicates a wild-type allele; * indicates a mutant allele. Note that only 1 somatic CCM mutation was identified in samples 5038 and 5079. P values were determined by two-tailed chi-squared test between the observed and expected triple mutant nuclei (or double mutant for lesions 5038 and 5079) determined by Poisson distribution (see Methods).

Article Snippet: These steps were repeated for three assays testing the presence of the three most common PIK3CA mutations: E542K, E545K, and H1047 (ThermoFisher assay IDs: Hs000000085_rm, Hs000000086_rm, Hs000000088_rm, respectively).

Techniques: DNA Sequencing, Sequencing, Staining, Mutagenesis, Amplification, Comparison, Two Tailed Test

Journal: Nature

Article Title: PIK3CA and CCM mutations fuel cavernomas through a cancer-like mechanism

doi: 10.1038/s41586-021-03562-8

Figure Lengend Snippet: a, Schematic of neonatal endothelial induction of KLF4 expression in KLF4iBECGOF animals. b, Immunostaining for KLF4 and the endothelial cell marker PECAM1 in hindbrain sections from P6 control and KLF4iBECGOF animals is shown. Boxes in upper images denote area of magnified image immediately below. Immunofluorescence images representative of 6 tissue sections from n=4 individual animals/genotype. Scale bars, 50 microns. c, H-E stained sections of hindbrain from control and KLF4iBECGOF littermates. Boxes in upper images denote area of magnified image immediately below. Black arrows indicate lesions. Dotted lines outline the white matter of the cerebellum. wm, white matter. Note the dilated white matter venules similar to those observed with CCM loss of function and PIK3CA gain of function shown in Extended Data Figure 2. H-E histology representative of 6 tissue sections from n=4 animals/genotype. Scale bars, 0.1mm. d, Immunostaining for phospho-S6 ribosomal protein (p-S6) and the endothelial cell marker PECAM1 in hindbrain sections from P6 control and KLF4iBECGOF animals is shown. White arrows indicate p-S6 positive endothelial cells. Yellow arrowheads into non-endothelial p-S6 positive cells. Immunofluorescence images representative of 6 tissue sections from n=4 individual animals/genotype. Scale bars, 50 microns. e, Immunoblot detection of KLF4, KLF4-GFP, the KLF4 target gene eNOS in HUVECs without and with inducible lentiviral expression of KLF4-GFP (“iKLF4” cells) or control lentivirus. TUBULIN is shown as a loading control.

Article Snippet: These steps were repeated for three assays testing the presence of the three most common PIK3CA mutations: E542K, E545K, and H1047 (ThermoFisher assay IDs: Hs000000085_rm, Hs000000086_rm, Hs000000088_rm, respectively).

Techniques: Expressing, Immunostaining, Marker, Control, Immunofluorescence, Staining, Western Blot