col2a1  (Santa Cruz Biotechnology)

Journal: Gut microbes

Article Title: The cnf1 gene is associated with an expanding Escherichia coli ST131 H 30Rx/C2 subclade and confers a competitive advantage for gut colonization.

doi: 10.1080/19490976.2022.2121577

Figure Lengend Snippet: Figure 1. Prevalence overtime in representative E. coli sequence types bearing cnf1. Bar chart show number of E. coli strains from ST131, ST73, ST12 and ST127 isolated each year during the period 2002–2019, left y-axis. Percentages of cnf1-positive strains per year, right y-axis.

Article Snippet: Membranes were incubated with the primary antibody: CNF1 (Santa Cruz sc52655 clone NG8 1/1000), RNA Polymerase (Biolegend 699907 clone NT73 1/ 1000) and rabbit serum (1/1000) against the conserved amino acids 914–936 of HlyA, as previously described.79 Membranes were washed with TBS-T and incubated with horseradish peroxidase (HRP)- conjugated secondary antibodies for 1 h. Signals were observed using Immobion Western Chemiluminescent HRP Substrate (Merck).

Techniques: Sequencing, Isolation

Journal: Gut microbes

Article Title: The cnf1 gene is associated with an expanding Escherichia coli ST131 H 30Rx/C2 subclade and confers a competitive advantage for gut colonization.

doi: 10.1080/19490976.2022.2121577

Figure Lengend Snippet: Figure 2. Dynamic of CNF1-encoding gene in E. coli ST131 from EnteroBase. A) Maximum likelihood phylogeny of E. coli ST131 from EnteroBase (Sup. Figure S2 for extended information). The phylogeny was constructed with 5,231 genomes for a total of 37,304 non-recombinant core-genome SNPs. The different clades and subclades A, B, C0, C1, C2_0, C2_1, C2_2 are highlighted in blue, red, light green, green, pink, Orange and purple respectively. From inside to outside circles are indicated (1) fimH alleles, (2) gyrA and parC alleles conferring resistance to fluoroquinolones (shown in green), (3) positive strains for blaCTX-M-15 (shown in Orange) and (4) strains bearing cnf1 gene (shown in black).) Hierarchical clustering of strains from clade C (n = 3981 strains) based on their accessory gene content. The pan-genome is composed of 51,742 genes including 2,672 genes that are present in 98% of the strains. The graph displays the 7,678 genes identified as present in at least 50 and less than 3,930 genomes. The colored annotation indicates (from left to right) the presence of cnf1 (CNF1_status), subclades (C1, C1 CNF1_LL1, C2_0, C2_1, C2_1 CNF1_LL2, C2_2) and accessory genes cluster (AG_clusters). Large lineages of cnf1-positive strains in clades C1 and C2_1 are denoted CNF1_LL1 and CNF1_LL2, respectively. Red stars indicate the two large lineages of cnf1-positive strains.

Article Snippet: Membranes were incubated with the primary antibody: CNF1 (Santa Cruz sc52655 clone NG8 1/1000), RNA Polymerase (Biolegend 699907 clone NT73 1/ 1000) and rabbit serum (1/1000) against the conserved amino acids 914–936 of HlyA, as previously described.79 Membranes were washed with TBS-T and incubated with horseradish peroxidase (HRP)- conjugated secondary antibodies for 1 h. Signals were observed using Immobion Western Chemiluminescent HRP Substrate (Merck).

Techniques: Construct, Recombinant

Journal: Gut microbes

Article Title: The cnf1 gene is associated with an expanding Escherichia coli ST131 H 30Rx/C2 subclade and confers a competitive advantage for gut colonization.

doi: 10.1080/19490976.2022.2121577

Figure Lengend Snippet: Figure 4. Increase over the years in the proportion of cnf1-positive strains in E. coli ST131 H30Rx/C2. A) Distribution of fimH alleles (upper panel) or clades/subclades (lower panel) within the study population of E. coli ST131. Both figures show observed counts per year (dots) and data fitted lines (dashed lines) with a generalized linear model (Poisson regression). B) Increase of the proportion of cnf1-positive strains in the whole E. coli ST131 population along time (top panel, P = 7.41 10–7) and by clades and subclades. The black dots represent the observed proportion of cnf1-positive strains by year with fitted line of a logistic regression model (blue curves). Dashed gray lines display the 95% confidence intervals. The P-values are not significant for clade A (P = 0.287), B (P = 0.952), H30R/C1 (P = 0.992) and significant for H30Rx/C2 (P = 1.25 10−11).

Article Snippet: Membranes were incubated with the primary antibody: CNF1 (Santa Cruz sc52655 clone NG8 1/1000), RNA Polymerase (Biolegend 699907 clone NT73 1/ 1000) and rabbit serum (1/1000) against the conserved amino acids 914–936 of HlyA, as previously described.79 Membranes were washed with TBS-T and incubated with horseradish peroxidase (HRP)- conjugated secondary antibodies for 1 h. Signals were observed using Immobion Western Chemiluminescent HRP Substrate (Merck).

Techniques:

Journal: Toxins

Article Title: CTX-CNF1 Recombinant Protein Selectively Targets Glioma Cells In Vivo

doi: 10.3390/toxins13030194

Figure Lengend Snippet: CNF1 maintains its activity on glioma cells even when inserted in the CTX-CNF1 recombinant molecule. ( A ) Structure of CTX-CNF1. Chlorotoxin (CTX) is located at the N-terminus of the recombinant molecule and is followed by CNF1, formed by its three domains (binding, translocation and catalytic). ( B ) Percentage of multi- and mononucleated GL261 cells in control (grey, PBS) and after 48 h of CTX-CNF1 administration (green, 25 nM). Inset, representative images of the two conditions, scale bar = 4μm. ( C ) Percentage of vital GL261 cells in control (grey, PBS) and in 48 h treatment of CTX-CNF1 (green) at different concentrations (i.e., 1, 12, 25, 30, 40 and 50 nM). Data represent means ± SEM, one way ANOVA p < 0.001. ( D ) Percentage of betagalactosidase positive GL261 cells in control (grey, PBS) and after CTX-CNF1 administration (green, 25 nM) at different time points (i.e., 24 h, 48 h, 72 h, 6 d). Data represent means ± SEM, One Way ANOVA p < 0.001. ( E ) Quantitative RT-PCRs showing the relative expression of the senescence markers p21 (Cdkn1a, cyclin-dependent kinase inhibitor (1) and p16 (Cdkn2a, cyclin-dependent kinase inhibitor 2) in GL261 cells (vehicle, grey; CTX-CNF1, green). Data represent means ± SEM, t test (p16, p = 0.0001; p21, p = 0.0164). ( F ) Increased expression of p16 (Cdkn2a, cyclin-dependent kinase inhibitor 2) in U87 cells treated with CTX-CNF1 (green) with respect to vehicle (grey). Data represent means ± SEM, t test ( p = 0.013). Total cells ( G ) and spheroids ( H ) number of PDGF+ TRP53−/− cells in control (grey, PBS) and after 25 nM of CTX-CNF1 (green) at 72 h and 6 d. Data represent means ± SEM, One Way ANOVA p < 0.001. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: To quantify the localization of CTX-CNF1 we performed a staining for CNF1 (1:500, ThermoFisher).

Techniques: Activity Assay, Recombinant, Binding Assay, Translocation Assay, Control, Expressing

Journal: Toxins

Article Title: CTX-CNF1 Recombinant Protein Selectively Targets Glioma Cells In Vivo

doi: 10.3390/toxins13030194

Figure Lengend Snippet: Increased survival of CTX-CNF1 glioma-bearing mice. ( A , C , E , G ) Experimental protocols. GL261 cells were injected in visual cortex and, 12 days after glioma induction, different treatments were administered. ( A , C ) Glioma-bearing mice were treated intraventricularly ( A , B ) or intravenously ( C , D ) with CTX-CNF1 (80 nM) or vehicle solution (PBS). ( B , D ) Survival rate of untreated and treated glioma-bearing mice. Note the increased surviving percentage of both CTX-CNF1 treated glioma-bearing mice ( B , p = 0.0018; D , p = 0.013). * p < 0.05; ** p < 0.01. ( F , H ) Survival rate of untreated and intravenously treated ( F, with CNF1; H , with CTX) glioma-bearing animals. We did not find any significant increase in survival after CNF1 ( F , p = 0.1); conversely, CTX treatment was able to increase survival rate of glioma-bearing animals ( H , p = 0.03). ( I ) Normalized fraction of tumor area occupied by KI67+ cells in control and in animals intravenously treated with CTX-CNF1 or CTX alone, 24 h and 48 h after the toxin administration (one way ANOVA p < 0.001). Data represent mean ± SEM. *** p < 0.001. ( L , M ) Number of peritumoral Iba1+ ( L , p = 0.19) and Mac2+ ( M , p < 0.001) cells in mice injected with GL261 cells into the visual cortex and systemically treated with CTX-CNF1 with respect to controls. Data represent mean ± SEM. *** p < 0.001.

Article Snippet: To quantify the localization of CTX-CNF1 we performed a staining for CNF1 (1:500, ThermoFisher).

Techniques: Injection, Control

Journal: Toxins

Article Title: CTX-CNF1 Recombinant Protein Selectively Targets Glioma Cells In Vivo

doi: 10.3390/toxins13030194

Figure Lengend Snippet: CTX-CNF1 specificity of action on glioma cells. Representative image ( A ) and magnification ( B ) of a triple staining for chimeric protein (red), glioma PDGF+ TRP53 −/− GFP + cells (green) and cellular nuclei (blue). Note the high selectivity of CTX-CNF1 for recognizing and entering into GB mass. CTX-CNF1 was intravenously delivered and the pictures were taken 24 h after chimeric administration. ( A ) Scale bar = 1000 μm. ( B ) Scale bar = 50 μm. ( C ) In vivo image taken of PDGF + TRP53 −/− GFP + cells 24 h after CTX-CNF1 intravenous delivery. Scale bar = 5 μm ( D ) In vivo image taken of GL261 cells 6 days after CTX-CNF1 intravenous delivery. Scale bar = 10 μm. ( E ) Representative western blot: 12 days after GL261 injection, CTX-CNF1 administration were performed (left, intraventricular; right, intravenous). 48 h after the treatment, the chimeric protein is clearly detectable only in the tumoral mass, even when its administration was made through the caudal vein. Tum = tumor; PTum = peritumoral; Mot = motor area; CD = catalytic domain; GL261 = GL261 cells lysate untreated.

Article Snippet: To quantify the localization of CTX-CNF1 we performed a staining for CNF1 (1:500, ThermoFisher).

Techniques: Staining, In Vivo, Western Blot, Injection