Journal: Cancers

Article Title: CRISPR-Mediated Non-Viral Site-Specific Gene Integration and Expression in T Cells: Protocol and Application for T-Cell Therapy

doi: 10.3390/cancers12061704

Figure Lengend Snippet: Overview of the steps to generate transgene knock-in in T cell: ( a ) Molecular steps considering vector design, amplification, purification, and concentration DNA. ( b ) Electroporation of RNA ribonucleoproteins (RNPs) and donor template to T cells using Lonza instrument. ( c ) Human T-cell preparation before electroporation. Complete and detailed knock-in protocol can be found in the . ( d ) The 1% agarose gel showing PCR amplicons that were gel purified vs. non-gel purified. ( e ) Concentration of dsDNA template after purification and concentration ( n = 24).

Article Snippet: Electroporation solution , P3 + S1 supplement (Lonza).

Techniques: Knock-In, Plasmid Preparation, Amplification, Purification, Concentration Assay, Electroporation, Agarose Gel Electrophoresis

Journal: Cancers

Article Title: CRISPR-Mediated Non-Viral Site-Specific Gene Integration and Expression in T Cells: Protocol and Application for T-Cell Therapy

doi: 10.3390/cancers12061704

Figure Lengend Snippet: Optimization steps to increase transgene knock-in (KI) efficiency in T cells: ( a ) Three different dsDNA template concentrations were evaluated for the best knock-in efficiency and cell viability ( n = 3 for 1 and 2 µg, n = 2 for 3 µg; two-tailed t -test; ns—not significant). ( b ) Two different numbers of T cells were tested for electroporation ( n = 5, two-tailed paired t -test, * p = 0.041). ( c ) Four different lengths of homology arms flanking the transgene of interest were evaluated ( n = 2–4, one-way ANOVA, ns—not significant). ( d ) Knock-in efficiency was tested at early (4–6 days) and late (>9 days) time points post-electroporation ( n = 15, two-tailed paired t -test; ** p = 0.0015).

Article Snippet: Electroporation solution , P3 + S1 supplement (Lonza).

Techniques: Knock-In, Two Tailed Test, Electroporation

Journal: Cancers

Article Title: CRISPR-Mediated Non-Viral Site-Specific Gene Integration and Expression in T Cells: Protocol and Application for T-Cell Therapy

doi: 10.3390/cancers12061704

Figure Lengend Snippet: Validating expression of transgenes in gene-edited T cells: ( a ) Representative flow graph of GFP expression in gene-edited T cells 12 days post-electroporation; right panel, overall knock-in efficiency of the transgene after knock-in optimization as determined by flow cytometry of GFP+/TCRαβ- cells ( n = 7). ( b ) IL-15 production from gene-edited T cells was detected by ELISA 8–10 days post-electroporation ( n = 3, two-tailed t-test, * p = 0.024).

Article Snippet: Electroporation solution , P3 + S1 supplement (Lonza).

Techniques: Expressing, Electroporation, Knock-In, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Cancers

Article Title: CRISPR-Mediated Non-Viral Site-Specific Gene Integration and Expression in T Cells: Protocol and Application for T-Cell Therapy

doi: 10.3390/cancers12061704

Figure Lengend Snippet: IL-15.E2A.mClover3 knock-in into IL-13 locus: ( a ) Transgene expression was evaluated by flow cytometry of GFP+ T cells 10 days post-electroporation; left panel, representative flow plots (samples electroporated without template DNA (-DNA) served as controls); right panel, summary graph of left panel ( n = 8, two-tailed t -test; *** p = 0.0004). ( b ) Fold increase of knock-in efficiency without (-) or with (+) T-cell activation ( n = 6, two-tailed t -test, *** p = 0.0005) ( c ) Knock-out of IL-13 was confirmed by IL-13 secretion; Ctrl, control knock-out; -DNA, knock-in without DNA template; +DNA, knock-in with DNA template ( n = 3–11, one-way ANOVA, * p = 0.036 ** p = 0.007). ( d ) IL-15 expression from IL-13-edited (10 days post-electroporation) T cells 24 h post-T-cell activation ( n = 4, two-tailed t -test, ** p = 0.005).

Article Snippet: Electroporation solution , P3 + S1 supplement (Lonza).

Techniques: Knock-In, Expressing, Flow Cytometry, Electroporation, Two Tailed Test, Activation Assay, Knock-Out, Control

Journal: Cancers

Article Title: CRISPR-Mediated Non-Viral Site-Specific Gene Integration and Expression in T Cells: Protocol and Application for T-Cell Therapy

doi: 10.3390/cancers12061704

Figure Lengend Snippet: Electroporation checklist for large gene knock-in in human T cells.

Article Snippet: Electroporation solution , P3 + S1 supplement (Lonza).

Techniques: Electroporation, Gene Knock-In, Incubation, Stripping Membranes

Journal: Applied and Environmental Microbiology

Article Title: Spatiotemporal Distribution of the Environmental Microbiota in Food Processing Plants as Impacted by Cleaning and Sanitizing Procedures: the Case of Slaughterhouses and Gaseous Ozone

doi: 10.1128/AEM.01861-20

Figure Lengend Snippet: Average C and T scores for oligotype occurrence matrices generated from the 10 selected core OTUs

Article Snippet: The remaining 5 ml was added to 45 ml of BPW and used to perform microbiological analyses by counting TVCs of mesophilic bacteria on plate count agar (Biolife s.p.a., Milan, Italy) incubated at 30°C for 72 h, Brochothrix spp. on streptomycin-thallous acetate-actidione medium (Biolife) incubated at 25°C for 48 h, Pseudomonas spp. on Pseudomonas agar base with cetrimide-fucidin-cephaloridine selective supplement (Biolife) incubated at 25°C for 48 h, and Staphylococcaceae on mannitol salt agar (Oxoid) incubated at 30°C for 48 h. Microbiological data were expressed and statistically analyzed as log-transformed CFU per square decimeter.

Techniques: Generated

Journal: Applied and Environmental Microbiology

Article Title: Spatiotemporal Distribution of the Environmental Microbiota in Food Processing Plants as Impacted by Cleaning and Sanitizing Procedures: the Case of Slaughterhouses and Gaseous Ozone

doi: 10.1128/AEM.01861-20

Figure Lengend Snippet: Co-occurrence networks of bacterial oligotypes belonging to Achromobacter, Acinetobacter, Psychrobacter, Pseudomonas, Propionibacterium, Carnobacterium, Streptococcus, and Staphylococcus. Each network is based on the log-transformed relative abundance matrix of each genus, and the edges represent significant positive correlations (co-occurrence) between the oligotypes (nodes) by means of Spearman’s correlations (rho > 0.6; FDR, P < 0.001). Nodes were made proportional to the weighted degree (total occurrence of an oligotype in the whole data set) and are colored in relation to the species of belonging. Edges were made proportional to the Spearman’s rho value. The M and D values shown in the box represent the modularity (clustering coefficient) and density (calculated using the ratio of the number of edges) of each genus-based network.

Article Snippet: The remaining 5 ml was added to 45 ml of BPW and used to perform microbiological analyses by counting TVCs of mesophilic bacteria on plate count agar (Biolife s.p.a., Milan, Italy) incubated at 30°C for 72 h, Brochothrix spp. on streptomycin-thallous acetate-actidione medium (Biolife) incubated at 25°C for 48 h, Pseudomonas spp. on Pseudomonas agar base with cetrimide-fucidin-cephaloridine selective supplement (Biolife) incubated at 25°C for 48 h, and Staphylococcaceae on mannitol salt agar (Oxoid) incubated at 30°C for 48 h. Microbiological data were expressed and statistically analyzed as log-transformed CFU per square decimeter.

Techniques: Transformation Assay

Journal: Applied and Environmental Microbiology

Article Title: Spatiotemporal Distribution of the Environmental Microbiota in Food Processing Plants as Impacted by Cleaning and Sanitizing Procedures: the Case of Slaughterhouses and Gaseous Ozone

doi: 10.1128/AEM.01861-20

Figure Lengend Snippet: (a) Pseudo-heatmap summarizing the abundance variations of the 10 core OTUs (>50% of the total abundance in >80% of the samples) occurred during the ozone treatments. Asterisks highlight significant decreases in relative abundances in each environment after ozone treatment at 40, 20, or 4 ppm (FDR-adjusted P value from Wilcoxon’s test). *, 0.05; **, 0.01; ***, 0.001. (b) Viable counts of Brochothrix, Pseudomonas, and Staphylococcus before (ACS) and after (AOT 20) the 20-ppm ozonation carried out in the environment B-PR; box plot colors highlight significant differences between ACS and AOT 20 counts (P < 0.05 [FDR adjusted], Wilcoxon’s test).

Article Snippet: The remaining 5 ml was added to 45 ml of BPW and used to perform microbiological analyses by counting TVCs of mesophilic bacteria on plate count agar (Biolife s.p.a., Milan, Italy) incubated at 30°C for 72 h, Brochothrix spp. on streptomycin-thallous acetate-actidione medium (Biolife) incubated at 25°C for 48 h, Pseudomonas spp. on Pseudomonas agar base with cetrimide-fucidin-cephaloridine selective supplement (Biolife) incubated at 25°C for 48 h, and Staphylococcaceae on mannitol salt agar (Oxoid) incubated at 30°C for 48 h. Microbiological data were expressed and statistically analyzed as log-transformed CFU per square decimeter.

Techniques: