Journal: MethodsX

Article Title: DNA extraction of bacterial cells using a semi-automated filtration system

doi: 10.1016/j.mex.2022.101785

Figure Lengend Snippet: Example of agarose gel picture to illustrate quality of PCR amplified genes from the DNA extracted from the enriched river sample with the various methods. Shown in the gel is the 100 bp DNA ladder (lane 1), Negative control (NTC; lane 2), in-house DNA extraction method, celite 1:420 dil. (Lane3), QIAamp 96 DNA QIAcube HT DNA extraction kit (Qiagen®) (Lane 4), QIAamp 96 DNA QIAcube HT DNA extraction kit (Qiagen®) with proteinase K (Lane 5), adapted in-house method, with binding buffer and proteinase K and no celite (Lane 6), Adapted in-house method, with binding buffer and proteinase K, no celite and no ethanol (Lane 7), adapted in-house method, with binding buffer, no celite, no ethanol and no proteinase K (Lane 8), E. coli DNA ladder (Lane 9), E. coli positive control PCR (Lane 10).

Article Snippet: A commensal non-pathogenic E. coli (ComEC) strain (ATCC 25922) was used in this study for the loading capacity experiment.

Techniques: Agarose Gel Electrophoresis, Amplification, Negative Control, DNA Extraction, Binding Assay, Positive Control

Journal: MethodsX

Article Title: DNA extraction of bacterial cells using a semi-automated filtration system

doi: 10.1016/j.mex.2022.101785

Figure Lengend Snippet:

Article Snippet: A commensal non-pathogenic E. coli (ComEC) strain (ATCC 25922) was used in this study for the loading capacity experiment.

Techniques: DNA Extraction, Purification

Journal: bioRxiv

Article Title: Generation of a porcine cell line stably expressing pig TMPRSS2 for efficient isolation of viruses from pigs with respiratory diseases

doi: 10.1101/2023.10.14.562371

Figure Lengend Snippet: ( a ) PK-15/TMPRSS2 #23 or normal PK-15 cells were infected with 15,000, 1,500, or 150 copies of IAV (H1N1), and vRNA levels in the culture supernatant were measured with RT-qPCR 2 days later. Results are presented as the mean and standard deviation of six measurements from one assay, representing at least three independent experiments. Cells treated with 100 ng/mL pig IFNβ served as negative control. ( b ) The relative value was calculated according to the values in normal PK-15 cells. Results are presented as the mean and standard deviation of six measurements from one assay and represent at least two independent experiments. Differences were examined by a two-tailed, unpaired Student’s t -test. **** p < 0.0001, * p < 0.05. ( c ) PK-15/TMPRSS2 #23 or normal PK-15 cells were infected with 15,000 copies of IAV (H1N1), and intracellular vRNA levels were measured with RT-qPCR 2 days later. The relative value was calculated according to the values in normal PK-15 cells. Results are presented as the mean and standard deviation of six measurements from one assay and represent at least two independent experiments. Differences were examined by a two-tailed, unpaired Student’s t -test. ** p < 0.01. ( d ) PK-15 ( Stat2 k/o)/TMPRSS2 #15, PK-15 ( Stat2 k/o), PK-15/TMPRSS2 #23, or normal PK-15 cells treated with one ng/mL pig IFNβ or left untreated for 24 hr and then infected with 15,000 copies of IAV (H1N1). vRNA levels in the culture supernatant were measured with RT-qPCR 2 days after infection. The relative value was calculated according to the values in normal PK-15 cells without pig IFNβ treatment. Results are presented as the mean and standard deviation of six measurements from one assay and represent at least two independent experiments.

Article Snippet: Influenza virus IAV (H1N1) strain A/PR/8/34 (American Type Culture Collection, Manassas, VA, USA, Cat# VR-95) was propagated in specific pathogen-free chicken embryonated eggs.

Techniques: Infection, Quantitative RT-PCR, Standard Deviation, Negative Control, Two Tailed Test

Journal: bioRxiv

Article Title: Generation of a porcine cell line stably expressing pig TMPRSS2 for efficient isolation of viruses from pigs with respiratory diseases

doi: 10.1101/2023.10.14.562371

Figure Lengend Snippet: ( a ) PK-15/TMPRSS2 #23 or normal PK-15 cells were infected with 80,000, 8,000, or 800 copies of SIV (H1N1). and vRNA levels in the culture supernatant were measured with RT-qPCR 2 days later Results are presented as the mean and standard deviation of six measurements from one assay, representing at least three independent experiments. Cells treated with 100 ng/mL pig IFNβ served as the negative control. ( b ) The relative value was calculated according to the values in normal PK-15 cells. Results are presented as the mean and standard deviation of six measurements from one assay and represent at least two independent experiments. Differences were examined by a Mann–Whitney U -test. ** p < 0.01.

Article Snippet: Influenza virus IAV (H1N1) strain A/PR/8/34 (American Type Culture Collection, Manassas, VA, USA, Cat# VR-95) was propagated in specific pathogen-free chicken embryonated eggs.

Techniques: Infection, Quantitative RT-PCR, Standard Deviation, Negative Control, MANN-WHITNEY

Journal: bioRxiv

Article Title: Generation of a porcine cell line stably expressing pig TMPRSS2 for efficient isolation of viruses from pigs with respiratory diseases

doi: 10.1101/2023.10.14.562371

Figure Lengend Snippet: ( a ) PK-15/TMPRSS2 #23 or VeroE6/TMPRSS2 cells were infected with 75,000 copies of IAV(H1N1), and vRNA levels in the culture supernatant were measured by RT-qPCR 2 days later. Results are the mean and standard deviation of six measurements from one assay, representing at least three independent experiments. Cells treated with 100 ng/mL pig IFNβ served as the negative control. The relative value was calculated according to the values in normal PK-15 cells. Results are the mean and standard deviation of six measurements from one assay and represent at least two independent experiments. Differences were examined by a two-tailed, unpaired Student’s t -test. **** p < 0.0001, *** p < 0.001.

Article Snippet: Influenza virus IAV (H1N1) strain A/PR/8/34 (American Type Culture Collection, Manassas, VA, USA, Cat# VR-95) was propagated in specific pathogen-free chicken embryonated eggs.

Techniques: Infection, Quantitative RT-PCR, Standard Deviation, Negative Control, Two Tailed Test

Journal: bioRxiv

Article Title: Generation of a porcine cell line stably expressing pig TMPRSS2 for efficient isolation of viruses from pigs with respiratory diseases

doi: 10.1101/2023.10.14.562371

Figure Lengend Snippet: ( a ) PK-15/TMPRSS2 #23 or normal PK-15 cells were infected with 15,000 copies of IAV(H1N1) in the presence of different concentrations of nafamostat mesylate, and vRNA levels in the culture supernatant were measured by RT-qPCR 2 days later. Results are the mean and standard deviation of measurements (n - 6) from one assay and are representative of at least three independent experiments. ( b ) The EC 50 of nafamostat mesylate against IAV (H1N1) in PK-15/TMPRSS2 #23 cells was calculated using Prism.

Article Snippet: Influenza virus IAV (H1N1) strain A/PR/8/34 (American Type Culture Collection, Manassas, VA, USA, Cat# VR-95) was propagated in specific pathogen-free chicken embryonated eggs.

Techniques: Infection, Quantitative RT-PCR, Standard Deviation

Journal:

Article Title: Association of Actinobacillus pleuropneumoniae Capsular Polysaccharide with Virulence in Pigs

doi: 10.1128/IAI.71.6.3320-3328.2003

Figure Lengend Snippet: Bacterial strains and plasmids used

Article Snippet: Kanamycin and streptomycin were used at 85 and 80 μg/ml, respectively, for selection of A. pleuropneumoniae recombinant strains. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain or plasmid Relevant genotype(s) or characteristic(s) Source or reference A. pleuropneumoniae strains 4074 Serotype 1 (ATCC 27088) ATCC a J45-100 Nonencapsulated mutant of serotype 5a 55 4074Δ cps 1N Nonencapsulated, nontypeable mutant of 4074; cps1AB Kan r This work 4074Δ cps 1B Nonencapsulated, nontypeable mutant of 4074; cps1B Kan r This work 4074Δ cps 1N(pAB cps 101) 4074Δ cps 1N containing pAB cps 101; serotype 1 + Str r This work 4074Δ cps 1N(pAB cps 55) 4074Δ cps 1N containing pAB cps 55; serotype 1 + Str r This work 4074Δ cps 1N(pJML cps 53) 4074Δ cps 1N containing pJML cps 53; serotype 1 + Str r This work E. coli XL1-Blue recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac ( F + proABlacI q ZΔM15 Tn 10 ); host for recombinant plasmids Stratagene Plasmids pBluescript II SK(+/−) 2.96-kb cloning vector; Amp r Stratagene pGSAp11 4.8-kb EcoR I fragment of 4074 cloned into pBluescript This work pMLAp53 8-kb EcoR I fragment of J45 cloned into pGEM-3z 55 pUC4K E. coli cloning vector; Amp r Kan r Pharmacia pAp12KB pGSAp11 with 0.7-kb Bgl II fragment deleted and blunt ended and 1.2-kb Sal I-cut and blunt-ended Kan r cassette from pUC4K ligated This work pGSAp11K pGSAp11 with 0.5-kb Nde I- SnaB I fragment deleted and blunt ended and 1.2-kb Sal I-cut and blunt-ended Kan r cassette from pUC4K ligated This work pLS88 Broad-host-range shuttle vector from H. ducreyii ; Str r Kan r 58 pAB cps 101 pLS88 containing cps1ABC in the Kan r site; Str r This work pAB cps 55 pLS88 containing cps5ABCDE in the Kan r site; Str r This work pJML cps 53 pLS88 containing cps5ABC in the Kan r site; Str r 55 Open in a separate window a American Type Culture Collection, Rockville, Md.

Techniques: Plasmid Preparation, Mutagenesis, Recombinant, Clone Assay

Journal:

Article Title: Association of Actinobacillus pleuropneumoniae Capsular Polysaccharide with Virulence in Pigs

doi: 10.1128/IAI.71.6.3320-3328.2003

Figure Lengend Snippet: Deletion of cps1AB in strain 4074Δcps1N. (A) The primers CPS1U2 and CPS1L3 were used to amplify the region deleted by mutagenesis of cps1AB. (B) The primers CPS1U3 and CPS1L4 were used to amplify the region not deleted by mutagenesis. The strains used were the serotype 5a knockout mutant strain J45-100 as a negative control (lanes 2), the serotype 1 parent strain 4074 (lanes 3), and the serotype 1 knockout mutant strain 4074Δcps1N (lanes 4).

Article Snippet: Kanamycin and streptomycin were used at 85 and 80 μg/ml, respectively, for selection of A. pleuropneumoniae recombinant strains. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain or plasmid Relevant genotype(s) or characteristic(s) Source or reference A. pleuropneumoniae strains 4074 Serotype 1 (ATCC 27088) ATCC a J45-100 Nonencapsulated mutant of serotype 5a 55 4074Δ cps 1N Nonencapsulated, nontypeable mutant of 4074; cps1AB Kan r This work 4074Δ cps 1B Nonencapsulated, nontypeable mutant of 4074; cps1B Kan r This work 4074Δ cps 1N(pAB cps 101) 4074Δ cps 1N containing pAB cps 101; serotype 1 + Str r This work 4074Δ cps 1N(pAB cps 55) 4074Δ cps 1N containing pAB cps 55; serotype 1 + Str r This work 4074Δ cps 1N(pJML cps 53) 4074Δ cps 1N containing pJML cps 53; serotype 1 + Str r This work E. coli XL1-Blue recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac ( F + proABlacI q ZΔM15 Tn 10 ); host for recombinant plasmids Stratagene Plasmids pBluescript II SK(+/−) 2.96-kb cloning vector; Amp r Stratagene pGSAp11 4.8-kb EcoR I fragment of 4074 cloned into pBluescript This work pMLAp53 8-kb EcoR I fragment of J45 cloned into pGEM-3z 55 pUC4K E. coli cloning vector; Amp r Kan r Pharmacia pAp12KB pGSAp11 with 0.7-kb Bgl II fragment deleted and blunt ended and 1.2-kb Sal I-cut and blunt-ended Kan r cassette from pUC4K ligated This work pGSAp11K pGSAp11 with 0.5-kb Nde I- SnaB I fragment deleted and blunt ended and 1.2-kb Sal I-cut and blunt-ended Kan r cassette from pUC4K ligated This work pLS88 Broad-host-range shuttle vector from H. ducreyii ; Str r Kan r 58 pAB cps 101 pLS88 containing cps1ABC in the Kan r site; Str r This work pAB cps 55 pLS88 containing cps5ABCDE in the Kan r site; Str r This work pJML cps 53 pLS88 containing cps5ABC in the Kan r site; Str r 55 Open in a separate window a American Type Culture Collection, Rockville, Md.

Techniques: Mutagenesis, Knock-Out, Negative Control

Journal:

Article Title: Association of Actinobacillus pleuropneumoniae Capsular Polysaccharide with Virulence in Pigs

doi: 10.1128/IAI.71.6.3320-3328.2003

Figure Lengend Snippet: Qualitative determination of CP production. Latex beads conjugated to IgG specific for serotype 1 CP was used. The parent strain, 4074, producing serotype 1 CP, agglutinated with the beads, while the nonencapsulated mutant 4074Δcps1N did not.

Article Snippet: Kanamycin and streptomycin were used at 85 and 80 μg/ml, respectively, for selection of A. pleuropneumoniae recombinant strains. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain or plasmid Relevant genotype(s) or characteristic(s) Source or reference A. pleuropneumoniae strains 4074 Serotype 1 (ATCC 27088) ATCC a J45-100 Nonencapsulated mutant of serotype 5a 55 4074Δ cps 1N Nonencapsulated, nontypeable mutant of 4074; cps1AB Kan r This work 4074Δ cps 1B Nonencapsulated, nontypeable mutant of 4074; cps1B Kan r This work 4074Δ cps 1N(pAB cps 101) 4074Δ cps 1N containing pAB cps 101; serotype 1 + Str r This work 4074Δ cps 1N(pAB cps 55) 4074Δ cps 1N containing pAB cps 55; serotype 1 + Str r This work 4074Δ cps 1N(pJML cps 53) 4074Δ cps 1N containing pJML cps 53; serotype 1 + Str r This work E. coli XL1-Blue recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac ( F + proABlacI q ZΔM15 Tn 10 ); host for recombinant plasmids Stratagene Plasmids pBluescript II SK(+/−) 2.96-kb cloning vector; Amp r Stratagene pGSAp11 4.8-kb EcoR I fragment of 4074 cloned into pBluescript This work pMLAp53 8-kb EcoR I fragment of J45 cloned into pGEM-3z 55 pUC4K E. coli cloning vector; Amp r Kan r Pharmacia pAp12KB pGSAp11 with 0.7-kb Bgl II fragment deleted and blunt ended and 1.2-kb Sal I-cut and blunt-ended Kan r cassette from pUC4K ligated This work pGSAp11K pGSAp11 with 0.5-kb Nde I- SnaB I fragment deleted and blunt ended and 1.2-kb Sal I-cut and blunt-ended Kan r cassette from pUC4K ligated This work pLS88 Broad-host-range shuttle vector from H. ducreyii ; Str r Kan r 58 pAB cps 101 pLS88 containing cps1ABC in the Kan r site; Str r This work pAB cps 55 pLS88 containing cps5ABCDE in the Kan r site; Str r This work pJML cps 53 pLS88 containing cps5ABC in the Kan r site; Str r 55 Open in a separate window a American Type Culture Collection, Rockville, Md.

Techniques: Mutagenesis

Journal:

Article Title: Association of Actinobacillus pleuropneumoniae Capsular Polysaccharide with Virulence in Pigs

doi: 10.1128/IAI.71.6.3320-3328.2003

Figure Lengend Snippet: Capsule production and degree of virulence in pigs of Ap serotype 1 isogenic strains

Article Snippet: Kanamycin and streptomycin were used at 85 and 80 μg/ml, respectively, for selection of A. pleuropneumoniae recombinant strains. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain or plasmid Relevant genotype(s) or characteristic(s) Source or reference A. pleuropneumoniae strains 4074 Serotype 1 (ATCC 27088) ATCC a J45-100 Nonencapsulated mutant of serotype 5a 55 4074Δ cps 1N Nonencapsulated, nontypeable mutant of 4074; cps1AB Kan r This work 4074Δ cps 1B Nonencapsulated, nontypeable mutant of 4074; cps1B Kan r This work 4074Δ cps 1N(pAB cps 101) 4074Δ cps 1N containing pAB cps 101; serotype 1 + Str r This work 4074Δ cps 1N(pAB cps 55) 4074Δ cps 1N containing pAB cps 55; serotype 1 + Str r This work 4074Δ cps 1N(pJML cps 53) 4074Δ cps 1N containing pJML cps 53; serotype 1 + Str r This work E. coli XL1-Blue recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac ( F + proABlacI q ZΔM15 Tn 10 ); host for recombinant plasmids Stratagene Plasmids pBluescript II SK(+/−) 2.96-kb cloning vector; Amp r Stratagene pGSAp11 4.8-kb EcoR I fragment of 4074 cloned into pBluescript This work pMLAp53 8-kb EcoR I fragment of J45 cloned into pGEM-3z 55 pUC4K E. coli cloning vector; Amp r Kan r Pharmacia pAp12KB pGSAp11 with 0.7-kb Bgl II fragment deleted and blunt ended and 1.2-kb Sal I-cut and blunt-ended Kan r cassette from pUC4K ligated This work pGSAp11K pGSAp11 with 0.5-kb Nde I- SnaB I fragment deleted and blunt ended and 1.2-kb Sal I-cut and blunt-ended Kan r cassette from pUC4K ligated This work pLS88 Broad-host-range shuttle vector from H. ducreyii ; Str r Kan r 58 pAB cps 101 pLS88 containing cps1ABC in the Kan r site; Str r This work pAB cps 55 pLS88 containing cps5ABCDE in the Kan r site; Str r This work pJML cps 53 pLS88 containing cps5ABC in the Kan r site; Str r 55 Open in a separate window a American Type Culture Collection, Rockville, Md.

Techniques: Produced

Journal:

Article Title: Association of Actinobacillus pleuropneumoniae Capsular Polysaccharide with Virulence in Pigs

doi: 10.1128/IAI.71.6.3320-3328.2003

Figure Lengend Snippet: Bactericidal activity of precolostral calf serum for strains 4074, 4074Δcps1N, and J45-100. The percent viability of each strain was evaluated 0 and 60 min after incubation at 37°C with 25% precolostral calf serum. Each data point represents the mean for three separate experiments performed in duplicate.

Article Snippet: Kanamycin and streptomycin were used at 85 and 80 μg/ml, respectively, for selection of A. pleuropneumoniae recombinant strains. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain or plasmid Relevant genotype(s) or characteristic(s) Source or reference A. pleuropneumoniae strains 4074 Serotype 1 (ATCC 27088) ATCC a J45-100 Nonencapsulated mutant of serotype 5a 55 4074Δ cps 1N Nonencapsulated, nontypeable mutant of 4074; cps1AB Kan r This work 4074Δ cps 1B Nonencapsulated, nontypeable mutant of 4074; cps1B Kan r This work 4074Δ cps 1N(pAB cps 101) 4074Δ cps 1N containing pAB cps 101; serotype 1 + Str r This work 4074Δ cps 1N(pAB cps 55) 4074Δ cps 1N containing pAB cps 55; serotype 1 + Str r This work 4074Δ cps 1N(pJML cps 53) 4074Δ cps 1N containing pJML cps 53; serotype 1 + Str r This work E. coli XL1-Blue recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac ( F + proABlacI q ZΔM15 Tn 10 ); host for recombinant plasmids Stratagene Plasmids pBluescript II SK(+/−) 2.96-kb cloning vector; Amp r Stratagene pGSAp11 4.8-kb EcoR I fragment of 4074 cloned into pBluescript This work pMLAp53 8-kb EcoR I fragment of J45 cloned into pGEM-3z 55 pUC4K E. coli cloning vector; Amp r Kan r Pharmacia pAp12KB pGSAp11 with 0.7-kb Bgl II fragment deleted and blunt ended and 1.2-kb Sal I-cut and blunt-ended Kan r cassette from pUC4K ligated This work pGSAp11K pGSAp11 with 0.5-kb Nde I- SnaB I fragment deleted and blunt ended and 1.2-kb Sal I-cut and blunt-ended Kan r cassette from pUC4K ligated This work pLS88 Broad-host-range shuttle vector from H. ducreyii ; Str r Kan r 58 pAB cps 101 pLS88 containing cps1ABC in the Kan r site; Str r This work pAB cps 55 pLS88 containing cps5ABCDE in the Kan r site; Str r This work pJML cps 53 pLS88 containing cps5ABC in the Kan r site; Str r 55 Open in a separate window a American Type Culture Collection, Rockville, Md.

Techniques: Activity Assay, Incubation

Journal:

Article Title: Association of Actinobacillus pleuropneumoniae Capsular Polysaccharide with Virulence in Pigs

doi: 10.1128/IAI.71.6.3320-3328.2003

Figure Lengend Snippet: Qualitative determination of CP production by latex beads conjugated to IgG specific for serotype 5 CP. Strains 4074 and 4074Δcps1N did not produce serotype 5 CP and did not agglutinate the beads, while the serotype 5 CP-producing chimeric strains 4074Δcps1N(pABcps55) and 4074Δcps1N(pJMLcps53) did agglutinate.

Article Snippet: Kanamycin and streptomycin were used at 85 and 80 μg/ml, respectively, for selection of A. pleuropneumoniae recombinant strains. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain or plasmid Relevant genotype(s) or characteristic(s) Source or reference A. pleuropneumoniae strains 4074 Serotype 1 (ATCC 27088) ATCC a J45-100 Nonencapsulated mutant of serotype 5a 55 4074Δ cps 1N Nonencapsulated, nontypeable mutant of 4074; cps1AB Kan r This work 4074Δ cps 1B Nonencapsulated, nontypeable mutant of 4074; cps1B Kan r This work 4074Δ cps 1N(pAB cps 101) 4074Δ cps 1N containing pAB cps 101; serotype 1 + Str r This work 4074Δ cps 1N(pAB cps 55) 4074Δ cps 1N containing pAB cps 55; serotype 1 + Str r This work 4074Δ cps 1N(pJML cps 53) 4074Δ cps 1N containing pJML cps 53; serotype 1 + Str r This work E. coli XL1-Blue recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac ( F + proABlacI q ZΔM15 Tn 10 ); host for recombinant plasmids Stratagene Plasmids pBluescript II SK(+/−) 2.96-kb cloning vector; Amp r Stratagene pGSAp11 4.8-kb EcoR I fragment of 4074 cloned into pBluescript This work pMLAp53 8-kb EcoR I fragment of J45 cloned into pGEM-3z 55 pUC4K E. coli cloning vector; Amp r Kan r Pharmacia pAp12KB pGSAp11 with 0.7-kb Bgl II fragment deleted and blunt ended and 1.2-kb Sal I-cut and blunt-ended Kan r cassette from pUC4K ligated This work pGSAp11K pGSAp11 with 0.5-kb Nde I- SnaB I fragment deleted and blunt ended and 1.2-kb Sal I-cut and blunt-ended Kan r cassette from pUC4K ligated This work pLS88 Broad-host-range shuttle vector from H. ducreyii ; Str r Kan r 58 pAB cps 101 pLS88 containing cps1ABC in the Kan r site; Str r This work pAB cps 55 pLS88 containing cps5ABCDE in the Kan r site; Str r This work pJML cps 53 pLS88 containing cps5ABC in the Kan r site; Str r 55 Open in a separate window a American Type Culture Collection, Rockville, Md.

Techniques:

Journal:

Article Title: Evaluation of Three Techniques for Detection of Low-Level Methicillin-Resistant Staphylococcus aureus (MRSA): a Disk Diffusion Method with Cefoxitin and Moxalactam, the Vitek 2 System, and the MRSA-Screen Latex Agglutination Test

doi: 10.1128/JCM.40.8.2766-2771.2002

Figure Lengend Snippet: Gel image of representative PCR mec gene products, mecA (528 bp), mecRA (310 bp), mecRB (236 bp), and mecI (481 bp), from different MRSA isolates, obtained with specific primers. Lane M, DNA molecular weight markers. Lanes 1, 7, 12, and 17, MRSA strain ATCC 44300; lanes 2, 8, and 13, MRSA strain CCUG 31966; lane 3, BORSA strain CCUG 35302; lanes 4, 9, 14, and 18, MRSA isolate 88; lanes 5, 10, and 15, MRSA isolate 112; lanes 6, 11, and 16, MRSA isolate 105.

Article Snippet: Control strains used for all assays included the MSSA β-lactamase-negative strain ATCC 25923, the BORSA (β-lactamase-positive, borderline methicillin-resistant, mecA -negative S. aureus ) strain CCUG 35302, the heterogeneous class 1 MRSA strain ATCC 43300 T , and the homogeneous MRSA strain CCUG 31966 from the Swedish Collection.

Techniques: Molecular Weight

Journal: Molecular plant

Article Title: DET1-mediated COP1 regulation avoids HY5 activity over second-site gene targets to tune plant photomorphogenesis.

doi: 10.1016/j.molp.2021.03.009

Figure Lengend Snippet: Figure 1. DET1- and COP1-associated proteins. (A) Schematic representation of proteins found to associate with DET1 and COP1 in TAP assays. Color code represents the maximum number of peptides for each represented protein found in a TAP assay as detailed in Supplemental Table 1. DET1 and COP1 proteins were expressed in Arabidopsis cell cultures. Five independent TAP experiments were performed for DET1 and two for COP1 (Supplemental Table 2). (B) MBP-COP1 and MBP-HY5 recombinant proteins expressed in E. coli pulled-down MYC-DET1 from 7-day-old Arabidopsis seedlings. MBP re- combinant protein was used as a control. Anti-MYC and anti-MBP antibodies were used for the immunoblots. (C and D) F€orster resonance energy transfer-fluorescence lifetime imaging microscopy (FRET–FLIM) analysis of the interaction between COP1 or HY5 fused to GFP and DET1 (C), and HY5 or COP1 fused to RFP (D). Box plots show the distribution of 5–9 measurements ±SD. (E) FRET–FLIM analysis of the interaction between GFP-COP1 and RFP-HY5 upon cluc-DET1 co-expression. Box plots show the distribution of 10 measurements ±SD. All FRET assays were performed following transient expression in N. benthamiana leaves. Free RFP was used as negative control. FE, FRET efficiency. Asterisks indicate statistically significant differences according to Student’s t-test (****p < 0.0001; ***p < 0.001; *p < 0.01). For all FRET experiments three independent biological replicates were performed, and results from one replicate are shown.

Article Snippet: Pull-down assays MBP recombinant protein fusions were expressed in the Escherichia coli BL21 (DE3) strain carrying the corresponding coding sequence cloned into the pKM596 plasmid, a gift from David Waugh (Addgene plasmid #8837).

Techniques: Recombinant, Control, Western Blot, Förster Resonance Energy Transfer, Imaging, Microscopy, Expressing, Negative Control

Journal: bioRxiv

Article Title: CARD-like domains mediate anti-phage defense in bacterial gasdermin systems

doi: 10.1101/2023.05.28.542683

Figure Lengend Snippet: a. Time-lapse microscopy of live E. coli cells expressing GFP (green) mixed with cells expressing the Lysobacter gasdermin system (black). Cells were infected with phage T6 at an MOI of 2 in the presence of propidium iodide (PI) and incubated at room temperature on an agar pad. Overlay images of phase contrast, green channel (GFP), and magenta channel (PI) are presented (scale bar = 0.5 µm). b. Phage replication assay. Plaque-forming units of phage T6 were sampled from the supernatant of E. coli cells containing an empty vector as a negative control or expressing the gasdermin system. Data show measured phage titer divided by the titer measured prior to infection. Cells were infected at an MOI of 0.01. Bars represent the average of three replicates with individual data points overlaid. c. Gasdermin cleavage during phage infection. Western blot analysis of N-terminally HA- tagged gasdermin following infection by phage T6 at an MOI of 2, in the WT or protease-mutated Lysobacter gasdermin system. d . Time-lapse microscopy of live cells expressing the WT or mutated Lysobacter gasdermin system in which gasdermin was N-terminally fused to GFP. Cells were infected with phage T6 in the presence of propidium iodide (PI) and incubated at room temperature on an agar pad. Overlay images of phase contrast, green channel (GFP), and magenta channel (PI) are presented.

Article Snippet: The strain Escherichia coli NEB 5-alpha (New England Biolabs, NEB C2987H) was used as a cloning strain.

Techniques: Time-lapse Microscopy, Expressing, Infection, Incubation, Plasmid Preparation, Negative Control, Western Blot

Journal: bioRxiv

Article Title: CARD-like domains mediate anti-phage defense in bacterial gasdermin systems

doi: 10.1101/2023.05.28.542683

Figure Lengend Snippet: Efficiency of plating of phages infecting E. coli cells that express the WT Lysobacter gasdermin system, as well as systems in which gasdermin was N-terminally fused to HA-tag or to GFP. Negative control is a strain in which GFP is expressed instead of the gasdermin system. Data represent plaque-forming units (PFU) per ml. Average of three independent replicates, with individual data points overlaid.

Article Snippet: The strain Escherichia coli NEB 5-alpha (New England Biolabs, NEB C2987H) was used as a cloning strain.

Techniques: Negative Control

Journal: bioRxiv

Article Title: CARD-like domains mediate anti-phage defense in bacterial gasdermin systems

doi: 10.1101/2023.05.28.542683

Figure Lengend Snippet: a. Domain architecture of the gasdermin defense system from Lysobacter enzymogenes . b. Alphafold2 prediction of the protease with the CARD-like domain. c . Crystal structure of the CARD-like domain encoded in the N-terminus of the Lysobacter gasdermin-associated protease. Also shown, for comparison, are the human ICEBERG CARD domain (PDB ID 1DGN), the PYD domain from human AIM2 (PDB ID 2N00) and the CARD domain from human NOD1 (PDB ID 2DBD) as well as topology diagrams of the Lysobacter CARD-like domain and the ICEBERG CARD domain. d. DALI Z scores of protein structures similar to the structure of the Lysobacter CARD-like domain. e . The CARD like domain is essential for gasdermin-mediated defense. Efficiency of plating of phages infecting E. coli cells that express either the WT gasdermin system or the system in which the CARD-like domain (residues 1-91) was deleted. Data represent plaque-forming units (PFU) per ml. Average of three independent replicates, with individual data points overlaid. Negative control is a strain in which GFP is expressed instead of the gasdermin system. f. A mutation in a conserved residue in the CARD-like domain abolishes defense. Experiment was performed as in panel E. g . Liquid culture growth of E. coli cells with WT or CARD-mutated gasdermin system, infected by phage T6 at 25°C. Bacteria were infected at time 0 at an MOI of 2 or 0.02. Three independent replicates are shown for each MOI, and each curve represents an individual replicate. h. Time-lapse microscopy of live E. coli cells expressing the WT or mutated Lysobacter gasdermin system in which gasdermin was N-terminally fused to GFP. Cells were infected with phage T6 in the presence of propidium iodide (PI) and incubated at room temperature on an agar pad. Overlay images of phase contrast, green channel (GFP), and magenta channel (PI) are presented. i. Gasdermin cleavage depends on integrity of a CARD-like domain. Western blot analyses of N-terminally HA-tagged gasdermin following infection by phage T6 at an MOI of 2, in the WT or CARD-mutated Lysobacter gasdermin system.

Article Snippet: The strain Escherichia coli NEB 5-alpha (New England Biolabs, NEB C2987H) was used as a cloning strain.

Techniques: Comparison, Negative Control, Mutagenesis, Residue, Infection, Bacteria, Time-lapse Microscopy, Expressing, Incubation, Western Blot

Journal: bioRxiv

Article Title: CARD-like domains mediate anti-phage defense in bacterial gasdermin systems

doi: 10.1101/2023.05.28.542683

Figure Lengend Snippet: a . Domain architecture of operons homologous to the Lysobacter gasdermin system. b. Defense profiles of the Lysobacter and the Pedobacter operons transformed into E. coli under an inducible promoter. Fold defense was measured using serial dilution plaque assays, comparing the efficiency of plating (EOP) of phages on the system-containing strain with the EOP on a control strain that lacks the system. Data represent an average of three replicates (see ). c. EOP of phages infecting E. coli cells that express either the WT or mutated system from Pedobacter rhizosphaerae . Data represent PFU per ml. Average of three independent replicates, with individual data points overlaid. Negative control is a strain in which GFP is expressed instead of the defense system. d. Domain architecture of operons in which structural homologs of the Lysobacter and Pedobacter proteases were detected. e. Multiple sequence alignment of the CARD-like domain from the protease of the Lysobacter , Pedobacter and Azospirillum systems. Shading indicates residue conservation and secondary structure elements are based on the Lysobacter CARD-like domain. f. Crystal structure of the CARD-like domain encoded in the N-terminus of the Azospirillum protease, and superposition of the Azospirillum and Lysobacter CARD-like domains.

Article Snippet: The strain Escherichia coli NEB 5-alpha (New England Biolabs, NEB C2987H) was used as a cloning strain.

Techniques: Transformation Assay, Serial Dilution, Control, Negative Control, Sequencing, Residue

Journal: bioRxiv

Article Title: CARD-like domains mediate anti-phage defense in bacterial gasdermin systems

doi: 10.1101/2023.05.28.542683

Figure Lengend Snippet: a. Efficiency of plating of phages infecting E. coli cells that express the Lysobacter or Pedobacter defense systems. Negative control is a strain in which GFP is expressed instead of the gasdermin system. Data represent plaque-forming units (PFU) per milliliter. Average of three independent replicates, with individual data points overlaid. b. Representative instances of homologous systems in their genomic neighborhood. Genes known to be involved in anti-phage defense are shown in yellow (RM, restriction-modification; CBASS, cyclic-oligonucleotide-based antiphage signaling system; AIPR, abortive infection phage resistance; REase, restriction endonuclease). c. DALI Z score of protein structures similar to the structure of the Azospirillum CARD-like structure.

Article Snippet: The strain Escherichia coli NEB 5-alpha (New England Biolabs, NEB C2987H) was used as a cloning strain.

Techniques: Negative Control, Modification, Infection

Journal: bioRxiv

Article Title: CARD-like domains mediate anti-phage defense in bacterial gasdermin systems

doi: 10.1101/2023.05.28.542683

Figure Lengend Snippet: a. T6 mutants that escape gasdermin-mediated defense. Data represent PFU per ml of phages infecting cells expressing the Lysobacter gasdermin system or control cells expressing GFP instead. b. Positions mutated in the rIIB gene of phage T6 escaper phages. Mutant numbers correspond to the numbers in panel A. c . Phage RIIB activates gasdermin aggregation in the absence of phage infection. Time-lapse microscopy of live cells co-expressing RIIB with the WT or mutated Lysobacter gasdermin system, in which gasdermin was N-terminally fused to GFP. Cells were visualized at room temperature on an agar pad in the presence of propidium iodide (PI). Overlay images of phase contrast, green channel (GFP), and magenta channel (PI) are presented. d. Gasdermins are cleaved when the system is co-expressed with RIIB. Western blot analyses of N-terminally HA-tagged gasdermin following induction of RIIB expression, in the WT or CARD-mutated Lysobacter gasdermin system. e. EOP of T6 mutant phages infecting cells that express the Pedobacter defense system. f. EOP of phages infecting E. coli cells that express either the Lysobacter gasdermin system or the RexAB system from the phage Lambda. Data represent PFU per ml. Average of three independent replicates, with individual data points overlaid. Negative control is a strain in which GFP is expressed instead of the defense system. g. Genomic neighborhoods of a phage CARD-only (pCARD) protein. h. The phage CARD-only protein interferes with gasdermin-mediated defense. Data represent PFU per milliliter of phages infecting cells expressing the Lysobacter gasdermin system together with a plasmid expressing pCARD or RFP, as well as cells expressing the pCARD gene alone. Control cells express GFP. i. Gasdermin cleavage is reduced in the presence of pCARD. Western blot analyses of N-terminally tagged gasdermin expressed together with pCARD or RFP following infection with phage T6 at an MOI of 2. j. Liquid culture growth of gasdermin-expressing E. coli cells infected with WT T4 or with T4 engineered to encode the pCARD gene instead of its IPI gene. Bacteria were infected at time 0 at an MOI of 2 or 0.02 at 25 °C. Three independent replicates are shown for each MOI, and each curve represents an individual replicate.

Article Snippet: The strain Escherichia coli NEB 5-alpha (New England Biolabs, NEB C2987H) was used as a cloning strain.

Techniques: Expressing, Control, Mutagenesis, Infection, Time-lapse Microscopy, Western Blot, Negative Control, Plasmid Preparation, Bacteria

Journal: bioRxiv

Article Title: CARD-like domains mediate anti-phage defense in bacterial gasdermin systems

doi: 10.1101/2023.05.28.542683

Figure Lengend Snippet: a. Bacteria expressing RIIB or RIIB together with the Lysobacter gasdermin system were plated in 10-fold serial dilution on LB-agar plates in conditions that repress expression (1% glucose) or induce expression (0.2% arabinose and 0.1 mM IPTG). b. Transformation efficiency assays of plasmids encoding rIIB or RFP into cells that contain the Lysobacter or Pedobacter systems on a pBAD plasmid. c. DALI Z scores of protein structures similar to the AlphaFold2 model of the phage CARD-only protein (pCARD). d. Liquid culture growth of E. coli control cells infected with WT T4 or with T4 engineered to encode the phage CARD-only gene (pCARD) instead of its IPI gene. Control bacteria, not encoding the defense system, were infected at time 0 at an MOI of 2 or 0.02 at 25 °C. Three independent replicates are shown for each MOI, and each curve represents an individual replicate.

Article Snippet: The strain Escherichia coli NEB 5-alpha (New England Biolabs, NEB C2987H) was used as a cloning strain.

Techniques: Bacteria, Expressing, Serial Dilution, Transformation Assay, Plasmid Preparation, Control, Infection