Journal: Applied and Environmental Microbiology

Article Title: Involvement of Two-Component System CBO0366/CBO0365 in the Cold Shock Response and Growth of Group I (Proteolytic) Clostridium botulinum ATCC 3502 at Low Temperatures

doi: 10.1128/AEM.00555-12

Figure Lengend Snippet: Bacterial strains and plasmids

Article Snippet: While constitutive expression and delicately balanced control through a phosphorelay system have been reported for most TCSs under normal growth conditions ( 18 , 19 , 24 , 30 ), induced TCS expression under cold stress conditions has been reported for B. subtilis and the psychrotrophic Y. pseudotuberculosis ( 4 , 31 ). table ft1 table-wrap mode="anchored" t5 caption a7 Strain or plasmid Relevant properties Source a (reference) Bacterial strains C. botulinum ATCC 3502 Wild type ATCC ( 34 ) C. botulinum ATCC 3502 cbo0365 ::intron- erm Insertion deletion in cbo0365 This study C. botulinum ATCC 3502 cbo0366 ::intron- erm Insertion deletion in cbo0366 This study E. coli TOP10 Electrocompetence Invitrogen, Paisley, UK E. coli CA434 Conjugation donor UNOTT ( 33 ) Plasmids pMTL82153 pBP1 g-positive replicon, catP , ColE1 g-negative replicon, tra , fdx promoter UNOTT ( 22 ) pMTL82153- cbo0366 pMTL82153 with cbo0366 under transcriptional control of fdx promoter This study pMTL007 ClosTron plasmid, catP , intron with ermB RAM UNOTT ( 21 ) pMTL007- cbo0365- 48s Derived from pMTL007 by retargeting to cbo0365 This study pMTL007- cbo0366- 267s Derived from pMTL007 by retargeting to cbo0366 This study Open in a separate window a ATCC, American Type Culture Collection; UNOTT, University of Nottingham, United Kingdom.

Techniques: Plasmid Preparation, Conjugation Assay, Control, Derivative Assay

Journal: Applied and Environmental Microbiology

Article Title: Involvement of Two-Component System CBO0366/CBO0365 in the Cold Shock Response and Growth of Group I (Proteolytic) Clostridium botulinum ATCC 3502 at Low Temperatures

doi: 10.1128/AEM.00555-12

Figure Lengend Snippet: Oligonucleotide primers

Article Snippet: While constitutive expression and delicately balanced control through a phosphorelay system have been reported for most TCSs under normal growth conditions ( 18 , 19 , 24 , 30 ), induced TCS expression under cold stress conditions has been reported for B. subtilis and the psychrotrophic Y. pseudotuberculosis ( 4 , 31 ). table ft1 table-wrap mode="anchored" t5 caption a7 Strain or plasmid Relevant properties Source a (reference) Bacterial strains C. botulinum ATCC 3502 Wild type ATCC ( 34 ) C. botulinum ATCC 3502 cbo0365 ::intron- erm Insertion deletion in cbo0365 This study C. botulinum ATCC 3502 cbo0366 ::intron- erm Insertion deletion in cbo0366 This study E. coli TOP10 Electrocompetence Invitrogen, Paisley, UK E. coli CA434 Conjugation donor UNOTT ( 33 ) Plasmids pMTL82153 pBP1 g-positive replicon, catP , ColE1 g-negative replicon, tra , fdx promoter UNOTT ( 22 ) pMTL82153- cbo0366 pMTL82153 with cbo0366 under transcriptional control of fdx promoter This study pMTL007 ClosTron plasmid, catP , intron with ermB RAM UNOTT ( 21 ) pMTL007- cbo0365- 48s Derived from pMTL007 by retargeting to cbo0365 This study pMTL007- cbo0366- 267s Derived from pMTL007 by retargeting to cbo0366 This study Open in a separate window a ATCC, American Type Culture Collection; UNOTT, University of Nottingham, United Kingdom.

Techniques: Sequencing, Binding Assay, Over Expression, Plasmid Preparation, Control, Mutagenesis

Journal: Applied and Environmental Microbiology

Article Title: Involvement of Two-Component System CBO0366/CBO0365 in the Cold Shock Response and Growth of Group I (Proteolytic) Clostridium botulinum ATCC 3502 at Low Temperatures

doi: 10.1128/AEM.00555-12

Figure Lengend Snippet: Relative expression levels of cbo0365 and cbo0366 in ATCC 3502 induced at 15°C. (A) ATCC 3502 was grown at 37°C, exposed to a temperature downshift (cold shock [gray curve]) at 15°C at an optical density at 600 nm (OD600) of 1.5, and sampled for qRT-PCR analysis before cold shock (T0) and 1 min, 30 min, 2 h, and 5 h after cold shock. A non-cold-shocked culture (black curve) served as a control. (B) Relative expression levels of cbo0365 (light gray) and cbo0366 (dark gray) in non-cold-shocked cultures calibrated at T0. (C) Relative expression levels of cbo0365 (light gray) and cbo0366 (dark gray) in cold-shocked cultures calibrated at T0. (D) Relative expression levels of cbo0365 (light gray) and cbo0366 (dark gray) in cold-shocked cultures calibrated to non-cold-shocked cultures at the corresponding time points. The normalization reference was 16S rrn. *, P < 0.05 (one-way analysis of variance). Error bars indicate standard deviations of three replicates.

Article Snippet: While constitutive expression and delicately balanced control through a phosphorelay system have been reported for most TCSs under normal growth conditions ( 18 , 19 , 24 , 30 ), induced TCS expression under cold stress conditions has been reported for B. subtilis and the psychrotrophic Y. pseudotuberculosis ( 4 , 31 ). table ft1 table-wrap mode="anchored" t5 caption a7 Strain or plasmid Relevant properties Source a (reference) Bacterial strains C. botulinum ATCC 3502 Wild type ATCC ( 34 ) C. botulinum ATCC 3502 cbo0365 ::intron- erm Insertion deletion in cbo0365 This study C. botulinum ATCC 3502 cbo0366 ::intron- erm Insertion deletion in cbo0366 This study E. coli TOP10 Electrocompetence Invitrogen, Paisley, UK E. coli CA434 Conjugation donor UNOTT ( 33 ) Plasmids pMTL82153 pBP1 g-positive replicon, catP , ColE1 g-negative replicon, tra , fdx promoter UNOTT ( 22 ) pMTL82153- cbo0366 pMTL82153 with cbo0366 under transcriptional control of fdx promoter This study pMTL007 ClosTron plasmid, catP , intron with ermB RAM UNOTT ( 21 ) pMTL007- cbo0365- 48s Derived from pMTL007 by retargeting to cbo0365 This study pMTL007- cbo0366- 267s Derived from pMTL007 by retargeting to cbo0366 This study Open in a separate window a ATCC, American Type Culture Collection; UNOTT, University of Nottingham, United Kingdom.

Techniques: Expressing, Quantitative RT-PCR, Control

Journal: Applied and Environmental Microbiology

Article Title: Involvement of Two-Component System CBO0366/CBO0365 in the Cold Shock Response and Growth of Group I (Proteolytic) Clostridium botulinum ATCC 3502 at Low Temperatures

doi: 10.1128/AEM.00555-12

Figure Lengend Snippet: Growth of cbo0365 and cbo0366 mutants under cold temperatures is impaired compared to ATCC 3502. ATCC 3502 wild type (black diamonds) and cbo0365 (open squares) and cbo0366 (multiplication symbols) mutants were grown in tryptose-peptone-glucose-yeast extract (TPGY) medium at 15°C, 20°C, and 37°C. Results for a negative-control sample (fresh TPGY) are marked with a dashed line. Error bars indicate standard deviations of three replicates.

Article Snippet: While constitutive expression and delicately balanced control through a phosphorelay system have been reported for most TCSs under normal growth conditions ( 18 , 19 , 24 , 30 ), induced TCS expression under cold stress conditions has been reported for B. subtilis and the psychrotrophic Y. pseudotuberculosis ( 4 , 31 ). table ft1 table-wrap mode="anchored" t5 caption a7 Strain or plasmid Relevant properties Source a (reference) Bacterial strains C. botulinum ATCC 3502 Wild type ATCC ( 34 ) C. botulinum ATCC 3502 cbo0365 ::intron- erm Insertion deletion in cbo0365 This study C. botulinum ATCC 3502 cbo0366 ::intron- erm Insertion deletion in cbo0366 This study E. coli TOP10 Electrocompetence Invitrogen, Paisley, UK E. coli CA434 Conjugation donor UNOTT ( 33 ) Plasmids pMTL82153 pBP1 g-positive replicon, catP , ColE1 g-negative replicon, tra , fdx promoter UNOTT ( 22 ) pMTL82153- cbo0366 pMTL82153 with cbo0366 under transcriptional control of fdx promoter This study pMTL007 ClosTron plasmid, catP , intron with ermB RAM UNOTT ( 21 ) pMTL007- cbo0365- 48s Derived from pMTL007 by retargeting to cbo0365 This study pMTL007- cbo0366- 267s Derived from pMTL007 by retargeting to cbo0366 This study Open in a separate window a ATCC, American Type Culture Collection; UNOTT, University of Nottingham, United Kingdom.

Techniques: Negative Control

Journal: Nature Communications

Article Title: Discovery of a signaling feedback circuit that defines interferon responses in myeloproliferative neoplasms

doi: 10.1038/s41467-022-29381-7

Figure Lengend Snippet: a Immunoblotting analysis of ROCK1 and ROCK2 in lysates from control (Ctrl) siRNA or ROCK1 and ROCK2 ( ROCK1/2 ) siRNA-transfected JAK2 V617F -positive SET-2 cells. b Proliferation and cell viability of control (Ctrl) siRNA or ROCK1 and ROCK2 ( ROCK1/2 ) siRNA-transfected JAK2 V617F -positive SET-2 cells either left untreated, or treated with IFNα was measured using a WST assay. Data are expressed as percent cell viability relative to Ctrl siRNA-transfected untreated cells (Ctrl siRNA), and represent means ± SEM of five independent experiments. Each symbol on the graphs represents an independent biological replicate. c Proliferation of JAK2 V617F -positive ( left panel ) SET-2 or ( middle and right panels ) HEL cells treated with ROCK1/2 inhibitor (GSK429286A or Fasudil) and/or IFNα vs. DMSO-vehicle control (Ctrl) was measured using a WST assay. Each symbol on the graphs represents an independent biological replicate. Data are expressed as percent cell viability relative to Ctrl-treated cells and represent means ± SEM of four, five and three independent experiments, as indicated. d Clonogenic capability of JAK2 V617F -positive HEL cells treated with the ROCK1/2 inhibitor GSK429286A and/or IFNα vs. DMSO-vehicle control (Ctrl). Each symbol on the graphs represents an independent biological replicate. Data are expressed as percent colony formation relative to Ctrl-treated cells and represent means ± SEM of three independent experiments. e Clonogenic capability of Ctrl siRNA or ROCK1/2 siRNA-transfected peripheral blood mononuclear cells from patients with PV, either left untreated or treated with IFNα. Data are expressed as percent colony formation relative to control siRNA-transfected untreated cells (Ctrl siRNA) and represent means ± SEM of four independent experiments, using cells from four different patients with PV. f Clonogenic capability of peripheral blood mononuclear cells from patients with PV treated with the ROCK1/2 inhibitor Fasudil and/or IFNα vs. DMSO-vehicle control (Ctrl). Data are expressed as percent colony formation relative to Ctrl-treated cells and represent means ± SEM of four independent experiments, as indicated, using cells from four different patients with PV. e , f Each data point on the graphs represents an independent biological replicate and data for an individual patient is represented by the same symbol for each experimental condition. b – d Data were normalized to the control group (Ctrl siRNA or Ctrl) and statistical analyses comparing the other three groups were performed using one-way ANOVA followed by Tukey’s multiple comparisons test. Adjusted p -values are reported. e , f Data were analyzed using linear mixed effects models, with % colony formation relative to the control group as the outcome, treatment (the three remaining groups) as the fixed effect, and subject as a random effect to account for within-subject correlation between multiple conditions. Kenward-Roger degrees of freedom adjustment was used, which improves performance when sample size is small. Pairwise group comparison tests were adjusted for multiple comparisons using Tukey’s method. p -values are reported. Source data are provided as a Source Data file.

Article Snippet: In the experiments to assess the effects of drug-targeted inhibition of ROCK1/2 on IFNα-induced anti-proliferative responses, SET-2 cells were seeded (10,000 cells/well) in quadruplicate in wells of 96-well plates and treated with vehicle-control (DMSO), GSK429286A (20 μM; #73182; StemCell Technologies), and/or human IFNα (100 IU/mL) for 5 days.

Techniques: Western Blot, Control, Transfection, WST Assay, Comparison