Journal: eLife

Article Title: A genetic selection reveals functional metastable structures embedded in a toxin-encoding mRNA

doi: 10.7554/eLife.47549

Figure Lengend Snippet: ( A ) Predicted hairpins involving the sequestration of the SD sequence by the aSD1 motif. Prediction of these secondary structures were carried out on the aapA3 -Tr transcript (see for visualizing the whole structure) using the same software as in , but with no additional constraint. The A28C mutation (dark orange) generates an extra G-C base-pair; in the A40T mutant (purple), two additional A-U pairs stabilize the hairpin; the A40T antagonist mutation A33T is shown in red; the SD and aSD1 sequences are shown in turquoise and yellow, respectively. ( B ) (left) PCR constructs used to assess the SD sequestering structure by transformation assay. (right) For each transformation with the indicated PCR constructs, the number of Str R obtained per total number of transformed cells was calculated and plotted on a log scale. Error bars represent s.d; n = 3 biological replicates. (***p<0.0001; *p=0.001 according to unpaired t -test). ( C ) Graph representing the RNase H assays. The position of the oligonucleotides (FA644 for WT and A40T; FA651 for A33T/A40T; and FA652 for A28C, see ) used in the RNase H protection assay is indicated by a black arrow along the first 45 nucleotides of the aapA3 mRNA . 30 fmol of internally labeled WT and mutated aapA3 -Tr transcripts were incubated with 0 to 100 pmoles of each specific DNA oligonucleotide and subjected to digestion by E. coli RNase H1. Digestion products were analyzed on an 8% PAA denaturing gel . Substrate consumption was quantified as relative substrate band intensity, 100% corresponding to the intensity obtained in absence of oligonucleotide. Error bars represent the s.d; n = 2 technical replicates. 10.7554/eLife.47549.018 Figure 5—source data 1. Raw data to determine the transformation efficiency in . 10.7554/eLife.47549.019 Figure 5—source data 2. Numerical values of the graph shown in in .

Article Snippet: To determine the secondary structure of RNA, 1 μl RNase T1 (0.01 U.μl-1; Ambion) was added to the labeled RNA and incubated in 1X Sequencing Buffer (20 mM Sodium Citrate, pH 5.0, 1 mM EDTA, 7M Urea) for 5 min at 37°C.

Techniques: Sequencing, Software, Mutagenesis, Construct, Transformation Assay, Labeling, Incubation

Journal: eLife

Article Title: A genetic selection reveals functional metastable structures embedded in a toxin-encoding mRNA

doi: 10.7554/eLife.47549

Figure Lengend Snippet: 30 fmol of internally labeled RNA were used. DNA oligonucleotides were used to a final concentration of 0 to 10 μM. Reactions were incubated for 30 min at 30°C in the presence or absence (C, control) of 0.25 U E. coli RNase H1. Reactions were stopped with 10 μl of 2X Loading Buffer and products were analyzed in an 8% denaturing PAA gel. See for relative band quantification. P1 and P2 indicate the two RNase H-oligonucleotide-mediated RNA cleavage products.

Article Snippet: To determine the secondary structure of RNA, 1 μl RNase T1 (0.01 U.μl-1; Ambion) was added to the labeled RNA and incubated in 1X Sequencing Buffer (20 mM Sodium Citrate, pH 5.0, 1 mM EDTA, 7M Urea) for 5 min at 37°C.

Techniques: Labeling, Concentration Assay, Incubation, Control

Journal: eLife

Article Title: A genetic selection reveals functional metastable structures embedded in a toxin-encoding mRNA

doi: 10.7554/eLife.47549

Figure Lengend Snippet: ( A ) The cell lysate of the T78C strain was subjected to ultracentrifugation through a sucrose gradient. RNA was analyzed as in . The different transcripts aapA3 -FL, aapA3 -Tr, and 5S rRNA (loading control) are indicated. M+D, monosomes + disomes. ( B ) Prediction of the secondary structure involving the second aSD sequence (aSD2). Prediction was carried out on the biologically relevant mRNA ( aapA3 -Tr) (see ). No additional constrains were used. The location of the oligonucleotide used in this assay (FA633) is shown by a black arrow in . The T78C mutation is shown in dark blue, SD sequence in turquoise, anti-SD sequence in yellow and start codon in green. ( C ) A typical RNase H protection assay is shown. A total of 30 fmol of internally labeled aapA3- FL and aapA3 -Tr RNA (WT or T78C) were incubated in presence of 0 to 100 pmoles of DNA oligonucleotide (FA633) and subjected to digestion by E. coli RNase H1. Lane C contains only the labeled substrate in absence of the enzyme. Two digestion products, P1 and P2, are indicated. ( D ) Substrate consumption was quantified as the relative substrate band intensity and plotted as a function of DNA oligonucleotide concentration. Error bars represent the s.d; n = 2 technical replicates. 10.7554/eLife.47549.024 Figure 6—source data 1. Numerical values of the graph shown in in .

Article Snippet: To determine the secondary structure of RNA, 1 μl RNase T1 (0.01 U.μl-1; Ambion) was added to the labeled RNA and incubated in 1X Sequencing Buffer (20 mM Sodium Citrate, pH 5.0, 1 mM EDTA, 7M Urea) for 5 min at 37°C.

Techniques: Control, Sequencing, Mutagenesis, Labeling, Incubation, Concentration Assay

Journal: eLife

Article Title: A genetic selection reveals functional metastable structures embedded in a toxin-encoding mRNA

doi: 10.7554/eLife.47549

Figure Lengend Snippet: 2D structure was predicted using the RNAfold Web Server with no additional constraints. VARNA was used for drawing. Translation start and stop codons are highlighted in green and red, respectively. Shine-Dalgarno (SD) and anti-SD (aSD) sequences are shown in turquoise and yellow, respectively. The suppressor mutation T78C is highlighted in dark blue. The predicted delta G value (kcal/mol) is shown under the structure. The small black arrow indicates the binding site of the oligonucleotide used in the RNase H assay shown in .

Article Snippet: To determine the secondary structure of RNA, 1 μl RNase T1 (0.01 U.μl-1; Ambion) was added to the labeled RNA and incubated in 1X Sequencing Buffer (20 mM Sodium Citrate, pH 5.0, 1 mM EDTA, 7M Urea) for 5 min at 37°C.

Techniques: Mutagenesis, Binding Assay, Rnase H Assay

Journal: eLife

Article Title: A genetic selection reveals functional metastable structures embedded in a toxin-encoding mRNA

doi: 10.7554/eLife.47549

Figure Lengend Snippet:

Article Snippet: To determine the secondary structure of RNA, 1 μl RNase T1 (0.01 U.μl-1; Ambion) was added to the labeled RNA and incubated in 1X Sequencing Buffer (20 mM Sodium Citrate, pH 5.0, 1 mM EDTA, 7M Urea) for 5 min at 37°C.

Techniques: Plasmid Preparation, Bacteria, DNA Extraction, Recombinant, Sequencing, Software

Journal: Vaccines

Article Title: Adenosine 2B Receptor Signaling Impairs Vaccine-Mediated Protection Against Pneumococcal Infection in Young Hosts by Blunting Neutrophil Killing of Antibody-Opsonized Bacteria

doi: 10.3390/vaccines13040414

Figure Lengend Snippet: The absence of A2B receptor increases intracellular killing by PMNs following antibody-mediated uptake. BM PMNs were isolated from young wild-type (WT) C57BL/6J or A2BR −/− mice. PMNs were infected with S. pneumoniae Δply TIGR4 that was pre-opsonized with naïve or HI immune sera. Gentamicin was added to kill extracellular bacteria not taken in. The percentage of intracellular bacteria killed was then calculated following plating of the reactions on blood agar to determine CFU. Fold changes in bacterial killing of A2BR −/− versus WT PMNs are shown. Data from five separate experiments with n = 5 biological replicates are pooled. * denotes significantly different from one ( p < 0.05) as determined by One Sample t - and Wilcoxon tests.

Article Snippet: Old mice were treated with specific A2B receptor antagonist MRS 1754 (Tocris Bioscience) dissolved in DMSO, which was filter sterilized by being passed through a 0.22 μm filter prior to use and was given to mice via intraperitoneal injection at a volume of 100 μL and a concentration of 2.5 mg/kg.

Techniques: Isolation, Infection, Bacteria

Journal: Vaccines

Article Title: Adenosine 2B Receptor Signaling Impairs Vaccine-Mediated Protection Against Pneumococcal Infection in Young Hosts by Blunting Neutrophil Killing of Antibody-Opsonized Bacteria

doi: 10.3390/vaccines13040414

Figure Lengend Snippet: A2BR signaling blunts intracellular CRAMP levels in PMNs following antibody-mediated bacterial uptake. PMNs were isolated from the bone marrow of young wild-type C57BL/6J or A2BR −/− mice. PMNs were infected with S. pneumoniae opsonized with HI immune sera. ( A , B ) Mitochondrial ROS production was determined using MitoSOX. ( A ) Mitochondrial ROS production over the course of 1 hour following infection. Representative data of one of three separate experiments are shown. ( B ) Fold changes in mitochondrial ROS production by A2BR −/− PMNs from wild-type controls were calculated using area under the curve. Data are pooled from separate experiments and * indicates significantly different from one as determined by One Sample t - and Wilcoxon tests. ( C , D ) Cell pellets were collected and lysed and CRAMP ELISA was performed. Pooled data from separate mice ( C ) and corresponding fold changes from uninfected baselines ( D ) are shown. ( C ) number indicates the p value as determined by unpaired Student’s t -test and ( D ) * indicates significantly different from one as determined by One sample t - and Wilcoxon tests.

Article Snippet: Old mice were treated with specific A2B receptor antagonist MRS 1754 (Tocris Bioscience) dissolved in DMSO, which was filter sterilized by being passed through a 0.22 μm filter prior to use and was given to mice via intraperitoneal injection at a volume of 100 μL and a concentration of 2.5 mg/kg.

Techniques: Isolation, Infection, Enzyme-linked Immunosorbent Assay

Journal: Vaccines

Article Title: Adenosine 2B Receptor Signaling Impairs Vaccine-Mediated Protection Against Pneumococcal Infection in Young Hosts by Blunting Neutrophil Killing of Antibody-Opsonized Bacteria

doi: 10.3390/vaccines13040414

Figure Lengend Snippet: A2BR activation in vivo worsens disease outcome in young, vaccinated mice. Young C57BL/6J mice were vaccinated with PCV and, 4 weeks later, infected with 5 × 10 6 CFU S. pneumoniae TIGR4. Eighteen hours prior to, at the time of, and 18 h post-infection, mice were treated i.p with a specific A2BR agonist or VC. twenty-four hours post-infection, mice were assessed for clinical signs of disease ( A ). Organs were harvested 24 hpi and CFU in the lungs ( B ) was determined by plating on blood agar. * denotes significant differences ( p < 0.05) determined by Mann–Whitney test. The limit of detection (LOD) is denoted by the dashed line. Data are pooled from two separate experiments and each point represents an individual mouse.

Article Snippet: Old mice were treated with specific A2B receptor antagonist MRS 1754 (Tocris Bioscience) dissolved in DMSO, which was filter sterilized by being passed through a 0.22 μm filter prior to use and was given to mice via intraperitoneal injection at a volume of 100 μL and a concentration of 2.5 mg/kg.

Techniques: Activation Assay, In Vivo, Infection, MANN-WHITNEY

Journal: Vaccines

Article Title: Adenosine 2B Receptor Signaling Impairs Vaccine-Mediated Protection Against Pneumococcal Infection in Young Hosts by Blunting Neutrophil Killing of Antibody-Opsonized Bacteria

doi: 10.3390/vaccines13040414

Figure Lengend Snippet: The absence of A2B receptor signaling does not boost the ability of PMNs from aged mice to kill bacteria intracellularly. BM PMNs isolated from old (18 months) wild-type (WT) C57BL/6J or A2BR −/− mice were infected with S. pneumoniae Δply TIGR4 that was pre-opsonized with naïve or HI immune sera. Gentamicin was added to kill extracellular bacteria not taken in. Reactions were plated on blood agar to enumerate CFU. The % of engulfed bacteria that were killed was then calculated. Fold changes in bacterial killing of A2BR −/− versus WT PMNs are shown. Data from five separate experiments with n = 5 biological replicates are pooled.

Article Snippet: Old mice were treated with specific A2B receptor antagonist MRS 1754 (Tocris Bioscience) dissolved in DMSO, which was filter sterilized by being passed through a 0.22 μm filter prior to use and was given to mice via intraperitoneal injection at a volume of 100 μL and a concentration of 2.5 mg/kg.

Techniques: Bacteria, Isolation, Infection

Journal: Vaccines

Article Title: Adenosine 2B Receptor Signaling Impairs Vaccine-Mediated Protection Against Pneumococcal Infection in Young Hosts by Blunting Neutrophil Killing of Antibody-Opsonized Bacteria

doi: 10.3390/vaccines13040414

Figure Lengend Snippet: A2BR inhibition in vivo has no effect on disease outcome in aged, vaccinated mice. Old (20–22 months) C57BL/6JN mice were vaccinated with PCV and, 4 weeks later, infected with 5 × 10 6 CFU S. pnuemoniae TIGR4. Eighteen hours prior to, at the time of, and 18 h post-infection, mice were treated i.p with either a specific A2BR inhibitor alone, a combination of specific A2BR inhibitor and A2AR inhibitor, or VC. Twenty-four hours post-infection, mice were assessed for clinical signs of disease ( A ). Organs were harvested 24 hpi and CFU in the lungs ( B ), blood ( C ), and brain ( D ) was enumerated by plating on blood agar. The dashed line indicates the limit of detection (LOD). Data are pooled from two separate experiments and each data point represents an individual mouse.

Article Snippet: Old mice were treated with specific A2B receptor antagonist MRS 1754 (Tocris Bioscience) dissolved in DMSO, which was filter sterilized by being passed through a 0.22 μm filter prior to use and was given to mice via intraperitoneal injection at a volume of 100 μL and a concentration of 2.5 mg/kg.

Techniques: Inhibition, In Vivo, Infection

Journal:

Article Title: Biological Characterization of Lipopolysaccharide from Treponema pectinovorum

doi: 10.1128/IAI.70.1.211-217.2002

Figure Lengend Snippet: IL-6, IL-8, and MCP-1 secretion by HGFs (Gin-7) challenged with purified T. pectinovorum ATCC 33768 LPS at 0.1, 1, 10, and 100 μg per ml. Supernatants were collected at 1, 3, 6, 12, 24, and 48 h after LPS challenge. Each cytokine concentration was determined by sequencial ELISA. The cells were stimulated with 5 × 109 bacteria. The bars denote the mean level of cytokine detected during 12-and 24-h culture periods from triplicate determinations.

Article Snippet: Following incubation of the serum, the system was developed with biotinylated goat polyclonal antibody specific for each cytokine (IL-1β, TNF-α, and IL-6; R & D Systems), streptavidin-alkaline phosphatase (Zymed, San Francisco, Calif.), and p -nitrophenylphosphate (Sigma).

Techniques: Purification, Concentration Assay, Enzyme-linked Immunosorbent Assay, Bacteria

Journal:

Article Title: Biological Characterization of Lipopolysaccharide from Treponema pectinovorum

doi: 10.1128/IAI.70.1.211-217.2002

Figure Lengend Snippet: Cytokine (IL-1β, TNF-α, and IL-6) pattern in C3H/HeN Tac-MTV (LPS responder) mice after LPS treatment. Mice (n = 3) were injected i.p. with 5, 0.5, and 0.05 μg of T. pectinovorum ATCC 33768 LPS with GalN as indicated in Materials and Methods. Serum samples were taken at 3, 8, and 12 h after LPS administration. Each cytokine concentration was determined by ELISA. Results are the means and standard deviations for three mice at 12 h.

Article Snippet: Following incubation of the serum, the system was developed with biotinylated goat polyclonal antibody specific for each cytokine (IL-1β, TNF-α, and IL-6; R & D Systems), streptavidin-alkaline phosphatase (Zymed, San Francisco, Calif.), and p -nitrophenylphosphate (Sigma).

Techniques: Injection, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Biological Characterization of Lipopolysaccharide from Treponema pectinovorum

doi: 10.1128/IAI.70.1.211-217.2002

Figure Lengend Snippet: Proinflammatory-cytokine (IL-1β, TNF-α, and IL-6) patterns in C3H/HeN Tac-MTV (LPS responder) mice were analyzed after i.p. challenge infection with live (108 cells) and heat-killed (H-K) (108 cells) T. pectinovorum ATCC 33768 and live E. coli O111 (105 cells) with and without GalN as described in Materials and Methods. Serum samples were taken at 3, 8, and 12 h postinfection. Each cytokine concentration was determined by sequential ELISA. Results are the means and standard deviations for three mice at 12 h.

Article Snippet: Following incubation of the serum, the system was developed with biotinylated goat polyclonal antibody specific for each cytokine (IL-1β, TNF-α, and IL-6; R & D Systems), streptavidin-alkaline phosphatase (Zymed, San Francisco, Calif.), and p -nitrophenylphosphate (Sigma).

Techniques: Infection, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Investigative Ophthalmology & Visual Science

Article Title: Long-Term Durability of Ciliary Neurotrophic Factor–Releasing Revakinagene Taroretcel-lwey in Individuals With Retinal Degenerative Disorders

doi: 10.1167/iovs.66.11.3

Figure Lengend Snippet: Ciliary neurotrophic factor (CNTF) pharmacokinetics from explanted revakinagene taroretcel-lwey devices. Secretion of CNTF (ng/day) by revakinagene taroretcel-lwey devices explanted over 14.5 years of implant durations, grouped to the nearest half-year for durations <3 years. The solid line indicates the weighted mean CNTF output level, and the dotted lines indicate the range of the 95% confidence interval of the slope. CNTF, ciliary neurotrophic factor; ng, nanogram.

Article Snippet: An ELISA was also used to quantify specific anti-human CNTF antibody titers after incubating participant serum on a plate coated with rhCNTF (R&D Systems).

Techniques: Drug discovery

Journal: Investigative Ophthalmology & Visual Science

Article Title: Long-Term Durability of Ciliary Neurotrophic Factor–Releasing Revakinagene Taroretcel-lwey in Individuals With Retinal Degenerative Disorders

doi: 10.1167/iovs.66.11.3

Figure Lengend Snippet: Units of activity of CNTF produced by revakinagene taroretcel-lwey explants over 14.5 years of implant duration. Seven samples were analyzed, 2 of which were analyzed twice, and the results were averaged. The solid line indicates the slope of units of activity, and the dotted lines indicate the range of the 95% confidence interval of the slope. CNTF, ciliary neurotrophic factor; EC 50 , half-maximal effective concentration.

Article Snippet: An ELISA was also used to quantify specific anti-human CNTF antibody titers after incubating participant serum on a plate coated with rhCNTF (R&D Systems).

Techniques: Activity Assay, Produced, Concentration Assay

Journal: Investigative Ophthalmology & Visual Science

Article Title: Long-Term Durability of Ciliary Neurotrophic Factor–Releasing Revakinagene Taroretcel-lwey in Individuals With Retinal Degenerative Disorders

doi: 10.1167/iovs.66.11.3

Figure Lengend Snippet: Electropherograms of CNTF from explanted revakinagene taroretcel-lwey. ( A ) Electropherograms and ( B ) corresponding bands from CNTF-conditioned media collected from devices explanted from 6 participants over 14.5 years. The explant-derived CNTF is a mixture of 29 kDa human CNTF and the 33 kDa glycosylated CNTF unique to NTC-201-6A cells. Electropherograms of ( C ) controls and ( D ) corresponding bands are shown. The controls used in this analysis include purified CNTF produced in bacteria, purified revakinagene taroretcel-lwey–secreted CNTF, and culture media. Purified CNTF protein produced in bacteria consistently has a high MW shoulder. CNTF, ciliary neurotrophic factor; kDa, kilodalton; MW, molecular weight.

Article Snippet: An ELISA was also used to quantify specific anti-human CNTF antibody titers after incubating participant serum on a plate coated with rhCNTF (R&D Systems).

Techniques: Derivative Assay, Purification, Produced, Bacteria, Molecular Weight

Journal: Nanomaterials

Article Title: Photocatalytic Inactivation of Plant Pathogenic Bacteria Using TiO 2 Nanoparticles Prepared Hydrothermally

doi: 10.3390/nano10091730

Figure Lengend Snippet: Antibacterial activity of A310C and P25 TiO 2 photocatalysts against Xanthomonas arboricola pv. juglandis , Allorhizobium vitis , Erwinia amylovora , and Pseudomonas syringae under UV-A irradiation. The concentration of aqueous TiO 2 dispersions was 0.5 mg/mL. Before UV-A irradiation, dispersions containing bacteria were kept in the dark for 15 min.

Article Snippet: For the pathogen inactivation study, four Gram-negative bacterium species such as the ATCC 49946 strain Erwinia amylovora (American Type Culture Collection, Manassas, VA, USA), the DSMZ 1049 strain Xanthomonas arboricola pv. juglandis (DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany), the DC3000 strain Pseudomonas syringae pv. tomato and the Allorhizobium vitis isolates were used.

Techniques: Activity Assay, Irradiation, Concentration Assay, Bacteria

Journal: Lab on a chip

Article Title: LabDisk with complete reagent prestorage for sample-to-answer nucleic acid based detection of respiratory pathogens verified with influenza A H3N2 virus.

doi: 10.1039/c5lc00871a

Figure Lengend Snippet: Fig. 3 Fluorescence signal for parallel RT-PCR reactions acquired after the annealing step in each reaction cavity. Except reaction cavity 4 (parainfluenza panel) and reaction cavity 8 (atypical bacteria panel) each reaction cavity contained primers and fluorescence probe also detecting RNA bacteriophage MS2 as internal control. Missing primer and fluorescence probes for internal control led to the negative signal in these two reaction cavities.

Article Snippet: As initial sample material we used the RNA bacteriophage MS2 (DSM-13767, type: Levivirus, species: Enterobacteria phage MS2, DSMZ GmbH, Germany) acting as internal control of the used respiratory panels and was diluted with SM buffer, pH 7.5 (Alfa Aesar GmbH & Co KG, Germany) and applied with the concentration of 75 pfu ml−1.

Techniques: Fluorescence, Reverse Transcription Polymerase Chain Reaction, Bacteria, Control