Journal: Biosensors

Article Title: Simple Detection of DNA Methyltransferase with an Integrated Padlock Probe

doi: 10.3390/bios12080569

Figure Lengend Snippet: Feasibility of M.SssI detection with RCA. ( A ) Fluorescence emission spectra with or without M.SssI following the addition of SYBR TM Gold. ( B ) Polyacrylamide gel electrophoresis supports the feasibility of the strategy: (Lane M) 20 bp DNA marker; (Lane a) PPs; (Lane b) PPc; (Lane c) PPs + PPc + T4 DNA ligase; (Lane d) PPs + PPc + T4 DNA ligase + HpaII + lambda exo + ExoI; (Lane e) PPs + PPc + T4 DNA ligase + M.SssI + HpaII + lambda exo+ ExoI.

Article Snippet: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/bios12080569/s1 , Table S1: Oligonucleotides used in this study; Table S2: Detection limits and real sample applications of some MTase detection methods; Figure S1: Electrophoresis analysis of the products of RCA of the detection system with (Lane a) and without (Lane b) M.SssI by RCA (Lane M) 15,000 bp DNA marker; Figure S2: Optimize the methylation and cleavage buffers. (A) F is the fluorescence intensity with M.SssI and F 0 is fluorescence intensity without M.SssI. (B) The fluorescence intensities of the sensing systems with or without M.SssI. (a) CutSmart buffer (methylation process) + CutSmart buffer (cleavage process); (b) NEBuffer 2 (methylation process) + CutSmart buffer (cleavage process); (c) NEBuffer 2(methylation process) + CutSmart buffer + lambda reaction buffer (cleavage process); (d) CutSmart buffer (methylation process) + lambda reaction buffer (cleavage process); Figure S3: Optimize the time of methylation process; Figure S4: Optimize the time of cleavage of HpaII and lambda exo; Figure S5: Optimize the time of digestion of ExoI; Figure S6: Eliminate the probable impact of detection system.

Techniques: Fluorescence, Polyacrylamide Gel Electrophoresis, Marker

Journal: Scientific reports

Article Title: Altered functional properties of the codling moth Orco mutagenized in the intracellular loop-3.

doi: 10.1038/s41598-021-83024-3

Figure Lengend Snippet: Figure 3. Comparison of HEK cell responses expressing wild-type and mutagenized form of Orco. (a)— Universal Orco agonist, VUAA1, elicits dose-dependent Ca++ i increase in HEK293A cells expressing either CpomOrco (blue) or CpomOrcoQ417H (red). (b)—VUAA1 concentration dependencies. Data points represent the mean response amplitudes (± SE). Data were fit to a Hill equation for CpomOrco (blue smooth line) or CpomOrcoQ417H (red smooth line) respectively, providing maximum responses of ~ 6.3 ± 0.1 and ~ 3.9 ± 1.3 ∆F, EC50s of ~ 157.1 ± 3.58 and ~ 261.8 ± 165.6 µM and Hill coefficients of ~ 2.4 ± 0.1 and ~ 2.1 ± 1.9; total number of cells analysed: N = 123 and 94. (c)—Effects of Pear ester on the activity of CpomOrco+OR3 (blue) and CpomOrcoQ417H+OR3 (red) heteromers. (d)—Pear ester concentration dependencies. Data points represent the mean response amplitudes (± SE) of cells from at least three experiments. Data were fit to a Hill equation providing the following parameters: maximum responses of ~ 6.6 ± 0.3 and 3.7 ± 0.1 ∆F; EC50s of ~ 210 ± 14.2 and ~ 733.5 ± 26.9 µM; Hill coefficients of ~ 4.6 ± 2.4 and ~ 4.6 ± 0.4, for CpomOR3/CpomOrco (blue smooth line) or CpomOR3/CpomOrcoQ417H (red smooth line) respectively. Total number of cells analysed: N = 160 and 262. Traces in A and C represent the mean responses of cells from one experiment. Data in B and D were not normalized. Scales in B and D are different.

Article Snippet: HEK293 cells lines (HEK293A/HEK293T) were grown in HEK cell media [Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (MP Biomedicals, Solon, OH, USA), 2.0 mM L-glutamine, and 100 μg/mL penicillin/streptomycin (Invitrogen)] at 37 °C and 5% CO2.

Techniques: Comparison, Expressing, Concentration Assay, Activity Assay

Journal: Scientific reports

Article Title: Altered functional properties of the codling moth Orco mutagenized in the intracellular loop-3.

doi: 10.1038/s41598-021-83024-3

Figure Lengend Snippet: Figure 4. Testing pH sensitivity of HEK cell expressing wild-type and mutagenized form of Orco. (a)— Decrease in fluorescence intensity of the pH sensitive probe, BCECF, possibly reflects acidification of cytoplasm in response to low pH extracellular conditions. (b)—Comparison of VUAA1 concentration dependencies obtained after 30 min incubation at low pHe for CpomOrco or CpomOrcoQ417H. Left panels—VUAA1 activated calcium responses. Right panels—VUAA1 concentration dependencies. Data points represent the mean response amplitudes (± SE). Data were fit to a Hill equation with the following parameters: maximum responses of ~ 1.3 ± 0.7 and ~ 1.8 ± 0.2 ∆F; EC50s of ~ 260 ± 187 and ~ 263.3 ± 45.3 µM; Hill coefficients of ~ 2.5 ± 4.3 and ~ 2.2 ± 0.5, for CpomOrco (blue smooth line, n = 84) or CpomOrcoQ417H (red smooth line, n = 63) respectively. Constraints were applied to fit greatly scattered data in B. Traces in B (left panels) represent the mean responses of cells from one experiment. Data in B (right panels) were not normalized. Concentration dependencies obtained in physiologically relevant control conditions were taken from Fig. 3.

Article Snippet: HEK293 cells lines (HEK293A/HEK293T) were grown in HEK cell media [Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (MP Biomedicals, Solon, OH, USA), 2.0 mM L-glutamine, and 100 μg/mL penicillin/streptomycin (Invitrogen)] at 37 °C and 5% CO2.

Techniques: Expressing, Fluorescence, Comparison, Concentration Assay, Incubation, Control