Journal: Journal of translational medicine

Article Title: Photothermal therapy combined with a STING agonist induces pyroptosis, and gasdermin D could be a new biomarker for guiding the treatment of pancreatic cancer.

doi: 10.1186/s12967-025-06247-2

Figure Lengend Snippet: Scheme 1 A schematic summary of this study, showing that PTT induces pyroptosis via caspase-1/GSDMD pathway and arouses antitumour immune response. PTT combined with a STING agonist (c-di-GMP) as an immune adjuvant can amplify the aroused antitumour immunity

Article Snippet: Evaluation of synergistic therapeutic effect of PTT combined with a STING agonist For further evaluation of the therapeutic effect of PTT combined with STING agonist c-di-GMP (HY-110382, MedChemExpress, NJ, USA), C57BL/6 N mice were injected subcutaneously with 5 × 106 Pan02-luc cells per mouse.

Techniques: Adjuvant

Journal: Journal of translational medicine

Article Title: Photothermal therapy combined with a STING agonist induces pyroptosis, and gasdermin D could be a new biomarker for guiding the treatment of pancreatic cancer.

doi: 10.1186/s12967-025-06247-2

Figure Lengend Snippet: Fig. 3 Pyroptosis induced by PTT combined with STING agonist and evaluation of anti-tumour effects of different therapies in Pan02-luc tumour-bearing mice. (a) Pan02-luc cells were divided into the Blank, PTT, c-di-GMP and c-di-GMP + PTT groups. Total proteins were extracted and subjected to western blotting analysis. (b) Morphological images of Pan02-luc after different treatments under a bright field microscope. Scale bar: 100 μm. (c) Schematic dia gram of the pancreatic tumour model and administration method. (d) The tumour volume during a 14-day observation post treatment (n = 5). (e) Survival analysis of mice during a 90-day observation post treatment (n = 5). (f) The body weight of mice during a 14-day observation post treatment (n = 5). (g) H&E staining of the major organs from the c-di-GMP + PTT treated mice. Scale bar: 100 μm. For analysis of tumour volume and body weight at 14 days post treatment, data were represented as mean ± SD if they accord with normal distribution. The comparison among different groups was carried out using one-way ANOVA followed by post hoc test. For survival analysis, Kaplan-Meier survival curves were conducted and the comparison between different groups was performed by Mantel-Cox test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001

Article Snippet: Evaluation of synergistic therapeutic effect of PTT combined with a STING agonist For further evaluation of the therapeutic effect of PTT combined with STING agonist c-di-GMP (HY-110382, MedChemExpress, NJ, USA), C57BL/6 N mice were injected subcutaneously with 5 × 106 Pan02-luc cells per mouse.

Techniques: Western Blot, Microscopy, Staining, Comparison

Journal: Science Advances

Article Title: How sensor Amt-like proteins integrate ammonium signals

doi: 10.1126/sciadv.adm9441

Figure Lengend Snippet: ( A ) The conversion of two Mg-GTP into one cyclic-di-GMP followed Michaelis-Menten kinetics under steady-state conditions. Below 200 μM GTP, product inhibition was not apparent and a fit to the data yielded V max (μM min −1 ) = 0.240 ± 0.004 (black), 0.097 ± 0.002 (cyan), 0.050 ± 0.004 (purple), and 0.038 ± 0.003 (green) and K M (μM) = 7.90 ± 0.76 (black), 3.80 ± 0.165 (cyan), 5.83 ± 2.87 (purple), and 6.98 ± 4.53 (green), with R 2 (COD) of 0.994, 0.978, 0.928, and 0.666, respectively. Lower k cat and k cat /K M were recorded for the DGC domain (0.038 min −1 and 0.0054 μM −1 min −1 , respectively; green) and inactive Sd-Amt1 H169F/H323F (0.050 min −1 and 0.0086 μM −1 min −1 , respectively; purple). Protein isolated from M9 cultures yielded an intermediate situation [cyan; see (C) and (G)], while protein from rich cultures had the highest k cat /K M (0.0304 μM −1 min −1 ) [black; see (C) and (F)]. ( B ) Lineweaver-Burk fit of initial velocities for distinct GTP concentrations, in the absence (square) or presence (circle) of 50 μM cyclic-di-GMP. The second quadrant intercept indicates mixed competitive/noncompetitive inhibition. The presence of ammonium did not alter this profile. ( C ) The influence of NH 4 + on the reaction rates was analyzed under nonlimiting, 100 μM GTP concentrations [see (A)]. The results show that Sd-Amt1 from LB cultures (black) is largely unresponsive, in line with a full occupancy of N1/N2 (≈100% ON state). The Sd-Amt1 H169F/H323F (purple) is also unresponsive as it does not conduct ammonium into N1/N2 (≈100% OFF state). In contrast, protein from M9 cultures (cyan) showed concomitant increases in product formation rates. The DGC domain activity (green; negative control) is also shown. ( D to G ) Cartoon summary of the behavior for (D) Sd-Amt1 453–644 , (E) Sd-Amt1 1–644 H169F-H323F, and (F) Sd-Amt1 1–644 produced in rich and (G) Sd-Amt1 1–644 in M9 medium.

Article Snippet: To validate the DGC reactivity of Sd-Amt1 and Sd- amt1 453–644 and confirm cyclic-di-GMP as product, a discontinuous assay was used in which 10 to 20 μM of pure protein was incubated with 10 to 1000 μM GTP (Jena Biosciences) in the presence of 1 mM MgCl 2 with/without 1 mM NH 4 Cl.

Techniques: Inhibition, Isolation, Activity Assay, Negative Control, Produced

Journal: Science Advances

Article Title: How sensor Amt-like proteins integrate ammonium signals

doi: 10.1126/sciadv.adm9441

Figure Lengend Snippet: ( A ) The AlphaFold2 prediction for the entire Sd-Amt1 monomer is in very good agreement with the experimental structure for the membrane-integral portion of the NH 4 + -bound protein up to residue 405. In the cytoplasm, it reveals an HAMP domain in the connection to the DGC module. ( B ) Surface representations of the two sides of the HAMP domain colored red for increasing hydrophobicity. The domain has a more hydrophobic side (top) and one more hydrophilic (bottom). ( C ) Only when a concatenate of three copies of Sd-Amt1 cropped after the HAMP domain was used with AlphaFold2 in monomer mode, the program consistently predicted the dimerization of two HAMP domains as known from other structures, breaking the trimeric symmetry of the Amt module. ( D ) In the conformation obtained in (C), the addition of the cropped C terminus juxtaposed two DGC domains in a functional arrangement following the dimerized HAMP modules. ( E ) Superposition of the DGC domains from the completed Sd-Amt1 model [colored as in (D)] with the functional dimer of the DGC domain of Caulobacter crescentus DgcB (PDB-ID 6TTS, dark blue and orange). ( F ) In the structure of Cc-DgcB, the product cyclic-di-GMP is bound at the interface of the two DGC domains, implying that this particular arrangement represents the product-inhibited structure [chains colored as in (E)].

Article Snippet: To validate the DGC reactivity of Sd-Amt1 and Sd- amt1 453–644 and confirm cyclic-di-GMP as product, a discontinuous assay was used in which 10 to 20 μM of pure protein was incubated with 10 to 1000 μM GTP (Jena Biosciences) in the presence of 1 mM MgCl 2 with/without 1 mM NH 4 Cl.

Techniques: Membrane, Residue, Functional Assay

Journal: mBio

Article Title: Global Transcriptional Repression of Diguanylate Cyclases by MucR1 Is Essential for Sinorhizobium -Soybean Symbiosis

doi: 10.1128/mBio.01192-21

Figure Lengend Snippet: Overview of c-di-GMP signaling in Sinorhizobium fredii SF45436. (A) Synthesis of c-di-GMP by diguanylate cyclase (DGC) harboring the GGDEF domain and degradation of c-di-GMP by phosphodiesterase (PDE) containing the EAL domain. X indicates various sensory domains. The number of proteins, with each corresponding domain organization form, encoded by the SF45436 genome is shown (No.). Among them, the number of proteins with a conserved active motif [D X(7 aa) N X(8 aa) D X(21 aa) R X G /S/A GD /E EF] is indicated (Act). (B) The number of genes encoding proteins with putative activity of DGC or PDE, DGC-PDE bifunctional proteins, or PilZ-like proteins in Sinorhizobium sibling species. The point in orange corresponds to the value of SF45436. The other test Sinorhizobium strains include S. fredii SF25509, S. fredii SF83666, S. sojae SJ05684, and Sinorhizobium sp. strain SS05631. Error bars represent SD.

Article Snippet: The resultant 600-μl extraction solution was analyzed by HPLC-MS/MS with c-di-GMP (MedChemExpress, USA) as an internal standard.

Techniques: Activity Assay

Journal: mBio

Article Title: Global Transcriptional Repression of Diguanylate Cyclases by MucR1 Is Essential for Sinorhizobium -Soybean Symbiosis

doi: 10.1128/mBio.01192-21

Figure Lengend Snippet: Characterization of the GGDEF-containing proteins from S. fredii SF45436. (A) A sequence alignment of the GGDEF domain. A reported conserved motif essential for functional DGCs is shown. Gray background indicates other conserved amino acids in this sequence alignment (present in more than 80% sequences). Residues in black background form the reported allosteric I site involved in product inhibition of c-di-GMP synthesis. (B) Congo red binding ability of E. coli strains harboring various GGDEF-containing proteins from SF45436. Western blotting with anti-His monoclonal antibody shows the induced expression of corresponding proteins by IPTG. (+) and (−) indicate proteins with/without a conserved active motif [D X(7 aa) N X(8 aa) D X(21 aa) R X G /S/A GD /E EF]. YdeH is a known functional DGC from E. coli . For proteins not expressed in BL21(DE3) harboring pET28a(+) derivatives, the Rosetta strains (#) containing the same expressing plasmids or BL21(DE3) carrying pET30a derivatives ($) were tested. (C and D) HPLC-MS determination of c-di-GMP (C) or pGpG (D) content in 1 μM purified protein prepared from E. coli strains harboring corresponding vectors derived from pET30a or pET28a(+). Error bars represent standard deviations. Different letters above error bars indicate significant difference between means based on three independent experiments (ANOVA followed by Bonferroni’s multiple-comparison test, α = 0.05). (E) HPLC-MS determination of c-di-GMP content in E. coli strains expressing the indicated proteins relative to that of strains carrying the empty vector (*, P < 0.05; **, P < 0.01; ***, P < 0.001; t test). Error bars represent SD. Protein purification for these five proteins was not successful under test conditions, and in vitro enzymatic assay was not done.

Article Snippet: The resultant 600-μl extraction solution was analyzed by HPLC-MS/MS with c-di-GMP (MedChemExpress, USA) as an internal standard.

Techniques: Sequencing, Functional Assay, Inhibition, Binding Assay, Western Blot, Expressing, Purification, Derivative Assay, Comparison, Plasmid Preparation, Protein Purification, In Vitro, Enzymatic Assay

Journal: mBio

Article Title: Global Transcriptional Repression of Diguanylate Cyclases by MucR1 Is Essential for Sinorhizobium -Soybean Symbiosis

doi: 10.1128/mBio.01192-21

Figure Lengend Snippet: Symbiotic defects mediated by c-di-GMP overload in soybean nodules. (A) Chlorophyll content. (B) Nodule number per plant. Test strains include deletion (Δ) or complementary (.c) strains for DGC genes ( SFc17580 and SFb52570 ), the deletion mutant of PDE gene ( SFc33230 ), and derivatives carrying PDE ( SFc33230 EAL ) and DGC ( SFc17580 ) driven by the nifH ( SFa46030 ) promoter P nifH . The Δ mucR1 mutant was used for comparison. Error bars represent SEM. Different letters indicate significant differences between means based on more than 12 plants (ANOVA followed by Turkey’s multiple-comparison test; α = 0.05). (C) Pictures indicating yellow leaves and the increased nodule number of soybean plants inoculated with the P nifH -DGC and the Δ mucR1 mutant. (D) c-di-GMP levels in nodules (three biological replicates; error bars represent SD). (E) Transcriptional changes of SFc17580 and SFc33230 EAL in test strains (three biological replicates, ANOVA followed by Dunnett’s multiple comparisons; ***, P < 0.001; ****, P < 0.0001; error bars represent SD).

Article Snippet: The resultant 600-μl extraction solution was analyzed by HPLC-MS/MS with c-di-GMP (MedChemExpress, USA) as an internal standard.

Techniques: Mutagenesis, Comparison

Journal: mBio

Article Title: Global Transcriptional Repression of Diguanylate Cyclases by MucR1 Is Essential for Sinorhizobium -Soybean Symbiosis

doi: 10.1128/mBio.01192-21

Figure Lengend Snippet: Transcriptional changes of c-di-GMP responsive genes of the Δ mucR mutant and the P nifH -DGC and P nifH -PDE strains in soybean nodules. An iron-responsive regulator gene, rirA , is used as a control. Significant differences compared to the wild-type SF45436 are indicated (three biological replicates, ANOVA followed by Dunnett’s multiple comparisons; **, P < 0.01; ***, P < 0.001).

Article Snippet: The resultant 600-μl extraction solution was analyzed by HPLC-MS/MS with c-di-GMP (MedChemExpress, USA) as an internal standard.

Techniques: Mutagenesis, Control

Journal: mBio

Article Title: Global Transcriptional Repression of Diguanylate Cyclases by MucR1 Is Essential for Sinorhizobium -Soybean Symbiosis

doi: 10.1128/mBio.01192-21

Figure Lengend Snippet: Global repression of functional DGC genes located on chromosome and chromid by MucR in efficient soybean nodules. Genes encoding functional and nonfunctional DGCs are in red and blue, respectively. Artificial accumulation of c-di-GMP in nodules by using PnifH-c17580 leads to upregulation of various c-di-GMP responsive processes and impairs symbiotic performance. Other regulators can be involved in the coordinated regulation of these DGC genes under different conditions but have not been identified yet. Circular replicons (chromosome and chromid) are linearized here for presentation purpose.

Article Snippet: The resultant 600-μl extraction solution was analyzed by HPLC-MS/MS with c-di-GMP (MedChemExpress, USA) as an internal standard.

Techniques: Functional Assay