Journal: Communications Biology

Article Title: Porcine intraepithelial lymphocytes undergo migration and produce an antiviral response following intestinal virus infection

doi: 10.1038/s42003-022-03205-2

Figure Lengend Snippet: a Sankey diagram depicts the chemokine secretion in the host cells and the intestinal mucosa infected with variant and classical PEDV strains. b The protein expression of CCL2 in the ligated terminal jejunum injected with PEDV and in the jejunum from piglets orally inoculated with PEDV was detected via immunofluorescence analysis. CCL2 and PEDV were immunolabeled with anti-CCL2 mAb (green) and anti-PEDV polyclonal antibody (Red), respectively; the cell nuclei were stained with DAPI (blue). Scale bars, 50 μm. c The protein expression of CCL2 and CCL5 in the medium of Marc-145 cells was detected using ELISA kits. d Schematic of experimental setting used to study the migration of IELs influenced by chemokines induced by PEDV infection. e Chemokines CCL2, CCL5, and the CCL2 inhibitor TC1 were added into the medium of the basolateral side. Filters from the co-culture system were determined via CLSM, and the intraepithelial and transepithelial (DAPI, blue) migration of IELs (CFSE, green) in response to different treatments was shown by a three-dimensional (3D) rendering of representative fields. f Quantitative analysis of transepithelial IELs was performed. The number of transepithelial IELs was counted from five random fields of view at a unit area (0.078 mm 2 ) for each of the three individual filters. g The IELs/IPEC-J2 co-culture model was pretreated with an CCL2 inhibitor TC1 for 2 h (DMSO as a negative control) by upper compartment inoculation, followed by a culture with PEDV-infected or whole inactivated PEDV-treated Marc-145 cells. The intraepithelial and transepithelial (DAPI, blue) migration of IELs (CFSE, green) was detected via CLSM. h Quantitative analysis of transepithelial IELs in each experimental group. All data are the mean ± SD, and comparisons were performed using one-way ANOVA. * P < 0.05, ** P < 0.01. The results are from at least three different experiments.

Article Snippet: To inhibit the CCR2 receptor activity of IELs, we used CCR2 antagonist 4 (Teijin compound 1, TC1; MedChemExpress, China).

Techniques: Infection, Variant Assay, Expressing, Injection, Immunofluorescence, Immunolabeling, Staining, Enzyme-linked Immunosorbent Assay, Migration, Co-Culture Assay, Negative Control

Journal: Acta Biochimica et Biophysica Sinica

Article Title: A novel small positive allosteric modulator of neuropeptide receptor PAC1-R exerts neuroprotective effects in MPTP mouse Parkinson’s disease model

doi: 10.3724/abbs.2022126

Figure Lengend Snippet: The binding sites and the details of predicted binding modes of DOX and SPAM1−5 and the cytoprotective activity screening of SPAM1–5 (A−F) The protein PAC1-R is shown as a cartoon, and the molecule is shown as sticks. The blue stick illustrates the docked DOX using Schrödinger software, and the yellow stick is the DOX using DS software. The contact residues are shown and labelled by type and number. The red/green dotted line illustrates the hydrogen bond interaction. (G) Cell viability assayed by MTT showed that only SPAM1 at concentrations from 1 μM to 100 μM had a significant cytoprotective effect. * P<0.05, ** P<0.01. Data are presented as the mean±SEM of three experiments.

Article Snippet: In the present study, we firstly used computer virtual screening to search for novel small molecular chemical candidates from the TargetMol small molecule library, which can target the same positive allosteric modulation site in the PAC1-EC1 domain recognized by DOX.

Techniques: Binding Assay, Activity Assay, Software

Journal: Acta Biochimica et Biophysica Sinica

Article Title: A novel small positive allosteric modulator of neuropeptide receptor PAC1-R exerts neuroprotective effects in MPTP mouse Parkinson’s disease model

doi: 10.3724/abbs.2022126

Figure Lengend Snippet: Table 1 List of five chemicals with the top highest affinities in the virtual screening results targeting the positive allosteric binding site of PAC1-R

Article Snippet: In the present study, we firstly used computer virtual screening to search for novel small molecular chemical candidates from the TargetMol small molecule library, which can target the same positive allosteric modulation site in the PAC1-EC1 domain recognized by DOX.

Techniques: Binding Assay

Journal: Acta Biochimica et Biophysica Sinica

Article Title: A novel small positive allosteric modulator of neuropeptide receptor PAC1-R exerts neuroprotective effects in MPTP mouse Parkinson’s disease model

doi: 10.3724/abbs.2022126

Figure Lengend Snippet: Molecular docking and details of the predicted binding mode of DOX and SPAM1 targeting PAC1-R (A) The predicted binding site of DOX to the N-terminal extracellular domain of PAC1-R is different from the binding site of hydrazide as an antagonist of PAC1-R. (B) The predicted binding site of SPAM1 targeting PAC1-R involves the site recognized by DOX, but the binding site of SPAM1 was slightly larger than that recognized by DOX. As shown in the predicted binding modes (lower), SER120, GLY123 and ASP124 are involved in the interaction of SPAM1 with PAC1-R but are not included in the residues mediating the binding of DOX with PAC1-R. PAC1 and PACAP(6–38) are shown as cartoon styles, and the chemicals are shown as sticks. The binding site was represented by surface modelling. Dark blue represents the N-terminal extracellular domain of PAC1, light blue corresponds to the transmembrane domain of PAC1-R, orange corresponds to PACAP(6–38), and green represents DOX and SPAM1. The contacting residues are labelled by type and numbers, and the red dotted line illustrates the hydrogen bond interactions. (C) Both the predicted binding site of DOX and SPAM1 to the N-terminal extracellular domain of PAC1-R is similar to the binding site of PACAP(30−37).

Article Snippet: In the present study, we firstly used computer virtual screening to search for novel small molecular chemical candidates from the TargetMol small molecule library, which can target the same positive allosteric modulation site in the PAC1-EC1 domain recognized by DOX.

Techniques: Binding Assay

Journal: Acta Biochimica et Biophysica Sinica

Article Title: A novel small positive allosteric modulator of neuropeptide receptor PAC1-R exerts neuroprotective effects in MPTP mouse Parkinson’s disease model

doi: 10.3724/abbs.2022126

Figure Lengend Snippet: Table 2 The binding affinities of SPAM1 and DOX with PAC1-R predicted by computer molecular docking using DS software

Article Snippet: In the present study, we firstly used computer virtual screening to search for novel small molecular chemical candidates from the TargetMol small molecule library, which can target the same positive allosteric modulation site in the PAC1-EC1 domain recognized by DOX.

Techniques: Binding Assay, Software

Journal: Acta Biochimica et Biophysica Sinica

Article Title: A novel small positive allosteric modulator of neuropeptide receptor PAC1-R exerts neuroprotective effects in MPTP mouse Parkinson’s disease model

doi: 10.3724/abbs.2022126

Figure Lengend Snippet: Affinities of SPAM1, DOX and competitive binding of PACAP(28−38) with recombinant PAC1-EC1 protein measured by ITC assay and the antibiotic assay of SPAM1 and DOX (A‒C) The thermogram is shown in the upper panel, and the binding isotherm obtained by plotting the reaction heat vs the molar ratio of each chemical to PAC1-EC1 protein is shown in the lower panel. (D) The antibiotic assay of SPAM1 (left) and DOX (right). Small round pieces of paper with a diameter of 0.5 cm soaked with 200 μL of SPAM1 or DOX at concentrations of 1 μM (I), 10 μM (II) and 100 μM (III) were placed on LB agarose after the suspension of E. coli DH5α was planked, and dimethylsulfoxide (DMSO) (IV) was used as a control. After incubation at 37°C for 24 h, there was no observable antibacterial circle surrounding the paper pieces with SPAM1 at any detected concentration and DMSO, while there was a significant antibacterial circle surrounding the paper pieces with DOX, and the antibacterial circle enlarged with increasing DOX concentration. Data are presented as the mean±SEM, n=3.

Article Snippet: In the present study, we firstly used computer virtual screening to search for novel small molecular chemical candidates from the TargetMol small molecule library, which can target the same positive allosteric modulation site in the PAC1-EC1 domain recognized by DOX.

Techniques: Binding Assay, Recombinant, Isothermal Titration Calorimetry, Incubation, Concentration Assay

Journal: Acta Biochimica et Biophysica Sinica

Article Title: A novel small positive allosteric modulator of neuropeptide receptor PAC1-R exerts neuroprotective effects in MPTP mouse Parkinson’s disease model

doi: 10.3724/abbs.2022126

Figure Lengend Snippet: Effects of L-SPAM1, H-SPAM1, DOX, and PACAP38 on the levels of PAC1-R in MPTP-treated mouse substantia nigra and striatum (A) Immunoreactivity and quantitative measurement of PAC1-R staining intensity in the mouse substantia nigra and striatum. SPAM1 and DOX treatment significantly increased the levels of PAC1-R in the substantia nigra and striatum compared with the MPTP group, while SPAM1 worked in a dose-dependent manner. (B) The western bolt images of PAC1-R in striatum and the corresponding statistical analysis showed that L-SPAM1, H-SPAM1 and DOX increased the levels of PAC1-R in striatum compared with the MPTP group. & P<0.05, MPTP group vs control group (NOR); * P<0.05, vs MPTP group; ** P<0.01, vs MPTP group. Data are presented as the mean±SEM of three experiments. Scale bars: left, 500 μm; right, 100 μm. STR (striatum), and ST (substantia nigra).

Article Snippet: In the present study, we firstly used computer virtual screening to search for novel small molecular chemical candidates from the TargetMol small molecule library, which can target the same positive allosteric modulation site in the PAC1-EC1 domain recognized by DOX.

Techniques: Staining, Western Blot