Journal: PLOS ONE

Article Title: Deep learning based predictive modeling to screen natural compounds against TNF-alpha for the potential management of rheumatoid arthritis: Virtual screening to comprehensive in silico investigation

doi: 10.1371/journal.pone.0303954

Figure Lengend Snippet: The model was developed using the ChEMBL dataset, with 2AZ5 as the target protein. After validation, the model was used to screen the Selleckchem Natural Compounds database. Compounds were prioritized based on their predicted pIC 50 values, and after additional filtering, four compounds were chosen for MD simulation studies.

Article Snippet: From the approximately 2563 substances within the Selleckchem natural product library, our study employed a DL-based model for screening, followed by assessment through ADMET prediction and docking to initially identify four potential candidates: Imperialine, Veratramine, Jervine, and Gelsemine.

Techniques: Biomarker Discovery

Journal: Frontiers in Veterinary Science

Article Title: Detection of spermatogonial stem cells in testicular tissue of dogs with chronic asymptomatic orchitis

doi: 10.3389/fvets.2023.1205064

Figure Lengend Snippet: Sequences of primers for RT-PCR and RT-qPCR, amplicon length, efficiency, and accession number (for, forward; rev, reverse).

Article Snippet: For PGP9.5, non-specific protein binding was blocked with 3% bovine serum albumin (BSA, VWR Life Science, Solon, OH, USA) and 10% horse serum (S-2000, RRID: AB_2336617, Vector Laboratories), and slides were subsequently incubated with the PGP9.5 monoclonal mouse anti-human antibody (7863-1004, RRID: AB_620256, AbD Serotec, Raleigh, NC, USA, dilution 1 x 10 −3 μg/μl) overnight at 4°C.

Techniques: Amplification, Sequencing

Journal: Frontiers in Veterinary Science

Article Title: Detection of spermatogonial stem cells in testicular tissue of dogs with chronic asymptomatic orchitis

doi: 10.3389/fvets.2023.1205064

Figure Lengend Snippet: Overview of reagents and dilutions used in the Western blot; SKU, Stock Keeping Unit; IC, Isotype control; * Abcam, Cambridge, UK; ** Bio-Rad Laboratories, Hercules, California, USA; *** Agilent Technologies, Santa Clara, California, USA; **** Cell Signaling Technology, Danvers, Massachusetts, USA.

Article Snippet: For PGP9.5, non-specific protein binding was blocked with 3% bovine serum albumin (BSA, VWR Life Science, Solon, OH, USA) and 10% horse serum (S-2000, RRID: AB_2336617, Vector Laboratories), and slides were subsequently incubated with the PGP9.5 monoclonal mouse anti-human antibody (7863-1004, RRID: AB_620256, AbD Serotec, Raleigh, NC, USA, dilution 1 x 10 −3 μg/μl) overnight at 4°C.

Techniques: Western Blot, Control, Concentration Assay, Positive Control

Journal: Science Advances

Article Title: The polymerase-associated factor 1 complex modulates the growth-defense tradeoff in Arabidopsis

doi: 10.1126/sciadv.ady5876

Figure Lengend Snippet: ( A ) Volcano plot analysis of gene expression in paf1-1 and Col-0 in RNA-seq assays. Up, up-regulated. Down, down-regulated. No-diff, no difference. ( B ) GO analysis of the up-regulated genes in paf1-1 . The top 14 significantly enriched GO terms are shown. ( C ) Relative expression of PR1 determined through RT-qPCR analysis. The data are represented as means ± SD ( n = 3). ( D ) Representative leaves at 3-days postinfection (dpi) by Psm ES4326 [optical density at 600 nm (OD 600 ) = 0.001]. Scale bar, 0.5 cm. ( E ) Growth of Psm ES4326 in the leaves at 3 dpi. The data are represented as means ± SD ( n = 8). ( F ) Schematic representation of the constructs used in the dual-luciferase reporter assays. 35 S, the 35 S promoter; proPR1 , the PR1 promoter; LUC, firefly luciferase; REN, renilla luciferase. ( G ) Dual-luciferase reporter assays. The reporter and effector were co-expressed in Arabidopsis protoplasts. The relative LUC activities normalized to the REN activities are shown (LUC/REN). The data are represented as means ± SD ( n = 3). ( H ) eChIP-seq assays. The 35S:PAF1-GFP /Col-0 seedlings were used for eChIP-seq analysis. The immunoprecipitation was carried out using an α-GFP antibody. Both the immunoprecipitated DNA and the input DNA were subjected to next-generation sequencing analysis. P1, P2, and P3 represent the regions used in the enhanced chromatin immunoprecipitation (eChIP)–quantitative polymerase chain reaction (qPCR) assays. ( I ) eChIP-qPCR assays. The ratios of eChIP and input are shown. UBQ5 serves as a negative control. The data are represented as means ± SD ( n = 3). The statistical significance was determined using a two-tailed Student’s t test. ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: The 35S:PAF1-GFP/ Col-0 transgenic Arabidopsis were treated with cycloheximide (50 μg/ml; #T1225, TargetMol, USA), 1 mM SA, and/or 50 μM MG132 (#133407-82-6, Aladdin) for 0, 4, or 16 hours.

Techniques: Gene Expression, RNA Sequencing, Expressing, Quantitative RT-PCR, Construct, Luciferase, Immunoprecipitation, Next-Generation Sequencing, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Negative Control, Two Tailed Test

Journal: Science Advances

Article Title: The polymerase-associated factor 1 complex modulates the growth-defense tradeoff in Arabidopsis

doi: 10.1126/sciadv.ady5876

Figure Lengend Snippet: ( A ) Representative images of PAF1-GFP in the 35S:PAF1-GFP /Col-0 transgenic Arabidopsis treated with or without 1 mM SA for 4 hours. Scale bar, 5 μm. ( B ) Protein level of PAF1-FLAG in the 35S:PAF1-FLAG /Col-0 transgenic Arabidopsis treated with 1 or 5 mM SA for 1 hour. The total proteins were subjected to immunoblotting analysis. ( C ) In vitro protein degradation assays. The recombinant His-PAF1 proteins were incubated with total protein extracts of Col-0 treated with or without 1 mM SA for 4 hours. ( D ) In vivo protein degradation assays. The 35S:PAF1-GFP /Col-0 transgenic Arabidopsis were treated with cycloheximide (CHX; 50 μg/ml), 1 mM SA, or 50 μM MG132. ( E ) Semi–in vitro ubiquitination assays. The recombinant GST and GST-PAF1 proteins coupled with glutathione beads were incubated with the total protein extracts of Col-0 treated with or without 1 mM SA in ubiquitination buffer for 4 hours. After washing, GST and GST-PAF1 proteins were eluted and subjected to immunoblotting analysis using α-Ubiquitin (Ub) antibody. ( F ) In vivo ubiquitination assays. The 35S:PAF1-GFP/ Col-0 transgenic Arabidopsis were treated with 50 μM MG132 and/or 1 mM SA for 4 hours. The proteins immunoprecipitated by α-GFP beads were subjected to immunoblotting analysis. ( G to I ) eChIP-qPCR assays. The plants were treated with or without 1 mM SA for 4 hours. The immunoprecipitation was carried out using α-GFP [(G) and (H)] or α-H3K9ac (I) antibodies, and the primers used for qPCR are shown in . The ratios of eChIP and input are shown. UBQ5 serves as a negative control. The data are represented as means ± SD ( n = 3). The statistical significance was determined using a two-tailed Student’s t test. ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: The 35S:PAF1-GFP/ Col-0 transgenic Arabidopsis were treated with cycloheximide (50 μg/ml; #T1225, TargetMol, USA), 1 mM SA, and/or 50 μM MG132 (#133407-82-6, Aladdin) for 0, 4, or 16 hours.

Techniques: Transgenic Assay, Western Blot, In Vitro, Recombinant, Incubation, In Vivo, Ubiquitin Proteomics, Immunoprecipitation, Negative Control, Two Tailed Test

Journal: Science Advances

Article Title: The polymerase-associated factor 1 complex modulates the growth-defense tradeoff in Arabidopsis

doi: 10.1126/sciadv.ady5876

Figure Lengend Snippet: ( A ) Schematic representation of the constructs used in the dual-luciferase reporter assays. ( B ) Dual-luciferase reporter assays. The reporter and effectors were coexpressed in Arabidopsis protoplasts. The relative LUC activities normalized to the REN activities are shown (LUC/REN). The data are represented as means ± SD ( n = 3). Different letters above each bar indicate significant differences determined by one-way ANOVA analysis followed by Tukey’s multiple comparison test ( P < 0.05). ( C ) Relative expression of PR1 in Arabidopsis treated with or without 1 mM SA for 4 hours. The data are represented as means SD ( n = 3). Different letters above each bar indicate significant differences determined by one-way ANOVA analysis followed by Tukey’s multiple comparison test ( P < 0.05). ( D ) eChIP-qPCR assays. 35S:PAF1-GFP/ Col-0 and 35S:PAF1-GFP/npr1-1 were treated with 1 mM SA for 4 hours. The immunoprecipitation was carried out using α-GFP antibody, and the primers used for qPCR are shown in . The ratios of eChIP and input are shown. UBQ5 serves as a negative control. The data are represented as means ± SD ( n = 3). The statistical significance was determined using a two-tailed Student’s t test. * P < 0.05 and *** P < 0.001. ( E ) Representative leaves at 3 dpi by Psm ES4326 (OD 600 = 0.0001). Scale bar, 0.5 cm. ( F ) Growth of Psm ES4326 in the leaves at 3 dpi. The data are represented as means ± SD ( n = 8). Different letters above each bar indicate significant differences determined by one-way ANOVA analysis followed by Tukey’s multiple comparison test ( P < 0.05). ( G ) Proposed working model.

Article Snippet: The 35S:PAF1-GFP/ Col-0 transgenic Arabidopsis were treated with cycloheximide (50 μg/ml; #T1225, TargetMol, USA), 1 mM SA, and/or 50 μM MG132 (#133407-82-6, Aladdin) for 0, 4, or 16 hours.

Techniques: Construct, Luciferase, Comparison, Expressing, Immunoprecipitation, Negative Control, Two Tailed Test