Journal: Frontiers in Pharmacology

Article Title: Enhanced therapeutic potential of paeoniflorin and vitamin B12 in intracerebropeduncle ethidium bromide-induced multiple sclerosis-like pathology

doi: 10.3389/fphar.2026.1792674

Figure Lengend Snippet: (A–E) PNN neuroprotective role in mitigating EBRO-induced alterations in neurotransmitter levels, including Acetylcholine (A) ; Dopamine (B) ; GABA (C) ; Glutamate (D) , and Serotonin (E) in brain homogenates. To identify significant differences between groups, a one-way ANOVA and Tukey’s post hoc test were used for statistical analysis (A–D) . The statistical significance level was set at p < 0.01, and the data were displayed as mean ± standard deviation (SD). There were eight wistar rats (n = 8) in each experimental group. β v/s Sham Control, Vehicle Control, and PNN Perse; δ v/s EBRO; δα1 v/s EBRO + PNN50; δα2 v/s EBRO + PNN100, EBRO + PNN50; and δα3 v/s EBRO + VB12 (30), EBRO + PNN100, EBRO + PNN50.

Article Snippet: Additionally, neurotransmitters like dopamine [KLR0219; Krishgen Biosystem, Mumbai, India], glutamate [KLR1474; Krishgen Biosystem, Mumbai, India], serotonin [KLR0866; Krishgen Biosystem, Mumbai, India], acetylcholine [KLR0722; Krishgen Biosystem, Mumbai, India] , and GABA [KLR0102; Krishgen Biosystem, Mumbai, India] were evaluated ( ).

Techniques: Standard Deviation, Control

Journal: bioRxiv

Article Title: Arginine synthesis pathway and ASS1 play a critical role in mRNA translation reprogramming and ICI resistance in cutaneous melanoma

doi: 10.64898/2026.03.17.712479

Figure Lengend Snippet: A , C57/BL6 mice were inoculated with murine Braf V600E Pten -/- melanoma cells (YUMM1.1), expressing a shRNA targeting ASS1 or a shRNA control. When tumors reached a mean of 100mm3, mice were treated with anti-PD-1 (4mg/kg-1) or isotype IgG control. The data are presented as the mean ± SEM for each group (n=10 mice per group). p-values were calculated using two-way ANOVA. B , Percentage of CD45-positive cells in tumors was determined by flow cytometry in tumors presented in B. The data are presented as the mean ± SEM (YUMM1.1shCTL n=9 tumors ; YUMM1.1shASS1 n=10 tumors). C , Deconvolution performed using CIBERSORTx performed on tumors from the experiment presented in A. D , Ratio CD8+ cells to malignant cells (Mal.) from the panel C is presented in tumor from the experiment presented in the panel A. E , Representative immunostaining of CD8+ T lymphocytes (red, CD8a) and nuclei (blue, DAPI) on frozen tumor sections of mice from panel A. F, Representation of CD8+ cells shown in D, the data are presented as the mean ± SEM (n=9 tumors). G. Representative immunostaining of CD8+ T cells and granzyme B on frozen tumor sections of mice from panel A.

Article Snippet: Antibodies InVivo Mab anti-mouse PD-1 (CD279), clone 29F.1A12 or InVivo Mab rat IgG2a isotype control, clone 2A3 were purchased from BioXCell, diluted in PBS and injected intraperitoneally (100μg in 100μL of PBS; 4mg.kg -1 per mouse).

Techniques: Expressing, shRNA, Control, Flow Cytometry, Immunostaining

Journal: bioRxiv

Article Title: Arginine synthesis pathway and ASS1 play a critical role in mRNA translation reprogramming and ICI resistance in cutaneous melanoma

doi: 10.64898/2026.03.17.712479

Figure Lengend Snippet: A , Immunoblot showing the expression levels of indicated protein in ICI-resistant YUMM1.1 cell line after 3 days of treatment with MDLA. HSP90 serves as a loading control. B , ASS1 enzymatic activity was performed in ICI-resistant YUMM1.1 cell line after 3 days of treatment with (n=3; mean ± SEM). C , Intracellular concentration of arginine was measure in ICI-resistant YUMM1.1 cells after 3 days of treatment with MDLA. (n=3; mean ± SEM). D. Schematic representation of the impact of arginine on the control upstream of mTORC1. E. Immunoprecipaption was performed on WM9 cells transfected with HA-tagged CASTOR1 and cultured for 3 days in the presence of different concentration of arginine and MDLA. Immunoblot showing the expression levels of indicated protein. F , Immunoblot showing the expression levels of indicated protein in ICI-resistant YUMM1.1 cell line. β-actine was use as a loading control. G , Immunoblot showing the expression levels of indicated protein in WM9 melanoma cell line. H , Renilla over Firefly luminescent ratio quantification in ICI-resistant YUMM1.1 cell line. I , Renilla over Firefly luminescent ratio quantification in WM9 melanoma cell line. J , YUMM1.1 cells treated or not with MDLA were then pulsed for 30 min with [ 35 S]Cys/Met, and the incorporation of 35 S into proteins was quantified and normalized to the total protein amount. Cells treated 30 minutes with 100µg/mL of cycloheximide (CHX) are used as a positive control. The data are presented as the mean (n = 3). K , WM9 cells treated or not with MDLA were then pulsed for 30 min with [ 35 S]Cys/Met, and the incorporation of 35 S into proteins was quantified and normalized to the total protein amount. Cells treated 30 minutes with 100µg/mL of cycloheximide (CHX) are used as a positive control. The data are presented as the mean (n = 3). L , Immunoblot showing the expression levels of indicated protein in cells from tumors of patients with metastatic melanoma. HSP90 serves as a loading control. M , Renilla over Firefly luminescent ratio quantification in cells from tumors of patients with metastatic melanoma (n=3; mean ± SEM). N , Cells isolated from tumors of patients with metastatic melanoma were treated or not with MDLA were then pulsed for 30 min with [ 35 S]Cys/Met, and the incorporation of 35 S into proteins was quantified and normalized to the total protein amount. Cells treated 30 minutes with 100µg/mL of cycloheximide (CHX) are used as a positive control. The data are presented as the mean (n = 3). O , C57BL/6 mice were inoculated with murine Braf V600E Pten -/- melanoma cells (YUMM1.1). When tumors reached a mean of 100mm 3 , mice were treated with MDLA (700mg.kg-1) or PBS, twice a day. Two days after the beginning of the MDLA treatment, mice were treated with anti-PD-1 (4mg/kg-1) or isotype IgG control, once per day, every two days, 3 times. Mice were treated with an additional dose of anti-PD-1 or isotype IgG control at day 24. Tumor growth was monitored for 27 days. The data representing the tumor growth are presented as the mean ± SEM for each group (n=10 mice per group). p -values were calculated using two-way ANOVA. P , Percentage of CD45-positive cells in tumors was determined by flow cytometry in tumors presented in P. The data are presented as a ratio of percentage between CD45-positive cells versus CD45-negative cells. (n=10 tumors per group, mean ± SEM). Q , Percentage of CD45 and CD8a-double positive cells in tumors, determined by flow cytometry in tumors presented in P. (n=10 tumors per group, mean ± SEM).

Article Snippet: Antibodies InVivo Mab anti-mouse PD-1 (CD279), clone 29F.1A12 or InVivo Mab rat IgG2a isotype control, clone 2A3 were purchased from BioXCell, diluted in PBS and injected intraperitoneally (100μg in 100μL of PBS; 4mg.kg -1 per mouse).

Techniques: Western Blot, Expressing, Control, Activity Assay, Concentration Assay, Transfection, Cell Culture, Positive Control, Isolation, Flow Cytometry

Journal: npj Viruses

Article Title: Site-1 protease mediated GPC processing is required for persistence of LCMV Clone 13

doi: 10.1038/s44298-026-00184-7

Figure Lengend Snippet: a – c Multi-step growth kinetics of rCl13, rCl13-RRRR, and rCl13-RRLL in WT ( a ), S1P-KO ( b ), and SP2-KO ( c ) HAP1 cells. Cells were seeded at 3 × 10 5 cells/well in a 24-well plate and 24 h later infected (MOI = 0.01) with the indicated virus. Cell culture supernatants (CCSs) were collected at indicated hours post infection (hpi), and infectious virus titers were determined by focus-forming assay (FFA). d Processing of GPC in WT, S1P-KO, and SP2-KO HAP1 cells infected (MOI = 1) with rCl13, rCl13-RRRR, or rCl13-RRLL. At 24 hpi, cell lysates were prepared and analyzed by western blotting using the mouse monoclonal G204 to GPC. e Multi-step growth kinetics of rCl13, rCl13-RRRR, and rCl13-RRLL in furin-deficient LoVo cells and LoVo-furin cells with reconstituted furin expression. Cells were seeded at 3 × 10 5 cells/well in a 24-well plate and 24 h later infected (MOI = 0.01) with the indicated virus. CCSs were collected at the indicated hpi, and infectious virus titers were determined by FFA. f , g Susceptibility of rCl13 and rCl13-RRRR to furin inhibitor BOS-318 ( f ) and S1P inhibitor PF-429242 ( g ). A549 cells were seeded in 96-well plates at 2 × 10 4 cells/well and 24 h later infected (MOI = 0.01) with the indicated virus and treated with serial dilutions (1:2, 6 replicates per dilution) of either BOS-318 (starting at 2 µM), or PF-429242 (starting at 40 µM). At 72 hpi, cell viability was determined by CellTiter 96 Aqueous One Solution (purple), then cells were fixed with 4% PFA. The level of infection was assessed by IF using the rat monoclonal antibody VL4 to NP (green), and cell viability was also alternatively determined by DAPI staining (blue). Raw values of CellTiter, NP, and DAPI staining were normalized to vehicle-treated and infected control cells present in each plate. Dose-response curves for BOS-318 and PF-429242 were generated using a nonlinear regression model with log(inhibitor) vs response – variable response (four parameters) equation in GraphPad Prism. The dashed line represents 50% of vehicle control. h Effect of PF-429242 on multi-step growth kinetics of rCl13 and rCl13-RRRR in A549 cells. A549 cells were seeded at 2.5 × 10 5 cells/well in a 24-well plate and 24 h later infected (MOI = 0.01) with the indicated virus. After 90 min adsorption, the virus inoculum was removed, and medium containing the indicated concentration of PF-429242 was added to the cells. At the indicated hpi, CCSs were collected and virus titers determined by FFA. Cells lacking S1P or treated with PF-429242 were grown in media supplemented with lipids (1 mM sodium mevalonate, 20 μM sodium oleate, 5 μg/ml cholesterol).

Article Snippet: Cells were fixed with 4% PFA at 72 hpi and stained with rat mAb VL4 against LCMV NP (Bio X Cell), then with anti-rat AF488-conjugated antibody.

Techniques: Infection, Virus, Cell Culture, Focus Forming Assay, Western Blot, Expressing, Staining, Control, Generated, Adsorption, Concentration Assay

Journal: npj Viruses

Article Title: Site-1 protease mediated GPC processing is required for persistence of LCMV Clone 13

doi: 10.1038/s44298-026-00184-7

Figure Lengend Snippet: a Eight-week-old B6 mice were infected with rCl13, rCl13-RRRR ( n = 5/group, 2 × 10 6 FFU/mouse, IV), or mock-infected ( n = 2, 200 µL PBS, IV). Spleens were harvested at 24 hpi and processed to generate single-cell suspensions. Specific cell types were identified by flow cytometry through surface marker staining: B cells: B220+, CD3−, CD4−, Siglec-H−; CD4 T cells: B220−, CD3+, CD4+; CD8 T cells: B220−, CD3+, CD8+; plasmacytoid dendritic cells (pDCs): B220+, CD3−, CD4+, Siglec-H+; natural killer (NK) cells: B220−, CD3−, NK1.1+; CD8+ dendritic cells (DCs): B220−, CD3−, NK1.1−, Ly6G−, CD11c high , MHC-II+, CD11b low , CD8+; CD11b+ cDCs: B220−, CD3−, NK1.1−, Ly6G−, CD11c high , MHC-II+, CD11b+, CD8−; macrophages: B220−, CD3−, NK1.1−, Ly6G−, CD11c low , CD11b−, F4/80+; Ly6C+ monocytes: B220−, CD3−, NK1.1−, Ly6G−, CD11c low , CD11b high , Ly6C high ; Ly6C- monocytes: B220−, CD3−, NK1.1−, Ly6G−, CD11c low , CD11b high , Ly6C low . Intracellular NP was stained with AF488-conjugated VL4 rat monoclonal antibody. b shows the representative contour plots of NP + gates from the macrophage populations. c , d Infection of B6 mice and processing of spleen samples were done as in ( a ). Specific cell types were identified by flow cytometry through surface marker staining: metallophilic marginal-zone macrophages (MMM): lin-(CD19−, CD3−, NK1.1−), Ly6G−, Ly6C−, F4/80+, CD11b high , CD169 high ; red pulp macrophages (RPM): lin-(CD19−, CD3−, NK1.1−), Ly6G−, Ly6C−, F4/80+, CD11b low , CD169−. Intracellular NP was stained with AF488-conjugated VL4 rat monoclonal antibody. Representative contour plots of NP + gates from the parental gates are shown. A statistical test (unpaired t -test) was done between rCl13 and rCl13-RRRR groups in GraphPad Prism. P values are shown or represented by asterisks (ns not significant; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001). e , f Eight-week-old B6 mice were infected as in ( a ). Spleens were collected at 24 hpi and split in half. One half was flash frozen in OCT and 8 µm tissue sections were stained with human anti-GPC antibody (green), rat anti-CD169 antibody (red) and DAPI (blue). Sections were prepared for four mice per group, and representative images from each group are shown. The other half of the spleen was homogenized and virus titers determined by FFA. A statistical test (unpaired t -test) was done between rCl13 and rCl13-RRRR groups in GraphPad Prism. P values are represented by asterisks (ns not significant; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001).

Article Snippet: Cells were fixed with 4% PFA at 72 hpi and stained with rat mAb VL4 against LCMV NP (Bio X Cell), then with anti-rat AF488-conjugated antibody.

Techniques: Infection, Single Cell, Flow Cytometry, Marker, Staining, Virus